Changes in Cardiotrophin-1 and Fibroblast Growth Factor-21 with Weight Loss by Robert Lawrence Bowers A disertation submited to the Graduate Faculty of Auburn University in partial fulfilment of the requirements for the Degre of Doctor of Philosophy Auburn, Alabama December 18, 2009 Approved by Peter Grandjean, Chair, Asociate Profesor of Kinesiology L. Bruce Gladden, Profesor of Kinesiology Lawrence Wit, Profesor of Biological Sciences Suresh Mathews, Asistant Profesor of Nutrition and Food Science i Abstract The purpose of this investigation was to examine the efects of modest weight loss on circulating concentrations of cardiotrophin-1 (CT-1) and fibroblast growth factor-21 (FGF-21) in obese men. Nine obese men (age = 41.5 ? 7.1; weight = 101.7 ? 21.0 kg; BMI = 32.8 ? 3.6 kg/m 2 ; % fat = 35.2 ? 4.3) were asigned to either an exercise or dietary restriction intervention. Seven age-matched controls (age = 42.3 ? 8.5; weight = 74.5 ? 5.0 kg; BMI = 24.8 ? 1.4 kg/m 2 ; % fat = 25.2 ? 5.5) served for comparison purposes. Control individuals were not asigned to an intervention. The overal targeted weight loss for both interventions was 8 to 10% of initial body weight over a 6 to 10 month period. A blood sample and DEXA scan were obtained for each participant at baseline and every four weks during the intervention in the obese group. Blood samples were analyzed for CT-1, FGF-21, glucose, insulin, non-esterified faty acids (NEFA), adiponectin, IL-6, TNF-!, myeloperoxidase (MPO), and total antioxidant capacity (TAC). Glucose to insulin ratio (GIR), homeostasis model index (HOMA) and the quantitative insulin sensitivity check index (QUICKI) were used as clinical indexes of metabolic health. Diferences betwen groups were determined by independent t-test. Changes in blood variables were determined by repeated measures ANOVA and paired t-test. Pearson product moment correlation coeficients were used to determine the relationship betwen baseline physiological and metabolic characteristics and dependent variables (p < 0.05 for al). Neither CT-1 nor FGF- 21 concentrations difered betwen groups at baseline. No significant change in CT-1 concentrations occurred with 8 to 10% weight loss. FGF-21 concentrations significantly decreased by 57.3% after weight loss of 8 to 10%. Significant reductions in total and regional body fat, lean mas, insulin, GIR, HOMA and QUICKI also occurred with weight loss. Reductions in FGF-21 occurred along with those in total and regional body fat and improvements in insulin sensitivity. These results indicate that FGF-21 may be a potential clinical indicator of improvements in metabolic regulation that occur with modest weight loss. ii Table of Contents Abstract??????????????????????????????????...i List of Tables????????????????????????????????..v List of Figures????????????????????????????????.vi Introduction?????????????????????????????????..1 Obesity and Metabolic Syndrome?????????????????????...1 Cardiotrophin-1 and Fibroblast Growth Factor-21???????????????..3 Lifestyle Modification and Weight Loss???????????????????.5 Purpose????????????????????????????????.6 Questions, Hypotheses and Rationale????????????????????..6 Asumptions, Limitations and Delimitations?????????????????.11 Significance of Study??????????????????????????.12 Review of Literature?????????????????????????????..14 Obesity and Metabolic Syndrome?????????????????????..14 Pathophysiology of Obesity???????????????????????..18 Cytokines, Cardiotrophin-1 and Fibroblast Growth Factor-21??????????.34 Weight Loss and Lifestyle Modification??????????????????..72 Methods??????????????????????????????????..81 Subjects???????????????????????????????..81 Preliminary Screning and Baseline Procedures??????????????..81 Weight Loss Procedures?????????????????????????82 iv Exercise Intervention??????????????????????????.83 Diet Intervention????????????????????????????84 Biochemical Procedures and Analysis???????????????????..84 Statistical Analysis???????????????????????????.85 Results???????????????????????????????????86 Baseline Physiological Characteristics???????????????????..86 Weight Loss and Tisue Loss???????????????????????89 Humoral Indices of Metabolic Homeostasis?????????????????..92 Cardiotrophin-1, Fibroblast Growth Factor-21 and Cytokine Responses?????...94 Oxidative Stres Responses???????????????????????..97 Physical Activity and Diet???????????????????????...98 Coeficients of Variation for Biochemical Analyses?????????????...99 Discussion?????????????????????????????????100 Changes in Fibroblast Growth Factor-21 with Weight Loss??????????..100 Changes in Cardiotrophin-1 with Weight Loss???????????????..103 Weight Loss and Insulin Sensitivity????????????????????104 Outside Influences on These Findings???????????????????.106 Overal Findings???????????????????????????..107 Clinical Significance and Conclusions???????????????????108 References?????????????????????????????????110 v List of Tables Table 1. NCEP ATP II: the metabolic syndrome criteria???????????????.16 Table 2. Factors regulating CT-1 expresion????????????????????.41 Table 3. Factors regulated by CT-1???????????????????????..42 Table 4a. Baseline physiological characteristics??????????????????..87 Table 4b. Baseline humoral and metabolic parameters????????????????.88 Table 5a. Changes in body fat and lean tisue distribution???????????????91 Table 5b. Change values for body fat and lean tisue?????????????????92 Table 6a. Weight los responses in humoral indices of metabolic homeostasis???????93 Table 6b. Change values for humoral indices of metabolic homeostasis?????????..94 Table 7a. CT-1 and FGF-21 responses to weight los????????????????..95 Table 7b. Change values for CT-1 and FGF-21???????????????????95 Table 8a. Cytokine responses to weight loss????????????????.........96 Table 8b. Cytokine change values????????????????????????.97 Table 9a. Weight loss responses of MPO and TAC?????????????????..97 Table 9b. Change values for MPO and TAC????????????????????.98 vi List of Figures Figure 1. JAK/STAT signaling?????????????????????????..38 Figure 2. Overview of cytokine signaling?????????????????????..40 Figure 3. Cardiotrophin-1 signaling???????????????????????..44 Figure 4. FGF-21 signaling in adipose tisue????????????????????55 Figure 5. Percent changes in body weight?????????????????????.90 Figure 6. Changes in FGF-21 with weight loss???????????????????.96 Figure 7. Changes in MPO with weight loss????????????????????.98 1 Chapter I. Introduction Obesity and Metabolic Syndrome Obesity is a major public health isue leading to a greater risk of al cause mortality and a predisposition to chronic diseases including atherosclerosis, hypertension, diabetes melitus and cancer (37, 76, 126). Until recently the role of fat cels in the pathogenesis of obesity and metabolic syndrome was thought to be a pasive one. Now adipose tisue is known to be an active endocrine organ, playing a major role in metabolic control (173, 177, 257, 440, 550). Obesity develops due to an imbalance betwen energy intake and energy expenditure (341). Adipose tisue normaly functions as the main storage area for dietary lipids. Increased dietary consumption without an increase in energy expenditure leads to adipose tisue growth and a reduction in adipose tisue lipid storage potential (173). Tisues that, under normal circumstances, do not function as lipid storage sites are forced to acommodate the exces energy causing ectopic fat acumulation at the liver, skeletal muscle, and pancreas (173, 177, 440). Consequently, increased hepatic glucose and very- low density lipoprotein production, elevated insulin secretion, as wel as decreased skeletal muscle glucose uptake occur (173, 177). Excesive faty acids in circulation and ectopic fat acumulation disrupt metabolic pathways and endocrine function in many tisues (257, 352). Moreover, the rapid acumulation of adipose tisue outpaces tisue circulation requirements, leading to a hypoxic state and eventual tisue necrosis (173). Macrophage infiltration and dysfunctional cytokine secretion take place in response to the hypoxic environment and perishing adipocytes, further leading to an insulin resistant state (173, 440). As such, obesity has been characterized by systemic oxidative stres caused by hyperglycemia, elevated tisue lipid levels, chronic inflamation, inadequate antioxidant defenses and reactive oxygen species (ROS) formation (571, 573). 2 Metabolic syndrome (MetS) is a constelation of interelated conditions including central obesity, dyslipidemia, hypertension and hyperglycemia that directly promote cardiovascular disease (CVD), insulin resistance and type 2 diabetes (179, 182, 222). Obesity appears to contribute to MetS via abdominal body fat distribution as opposed to the absolute amount of adipose tisue (37). Therefore, exces intra-abdominal adiposity is key in the pathogenesis of obesity and a host of acompanying cardiovascular and metabolic disorders (35, 173, 177, 550). Adipose tisue secretes a wide aray of biologicaly active proteins caled cytokines, or more specificaly, adipocytokines or adipokines (37, 486). Since some of these factors greatly influence insulin sensitivity, glucose metabolism, inflamation and atherosclerosis, they may provide a molecular link betwen obesity, insulin resistance and the development of type 2 diabetes and cardiovascular disease (222). Some of the most wel characterized adipocytokines are leptin, adiponectin, TNF-! and IL-6 (159, 173, 177, 222, 440, 550). Adiponectin exerts insulin-sensitizing and anti-atherogenic efects that include lowering of hepatic glucose production and increasing glucose uptake and faty acid oxidation in skeletal muscle (35, 222, 331). Adiponectin is down-regulated in obesity and a significant inverse relationship exists betwen adiponectin and markers of oxidative stres that are observed in states of exces obesity (159, 222, 331, 571, 573). TNF-! plays a key role in mediating imune system response (372). TNF-! regulates the production of other cytokines such as IL-1 and IL-6, inflamation and cel apoptosis and promotes cel survival (372). TNF-! expresion is increased in obesity and contributes to insulin resistance by directly inhibiting insulin-signaling (5, 550). Increased TNF-! levels induce the production of reactive oxygen species as part of its activity in the inflamatory response (634). IL-6 is closely asociated with obesity and insulin resistance (550). IL-6 stimulates an increased production of adhesion molecules in inflamed and dysfunctional tisue resulting in the recruitment of leukocytes to inflamed sites. IL-6 may also increase IL-1 and TNF-! synthesis (531). Elevated IL-6 expresion leads to insulin resistance through a down-regulation of insulin receptor substrate (440, 457). Increased circulating concentrations of IL-6 are demonstrated with impaired glucose tolerance and 3 hyperlipidemia and are implicated in development of type 2 diabetes (31, 440, 575). Furthermore, reactive oxygen species raise the mRNA expresion levels of IL-6 (159). There are 50 to 100 adipocytokines that have been identified to date (486). The physiology of many of these proteins remains poorly understood and the functional roles are currently being characterized. Two novel bioactive peptides secreted from adipose tisue that have received considerable recent atention and may further explain the link betwen obesity, insulin resistance, cardiovascular disease and oxidative stres are cardiotrophin-1 and fibroblast growth factor-21 (73, 268, 308, 365, 636, 645). Cardiotrophin-1 and Fibroblast Growth Factor-21 Cardiotrophin-1 (CT-1) was first identified in 1995 based on its ability to induce cardiac myocyte hypertrophy (422, 423). CT-1 expresion is high in heart, skeletal muscle, prostate, ovaries, liver, lung, kidney and adipose tisues (365, 424). Expresion of CT-1 is up-regulated in states of obesity and in individuals with MetS (334, 365). CT- 1 has wel documented efects on the myocardium that include hypertrophy and prevention of apoptosis in response to myocardial ischemia (299). Elevated CT-1 levels are found along with failing left ventricular myocardium, left ventricular hypertrophy, treated and untreated hypertension, and vascular dysfunction (319, 320, 334, 421, 453, 644). CT-1 contributes to the proposed etiology of MetS by inhibiting adipocyte insulin signaling and insulin-stimulated glucose uptake (525, 645). CT-1 and some ROS regulate one another through a positive fedback loop as CT-1 potentiates ROS production, which in turn, can lead to further CT-1 expresion (21, 482). These data suggest that the close asociation betwen obesity, hypertension, insulin resistance, vascular inflamation and hypoxia may acount for the high circulating concentrations of CT-1 observed with obesity and conditions contributing to MetS. Therefore, CT-1 concentrations may present a possible link betwen obesity, oxidative stres, cardiovascular disease and type 2 diabetes (365, 525). In 2000, fibroblast growth factor-21 was first identified as a secreted protein preferentialy expresed in the liver (374). Recently FGF-21 expresion was found in adipose tisue (358, 636). It was not until 2005 that FGF-21 was determined to be a potent metabolic regulator (268). These metabolic actions include lowering blood 4 glucose through decreased insulin resistance and increased GLUT-1-mediated glucose uptake in adipocytes (268, 269, 355, 592). Reductions in fasting insulin, triglyceride and LDL-cholesterol levels are also reported with FGF-21 treatment (268, 269, 592). FGF-21 induces an increase in adipocyte PAR-" expresion; thereby, increasing adiponectin expresion and reducing oxidative stres by inhibiting the formation of reactive oxygen species (357, 358, 376, 389). FGF-21 administration in animals increased energy expenditure and fat oxidation, acelerated weight loss and decreased adiposity and body weight (89, 269). However, FGF-21 data in humans does not appear to be consistent with that sen in animal models. In humans, elevated levels of FGF-21 are observed in obesity, type 2 diabetes and MetS (73, 308, 636). FGF-21 concentrations are elevated along with BMI and body fat percentage as wel (73, 111, 308, 636). Individuals with hypertension, elevated triglycerides, hyperglycemia and increased fasting insulin also exhibit increased FGF-21 concentrations (73, 307, 308, 636) The elevated concentrations of FGF-21 in human obesity may be a result of FGF- 21 resistance. However, other hypotheses for the greater FGF-21 concentrations in obese humans versus their normal weight counterparts have been proposed (475). Lower adiponectin levels observed with elevated FGF-21 concentrations in human obesity sem counter-intuitive (111, 636). Both adiponectin and FGF-21 are gene targets of the nuclear receptor PAR-" (357, 358, 580). PAR-" is suppresed in obesity and with oxidative stres (376). This relationship would predict decreased levels of FGF-21 in human obesity and states of elevated oxidative stres. The opposite has actualy been observed suggesting a level of FGF-21 resistance (73, 308, 475, 636). At present, the manner in which exces adipose tisue and body weight and body fat loss influence concentrations of CT-1 and FGF-21 are not characterized. In addition, the metabolic efects obtained by changes in these adipocytokines with weight and body fat loss are not wel understood. 5 Lifestyle Modification and Weight Loss Weight loss through lifestyle modification (exercise and/or hypocaloric diet) is beneficial for treating excesive adiposity, dyslipidemia, hypertension, hyperglycemia and insulin resistance (87, 413). The magnitude of weight loss does not need to be large, as even modest weight loss of 5-10% significantly atenuates metabolic dysfunction (87). A decrease in caloric intake is an avenue by which to create a negative energy balance resulting in weight loss (476). It is prudent to recomend a reduced calorie diet low in saturated fat, higher in unsaturated fat, high in whole grains and low in sodium (87). However, more important than the composition of the diet, is the overal caloric intake. Regardles of which macronutrients are emphasized, hypo-caloric diets are key to inducing meaningful weight loss (476). A lifestyle aimed at increasing physical activity/energy expenditure and decreasing, or even maintaining, body weight is another important approach to reducing health risks (87). Increased physical activity and higher cardio-respiratory fitnes, independent of weight loss, are asociated with decreased CVD risk and lower incidence of type 2 diabetes and MetS (87, 298, 383, 585, 622). Exercise is also particularly efective at improving insulin sensitivity as wel as reducing dyslipidemia and hypertension (23, 87, 106, 474, 524). Cardiorespiratory fitnes sems to have an independent efect on metabolic function in some aspects; however, a change in body weight and body composition (particularly abdominal adiposity), is an important mediator in the ability of increased physical activity to modify chronic disease risk (87). Regular exercise appears to play an important role in abdominal fat loss and the metabolic changes that ensue (87, 466). The temporal efects of weight loss are not yet firmly established in the literature. However, evidence suggests that metabolic improvements may occur rapidly within the first few eks of lifestyle modification and continue at a slower rate as weight loss continues. Clinical markers of metabolic health that demonstrate this trend include triglycerides, glucose, insulin, hemoglobin A1c, insulin sensitivity and biomarkers of oxidative stres (96, 262, 318, 345, 478, 513). 6 Purpose Enlargement of adipocytes, impaired adipose tisue blood flow, adipose tisue hypoxia and macrophage infiltration of adipose tisue are interelated and may lead to dysregulated adipokine secretion and ectopic fat storage, which together may result in insulin resistance and further chronic disease (173). The purpose of the present investigation is to examine the temporal efects of modest weight loss on circulating levels of CT-1 and FGF-21 in obese men. Both of these cytokines appear to afect insulin sensitivity and may possibly play a role in the link betwen obesity, insulin resistance and chronic disease. A secondary purpose is to characterize changes in CT-1 and FGF-21 with those observed for oxidative stres, cytokine responses and clinical markers of metabolic health. Questions, Hypotheses and Rationale Question: 1. What are the diferences in circulating concentrations of CT-1 and FGF-21 betwen the control and obese individuals in our cohort? Hypotheses: Null: There are no diferences in CT-1 and FGF-21 levels betwen obese middle-aged men and age matched controls. Alternative: Circulating CT-1 and FGF-21 levels are significantly higher in obese individuals as compared to age-matched controls. Rationale: Obesity is characterized by excesive adipose tisue size, decreased adipose tisue storage capabilities, ectopic fat distribution, adipose tisue hypoxia, and dysregulated adipokine secretion (173, 177, 440). It has recently been determined that adipose tisue is a source of CT-1 and FGF-21 expresion (365, 636). Furthermore, previous work demonstrates that there are diferences in CT-1 and FGF-21 concentrations betwen 7 obese and lean individuals (73, 308, 365, 636). CT-1 expresion is up-regulated in obese individuals and in persons with MetS (334, 365). Elevated levels of FGF-21 are also reported in states of obesity and in persons with type 2 diabetes and MetS (73, 308, 636). It appears that in obese individuals, levels of the cytokines CT-1 and FGF-21 wil be elevated significantly versus concentrations observed in lean controls due to an increased amount of adipose tisue and ectopic fat acumulation contributing to vascular and tisue inflamation (37, 173, 177, 550). Question: 2. What is the efect of 8-10% weight loss on circulating levels of CT-1 and FGF-21? Hypotheses: Null: Weight loss of 8-10% has no efect on circulating amounts of CT-1 and FGF-21. Alternative: Weight loss of 8-10% significantly decreases circulating concentrations of CT-1 and FGF-21. Rationale: Weight loss through lifestyle modification is beneficial for treating excesive adiposity, dyslipidemia, hypertension, hyperglycemia and insulin resistance (87, 413). Modest weight reductions of 5-10% significantly atenuate metabolic dysfunction (87). Currently, there is no evidence in the literature that we know of regarding the efect of weight loss on CT-1 and FGF-21 concentrations. However, data show that adipose tisue is a source of CT-1 and FGF-21, which may acount for the high circulating concentrations of these bioactive peptides observed in obese humans (73, 308, 365, 525, 636). Moreover, it is likely that vascular and tisue inflamation exists as a result of ectopic fat acumulation (37, 173, 177, 550). Since several tisues produce CT-1 and FGF-21, circulating levels may also be reflective of this vascular and non-adipose tisue inflamation (268, 365, 374, 424). As previously noted, weight loss, through diet or exercise, is efective in reducing body fat and excesive adiposity and in normalizing adipokine secretion (87, 413, 462). Thus, it appears prudent to predict that weight loss- 8 induced reductions in adipose tisue, ectopic fat acumulation and atenuation of dysregulated adipokine secretion wil serve to lower circulating concentrations of CT-1 and FGF-21. Along with the possibility of increased adipose tisue expresion of FGF-21, there are thre rational explanations for why FGF-21 concentrations are increased in obesity. The increase may be an indirect sign of FGF-21 resistance, a protective mechanism or possibly indicates the presence of truncated, inactive form of the protein in circulation (475). If inactive isoforms are present in obesity, it may be that with weight loss FGF-21 levels do not decrease, but normal FGF-21 production and/or signaling is restored. Question: 3. When do changes in circulating concentrations of CT-1 and FGF-21 occur and does the magnitude of change continue as weight reduction progreses? Hypotheses: Null: Progresive weight loss does not have an efect on circulating concentrations of CT-1 and FGF-21. Alternative: Lifestyle intervention and weight loss initialy causes a rapid change in circulating concentrations of CT-1 and FGF-21, which continues at a slower rate over time until the desired 8-10% weight loss is achieved. Rationale: In order to experience the health benefits asociated with weight loss, the magnitude of weight loss does not need to be large, as even modest reductions of 5-10% significantly atenuate metabolic dysfunction (87). However, do these changes occur linearly over time or do they occur rapidly at first and then continue at a slower rate until the desired weight loss is achieved? Furthermore, are the changes related to total body fat or regional body fat reduction? In the literature, the answer to this question is not clear. A hypothesis regarding the temporal responses of FGF-21 and CT-1 to weight loss can be formulated through the responses of oxidative stres, glucose, insulin and insulin 9 sensitivity. Rapid reductions in markers of oxidative stres are sen before weight loss and changes in body composition occur with lifestyle modification (96, 318, 412, 478, 513, 571, 573). Reductions continue; however, at a slower rate throughout the intervention (96, 318, 345, 478, 513). In addition, total fat mas and abdominal visceral adipose tisue decrease in line with biomarkers of oxidative stres during weight loss (168). FGF-21 is a gene target of PAR-", which is up-regulated by weight loss and also known to reduce oxidative stres (357, 358, 376, 389, 568). Through the activity of PAR-", oxidative stres and FGF-21 appear to be linked. This relationship suggests that FGF-21 levels wil increase with reductions in oxidative stres occurring with weight loss. On the other hand, it is known that FGF-21 concentrations and oxidative stres levels are increased in obese humans (73, 308, 342, 571, 573, 636). A relevant hypothesis can be formulated for each case. However, based on human data it appears that FGF-21 concentrations wil most likely decrease along with reductions in oxidative stres, increases in adiponectin and enhancements in insulin sensitivity during weight loss. Furthermore, if it is true that in obesity truncated, inactive forms of the protein are present, it could be postulated that FGF-21 levels wil not decrease, but normal FGF-21 signaling/production wil be restored with weight loss. CT-1 and reactive oxygen species directly regulate one another through a positive fedback loop as elevated concentrations of CT-1 induce production of ROS and vice versa (21, 482). Therefore, it appears that CT-1 levels wil miror reductions in markers of oxidative stres based on these data and data from cases of human obesity (21, 365, 482). Reductions in glucose and insulin and enhancements in insulin sensitivity with weight loss appear to follow the same trend as do markers of oxidative stres (73, 87, 262, 292, 516). Based on interactions demonstrated in the literature, CT-1 and FGF-21 responses to weight loss may miror those of insulin, glucose and insulin sensitivity (73, 111, 365, 636, 645). CT-1 expresion is enhanced by glucose in a dose dependent manner and CT-1 levels are significantly higher in subjects with hyperglycemia (365). Furthermore, chronic administration of CT-1 causes insulin resistance and hyperinsulinemia (645). Also, CT-1 inhibits insulin action through decreasing the expresion of IRS-1 (645). Fasting blood glucose and plasma insulin concentrations were 10 found to independently influence plasma FGF-21 levels (73). Increased FGF-21 concentrations were also reported along with elevated fasting insulin levels and decreased insulin sensitivity (73, 111, 636). These relationships with oxidative stres, glucose, insulin and insulin sensitivity provide evidence that the temporal changes in CT-1 and FGF-21 may occur rapidly at the outset of the weight loss intervention and then continue at a slower pace as weight loss progreses. Question: 4. Do changes in circulating concentrations of CT-1 and FGF-21 occur along with improvements in clinical markers of metabolic health (glucose, insulin, NEFA, adiponectin, biomarkers of oxidative stres) known to occur with weight loss? Hypotheses: Null: Circulating amounts of CT-1 and FGF-21 are not asociated with improvements in clinical markers of metabolic health known to occur with weight loss. Alternative: Circulating concentrations of CT-1 and FGF-21 wil decrease in conjunction with reductions in glucose, insulin, NEFAs and biomarkers of oxidative stres and increases in adiponectin levels. Rationale: Weight loss through lifestyle modification is efective in causing changes in clinical markers of metabolic health such as glucose, insulin, non-esterified faty acids, adiponectin and reactive oxygen species (ROS) (87, 96, 142, 318, 373, 403, 412, 413, 439, 513, 568, 571). It is possible that alterations in circulating levels of CT-1 and FGF- 21 may miror these changes based on mechanisms and relationships presented in the literature. For example, CT-1 concentrations are elevated along with increased levels of insulin and glucose (365). CT-1 also down-regulates the nuclear receptor PAR-" most likely resulting in decreased expresion of the PAR-" target and insulin sensitizing agent adiponectin (358, 645). Furthermore, CT-1 and reactive oxygen species appear to 11 regulate one another through a positive fedback loop in that CT-1 potentiates ROS production, which in turn, leads to further CT-1 expresion (21, 482). In humans, FGF-21 concentrations are elevated along with circulating faty acids, glucose and insulin (73, 111, 308, 636). FGF-21 is also a gene target of PAR-", which up-regulates adiponectin expresion and inhibits the formation of ROS (357, 358, 580). Weight loss results in an up-regulation of PAR-" that should cause an increase in FGF- 21 and adiponectin and a decrease in ROS (568). However, FGF-21 levels are already increased in states of human obesity hinting at possible FGF-21 resistance (73, 308, 475, 636). Based on these data, FGF-21 levels may decrease with weight loss, despite being a gene target of PAR-". In other words, reductions in FGF-21 hypothesized here to occur with weight loss wil likely occur along with elevations in adiponectin and decreases in biomarkers of oxidative stres. It is possible that the link betwen FGF-21 and levels of faty acids, glucose and insulin may be stronger than the efects levied by weight loss- induced up-regulation of PAR-". Based on these specific relationships, CT-1 and FGF- 21 concentrations should decrease along with weight loss-induced reductions in glucose, insulin, NEFAs and biomarkers of oxidative stres and elevations in adiponectin demonstrated in the literature (87, 96, 142, 318, 373, 403, 412, 413, 439, 513, 568, 571). Asumptions 1. A major contribution to changes in blood variables reflected metabolic shifts resulting from improvements in cardiovascular fitnes and decreases in body weight, adipose tisue, hypoxia and inflamation. 2. The diet group remained physicaly inactive throughout the duration of the study. 3. The exercise group did not engage in a hypo-caloric diet during the study. 