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.

Trial registration: ClinicalTrials.gov NCT01129297.

Figure 1. Identification of FXR-associated cofactors.

(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.

Figure 2. FXR interacts with AMPK in living cells.

(A) A GFP-FXR fusion protein colocalizes with AMPK in immortalized mouse hepatocytes. The doxycyclin-dependent induction of GFP-FXR expression was induced or not in AML12 cells, and the localization of FXR and AMPKα was determined by (immuno)fluorescence. (B) FXR interacts with AMPK in the cytoplasm of living cells. Top panel: Western blot analysis of HepG2 cell extracts. Bottom panels: A PLA was performed in fixed HepG2 cells after indicated treatment with 2 μM GW4064 and/or 1 mM AICAR for 2 hours.

Figure 3. AMPK activation represses FXR transcriptional activity in human or mouse hepatocytes in vitro and in vivo.

(A) FXR phosphorylation on Serine residues. HepG2 cells were treated with vehicle (DMSO), 2 μM GW4064 (GW), or GW4064 and 1 mM AICAR (GW+AICAR). Whole-cell extracts were immunoprecipitated with an anti-FXR antibody or nonspecific IgG. Precipitates were analyzed by Western blot analysis as indicated. (B) AMPK activation represses FXR-mediated gene expression. HepG2 cells were treated as in A, and total RNA was analyzed after 18 hours by RT-qPCR. Results are expressed relative to the mRNA level in untreated cells (set to 1). (C) Gene mRNA levels were measured in AML12 cells treated under conditions similar to those in B, and results are expressed as in B. (D) Mouse primary hepatocytes (PH) were isolated from 12-week-old male mice and stimulated for 6 hours. RNA levels were analyzed as in C, and results are expressed as in B. *P < 0.05; **P < 0.01; ***P < 0.005.

Figure 4. AMPK activation represses FXR transcriptional activity in human and mouse intestinal cells in vitro and in vivo.

(A) FXR target gene expression in differentiated enterocyte-like Caco-2/TC7. Differentiated Caco-2/TC7 cells were treated with 2 μM GW4064 and/or 1 mM metformin for 2 hours. Gene expression levels were assessed by RT-qPCR assays. (B) Left panel: FXR target gene expression in C57BL/6 mice. GW4064 (30 mpk/d by gavage in 1% CMC, 1% Tween 80) and/or AICAR (200 mpk/d by intraperitoneal injection) were administered to mice (n = 6–8) for 5 days. Basal gene expression was arbitrarily set to 1 for a randomly selected mouse fed a chow diet (control), and all results are expressed relative to this basal level. Right panel: AMPK activation in AICAR-treated mice. Whole-cell extracts from mouse ilea were analyzed by Western blotting using the indicated antibodies. *P < 0.05; **P < 0.01.

Figure 5. AMPK phosphorylates FXR on a single serine residue.

(A) Mouse FXR is phosphorylated by AMPK in vitro. Purified GST-ACC (positive control), His₆-tagged mFXRα3 were incubated together with purified wtAMPK or constitutively active (CA) AMPK and ³²P-ATP and resolved by SDS-PAGE. (B) The ETD MS/MS spectrum of chymotrypsin peptide 246–254 (RQVTSTTKF, 383.16³⁺) from phosphorylated mFXRα3 showing phosphorylation at Ser250. (C) Sequence conservation of the hinge region of mouse and human FXR isoforms. (D) An anti–phospho-S250 peptide recognizes specifically AMPK-phosphorylated FXR. Purified His₆-tagged mFXRα3 was phosphorylated as in A and analyzed by Western blotting using anti-FXR (FXR) or the anti-S250P FXR antibody. (E) The S250A mutation protects FXR from AICAR-induced inactivation. HepG2 cells were transfected with a Gal4-responsive luciferase reporter gene and an expression vector coding for a Gal4 DBD-wtFXR (mFXRα3) fusion protein or its mutated counterpart. ***P < 0.005.

Figure 6. FXR activity is specifically inhibited by the LKB1/AMPK pathway.

