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Loss of hepatic Lgr4 and Lgr5 promotes nonalcoholic fatty liver disease

Enrica Saponara, Carlos Penno, Meztli L. Matadamas Guzmán, Virginie Brun, Benoit Fischer, Margaret Brousseau, Zhong-Yi Wang, Peter ODonnell, Jonathan Turner, Alexandra Graff Meyer, Laura Bollepalli, Giovanni d’Ario, Guglielmo Roma, Walter Carbone, Vanessa Orsini, Stefano Annunziato, Michael Obrecht, Nicolau Beckmann, Chandra Saravanan, Arnaud Osmont, Philipp Tropberger, Shola Richards, Christel Genoud, Alexandra Aebi, Svenja Ley, Iwona Ksiazek, Florian Nigsch, Luigi Terraciano, Tewis Bouwmeester, Jan Tchorz, View ORCID ProfileHeinz Ruffner
doi: https://doi.org/10.1101/2021.11.22.469602
Enrica Saponara
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Carlos Penno
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Meztli L. Matadamas Guzmán
2Instituto Nacional de Medicina Genomica, UNAM, INMEGEN, 14610 México City
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Virginie Brun
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Benoit Fischer
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Margaret Brousseau
3Novartis Institutes for BioMedical Research, Novartis Pharma AG, Cambridge, Massachusetts 02139, USA
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Zhong-Yi Wang
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Peter ODonnell
3Novartis Institutes for BioMedical Research, Novartis Pharma AG, Cambridge, Massachusetts 02139, USA
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Jonathan Turner
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Alexandra Graff Meyer
4Friedrich Miescher Institute for BioMedical Research (FMI), 4058 Basel, Switzerland
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Laura Bollepalli
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Giovanni d’Ario
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Guglielmo Roma
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Walter Carbone
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Vanessa Orsini
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Stefano Annunziato
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Michael Obrecht
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Nicolau Beckmann
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Chandra Saravanan
3Novartis Institutes for BioMedical Research, Novartis Pharma AG, Cambridge, Massachusetts 02139, USA
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Arnaud Osmont
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Philipp Tropberger
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Shola Richards
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Christel Genoud
4Friedrich Miescher Institute for BioMedical Research (FMI), 4058 Basel, Switzerland
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Alexandra Aebi
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Svenja Ley
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Iwona Ksiazek
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Florian Nigsch
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Luigi Terraciano
5IRCCS Humanitas Research Hospital, Rozzano (Milan), Italy
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Tewis Bouwmeester
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Jan Tchorz
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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Heinz Ruffner
1Novartis Institutes for BioMedical Research, Novartis Pharma AG, 4056 Basel, Switzerland
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  • ORCID record for Heinz Ruffner
  • For correspondence: heinz.ruffner@novartis.com
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Abstract

Background & Aims The Rspo-Lgr4/5-Znrf3/Rnf43 module is a master regulator of hepatic Wnt/β-catenin signaling and metabolic zonation, but its impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We studied whether liver-specific loss of the Wnt/β-catenin modulators Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4/5 (Lgr4/5) promotes nonalcoholic fatty liver disease (NAFLD).

Methods Mice with liver-specific deletion of both receptors Lgr4/5 (Lgr4/5dLKO) were fed with normal diet (ND) or high fat diet (HFD). Livers of these mice were analyzed for lipid and fibrotic content by tissue staining and immunohistochemistry (IHC), and lipoproteins, inflammation and liver enzyme markers were measured in blood. Mechanistic insights into hepatic lipid accumulation were obtained by using ex vivo primary hepatocyte cultures derived from the Lgr4/5dLKO mice. Lipid analysis of mouse livers was performed by mass spectrometry (MS)-based untargeted lipidomic analysis.

Results We demonstrated that liver-specific ablation of Lgr4/5-mediated Wnt signaling resulted in hepatic steatosis, impaired bile acid (BA) secretion and predisposition to liver fibrosis. Under HFD conditions, we observed progressive intrahepatic fat accumulation, developing into macro-vesicular steatosis. Serum lipoprotein levels in HFD-fed Lgr4/5dLKO mice were decreased, rather than increased, suggesting that accumulation of fat in the liver was due to impaired lipid secretion by hepatocytes. Our lipidome analysis revealed a severe alteration of several lipid species in livers of Lgr4/5dLKO mice, including triacylglycerol estolides (TG-EST), a storage form of bioactive free fatty acid (FA) esters of hydroxy FAs (FAHFAs).