4. The exercise group exercised as instructed outside of the lab. 5. Both groups acurately reported their diet and physical activity. Limitations 1. We could not control the participant?s slep habits, stres levels and outside physical activities. 12 2. There are inherent limitations to the diet recording proces such as under estimating portion sizes and inacurate reporting. 3. Nutrient composition and caloric intake were not controlled. 4. We were limited in the number of dependent blood variables that we could measure. Delimitations 1. Only physicaly inactive males betwen the ages of 30 and 65 years were recruited for the study. 2. Al participants had no prior history of extreme weight loss and had been relatively weight stable over the previous 6 months. 3. Al participants were recruited from Auburn, Alabama and the surrounding areas. 4. An intensity of 60-70% of VO 2 max was used to expend the desired amount of calories for al exercise sesions. Significance of Study Excesive adiposity reflects a chronic imbalance of energy intake exceding energy expenditure. In obese individuals, this excesive adiposity contributes to the development of metabolic dysfunction, in part, due to the inability to store additional lipids, ectopic fat distribution, the creation of a hypoxic state within the tisue, and abnormal adipokine secretion (173). On the other hand, weight loss through lifestyle modification is beneficial for treating excesive adiposity, dyslipidemia, hypertension, hyperglycemia and insulin resistance (87, 413). Weight loss generated by hypo-caloric diets and regularly practiced exercise can create the negative energy balance necesary to generate these beneficial efects. Recent evidence supports the theoretical role of cytokines and adipokines in obesity and weight loss (37, 126, 143, 222). Both CT-1 and FGF-21 may be related to the metabolic dysfunction occurring with obesity as wel as play a role in the metabolic and cardiovascular health improvements asociated with weight loss (268, 365, 636, 645). However, there appear to be limited data in the literature that directly addreses the response of CT-1 and FGF-21 to weight loss in humans. Therefore, the importance of this study lies in its potential to contribute 13 foundational information that wil be useful and relevant to our understanding of the responses of CT-1 and FGF-21 to obesity and modest weight loss. 14 Chapter I. Review of Literature Obesity and Metabolic Syndrome Overweight and obesity are a major public health isue afecting nearly half a bilion of the world?s population (469). The World Health Organization estimates that by 2015, the number of overweight people globaly wil increase to 2.3 bilion and greater than 700 milion wil be obese (3). Once considered only a problem in high-income countries, obesity rates are now rising dramaticaly in developing nations (3). In the United States 30% of al adults are presently overweight and 32% are obese (178, 382). Obesity rates in Europe range from 10 to 20% in men and 10 to 25% in women (469). As a result, the cost of obesity is betwen 2 and 8% of total health care budgets in al parts of the world, irespective of the health care system (469). In the United States alone, annual medical spending due to overweight and obesity is estimated as $92.6 bilion, which represents 9.1% of US health expenditures (135). Hospital costs and the use of medication increase with increasing BMI. In a large health maintenance organization, annual costs were 25% higher for individuals with a BMI betwen 30 and 35 kg/m 2 and 44% higher for a BMI greater than 35 kg/m 2 than in those with a BMI betwen 20 and 25 kg/m 2 (54, 443). In addition, complications asociated with obesity contribute to 100,000 to 400,000 deaths per year (350). Data show that a BMI of greater than 30 reduces life expectancy by 3 to 5 years (418). Moreover, a 30% increase in al cause mortality was reported in persons with a BMI betwen the 85 th and 95 th percentiles (117). Both elevated healthcare costs and increased mortality are a result of the fact that obesity causes a predisposition to chronic diseases including atherosclerosis, hypertension, diabetes melitus, cancer and slep apnea (37, 76, 126, 434). 15 The two most common and alarming disorders resulting from obesity are type 2 diabetes and cardiovascular disease. Type 2 diabetes is tightly asociated with obesity due to the combination of the modern diet and sedentary lifestyle (340). Adults with a BMI of 35 or more are roughly 20 times more likely to develop diabetes over a ten year period than their normal weight counterparts (134). As a result of skyrocketing obesity rates, the prevalence of diabetes is rising dramaticaly throughout the world. The number of adults with diabetes is predicted to increase by 46% from 151 milion in 2000 to 221 milion in 2010 (641). Furthermore, by 2025, the global number of individuals with type 2 diabetes could double from the number observed in 1995 (273). Almost 81 milion (1 in 3) American adults have 1 or more types of cardiovascular disease (CVD). Total CVD includes the disorders of hypertension, coronary heart disease (myocardial infarction and angina pectoris), heart failure and stroke. Mortality data show that CVD acounted for 36.3% of al deaths in 2004, or 1 of every 2.8 deaths in the United States. An average of 2,400 Americans die from CVD each day, an average of 1 death every 37 seconds (464). Acording to the NCHS, if al forms of major CVD were eliminated, life expectancy would rise by almost 7 years (10, 464). The estimated direct and indirect cost of CVD for 2008 was $448.5 bilion. Moreover, the total healthcare spending for CVD increased 8.06% from 1987 to 2000 (464, 548). The relation betwen obesity and CVD is complicated. Some suggest that the connection is indirect and dependent on the increased prevalence of diabetes, hypertension and dyslipidemia, while others suggest an independent asociation betwen obesity and CVD risk (147, 180, 336). A recent meta-analysis of 57 studies including over 900,000 adults showed that for each 5 kg/m 2 increase above a BMI of 25 kg/m 2 , cardiovascular mortality increases by 40% (597). However, the use of BMI as a CVD risk factor has been debated as BMI fails to consider body fat distribution and storage paterns (336). BMI is not a good index of visceral fat, which serves as the major fat depot contributing to increased metabolic and cardiovascular risk (145, 336). Waist-to- hip ratio (WHR) and waist circumference (WC) might be superior to BMI as risk asesment tools (336, 631). A recent meta-regresion analysis concluded that a 1 cm increase in WC or a 0.01 increase in WHR elevated relative CVD risk by 2% and 5% 16 respectively (100). The INTERHEART study, on almost 30,000 acute myocardial infarction (AMI) patients from 52 countries, demonstrates a strong asociation betwen abdominal obesity and AMI risk worldwide. Furthermore, a WHR greater than 0.85 in women and greater than 0.90 in men is a risk factor for AMI (631). The major pathophysiologic conditions asociated with obesity are also involved in the pathogenesis of the metabolic syndrome (MetS) (87, 95, 181, 184). Metabolic syndrome is a constelation of interelated conditions including abdominal obesity, dyslipidemia, hypertension and hyperglycemia that directly promote cardiovascular disease (CVD) and type 2 diabetes (179, 182, 222). Over the past decade a number of organizations have proposed diferent sets of criteria for defining MetS. The organizations include the World Health Organization (WHO), International Diabetes Federation, American Heart Asociation (AHA) and National Heart, Lung and Blood Institute (NHLBI). However, the most widely used diagnosis criteria are those from the National Cholesterol Education Program (NCEP) Adult Treatment II (ATP II) endorsed by the AHA and NHLBI (182). These criteria are based upon common clinical measures such as waist circumference, triglycerides, HDL-C, blood presure, and fasting glucose level (se Table 1) (182, 184). Table 1. NCEP ATP II: the metabolic syndrome criteria Risk Factor Defining Level Adominal obesity (waist circumference) Men > 102 cm (40 in) Women > 88 cm (35 in) Triglycerides > 150 mg/dL HDL-C Men < 40 mg/dL Women < 50 mg/dL Blood presure Systolic > 130 mHg Diastolic > 85 mHg Fasting glucose > 100 mg/dL *Diagnosis = when # 3 of these risk factors are present The presence of defined abnormalities in any 3 of the 5 measures constitutes diagnosis of MetS (182). Although the criteria difer slightly from one organization to the other, the respective definitions al serve the clinical function of identifying obese individuals who are at a greater risk of developing co-morbid metabolic conditions such as type 2 diabetes and cardiovascular disease (293, 603). 17 The prevalence of the metabolic syndrome is relatively high worldwide. The Third National Health and Nutrition Examination Survey (NHANES) reported that the overal prevalence of MetS in adults over the age of 20 years was 24% in the United States. This prevalence increased to greater than 30% in 50-year-old individuals and greater than 40% in individuals aged 60 years and over (184). When using the NCEP criteria, the prevalence of MetS is lower in Black Americans (140, 182). This is most likely due to lower average waist circumferences, lower triglycerides and higher HDL-C levels. On the other hand, Blacks are more susceptible to insulin resistance, hypertension and diabetes (178). Thus, the NCEP criteria may not provide a clear picture of the metabolic abnormalities occurring in this population (178). The greatest prevalence of MetS among any racial group in the United States was found in Hispanics, with 32% estimated to have MetS (139). The most likely reason for this is greater insulin resistance, glucose intolerance and hyperglycemia in this ethnic group (306). This is manifest in the fact that Hispanics have the highest rate of type 2 diabetes in the US (185). Furthermore, from a worldwide perspective, approximately one fourth of the European and Latin American populations are thought to exhibit MetS (178). As previously noted, the risk for cardiovascular disease and type 2 diabetes rises dramaticaly in individuals with MetS (182, 293, 296). Prospective population studies show that MetS results in a ~ 2 fold increase in the relative risk for CVD as opposed to individuals without metabolic syndrome (182, 296). In the Framingham Heart Study population, MetS predicted roughly 25% of al new-onset CVD (600). The ten-year risk for developing CVD in men with MetS was in the range of 10-20% (181). Furthermore, Lakka and colleagues (296) reported that men with metabolic syndrome, as defined by NCEP ATP II, were 2.9 to 4.2 times more likely to die of CHD. MetS as defined by the WHO was asociated with 2.6 to 3.0 times higher CVD mortality (296). Prospective population studies also show a ~ 5 fold increase in risk for developing type 2 diabetes as compared to people without the syndrome (182). The presence of METs, in both men and women in the Framingham cohort, was highly predictive of new-onset diabetes (182). Moreover, almost half of the atributable risk for type 2 diabetes could be 18 explained by the presence of MetS as defined by NCEP ATP II standards (181). As reported by Laksonen and colleagues (293), men who met the WHO definition of MetS in which adiposity was defined as waist-to-hip-ratio > 0.90 or body mas index # 30 kg/m 2 had a ~ 9 fold greater risk of developing diabetes than men without the syndrome. Men fulfiling the WHO definition of MetS where adiposity was defined as # 94 cm were 7 times more likely to develop diabetes (293). When using the NCEP ATP II criteria with abdominal adiposity defined as # 102 cm, the risk for developing diabetes was 5 times greater than that for men without MetS (293) Pathophysiology of Obesity Although the relationship betwen obesity, insulin resistance and cardiovascular disease is wel-recognized, the mechanisms involved remain relatively poorly understood (173, 181). It is evident that adipocytes are very important for health as shown using a lipoatrophic mouse model. These mice have almost no white fat tisue and had characteristics similar to those sen in humans with lipoatrophic diabetes, insulin resistance, hyperglycemia, hyperlipidemia, and faty livers (164, 507). Transplantation of adipose tisue from healthy mice resulted in atenuated hyperglycemia, acompanied by lowered insulin concentrations, improved muscle insulin sensitivity, decreased serum triglycerides, decreased hepatic gluconeogenesis and decreased amounts of fat deposited in the muscle and liver (164). Thus, fat cels obviously play a role in health and the absence of adipocytes is ?metabolicaly detrimental? (177). Adipose tisue dysfunction appears to contribute to obesity related insulin resistance and type 2 diabetes (485, 508). Evidence suggests that enlarged adipocytes, impaired adipose tisue blood flow, adipose tisue hypoxia, local inflamation in adipose tisue and adipose tisue macrophage infiltration sem to be interelated and may lead to disturbances in adipokine secretion, lipid overflow, and ectopic fat storage, which together may result in the development and progresion of insulin resistance and type 2 diabetes (173). 19 Adipose Tisue Size The enlargement of adipocytes is frequently observed in obesity and is proposed to be involved in the pathogenesis of insulin resistance and type 2 diabetes (118, 173, 407, 593). Increased adipocyte size may be the result of impaired diferentiation, which appears to be a precipitating factor in the development of type 2 diabetes and an independent marker of insulin resistance (326, 595). A possible link betwen adipocyte size and insulin resistance may be the release of faty acids from adipose tisue (173). The mobilization of faty acids from stored adipocyte triglycerides is mediated by hormone sensitive lipase and adipose triglyceride lipase and is strongly inhibited by insulin (207, 640). Some have suggested that adipocytes of obese individuals have become resistant to the anti-lipolytic efects of insulin, thus resulting in the increased release of faty acids from the tisue into circulation (173). However, non-esterified faty acid release into circulation may not be the only link betwen adipocyte size and insulin resistance as diabetic subjects are equaly responsive to the anti-lipolytic efects of insulin compared to control subjects despite the presence of systemic insulin resistance (17, 317, 326, 593). Since obese adipose tisue may be normaly responsive to the anti-lipolytic efects of insulin, the hyperinsulinemia often present in insulin resistant conditions might decrease fasting lipolysis; thereby, protecting obese individuals from the deleterious efects of high circulating faty acid concentrations (256). On the other hand, decreased lipolysis may also contribute to a continuous increase in adipocyte size, which may further impair the adispose tisue dynamic storage capabilities (173). Impaired adipose tisue lipid buffering capacity, rather that increased fasting adipocyte lipolysis, may play a greater role in the prolonged elevation of circulating non-esterified faty acids, thus serving as the link betwen adipocyte size and insulin resistance (86). Furthermore, studies report that the insulin-sensitizing agents TZDs, acting via activation of PAR", stimulate adipocyte diferentiation resulting in an increase in the number of smal adipocytes with large capacitance (102, 187, 388). In addition, TZDs promote the traficking of bone marow-derived circulating progenitor cels to adipose tisue and stimulate their diferentiation into adipocytes (92). A novel TZD has been shown to induce adipocyte diferentiation in Zucker diabetic faty rats by stimulating 20 PAR", thus increasing the number of smal adipocytes and improving insulin sensitivity (347). In summary, one theory as to how adipocyte size is linked to insulin resistance is based on the ability of enlarged adipocytes to efectively store dietary faty acids. Consequently, lipid overflow, ectopic fat distribution and insulin resistance may develop (173). Adipose Tisue Storage Capability and Ectopic Fat Deposition Adipose tisue is the main lipid storage depot. In obese individuals, whose energy intake exceds energy expenditure, adipose tisue is overloaded with triglycerides and the additional lipid storage capability is diminished. In other words, adipose tisue is a ?metabolic sink? that can reach capacity. Triglyceride storage in the adipocytes of obese subjects has reached a near maximum levels, thus these adipocytes cannot efectively store exces lipids. Consequently, non-adipose tisues such as skeletal muscle, the liver and the pancreas are exposed to fre faty acids that remain in circulation leading to lipid storage redistribution in these tisues (146, 173). There is a plethora of information suggesting that ectopic fat distribution in non- adipose tisue plays a crucial role in the development of insulin resistance and impaired insulin secretion (28, 41, 141, 234, 280, 404). Animal studies have shown a direct relationship betwen the acumulation of faty acid derived metabolites in the liver or skeletal muscle and insulin resistance mediated by alterations in the insulin-signaling pathway (271). In the liver and skeletal muscle, imaging studies have ilustrated an inverse linear relationship betwen insulin-mediated glucose disposal and the presence of intra-myocelular and hepatic fat (340). Similarly Greco and colleagues (176) demonstrated that lipid deprivation in skeletal muscle selectively depletes inter- and intra-myocelular lipid stores and reverses insulin resistance. Work from a number of laboratories has shown that in both rodents and humans, the triglyceride content of muscle bears a negative relationship to whole-body insulin sensitivity (404, 488). Increased delivery of faty acids to the liver leads to increased hepatic glucose production, elevated hepatic very low-density lipoprotein and triglyceride output, and reduced insulin clearance by the liver, resulting in conditions asociated with insulin resistance, such as glucose intolerance, hyperlipidemia, and hyperinsulinemia (63, 125, 21 133, 149, 201, 305, 534, 598). Moreover, there is evidence that chronic exposure of pancreatic $-cels to elevated fre faty acid (FA) levels can be damaging to their function (44, 479, 504, 638). Isolated islets exposed to high concentrations of FA for periods of 24-48 hours typicaly show enhanced insulin secretion at low glucose concentrations, depresed proinsulin biosynthesis, depletion of insulin stores, and an impaired response of the $-cel to stimulatory concentrations of glucose (44, 638). Similar findings were also found in intact rats and Zucker diabetic faty rats (479, 504). The mechanism by which elevated fre faty acids in circulation and ectopic fat deposition result in insulin resistance is not fully understood. However, it is possible that faty acids and potentialy several metabolites including acyl-CoAs, ceramides, and diacylglycerol serve as signaling molecules that activate protein kinases such as protein kinase C (PKC), Jun kinase (JNK), and the inhibitor of nuclear factor-%B (NF-%B) kinase-$ (IK$). These kinases can subsequently impair insulin signaling by stimulating the inhibitory serine phosphorylation of insulin receptor substrates (IRS), the key mediators of insulin receptor signaling (430, 440). Based on these data, it appears that an impaired storage potential of dietary fat in adipose tisue may result in ectopic fat deposition and insulin resistance in situations where energy intake exceds energy expenditure (173). Adipose Tisue Blood Flow Blood flow may be an important regulator of metabolism in adipose tisue (60, 160, 480). It is possible that disturbances in normal adipose tisue blood flow might contribute to an increased lipid supply to non-adipose tisues resulting in ectopic fat deposition and insulin resistance (173). In lean healthy individuals, adipose tisue blood flow is responsive to nutrient intake, which may be important in the regulation of metabolism by facilitating signaling betwen adipose tisue and other tisues (12, 13, 86, 123, 148, 175). Blood flow to the adipose tisue controls the interaction betwen circulating triglyceride rich lipoprotein particles and lipoprotein lipase, which hydrolyzes these particles into faty acids and glycerol (480). Evidence suggests that both fasting and postprandial adipose tisue blood flow is diminished in obese states and may contribute to the reduced fasting and postprandial triglyceride extraction/clearance in 22 adipose tisue in obese compared to lean individuals (42, 174, 239, 436, 533, 574). Moreover, intravenous adrenaline infusion causing an elevation of adipose tisue blood flow resulted in the increased clearance of circulating triglycerides into adipose tisue (480). Therefore, decreased adipose tisue blood flow may play a role in decreased triglyceride extraction, lipid overflow, ectopic fat distribution and lipid-induced insulin resistance (173). Adipose Tisue Hypoxia Adipose tisue hypoxia, which is closely related to adipose tisue blood flow, may contribute to obesity related impairments in adipokine expresion/secretion and subsequent insulin resistance (173). Adipocyte size increases up to 140-180 ?m in diameter during the development of obesity (57). These enlarged adipocytes encounter les than adequate oxygen supply since the maximum difusion distance for oxygen is 100 ?m (200). Hosogai et al. (212) demonstrated that white adipose tisue is hypoxic and exhibits markedly increased lactate concentration in obese versus lean mice. Furthermore, weight loss appears to decrease the expresion of hypoxia responsive genes (66). A likely explanation for adipose tisue hypoxia is based on blood flow through the tisue (173). Trayhurn and colleagues (555) have recently proposed that the expansion of adipose tisue mas during the progresion of obesity may lead to hypoxia in parts of the tisue because angiogenesis is insufficient to maintain normoxia in the entire adipose tisue depot. This may lead to increased production of inflamatory factors, acute phase proteins and angiogenic factors by adipose tisue (173). These events most likely involve the key controller of the celular response to hypoxia, the transcription factor hypoxia-inducible factor-1 (HIF-1) (554-556). Therefore, decreased adipose tisue blood flow and the resultant hypoxia in obesity appear to be integral to the development of adipocyte dysfunction and the subsequent metabolic and cardiovascular complications described previously. Hypoxic cels respond by altering gene expresion in an atempt to ensure adaptation (173). Hypoxia causes the expresion of HIF-1!, which combines with HIF- 1$ to form the transcription factor HIF-1 (211, 495, 591). This transcription factor 23 regulates the response to alterations in oxygen supply and the expresion of genes that are involved in angiogenesis, erythropoiesis, inflamation and glucose metabolism (211, 316, 496, 591). Hypoxia may lead to disturbances in adipose tisue glucose homeostasis through inducing changes in the expresion of glucose transporters (607). Furthermore, recent data suggest that hypoxia dysregulates the expresion of several adipokines known to afect glucose homeostasis and insulin sensitivity. For example, adiponectin and PAR" mRNA expresion were reduced while PAI-1 and visfatin mRNA expresion were elevated in hypoxic 3T3-L1 adipocytes when compared to normoxic cels (212, 493). Similarly, hypoxia induces PAI-1 production and inhibits adiponectin synthesis in 3T3-L1 adipocytes (71). Adipose tisue hypoxia in obese mice was also asociated with elevations in inflamatory genes and decreased expresion of adiponectin (623). Recent data show that hypoxia induces HIF-1! protein synthesis, while increasing the expresion of inflamatory adipokines in human adipocytes (579). HIF-1! expresion is also related to macrophage infiltration of obese adipose tisue (66). When macrophages experience hypoxic conditions, they signal for further macrophage recruitment and the activation of other inflamatory cels (304). Furthermore, cel death may occur in response to hypoxia. The severity of hypoxia determines whether apoptosis occurs or if adipocytes adapt and survive (173). Adipose tisue macrophages are reportedly predominantly localized to necrotic adipocytes (79). Adipocyte death was positively asociated with adipocyte size in obese mice and humans and in hormone-sensitive lipase deficient mice (79). Thus, adipocyte death may induce exces macrophage recruitment to adipose tisue of obese individuals (173). Hypoxia also inhibits adipocyte diferentiation, which may be regulated by the production of mitochondrial reactive oxygen species (68, 69, 630). As mentioned previously, impaired adipocyte diferentiation appears to be causaly related to the development of type 2 diabetes (407, 593). Based on these presented data, adipose tisue hypoxia, most likely due to inadequate adipose tisue blood flow in response to increased adipose tisue size, may play an important role in the link betwen obesity and insulin resistance through efects on macrophage infiltration, adipokine expresion and/or adipocyte diferentiation. 24 Adipose Tisue Inflammation, Macrophage Infiltration and Dysregulated Adipokine Secretion Obesity is asociated with a state of chronic, low-grade inflamation characterized by abnormal cytokine production and the activation of inflamatory signaling pathways in adipose tisue (213, 222). Obese hypertrophic adipocytes and stromal cels within the white adipose tisue directly augment systemic inflamation (222). White adipose tisue typicaly consists of a 5-10% macrophage population, however, a rodent model of diet-induced obesity causes a significant increase in macrophage infiltration, with macrophages consisting of 60% of al cels found in white adipose tisue (587). Macrophages are now recognized as important non-adipocyte cels that contribute to the production of adipose tisue inflamatory factors (173). It has been reported that non-adipose cels in adipose tisue are responsible for the majority of secreted inflamatory factors, except for leptin and adiponectin, which are primarily produced by adipocytes (124). Adipose tisue macrophages are responsible for nearly al TNF-! expresion and a significant portion of IL-6 expresion (101, 587). Enhancements in inflamatory cytokine expresion by white adipose tisue are asociated with a paralel increase in white adipose tisue macrophage content (66, 587, 610). Thus, it appears that several adipokines implicated in systemic inflamation are cytokines produced by macrophages in the adipose tisue. Obese adipose tisue is characterized by progresive infiltration by macrophages as obesity develops (173, 587, 589, 610). Macrophage infiltration in adipose tisue is positively asociated with body mas index, adipocyte size and insulin resistance (66, 94, 108, 587, 610). Moreover, weight loss results in a regresion of adipocyte hypertrophy and macrophage infiltration and an improvement in the inflamatory profile of gene expresion (66, 80). It has been reported that macrophage-secreted factors impair human fat cel diferentiation and induce inflamatory events in 3T3-L1 adipocytes by activating the NF-%B pathway (294, 427). Thus, it sems that macrophage infiltration may play an important role in the inflamatory response of obese adipose tisue (173). Initial studies focusing on the role of macrophages in insulin signaling ilustrated that inhibition of the macrophage inflamatory pathway IkkB protects mice from obesity-induced insulin resistance (15). Arkan and colleagues(15) found that liver- 25 specific IkkB deletion in mice resulted in protection from high fat diet-induced hepatic insulin resistance. Moreover, these mice demonstrated a significant reduction in the expresion of inflamatory markers in the liver (15). This study also showed that tisue- specific deletion of myeloid cel IkkB led to enhanced systemic insulin sensitivity with improved insulin action in skeletal muscle, liver and fat (15). Therefore, inactivation of the inflamatory pathway IkkB sems to prevent local and systemic insulin resistance, most likely by interupting the local paracrine efects betwen resident macrophages and insulin target tisues (15). Furthermore, JNK-1 is an important component of obesity- induced insulin resistance (101). Solinas et al. (514) show that JNK-1 signaling in macrophages is a key component of macrophage function and a mediator of the macrophage inflamatory response, which subsequently leads to insulin resistance. It has recently been determined that monocyte chemoatractant protein-1 (MCP- 1), a chemokine and member of the smal inducible cytokine family, may play an important role in macrophage infiltration of adipose tisue (173). Macrophages, endothelial cels and adipocytes produce MCP-1 and its expresion is closely related to the number of resident macrophages (58, 75, 165). MCP-1 expresion is increased in obese rodents and concentrations are elevated in obese and diabetic humans (75, 270, 375, 431, 481, 536). Furthermore, concentrations of MCP-1 have been found to decrease with weight-loss (487). Elevated MCP-1 expresion is found in the early stages of obesity, suggesting that MCP-1 is initialy produced by cels other than recruited macrophages (610). In addition, MCP-1 expresion/secretion can be induced by TNF-!, IL-6 and insulin in pre-adipocytes and 3T3-L1 adipocytes, while its secretion can be suppresed by the PAR-" agonists, TZD?s, and adiponectin (110, 127, 432, 481, 494, 610). Therefore, dysregulation of adipokine secretion in obesity may alter MCP-1 expresion/secretion, which in turn can influence the extent of macrophage infiltration in adipose tisue (173). MCP-1 knockout mice are characterized by decreased adipose tisue macrophage infiltration, reduced pro-inflamatory gene expresion in adipose tisue, decreased hepatic triglyceride content and improved insulin sensitivity as compared to wild type animals (250, 586). On the other hand, adipose tisue-specific overexpresion of MCP-1 increases adipose tisue macrophage infiltration and decreases insulin sensitivity (248, 26 250). It sems that MCP-1 is important in the proces of macrophage infiltration in obese adipose tisue and may contribute to the development of obesity-related insulin resistance. It appears to be improbable that macrophages initiate inflamation and dysregulated adipokine secretion in obese adipose tisue. Macrophages most likely augment the inflamatory signals that have already been established (173). Whatever the initial stimulus to recruit macrophages into adipose tisue is, once these cels are present and active, they, along with adipocytes and other cel types, could perpetuate a cycle of macrophage recruitment, production of inflamatory cytokines, and impairment of adipocyte function (589). The exact mechanisms underlying the disturbances in adipokine secretion and macrophage infiltration in obesity are yet to be fully elucidated. However, it is highly likely that these events relate to disruption within the adipose tisue itself such as the previously discussed increase in adipose tisue size, decrease in adipose tisue blood flow and adipose tisue hypoxia (173). Oxidative Stres Oxidative stres is an imbalance betwen reactive oxygen species (ROS), tisue fre radicals and antioxidants that may possibly be a mechanism underlying obesity related disorders (571). Low levels of fre radicals and ROS are necesary for normal cel redox state, cel function and intracelular signaling (370). High concentrations of fre radicals and ROS damage DNA, proteins, carbohydrates and lipid constituents and compromise cel function (573, 626). Furthermore, ROS might be a key feature in the pathogenesis of the obesity asociated metabolic disease, as recent evidence suggests that increased oxidative stres in acumulated fat is an early instigator of insulin resistance (159, 209, 218). Oxidative stres correlates with fat acumulation in humans and mice and may be a causative factor in the development of insulin resistance (122, 188, 440, 465). This is supported by studies showing that a reversal of the imbalance betwen ROS and antioxidants improves insulin resistance in humans and rodents (159, 183, 264, 330). Possible mechanisms that generate oxidative stres/ROS in obesity include hyperglycemia, tisue lipids, inadequate antioxidant defenses, chronic inflamation, and hyperleptinemia. Several oxidative pathways are activated by hyperglycemia(573). 