(A) hFXR is phosphorylated by AMPK in vitro. Partially purified GST-hFXRα2 or His₆-tagged hFXRα3 and wtAMPK were incubated with ³²P-ATP. (B) KNG, BSEP, FGF19, and SHP mRNA levels in FXR-, PGC1α-, SMRT-, SIRT1, or AMPK-depleted HepG2 cells. mRNA levels in siRNA-transfected cells treated with 2 μM GW4064 are expressed relative to control levels in siRNA-treated cells (set at 100%). (C) AMPK activation in HepG2 cells. Whole-cell extracts from AICAR and/or GW4064-treated cells were analyzed by Western blot using the indicated antibodies. (D) Repression of FXR activity requires a functional AMPK pathway. KNG mRNA levels were monitored in siRNA-transfected HepG2 cells. Results are expressed relative to the basal level (DMSO) set at 1. (E) AMPK activators inhibits FXRE-bound RXR/FXR heterodimers. HepG2 cells were transfected with either the reference pGL3-tk Luc reporter vector, or the same vector driven by the human SHP (pSHP-tk Luc) or KNG (pKNG-tk Luc) FXREs and expression plasmids encoding human flhRXRα and flFXRα2. Values represent the fold activation over the activity of pGL3-tk Luc alone set to 1. (F) AMPK activators block Gal4-FXR activity. HepG2 cells were transfected with the Gal4-tk Luc reporter gene and Gal4-DBD, or Gal4-DBD fused to human flFXRα2 (Gal4-FXR), flRXRα (Gal4-RXRα), or flLXRα (Gal4-LXRα) and flLXRβ (Gal4-LXRβ) Gal4 expression vectors. Cells were treated for 24 hours after transfection, and results are expressed as in E. *P < 0.05; **P < 0.01; ***P < 0.005.

Figure 7. AMPK blocks FXR transcriptional activity through inhibition of coactivator loading.

(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.

Figure 8. Metformin inhibits FXR target gene induction by TCA and BA excretion in vivo.

(A) FXR target gene expression in liver. C57BL/6 mice were treated with 0.5% TCA and/or metformin (ca. 50–80 mpk/d). FXR target gene expression was monitored by RT-qPCR, and results are expressed relative to control samples (chow diet) arbitrarily set to 1. (B) Biometric and plasma biochemical parameters. (C) BA content in plasma, gallbladder, and feces. BA content was assayed as described in Methods. Results are expressed as in C. (D) Phosphorylation state of FXR in mouse livers. Bar graph represents the densitometric quantification of Western blot analysis (see Supplemental Figure 16), which is expressed as values of the phospho signal normalized to that of total FXR, relative to the chow diet signal. which was arbitrarily set to 1. *P < 0.05; **P < 0.01; ***P < 0.005.

Figure 9. FXR is required to observe the inhibitory effect of metformin on FXR target genes and disrupted BA metabolism.

(A) FXR target gene expression in liver of WT or FXR KO mice after TCA and/or metformin treatment. Liver FXR-KO (LFxr^–/–) or WT littermates (LFxr^+/+) were treated for 5 days with a chow diet supplemented (TCA) or not (chow) with 0.5% TCA. Metformin was administered per oral administration at ca. 50–80 mpk/d. (B) Ileal gene expression in WT or LFxr^–/– mice after TCA and/or metformin treatment. Results are expressed as in B. (C) BA excretion in LFxr^–/– mice and WT littermates. Biometrics and biochemical assays were carried out as described in the Figure 8 legend. *P < 0.05, **P < 0.01; ***P < 0.005.

Figure 10. Metformin aggravates intrahepatic cholestasis.

C57BL/6 mice were treated or not with metformin for 7 days, then gavaged with ANIT. (A) Liver histology in ANIT-induced cholestasis. H&E-stained liver sections from each treatment group are shown. Right panel; quantification of lesion area in each treatment group. Results are expressed as lesion area normalized to slice area (n = 6–8). Original magnification, ×20. (B) Biometric and plasma biochemical parameters in control (chow), metformin-, ANIT-, or ANIT and metformin–treated mice. D₀: day 0; D₉: day 9 (end of treatment). (C) FXR expression and phosphorylation levels. Total RNA or whole-cell extracts were used to assess Fxr mRNA expression by RT-qPCR or the phospho-FXR levels by IP/Western-blotting respectively. Results are expressed relative to control conditions (chow) set to 1. (D and E) FXR target gene expression in liver. mRNA levels were quantified by RT-qPCR, and results are expressed relative to a control sample (chow diet) arbitrarily set to 1. *P < 0.05, **P < 0.01; ***P < 0.005.