Advertisement

Impaired hepatic glucose metabolism and liver-α-cell axis in mice with liver-specific ablation of the Hepatocyte Nuclear Factor 4α (Hnf4a) gene

  • Efstathia Thymiakou
    Affiliations
    Laboratory of Biochemistry, University of Crete Medical School, Heraklion 71003, Greece

    Gene Regulation and Epigenetics group, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion 71003, Greece
    Search for articles by this author
  • Maria Tzardi
    Affiliations
    Department of Pathology, University of Crete Medical School, Heraklion, Crete, Greece
    Search for articles by this author
  • Dimitris Kardassis
    Correspondence
    Corresponding author at: Laboratory of Biochemistry, University of Crete Medical School, Heraklion 71003, Greece.
    Affiliations
    Laboratory of Biochemistry, University of Crete Medical School, Heraklion 71003, Greece

    Gene Regulation and Epigenetics group, Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion 71003, Greece
    Search for articles by this author
Published:November 30, 2022DOI:https://doi.org/10.1016/j.metabol.2022.155371

      Highlights

      • Insulin and glucagon signaling are perturbed in H4LivKO mice.
      • Liver-α-cell axis is impaired in H4LivKO mice.
      • Oral administration of glucose significantly improves survival of H4LivKO mice.
      • H4LivKO mice show increased hepatic gene expression of fibrotic markers.

      Abstract

      Background

      Hnf4a gene ablation in mouse liver causes hepatic steatosis, perturbs HDL structure and function and affects many pathways and genes related to glucose metabolism. Our aim here was to investigate the role of liver HNF4A in glucose homeostasis.

      Methods

      Serum and tissue samples were obtained from Alb-Cre;Hnf4afl/fl (H4LivKO) mice and their littermate Hnf4afl/fl controls. Fasting glucose and insulin, glucose tolerance, insulin tolerance and glucagon challenge tests were performed by standard procedures. Binding of HNF4A to DNA was assessed by chromatin immunoprecipitation assays. Gene expression analysis was performed by quantitative reverse transcription PCR.

      Results

      H4LivKO mice presented lower blood levels of fasting glucose, improved glucose tolerance, increased serum lactate levels and reduced response to glucagon challenge compared to their control littermates. Insulin signaling in the liver was reduced despite the increase in serum insulin levels. H4LivKO mice showed altered expression of genes involved in glycolysis, gluconeogenesis and glycogen metabolism in the liver. The expression of the gene encoding the glucagon receptor (Gcgr) was markedly reduced in H4LivKO liver and chromatin immunoprecipitation assays revealed specific and strong binding of HNF4A to the Gcgr promoter. H4LivKO mice presented increased amino acid concentration in the serum, α-cell hyperplasia and a dramatic increase in glucagon levels suggesting an impairment of the liver-α-cell axis. Glucose administration in the drinking water of H4LivKO mice resulted in an impressive extension of survival. The expression of several genes related to non-alcoholic fatty liver disease progression to more severe liver pathologies, including Mcp1, Gdf15, Igfbp-1 and Hmox1, was increased in H4LivKO mice as early as 6 weeks of age and this increased expression was sustained until the endpoint of the study.

      Conclusions

      Our results reveal a novel role of liver HNF4A in controlling blood glucose levels via regulation of glucagon signaling. In combination with the steatotic phenotype, our results suggest that H4LivKO mice could serve as a valuable model for studying glucose homeostasis in the context of non-alcoholic fatty liver disease.

      Graphical abstract

      Unlabelled Image
      Graphical AbstractSchematic representation of the effect of hepatic Hnf4a gene ablation on glucose and amino acid metabolism in the liver and the impairment of the liver-α-cell axis.