27 Advanced glycosylation end products (AGE) formed from proteins, lipids and nucleic acids are diabetic precursors. AGEs bind to specific cel surface receptors and lead to post-receptor signaling and generation of ROS (573). AGEs also activate intracelular transcription factors such as NF-%B, which activates protein kinase C and sorbitol and transcription of vascular cel adhesion molecule-1 (VCAM-1) and intracelular adhesion molecule-1 (ICAM-1). Activation of these celular molecules can produce ROS, as shown in rodent vesel tisues (460). As a result, oxidative damage and acelerated monocyte binding to the endothelium occur (19, 122). Hyperglycemia also stimulates the polyol pathway through which aldose reductase mediates the conversion of glucose to sorbitol (122). Elevated sorbitol levels in animal models cause oxidative damage and activate stres genes (122). Moreover, hyperglycemia induces NADPH oxidase activity, which serves to produce the ROS, superoxide, especialy in the endothelium (635). Finaly, recent data suggest that diet-induced increases in adipocyte glucose uptake in obese mice result in elevated ROS production (540). Oxidative stres may partly be the result of the metabolic impact of intracelular triglycerides, which are elevated in obesity. Excesive triglycerides, by suppresing mitochondrial adenine nucleotide transporters, may increase ROS production within the mitochondrial electron transport chain (25). This decreases intramitochondrial adenine diphosphate causing electrons to build up within the electron transport chain, alowing them to react with oxygen to form superoxide (25). Visceral adiposity is also linked to elevated circulating FA concentrations. FA can acutely increase ROS formation in culture (224). Lipids, such as LDL-C, serve as a major substrate for oxidation, stimulate radical formation, and enhance the acumulation of oxidative by products, especialy in white adipose tisue (159). Exces lipid oxidation enhances the risk for thrombosis, endothelial dysfunction, and atherosclerosis (327, 461). Elevated lipid concentrations present in obesity may simply serve as an enlarged target for oxidative modification by ROS (391, 572). Circulating concentrations of biomarkers of oxidative stres were significantly elevated in obese vs lean Zucker rats. The major contributor to lipid peroxidation in the high fat fed Zucker rat was tisue lipid content (572). Furukawa and colleagues (159) discovered that in several obese mice models (KAy, db/db, diet-induced, C57BL/6) 28 acumulation of fat in white adipocytes increased oxidative stres in white adipose tisue. Moreover, the KAy mouse exhibited increased NADPH oxidase activity (generating ROS) and reduced activity and mRNA of the antioxidant enzymes superoxide dismutase, glutathione peroxidase and catalase in white adipose tisue (159). Based on these data, it appears that obesity induces ROS formation and lowers antioxidant defenses, thus promoting oxidative damage in the adipose tisue (573). Furukawa and colleagues (159) further explain that ROS and biomarkers of lipid peroxidation enter into circulation and initiate a vicious cycle of systemic oxidative stres in obesity. Many human studies based on the link betwen oxidative stres and insulin resistance focus on the generation of ROS by hyperglycemia in diabetic patients. This suggests ROS as a consequence of diabetes-induced hyperglycemia and not a causative factor for insulin resistance (121, 122, 440). However, insulin resistance first presents itself long before the development of chronic fasting hyperglycemia, thus it is unlikely that insulin resistance in the pre-diabetic stage results from oxidative stres triggered by hyperglycemia (103, 440, 451). The increase in ROS in the pre-diabetic stage is more likely a result of obesity-induced elevations in FAs that cause increased mitochondrial uncoupling (se following section) and $ oxidation, leading to increased production of ROS and oxidative stres (67, 448, 604, 614). In healthy individuals, infusion of FAs causes increased oxidative stres and insulin resistance that is reversed by infusion of antioxidants (405, 406). Adequate tisue, dietary, enzymatic and non-enzymatic antioxidant defenses are critical to maintain antioxidant-prooxidant balance in tisues (573). Disturbances in this balance occur in obesity. Inadequate antioxidant defenses in obesity may begin with a low intake of antioxidant and phytochemical rich foods such as fruits, vegetables, whole grains, legumes, etc. Phytochemical intake is inversely correlated with waist circumference, BMI, and plasma lipid peroxidation (240). Moreover, dietary antioxidant levels are inversely related to the degre of adiposity (455, 577). Plasma vitamin concentrations also progresively decline as BMI increases (354, 532). In adults, blood retinol, tocopherol, vitamin C and carotene concentrations were 18-37% lower in obese women as compared to lean counterparts (354). Similarly, zinc levels were 38% lower in obese versus lean men (399). These data suggest that elevated BMI and obesity are 29 related to an increasing imbalance in the antioxidant-prooxidant status (573). In addition, dietary antioxidants may be used more rapidly in combating excesive prooxidant proceses in obese individuals, leaving these subjects susceptible to fre radical damage (573). Activities of the major antioxidant enzymes may also be decreased in obesity (573). Erythrocyte superoxide dismutase and glutathione peroxidase activities were 29- 42% lower in obese, high fat fed rats as opposed to control animals (33). On the other hand, in the early stages of obesity there may actualy be increased activity of antioxidant enzymes in order to combat oxidative stres. However, as obesity progreses the sources of antioxidant enzymes are depleted (391, 572). Moreover, the degre of adiposity sems to afect enzyme activities. Olusi and colleagues (391) found that erythrocyte superoxide dismutase activity and glutathione peroxidase activity were lower in obese individuals versus non-obese controls. Furthermore, Ozata et al. (399) found 75 and 42% lower erythrocyte glutathione peroxidase and superoxide dismutase activities respectively in obese men compared to non-obese men. The combination of inadequate dietary, blood and enzymatic antioxidants and increased production of ROS creates an imbalance that favors oxidative stres in obesity (573). Obesity in humans is characterized by a chronic state of low-grade inflamation (279, 596). Inflamation is defined by increased inflamatory cytokine expresion and increased white blood cel counts and white cel activity (573). Fat is an active endocrine organ that expreses pro-inflamatory cytokines such as TNF-! and IL-6. The expansion of adipose tisue in obesity is asociated with increased concentrations of TNF-! and IL-6, which may be released by adipocytes themselves or resident macrophages, as discussed previously (279, 328). Reduction of fat mas through weight loss is directly related to reductions in inflamatory cytokines in both humans and animals (97, 327). The cytokine profile may shift toward a prooxidant state in obesity and toward a balanced prooxidant-antioxidant state with optimal weight (573). White blood cel counts are higher in obese individuals with elevations occurring in the monocyte and neutrophil subfractions (282). Monocytes produce ROS and oxidative enzymes, and when developed into macrophages they produce TNF-! and IL-6 30 (573). Neutrophils generate superoxide via NADPH oxidase (163). Both monocytes and neutrophils can convert hydrogen peroxide into ROS via myeloperoxidase (163). As explained earlier, there is a progresive infiltration of macrophages into adipose tisue as obesity develops (173). This is in part due to the chemoatraction of leukocytes into adipose tisue by MDA and 4HNE (by-products of fat induced ROS generation) (159). It has been proposed that in obesity adipocyte-initiated macrophage recruitment and cytokine/ROS production by these macrophages occurs, which could potentialy lead to oxidative damage and disease proceses such as atherosclerosis (573, 589). In summary, obese individuals display increased inflamatory cytokine levels, indirectly causing ROS formation via several intracelular signaling pathways (NF-%B, NADPH), hyperglycemia and insulin receptor impairment. Leukocyte infiltration causes the enzymatic formation of ROS via NADPH oxidase. Both pathways (cytokines, leukocytes) generate ROS and induce oxidative stres (573). Plasma leptin concentrations are proportional to the amount of adipose tisue and have a potential role in obesity-induced oxidative stres (84, 573). Leptin can directly stimulate production of ROS in cultured endothelial cels (49). Wistar rats injected with leptin displayed elevated plasma levels of various biomarkers of oxidative stres as compared to non-treated controls (34). Moreover, leptin indirectly stimulates production of inflamatory cytokines such as IL-6 and TNF-! (377). These cytokines increase NADPH oxidase production, which in turn generates ROS (377). Finaly, leptin reduces the activity of the celular antioxidant, paranoxase-1 (PON-1). This reduction is related to increased levels of various biomarkers of oxidative stres (34). Therefore, hyperleptinemia may be involved in several mechanisms that promote oxidative stres in obesity (573). Recent investigations have explored the molecular mechanisms through which oxidative stres might lead to insulin resistance (440). ROS and oxidative stres lead to the activation of several serine/threonine kinase signaling cascades in vitro (121, 291). These activated kinases act on a number of potential targets including the insulin receptor and the family of IRS proteins. An increase in serine phosphorylation on IRS-1 and IRS- 2 inhibits the insulin signal that normaly induces activating tyrosine phosphorylation (40, 31 121). The kinases found to be activated by oxidative stres include JNK, p38 MAPK, and IK$ (5, 43, 206, 218, 330, 627). Specific atention has been paid to the activity of JNK. ROS-induced insulin resistance may be mediated by JNK as increased ROS levels are known to stimulate threonine phosphorylation of JNK (206, 218, 252). This postulation is supported by evidence showing that the inhibition of JNK activity, through genetic knockout or an inhibitory peptide, improves insulin sensitivity in mice (206, 247, 251, 252). Other Mechanisms Involved in the Pathophysiology of Obesity Toll-like receptor 4 (TLR-4) is stimulated by lipopolysacharide, an endotoxin released by gram-negative bacteria (101). TLR-4 stimulation results in the activation of IkkB/NF%B and JNK/AP-1 signaling, up-regulating the expresion and secretion of proinflamatory cytokines in adipose tisue and resident macrophages, including, IL-6, TNF-! and MCP-1 among others (231). TLR-4 was first shown to be a component in faty acid-induced inflamation in studies examining the ability of dietary faty acids to activate the inflamatory response via TLR-4 signaling in cultured macrophages (303). Shi and colleagues (499) discovered that fre faty acids (FA) activate the NF%B signaling pathway in primary macrophages from wild type mice, but not in macrophages harvested from TLR-4 deficient mice. Furthermore, in vivo, obese mice have increased TLR-4 expresion in adipose tisue compared to lean controls (499). TLR-4 deficient mice are protected from lipid infusion-induced insulin resistance, due to decreased FA- induced NF%B transcriptional activity, as wel as reduced expresion of TNF-!, IL-6 and MCP-1 in adipose tisue (499). The work of Shi et al. (499) implicates TLR-4 in the development of lipid and obesity-induced insulin resistance and suggests that TLR-4 can act as a potential intracelular sensor of elevated tisue or serum FA concentrations that are commonly found in obese individuals. Furthermore, recent data suggests that TLR-4 specificaly located in macrophages and hepatic Kupffer cels participates in a sensing mechanism facilitating faty acid-induced inflamation and insulin resistance (104, 109). Another potential mechanism of inflamation and insulin resistance in obesity is endoplasmic reticulum (ER) stres (101, 173, 440). This is based upon the idea that overnutrition causes mechanical stres, exces lipid acumulation and protein synthesis, 32 and abnormal energy metabolism, al of which lead to an overburdened ER (101). It has been reported that ER stres is elevated in the adipose tisue and liver of obese mice (364, 401). This ER stres disrupts the normal folding of proteins and activates the unfolded protein response (UPR), an HIF-1-independent signaling pathway (39). Many disturbances, including hypoxia, cause acumulation of unfolded proteins in the ER, leading to ER stres (39, 129). Ozcan et al. (401) demonstrated that obesity imposes a strain on the ER machinery, triggering an ER stres response that activates JNK-mediated serine phosphorylation of IRS-1, resulting in an inhibition of the insulin-signaling pathway. Newly synthesized proteins are regularly folded and asembled by chaperones in the ER (260). Recent studies have shown protection against obesity-induced type 2 diabetes in mice through overexpresion of ER chaperones, while a knockdown of these chaperones resulted in an increased incidence of T2D (364, 400). Similarly, obese mice deficient for one alele of X-box binding protein-1, a transcription factor that promotes expresion of molecular chaperones in response to ER stres, are more severely insulin resistant compared to obese controls (401). Oral administration of active chemical chaperones reduces ER stres and improves glucose homeostasis in obese mice (402). The mechanism that leads to ER stres is not fully understood (440). Ectopic lipid deposition may trigger ER stres by sheer mechanical stres, perturbations in intracelular nutrient and energy fluxes or severe changes in tisue architecture (401). In addition, chronic increases in FA concentration may induce ER stres (254). Finaly, ER stres might lead to an increase in oxidative stres that in turn may contribute to insulin resistance (193). Insulin resistance and type 2 diabetes melitus are asociated with a decrease in mitochondrial function that contributes to ectopic fat acumulation (430, 440). Petersen and colleagues (429) discovered that in elderly subjects severe insulin resistance is asociated with significantly higher levels of triglycerides in both muscle and liver. This was acompanied by decreases in mitochondrial oxidative activity and mitochondrial ATP synthesis (429). Studies further suggest that insulin resistant individuals acumulate intramyocelular fat due to a decrease in the number of muscle mitochondria caused by reductions in the expresion of nuclear-encoded genes that regulate mitochondrial 33 biogenesis, such as PGC-1! and PGC-1$ (520, 609). Microaray studies show that PGC- 1-responsive genes are down-regulated in obese Caucasians and Mexican-Americans with impaired glucose tolerance and type 2 diabetes (356, 415). Finaly, activation of PGC-1! is asociated with improved mitochondrial function and insulin sensitivity in both animals and humans (295, 343). Therefore, insulin resistance might arise from defects in mitochondrial function, which in turn lead to increases in intracelular faty acid metabolites (diacylglycerol, faty acyl-CoA, ceramides) that disrupt insulin signaling (440). A decrease in mitochondrial function in obese and insulin resistant individuals may sem counter-intuitive given that it is known that functional mitochondria are needed for the faty acid-induced increase in ROS (122). It may be possible that the increase in ROS from faty acid oxidation occurs early in the development of insulin resistance before mitochondrial dysfunction occurs (440). In the later stages of insulin resistance, ROS might promote a decrease in mitochondrial function that then leads to ectopic fat deposition, thus exacerbating the insulin resistant phenotype in obese individuals (440). Some endocrinologists have proposed that obesity results from an increased endogenous production of the glucocorticoid hormone cortisol (177). Endogenous production as wel as exogenous administration of cortisol results in weight gain and an increase in visceral fat deposition (177). Furthermore, elevated glucocorticoid levels cause insulin resistance and type 2 diabetes, primarily by opposing the anti- gluconeogenic efects of insulin in the liver (492). Hypercortisolinemia, additionaly, results in hyperphagia, central obesity and high concentrations of VLDL (177). Circulating cortisol levels are near normal in obese individuals (192). However, adipose tisue does contain 11$HSD-1, which converts the inactive metabolite, cortisone, to cortisol (440). Concentrations and activity of 1$HSD-1 are elevated in adipose tisue of obese humans and rodents (313, 416, 449, 576). Transgenic overexpresion, to about the same extent as is sen in obese humans, of 11$HSD-1 selectively in mouse adipose tisue results in visceral obesity, insulin resistance and diabetes, increased cytokine expresion, hyperphagia, hyperlipidemia and hypertension (338). Moreover, liver- specific antagonism of glucocorticoid action reduces hepatic glucose output and improves 34 glucose homeostasis in animal models of obesity-induced insulin resistance (235). Therefore, increases in endogenous 11$HSD-1 appear to contribute to obesity-asociated insulin resistance, possibly due to the delivery of cortisol/glucocorticoids to the liver via the portal vein (440). Cytokines, Cardiotrophin 1 and Fibroblast Growth Factor-21 Adipose tisue secretes a wide aray of biologicaly active proteins caled cytokines, or more specificaly, adipocytokines or adipokines (37, 486). Cytokines are a diverse group of proteins produced by a variety of cels that exert their influences through autocrine, paracrine and endocrine manners (45, 128). Stanley Cohen (82) first introduced the term ?cytokine? in 1974. Until then the term ?lymphokine? was used to describe proteins secreted from a variety of cel sources, afecting the growth and function of many types of cels (112, 547). In 1989, Balkwil and Burke (27) further defined cytokines as a group of protein regulators, which are produced by a wide variety of cels in the body, play an important role in many physiological responses, are involved in the pathophysiology of a range of diseases, and have therapeutic potential. Currently, over 50 cytokines have been identified and characterized (486, 547). Cytokines are clasified into several groups: interleukins, tumor necrosis factors, interferons, colony stimulating factors, transforming growth factors, and chemokines (547). These bioactive proteins are involved in intercelular communication, which regulates fundamental biological proceses such as body growth, lactation, adiposity and hematopoiesis (48). They are especialy important in regulating inflamatory and imune responses and have crucial functions in controlling both innate and adaptive imunity. The actions of cytokines are redundant, both functionaly and temporaly as they share a number of specific functions and features. The fact that cytokines have a local mode of action sets them apart from clasical hormones (547). Cytokines show a wide variety of activities and can trigger several diferent celular responses depending on cel type, timing and context. Also, they act synergisticaly in that the asociation of two cytokines amplifies their activity. Cytokines may also function in a juxtacrine manner stimulating cels that produce them, adjacent cels and cels throughout the body, or intervene in direct cel-to-cel interaction (128, 547). Lastly, these bioactive peptides 35 commonly share cytokine receptor subunits (547). For example, the IL-6 family of cytokines shares the gp130 subunit (359). Based on this redundancy and these shared functions and features, no single cytokine alone is likely to be solely responsible for controlling a specific celular function or physiologic proces (128). A bulk of the current literature focuses on the roles of cytokines in the pathogenesis of disease. However, we know this group of proteins is involved in regulating normal physiologic responses. Even though elevated or suppresed cytokine production may be related to diseased states, cytokines are also involved in normal regulation of physiologic proceses (128). With this being said, abnormalities in cytokines, their receptors and the signaling pathways that they initiate are involved in a wide variety of diseases (130). The cytokines, and possibly more appropriately adipokines or adipocytokines, that may provide a molecular link betwen obesity and the development of MetS, type 2 diabetes and cardiovascular disease are of particular interest. Some of the beter-characterized cytokines involved with adipose tisue dysfunction include adiponectin, leptin, TNF-!, and IL-6. General Cytokine Signaling Pathways (se Figure 2) 1. IK!/ NF-"B Cytokine-mediated inflamatory signals can impair insulin receptor and insulin receptor substrate (IRS) signaling through multiple signal transduction cascades (37, 603). Until recently the exact intracelular mechanism by which this occurred was not clear. Experiments using in vitro models of insulin resistance have shown that serine/threonine kinases phosphorylate insulin receptor and IRS molecules such that the necesary activation of the insulin signal by tyrosine phosphorylation is prevented (441). Considerable atention has now focused on the IK$ complex as a mediator of insulin resistance (603). IK$ can impact insulin signaling by directly phosphorylating inhibitory serine residues on IRS-1 and by phosphorylating inhibitor of NF-%B (I%B), thus activating the NF-%B pathway, a crucial second mesenger system for inflamatory cytokine signaling (440, 509, 603). More specificaly, when stimulated, cytokine receptors recruit kinases that activate the IK complex. IK2, a catalytic subunit within the IK complex, then phosphorylates I%B thus alowing the transcription factor NF-%B 36 to translocate into the nucleus and activate inflamatory target genes (255). This might trigger inflamatory responses that further dysregulate proper insulin signaling (440). Two groups have recently shown the relationship betwen IK$ expresion and insulin resistance. Cai et al. (64) created a state of chronic low-grade inflamation in the liver in a transgenic mouse model via selective activation of NF-%B. This caused continuous expresion of IK$. These mice exhibited a type 2 diabetic phenotype with evidence of moderate systemic insulin resistance (64). Similarly, Arkan and colleagues (15) used mice lacking IK$ in hepatocytes or myeloid cels. The deletion in hepatocytes resulted in increased insulin sensitivity in the mice when placed on a high fat diet or intercrossed with the ob/ob model of genetic obesity. Mice deficient in myeloid IK$ also demonstrated enhanced insulin sensitivity (15). Through this pathway pro-inflamatory cytokines such as TNF-! stimulate the transcription of cytokines and adhesion molecules in peripheral tisues. These adhesion molecules, in turn, are elevated along with plasma lipids in atherosclerosis and diabetes, suggesting a role for NF-%B in the pathogenesis of these chronic diseases (437, 535). In addition, activated NF-%B has been found in smooth muscle cels and macrophages in human atherosclerotic lesions (46, 50, 351). NF-%B activation in these cels controls the expresion of the pro-inflamatory cytokines TNF-!, IL-6 and IL-8, as shown by their selective inhibition following NF-%B blockade (351). Adiponectin suppreses TNF-! induced NF-%B signaling at a step just before IK$ activation, thereby protecting against the development of long term comorbidities such as CVD and type 2 diabetes (397). NF-%B is a redox sensitive transcription factor, as the intracelular redox status of the cel is extremely important in the regulation of NF-%B activity (238). Cytokine- stimulated activation of NF-%B increases production of NO, which serves as a substrate for the formation of reactive oxygen species (ROS) (83). As described previously, ROS contribute to obesity related oxidative stres and likely mediate the pathogenesis of insulin resistance and atherosclerosis (22, 83, 122, 310, 515). Antioxidants, such as aspirin, NAC and flavanoids can inhibit cytokine-induced activation of NF-%B and subsequent formation of ROS (546) (se figure 2). 37 2. JNK c-Jun N-terminal kinase (JNK) has recently emerged as an important regulator of insulin resistance in obesity (206, 550). The JNK group controls celular functions through control of the transcription factor activator protein-1 (AP-1) (550). A number of proimflamatory cytokines such as TNF-!, IL-6, IL-2 and MCP-1 are regulated by the JNK pathway through the interaction of AP-1 and sequences in their promoters (369). JNK phosphorylation is mediated by two MAPK kinases, MAP2K4 and MAP2K7, which serve to cooperatively activate JNK. Gene disruption studies in mice show that both of these MAPKs are necesary for full activation of JNK and that MK7 is esential for JNK activation by proinflamatory cytokines (553). In both genetic and dietary animal models of obesity, JNK1 activity is increased in the liver, skeletal muscle and adipose tisue (206). Specificaly, JNK1 is thought to likely mediate the crosstalk betwen inflamatory and metabolic signaling through activation by inflamatory stimuli. Moreover, the loss of JNK1 prevents the development of insulin resistance in these models (206). Liver specific knockout of JNK1 lowers circulating glucose and insulin concentrations, further solidifying its role in the development of insulin resistance (620) (se figure 2). 3. JAK/STAT A number of cytokines also activate JAK and/or STAT proteins. Cytokines induce activation of their cognate receptors, resulting in activation of asociated JAK kinases (JAK1, JAK2, JAK3) (378). Activated JAKs phosphorylate receptor cytoplasmic domains. Among the phosphorylated substrates are members of the STAT family of proteins (378). Receptor engagement and tyrosine phosphorylation activate the cytosolic STATs, resulting in nuclear translocation and gene activation. In particular, IL-6 binds to its receptor and activates JAK1 and STAT3 (547). STAT3 can be activated by a number of cytokines, especialy those of the IL-6 family, mediating the expresion of several acute phase response genes. STAT3 appears to play a critical negative role in controling inflamation as shown in mice with STAT3 deletion (253, 538, 539, 590). Se figure 1 for an overview of JAK/STAT signaling (230). 