AMP-activated Protein Kinase Is Required for the Lipid-lowering Effect of Metformin in Insulin-resistant Human HepG2 Cells

Abstract

The antidiabetic drug metformin stimulates AMP-activated protein kinase (AMPK) activity in the liver and in skeletal muscle. To better understand the role of AMPK in the regulation of hepatic lipids, we studied the effect of metformin on AMPK and its downstream effector, acetyl-CoA carboxylase (ACC), as well as on lipid content in cultured human hepatoma HepG2 cells. Metformin increased Thr-172 phosphorylation of the ␣ subunit of AMPK in a dose-and time-dependent manner. In parallel, phosphorylation of ACC at Ser-79 was increased, which was consistent with decreasing ACC activity. Intracellular triacylglycerol and cholesterol contents were also decreased. These effects of metformin were mimicked or completely abrogated by adenoviralmediated expression of a constitutively active AMPK␣ or a kinase-inactive AMPK␣, respectively. An insulinresistant state was induced by exposing cells to 30 mM glucose as indicated by decreased phosphorylation of Akt and its downstream effector, glycogen synthase kinase 3␣/␤. Under these conditions, the phosphorylation of AMPK and ACC was also decreased, and the level of hepatocellular triacylglycerols increased. The inhibition of AMPK and the accumulation of lipids caused by high glucose concentrations were prevented either by metformin or by expressing the constitutively active AMPK␣. The kinase-inactive AMPK␣ increased lipid content and blocked the ability of metformin to decrease lipid accumulation caused by high glucose concentrations. Taken together, these results indicate that AMPK␣ negatively regulates ACC activity and hepatic lipid content. Inhibition of AMPK may contribute to lipid accumulation induced by high concentrations of glucose associated with insulin resistance. Metformin lowers hepatic lipid content by activating AMPK, thereby mediating beneficial effects in hyperglycemia and insulin resistance. AMP-activated protein kinase (AMPK)1 is a phylogenetically conserved intracellular energy sensor that has been implicated in the regulation of glucose and lipid homeostasis (1-4). AMPK is activated by physiological stimuli, such as exercise, muscle contraction, and hormones including adiponectin and leptin, as well as by pathological stresses, glucose deprivation, hypoxia, oxidative stress, and osmotic shock (2, 5). AMPK serine/threonine protein kinase is a heterotrimeric complex consisting of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥) (5). Regulation of AMPK activity is complex; it involves allosteric activation by AMP, which increases during states of stress where ATP is depleted, and phosphorylation via the presumptive upstream activator AMPK kinase (6 -9), which may also be allosterically activated by AMP (5). Moreover, phosphorylation of Thr-172 within the activation loop of the catalytic domain of the ␣ subunit is necessary for AMPK activity because sitedirected mutagenesis of Thr-172 to Ala completely abolishes AMPK activity (10, 11). Once activated, AMPK phosphorylates its downstream substrates to reduce ATP-consuming anabolic pathways, including ...

Glucose inhibits the insulin-induced activation of the insulin-degrading enzyme in HepG2 cells

“…Hence, the cause of this effect remains unidentified. Incubation of HepG2 cells in a high glucose concentration was shown to induce insulin resistance [27], which may result in the observed disturbance of regulation of IDE activity by insulin.…”

“…FBS and either normal (1.0 g/l) or high (4.5 g/l) concentrations of D-glucose for 48-72 h to 80% confluence. Incubation of cells in high-glucose medium was used as a model of insulin resistance [27]. After 24 h of serum starvation cells were treated with 0.1, 1, 10, 100 or 200 nmol/l insulin (Sigma Aldrich, Taufkirchen, Germany) for 24 h. As an osmotic control, 3.64 g/l mannitol (Sigma Aldrich) was added to normal glucose medium in simultaneous wells.…”

Regulation of HMGCoA Reductase Activity by Policosanol and Octacosadienol, a New Synthetic Analogue of Octacosanol

“…The treatment of HepG2 and HUVEC cells with metformin did not show any detectable cell toxicity. After 6 hours cells were washed twice with phosphate-buffered saline, and lysed in ice-cold buffer (20 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM phenylmethyl sulfonyl fluoride and 1 mM protease inhibitor cocktails (Sigma-Aldrich) [36]. Cells were scraped from the plates and collected in a micro-centrifuge tube.…”

General aspects of muscle glucose uptake

“…This was strengthened by showing that the metabolic effect of the drug is preserved in liver-specific AMPK-deficient mice (Viollet et al 2012). It is worth remembering that AMPK inhibits gluconeogenesis and glycogenolysis, stimulates glycogenesis in hepatocytes, and increases glucose uptake in peripheral tissues (Zang et al 2004, Zhou et al 2001. Besides the effects on the liver and skeletal muscle, studies have shown that metformin activates AMPK in aortic endothelial tissue in vivo (Zou et al 2004).…”