      Abbreviations:

      AUC (area under the curve), CTR (control), GCT (glucagon challenge test), GCGR (glucagon receptor), GLUT (glucose transporter), H4LivKO (HNF4A liver-specific knock out), H&E (hematoxylin and eosin), HCC (hepatocellular carcinoma), HNF4 (hepatocyte nuclear factor 4), HOMA-IR (homeostatic model assessment of insulin resistance), IPGTT (intraperitoneal glucose tolerance test), IR (Insulin resistance), ITT (insulin tolerance test), MODY1 (maturity-onset diabetes of the young type 1), NAFLD (non-alcoholic Fatty Liver Disease), NASH (non-alcoholic steatohepatitis), RT-qPCR (quantitative reverse transcription PCR), SGOT (aspartate transaminase), SGPT (alanine transaminase), T2D (type 2 diabetes), ZT (Zeitgeber time)

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Metabolism - Clinical and Experimental
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Stoffel M.
        • Duncan S.A.
        The maturity-onset diabetes of the young (MODY1) transcription factor HNF4alpha regulates expression of genes required for glucose transport and metabolism.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13209-13214
        • Chen W.S.
        • Manova K.
        • Weinstein D.C.
        • Duncan S.A.
        • Plump A.S.
        • Prezioso V.R.
        • et al.
        Disruption of the HNF-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos.
        Genes Dev. 1994; 8: 2466-2477
        • Shih D.Q.
        • Dansky H.M.
        • Fleisher M.
        • Assmann G.
        • Fajans S.S.
        • Stoffel M.
        Genotype/phenotype relationships in HNF-4alpha/MODY1: haploinsufficiency is associated with reduced apolipoprotein (AII), apolipoprotein (CIII), lipoprotein(a), and triglyceride levels.
        Diabetes. 2000; 49: 832-837
        • Thymiakou E.
        • Othman A.
        • Hornemann T.
        • Kardassis D.
        Defects in high density lipoprotein metabolism and hepatic steatosis in mice with liver-specific ablation of hepatocyte nuclear factor 4A.
        Metab Clin Exp. 2020; 110154307
        • Anstee Q.M.
        • Reeves H.L.
        • Kotsiliti E.
        • Govaere O.
        • Heikenwalder M.
        From NASH to HCC: current concepts and future challenges.
        Nat Rev Gastroenterol Hepatol. 2019; 16: 411-428
        • Xu Y.
        • Zalzala M.
        • Xu J.
        • Li Y.
        • Yin L.
        • Zhang Y.
        A metabolic stress-inducible miR-34a-HNF4alpha pathway regulates lipid and lipoprotein metabolism.
        Nat Commun. 2015; 6: 7466
        • Gunewardena S.
        • Huck I.
        • Walesky C.
        • Robarts D.
        • Weinman S.
        • Apte U.
        Progressive loss of hepatocyte nuclear factor 4 alpha activity in chronic liver diseases in humans.
        Hepatology. 2022; 76: 372-386
        • Yang T.
        • Poenisch M.
        • Khanal R.
        • Hu Q.
        • Dai Z.
        • Li R.
        • et al.
        Therapeutic HNF4A mRNA attenuates liver fibrosis in a preclinical model.
        J Hepatol. 2021; 75: 1420-1433
        • Abd El-Kader S.M.
        • El-Den Ashmawy E.M.
        Non-alcoholic fatty liver disease: the diagnosis and management.
        World J Hepatol. 2015; 7: 846-858
        • Adams L.A.
        • Anstee Q.M.
        • Tilg H.
        • Targher G.
        Non-alcoholic fatty liver disease and its relationship with cardiovascular disease and other extrahepatic diseases.
        Gut. 2017; 66: 1138-1153
        • Xia M.F.
        • Bian H.
        • Gao X.
        NAFLD and diabetes: two sides of the same Coin? Rationale for gene-based personalized NAFLD treatment.
        Front Pharmacol. 2019; 10: 877