38 Figure 1. JAK/STAT signaling. The cytokine binds to the receptor resulting in activation of Jak-kinases and phosphorylation of receptor tyrosine residues. STATs dock on the phosphorylated tyrosine residues. STATs are then phosphorylated and translocate to the nucleus to activate gene transcription. Cytokine signaling by the JAK/STAT pathway is regulated, in part, by a family of endogenous JAK kinase inhibitor proteins caled suppresors of cytokine signaling (SOCS) (608). These inflamatory mediators constitute a negative fedback pathway in cytokine signaling and contribute to obesity-induced insulin resistance (440). At least thre members of the SOCS family (SOCS-1, SOCS-3, SOCS-6) have been implicated in cytokine-mediated inhibition of insulin signaling (116, 353, 472). This occurs either by interference with IRS-1 and IRS-2 tyrosine phosphorylation, or by targeting IRS-1 and IRS-2 for proteosomal degradation (472, 564). Recent studies report that SOCS-3 levels were elevated in obese rodents and reductions in SOCS-3 expresion resulted in resistance to high-fat-diet-induced obesity and insulin resistance (219, 500, 564). Furthermore, overexpresion of SOCS-1 and SOCS-3 in the liver caused systemic insulin resistance (565) (se figure 2). 4. PAR-# 39 The peroxisomal proliferating-activated receptors (PARs) are lipid sensing transcription factors that are primarily known to modulate energy metabolism, lipid storage/transport, inflamation and wound healing, as wel as reducing oxidative stres (376). PAR-" is a member of the PAR family that is most abundantly expresed in adipose tisue and heterodimerizes with retinoid X receptor (RXR). The heterodimer of RXR and PAR-" binds to a specific DNA sequence of PAR responsive elements (PRE) to activate several genes, especialy the group involved in adipocyte diferentiation and lipid metabolism (77, 228). Furthermore, PAR-" is intimately involved in the regulation of lipid and glucose homeostasis and insulin sensitivity as mutations in PAR-" result in insulin resistance, hypertension and diabetes (30). PAR-" is the molecular target of TZDs, pharmacological agents that exert insulin-sensitizing efects in adipose tisue, skeletal muscle and liver and negatively regulate the production of various pro-inflamatory cytokines that promote insulin resistance (550) (se figure 2). In macrophages, that prominently invade adipose tisue in states of obesity as discussed earlier, PAR-" expresion inhibits toll like receptor and IFN-" mediated inflamatory responses (550). Resident macrophages in adipose tisue of lean mice display the alternatively activated or M2 phenotype characterized by activated genes for such anti-inflamatory cytokines as IL-10 (323). However, clasic M1 pro- inflamatory macrophages are recruited to sites of tisue damage in the adipose tisue as in obesity. M1 macrophages produce enhanced levels of pro-inflamatory cytokines such as TNF-! (550). Obesity forces the state of adipose tisue macrophages from an M2 state that protects adipocytes from inflamation, to an M1 pro-inflamatory state leading to insulin resistance (550). Recent evidence suggests that this phenotypic alteration in adipose tisue macrophage polarization is governed by PAR-", as PAR-" is required for the maturation of M2 macrophages (380). Based on these discussed data, the transcription factor PAR-" appears to play a role in cytokine production and signaling. 40 Figure 2. Overview of cytokine signaling. The engagement of TNFR by TNF-! activates IK$/NF-%B and JNK pathways, two major intracelular regulators of insulin resistance. Adiponectin signals via adiporeceptors to enhance PAR activity. IL-1 and IL-18 can reduce IRS-1 expresion via ERK 1/2 and can activate the IK$/NF-%B pathway. IL-6 can induce SOCS1 and SOCS3 to inhibit IRS-1. ER stres and oxidative stres are both involved in the activation of JNK and IK$ (50). Cardiotrophin-1 Cardiotrophin-1 (CT-1), a 201 amino acid protein, was first identified in 1995 through expresion cloning based on its ability to induce cardiac myocyte hypertrophy in vitro (422, 423). Amino acid sequence similarity shows that cardiotrophin-1 is a member of the interleukin-6 cytokine family (423). This family of cytokines has a wide range of growth and diferentiation activities on many cel types including those from the blood, liver and nervous system (7, 274, 423). Cardiotrophin-1 is expresed in the adult human heart, skeletal muscle, ovary, colon, prostate and adipose tisue as wel as fetal kidney and lung (365, 422, 424). The expresion patern of CT-1 and its range of activities in hematopoietic, neuronal, and developmental asays suggest that CT-1 may play an important role in other organ systems, in addition to its actions in cardiac development 41 and hypertrophy (425, 525). Se tables 2 and 3 for factors regulating CT-1 expresion and regulated by CT-1 (525). Table 2. Factors regulating CT-1 expresion Angiotensin I Increases CT-1 in cardiac fibroblasts. CT-1 increases angiotensinogen mRNA expresion in cardiac myocytes. Up- regulation of angiotensinogen and angiotensin I production contribute to CT-1-induced cardiac myocyte hypertrophy (157). Norepinephrine Increases the expresion of CT-1 mRNA in cardiac myocytes in vivo and in vitro (158). Urocortin Increases expresion of CT-1 protein and mRNA (237). FGF-2 Increases CT-1 in cardiomyocytes (241). Glucose and Insulin Increase CT-1 expresion in human and murine adipocytes and neonatal rat cardiomyocytes (312, 365) Reactive Oxygen Species Increase CT-1 expresion in murine embryonic stem cels (21, 482). 42 Table 3. Factors regulated by CT-1 HSP CT-1 increases synthesis of hsp70 and hsp90. CT-1 increases hsp56 protein and mRNA (166, 236, 445). PAR-" CT-1 causes a transient decrease in PAR-" mRNA in 3T3-L1 adipocytes (645). IL-6 CT-1 increases IL-6 mRNA and protein (154, 459). STAT-1, -3, -5A, - 5B, ERK1 and -2 CT-1 increases activation and nuclear translocation of STAT-1, -3, -5A, -5B, ERK-1 and -2 (482, 645). MCP-1 CT-1 increases MCP-1 mRNA; STAT-3 phosphorylation, activation of JAK-2 and NF-%B are involved in this mechanism (153). Faty acid synthase and IRS-1 CT-1 decreases faty acid synthase and IRS-1 protein expresion (645). SOCS-3 CT-1 increases SOCS-3 protein and mRNA expresion in 3T3- L1 adipocytes (194, 645). Endothelin-1 CT-1 increases ET-1 gene expresion in and secretion from vascular endothelial cels (243). Vascular Endothelial Growth Factor CT-1 induces a 3-fold increase in VEGF production in human visceral adipocytes (453). 1. Cardiotrophin-1 Signaling CT-1 causes pleiotropic biological responses through the LIFR complex, consisting of the gp130/LIFR-$ heterodimer (65). The signaling pathway downstream from gp130 is reported to consist of at least thre distinct pathways: JAK/STAT, p42/44 MAPK or ERK1/2, and P13K/Akt (65, 199, 289). Stimulation of the JAK/STAT pathway results in the tyrosine phosphorylation of STAT-3, causing its dimerization and translocation to the nucleus where it can activate target genes (6, 584). Activation of MAPK causes the threonine phosphorylation and activation of NF-IL6, a transcription factor involved in cytokine signal transduction (362). Finaly, activation of P13K results in the phosphorylation of Akt and the pro-apoptotic gene BAD (289). 43 The protective efect of CT-1 is likely to be dependent upon its ability to activate the p42/44 MAPK pathway (52, 309, 446, 497). Although there is agrement on the efects mediated through MAPK, opinions regarding the actions mediated by JAK/STAT vary (65). Acording to the findings of Sheng et al. (497), Latchman explains that the phosphorylation of the STAT-3 transcription factor was entirely unafected by inhibition of the MAPK pathway and, therefore, is likely to mediate the hypertropic efect of CT-1 (299). In line with this statement, the majority of works propose that the activation of STAT-3 promotes myocardial hypertrophy (283, 284, 446, 497). However, acording to Takahashi and colleagues (537) the major pathway responsible for the hypertrophic response to CT-1 is MEK-5/ERK-5. Furthermore, the JAK/STAT pathway appears to also be involved in the beneficial mechanism that transduces the protective signal against doxorubicin-induced and postpartum cardiomyopathy, protecting cardiomyocytes from ischemic and oxidative stres, promoting myocardial angiogenesis and tisue oxygenation during reperfusion, and controlling interstitial collagen metabolism with a reduction in cardiac fibrosis (204, 205, 283, 366, 367). Finaly, the P13K/Akt pathway appears to, along with the p42/44 MAPK pathway, promote cardiac myocyte survival against apoptosis (368). Neither MEK1, p42/44 MAPK nor P13K/Akt pathway activation alone is sufficient to induce CT-1 mediated cardioprotection against non-ischemic stimuli and re-oxygenation (53, 321). Thus, the MAPK and P13K/Akt pathways may cooperate to produce the cardioprotective efects of CT-1 (53, 65, 289, 321). Se figure 3 for an overview of cardiotrophin-1 signaling (65). 44 Figure 3. Cardiotrophin-1 signaling. Structure of the CT-1 receptor, made up of the LIFR/gp130 heterodimer and the intracelular pathways activated by CT-1. 2. CT-1 and the Heart As stated previously, cardiotrophin-1 has two wel-documented efects on the heart: myocardial hypertrophy and cardioprotection/cardiac myocyte survival. CT-1 was first isolated as a factor capable of inducing cardiac myocyte hypertrophy, one of the most important adaptive responses of the heart and a central feature of many cardiac diseases in man (65). Cardiomyocyte hypertrophy eventualy leads to cardiac muscle failure at it latest stages. The original report on CT-1 from Pennica et al. (422) showed that CT-1 was a potent inducer of hypertrophy, with activity being detected at concentrations of 0.1 nM or lower. Further in vitro studies showed that the hypertrophy induced by CT-1 was distinct in terms of cel morphology and gene expresion patern from that induced by, for example, !-adrenergic stimulation (606). CT-1 promotes an increase in cardiac cel size caused by an increase in cel length (sarcomeres in series) without a significant change in cel width (sarcomeres in paralel) (606). Studies also indicate that CT-1 induces a 45 distinct gene expresion patern as it up-regulates the atrial naturiuretic peptide gene, a marker activated during hypertrophyin vivo. However, CT-1 does not afect skeletal !- actinin or myosin light chain-2 synthesis (606). This is indicative of volume overload- induced hypertrophy as opposed to the presure overload-induced hypertrophy that is characteristic of !-adrenergic stimulation (606). These findings led Wollert and colleagues (606) to conclude that CT-1-induced hypertrophy is closely related to the volume overload-induced hypertrophy that occurs during valvular insufficiency and results in ireversible loss of cardiac function in humans. Jin et al. (242) further confirmed the hypertrophic efects of CT-1 through experiments in vivo. Chronic administration of CT-1 to mice caused a dose-dependent increase in both heart weight to body ratio and ventricular weight to body ratio (242). Based on this evidence, CT-1 can clearly induce cardiac hypertrophy both in vivo and in vitro (242, 299, 606). Cardiac muscle cel survival plays a critical role in maintaining the normal function of the heart. Adult cardiac muscle cels are fully developed and no longer diferentiate. Thus, they have lost their proliferative capacity, and a heart injury might result in scaring and an eventual decrease in global cardiac function (65). The literature has widely shown that CT-1 has a cardioprotective efect. Sheng and colleagues (497) demonstrated that treatment with CT-1 was able to enhance the survival of cultured neonatal rat cardiac myocytes. Furthermore, Stephanou et al. (526) found that pretreatment with CT-1 was able to protect cultured neonatal cardiac myocytes against subsequent exposure to either heat shock or simulated ischemia/hypoxia. Similarly, CT-1 was also able to enhance levels of heat shock proteins hsp70 and hsp90, whose over- expresion has been shown to protect cardiac myocytes against both thermal and ischemic stres (93, 195, 196). CT-1 appears to exert its cardioprotective efects by minimizing the degre of programed cel death (apoptosis) that is induced by serum removal or by thermal and ischemic stres (52, 53, 497, 526). More recent studies have further documented, both in neonatal and adult cardiac cels, the cardioprotective efects of CT-1 against ischemia, when added both prior to and after hypoxic stimulus (52, 309). In addition, CT-1 promoted cardiac myocyte survival against non-ischemic death stimuli, such as angiotensin I and hydrogen peroxide (H 2 O 2 ) (321). Therefore, myocardial CT-1 expresion is a clasic example of a cardiac compensatory mechanism that can be helpful 46 of harmful. CT-1 expresion is anti-apoptotic and promotes hypertrophy, both of which my initialy be beneficial. However, CT-1 appears to be involved in the induction of pathological hypertrophy known to have an adverse efect on left ventricular systolic function (56). Due to these diferent efects on the heart, CT-1 may be a relevant marker of disease and may play a role in the pathological changes typical of such cardiovascular diseases as hypertension, congestive heart failure, and ischemic heart disease (65). Hypertensive heart disease is largely characterized by the presence of left ventricular hypertrophy (LVH) (517). Systemic hypertension places a mechanical overload on the left ventricle and activates several stres pathways that induce an increase in left ventricular mas (65). It has been proposed that some of these pathways involve gp-130 dependent ligands (420). Furthermore, a recent study indicates that IL-6-related cytokines may participate in the development of hypertensive LVH (285). Concentrations of cardiotrophin-1, a member of the IL-6 family of cytokines, are elevated in humans with both treated and untreated hypertension as compared to normotensive individuals (320, 421). Moreover, in hypertensives, CT-1 levels are higher in those with LVH as opposed to those with normal ventricular thickening (320). Lopez and colleagues (319) further report that plasma levels of CT-1 in subjects with esential hypertension are correlated to inappropriate left ventricular mas (ILVM), defined as left ventricular mas/predicted left ventricular mas > 128%. In treated hypertensives, normalization of CT-1 concentrations is asociated with a regresion of ILVM/LVH, whereas an increase in CT-1 levels results in persistent ILVM/LVH (171, 319). Congestive heart failure is a condition in which the heart?s ability to deliver oxygen rich blood to the body is not sufficient to met the body?s needs (65). Both atrial and ventricular gene expresion of CT-1 is increased in animal models of CHF (11, 245). Also, in animal models, CT-1 expresion precedes the development of the pathological hypertrophy that characterizes CHF (225). In humans with congestive heart failure circulating concentrations of CT-1 are elevated (541). However, the exact role of CT-1 in the pathophysiology of CHF or as a marker of CHF remains unclear (65). Plasma concentrations of CT-1 increase with the severity of CHF and CT-1 levels are significantly higher in hearts explanted from patients with end-stage heart failure as 47 opposed to donor hearts (644). These explanted hearts expresed 142% higher levels of CT-1 mRNA and 68% higher CT-1 protein concentrations (644). In patients with dilated cardiomyopathy, characterized by volume overload, CT-1 concentrations correlate with left ventricle mas index, suggesting a role for CT-1 in left ventricular remodeling and left ventricular hypertrophy (562). Brain naturietic peptide (BNP) is a marker for overt CHF (65). Ventricular CT-1 expresion is elevated, while ventricular BNP gene expresion is not augmented in early stage left ventricular dysfunction (244). These findings suggest that CT-1 is a biomarker for detecting early ventricular dysfunction as compared to BNP (65). Furthermore, Tsutamoto et al. report on the additional prognostic value of CT-1 alone or combined with BNP in patients with CHF (561). Ischemic heart disease is the most common cause of mortality worldwide (65). Elevated serum concentrations of CT-1 are clearly correlated with the degre of left ventricular systolic dysfunction and are observed in individuals with unstable angina, acute myocardial infarction and heart failure (542-544). In addition, CT-1 expresion was elevated in the post-myocardial infarction heart from 24 hours to 8 weks (151). Evidence suggests that CT-1 has beneficial efects during the early phases of post-MI wound healing. These efects include: promoting myocardial cel survival, inducing hypertrophy of remaining myocytes, and inducing proliferation and migration of fibroblasts from adjacent viable myocardium (outside the infarct zone). In this way, CT- 1 can help reduce myocyte death and improve ventricular performance (150, 151). However, in the chronic post-MI stages, CT-1 may actualy contribute to the deterioration of ventricular function due to the stimulation of ventricular hypertrophy (65). Therefore, chronicaly increased synthesis and release of CT-1 could acelerate contractile dysfunction, whereas acute synthesis could preserve contractility (643). The role of CT-1 as a marker and as a cause of structural and functional changes typical of advanced cardiovascular disease is becoming wel documented. CT-1 has a wide variety of functions that sometimes have opposite results. Thus, at this time it is dificult to say whether CT-1 is a favorable or adverse molecule in the context of cardiovascular disease (65). 48 3. CT-1, Adipose Tisue, and Metabolic Disease Two recent investigations have explored the efect of cardiotrophin-1 in adipose tisue and metabolic disease (365, 645). Zvonic and colleagues (645) sought to determine the role of CT-1 on adipocyte physiology. CT-1 administration results in a dose- and time-dependent activation and nuclear translocation of STAT-1, -3, -5A and - 5B as wel as ERK-1 and -2 MAPKs in 3T3-L1 adipocytes in vitro. Furthermore, acute CT-1 administration was able to activate STAT-1 and -3, as wel as ERK-1 and -2 in the ependymal fat pads of C57B1/6J mice in vivo (645). Acute treatment of CT-1 in 3T3-L1 adipocytes resulted in the induction of SOCS-3 mRNA as wel as a transient down- regulation of PAR-" mRNA. These efects were independent of MAPK activity, providing evidence that they involve the same signaling pathways that induce CT-1?s hypertrophic efects. The ability of CT-1 to regulate PAR-" and SOCS-3 may prove to be interesting in its relation to afecting adipocyte signaling and metabolism (645). Chronic administration of CT-1 to 3T3-L1 adipocytes resulted in decreased protein levels of IRS-1 after 72 and 96 hours of exposure (645). Based on this result, Zvonic et al. (645) examined the efects of CT-1 on insulin stimulated glucose uptake in 3T3-L1 adipocytes. One hour pretreatment with CT-1 did not significantly inhibit glucose uptake. Following a 24 and 96 hour pretreatment, insulin-stimulated glucose uptake was significantly decreased. The 96-hour pretreatment specificaly resulted in a 4-fold decrease as compared to control cels (645). Therefore, the down-regulation of IRS-1 expresion by CT-1 could be a possible marker of impaired insulin sensitivity in CT-1 treated adipocytes. The observation that chronic CT-1 exposure results in a statisticaly significant decrease in insulin-stimulated glucose uptake supports the hypothesis that CT- 1 may be a mediator of impaired insulin sensitivity (645). Elevated circulating levels of CT-1 are found in patients with ischemic heart disease and valvular heart disease (151). Clinical aspects of these diseases are tightly linked to obesity and type 2 diabetes (450, 518). Therefore, CT-1 may serve as a link betwen obesity-related complications and cardiovascular disease (645). Natal and colleagues (365) sought to test whether adipose tisue expreses CT-1 and whether CT-1 expresion can be modulated as wel as to compare serum CT-1 concentrations in subjects with and without metabolic syndrome as diagnosed by the 49 NCEP ATP II criteria. These investigators found that both mouse and human adipocytes expres cardiotrophin-1. Moreover, in 3T3-L1 adipocytes, CT-1 expresion progresively increases along with diferentiation time from preadipocyte to mature adipocyte. In other words, mature adipocytes expres higher levels of CT-1 than do undiferentiated cels. The analysis of various murine tisues including the heart, thymus, splen, kidney, pancreas, liver and adipose tisues confirmed this novel finding that adipose tisue is an important source of CT-1. In fact, CT-1 expresion in fat tisue exceded by thre-fold that found in other tisues tested. However, it has not yet been determined whether CT-1 release by adipose tisue is due to resident cels (macrophages, etc.) rather than adipocytes (365). Furthermore, Natal et al. (365) screned glucose and some cytokines/nuerohumoral factors implicated in obesity and inflamation for their ability to induce CT-1 expresion. Al tested stimuli positively increased CT-1 expresion in 3T3- L1 cels, however glucose significantly increased adipocyte-derived CT-1 mRNA and protein expresion in a dose-dependent manner. This efect of glucose could acount for the elevated circulating CT-1 concentrations observed in individuals with hyperglycemia and metabolic syndrome (MetS) (365). Natal and colleagues (365) observed significantly elevated serum CT-1 concentrations in obese subjects and in individuals with hyperglycemia and MetS as opposed to normal-weight controls and those with normal fasting blood glucose concentrations. Furthermore, in the entire population of individuals both with and without MetS, CT-1 concentrations positively correlated with glucose but not with blood presure, triglycerides, HDL-C or BMI. Similarly, CT-1 concentrations were significantly higher in subjects with hyperglycemia (fasting glucose > 100 mg/dL), but not significantly diferent in patients with hypertension, hypertriglyceridemia or low HDL-C (365). In other words, increased serum levels of CT-1 were observed in individuals with MetS, particularly in obese and diabetic subjects, which could be explained by overexpresion of the cytokine from adipose tisue (365). The altered release of cytokines by adipose tisue in obesity may have negative efects on the cardiovascular system (90, 126, 554). The novel observation from Natal and colleagues (365) that adipose tisue expreses CT-1, a cytokine with demonstrated cardiovascular 50 and metabolic actions, presents the possibility that CT-1 may play a pathophysiological role in MetS, serving as a link betwen obesity-related complications and cardiovascular disease. Moreover, glucose-induced adipose tisue expresion of CT-1 may further explain the relationship betwen obesity and insulin resistance in MetS (365). 4. CT-1 and Reactive Oxygen Species/Oxidative Stres Several recent studies have demonstrated a direct relationship betwen cardiotropin-1 and reactive oxygen species (ROS)/oxidative stres (21, 482). It has previously been discused that CT-1 activates the Jak/STAT signaling cascade as wel as the ERK/Akt (or protein kinase B) pathways, which are known to be involved in the regulation of cel proliferation and cytoprotection (289, 497). It has also recently been demonstrated that CT-1 activates NF-%B and p38, which may partialy mediate the cytoprotective and anti-apoptotic efects of CT-1 in cardiomyocytes (91). The Jak/STAT, ERK and NF-%B signaling pathways have previously been shown to be regulated by intracelular reactive oxygen species. Thus, CT-1 may exert its biological efects through an elevation of ROS, which act as signaling molecules in CT-1 induced signal transduction cascades (238, 482-484, 511). Sauer et al. (482) investigated the involvement of ROS in CT-1 mediated signaling cascades in cardiomyocytes diferentiated from murine embryonic stem cels. This group found that upon stimulation of cels with CT-1, intracelular ROS levels increased rapidly via the elevated activity of NADPH-oxidase, which is regulated by PI3- kinase. These reactive oxygen species are then utilized as signaling molecules to ensure the proper functioning of the CT-1-induced signaling pathways (482). Furthermore, Sauer and colleagues (482) found that CT-1?s stimulation of cardiac cel proliferation was regulated by ROS, as in the presence of the fre radical scavenger vitamin E, the stimulation of cardiomyocyte proliferation was abolished. In summary, CT-1 elicits an increase in intracelular ROS via NADPH-oxidase, regulated by PI3-kinase (482, 484). ROS generated by NADPH-oxidase in response to stimulation with CT-1 may be utilized to initiate and maintain CT-1-mediated signaling cascades (482). The properties of CT-1 as a cardioprotective and hypertrophic cytokine in stres conditions predict up-regulation during cardiac diseases that are characterized by an 51 environment of hypoxia, inflamation and oxidative stres (21). Ateghang and colleagues (21) have recently shown that CT-1 expresion is regulated by reactive oxygen species and hypoxia in diferentiating mouse embryonic stem cels. ROS and hypoxia use a common HIF-1-regulated signaling pathway that results in increased CT-1 expresion. HIF-1 is a heterodimer composed of the constitutively expresed $-subunit and the oxygen-dependent !-subunit (HIF-1!) that is stabilized under hypoxic conditions (211). Treatment with the antioxidant vitamin E down-regulated CT-1 as wel as HIF- 1!, indicating regulation by ROS endogenously generated in diferentiating embryonic stem cels (21). Treatment of these same cels with the pro-oxidant hydrogen peroxide resulted in a dose dependent increase in CT-1 protein expresion. Under the same experimental conditions, increased protein and mRNA expresion of HIF-1! was also observed, thus corroborating the idea of paralel regulation of CT-1 and HIF-1! by pro- oxidants. However, statistical significance was achieved at earlier time points for HIF- 1! expresion compared with CT-1 expresion, suggesting that HIF-1! precedes CT-1 expresion (21). This hints that the up-regulation of CT-1 by ROS is mediated by HIF-1 signaling. The up-regulation of CT-1 mRNA and protein was completely abolished in HIF-1!-deficient embryonic stem cels. This further supports the notion that ROS and hypoxia regulated CT-1 expresion is mediated by HIF-1 (21). Furthermore, pro- oxidants appear to increase the activity of ROS generating NADPH-oxidase thereby providing a fed-forward loop of increased ROS generation even in the absence of external pro-oxidants. This, in turn, wil help to induce an increased expresion of CT-1 via HIF-1. On the other hand, an inhibition of NADPH-oxidase significantly reduced the increase in CT-1 expresion following treatment with pro-oxidants, thus indicating that NADPH-oxidase derived ROS are involved in up-regulation of CT-1 by pro-oxidants and hypoxia (21). Hypoxia is a pathophysiological situation occurring during conditions where increased expresion of CT-1 has been reported, such as angina pectoris, myocardial infarction and heart failure (56, 65, 151). It has recently been demonstrated that hypoxia is asociated with increased ROS generation produced either through the mitochondrial respiratory chain or NADPH-oxidase (605). Therefore, CT-1 is asociated with hypoxic 52 conditions asociated with increased generation of ROS. Based on the studies from Sauer et al. (482) and Ateghang et al. (21), it appears that CT-1 elicits an increase in ROS via NADPH-oxidase regulated by PI3-kinase. This elevation in ROS, in turn, induces an up- regulation of CT-1 mediated by HIF-1 signaling (21). Thus, it is possible that CT-1 and ROS regulate one another through a positive fedback loop initiated by pathophysiological hypoxia. Fibroblast Growth Factor-21 Fibroblast growth factor-21 was first identified in 2000 as a secreted protein preferentialy expresed in the liver (374). The fibroblast growth factor family consists of 22 members, divided into 7 subfamilies based on structural similarities and modes of action (438). A majority of FGFs serve as paracrine factors regulating cel growth and diferentitation, including angiogenesis and transformation (229). Members of the FGF- 19 subfamily, including FGF-21 and FGF-23, difer in that they have no or very smal mitogenic efects and they exert important metabolic efects via systemic endocrine mechanisms (374, 475, 506, 615). The thre members of the FGF-19 subfamily share approximately 30% amino acid sequence homology (267). FGF-19 subfamily members regulate diverse physiological proceses that are not afected by other FGFs. These metabolic activities include the regulation of lipid and carbohydrate metabolism, as wel as bile acid, phosphate, calcium and vitamin D homeostasis (208, 268, 324, 505, 552). FGF-21 is predominantly expresed in the liver and has beneficial efects in various animal models of obesity and metabolic disease (89, 268, 269). FGF-21 expresion in the liver was shown to be under the control of the transcription factor PAR-! (325). However, FGF-21 expresion is rather plastic and can be induced by PAR-" activation in adipocytes and muscle cels (233, 357, 358, 580). The human FGF-21 gene is located on chromosome 19 and encodes a 209 amino acid protein. Human and mouse FGF-21 share a 75% amino acid sequence identity (267). Results from recent studies show that FGF-21 expresion and regulation in human subjects is considerably diferent than that observed in animal models, thus provoking discussion of the possible clinical significance of this cytokine (73, 89, 268, 269, 308, 636). 