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 7

AMPK blocks FXR transcriptional activity through inhibition of coactivator loading.

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AMPK blocks FXR transcriptional activity through inhibition of coactivat...
(A and B) AMPK activation prevents coactivator recruitment to FXR in a mammalian 2-hybrid assay. HepG2 cells were transfected with the Gal4-tk Luc reporter gene and a Gal4-DBD fused to human flFXRα2 (Gal4-FXR) without (–) or with (+) an expression vector coding for flSRC-2 (A) or SRC2 RID (B) and treated for 24 hours after transfection as indicated, after which luciferase activities were assayed. Results are expressed as above. (C) AMPK activation prevents coactivator recruitment to DNA-bound FXR/RXR heterodimers. HepG2 were transfected with the pKNG-tk Luc and expression vectors coding for human flRXRα and hFXRα2, VP16 activating domain (VP16-AD), or VP16 coupled to SRC2-RID (VP16-SRC2). Cells were treated and results expressed as in A. (D) HepG2 cells were incubated with or without GW4064/AICAR for 90 minutes, crosslinked chromatin was immunoprecipitated with the indicated antibodies, and the precipitated genomic DNA purified. The KNG promoter fragment, including its FXRE, was amplified by PCR. Results were normalized to myoglobin as a negative control and are plotted as the fold enrichment over background (n = 2–3). (E) Mouse FXRα3 S250 mediates the inhibitory effect of AMPK activation on coactivator recruitment. The 2-hybrid assay was carried out as in B using Gal4-mFXRα3 or Gal4-mFXRα3 S250A and VP16-SRC2 RID expression vectors. (F) The mouse FXRα2 isoform is sensitive to AMPK-mediated inhibition. Transactivation assays were carried out as in C. *P < 0.05; **P < 0.01; ***P < 0.005.