        • Gastaldelli A.
        • Cusi K.
        From NASH to diabetes and from diabetes to NASH: Mechanisms and treatment options.
        in: JHEP reports: innovation in hepatology. 1. 2019: 312-328
        • Wewer Albrechtsen N.J.
        • Pedersen J.
        • Galsgaard K.D.
        • Winther-Sorensen M.
        • Suppli M.P.
        • Janah L.
        • et al.
        The liver-alpha-cell Axis and type 2 diabetes.
        Endocr Rev. 2019; 40: 1353-1366
        • Janah L.
        • Kjeldsen S.
        • Galsgaard K.D.
        • Winther-Sorensen M.
        • Stojanovska E.
        • Pedersen J.
        • et al.
        Glucagon receptor signaling and glucagon resistance.
        Int J Mol Sci. 2019; : 20
        • Sammons M.F.
        • Lee E.C.
        Recent progress in the development of small-molecule glucagon receptor antagonists.
        Bioorg Med Chem Lett. 2015; 25: 4057-4064
        • Richter M.M.
        • Galsgaard K.D.
        • Elmelund E.
        • Knop F.K.
        • Suppli M.P.
        • Holst J.J.
        • et al.
        The liver-alpha-cell Axis in health and in disease.
        Diabetes. 2022; 71: 1852-1861
        • Negi C.K.
        • Babica P.
        • Bajard L.
        • Bienertova-Vasku J.
        • Tarantino G.
        Insights into the molecular targets and emerging pharmacotherapeutic interventions for nonalcoholic fatty liver disease.
        Metab Clin Exp. 2022; 126154925
        • Hayhurst G.P.
        • Lee Y.H.
        • Lambert G.
        • Ward J.M.
        • Gonzalez F.J.
        Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis.
        Mol Cell Biol. 2001; 21: 1393-1403
        • Wang T.
        • Chen K.
        • Yao W.
        • Zheng R.
        • He Q.
        • Xia J.
        • et al.
        Acetylation of lactate dehydrogenase B drives NAFLD progression by impairing lactate clearance.
        J Hepatol. 2021; 74: 1038-1052
        • Yu S.
        • Meng S.
        • Xiang M.
        • Ma H.
        Phosphoenolpyruvate carboxykinase in cell metabolism: roles and mechanisms beyond gluconeogenesis.
        Mol Metab. 2021; 53101257
        • Zoka A.
        • Barna G.
        • Somogyi A.
        • Muzes G.
        • Olah A.
        • Al-Aissa Z.
        • et al.
        Extension of the CD4(+)Foxp3(+)CD25(-/low) regulatory T-cell subpopulation in type 1 diabetes mellitus.
        Autoimmunity. 2015; 48: 289-297
        • Longuet C.
        • Robledo A.M.
        • Dean E.D.
        • Dai C.
        • Ali S.
        • McGuinness I.
        • et al.
        Liver-specific disruption of the murine glucagon receptor produces alpha-cell hyperplasia: evidence for a circulating alpha-cell growth factor.
        Diabetes. 2013; 62: 1196-1205
        • Solloway M.J.
        • Madjidi A.
        • Gu C.
        • Eastham-Anderson J.
        • Clarke H.J.
        • Kljavin N.
        • et al.
        Glucagon couples hepatic amino acid catabolism to mTOR-dependent regulation of alpha-cell mass.
        Cell Rep. 2015; 12: 495-510
        • Fekry B.
        • Ribas-Latre A.
        • Baumgartner C.
        • Mohamed A.M.T.
        • Kolonin M.G.
        • Sladek F.M.
        • et al.
        HNF4alpha-deficient fatty liver provides a permissive environment for sex-independent hepatocellular carcinoma.
        Cancer Res. 2019; 79: 5860-5873
        • Govaere O.
        • Cockell S.
        • Tiniakos D.
        • Queen R.
        • Younes R.
        • Vacca M.
        • et al.
        Transcriptomic profiling across the nonalcoholic fatty liver disease spectrum reveals gene signatures for steatohepatitis and fibrosis.
        Sci Transl Med. 2020; : 12