53 1. FGF-21 Signaling Fibroblast growth factors mediate their action via a set of membrane-bound FGF receptors (FGFR). Upon binding, FGF-21 stimulates tyrosine phosphorylation of FGFRs, which appears to be critical to FGF-21 activity. This leads to the activation of a number of downstream signals including MAPKs, RAF1, AKT1, and STATs. The same efects can be caused by archetypal FGFs such as FGF1, providing evidence that FGF21 signals through activation of a conventional FGFR-mediated pathway (268, 357, 381, 592). However, FGFs cannot interact with FGFRs directly as they require a co-factor to bind and activate FGFR signaling. For most, this co-factor is heparin/heparin sulfate (169, 475). FGF-21 is unique in that it does not bind or require heparin for its bioactivity, fails to directly interact with soluble FGFRs and shows an activity profile restricted to adipose and pancreatic cels (169, 268, 357, 381). The weak heparin sulfate binding afinity prevents FGF-21 from being captured in the extracelular matrix, thus alowing it to function as an endocrine factor (169, 349). FGF-21, along with the other members of the FGF-19 subfamily, require the presence of specific transmembrane proteins from the klotho family for FGFR binding and activation (288, 566). $-klotho, a protein that shares 41% amino acid identity with klotho, is the critical component of the FGF-21 receptor complex (288, 381). $-klotho is a type I transmembrane protein whose function is not wel understood. A recent report links this molecule to cholesterol and bile acid metabolism; however, the exclusive expresion of $-klotho in the pancreas, liver and adipose tisue suggests a broader biological role (226, 227, 267, 286). An investigation in murine 3T3-L1 cels showed that $-klotho forms a preformed complex with FGFR1 or FGFR2, which is then activated by FGF-21 binding (266). Two studies concluded that the carboxy-terminus region of FGF-21 is esential for $-klotho binding, while the amino-terminus is important for FGFR activation (344, 624). Furthermore, several reports suggest that the c-receptor splice isoforms of FGFR1-3 exhibit an afinity to $-klotho and most likley act as endogenous receptors for FGF-21 (266, 287, 381). FGF-21 was shown to be bioactive in murine BaF3 cels stably expresing both $-klotho and FGFR1c or FGFR3c, but not in those that do not expres any endogenous FGFRs (267). Similarly, cels lacking $-klotho do not respond to FGF- 54 21 and the introduction of $-klotho to these cels confers FGF-21 responsivenes (266). Moreover, in 3T3-L1 cels, transient overexpresion of FGFR4 resulted in interation with $-klotho and the binding of FGF-19 and FGF-21 (267). These results together suggest that FGF-21 may interact with diferent FGFRs in diferent celular environments as long as $-klotho stimulates receptor activation (475). $-klotho is almost exclusively expresed in the pancreas, liver and adipose tisue (227). The main efects of FGF-21 have been observed in the adipose tisue and pancreas, however, no direct efects of FGF-21 have been observed in the liver (475). This may be due to the fact that the liver predominantly expreses FGFR4. Although both FGF-19 and FGF-21 can bind to the $-klotho-FGF-4 complex, only interactions with FGF-19 result in sufficient receptor activation (287). On the other hand, Kharitonenkov and colleagues (267) explain that FGF-21 likely does have an efect on liver-derived cels, given the profound impact of FGF-21 on lipid metabolism in animals. FGFR1 is the most abundant receptor in 3T3-L1 cels and in white adipose tisue. Thus, in adipose tisue, it is likely that the main functional receptors of FGF-21 are preformed $-klotho- FGFR1c complexes (266, 287) (se figure 4). However, although the initial steps in FGF-21 signaling are becoming beter characterized, the afected downstream target genes remain to be defined (475). Furthermore, molecular specifics about the initial FGF-21 mechanism of action need to be further clarified (267). In native conditions, FGF-21 functions through FGFR1 and FGFR2 in adipocytes and pancreatic cels. Multiple splice isoforms of these receptors have been detected, and it is currently unclear which particular variants serve as the specific FGF-21 receptors within the FGF-21/FGFR/$-klotho complex (268, 381, 592). Also, no FGFR4 and insignificant FGFR3 levels were detected in 3T3-L1 adipocytes (357). Thus, it remains unclear whether FGF-21 can signal through these FGFRs when they are present at elevated levels (267). 55 FGF-21 Carbohydrate and Lipid Metabolism Figure 4. FGF-21 signaling in adipose tisue. The interaction of $-klotho and FGFR1 stimulates receptor activation. FGF-21 binds to the $-klotho-FGFR1 complex to induce changes in carbohydrate and lipid metabolism. 2. FGF-21 in Animals Mounting evidence from animal-based studies suggests FGF-21 as a potent metabolic regulator with multiple beneficial efects on obesity and diabetes (355, 456). Kharitonenkov and colleagues (268) first identified these efects in 2005. FGF-21 stimulated insulin-independent glucose uptake through increasing GLUT-1 expresion in murine 3T3-L1 adipocytes in vitro. Furthermore, FGF-21 administration in ob/ob and db/db mice lowered blood glucose and triglyceride levels, while transgenic mice that overexpres FGF-21 were protected against diet induced obesity and insulin resistance. Four hours after the administration of FGF-21, GLUT-1 mRNA upregulation was observed in the white adipose tisue, but not in the muscle, liver, kidney, or brain of ob/ob mice. Importantly, FGF-21 did not induce mitogenicity, hypoglycemia, or weight gain at any dose tested in diabetic, obese or healthy animals or when overexpresed in transgenic mice (268). $-klotho FGFR1c 56 Several studies employing murine models of diet-induced obesity found that FGF- 21 administration can reduce body weight by up to 20%, increase energy expenditure and reverse insulin resistance and hepatic steatosis (89, 612). Coskun and colleagues (89) reported that two wek systemic administration of FGF-21 in diet-induced obese and ob/ob mice resulted in a 20% reduction in body weight predominantly due to a reduction in adiposity. Also, via transcriptional and blood cytokine profiling, FGF-21 treated animals demonstrated increased energy expenditure and fat utilization and reduced hepatosteatosis. In addition, FGF-21 exhibited the ability to ameliorate insulin and leptin resistance, enhance fat oxidation and suppres de novo lipogenesis in the liver (89). Recently, Xu et al. (612) reported similar findings in diet-induced obese mice. FGF-21 dose dependently reduced body weight and fat mas in these animals due to marked increases in total energy expenditure and physical activity levels. Administration of FGF-21 reduced blood glucose, insulin, and lipid levels and reversed hepatic steatosis. FGF-21 also dramaticaly improved hepatic and peripheral insulin sensitivity in both lean and diet-induced obese mice independently of reductions in body weight and adiposity (612). The findings of these two studies are quite similar, yet they do have their diferences. Coskun and colleagues (89) reported a reduced respiratory quotient that is indicative of a preferential utilization of fat as an energy store. Xu et al. (612) found similar reductions in body weight (~20%) via decreased adiposity and increased energy expenditure. However, no changes in respiratory quotient were observed, suggesting that there were no alterations in fuel selection betwen faty acids and carbohydrates. Instead these mice demonstrated a significant increase in physical activity (612). FGF-21 caused a dramatic decline in fasting plasma glucose, triglycerides, insulin and glucagon when administered for 6 weks to diabtetic rhesus monkeys (269). FGF-21 administration also led to beneficial changes in lipid profiles including the lowering of LDL-C and elevation of HDL-C and the induction of smal but significant weight loss. Of significant importance from a safety standpoint, hypoglycemia was not observed at any point after FGF-21 administration (269). The celular mechanisms through which these efects are elicited are not entirely clear. As mentioned earlier, in murine adipocytes in vitro FGF-21 increases glucose uptake by an upregulation of GLUT-1 (268). However, it is unlikely that an upregulation 57 of GLUT1 is the main mechanism through which FGF-21 exerts its insulin sensitizing efects (475). The enhancement of GLUT-1 following FGF-21 administration in mice is modest and limited to adipose tisue (268). Also, an upregulation of GLUT-1 does not explain the FGF-21-induced amelioration of dyslipidemia or weight reduction (475). FGF-21 inhibited pancreatic glucagon secretion and increased islet insulin content and glucose-induced insulin release by increasing islet survival through a protection from glucolipotoxicity and cytokine-mediated apoptosis (592). Yet, these efects on islet survival and $-cel function most likely are not the primary mechanism, as they cannot explain the reductions in weight or hepatosteatosis (475). Furthermore, considering the results from Coskun and colleagues (89), the anti-obesity efect of FGF-21 is probably not mediated via changes in circulating factors since out of 67 studied hormones and cytokines, FGF-21 treatment only led to a significant reduction in leptin and insulin. Based on the contrasting findings from Coskun et al. (89) and Xu et al. (612) regarding whether FGF-21 administration afects physical activity, it is stil unclear whether FGF- 21 increases locomotor activity or not. This should be addresed in future studies as it could provide a mechanism through which FGF-21 exerts its insulin sensitizing and anti- obesity efects in animals (475). In addition to its insulin sensitizing efects in animals, FGF-21 also appears to mediate the physiological response to food deprivation/starvation and to be a primary factor in intitating the production of ketone bodies (24, 223, 456). As noted earlier, FGF- 21 sems to be under the control of PAR-! in the liver, which is activated during starvation (24, 223). The activation of PAR-! (via starvation, fibrates or ketogenic diet) in male mice directly results in an increase in circulating FGF-21 concentrations, which promotes lipolysis in adipose tisue. Subsequently, faty acids are taken up by the liver and converted into ketone bodies, which are crucial energy sources during fasting and starvation (24, 223). Also, Inagaki and colleagues (223) reported that FGF-21 reduces physical activity and promotes torpor, a short-term hibernation-like state of regulated hypothermia that conserves energy. These findings are in direct contrast to previous reports that FGF-21 increases energy ependiture and physical activity (89, 612). However, these contrasting findings could be due to the fact that they were found in response to starvation, as opposed to data from animal models of obesity/energy exces. 58 Thus, in states of energy exces FGF-21 appears to induce an insulin sensitizing and anti- obesity efect, while in states of starvation/energy deficit FGF-21 promotes energy conservation through ketogenesis and torpor. As mentioned previously, recent data show that FGF-21 expresion can be induced not only in the liver, but also in adipose tisue (358, 580, 636). This induction is most likely regulated by PAR-" activity. Wang and colleagues (580) identified an amino acid within helix 7 of the PAR-" ligand binding domain that is required for the ability of PAR-" to activate a novel group of adipocyte genes, including FGF-21. Furthermore, exposure of 3T3-L1 adipocytes to the PAR-" agonist troglitazone resulted in rapid induction of FGF-21 mRNA expresion. These results were confirmed by a series of FGF-21 gene promoter/luciferase reporter asays. It was thus determined that PAR-" directly regulates expresion of FGF-21 (580). Moreover, Muise et al. (358) sought to identify potential secreted proteins regulated by PAR-" in diabetic animal models. This group identified 33 genes coding for secreted proteins regulated by PAR-" agonists in epididymal white adipose tisue of db/db mice, one of which was FGF-21 (358). This indicates that FGF-21 gene expresion results from PAR-" activation in adipose tisue. Expresion of FGF-21 in vivo was also found to be regulated by PAR-", as PAR-" agonist treatment in db/db mice resulted in a 2-3 fold elevation of FGF-21 mRNA in adipose tisue coincident with an elevation of plasma FGF-21 concentrations (358). Lastly, Zhang and colleagues (636) observed that chronic treatment with the PAR-" agonist rosiglitazone caused a dramatic induction of FGF-21 production in 3T3- L1 adipocytes. These results suggest a role for FGF-21 in mediating the anti-dibetic efects of PAR-" agonists (358). In addition, it appears that FGF-21 also regulates PAR-" expresion. Continuous treatment of 3T3-L1 cels with FGF-21 induces an increase in PAR-" protein expresion. Treatment of cels with FGF-21 and the PAR-" agonist rosiglitazone in combination leads to a pronounced expresion of GLUT-1 and a marked stimulation of glucose uptake. Treatment with either FGF-21 or PAR-" alone did not significantly increase glucose transport (357). These results reveal a novel synergy betwen FGF-21 and PAR-" in regulating glucose homeostasis. This synergy demonstrated a marked 59 functional interplay betwen FGF-21 and PAR-" pathways. In summary, FGF-21 stimulation of 3T3-L1 adipocytes elevates PAR-" expresion and the PAR-" agonist rosiglitazone modulates FGF-21 action, sensitizing its ability to activate/induce tyrosine phophorylation of FGFR-2 (357). These non-human data suggest that FGF-21 and PAR-" regulate one another as a possible protective pathway in diabetic and obese states. 3. FGF-21 in Humans There are no published data to date regarding the efects of FGF-21 in humans in vivo. Only studies investigating the efects in human cels in vitro and cross-sectional analysis of FGF-21 concentrations in various subpopulations show that the efects of FGF-21 in humans do not appear to be consistent with those observed in animal models (18, 73, 111, 161, 307, 308, 636). For example, murine FGF-21 sems to promote lipolysis in 3T3-L1 adipocytes (223, 268). However, FGF-21 has no efects on basal lipolysis, and actualy atenuates hormone-stimulated lipolysis in primary cultures of human adipocytes. It must be noted though that these efects were only elicited at supraphysiological concentrations (18). Based on these data, FGF-21 may have a beneficial efect on insulin sensitivity in man. The increased release of faty acids into circulation is a wel-established factor underlying the development of insulin resistance (16). Although it remains to be shown, the FGF-21 mediated atenuation of lipolysis could result in reduced levels of circulating fre faty acids in vivo thus positively influencing insulin sensitivity (475). Several recent investigations have asesed circulating concentrations of FGF-21 in various human cohorts (73, 111, 161, 307, 308, 636). Zhang et al. (636) reported that serum FGF-21 levels were significantly higher in overweight/obese subjects than in lean individuals. Serum FGF-21 concentrations also corelated positively with BMI, waist circumference, waist-to-hip ratio, fat percentage, fasting insulin, HOMA and triglycerides and negatively with HDL-C, QUICKI and adiponectin after adjusting for age and BMI. Moreover, logistic regresion analysis showed an independent asociation betwen serum FGF-21 levels and the metabolic syndrome. The increased risk of the metabolic syndrome asociated with elvated serum FGF-21 was over and above the efects of 60 individual components of MetS (636). However, interestingly, no significant diference in serum FGF-21 levels was observed betwen non-diabetic and diabetic subjects. This study ilustrates an asociation betwen elevated circulating concentrations of FGF-21 and metabolic complications, which directly conflicts with data gathered from animal models. The paradoxical increase in serum FGF-21 in obese individuals may possibly be explained by a compensatory response or a resistance to the protein similar to that sen with leptin (636). Li and colleagues (308) investigated whether plasma FGF-21 levels were diferent in patients with type 2 diabetes (T2DM) or diabetic ketosis (T2DK) and in sex- and age- matched normal controls. Plasma concentrations of FGF-21 were markedly higher in patients with T2DK and T2D than in controls. Increasing plasma concentrations of FGF- 21 were independently and significantly asociated with T2DM and T2DK even after controlling for anthropometric variables, blood presure and lipid profile. Fasting plasma FGF-21 was positively correlated with systolic blood presure, diastolic blood presure, HbA 1 c, HDL-C, and FA and negatively correlated with fasting plasma insulin and insulin sensitivity. Multiple regresion analysis demonstrated that diastolic blood presure, waist-to-hip ratio and FA were independent related factors regulating plasma FGF-21 concentrations. These results further suggest that FGF-21 is related to metabolic complications in humans and may play a role in the pathogenesis of type 2 diabetes (308). It must be noted, however, that the findings of this study conflict with the results from Zhang et al. (636) that showed no elevation in FGF-21 concentrations in diabetic subjects. The diferences in study design, including subject selection and experimental conditions is likely the contributing factor to this discrepancy. Chen et al. (73) investigated whether or not plasma FGF-21 levels were diferent in patients with type 2 diabetes melitus (T2DM) and non-diabetic controls. Fasting FGF-21 concentrations were significantly higher in patients with T2DM as compared to controls. There were no gender diferences and plasma FGF-21 levels remained significantly higher in diabetics after adjustment for age, sex and BMI and after controlling for anthropometric variables and diferences in blood presure and lipids (73). This result is in line with the findings of Li and colleagues (308), yet in contrast to the findings of Zhang and colleagues (636). Furthermore, plasma FGF-21 concentrations 61 inversely correlated with fasting plasma glucose in simple regresion analysis. After multiple regresion analysis, fasting plasma glucose, insulin and insulin sensitivity as asesed by HOMA, negatively correlated with plasma FGF-21 levels. These inverse correlations suggest that insulin secretion or action may be an FGF-21 determining factor. Moreover, elevated FGF-21 levels in patients with T2DMmay suggest the impairment of FGF-21 signaling in target tisues or the dysregulation in biosynthesis/in response to hyperglycemia or hyperinsulinemia in a diabetic state (73). The results here further suggest a potential role for FGF-21 in the pathogenesis of insulin resistance and T2DM. The findings of Li and colleagues (307) are a bit diferent from those discussed previously (73, 308, 636). This group reported that serum FGF-21 concentrations were significantly higher in subjects with impaired glucose tolerance than in individuals with normal glucose tolerance after adjustment for age, gender and BMI. Serum FGF-21 levels correlated positively with alanine aminotransferase, "-glutamyltransferase, total cholesterol, triglycerides, LDL-C and several parameters of adiposity including BMI, waist circumference, fat percentage and fat mas after adjustment for age. A negative asociation betwen FGF-21 and HDL-c was also demonstrated. Multiple stepwise regresion anlysis showed an independent asociation of serum FGF-21 with serum triglycerides, total cholesterol and "-glutamyltransferase. However, unlike the previous thre studies discussed, FGF-21 did not correlate with insulin secretion and sensitivity as measured by hyperglycemic clamp and 75-g oral glucose tolerance test (307). Thus, these data demonstrate that serum concentrations of FGF-21 are elevated in subjects with impaired glucose tolerance, however, FGF-21 is not related to insulin secretion and sensitivity. Instead, serum FGF-21 levels in humans appear to be more related to lipid metabolism and early liver injury (307). Since litle information is available on the functions of FGF-21 in humans, Galman and colleagues (161) caried out studies to analyze circulating levels of FGF-21 in various conditions, such as in normal and hypertriglyceridemic humans and during metabolic perturbations induced by fasting, a ketogenic diet, and by fenofibrate (a PAR- ! agonist) treatment. Results demonstrated that FGF-21 circulates in humans at highly variable levels. Specificaly, serum FGF-21 varied 250-fold among 76 healthy 62 individuals and did not relate to age, gender, BMI, serum lipids, or plasma glucose. It should be noted that subjects with high or low FGF-21 concentrations maintained their individual levels when sampled for 25 hours. This indicates that there is an intrinsic variation in FGF-21 metabolism in healthy subjects. Therefore, variation in serum concentrations FGF-21 may not be important for metabolic regulation (161). No increases in serum FGF-21 were observed following a 48 hr fast or ketogenic diet treatment, despite the induction of ketosis. However, fasting for 7 days significantly increased serum FGF-21, yet these levels remained in the normal range. FGF-21 concentrations were also elevated by two-fold in hypertriglyceridmic patients as compared to healthy controls, while fenofibrate treatment lowered serum triglycerides and increased serum FGF-21 (161). In summary, Galman et al. (161) ascertain that there is a large interindividual variation in serum levels of FGF-21 in humans and that hypertriglyceridemia appears to be linked to elevated levels. Prolonged fasting and fenofibrate treatment both increase FGF-21, although ketogenesis can clearly be induced independent of serum FGF-21 concentrations. These results together hint that the physiological roles of FGF-21 in man may be far diferent from those observed in mice (161). Interestingly, Dostalova et al. (111) measured plasma concentrations of FGF-21 in patients with anorexia nervosa in order to explore a relationship with anthropometric and endocrine parameters. Plasma FGF-21 concentrations were significantly reduced in anerorexia nervosa relative to normal age-matched controls and were positively correlated with BMI, serum leptin and insulin. An inverse relationship to serum adiponectin was reported in both the anorexia nervosa and control groups. No significant relationship was found betwen FGF-21 and serum ketone bodies, FA or HOMA. Multiple regresion analysis showed that leptin and adiponectin were independent predictors of plasma FGF-21 concentrations (111). Similar to the previous studies discussed that measured FGF-21 concentrations in obese individuals, these results suggest that circulating levels of FGF-21 are strongly related to body weight. Moreover, in line with the results from Galman and colleagues (161) and contradictory to acute fasting animal studies, malnutrition (fasted state) in humans is asociated with markedly 63 reduced levels of FGF-21. These findings further demonstrate that the bioactivity of FGF-21 in humans is diferent relative to that in rodents. FGF-21 is primarily believed to act via endocrine mechanisms. Thus, a detailed asesment of tisue expresion is lacking in these studies. However, the results of these studies help characterizing the bioactivity of FGF-21 in humans. They show some similarities, but many diferences with previously published data in animals, suggesting that the biology of FGF-21 may be quite complex (475). Plasma FGF-21 levels are highly variable in normal weight subjects, yet a significant positive correlation is observed with obesity, the metabolic syndrome and type 2 diabetes (73, 161, 308, 636). This paradoxical increase of FGF-21 concentrations, as compared to animal data, might be a defensive response of the human body to counteract the stres imposed by obesity and metabolic complications. Alternatively, reminiscent of hyperinsulinemia and hyperleptinemia, obesity and metabolic disorders may cause a resistance to FGF-21?s actions leading to its compensatory up-regulation (636). Another possibility is that FGF- 21 in circulation may undergo truncations in either the amino- or carboxy-terminal region, thereby inactivating the protein. The elevated levels observed in various clinical conditions in man could be characterized by the presence of a non-functional FGF-21 molecule. These possible mechanisms as to why FGF-21 concentrations sem to be elevated in conditions asociated with the metabolic syndrome are of great interest and have yet to be investigated. Therefore, one can only speculate as to whether the increase in FGF-21 is a protective mechanism, an indirect sign of FGF-21 resistance or a phenomenon secondary to reduced plasma clearance, increased ectopic expresion or the presence of a truncated, inactive form of the protein in circulation (475). Adiponectin Adiponectin, a protein hormone also known as adipoQ or adipocyte complement related protein, is specificaly and very highly expresed in adipose tisue (177). Adiponectin is also the most abundantly expresed adipokine in white adipose tisue and circulates in concentrations of 5 to 10 ?g/mL in human serum (36, 222). Adiponectin exerts insulin-sensitizing and anti-atherogenic efects (76). The hormone enhances 64 insulin sensitivity in the muscle and liver and increases fre faty acid (FA) oxidation in several tisues (155, 618). It also lowers serum FA, glucose and triglyceride concentrations (155, 177). In circulation, adiponectin forms a variety of multimers such as trimers (low molecular weight ? LMW), hexamers (medium molecular weight ? MW) and dodecamers or 18-mers (high molecular weight ? HMW) (246). It has been shown that HMW adiponectin is the active form that results in the improvement of insulin sensitivity (136). Two receptors for adiponectin have been identified: adipoR1 and adipoR2 (616). Disruption of both prevents adiponectin binding, resulting in increased triglyceride levels, inflamation and oxidative stres (619). This further supports the role of adiponectin in regulating insulin sensitivity (550). Clinical studies implicate levels of adiponectin in the pathogenesis of obesity related disease. Unlike most cytokines, adiponectin concentrations are decreased in obesity (14, 81, 594). Plasma concentrations are negatively correlated with BMI (14). Moreover, a longitudinal study in primates demonstrated that adiponectin decreases with weight gain as the animals become obese (217). Conversely, plasma adiponectin levels increase significantly following weight loss (621). Adiponectin levels difer with the distribution of body fat as wel (222). Indeed, plasma concentrations exhibit strong negative correlations with intra-abdominal fat mas (81). Visceral, but not subcutaneous, fat stores were reported to be inversely asociated with plasma adiponectin levels in healthy women (290). A low waist to hip ratio is asociated with elevated circulating levels of adiponectin, independent of body fat percentage (521). Furthermore, plasma adiponectin concentrations are lower in individuals with type 2 diabetes melitus (216). Adiponectin levels correlate strongly with insulin sensitivity, suggesting a role for low plasma concentration in the development of insulin resistance (523). In a study of Pima Indians, who demonstrate a high incidence of obesity, insulin resistance and type 2 diabetes, individuals with high levels of adiponectin were les likely to develop diabetes (311). Adiponectin concentrations are also reported to be asociated with components of MetS. Elevated levels relate to an advantageous blood lipid profile (29, 557). Moreover, adiponectin concentrations are decreased in persons with hypertension, irespective of the presence of IR (232). A recent study reported that adiponectin levels might serve as a 65 biomarker for MetS, demonstrating that adiponectin is a key contributor within the pathogenesis of obesity and MetS (601). Several studies using experimental animal models have studied the metabolic actions of adiponectin. In obese animals, treatment with adiponectin decreases hyperglycemia and plasma FA levels and improves insulin sensitivity (36). Adiponectin knockout mice exhibit severe insulin resistance in response to a high fat/sucrose diet (332). This model also displays delayed clearance of plasma FA, elevated levels of TNF-! mRNA in adipose tisue and higher plasma TNF-! concentrations (332). Restoration of adiponectin expresion by adenovirus-mediated gene transfer resulted in a reversal of these conditions (281, 332). Furthermore, studies ilustrate that adiponectin regulates glucose metabolism and insulin sensitivity through the phosphorylation and activation of the 5?