Metformin interferes with bile acid homeostasis through AMPK-FXR crosstalk
Fleur Lien, … , Bart Staels, Philippe Lefebvre
Fleur Lien, … , Bart Staels, Philippe Lefebvre
Published February 17, 2014
Citation Information: J Clin Invest. 2014;124(3):1037-1051. https://doi.org/10.1172/JCI68815.
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Research Article Metabolism

Metformin interferes with bile acid homeostasis through AMPK-FXR crosstalk

Abstract

The nuclear bile acid receptor farnesoid X receptor (FXR) is an important transcriptional regulator of bile acid, lipid, and glucose metabolism. FXR is highly expressed in the liver and intestine and controls the synthesis and enterohepatic circulation of bile acids. However, little is known about FXR-associated proteins that contribute to metabolic regulation. Here, we performed a mass spectrometry–based search for FXR-interacting proteins in human hepatoma cells and identified AMPK as a coregulator of FXR. FXR interacted with the nutrient-sensitive kinase AMPK in the cytoplasm of target cells and was phosphorylated in its hinge domain. In cultured human and murine hepatocytes and enterocytes, pharmacological activation of AMPK inhibited FXR transcriptional activity and prevented FXR coactivator recruitment to promoters of FXR-regulated genes. Furthermore, treatment with AMPK activators, including the antidiabetic biguanide metformin, inhibited FXR agonist induction of FXR target genes in mouse liver and intestine. In a mouse model of intrahepatic cholestasis, metformin treatment induced FXR phosphorylation, perturbed bile acid homeostasis, and worsened liver injury. Together, our data indicate that AMPK directly phosphorylates and regulates FXR transcriptional activity to precipitate liver injury under conditions favoring cholestasis.

Authors

Fleur Lien, Alexandre Berthier, Emmanuel Bouchaert, Céline Gheeraert, Jeremy Alexandre, Geoffrey Porez, Janne Prawitt, Hélène Dehondt, Maheul Ploton, Sophie Colin, Anthony Lucas, Alexandre Patrice, François Pattou, Hélène Diemer, Alain Van Dorsselaer, Christophe Rachez, Jelena Kamilic, Albert K. Groen, Bart Staels, Philippe Lefebvre

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Figure 1

Identification of FXR-associated cofactors.

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Identification of FXR-associated cofactors. (A) Representative silver-st...
(A) Representative silver-stained acrylamide gel showing polypeptides from HepG2 extracts specifically bound to unliganded FXR LBD (–GW4064) or liganded LBD (+GW4064). Arrowheads indicate excised bands processed for mass spectrometry analysis. Selected proteins yielded a peptide coverage above 20% and were detected at least twice out of 3 analyses. (B) Detected interactions with FXR. Interactions validated by GST pull-downs are indicated here. Solid line: ligand-dependent interactions; dotted line: constitutive interactions. (C) FXR interacts with both AMPKα1 and AMPKγ1. GST pull-down experiments were carried out using GST-fused flFXR (GST-FXR) or FXR LBD (GST-FXR LBD) and 35S-labeled AMPKα1 and AMPKγ1. (D and E) AMPK coimmunoprecipitates with FXR. HepG2 cells were treated with 2 μM GW4064 and/or 1 mM AICAR for 2 hours. (D) Western blot analysis of whole-cell extracts was performed to assess the presence of the indicated proteins. (E) Whole-cell extracts were immunoprecipitated with either a nonspecific IgG or an anti-FXR antibody. Immunoprecipitated material was analyzed by Western blot using the indicated antibodies. Western blot analysis was carried out to assess AMPK and FXR expression levels in whole cell extracts (upper panel). (F) AMPK coimmunoprecipitates with FXR in mouse liver extracts. Mice received the indicated treatment (see Figure 6 for details), and FXR was immunoprecipitated as in E.