        • Haukeland J.W.
        • Damas J.K.
        • Konopski Z.
        • Loberg E.M.
        • Haaland T.
        • Goverud I.
        • et al.
        Systemic inflammation in nonalcoholic fatty liver disease is characterized by elevated levels of CCL2.
        J Hepatol. 2006; 44: 1167-1174
        • Pessoa J.
        • Teixeira J.
        Cytoskeleton alterations in non-alcoholic fatty liver disease.
        Metab Clin Exp. 2022; 128155115
        • Hagstrom H.
        • Stal P.
        • Hultcrantz R.
        • Brismar K.
        • Ansurudeen I.
        IGFBP-1 and IGF-I as markers for advanced fibrosis in NAFLD - a pilot study.
        Scand J Gastroenterol. 2017; 52: 1427-1434
        • Malaguarnera L.
        • Madeddu R.
        • Palio E.
        • Arena N.
        • Malaguarnera M.
        Heme oxygenase-1 levels and oxidative stress-related parameters in non-alcoholic fatty liver disease patients.
        J Hepatol. 2005; 42: 585-591
        • Ratziu V.
        • Francque S.
        • Sanyal A.
        Breakthroughs in therapies for NASH and remaining challenges.
        J Hepatol. 2022; 76: 1263-1278
        • Targher G.
        • Corey K.E.
        • Byrne C.D.
        • Roden M.
        The complex link between NAFLD and type 2 diabetes mellitus - mechanisms and treatments.
        Nat Rev Gastroenterol Hepatol. 2021; 18: 599-612
        • Pearson E.R.
        • Boj S.F.
        • Steele A.M.
        • Barrett T.
        • Stals K.
        • Shield J.P.
        • et al.
        Macrosomia and hyperinsulinaemic hypoglycaemia in patients with heterozygous mutations in the HNF4A gene.
        PLoS Med. 2007; 4e118
        • Gupta R.K.
        • Vatamaniuk M.Z.
        • Lee C.S.
        • Flaschen R.C.
        • Fulmer J.T.
        • Matschinsky F.M.
        • et al.
        The MODY1 gene HNF-4alpha regulates selected genes involved in insulin secretion.
        J Clin Invest. 2005; 115: 1006-1015
        • Miura A.
        • Yamagata K.
        • Kakei M.
        • Hatakeyama H.
        • Takahashi N.
        • Fukui K.
        • et al.
        Hepatocyte nuclear factor-4alpha is essential for glucose-stimulated insulin secretion by pancreatic beta-cells.
        J Biol Chem. 2006; 281: 5246-5257
        • Chella Krishnan K.
        • Floyd R.R.
        • Sabir S.
        • Jayasekera D.W.
        • Leon-Mimila P.V.
        • Jones A.E.
        • et al.
        Liver pyruvate kinase promotes NAFLD/NASH in both mice and humans in a sex-specific manner.
        Cell Mol Gastroenterol Hepatol. 2021; 11: 389-406
        • Jeon J.H.
        • Thoudam T.
        • Choi E.J.
        • Kim M.J.
        • Harris R.A.
        • Lee I.K.
        Loss of metabolic flexibility as a result of overexpression of pyruvate dehydrogenase kinases in muscle, liver and the immune system: therapeutic targets in metabolic diseases.
        J Diabetes Investig. 2021; 12: 21-31
        • de la Cruz-Lopez K.G.
        • Castro-Munoz L.J.
        • Reyes-Hernandez D.O.
        • Garcia-Carranca A.
        • Manzo-Merino J.
        Lactate in the regulation of tumor microenvironment and therapeutic approaches.
        Front Oncol. 2019; 9: 1143
        • Heller S.
        • Worona L.
        • Consuelo A.
        Nutritional therapy for glycogen storage diseases.
        J Pediatr Gastroenterol Nutr. 2008; 47: S15-S21
        • Holloway M.G.
        • Miles G.D.
        • Dombkowski A.A.
        • Waxman D.J.
        Liver-specific hepatocyte nuclear factor-4alpha deficiency: greater impact on gene expression in male than in female mouse liver.
        Mol Endocrinol. 2008; 22: 1274-1286
        • Bosch F.X.