-AMP-activated protein kinase (AMPK) in muscle and liver tisues (551, 617). Adiponectin also appears to protect against the development of various vascular diseases through its anti-atherogenic function (578). Adiponectin concentrations are reduced in patients with coronary artery disease (216). In addition, adiponectin inhibits TNF-! induced expresion of adhesion molecules and the transformation of macrophages into foam cels, both of which are key components of atherogenesis (395, 396). Administration of adiponectin reduces atherosclerotic lesion size by 30% in apolipoprotein E deficient mice, and this coincides with reductions in VCAM-1 and TNF-! expresion in the aortic sinus (386). On the other hand, adiponectin-deficiency leads to an increase in vascular lesion area n apolipoprotein E deficient mice (385). Adiponectin knockout mice also exhibit a greater incidence oh hypertension when placed on a high salt diet (384). Adiponectin stimulates the intracelular signaling kinases Akt and AMPK, which phosphorylate and activate endothelial nitric oxide synthase (398). It appears that the anti-hypertensive efects of adiponectin are mediated by its ability to increase the production of nitric oxide by endothelial cels (578). Adiponectin exerts beneficial efects on the heart under pathological conditions (578). Adiponectin knockout mice contract severe cardiac hypertrophy and overexpresion of adiponectin wil atenuate cardiac hypertrophy in response to presure overload in wild type and diabetic db/db mice (502). In response to angiotensin I 66 infusion, adiponectin deficient mice develop cardiac hypertrophy, while adiponectin overexpresion reduces the hypertrophic response (549). The anti-hypertrophic actions of adiponectin can be atributed to modulation of the intracelular AMPK cascade. Cel culture experiments in cardiac myocytes show that adiponectin activates AMPK, and inhibits the hypertrophic response to !-adrenergic receptor stimulation (502). Adiponectin?s anti-hypertrophic efects can be reversed by transduction with dominant negative AMPK (156). Furthermore, in response to myocardial ischemia, adiponectin deficient mice display increased myocardial infarct size, myocyte apoptosis and myocardial TNF-! expresion. On the other hand, overexpresion of adiponectin reduces infarct size, apoptotic cel frequency and TNF-! levels (503). It appears that, in cultured myocytes, adiponectin?s inhibition of apoptosis is mediated by its ability to activate AMPK signaling and that adiponectin also suppreses TNF-! production (501). In humans, it has been reported that elevated adiponectin levels are asociated with lower risk of myocardial infarction in men and that adiponectin concentrations rapidly decline following myocardial infarction (277, 433). Moreover, lower levels of adiponectin are also asociated with a further progresion of left ventricular hypertrophy in patients presenting with hypertension, left ventricular diastolic dysfunction and hypertrophy (210). Leptin Leptin, predominantly produced by adipose tisue, was the first adipocyte hormone identified (76, 177, 186, 637). Leptin influences appetite and food intake through a direct efect on the hypothalamus (302). Leptin is also a marker of nutrition, as low plasma leptin concentrations serve as a ?starvation signal?, decreasing energy expenditure and stimulating the search for food in food deprived rodents (137, 220, 394). Leptin receptors have been identified in peripheral tisues including endothelial cels, platelets, monocytes/macrophages and the brainstem (337, 444, 633). In the obese state leptin is thought to contribute to insulin resistance and is considered one of the links betwen obesity, insulin resistance and atherosclerosis (37). Plasma leptin concentrations are highly correlated with BMI (333). Mice deficient of the leptin coding gene, or ob/ob mice, are very obese and diabetic. If these 67 mice are treated with regular injections of leptin they reduce their food intake, their metabolic rate is increased and they lose weight (177, 186, 419). As animals and humans become obese, the role of leptin in regulating body weight becomes more complex. In most obese individuals leptin concentrations are high because of the increased amount of leptin secreting adipose tisue (84). There is also a suggested leptin resistance. It appears that as leptin concentrations rise, the hormone induces target cels to become resistant to its actions (360). As leptin concentrations increase, so does the expresion of SOCS-3, a potent inhibitor of leptin signaling (360). Leptin has important efects on peripheral metabolism as wel. Leptin is able to reverse hyperglycemia in ob/ob mice before body weight is corrected (419). Furthermore, in a mouse model of congenital lipodystrophy resulting in insulin resistance, hyperinsulinemia, hyperglycemia, and faty liver, leptin therapy reversed insulin resistnce and diabetes (507). Leptin also improves glucose homeostasis in humans with lipodystrophy or congenital leptin deficiency (392). Administration of exogenous leptin to individuals with lipoatrophic diabetes resulted in marked reductions in triglyceride concentrations, liver volume, and glycated hemoglobin and discontinuation or large reduction in anti-diabetes therapy (392). However, leptin failed to correct hyperglycemia in obese patients, further supporting the concept of ?leptin resistance? in these individuals (203). Leptin improves insulin sensitivity in muscle by reducing intramyocelular lipid levels and activating AMPK (346). Leptin also enhances insulin sensitivity in the liver by decreasing intracelular hepatic triglyceride levels (249). Interestingly, exercise activates AMPK, which also increases fat oxidation and reduces insulin resistance (471). Thus, leptin, adiponectin and exercise may act via the same signal transdusction pathway to increase fat oxidation and promote insulin sensitivity (177). Leptin is also apparently involved in the pathogenesis of cardiovascular disorders. Hyperleptinemia is asociated with atherosclerosis independent of insulin resistance (454). It has been shown that leptin deficient mice were protected from the development of atherosclerosis despite the contribution of al other metabolic factors that acelerate vascular disease (191). The mechanism of this is not completely understood, but the prolonged stimulation of the imune system sems to be one of the potential 68 mechanisms since leptin may enhance both proliferation and diferentiation of hemopoietic cels, including T cels (322). Furthermore, leptin has multiple efects on cels of the arterial wal. In endothelial cels leptin induces oxidative stres, increases production of MCP-1 and endothelin-1, and potentiates proliferation (49, 411, 442, 614). In addition, leptin increases platelet aggregation and arterial thrombosis via a leptin receptor-dependent pathway, stimulates macrophage recruitment by inducing the release of monocyte colony-stimulating factor, promotes cholesterol acumulation in macrophages under high glucose conditions and stimulates angiogenesis (85, 278, 314, 377, 510). Leptin also promotes calcification of cels in the vascular wal and facilitates thrombosis by increasing platelet aggregation (363, 408). Finaly, leptin appears to contribute to vascular disease via obesity-asociated hypertension, not through metabolic actions, but via action on central sympathoregulatory pathways (88). Leptin increases peripheral sympathetic tone, and lower arterial presures are found in leptin-deficient mice, thus suggesting a role for leptin in the development of hypertension (49, 442). These aforementioned efects of leptin may explain the epidemiological asociation betwen elevated levels and cardiovascular risk (37). TNF-$ TNF-! was first described as an endotoxin-induced serum factor that causes necrosis of tumors (70, 372). Two TNF-! receptors, type 1 (TNFR1) and type I (TNFR2), mediate the TNF-! signal by forming protein complexes with cytoplasmic adaptor proteins (372). TNF-! plays a key role in the mediation of the imune response as a multi-functional regulator of inflamation, cel apoptosis and survival, cytotoxicity, and production of other cytokines, such as IL-1 and IL-6 (372). Obesity-asociated TNF- ! is primarily secreted from macrophages infiltrating adipose tisue, whereas the adipocytes predominantly produce un-secreted, membrane bound TNF-! (37, 587, 611). The secreted adipose tisue TNF-! is specificaly increased in visceral adipose depots (558). TNF-! is thought to work mainly through autocrine or paracrine functions, where local concentrations would be more likely to exert its metabolic efects (222, 348, 629). TNF-! was the first cytokine to be implicated in the pathogenesis of obesity and insulin resistance (215). Adipose tisue expresion of TNF-! is increased in obese 69 rodents and humans and positively correlated with adiposity and insulin resistance (132, 215, 470). In obese humans, TNF-! expresion is increased and improvements in this increased expresion occur following weight loss (97, 263). Moreover, expresion of TNF-! in obese fa/fa rats and ob/ob mice was increased and shown to regulate insulin action (215, 550). Mice lacking TNF-! or the TNF receptors had improved insulin sensitivity and decreased circulating faty acids in both dietary and genetic models of obesity (567). At a molecular level, exposure of cels to TNF-! or elevated levels of fre faty acids stimulated inhibitory phosphorylation of serine residues on IRS-1, thus lending to insulin resistance (5, 417). Several potential mechanisms for TNF-!?s metabolic efects have been described. TNF-! activates serine kinases such as JNK and p38 mitogen-activates protein kinase (MAPK) that increase serine phosphorylation of IRS-1 and IRS-2, making them poor substrates for insulin receptor-activating kinases and increasing their degradation (214, 527). TNF-! not only initiates, but also propagates atherosclerotic lesion formation (300). It is known that TNF-! activates the transcription factor nuclear factor-%B (NF- %B), which acelerates experimental atherogenesis, in part by inducing the expresion of VCAM-1, ICAM-1, MCP-1, and E-selectin in aortic endothelial and vascular smooth muscle cels (395). This results in inflamatory and foam cel acumulation (38). Moreover, TNF-! reduces NO bioavalibility in endothelial cels and impairs endothelium-dependent vasodilation, promoting endothelial dysfunction (38, 581). Despite these intriguing in vitro data, animal studies focused on TNF-! and the development of atherosclerosis have produced mixed results (37). Reducing TNF-! concentrations in apoE deficient mice resulted in a significant decrease in atheromatous lesions, however, in wild-type mice reduced TNF-! levels produced no improvements (51, 490). In addition, mice deficient of the p55 TNF-! receptor exhibited acelerated atherosclerosis (489). Finaly, although TNF-! is thought to play a role in the progresion of ischemia-related congestive heart failure, anti- TNF-! therapy has produced no benefits for congestive heart failure progresion (265). Despite these conflicting data, TNF-! stil may very wel partake in the pathogenesis of atherosclerosis and vascular disease. 70 Interleukin-6 IL-6 belongs to a family of cytokines that activate target genes involved in cel diferentiation, survival, apoptosis and proliferation (76). IL-6 is one of the primary pro- inflamatory mediators secreted by imune system cels and is a key player in haematopiesis and acute phase and imune responses (198). IL-6 and its soluble receptor activate endothelial cel production of chemokines and upregulate expresion of adhesion molecules resulting in the recruitment of leukocytes to inflamatory sites, and may wel regulate TNF-! and IL-1 synthesis through autocrine and paracrine loops (463, 531). IL-6 binds to a plasma membrane receptor complex containing the common signal transducing receptor chain gp 130. Signal transduction involves the activation of JAK tyrosine kinase family members, leading to activation of transcription factors from the STAT family. IL-6 also appears to signal through the MAPK cascade (198). Circulating IL-6 concentrations are positively correlated with obesity, impaired glucose tolerance and insulin resistance (31). Several studies have reported the positive relationship betwen body mas index and plasma IL-6 concentrations (328, 409). 20-30 % of this cytokine in circulation is produced by adipose tisue. In obese subjects, with elevated waist-to-hip ratios, this participation is even greater (348). It has been documented that visceral adipose tisue releases 2 to 3 times more IL-6 than does subcutaneous adipose tisue (138, 152). This cytokine may also be involved in the pathogenesis of insulin resistance, as insulin sensitivity is enhanced in diet-induced obese mice treated with anti-IL-6 antibodies (275). In addition, plasma IL-6 concentrations predict the development of type 2 diabetes melitus (575). Fasting plasma IL-6 levels were negatively correlated with the rate of insulin-stimulated glucose disposal in Pima Indians (222, 575). Peripheral administration of IL-6 induces hyperlipidemia, hyperglycemia, and insulin resistance in rodents and humans (428, 529, 559). IL-6 impairs insulin signaling through a down-regulation of IRS and an up-regulation of SOCS-3 (457). Bastard and colleagues (31) have reported that IL-6 concentrations are more strongly corelated with obesity and insulin resistance than TNF-!, and that very low-calorie diet results in significant decreases in circulating IL-6 concentrations in obese women. A variety of other studies also demonstrate that weight loss results in decreased levels of circulating IL-6 (120, 167, 279). Moreover, circulating IL-6 stimulates the 71 hypothalamic-pituitary-adrenal axis, which is asociated with central obesity, hypertension and insulin resistance (628). Obesity asociated induction of adipose IL-6 production causes the secretion of hepatic acute phase response proteins including CRP, fibrinogen, serum amyloid-A and !-1 antichymotrypsine (642). It is wel known that CRP is both a marker of and an important risk factor for cardiac events and atherosclerosis in the general population (61, 76). Recent data suggest that CRP might directly elicit endothelial dysfunction and atherogenesis at the vesel wal (107, 414, 512). Furthermore, IL-6 stimulates the production of fibrinogen and platelet activity, which increase the risk of clot formation (62). There are data that suggest IL-6 decreases lipoprotein lipase activity, which results in elevated circulating FA and macrophage uptake of lipids. In young atheromatous lesions, macrophage foam cels and smooth muscle cels expres IL-6, suggesting a role for this cytokine in the early stages of atherosclerosis (628). These data suggest a role for the cytokine IL-6 in the pathogenesis of obesity related metabolic and cardiovascular disorders. Sumary It is recognized that adipose tisue functions as both an energy storage and secretory tisue producing a variety of bioactive peptides. Adipocytes from lean, healthy individuals secrete cytokines that coordinate systemic metabolic proceses and protect cardiovascular tisues from stres (578). Moreover, complex interactions exist betwen cytokines, inflamation and the adaptive responses in maintaining homeostasis and health (115). Obesity causes an imbalance in cytokine secretion (578). Abnormalities in cytokines, their receptors and the signaling pathways that they initiate such as IK$, JNK and JAK/STAT are involved in a wide variety of diseases (130). The cytokines, and possibly more appropriately adipokines or adipocytokines, that may provide a molecular link betwen obesity and the development of MetS, type 2 diabetes and cardiovascular disease are of particular interest. These cytokines include wel-characterized bioactive peptides such as adiponectin, TNF-! and IL-6, as wel as a wide variety of les understood cytokines including cardiotrophin-1 and fibroblast growth factor-21. 72 Weight Loss and Lifestyle Modification Weight loss through lifestyle modification is beneficial for treating excesive adiposity, dyslipidemia, hypertension, hyperglycemia and insulin resistance among other metabolic disorders (87, 413). The magnitude of weight loss does not need to be drastic, as even modest weight loss of 5-10% significantly atenuates metabolic dysfunction (87). The Finnish Diabetes Prevention Study demonstrated that lifestyle intervention with modest weight loss significantly reduced the prevalence of MetS (221). Furthermore, the intensive lifestyle intervention with moderate weight loss utilized in the Diabetes Prevention Program also resulted in a 41% reduction in the incidence of MetS (393). Current therapies that cause weight loss by inducing a negative energy balance include dietary intervention, physical activity, pharmacotherapy and surgery. Behavior modification to enhance dietary and activity compliance is an important component of al these treatments. At present, the therapeutic intervention used does not appear to be relevant to the benefit of weight reduction with a few exceptions (434). However, the use of caloric restriction through dietary modification and enhanced energy expenditure through physical activity are the most commonly prescribed therapies to induce modest weight loss. A decrease in caloric intake is an avenue by which to create a negative energy balance resulting in weight loss (476). It is prudent to recommend a reduced calorie diet low in saturated fat, higher in unsaturated fat, high in whole grains and low in sodium (87). However, more important than the composition of the diet, is the overal caloric intake. Regardles of which macronutrients are emphasized, hypo-caloric diets are key to inducing meaningful weight loss (476). A lifestyle aimed at increasing physical activity/energy expenditure and decreasing, or even maintaining, body weight is another important approach to reducing health risks (87). Increased physical activity and higher cardio-respiratory fitnes, independent of weight loss, are asociated with decreased CVD risk and lower incidence of type 2 diabetes and MetS (298, 383, 585, 622). Exercise is also particularly efective at improving insulin sensitivity as wel as reducing dyslipidemia and hypertension (23, 106, 474, 524). Cardiorespiratory fitnes sems to have an independent efect on metabolic function in some aspects; however, a change in body weight and body 73 composition (particularly abdominal adiposity), is an important mediator in the ability of increased physical activity to modify chronic disease risk (87). Regular exercise appears to play an important role in abdominal fat loss and the metabolic changes that ensue (466). Inflammation and Weight Loss Weight loss and a reduction in adipose tisue mas are asociated with a decrease in obesity related markers of inflamation regardles of whether weight loss is induced by caloric restriction (20, 80, 259, 462, 613), exercise (387, 462) or a combination of the two (462). However, when employing dietary restriction, studies using low-calorie diets as the method of intervention resulted in the highest mean weight loss, compared with those using other methods such as low-fat diets. The greatest improvements in circulating concentrations of obesity related markers of inflamation were observed in studies reporting at least 10% weight loss (47, 59, 84, 426, 447, 545). For example, Kasim-Karakas and coleagues failed to show any significant changes in serum CRP, IL- 6 or adiponectin with 8% (6 kg) weight loss after a 12-month low-fat diet (258). On the other hand, two low-calorie diet investigations reported a weight loss of approximately 15% with a 32% decrease in plasma CRP after 14 months and a 24% reduction in plasma IL-6 after only 6 months (59, 84). Moreover, You and colleagues showed that a 5% (5 kg) weight reduction was unable to induce significant improvements in markers of inflamation (625). Bougoulia et al. demonstrated a 22, 21, 86, and 32% decrease in plasma CRP, TNF-!, IL-6, and leptin, respectively, and a 72% increase in plasma adiponectin concentrations with a 19% (20 kg) weight loss (47). In addition, Hannum et al. failed to show a significant decrease in serum CRP with modest weight loss (4-7% or 4-6 kg) in either a self-selected or portion-controlled low-calorie, low-fat diet (190). Therefore, based on this data, the benefits of weight loss greater than 10% can clearly be sen (142). Yet, this does not discount the positive benefits asociated with a modest 5- 10% weight loss. For example, Heald and colleagues reported that a reduced-fat diet resulted in a smal but significant weight loss of 2 % (1 kg) and a significant 24% decrease in serum CRP (197). 74 Few studies have explored the efect of weight loss on inflamatory markers through increased physical activity alone (387, 462, 530, 560). In these studies, the resulting changes in obesity related markers of inflamation are fairly inconsistent. For example, TNF-! significantly decreased by 16% after 12 weks in one investigation (530), decreased by 83% after 5 months in another (560), and significantly increased by 12% in a third intervention (462). These diferences are not explained by changes in body composition or the degre of weight loss. However, difering intensities and/or frequencies of exercise training in each investigation, or possibly the duration of the intervention may explain the observed findings (142). Interventions combining both diet and physical activity modifications also report significant weight loss and improvements in obesity related markers of inflamation (55, 78, 97, 114, 119, 120, 335, 371, 379, 462, 473, 498, 519, 625, 632, 639). For example, Ryan and colleagues (473) observed reductions in plasma CRP and IL-6 of 7 and 16%, respectively with modest weight loss. Moreover, with 7-9% weight loss You et al. (625) found that plasma CRP, IL-6 and TNF-! concentrations decreased by 34, 27 and 6% respectively. The thre investigations that resulted in the greatest amount of weight loss were also the longest in duration (1 ? 2 years) (97, 120, 335). Significant improvements in inflamatory markers were also reported. The decrease in serum TNF-! concentrations ranged from 24 ? 31% (97, 335), the decrease in serum CRP ranged from 34 ? 44% and the decrease in serum IL-6 ranged from 33 ? 67 % (120, 335). Serum adiponectin concentrations were shown to increase by 48% (97). These thre studies were performed in women, however similar improvements in CRP and IL-6 were also found in men (119). These studies clearly demonstrate the benefit of weight loss through dietary and exercise modification in ameliorating obesity-induced inflamation (142). Insulin Resistance, Diabetes and Weight Loss A variety of studies have shown that modest weight loss through caloric restriction (20, 80, 361, 613), increased physical activity and a combination of both (315, 410, 452, 582, 588) serves to improve insulin sensitivity and ameliorate insulin resistance. Weight loss has a dramatic efect on blood glucose values in individuals with or without diabetes. It is estimated that for every 1 kg of weight loss, plasma glucose 75 decreaes by roughly 0.2 mM. Thus, a modest 5 kg weight loss would decrease average fasting plasma glucose by 1 mM or 18 mg/dL. This improvement is in the range that is provided by many of the oral hypoglycemic agents that are commonly approved by the FDA (9). The US Diabetes Prevention Program Research Group recently reported data on a large randomized clinical trial of type 2 diabetes prevention (276). Over 3200 overweight adults with impaired glucose tolerance were included in the study. The intensive lifestyle intervention group received diet, exercise and behaviour modification counseling, and aimed for a 7% reduction in body weight. Average weight loss was roughly 4 kg at the end of the follow-up period. This lifestyle intervention and subsequent weight loss was highly efective in preventing/delaying the appearance of type 2 diabetes. Compared with the placebo/control group, there was a 58% reduction in the incidence of diabetes in the lifestyle inervention group (276, 569). Furthermore, The DP showed that weight loss was the number one predictor of reduction in the incidence of diabetes. In fact, for every kilogram of weight loss, the risk of diabetes development was decreased by 16% (189). The Finnish Diabtetes Prevention Study (FDPS) also reported that modest weight reduction could limit the incidence of type 2 diabetes (563). This study included subjects with impaired glucose tolerance divided into a lifestyle intervention group and a control group. After 1 year the average weight loss in the intervention group was 4 kg (5% weight reduction), resulting in a 58% lower cumulative incidence of diabetes as compared to subjects in the control group. This weight loss further beneficial in reducing waist circumference, plasma triglycerides and systolic and diastolic blood presure, emphasizing the pleiotropic efects of modest weight loss (563, 569). Cardiovascular Disease Risk Factors and Weight Loss Weight loss systematicaly reduces risk factors for cardiovascular disease (8). A number of studies demonstrate that modest weight loss achieved by caloric restriction (20, 80, 259, 361, 435, 613), exercise and combination of both (410, 570) is efective in normalizing cardiovascular disease risk factors such as hypertension and dyslipidemia. For example, Dengel and colleagues (105) studied 12 overweight individuals with known diabetes or vascular disease. During weight loss, there were significant reductions in 76 BMI and body fat percentage along with improvements in total cholesterol, LDL-C, triglycerides and insulin sensitivity. After 6 months, there were also significant improvements in brachial artery compliance and distensibility (105). Acording to a meta-analysis conducted by Datilo and Kris-Etherton (99), a weight loss of just 1 kg reduces serum cholesterol values by 2.28 mg/dL, LDL-C by 0.91 mg/dL, and triglycerides by 1.54 mg/dL, and increases serum HDL-C by 0.07 mg/dL. Furthermore, Balkestein et al. (26) reported that 3 months of weight loss induced by negative caloric balance improved carotid distensibility in obese men. Thus, along with improvements in blood lipids, weight loss may also lead to enhanced vascular function. Moreover, blood presure sems to decrease in a linear manner with weight loss (8). Acording to a meta-analysis from MacMahon et al. a smal 1 kg weight loss results in an average systolic blood presure reduction of 0.49% or 0.68 mHg, whereas diastolic blood presure decreases 0.38% or 0.34 mHg. This same report observed a 6.3 mHg reduction in systolic blood presure and a 3.1 mHg reduction in diastolic blood presure with a 9.2 kg weight loss (329). Anderson and colleagues found that with a greater weight loss of, on average, 35.3 kg in morbidly obese subjects, the reductions of systolic and diastolic blood presure were 13 mHG and 9.6 mHg, respectively (8). This equates to a reduction in blood presure of roughly 10% (9). However, it should be noted that only about 85% of hypertensive obese individuals have significant reductions in blood presure with significant weight loss, indicating that other factors are at play a role in regulating hypertension in obese individuals (8). Furthermore, the US Trials of Hypertension Prevention, Phase I evaluated the impact of weight loss on the incidence of hypertension (528). It included 1,191 overweight individuals with borderline hypertension (systolic <140 mHg, diastolic 83- 89 mHg). These subjects were alocated to either a control group or a weight loss intervention group aiming at a 4.5 kg weight reduction during the first 6 months of the trial. This weight loss was to be maintained until the 36 months period. The weight loss intervention included behavior modification consisting of dietary change and increased physical activity. The control group experienced a slight weight gain during the study period. The intervention group had a mean weight reduction of 4.4 kg at 6 months, 2.0 kg and 18 months and 0.2 kg at 36 months. Although the initial weight loss was not 77 maintained, mean bosy weight in the intervention group was always significantly les than that of the control group. At the 6, 18 and 36 month follow-up periods, the cumulative incidence of hypertension was significantly lower in the intervention group. There was a dose response relationship betwen the degre of weight loss and the amount of blood presure reduction. Subjects who lost 4.5 kg or more at 6 months and maintained that weight loss over 36 months demonstrated a 65% reduction in hypertension risk as compared to those who did not lose weight (528, 569). Oxidative Stres and Weight Loss Weight loss and fat loss through dietary restriction and/or increased physical activity appears to have positive efects on oxidative stres as wel (573). A 4-wek dietary weight loss study in 9 obese patients measured oxidative stres before and after the weight loss program. Al subjects were restricted to a 1000 kcal/day diet. Weight loss (4.5 +/- 2.8 kg) was acompanied by a 13% reduction in plasma TBARS (a biomarker of oxidative stres) and a significant reduction in ROS. Reductions in al oxidative stres biomarkers occurred rapidly in obese subjects after only one wek of dietary restriction. This benefit persisted until wek 4, but disappeared after the cesation of treatment (96). Chen and colleagues examined the relationship betwen dietary paterns and oxidative stres. 