AMPK activation restores the stimulation of glucose uptake in an in vitro model of insulin-resistant cardiomyocytes via the activation of protein kinase B

Abstract

Diabetic hearts are known to be more susceptible to ischemic disease. Biguanides, like metformin, are known antidiabetic drugs that lower blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in muscle. Part of these metabolic effects is thought to be mediated by the activation of AMP-activated protein kinase (AMPK). In this work, we studied the relationship between AMPK activation and glucose uptake stimulation by biguanides and oligomycin, another AMPK activator, in both insulin-sensitive and insulin-resistant cardiomyocytes. In insulin-sensitive cardiomyocytes, insulin, biguanides and oligomycin were able to stimulate glucose uptake with the same efficiency. Stimulation of glucose uptake by insulin or biguanides was correlated to protein kinase B (PKB) or AMPK activation, respectively, and were additive. In insulin-resistant cardiomyocytes, where insulin stimulation of glucose uptake was greatly reduced, biguanides or oligomycin, in the absence of insulin, induced a higher stimulation of glucose uptake than that obtained in insulin-sensitive cells. This stimulation was correlated with the activation of both AMPK and PKB and was sensitive to the phosphatidylinositol-3-kinase/PKB pathway inhibitors. Finally, an adenoviral-mediated expression of a constitutively active form of AMPK increased both PKB phosphorylation and glucose uptake in insulin-resistant cardiomyocytes. We concluded that AMPK activators, like biguanides and oligomycin, are able to restore glucose uptake stimulation, in the absence of insulin, in insulin-resistant cardiomyocytes via the additive activation of AMPK and PKB. Our results suggest that AMPK activation could restore normal glucose metabolism in diabetic hearts and could be a potential therapeutic approach to treat insulin resistance.

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake

“…1, C and D). This insulin-sensitizing effect of AMPK activators was similar to that previously described (4).…”

“…Taking into account the negative effect of AMPK on mTOR/p70S6K, it is speculated that AMPK activation, by inactivating the mTOR/p70S6K-mediated negative feedback loop, should prevent IRS-1 inhibition and should, then, increase insulin sensi-tivity (14). In line with this hypothesis, we previously showed that activation of AMPK by various activators, including metformin, phenformin, and the non-antidiabetic drug oligomycin, increased insulin signaling (monitored by PKB/Akt phosphorylation state measurement) and overstimulated insulin-induced glucose uptake in both insulin-sensitive and -resistant primary cultured cardiomyocytes (4). The aim of the present work was to prolong this initial study by 1) investigating the interplay of insulin and AMPK activators on different elements of the insulin signaling pathway including IRS-1, p70S6K, and AS160; and 2) evaluating the role of the inhibition of the insulin-induced negative feedback loop in the insulin-sensitizing effect of AMPK activators by using rapamycin the specific inhibitor of the mTOR/p70S6K pathway.…”

“…Cultured adult cardiomyocytes were prepared from male Wistar rats as described previously (4). The cardiomyocytes were equally distributed in laminin-coated dishes and incubated at 37°C for 1 h in medium 199 (Invitrogen; 5.5 mM glucose), 100 U/ml penicillin, and 100 g/ml streptomycin.…”

“…7). Insulin resistance was already characterized in our previous study (4) and was obtained in vitro by treating cells during 24 h at supraphysiological concentration of insulin. Phenformin induced normal AMPK phosphorylation at all concentrations of insulin (Fig.…”

“…More recently, it has been shown that AMPK can be activated by the insulin-sensitizing and antidiabetic drug metformin (12,15,43). AMPK, independently of insulin, is able to phosphorylate AS160 (19,27) and to stimulate glucose uptake (4,29) in the heart. In addition, AMPK inhibits the mTOR/p70S6K pathway first discovered to be involved in the regulation of protein synthesis (5).…”