1Metformin, a drug widely used to treat type 2 diabetes, was recently shown to activate the AMP-activated protein kinase (AMPK) in intact cells and in vivo. In this study we addressed the mechanism for this effect. In intact cells, metformin stimulated phosphorylation of the key regulatory site (Thr-172) on the catalytic (␣) subunit of AMPK. It did not affect phosphorylation of this site by either of two upstream kinases in cell-free assays, although we were able to detect an increase in upstream kinase activity in extracts of metformintreated cells. Metformin has been reported to be an inhibitor of complex 1 of the respiratory chain, but we present evidence that activation of AMPK in two different cell types is not a consequence of depletion of cellular energy charge via this mechanism. Whereas we have not established the definitive mechanism by which metformin activates AMPK, our results show that the mechanism is different from that of the existing AMPK-activating agent, 5-aminoimidazole-4-carboxamide (AICA) riboside. Metformin therefore represents a useful new tool to study the consequences of AMPK activation in intact cells and in vivo. Our results also show that AMPK can be activated by mechanisms other than changes in the cellular AMP-to-ATP ratio. Diabetes 51:2420 -2425, 2002

The 5AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-␤-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). In muscle of mice with targeted deletion of the ␣2-AMPK gene, phosphorylation of GS site 2 was decreased under basal conditions and unchanged by AICAR treatment. In contrast, in ␣1-AMPK knockout mice, the response to AICAR was normal. Fuel surplus (glucose loading) decreased AMPK activation by AICAR, but the phosphorylation of the downstream targets acetyl-CoA carboxylase-␤ and GS was normal. Fractionation studies suggest that this suppression of AMPK activation was not a direct consequence of AMPK association with membranes or glycogen, because AMPK was phosphorylated to a greater extent in response to AICAR in the membrane/glycogen fraction than in the cytosolic fraction. Thus, the downstream action of AMPK in response to AICAR was unaffected by glucose loading, whereas the action of the kinase upstream of AMPK, as judged by AMPK phosphorylation, was decreased. The fact that ␣2-AMPK is a GS kinase that inactivates GS while simultaneously activating glucose transport suggests that a balanced view on the suitability for AMPK as an antidiabetic drug target should be taken. Diabetes 53:3074 -3081, 2004 T he 5ЈAMP-activated protein kinase (AMPK) system is a sensor of cellular energy status that adjusts the supply of ATP to the demand for the nucleotide (1). Activation of ␣2-AMPK stimulates muscle glucose transport (2,3). Once glucose has been taken up and converted to glucose-6-phosphate (G6P), it can be stored as glycogen or metabolized by glycolysis to generate ATP. It has been reported that AMPK phosphorylates muscle glycogen synthase (GS) in cell-free assays at site 2 (Ser 7) (4). Thus, AMPK activation may under some conditions decrease the potential for glycogen synthesis. Recently, we and others showed that GS activity decreases in response to acute 5-aminoimidazole-1-␤-4-carboxamide ribofuranoside (AICAR) treatment of muscle-like cells in culture (5), isolated and perfused skeletal muscle (6 -8), and fast twitch, but not slow twitch, muscle in vivo (7). AICAR treatment leads to decreased gel mobility of GS in perfused muscle, which together with the decreased activity, is reversed by protein phosphatase treatment (6). These observations indicate that regulation of GS activity by AICAR involves phosphorylation of GS. Although it has been suggested that AICAR-induced GS deactivation is mediated by AMPK due to the negative correlation between ␣2-AMPK and GS activity (6), these data do not prove a causal relation. Thus, by studying muscle from ␣-AMPK knockout (KO) mice in the present study, we aimed to verify that AMPK is a muscle GS kinase in vivo.AMPK activity decreases when muscle is exposed to fuel surplus. For example, glucose loading and glycogen accumulation suppress muscle AMPK phosphorylation/ activation at basal co...

A-769662 activates AMPK ␤1-containing complexes but induces glucose uptake through a PI3-kinase-dependent pathway in mouse skeletal muscle.