122 premenopausal women were randomized into one of four groups for a 12-month program. The groups included: no intervention, low-fat (15% energy from fat), high fruit and vegatable intake (nine servings per day), and combined low-fat, high fruit and vegetable intake group. Following the intervention, weight loss occurred only in the low-fat groups. Concentrations of 8-isoprostane were also only reduced in the low-fat groups. Moreover, changes in BMI were directly correlated with changes in 8-isoprostane following the intervention. Therefore, the authors concluded that weight loss directly resulted in reductions in oxidative stres (72). Controlled food intake also reduces blood insulin and glucose concentrations, thereby suppresing insulin- induced fre radical formation that may occur in obesity (297). Thus, caloric restriction and diet modification may reduce body weight and fre radical/ROS formation in obese individuals, both of which lower oxidative stres (573). 78 A 3-wek combined diet and exercise intervention consisting of high carbohydrate/low fat diets and 1.5 hours of daily exercise was administered to 80 obese individuals. Following the program, body weight was reduced by 4-5% and LDL shifted from smal pro-atherogenic to large les-atherogenic particles. Furthermore, the basal level of serum lipid oxidation was reduced by 21%. There was no change in LDL vitamin E content, but LDL $-carotene content increased by 46% (32). Therefore, diet and exercise intervention shifted the LDL particles to the les atherogenic large particle size and reduced oxidative stres in obese individuals (573). In a study caried out by Roberts and colleagues, obese men completed a 3-wek short-term Pritikin vegitarian diet regimen and walked for 45-60 min/day. Average weight loss was 4 kg or 3.8% of body weight. This weight loss resulted in reduced total cholesterol, LDL-C and triglycerides and increased HDL-C. Serum 8-isoprostane levels were also reduced from 210 to 150 pg/ml from baseline to post-treatment (458). Again, rapid reductions in oxidative stres were observed with short-term weight loss. These reductions may be asociated with improvements in blood lipids that acompany dietary change and weight loss (573). One recent study compared the efect of adding exercise to Orlistat treatment in ameliorating oxidative stres (403). Participants were placed into either and Orlistat treatment group or an Orlistat-exercise group (cycle ergometer, 3 days/wek, 45 min/sesion). Following the 12-wek intervention weight loss was 8.5% for the Orlistat group and 10.2% for the combined treatment group. MDA (a marker of oxidative stres) levels were 29.9% lowerin the Orlistat-exercise group as compared to the Orlistat-only intervention. Both groups showed reductions in serum vitamin A and E levels; however, these values remained higher in the Orlistat-exercise group. Therefore, the authors concluded that the combination of weight loss and exercise-induced up-regulation of antioxidant defenses in the combined treatment group were efective in lowering systemic oxidative stres (403, 573). In addition, animal studies provide evidence regarding exercise, weight loss and the reduction of oxidative stres. Saengsirisuwan et al. (477) separated obese Zucker rates into a control group, lipoic acid group (30 mg/kg body weight), exercise group (treadmil running for 60-75 min/day) and lipoic acid and exercise group. Following the 6-wek intervention, al treatment groups had lower body weights than the control 79 animals. Oxidative stres levels were significantly lower in al treatment groups and this was asociated with improvements in peripheral glucose uptake (477). Based on this and previous data, exercise-induced weight loss appears to be efective in reducing oxidative stres. Temporal Efects of Weight Loss and Significance of Study Modest weight loss of roughly 5 ? 10% of initial body weight has been demonstrated to have beneficial efects on metabolic and cardiovascular risk factors asociated with obesity. Hypo-caloric diets and regularly practiced exercise can create the negative energy balance necesary to generate these beneficial efects. However, the temporal efects of weight loss are not yet firmly established in the literature. Evidence suggests that metabolic improvements may occur rapidly within the first few eks of lifestyle modification and continue at a slower rate as weight loss continues. Clinical markers of metabolic health that demonstrate this trend include triglycerides, glucose, insulin, hemoglobin A1c, insulin sensitivity and biomarkers of oxidative stres (96, 262, 318, 345, 478, 513). It is possible that alterations in circulating levels of CT-1 and FGF-21 may miror these changes based on mechanisms and relationships presented in the literature. For example, CT-1 concentrations are elevated along with increased levels of insulin and glucose (365). CT-1 also down-regulates the nuclear receptor PAR-" most likely resulting in decreased expresion of the PAR-" target and insulin sensitizing agent adiponectin (358, 645). Furthermore, CT-1 and reactive oxygen species appear to regulate one another through a positive fedback loop in that CT-1 potentiates ROS production, which in turn, leads to further CT-1 expresion (21, 482). In obese humans, FGF-21 concentrations are elevated along with circulating faty acids, glucose and insulin (73, 111, 308, 636). FGF-21 is also a gene target of PAR-", which up-regulates adiponectin expresion and inhibits the formation of ROS (357, 358, 580). Weight loss results in an up-regulation of PAR-" that should cause an increase in FGF-21 and adiponectin and a decrease in ROS (568). However, FGF-21 levels are already increased in states of human obesity suggesting a possible FGF-21 resistance (73, 308, 475, 636). Based on this data, FGF-21 levels may decrease with weight loss, despite 80 being a gene target of PAR-". In other words, reductions in FGF-21 hypothesized here to occur with weight loss wil likely occur along with elevations in adiponectin and decreases in biomarkers of oxidative stres. It is possible that the link betwen FGF-21 and levels of faty acids, glucose and insulin may be stronger than the efects levied by weight loss-induced up-regulation of PAR-". Based on these specific relationships, CT- 1 and FGF-21 concentrations should decrease along with weight loss-induced reductions in glucose, insulin, NEFAs and biomarkers of oxidative stres and elevations in adiponectin demonstrated in the literature (96, 318, 373, 403, 412, 413, 439, 513, 568). Recent evidence supports the theoretical role of cytokines and adipokines in obesity and weight loss (37, 126, 143, 222). Both CT-1 and FGF-21 may be related to the metabolic dysfunction occurring with obesity as wel as play a role in the metabolic and cardiovascular health improvements asociated with weight loss (268, 365, 636, 645). However, there appears to be limited data in the literature that directly addreses the response of CT-1 and FGF-21 to weight loss in humans. Therefore, the importance of this study lies in its potential to contribute foundational clinical information that wil be useful and relevant to our understanding of the responses of CT-1 and FGF-21 to obesity, glucose metabolism and modest weight loss. 81 Chapter II. Methods Subjects This study used a sample cohort of subjects initialy recruited for participation in a larger investigation. We analyzed the data from 9 obese participants that completed a weight loss program and 7 aged matched controls. Al volunters met the following characteristics in order to be eligible for the weight loss portion of the study: 1) male betwen 30 to 65 years of age; 2) body mas index (BMI) was greater than 30 kg/m 2 and/or body fat was greater than 30% of total body weight and waist girth was greater than 40 inches; 3) weight stable over previous 6 months; 4) without previously diagnosed cardiovascular or metabolic disease; 5) exhibited no signs or symptoms of latent heart disease; 6) no regular exercise over the past six months and did not have a job requiring strenuous physical activity; 7) non-smoker; 8) did not take medication known to alter lipid or glucose metabolism, and; 9) did not exhibit conditions that would prevent regular treadmil walking. Control subjects also exhibited each of these characteristics excluding number 2. These individuals had a BMI below 27 kg/m 2 . Preliminary Screning and Baseline Procedures Volunters were recruited by flyers placed around the Auburn-Opelika area, as wel as through Auburn University?s daily faculty/staf announcement e-mail. Volunters were scheduled to visit the lab in order to undergo preliminary screning procedures. Initialy, volunters were given a health history questionnaire and physical activity questionnaire to complete. Subsequently, height, weight, waist girth and resting blood presure were measured. If al criteria for inclusion in the study were met, individuals were scheduled to return to the lab and underwent a physical exam by a physician. Following the physical exam, a blood sample was obtained via venipuncture 82 from an antecubital vein and conducted a body composition asesment using total body x-ray (DEXA) scan to determine body fat levels and fat distribution paterns. Volunters were then asked to perform a maximal exercise test (GXT), using the standard Bruce protocol, on a motor-driven treadmil to determine cardiovascular fitnes (301). For this test, a 12-lead electrocardiogram was administered at rest and throughout the exercise test. Blood presures were obtained periodicaly throughout the GXT to further determine cardiovascular responses to exercise. During the GXT, respiratory gases were measured by a Medical Graphics Ultima metabolic unit (MedGraphics, St. Paul, MN). Furthermore, heart rate and ratings of perceived exertion were monitored throughout the test. Following the preliminary screning procedures, volunters who met the necesary criteria were asked to report to the lab after two days of stable diet and physical activity and after a 10 to 12 hr overnight fast. After obtaining body weight and blood presure, a blood sample was collected via venipuncture in an antecubital vein. This fasting blood sample was used as the baseline sample for al biochemical variables. Participants also recorded their diet and physical activity for 7 days (starting 2 days before lab visit for fasting blood sample) for the purpose of baseline asesment. Weight Loss Procedures Volunters were randomly asigned to either an exercise or dietary restriction intervention in order to achieve an 8 to 10% weight loss, as recommended by the National Heart, Lung and Blood Institute (NHLBI) (1). The exercise and diet interventions were initialy designed to achieve a 2000 to 2500 kcal/wek energy expenditure or energy deficit determined from our initial energy requirement estimates. The overal targeted weight loss for both interventions was 8 to 10% of initial body weight over a 6 to 10 month period (1). Initialy, both groups underwent nutritional counseling and education regarding how to use the American Dietetic Asociation Food Exchange Program (2). The food exchange program was used to help participants maintain specific energy intake recommendations over the course of the weight loss period. 83 Every 4 weks each participant scheduled an appointment where a fasting blood sample was collected , DEXA scan performed, and current dietary and nutrient intake and exercise prescription was reviewed and modified. Also, subjects turned in 3-day diet and physical activity record. Recommendations at the monthly meting were based on the information provided in exercise logs (exercise intervention only), 3-day diet records and the rate of weight loss achieved over the previous month. Modifications to an intervention were based on adaptations in metabolic eficiency known to occur with caloric restriction and increased energy expenditure (341). Once participants achieved the targeted weight loss, we administered an OGT and collected a 3-day diet and physical activity log (starting 2 days before OGT) for the purpose of post-weight loss asesment. Exercise Intervention If asigned to the exercise intervention, participants achieved their wekly energy expenditure goal by exercising 4 to 5 times per wek. Each individual was asked to complete two exercise sesions per wek in the lab under direct supervision. The in lab exercise sesions included monitoring exercise intensity, obtaining a body weight and blood presure and reviewing outside physical activity records/exercise logs. These exercise sesions were monitored (respiratory gases and heart rates measured) and modified to achieve the desired work rates and corresponding heart rate range to met the exercise sesion energy expenditure goal (400 to 500 kcal/sesion). Each participant was trained and counseled in the use of a heart rate monitor and rating of perceived exertion (RPE) scale for monitoring exercise intensity. Further training included record keeping for maintaining an exercise log. Apart from the two exercise sesions per wek under laboratory supervision, participants completed the remaining exercise sesions on their own in order to achieve the recommended wekly caloric expenditure. Diet Intervention If asigned to the diet intervention, participants achieved their wekly energy deficit goal by initialy reducing daily caloric intake 300 to 350 kcal/day. The general counseling strategy for reducing daily caloric intake was to reduce portion sizes and to 84 replace caloricaly dense foods and those high in saturated fats with les-dense alternatives containing poly- and monounsaturated fats (87, 476). Subjects visited the lab two times per wek for body weight and blood presure measurment and review of their outside physical activity record. Any dietary isues or questions that subjects may have had were resolved during these regular visits. Biochemical Procedures and Analysis Fasting blood samples were collected into one 10 ml ?red top? serum vacutainer tube and one 4 ml ?purple top? potasium EDTA tube (Becton Dickinson Vacutainer, Franklin Lakes, NJ). Blood samples for each time point during the OGT were collected into one 10 ml ?red top? serum tube. Hemoglobin and hematocrit levels were measured from each blood sample collected in the potasium EDTA tubes. The remaining blood in al tubes was alowed to clot for 30 minutes and then centrifuged for 10 minutes to separate the serum and plasma. Aliquots of both serum and plasma were transfered into 2 ml ultracentrifuge tubes and subsequently stored at -70 degres centigrade for future analysis. FGF-21 (Milipore, St. Charles, MO), total adiponectin (Alpco Diagnostics, Salem, NH), TNF-! (R&D Systems, Minneapolis, MN), IL-6 (R&D Systems, Minneapolis, MN), and myeloperoxidase (MPO) (Alpco Diagnostics, Salem, NH) were determined by enzyme-linked imunosorbent asay (ELISA). Total antioxidant capacity (TAC) (BioVision Inc., Mountain View, CA), non-esterified faty acids (NEFA) (Wako Diagnostics, Richmond, VA), and glucose (Wako Diagnostics, Richmond, VA) concentrations were determined by an enzymatic colorimetric asay. CT-1 (Christchurch Cardio-Endocrine Research Group, Christchurch, NZ) (421) and insulin (Milipore, Bilerica, MA) were determined by radioimunoasay (RIA). The homeostasis model asesment (HOMA) using fasting insulin and glucose concentrations was used to estimate insulin resistance as follows: [fasting insulin !U/mL x fasting glucose (mol/L)]/22.5. The quantitative insulin sensitivity check index (QUICKI) was calculated as follows: 1 / (log (fasting insulin !U/mL) + log (fasting glucose mg/dL). 85 Statistical Analysis For the purpose of statistical analysis the data from both the diet and exercise groups were combined due to no observable diference in the extent or patern of weight loss betwen groups. The primary dependent variables of interest were plasma concentrations of CT-1 and serum concentrations of FGF-21. We measured myeloperoxidase and total antioxidant capacity (TAC) as biomarkers of oxidative stres. Cytokines measured were total adiponectin, TNF-!, and IL-6. Clinical markers of metabolic health that served as dependent variables were non-esterified faty acids (NEFA), glucose and insulin. Glucose to insulin ratio, HOMA and QUICKI were indexes used as surrogate markers of insulin resistance and insulin sensitivity. We used multiple statistical procedures to analyze our data. In order to addres question 1, an independent t-test was employed to determine diferences betwen obese and lean participants. A paired t-test was used to analyze changes that occured in the obese group with 8 ? 10% weight loss. Temporal changes with weight loss were analyzed using 1 x 6-10 repeated measures ANOVAs. Duncan?s New Multiple Range test was employed to describe significant diferences determined from ANOVAs. In order to examine the temporal response, weight loss was captured at each month and partitioned into percentages as follows: 2 ? 4 %, 4 ? 6 %, 6 ? 8 % and target/post-weight loss. The lowest monthly weight achieved and the corresponding variables from DEXA scans and fasting blood samples that fel within each range were used for statistical analysis. Pearson product moment correlation coeficients were used to determine the relationship betwen baseline physiological characteristics and dependent variables in the obese and lean groups as wel as changes observed with weight loss in the obese group. 86 Chapter IV. Results Baseline Physiological Characteristics The data from nine obese participants and seven age-matched controls are reported. 87 Table 4a. Baseline physiological characteristics Obese n = 9 Control n = 7 Variable Mean ? SD Min Max Mean ? SD Min Max Age 41.5 ? 7.1 30 54 42.3 ? 8.5 31 53 Height (in) 68.9 ? 3.6 64.5 74.0 68.3 ? 3.0 64.0 73.0 Weight (kg)* 101.7 ? 21.0 81.6 137.9 74.5 ? 5.0 64.5 78.6 BMI* 32.8 ? 3.6 30.1 39.2 24.8 ? 1.4 22.9 26.9 Waist girth (in)* 43.4 ? 4.7 38.0 52.0 34.9 ? 2.5 30.5 37.5 Total fat mas (kg)* 34.4 ? 9.2 24.8 55.7 18.2 ? 4.3 9.0 21.7 % fat* 35.2 ? 4.3 29.5 43.5 25.2 ? 5.5 14.4 31.4 Android fat mas (kg)* 8.6 ? 2.3 6.0 12.5 5.7 ? 0.8 4.2 6.6 % android fat* 47.4 ? 5.2 39.9 55.7 35.2 ? 9.3 16.4 43.8 Gynoid fat mas (kg)* 14.8 ? 3.0 11.4 20.3 11.3 ? 0.9 9.7 12.7 % gynoid fat* 36.0 ? 5.1 29.5 44.8 28.9 ? 5.9 21.0 38.5 Lean mas (kg) 62.8 ? 12.3 47.7 88.6 53.6 ? 3.6 47.5 59.2 % lean* 64.8 ? 4.3 56.5 70.5 74.8 ? 5.5 68.6 85.6 VO 2max (L/min) 3.03 ? 0.39 2.43 3.60 2.59 ? 0.47 1.82 3.08 VO 2max (mL/kg/min) 30.4 ? 3.8 24.9 37.4 35.2 ? 7.3 25.6 47.9 SBP (mHg)* 131 ? 6 124 144 118 ? 12 104 142 DBP (mHg) 82 ? 7 68 90 76 ? 6 70 88 88 Table 4b. Baseline humoral and metabolic parameters Obese n = 9 Control n = 7 Variable Mean ? SD Min Max Mean ? SD Min Max TC (mg/dL) 196 ? 30 154 238 191 ? 31 147 230 TG (mg/dL) 202 ? 102 74 344 157 ? 66 68 230 HDL-C (mg/dL) 36 ? 6 26 46 43 ? 7 35 52 LDL-C (mg/dL) 119 ? 27 82 159 116 ? 29 81 168 NEFA (mEq/L)* 0.70 ? 0.25 0.44 1.22 0.37 ? 0.20 0.13 0.69 Glucose (mg/dL) 91 ? 11 76 107 95 ? 5 87 104 Insulin (?U/mL)* 24.3 ? 9.9 17.0 46.5 7.5 ? 7.1 2.5 20.9 GIR* 4.09 ? 1.05 2.26 5.25 22.29 ? 13.85 4.55 41.60 HOMA* 5.60 ? 2.68 3.64 12.06 1.75 ? 1.65 0.64 4.90 QUICKI* 0.302 ? 0.015 0.271 0.316 0.373 ? 0.044 0.303 0.414 CT-1 (nmol/L) 0.295 ? 0.087 0.143 0.414 0.239 ? 0.099 0.049 0.382 FGF-21 (pg/mL) 433 ? 152 265 714 408 ? 219 72 727 Adiponectin (ng/mL)* 4047 ? 1712 1705 6315 2862 ? 788 1581 4064 TNF-! (pg/mL) 1.35 ? 0.34 1.08 2.13 1.04 ? 0.45 0.28 1.53 IL-6 (pg/mL) 1.98 ? 0.94 1.09 3.80 1.73 ? 1.39 0.72 4.62 Al values presented as means ? standard deviation along with minimum and maximum values in range. * indicates a significant diference betwen groups (p < 0.05). Total a regional tisue mas is expresed absent bone mineral content. VO 2max = the maximal oxygen consumption measured over one minute of a standard Bruce protocol graded exercise test; BMI = body mas index; SBP = systolic blod presure; DBP = diastolic blod presure; TC = total cholesterol; TG = triglycerides; HDL-C = high density lipoprotein cholesterol; LDL-C = low density lipoprotein cholesterol; GIR = glucose to insulin ratio; HOMA = homeostasis model score; QUICKI = quantitative insulin sensitivity check index; MPO = myeloperoxidase; TAC = total antioxidant capacity. Al obese participants were caucasian. Five of the nine obese individuals were clasified as having metabolic syndrome as described by NCEP ATP II (182). Eight subjects qualified for the MetS parameter of waist girth above 40 inches. Thre participants had 89 blood glucose levels above 100 mg/dl, five exhibited triglyceride levels above 250 mg/dl and six had HDL-C concentrations below 40 mg/dl. Control participants included in the study were sedentary, otherwise healthy individuals that maintained a BMI of les that 27 kg/m 2 . Four individuals in the control group were caucasian, one was asian, one was black and one was of Latin American decent. The baseline physiological and humoral characteristics of the study participants are provided above in Tables 4a and 4b, respectively. By design body weight, total and regional body fat, BMI and waist girth difered significantly betwen groups (p < 0.05). Humoraly, obese and age-matched control individuals did not difer significantly in a majority of the variables measured at baseline (p > 0.05). However, NEFA and insulin concentrations and indexes of insulin sensitivity (GIR, HOMA, QUICKI) were significantly diferent betwen groups, indicating greater insulin sensitivity in control individuals (p < 0.05). At baseline the obese group had significantly higher adiponectin levels when compared to controls (p < 0.05) Weight Loss and Tisue Loss Obese participants lost an average of 19.7 ? 4.2 lbs or roughly 8.9% of initial body weight. The weight loss ranged from 12.8 lbs to 27.9 lbs or from 7.6% to 10.5%. On average, target weight loss occurred at 6.3 ? 1.8 months. Percent changes in body weight are provided in Figure 5. 90 Figure 5. Percent changes in body weight. Data presented as the average percent weight los within a given range. Post-WL = post weight los. Al changes from baseline to post weight loss in lean mas and total and regional body fat were significant (p < 0.05). Total body fat mas decreased by approximatley 5.7 kg or 16.6% and total body fat percentage decreased by 3.6% from baseline values. Android fat mas decreased by an average of 1.1 kg or 12.8%, while android fat percentage decreased by 4.7%. Similarly, a reduction of 1.3 kg or 8.8% was observed in gynoid fat mas. Gynoid fat percentage decreased 2.6%. Lastly, lean mas decreased by 1.7 kg or 2.7%. However, obese individuals increased lean mas percentage by approximately 3.5% from baseline. Please se tables 5a and 5b. 91 Table 5a. Changes in body fat and lean tisue distribution Baseline 2 ? 4% 4 ? 6% 6 ? 8% Post-WL Total mass Lean (kg) 62.8 ? 4.1 a b 61.9 ? 3.9 b c 63.7 ? 4.1 a 61.5 ? 4.2 c 61.1 ? 4.2 c Lean % 64.8 ? 1.4 a 66.4 ? 1.7 b 67.8 ? 1.8 c 68.4 ? 1.8 c 68.3 ? 1.7 c Fat (kg) 34.4 ? 3.1 a 31.9 ? 3.3 b 30.7 ? 3.5 c 28.9 ? 3.4 d 28.7 ? 3.2 d Fat % 35.2 ? 1.4 a 33.6 ? 1.7 b 32.2 ? 1.8 c 31.6 ? 1.8 c 31.6 ? 1.7 c Android mass Fat (kg) 8.6 ? 0.8 a 8.1 ? 0.7 b 8.1 ? 0.7 b 7.5 ? 0.7 c 7.5 ? 0.7 c Fat % 47.4 ? 1.8 a 45.7 ? 1.9 b 43.0 ? 2.1 c 43.0 ? 2.1 c 42.7 ? 2.0 c Gynoid mass Fat (kg) 14.8 ? 1.0 a 14.2 ? 0.9 b 14.3 ? 0.9 b 13.6 ? 0.9 c 13.5 ? 0.9 c Fat % 36.0 ? 1.7 a 34.9 ? 1.8 b 33.5 ? 1.7 c 33.3 ? 1.7 c 33.4 ? 1.7 c Al values presented as means ? standard eror. Means with the same superscript are similar (p > 0.05). Post-WL = post weight los. Total mas = total body mas ? bone mineral content. Android mas is defined by the folowing boundaries: lower = pelvic cut/superior angle of iliac crest; uper = 20% of the distance betwen pelvis and the neck cut/cervical vertebrae 5; lateral = medial border of the arm cuts. Gynoid mas is defined by the folowing boundaries: uper/lower boundary = below pelvis by 1.5 x height of android ROI; lateral = outer leg cuts. Definition as described by GE Lunar Prodigy software. 92 Table 5b. Change values for body fat and lean tisue Baseline 2 ? 4% 4 ? 6% 6 ? 8% Post-WL Total mass Lean (kg) 0 a b -0.9 b c 0.9 a -1.3 c -1.7 c Lean % 0 a 1.6 b 3.0 c 3.6 c 3.5 c Fat (kg) 0 a -2.5 b -3.7 c -5.5 d -5.7 d Fat % 0 a -1.6 b -3.0 c -3.6 c -3.6 c Android mass Fat (kg) 0 a -0.5 b -0.5 b -1.1 c -1.1 c Fat % 0 a -1.7 b -4.4 c -4.4 c -4.7 c Gynoid mass Fat (kg) 0 a -0.6 b -0.5 b -1.2 c -1.3 c Fat % 0 a -1.1 b -2.5 c -2.7 c -2.6 c Al changes calculated from initial baseline values (se table 5a). Baseline values indicated as ?0?. Values with the same superscript are similar (p > 0.05). Post-WL = post weight los. Total mas = total body mas ? bone mineral content. Android mas is defined by the folowing boundaries: lower = pelvic cut/superior angle of iliac crest; uper = 20% of the distance betwen pelvis and the neck cut/cervical vertebrae 5; lateral = medial border of the arm cuts. Gynoid mas is defined by the folowing boundaries: uper/lower boundary = below pelvis by 1.5 x height of android ROI; lateral = outer leg cuts. Definition as described by GE Lunar Prodigy software. Despite significant reductions in total body fat, BMI and waist girth after modest weight loss, these measures remained significantly higher when compared to the control participants at baseline (p < 0.05). However, diferences in regional body fat that were significant betwen groups at baseline were no longer significant after modest weight loss in the obese group (p > 0.05). Humoral Indices of Metabolic Homeostasis NEFA?s, glucose, insulin, glucose to insulin ratio (GIR), the homeostasis model asesment (HOMA), and the quantitative insulin sensitivity check index (QUICKI) are included as markers of metabolic homeostasis. Glucose and NEFA concentrations were unaltered with modest 8 to 10% weight loss (p > 0.05). However, insulin, GIR, HOMA and QUICKI al were significantly improved by 8 to 10% weight loss (p < 0.05). Insulin 93 levels decreased by 5.9 ?U/mL or 24.3%. GIR increased by 1.31 or 32.0%. HOMA, a surrogate measure of insulin resistance, was reduced by 25.4% from 5.60 to 4.18. Finaly, QUICKI, an indicator of insulin sensitivity, increased by 4.3%. The weight loss reponses of these markers are provided in Tables 6a and 6b. Table 6a. Weight loss responses in humoral indices of metabolic homeostasis Variable Baseline 2 ? 4% 4 ? 6% 6 ? 8% Post-WL NEFA 0.70 ? 0.09 a 0.73 ? 0.15 a 0.68 ? 0.12 a 0.76 ? 0.15 a 0.67 ? 0.06 a Glucose 91 ? 4 a 92 ? 3 a 94 ? 4 a 89 ? 3 a 90 ? 3 a Insulin 24.3 ? 3.2 a 22.6 ? 2.9 a b 21.3 ? 2.9 b 18.2 ? 2.6 c 18.4 ? 2.3 c GIR 4.09 ? 0.35 a 4.43 ? 0.34 a 4.82 ? 0.44 a b 5.33 ? 0.55 b 5.40 ? 0.55 b HOMA 5.60 ? 0.89 a 5.34 ? 0.93 a 5.09 ? 0.86 a 4.00 ? 0.68 b 4.18 ? 0.66 b QUICKI 0.301 ? 0.004 a 0.304 ? 0.005 a 0.306 ? 0.006 a 0.316 ? 0.005 b 0.314 ? 0.006 b Al values presented as means ? standard eror. Means with the same superscript are similar (p > 0.05). Units: NEFA (mEq/L); glucose (mg/dL); insulin (?U/mL). Post-WL = post weight los; GIR = glucose to insulin ratio; HOMA = homeostasis model score [fasting insulin !U/mL x fasting glucose (mol/L)]/2.5; QUICKI = quantitative insulin sensitivity check index [1 / (log (fasting insulin !U/mL) + log (fasting glucose mg/dL)]. 94 Table 6b. Change values for humoral indices of metabolic homeostasis Variable Baseline 2 ? 4% 4 ? 6% 6 ? 8% Post-WL NEFA (mEq/L) 0 a 0.03 a -0.02 a 0.06 a -0.03 a Glucose (mg/dL) 0 a 1 a 3 a -2 a -1 a Insulin (?U/mL) 0 a -1.7 a b -3.0 b -6.1 c -5.9 c GIR 0 a 0.34 a 0.73 a b 1.24 b 1.31 b HOMA 0 a -0.26 a -0.51 a -1.6 b -1.42 b QUICKI 0 a 0.003 a 0.005 a 0.015 b 0.013 b Al changes calculated from initial baseline values (se table 6a). Baseline values indicated as ?0?. Values with the same superscript are similar (p > 0.05). Post-WL = post weight los; GIR = glucose to insulin ratio; HOMA = homeostasis model score [fasting insulin !U/mL x fasting glucose (mol/L)]/2.5; QUICKI = quantitative insulin sensitivity check index [1 / (log (fasting insulin !U/mL) + log (fasting glucose mg/dL)]. Although changes in insulin, GIR, HOMA and QUICKI were significant (p < 0.05) in the obese group post weight loss, levels of these variables were stil not similar to those observed in control participants at baseline (p < 0.05). Significant correlations betwen alterations in body tisue stores and markers of metabolic homeostasis with weight loss are as follows (p > 0.05): total fat % and NEFA (r = 0.78); fat mas and NEFA (r = 0.70); gynoid fat and NEFA (r = 0.83); lean % and NEFA (-0.78); fat mas and glucose (r = 0.73); android mas and glucose (r = 0.73); lean mas and insulin (r = 0.85); android mas and HOMA (r = 0.75); lean mas and HOMA (r = 0.89); android mas and QUICKI (r = -0.77); gynoid mas and QUICKI (r = -0.74); lean mas and QUICKI (r = -0.89). Cardiotrophin-1, Fibroblast Growth Factor-21 and Cytokine Responses No significant change in cardiotrophin-1 concentrations occurred with 8 to 10% weight loss (p > 0.05). CT-1 concentrations significantly correlated with the following variables (p < 0.05): waist circumference (r = .68), VO 2 max (r = -0.82), and glucose (r = 0.86). FGF-21 concentrations significantly decreased 57.3% (p < 0.05) after a weight 95 reduction of 8 to 10%. Please se figure 6. The changes in CT-1 and FGF-21 are described in Tables 7a and 7b. Table 7a. CT-1 and FGF-21 responses to weight los Variable Baseline 2 ? 