The Effects of 1-Week and 8-Month Ketogenic Dieting or Ketone Salt Supplementation on Markers of Tissue Oxidative Stress and Mitochondrial Quality in Rats

Abstract

Purpose: Herein we sought to examine the short-term (i.e., one-week) and long-term (i.e., 8-month) effects of a ketogenic diet (KD) or ketone salt supplementation on multi-organ markers of oxidative stress and mitochondrial function in male Fisher 344 in versus control animals fed standard rodent chow (SC). Methods: In AIM 1, 4 month old male Fisher 344 rats were provided isocaloric amounts of a KD (5.2 kcal/g, 23.1% protein, 9.6% carbohydrate, and 65.3% fat, n=10) or SC (3.1 kcal/g, 24% protein, 58% carbohydrate, 18% fat; n=20) and un-supplemented water ad libitum for 7 days. The SC rats were split into sub-groups such that, one group was provided ketone salts in their drinking water in addition to a SC diet (SC+KS ~1.2 g/day, n=10), and one group was un-supplemented (SC, n=10). In AIM 2, 4 month old male Fisher 344 rats were provided the KD (n=8), SC (n=7) and SC+KS (n=7) for 8 months. Following treatment completion for AIMs 1 and 2, rats were sacrificed and blood was obtained and serum was aliquoted and stored, and brain, liver and gastrocnemius muscle were harvested for mRNA and/or protein analyses. In addition for AIM 2, brain, muscle and liver mitochondria were freshly isolated for mitochondrial respiration and reactive oxygen species (ROS) assays. Results: Serum ketone levels were greatest in KD rats after one-week and 8-mo interventions relative to the SC and SC+KS groups both suggesting that diet, but not salt supplementation, induced a ketogenic state. Both short-term and long-term KD resulted in blunted weight gain and feed efficiency when compared to both SC and SC+KS rats (p<0.001 and p<0.001, respectively). Muscle, brain and liver expression of oxidative stress-related genes were not different between groups after one-week. Similarly, muscle/brain/liver protein expression of glutathione peroxidase, superoxide dismutase 2, and catalase as well as protein carbonyl and 4-hydroxynonenal levels were not different between groups following the 8 mo study. After 8 months, gastrocnemius mitochondrial ROS production was higher in KD animals versus all other treatments (p=0.007), and this may have been related to a decreased state III respiration and respiratory control ratio in this tissue relative to other groups (p=0.072 and p=0.018, respectively). Moreover, gastrocnemius citrate synthase activity (a surrogate of mitochondrial density) was lowest in KD rats versus the SC and SC+KS groups (p<0.001). However, despite these gastrocnemius deficits, rotarod performance was greatest in KD rats versus the all other groups after 2 mo, 4 mo and 8 mo of treatment or control diet. Conclusions: Our data suggest that ketogenic dieting, but not ketone salt supplementation, reduces feed efficiency and body mass acutely and chronically. No changes in antioxidant muscle/brain/liver gene or protein expression were elicited by short-term or long-term ketogenic dieting or ketone salt supplementation. Interestingly, chronic ketogenic dieting elicits an increase in skeletal muscle mitochondrial ROS formation and, despite the lack of oxidative damage (i.e., protein carbonyl and 4HNE production), this paralleled decreases in mitochondrial quantity and quality markers. Notwithstanding, these skeletal muscle deficits did not translate into a decline in muscular endurance and/or grip strength with long-term ketogenic dieting suggested that a long-term ketogenic diet may result in increased skeletal muscle metabolic efficiency and improved performance in aged (12 month old) rats.