The sand rat (Psammomys obesus) is an animal model of nutritionally induced diabetes. We report here that several protein kinase C (PKC) isoforms (␣, , and , representing all three subclasses of PKC) are overexpressed in the skeletal muscle of diabetic animals of this species. This is most prominent for the isotype of PKC. Interestingly, increased expression of PKC could already be detected in normoinsulinemic, normoglycemic (prediabetic) animals of the diabetes-prone (DP) line when compared with a diabetes-resistant (DR) line. In addition, plasma membrane (PM)-associated fractions of PKC␣ and PKC were significantly increased in skeletal muscle of diabetic animals, suggesting chronic activation of these PKC isotypes in the diabetic state. The increased PM association of these PKC isotypes revealed a significant correlation with the diacylglycerol content in the muscle samples. P sammomys obesus, often nicknamed "sand rat," is a herbivorous desert gerbil living in the eastern Mediterranean and North Africa. The Jerusalem colony was established by domesticating the animals collected from the shores of the Dead Sea (1,2). In its native habitat, the Psammomys feeds on the halophilic plant Atriplex halimus and has never been found to be hyperglycemic or hyperinsulinemic. In captivity, it remains nondiabetic when fed a low energy (LE) diet containing 2.4 cal/g. However, when transferred to a relatively high energy (HE) diet of 3.0 cal/g, similar to the regular rodent diet, it gradually develops hyperinsulinemia and hyperglycemia. Four generally consecutive stages of progression to diabetes have been described (3). The basal stage A is followed by hyperinsulinemia without hyperglycemia (stage B), which precedes hyperinsulinemia with hyperglycemia (stage C), and lastly, stage D, marked by pancreatic insulin secretion collapse with apoptosis (4) and dependence on external insulin supply for survival.Two Psammomys lines have been separated by selective breeding: the diabetes-resistant (DR) line, which remains normoglycemic and normoinsulinemic even on an HE diet, and the diabetes-prone (DP) line, which is susceptible to diabetes when exposed to the HE diet (4). The main difference between the two lines seems to be the cost of weight gain: the DP line uses 6.0 kcal/g for growth during 2 weeks after weaning, whereas the DR line requires 9.3 kcal/g (4). The DP animals exhibit insulin resistance even in the state of normoglycemia, as evidenced by the failure to induce hypoglycemia after external insulin administration and a minimal reduction of hepatic gluconeogenesis during the euglycemic-hyperinsulinemic clamp (5). Therefore, the insulin resistance in the Psammomys may be considered an innate characteristic of a desert animal according to the thrifty gene hypothesis (6).Alterations in the expression level and/or activity of several protein kinase C (PKC) isoforms were found to be associated with insulin resistance in type 2 diabetic patients, animal models of diabetes, and different cell models (7-10). The serine/threonine ...

Both hyperglycemia and tumor necrosis factor ␣ (TNF␣) were found to induce insulin resistance at the level of the insulin receptor (IR). How this effect is mediated is, however, not understood. We investigated whether oxidative stress and production of hydrogen peroxide could be a common mediator of the inhibitory effect. We report here that micromolar concentrations of H 2 O 2 dramatically inhibit insulin-induced IR tyrosine phosphorylation (pretreatment with 500 M H 2 O 2 for 5 min inhibits insulin-induced IR tyrosine phosphorylation to 8%), insulin receptor substrate 1 phosphorylation, as well as insulin downstream signaling such as activation of phosphatidylinositol 3-kinase (inhibited to 57%), glucose transport (inhibited to 36%), and mitogenactivated protein kinase activation (inhibited to 7.2%). Both sodium orthovanadate, a selective inhibitor of tyrosine-specific phosphatases, as well as the protein kinase C inhibitor Gö 6976 reduced the inhibitory effect of hydrogen peroxide on IR tyrosine phosphorylation. To investigate whether H 2 O 2 is involved in hyperglycemiaand/or TNF␣-induced insulin resistance, we preincubated the cells with the H 2 O 2 scavenger catalase prior to incubation with 25 mM glucose, 25 mM 2-deoxyglucose, 5.7 nM TNF␣, or 500 M H 2 O 2 , respectively, and subsequent insulin stimulation. Whereas catalase treatment completely abolished the inhibitory effect of H 2 O 2 and TNF␣ on insulin receptor autophosphorylation, it did not reverse the inhibitory effect of hyperglycemia. In conclusion, these results demonstrate that hydrogen peroxide at low concentrations is a potent inhibitor of insulin signaling and may be involved in the development of insulin resistance in response to TNF␣.