Objective—It has been shown that high extracellular glucose promotes ATP release in different cells, but its biological significance is not clear. Also, it has been reported that extracellular ATP either increase or decrease insulin signaling depending on cell types. Moreover, it has been known that insulin resistance could lead to hyperglycemia and diabetes; however, the exact cause effect relationship between hyperglycemia and insulin resistance is not fully understood. Since over nutrition often leads to energy excess with ATP being the major cellular energy source, and Metformin is a well-established anti-diabetic drug with its molecular mechanism being in debate, we hypothesized that abnormal high level of extracellular glucose promotes skeletal muscle cells to release ATP, which in turn activates the purinergic P2Y2 receptor, resulting to insulin resistance and P2Y2 nucleotide receptor signaling can be modulated by Metformin treatment in different cells, mainly human skeletal muscle cells.
Methods and Results— Real-time RT-PCR analysis indicated that the P2Y2 receptor mRNA was significantly up-regulated during human skeletal muscle cell differentiation, and functional P2Y2 receptor expression was confirmed by measuring intracellular [Ca2+]i mobilization in response to ATP or UTP in mature human skeletal muscle cells (HSKMC). Interestingly, incubation of HSKMC with high glucose (25 mM) triggered significant amount of ATP release as compared with normal glucose (6.1 mM). In addition, both ATP and UTP dramatically suppressed insulin-induced AKT activation in a dose-dependent manner in HSKMC, which could be reversed by AR-C118925, a selective P2Y2 receptor antagonist, suggesting a role of P2Y2 receptor in control of insulin receptor signaling. The P2Y2 receptor suppression of the AKT pathway was selective, since ATP/UTP treatment
potentiated insulin-induced ERK1/2 pathway in HSKMC. Furthermore, we found that activation of the P2Y2 receptor significantly inhibited insulin-induced glucose uptake in HSKMC. On the other hand, we confirmed that in primary human umbilical vein endothelial cells metformin inhibits P2Y2 receptor signaling. However, in immortalized human umbilical vein endothelial cells we found that metformin increased P2Y2 signaling.
Conclusions—We concluded that ATP release and P2Y2 receptor activation is a previously unrecognized cellular signaling node linking hyperglycemia and insulin resistance in HSKMC through the PI3K-AKT pathway, highlighting that P2Y2 receptor may be a new drug target for the prevention and/or treatment of diabetes. In addition, Metformin, at clinical relevant dose may affect P2Y2 receptor signaling in a cell type dependent manner, of which its inhibitory effect in HSKMCs may explain its anti-diabetic action. These findings will give us better understating of insulin resistance andMetformin mechanism of action and thus can help scientists to find new Antidiabetic drugs.||en_US