4% 4 ? 6% 6 ? 8% Post-WL CT-1 0.295 ? 0.029 a 0.294 ? 0.024 a 0.266 ? 0.029 a 0.271 ? 0.026 a 0.288 ? 0.039 a FGF-21 433 ? 50 a 280 ? 59 b c 332 ? 67 a b 282 ? 76 b c 185 ? 34 c Al values presented as means ? standard eror. Means with the same superscript are similar (p > 0.05). Units: CT-1 (nmol/L); FGF-21 (pg/mL). Post-WL = post weight los. Table 7b. Change Values for CT-1 and FGF-21 Variable Baseline 2 ? 4% 4 ? 6% 6 ? 8% Post-WL CT-1 (nmol/L) 0 a -0.001 a -0.029 a -0.024 a -0.007 a FGF-21 (pg/mL) 0 a -153 b c -101 a b -151 b c -248 c Al changes calculated from initial baseline values (se table 7a). Baseline values indicated as ?0?. Values with the same superscript are similar (p > 0.05). Post-WL = post weight los. 96 Figure 6. Changes in FGF-21 with weight los. Al values are means ? standard eror. Means with the same superscript are similar (p > 0.05). Post-WL = post weight los. Circulating concentrations of the cytokines adiponectin, TNF-! and IL-6 did not significantly change after an 8 to 10% weight reduction (p > 0.05). Adiponectin concentrations were significantly elevated (p < 0.05) post weight loss as compared to the 4 to 6% weight loss range; however, post weight loss concentrations were not significantly diferent from baseline values (p > 0.05). The responses of adiponectin as wel as TNF-! and IL-6 are described in Tables 8a and 8b. Table 8a. Cytokine responses to weight loss Variable Baseline 2 ? 4% 4 ? 6% 6 ? 8% Post-WL Adiponectin 4047 ? 571 a b 4032 ? 456 a b 3833 ? 483 b 3969 ? 515 a b 4546 ? 599 a TNF-! 1.35 ? 0.12 a 1.34 ? 0.08 a 1.15 ? 0.11 a 1.07 ? 0.14 a 1.11 ? 0.11 a IL-6 1.98 ? 0.31 a 1.69 ? 0.27 a 2.03 ? 0.24 a 1.79 ? 0.30 a 1.87 ? 0.42 a Al values presented as means ? standard eror. Means with the same superscript are similar (p > 0.05). Units: adiponectin (ng/mL); TNF-! (pg/mL); IL-6 (pg/mL). Post-WL = post weight los. 97 Table 8b. Cytokine change values Variable Baseline 2 ? 4% 4 ? 6% 6 ? 8% Post-WL Adiponectin (ng/mL) 0 a b -15 a b -214 b -78 a b 499 a TNF-! (pg/mL) 0 a -0.01 a -0.20 a -0.28 a -0.24 a IL-6 (pg/mL) 0 a -0.29 a 0.05 a -0.19 a -0.11 a Al changes calculated from initial baseline values (se table 8a). Baseline values indicated as ?0?. Values with the same superscript are similar (p > 0.05). Post-WL = post weight los. Oxidative Stres Responses Myeloperoxidase (MPO), an enzyme that catlyzes lipid peroxidation, thus contributing to oxidative stres, and total antioxidant capacity, a marker of defenses against reactive oxygen species and fre radical damage, were measured as biomarkers of oxidative stres. Although MPO levels fluctuated with modest weight loss and were significantly (p < 0.05) reduced from 4 to 6% weight loss to post-weight loss, the change was not significant from baseline to post-weight los (p > 0.05). These changes are ilustrated in figure 7. Total antioxidant capacity was unafected by modest weight reduction of 8 to 10% (p > 0.05). The changes in MPO and TAC are provided in Tables 9a and 9b. Table 9a. Weight loss responses of MPO and TAC Variable Baseline 2 ? 4% 4 ? 6% 6 ? 8% Post-WL MPO 257 ? 20 a b 255 ? 25 a b 282 ? 27 a 247 ? 23 b 225 ? 23 b TAC 334 ? 11 a 339 ? 8 a 336 ? 8 a 340 ? 11 a 336 ? 11 a Al values presented as means ? standard eror. Means with the same superscript are similar (p > 0.05). Units: MPO (ng/mL); TAC (mol trolox). Post-WL = post weight los; MPO = myeloperoxidase; TAC = total antioxidant capacity; trolox equivalents are used to standardize antioxidants. 98 Table 9b. Change values for MPO and TAC Variable Baseline 2 ? 4% 4 ? 6% 6 ? 8% Post-WL MPO (ng/mL) 0 a b -2 a b 25 a -10 b -32 b TAC (mol trolox) 0 a 5 a 2 a 6 a 2 a Al changes calculated from initial baseline values (se table 9a). Baseline values indicated as ?0?. Values with the same superscript are similar (p > 0.05). Post-WL = post weight los; MPO = myeloperoxidase; TAC = total antioxidant capacity; trolox equivalents are used to standardize antioxidants Figure 7. Changes in MPO with weight los. Al values presented as means ? standard eror. Means with the same superscript are similar (p > 0.05). Post-WL = post weight los. Physical Activity and Diet Six of the nine obese individuals were asigned to the exercise based weight loss group. Exercise sesions were performed on a Trackmaster treadmil and were progresive and interval in nature. Each subject completed two supervised exercise sesions per wek in the lab and was asked to aerobicaly exercise twice independently per wek at approximately the same intensity based on heart rate ranges. Participants completed an average of 48 supervised exercise sesions or 84.4% of al possible sesions. Exercise sesions for each participant began with an initial caloric expenditure of 500 kcals. Caloric expenditure progresively increased to an 800 kcal maximum (as calculated from absolute oxygen consumption measurements) depending on the subject?s 99 weight loss progresion, ratings of perceived exertion and improvements in cardio- respiratory fitnes. One participant completed the 8 ? 10% weight loss goal at a final caloric expenditure of 800 kcals, one at 750 kcals, one at 725 kcals, two at 650 kcals and one at 625 kcals. Data from the first six and final six exercise sesions for each participant were averaged and used to determine progresion and improvements that occurred due to the exercise intervention. The initial average duration for the first six exercise sesions was 46.4 minutes, while the average duration for the final six sesions was 52.8 minutes. Average intensity increased from 58.2% of heart rate reserve (HR) over the first six sesions to 60.13% of HR for the final six sesions. Moreover, intensity increased from, on average, 69.8% of VO 2 reserve (as calculated from initial VO 2max ) to 93.9% at the completion of the weight loss phase. Thre of the nine obese individuals asesed were asigned to the diet based weight loss group. Exercisers were instructed not to change their diet or nutrient intake. On the other hand, dieters were asked not to alter their physical activity habits. The diet group was initialy instructed to reduce daily caloric intake by 300 kcals. This caloric restriction increased to a maximum of 500 kcal/day through the intervention based on the individuals weight loss progres. For the purpose of statistical analysis the results from both the diet and exercise groups were combined due to no observable diference in the extent or patern of weight loss betwen groups. Coeficients of Variation for Biochemical Analyses Al samples for one individual were measured in single runs for each biochemical parameter. Therefore, inter-asay variation did not influence the temporal responses determined for each individual. The inter-asay coeficients of variation were as follows: FGF-21 ? 2.89%; MPO ? 2.41%; TAC ? 3.41%; adiponectin ? 29.93%; NEFA ? 2.78%; glucose ? 0.62%. The average intra-asay coeficients of variation were as follows: CT-1 ? 0.5% to 5.2%; FGF-21 ? 4.44%; MPO ? 2.45%; TAC ? 2.11%; adiponectin ? 3.11%; NEFA ? 3.07%; glucose ? 1.80%; insulin ? 2.93%. 100 Chapter V. Discusion Cytokines and adipokines influence physiological adaptations in obesity and during weight loss (37, 126, 143, 222). Both CT-1 and FGF-21 may be related to the metabolic dysfunction occurring with obesity as wel as the metabolic and cardiovascular health improvements asociated with weight loss (268, 365, 636, 645). Elevations in both CT-1 and FGF-21 in obese individuals have recently been described, yet changes in individuals undergoing weight loss are not curently known (365, 636). Therefore, our purpose was to determine changes in cardiotrophin-1 and fibroblast growth factor-21 in obese individuals undergoing modest weight loss of 8 to 10% of initial body weight. We hypothesized that 8 to 10% weight loss would significantly decrease circulating concentrations of CT-1 and FGF-21. Our findings indicate that FGF-21 concentrations are significantly reduced by 248 pg/mL or 57.3% after modest weight loss, while CT-1 levels were not significantly altered. The extent to which circulating CT-1 and FGF-21 concentrations reflect tisue metabolism or exert endocrine or paracrine efects similar to those observed in animal models and cel culture is yet to be determined. However, to our knowledge these are the first reported findings indicating the responses of circulating concentrations of CT-1 and FGF-21 to modest weight loss. Changes in Fibroblast Growth Factor-21 with Weight Loss There is currently litle evidence in the literature regarding the influence of modest weight loss on circulating concentrations of FGF-21 in obese individuals. We hypothesized that weight loss of 8 to 10% of initial body weight would significantly decrease FGF-21 concentrations. In fact, we found that 8 to 10% weight loss results in a significant 57.3% reduction in FGF-21 levels. It has recently been determined that 101 adipose tisue is a source of FGF-21 expresion posibly leading to elevated levels of this cytokine in obesity (636). Previous work demonstrates that there are diferences in FGF- 21 concentrations betwen obese and lean individuals (73, 308, 636). Elevated levels of FGF-21 are also reported in obese individuals and in persons with type 2 diabetes and MetS (73, 308, 636). Our findings suggest substantial individual variability in FGF-21 concentrations, such that FGF-21 levels were not diferent in obese individuals as compared to age-matched controls. We are the first to report, however, that modest weight loss of 8 to 10% of initial body weight results in significant reductions in FGF-21 concentrations. 2 to 4% weight loss caused an initial significant decrease in FGF-21 concentrations from baseline. Subsequently, FGF-21 levels increased through the 4 to 6% range followed by a continued reduction until the target weight loss of 8 to 10% of initial body weight was achieved. This response difered from our hypothesis that FGF- 21 concentrations would initialy decrease rapidly in response to our intervention followed by a more gradual decrease over time until the desired 8 to 10% weight loss was achieved. The rationale for this hypothesis was based on previous findings showing that modest weight loss caused initial rapid reductions in markers of oxidative stres that continued at a slower pace throughout the intervention (96, 318, 412, 478, 513, 571, 573). The possible mechanisms explaining elevated FGF-21 concentrations observed with obesity in previous investigations are not completely understood (73, 308, 636). The elevations in FGF-21 levels may be an indirect sign of FGF-21 resistance, a protective mechanism or possibly indicate the presence of a truncated, inactive form of the protein in circulation (475). These elevated concentrations of FGF-21 would signify a protective mechanism if, in fact, FGF-21 elicits potent metabolic efects in humans as is observed in animal models (89, 268, 269). An increased expresion of FGF-21 could be part of the body?s natural defense against increased caloric intake that is asociated with weight gain and obesity. The significant reduction in FGF-21 that occurred with modest weight loss in our cohort could be due to the restoration of normal FGF-21 production and/or signaling, as occurs with insulin. Yet, if this was the case it sems that reductions in FGF-21 would more closely follow changes in insulin, GIR, HOMA and QUICKI. Changes in FGF-21 102 concentrations also appear to miror the reductions in lean tisue mas, yet correlations betwen these two variables were not significant. Metabolic eficiency may be defined as a physiological bias towards weight gain or a decrease in metabolic rate acompanying weight loss in order to defend the original body mas and acount for increased caloric expenditure or decreased caloric intake. In other words, human bioenergetics are biased more to protect against weight loss than to prevent unwanted weight gain (341). It is known that lean tisue is more metabolicaly active than fat and that changes in metabolic eficiency occur with weight loss (341). FGF-21 also appears to be a potent metabolic regulator (89, 268, 269, 592). Thus, changes in both lean tisue and FGF-21 may serve as indicators of alterations in metabolic eficiency that occur with weight loss. As elevations or reductions in lean mas occur it is plausible that FGF-21 concentrations wil increase or decrease concomitantly in order to acommodate the changes in metabolic demand. Findings from Dostalova and colleagues (111) provide evidence for this postulation. This group found significantly reduced FGF-21 concentrations in individuals with anorexia nervosa as opposed to normal weight controls (111). Anorexic individuals have litle lean tisue mas and due to extremely low body weight and low caloric intake most likely demonstrate a higher degre of metabolic eficiency, which signifies a bias toward weight gain. Thus, the findings of Dostalova and colleagues (111) provide evidence supporting the possibility that FGF-21 concentrations may inded track changes in lean tisue mas and metabolic demand. As noted previously, significant diferences in circulating FGF-21 concentrations betwen obese and lean individuals have been reported (73, 308, 636). However, our data do not coroborate these findings. If adhering to the notion that changes in FGF-21 track alterations in lean tisue mas and metabolic demand, then the lack of a noteworthy diference betwen FGF-21 levels in the obese and control individuals in our cohort makes sense due to the fact that there also was not a significant diference in lean tisue mas betwen the two groups. This provides possible evidence that alterations in lean tisue mas may influence changes in FGF-21 with weight loss. 103 Changes in Cardiotrophin-1 with Weight Loss Recent data show that CT-1 is expresed in adipose tisue and is up-regulated in states of obesity and in individuals with MetS (334, 365). Moreover, CT-1 inhibits adipocyte insulin signaling and insulin-stimulated glucose uptake in cel culture (645). CT-1 also has wel documented efects on the myocardium that include left ventricular hypertrophy and prevention of cardiac myocyte death in response to myocardial ischemia (299). Elevated CT-1 levels are found along with failing left ventricular myocardium, left ventricular hypertrophy, treated and untreated hypertension, and vascular dysfunction (319, 320, 334, 421, 453, 644). Therefore, it was thought that cardiotrophin-1, through increased adipose tisue expresion in obese individuals, may take part in the link betwen obesity, insulin resistance and cardiovascular pathologies such as hypertension, left ventricular hypertrophy and congestive heart failure. Based on previous findings and this rationale, we hypothesized that CT-1 concentrations in our obese cohort would be elevated as compared to control individuals and that 8 to 10% weight loss would significantly reduce circulating levels of this bioactive peptide. However, it does not appear that CT-1 concentrations are significantly diferent betwen obese and control individuals in our cohort. Furthermore, CT-1 concentrations did not change with weight loss of 8 to 10% of initial body weight. Natal and colleagues (365) reported that adipose tisue expreses CT-1 in 3T3-L1 cel culture, murine and human adipose tisue. However, the adipose expresion as compared to other tisues was only investigated in the murine model. Therefore, human adipose tisue may in fact be a source of cardiotrophin-1, yet this bioactive protein may be expresed to a greater degre in other tisues such as the heart. Studies investigating the expresion of CT-1 in various human tisues need to be performed before it can be determined if adipose tisue is a major source of this cytokine in humans. Zvonic et al. (645) reported that 0.2 nM CT-1 treatment significantly reduced insulin-stimulated glucose uptake in 3T3-L1 adipocytes and hypothesized that CT-1 may be a mediator of impaired insulin sensitivity. The 0.2 nM treatment levels were similar to the fasting serum concentrations of CT-1 found in our cohort. However, our data do not indicate a relationship betwen CT-1 and insulin resistance. CT-1 concentrations did not change with weight loss or improvements in insulin sensitivity and did not significantly 104 correlate with any clinical surrogate markers of insulin sensitivity. This discrepancy may be due to the fact that Zvonic and colleagues (645) reported findings from 3T3-L1 adipocytes in vitro. Thus, future studies in vivo are necesary to determine if CT-1 does indeed contribute to obesity related insulin resistance in humans. Moreover, Natal et al. (365) showed that cardiotrophin-1 expresion in 3T3-L1 adipocytes was dose-dependently related to glucose and that CT-1 positively corelated with glucose concentrations in human serum. In fact, we too found a significant correlation (r = 0.86, P = 0.003) betwen cardiotrophin-1 and glucose levels. However, none of the individuals in our cohort had diabetes or severe impaired fasting glucose. Thus, it may be that CT-1 concentrations are elevated to a greater extent in obese individuals with impaired fasting glucose or type I diabetes melitus and that modest weight loss has a greater afect on CT-1 concentrations in these individuals. In other words, it is possible that since we observed no weight loss induced changes in fasting blood glucose concentrations, then CT-1 concentrations were unaltered as wel. Weight Loss and Insulin Sensitivity The modest weight loss achieved through this intervention caused significant reductions in overal body fat, particularly body fat in the android and gynoid regions. Lean mas was significantly reduced, although when expresed as a percentage of total body mas, lean mas increased from baseline to target weight loss. This indicates that a majority of the observed weight loss was through reductions in body fat. Body composition changes, apart from those observed in lean mas, occured sequentialy (se Table 5). Obese individuals lost an average of 8.9 kg with contributions of 5.7 kg, 1.7 kg, 1.1 kg and 1.3 kg coming from total fat mas, lean mas, android fat and gynoid fat, respectively. From a percentage standpoint losses in total fat mas, android and gynoid mas were much more substantial as lean mas percentage actualy increased. Yet, it stil remains that losses in both fat mas and lean tisue mas contributed to weight loss. These findings are similar to those reported previously for obese individuals (74, 172, 468, 522). It appears that modest weight loss consistently results in significant reductions in total body fat, android fat, gynoid fat and lean tisue. Exercise is often credited with preserving lean tisue if practiced while undergoing weight loss; however, this is an 105 equivocal finding (98, 272). In our cohort lean mas decreased in spite of the regularly practiced exercise regimen by most of our participants. The weight loss achieved through this intervention induced significant reductions in fasting insulin concentrations and clinical surrogate markers of insulin sensitivity. With weight loss, both GIR and QUICKI increased, indicating enhanced insulin sensitivy and HOMA decreased, indicating reduced insulin resistance. These findings are consistent with previous results in obese individuals, whereby HOMA decreased and QUCKI increased with modest weight loss (113, 452, 491). In the present cohort, significant reductions in insulin concentrations occur with only 4 to 6%. This indicates that metabolic improvements begin with only slight weight loss. However, significant changes in the GIR, HOMA, and QUICKI index are not evident until 6 to 8% weight loss is achieved. Enhanced insulin sensitivity after weight loss is partialy related to the loss of total body fat and highly correlated with the loss of android fat (172). Similarly, changes here were acompanied by significant decreases in total and android body fat. No significant correlations were found betwen total body fat reductions, insulin and surrogate markers of insulin sensitivity. However, significant relationships were observed betwen regional body fat stores, HOMA and QUICKI, as wel as betwen lean mas, HOMA and QUICKI. Thus, in our cohort it appears that changes in insulin sensitivity and insulin action reflect those sen in regional body fat and lean mas. The strongest relationships occur betwen alterations in markers of insulin sensitivity and lean body mas, which is not reported prevalently in the literature. The strong correlation betwen surrogate markers of insulin sensitivity and lean mas reduction may sem counter-intuitive, as muscle is more metabolicly active than fat tisue. However, this may be due to a concomitant decrease in intramuscular lipid content, known to promote insulin resistance and decrease with weight loss (131, 162). Yet, we curently have no data to support this postulation. Modest weight loss and changes in body composition frequently result in decreased fasting glucose concentrations (4, 144, 202, 599, 602). Yet, fasting glucose was unafected by our intervention. This may be due to the fact that the mean fasting glucose concentration of our cohort at baseline was 95 mg/dL, which is below the > 100 106 mg/dL indicator for impaired fasting glucose and within the normal fasting glucose range. The significant changes frequently reported in the literature usualy occur with weight loss in obese individuals with impaired fasting glucose or type 2 diabetes at baseline (4, 202, 261, 262, 339, 583, 599). Our findings are fairly consistent with previous reports that weight loss does not need to be drastic, as modest weight reductions of 5 to 10% can significantly improve insulin action, glucose metabolism and hyperinsulinemia in obese individuals (170, 276, 390, 413, 599, 602). The present data indicate that our intervention induced weight loss adequate to elicit beneficial changes in metabolic regulation and manifested as improvements in insulin concentrations and surrogate markers of insulin sensitivity. These changes are reflective of alterations in body composition and body tisue stores, notably regional body fat and lean mas. Modifications in body tisue stores and markers of metabolic homeostasis occured rapidly within the weight loss proces, usualy within the 4 to 8% weight loss range. This further solidifies the concept that only modest weight loss below 10% of initial body weight is necesary to induce positive anthropometric and metabolic outcomes. Outside Influences on These Findings Factors that may have influenced the results of this investigation include diet (nutrient composition and caloric intake), outside physical activity, stres levels and slep habits. Participants in the exercise group were advised throughout the study not to make significant changes in their diet. Each individual recorded al food and drink consumed for thre days each month during the intervention. Food record analysis did not show any major changes in dietary nutrient composition or caloric intake. Furthermore, changes in total antioxidant capacity would reflect a change in diet consisting of increased intake of fruits and vegetables, which contain high levels of antioxidants. This type of change in nutrient consumption is common in dieting individuals. The fact that TAC remained stable throughout the intervention indicates that changes in nutrient intake most likely did not occur. During the 6 to 10 month intervention period individuals in the diet group were asked to avoid regular physical activity as much as possible. These participants had relatively sedentary jobs and were capable of maintaining their physical activity to typical 107 activities of daily living. Al refrained from any formal exercise as indicated by monthly 3 day physical activity logs. It appears that no major changes in dietary composition occurred in the exercise group and that no significant elevations in physical activity level occurred in the diet group or were an appreciable influence on the magnitude or direction of blood variable changes observed with weight los. Stres levels and slep habits were not directly controlled or quantified during the intervention. Overall Findings The results of this investigation suggest that modest weight loss of 8 to 10% of initial body weight is efective in reducing circulating concentrations of FGF-21. However, weight loss did not alter cardiotrophin-1 concentrations. FGF-21 levels were significantly reduced by 57.3% from baseline to post weight loss. 2 to 4% weight loss caused an initial significant reduction in FGF-21 concentrations followed by an increase through the 4 to 6% range. Subsequently, FGF-21 levels were significantly reduced in the target weight loss range of 8 ? 10%. Furthermore, weight loss resulted in a 2.7% decrease in lean tisue mas. The fact that both lean tisue mas and FGF-21 concentrations were reduced after 8 ? 10% weight loss is interesting since lean tisue is more metabolicaly active than fat and FGF-21 is described as a potent metabolic regulator (89, 268, 269, 355, 592). In other words, fluctuations in FGF-21 and lean tisue may reflect alterations metabolic demand occurring with weight loss. Melby and Hickey (341) have previously described the concept of metabolic eficiency. Losses in lean tisue mas and reductions in FGF-21 may contribute to increased metabolic eficiency with weight loss. The elevated FGF-21 concentrations previously reported in obese individuals may be asociated with elevated lean tisue mas, not exces adipose tisue, FGF-21 resistance, a protective mechanism, or production of truncated, inactive forms of the protein (73, 308, 636). Thus, the observed reductions in FGF-21 concentrations with 8 to 10% weight loss may be a result of lean mas loss and enhanced metabolic eficiency. However, this is a postulation and is not the result of mechanistic data. Other possible explanations exist as to why FGF-21 decreases with weight loss. If there is in fact some level of FGF-21 resistance in obese individuals, much like is 108 known to occur with insulin, then the reductions in FGF-21 observed with modest weight loss may be reflective of the restoration of normal FGF-21 production and signaling. Furthermore, reductions in FGF-21 occurred along with significant body fat loss. Therefore, decreased adipose tisue mas and restoration of adipose tisue health may possibly cause fat tisue to become les resistant to the efects of FGF-21. Clinical Significance and Conclusions Finaly, to our knowledge we are the first to report the efects of modest weight loss on circulating concentrations of cardiotrophin-1 and fibroblast growth factor-21. CT-1 levels most likely are quite clinicaly important based on this bioactive peptide?s wel-documented efects on the heart (299, 319, 321, 334, 421, 453, 644). However, our data suggest that the efect of modest weight loss on concentrations of CT-1 holds litle clinical significance in obese individuals without impaired fasting glucose. If the hypothesis is true that FGF-21 concentrations may track changes in lean tisue and metabolic demand, the clinical relevance of this cytokine may be substantial. The increased concentrations of FGF-21 in obese individuals reported previously may be due to elevated lean tisue mas as opposed to increased adipose tisue expresion and subsequent FGF-21 resistance (73, 308, 636). Thus, FGF-21 could be a potential therapeutic and pharmaceutical target that along with proper diet and exercise may be capable of eliciting potent metabolic benefits in obese individuals. However, a wealth of further data is needed before this postulation can be solidified. From a clinical standpoint, it is wel documented that weight loss results in a number of metabolic benefits including reductions in body fat and enhanced insulin sensitivity (74, 113, 172, 452, 467, 491, 522). The conservative weight loss regimen employed in this study lead to significant reductions in total, android and gynoid body fat stores of 5.7 kg, 1.1 kg and 1.3 kg, respectively. Significant beneficial changes in fasting insulin concentrations and clinical surrogate markers of insulin sensitivity, such as GIR, HOMA and QUICKI were also observed. Some of these improvements in the clinical markers of insulin sensitivity began with as litle as 4 to 6% weight loss, indicating that only slight weight loss is necesary to induce metabolic benefits. Thus, our findings corroborate a wealth of previous information citing that modest weight loss through 109 lifestyle modifications is efective in causing changes in total and regional body fat as wel as clinical markers of metabolic health (74, 113, 170, 172, 276, 390, 413, 452, 467, 491, 522, 599, 602). These changes reflect a reduction in the risk for MetS, type I diabetes melitus and cardiovascular disease (100, 180, 182, 293, 296, 597, 631). In conclusion, modest weight loss is succesful in causing significant and beneficial changes in total and regional body fat as wel as in clinical markers of metabolic health. 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