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Metformin is a major oral anti-diabetic drug and is known as an insulin sensitizer. However, the mechanism by which metformin acts is unclear. In this study, we found that AICAR, an AMPK activator, and metformin increased the expression of Rab4 mRNA and protein levels in skeletal muscle C2C12 cells. The promoter activity of Rab4 was increased by metformin in an AMPK-dependent manner. Metformin stimulated the phosphorylation of AS160, Akt substrate, and Rab GTPase activating protein (GAP), and also increased the phosphorylation of PKC-zeta, which is a critical molecule for glucose uptake. Knockdown of AMPK blocked the metformin-induced phosphorylation of AS160/PKC-zeta. In addition, a colorimetric absorbance assay showed that insulin-induced translocation of GLUT4 was suppressed in Rab4 knockdown cells. Moreover, Rab4 interacted with PKC-zeta but not with GLUT4. The C-terminal-deleted Rab4 mutant, Rab4ΔCT, showed diffuse sub-cellular localization, while wild-type Rab4 localized exclusively to the perinuclear membrane. Unlike Rab4ΔCT, wild-type Rab4 co-localized with PKC-zeta. Together, these results demonstrate that metformin induces Rab4 expression via AMPK-AS160-PKC-zeta and modulates insulin-mediated GLUT4 translocation.

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.

Homocysteine sulfinic acid (HCSA) is a homologue of the amino acid cysteine and a selective metabotropic glutamate receptor (mGluR) agonist. However, the metabolic role of HCSA is poorly understood. In this study, we showed that HCSA and glutamate stimulated glucose uptake in C2C12 mouse myoblast cells and increased AMP-activated protein kinase ( Homocysteine, a sulfur-containing amino acid, is a potent and selective metabotropic glutamate receptor agonist (1). A number of epidemiologic studies have shown that moderate hyperhomocysteinemia is a risk factor for atherosclerosis (2, 3), stroke (4), Alzheimer disease (5), and schizophrenia (6). Hyperhomocysteinemia is common in patients with non-insulin-dependent diabetes mellitus (7) and nephritic diabetes (8), suggesting a possible relationship between homocysteine and the pathogenesis. Recent evidence has indicated the presence of glutamate receptors in non-neuronal tissues, including skeletal muscle cells; however, the functional implications involving homocysteine in diabetes are not fully understood.AMP-activated protein kinase (AMPK) 2 is a phylogenetically conserved intracellular energy sensor that plays a central role in the regulation of glucose and lipid metabolism. AMPK, a heterotrimeric complex composed of a catalytic subunit and two regulatory subunits, is activated when cellular energy is depleted (9). Upon activation by allosteric binding of AMP or phosphorylation at Thr 172 of the catalytic subunit by AMPK kinase, AMPK accelerates ATP-generating catabolic pathways, including glucose and fatty acid oxidation (10 -12), while simultaneously reducing ATP-consuming anabolic pathways, including cholesterol, fatty acid, and triacylglycerol synthesis (13).Some nonessential amino acids and derivatives, such as L-glutamate, L-aspartate, and ␥-aminobutyric acid, have long been known to act as neurotransmitters (14). In addition, a group of sulfur-containing amino acids has previously been shown to exhibit effects similar to those of L-glutamic acid and L-aspartic acid (15). Glutamate is the most prominent and excitatory neurotransmitter at the neuromuscular junction. Glutamate receptors can be subdivided into two classes: ionotropic and metabotropic. Ionotropic glutamate receptors are ligand-gated, nonselective cation channels that allow the flow of Na