Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state
Marc Foretz, … , Fabrizio Andreelli, Benoit Viollet
Marc Foretz, … , Fabrizio Andreelli, Benoit Viollet
Published June 23, 2010
Citation Information: J Clin Invest. 2010;120(7):2355-2369. https://doi.org/10.1172/JCI40671.
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Research Article

Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state

Abstract

Metformin is widely used to treat hyperglycemia in individuals with type 2 diabetes. Recently the LKB1/AMP-activated protein kinase (LKB1/AMPK) pathway was proposed to mediate the action of metformin on hepatic gluconeogenesis. However, the molecular mechanism by which this pathway operates had remained elusive. Surprisingly, here we have found that in mice lacking AMPK in the liver, blood glucose levels were comparable to those in wild-type mice, and the hypoglycemic effect of metformin was maintained. Hepatocytes lacking AMPK displayed normal glucose production and gluconeogenic gene expression compared with wild-type hepatocytes. In contrast, gluconeogenesis was upregulated in LKB1-deficient hepatocytes. Metformin decreased expression of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), while cytosolic phosphoenolpyruvate carboxykinase (Pepck) gene expression was unaffected in wild-type, AMPK-deficient, and LKB1-deficient hepatocytes. Surprisingly, metformin-induced inhibition of glucose production was amplified in both AMPK- and LKB1-deficient compared with wild-type hepatocytes. This inhibition correlated in a dose-dependent manner with a reduction in intracellular ATP content, which is crucial for glucose production. Moreover, metformin-induced inhibition of glucose production was preserved under forced expression of gluconeogenic genes through PPARγ coactivator 1α (PGC-1α) overexpression, indicating that metformin suppresses gluconeogenesis via a transcription-independent process. In conclusion, we demonstrate that metformin inhibits hepatic gluconeogenesis in an LKB1- and AMPK-independent manner via a decrease in hepatic energy state.

Authors

Marc Foretz, Sophie Hébrard, Jocelyne Leclerc, Elham Zarrinpashneh, Maud Soty, Gilles Mithieux, Kei Sakamoto, Fabrizio Andreelli, Benoit Viollet

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Figure 1

Metformin inhibits gluconeogenesis in AMPKα1α2-null (AMPK KO) mouse hepatocytes.

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Metformin inhibits gluconeogenesis in AMPKα1α2-null (AMPK KO) mouse hepa...
After attachment, WT and AMPK-deficient primary hepatocytes were cultured for 16 hours in M199 medium containing 100 nM dex. Hepatocytes were then incubated in glucose-free DMEM containing lactate/pyruvate (10:1 mM) and 100 nM dex alone or with 100 μM Bt2-cAMP and with or without 0.25, 0.5, 1, or 2 mM metformin. After 8 hours, medium was collected for glucose measurement and cells were harvested for Western blot and gluconeogenic gene expression analyses. (A) Glucose production was normalized to protein content and presented as a percentage of glucose produced by WT hepatocytes incubated in the absence of both Bt2-cAMP and metformin. Results are representative of 5 independent experiments. (B) Immunoblots were performed against phospho-AMPKα (Thr172), AMPKα, phospho-ACC (Ser79), ACC, CRTC2, G6Pase, and PEPCK. Blots are representative of at least 5 independent experiments. (C) Relative mRNA levels of Pgc-1α, Pepck, and G6Pase expressed as fold activation relative to levels in WT hepatocytes incubated in the absence of both Bt2-cAMP and metformin. Results are representative of 5 independent experiments. Data are mean ± SEM. §P < 0.001, ‡P < 0.001 compared with WT and AMPK-KO hepatocytes incubated without Bt2-cAMP; *P < 0.001, †P < 0.001 compared with WT and AMPK-KO hepatocytes incubated with Bt2-cAMP alone; #P < 0.01 compared with WT hepatocytes incubated under the same conditions.