        • Ribes J.
        • Diaz M.
        • Cleries R.
        Primary liver cancer: worldwide incidence and trends.
        Gastroenterology. 2004; 127: S5-S16
        • Pedersen J.S.
        • Rygg M.O.
        • Kristiansen V.B.
        • Olsen B.H.
        • Serizawa R.R.
        • Holst J.J.
        • et al.
        Nonalcoholic fatty liver disease impairs the liver-alpha cell Axis independent of hepatic inflammation and fibrosis.
        Hepatol Commun. 2020; 4: 1610-1623
        • Geisler C.E.
        • Renquist B.J.
        Hepatic lipid accumulation: cause and consequence of dysregulated glucoregulatory hormones.
        J Endocrinol. 2017; 234: R1-R21
        • Galsgaard K.D.
        The vicious circle of hepatic glucagon resistance in non-alcoholic fatty liver disease.
        J Clin Med. 2020; : 9
        • Gelling R.W.
        • Du X.Q.
        • Dichmann D.S.
        • Romer J.
        • Huang H.
        • Cui L.
        • et al.
        Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1438-1443
        • Zhu L.
        • Dattaroy D.
        • Pham J.
        • Wang L.
        • Barella L.F.
        • Cui Y.
        • et al.
        Intra-islet glucagon signaling is critical for maintaining glucose homeostasis.
        JCI Insight. 2019; : 5
        • Zhang Y.
        • Han C.
        • Zhu W.
        • Yang G.
        • Peng X.
        • Mehta S.
        • et al.
        Glucagon potentiates insulin secretion via beta-cell GCGR at physiological concentrations of glucose.
        Cells. 2021; : 10
        • Inoue Y.
        • Hayhurst G.P.
        • Inoue J.
        • Mori M.
        • Gonzalez F.J.
        Defective ureagenesis in mice carrying a liver-specific disruption of hepatocyte nuclear factor 4alpha (HNF4alpha ). HNF4alpha regulates ornithine transcarbamylase in vivo.
        J Biol Chem. 2002; 277: 25257-25265
        • Charbonneau A.
        • Couturier K.
        • Gauthier M.S.
        • Lavoie J.M.
        Evidence of hepatic glucagon resistance associated with hepatic steatosis: reversal effect of training.
        Int J Sports Med. 2005; 26: 432-441
        • Winther-Sorensen M.
        • Galsgaard K.D.
        • Santos A.
        • Trammell S.A.J.
        • Sulek K.
        • Kuhre R.E.
        • et al.
        Glucagon acutely regulates hepatic amino acid catabolism and the effect may be disturbed by steatosis.
        Mol Metab. 2020; 42101080
        • Bonzo J.A.
        • Ferry C.H.
        • Matsubara T.
        • Kim J.H.
        • Gonzalez F.J.
        Suppression of hepatocyte proliferation by hepatocyte nuclear factor 4alpha in adult mice.
        J Biol Chem. 2012; 287: 7345-7356
        • Abdelmalek M.F.
        NAFLD: the clinical and economic burden of NAFLD: time to turn the tide.
        Nat Rev Gastroenterol Hepatol. 2016; 13: 685-686
        • Dubois V.
        • Staels B.
        • Lefebvre P.
        • Verzi M.P.
        • Eeckhoute J.
        Control of cell identity by the nuclear receptor HNF4 in organ pathophysiology.
        Cells. 2020; : 9
        • Diaz-Aragon R.
        • Coard M.C.
        • Amirneni S.
        • Faccioli L.
        • Haep N.
        • Malizio M.R.
        • et al.
        Therapeutic potential of HNF4alpha in end-stage liver disease.
        Organogenesis. 2021; 17: 126-135
        • Colclough K.
        • Bellanne-Chantelot C.
        • Saint-Martin C.
        • Flanagan S.E.
        • Ellard S.
        Mutations in the genes encoding the transcription factors hepatocyte nuclear factor 1 alpha and 4 alpha in maturity-onset diabetes of the young and hyperinsulinemic hypoglycemia.
        Hum Mutat. 2013; 34: 669-685