Methods Measurements of AMPK, ACC, and fatty acid oxidation in primary hepatocytes.Hepatocytes were isolated from male Sprague Dawley (SD) rats by collagenase digestion (18). For the AMPK assay, cells were seeded in six-well plates at 1.5 × 10 6 cells/well in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FBS, 100 nM insulin, 100 nM dexamethasone, and 5 µg/ml transferrin for 4 hours. Cells were then cultured in serum-free DMEM for 16 hours followed by treatment for 1 hour or 7 hours with control medium, 5-amino-imidazole carboxamide ribo-

Methods Measurements of AMPK, ACC, and fatty acid oxidation in primary hepatocytes.Hepatocytes were isolated from male Sprague Dawley (SD) rats by collagenase digestion (18). For the AMPK assay, cells were seeded in six-well plates at 1.5 × 10 6 cells/well in DMEM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10% FBS, 100 nM insulin, 100 nM dexamethasone, and 5 µg/ml transferrin for 4 hours. Cells were then cultured in serum-free DMEM for 16 hours followed by treatment for 1 hour or 7 hours with control medium, 5-amino-imidazole carboxamide ribo-

Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is a key player in regulating energy metabolism, placing it at the center stage in studies of diabetes and related metabolic diseases. Expressed in key metabolically relevant organs, AMPK is activated in response to a variety of stimuli, including cellular stress, exercise, and a wide range of hormones and agents that exert impacts on cellular metabolism. Genetic and pharmacological studies demonstrate that AMPK is required for maintaining glucose homeostasis. Activation of AMPK by pharmacological agents presents a unique challenge, given the complexity of the biology, but holds a considerable potential to reverse the metabolic abnormalities associated with type 2 diabetes.

Metformin is an effective hypoglycemic drug that lowers blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in skeletal muscle; however, the molecular site of metformin action is not well understood. AMP-activated protein kinase (AMPK) activity increases in response to depletion of cellular energy stores, and this enzyme has been implicated in the stimulation of glucose uptake into skeletal muscle and the inhibition of liver gluconeogenesis. We recently reported that AMPK is activated by metformin in cultured rat hepatocytes, mediating the inhibitory effects of the drug on hepatic glucose production. In the present study, we evaluated whether therapeutic doses of metformin increase AMPK activity in vivo in subjects with type 2 diabetes. Metformin treatment for 10 weeks significantly increased AMPK ␣2 activity in the skeletal muscle, and this was associated with increased phosphorylation of AMPK on Thr172 and decreased acetyl-CoA carboxylase-2 activity. The increase in AMPK ␣2 activity was likely due to a change in muscle energy status because ATP and phosphocreatine concentrations were lower after metformin treatment. Metformin-induced increases in AMPK activity were associated with higher rates of glucose disposal and muscle glycogen concentrations. These findings suggest that the metabolic effects of metformin in subjects with type 2 diabetes may be mediated by the activation of AMPK ␣2.

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