Conclusions Loss of hepatic Wnt/β-catenin activity by Lgr4/5 deletion led to deregulation of lipoprotein pathways, loss of BA secretion, intrinsic alterations of lipid homeostasis and the onset of NAFLD.

Lay summary The Wnt/β-catenin pathway plays an important role during development and tissue homeostasis. Loss of Wnt/β-catenin activity in mouse liver leads to loss of liver zonation, but the impact on nonalcoholic fatty liver disease (NAFLD) remains unclear. We show that livers of mice developed steatosis upon deletion of the positive pathway regulators Lgr4/5. Livers of knock-out (KO) mice exhibited altered lipid composition due to impaired lipid secretion. Furthermore, livers of these mice developed a nonalcoholic steatohepatitis (NASH)-like phenotype and fibrotic features derived from activated hepatic stellate cells. Our data demonstrate a protective role of Wnt/β-catenin pathway activity towards the development of NAFLD.

Highlights

  • Abrogation of hepatic Wnt/β-catenin activity and liver zonation upon Lgr4/5 deletion in mice led to hepatic steatosis.

  • Liver fat accumulation was caused by impaired lipid secretion from hepatocytes.

  • Steatotic livers contained increased levels of diverse lipid species, including polyunsaturated fatty acids and triglycerol-estolides.

  • These data confirmed that a decrease in Wnt/β-catenin signaling led to the development of nonalcoholic fatty liver disease (NAFLD) in mice.

Competing Interest Statement

All authors except M.L.M.G, A.G.M, C.G. and L.T. are or have been employed by and/or shareholders of Novartis Pharma AG.

  • Abbreviations

    Alb-Cre
    Cre recombinase under the hepatocyte-specific albumin promoter
    αSMA
    Alpha Smooth Muscle Actin
    ALT
    alanine aminotransferase
    AST
    aspartate aminotransferase
    AXIN2
    Axis inhibition protein 2
    BA
    bile acid
    CDH1
    Cadherin 1
    c-HDL
    cholesterol-high density lipoprotein
    CLDN2
    Claudin-2
    CLF
    cholyl-L-lysyl-fluorescein
    CTNNB1
    Catenin beta 1
    DG
    diacylglycerol
    DNL
    de novo lipogenesis
    FA
    fatty acid
    FAHFA
    fatty acid esters of hydroxy fatty acids
    FPLC
    fast protein liquid chromatography-based sizeexclusion
    GTT
    glucose tolerance test
    HFD
    high fat diet
    HSC
    hepatic stellate cells
    IBA1
    Ionized Calcium-Binding Adapter Molecule 1
    IHC
    immunohistochemistry
    IL6
    Interleukin 6
    ISH
    in situ hybridization
    ITT
    insulin tolerance test
    KO
    knock-out
    KRT19
    Keratin 19
    LGR
    Leucine-Rich RepeatContaining G Protein-Coupled Receptor
    Lgr4/5dLKO
    Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 4 and 5 double liver knock-out
    LDL
    low density lipoprotein
    LRP6
    LDL-Receptor Related Protein 6
    MCD
    methionine- and choline-deficient diet
    NAFLD
    nonalcoholic fatty liver disease
    NASH
    nonalcoholic steatohepatitis
    ND
    normal diet
    OxTG
    oxidized triglycerol
    RNF43
    Ring Finger Protein 43
    RSPO
    Roof Plate-Specific Spondin
    SD, SD
    standard deviation
    SEM
    standard error of the mean
    SLC
    solute carrier
    SWE
    shear wave elastography
    t-BA
    total bile acids
    t-Chol
    total cholesterol
    TEM
    Transmission electron microscopy
    TG
    triglycerol
    TG-EST
    triacylglycerol estolides
    TJ
    tight junctions
    TNFα
    tumor necrosis factor alpha
    VLDL
    very low density lipoprotein
    VLDLR
    very low density lipoprotein receptor
    Wnt
    Wingless-related integration site
    WT
    wild-type
    ZNRF3
    Zinc And Ring Finger 3
    ZO-1
    Zona Occludens Protein-1
  • Copyright 
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    Posted November 22, 2021.
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    Loss of hepatic Lgr4 and Lgr5 promotes nonalcoholic fatty liver disease
    Enrica Saponara, Carlos Penno, Meztli L. Matadamas Guzmán, Virginie Brun, Benoit Fischer, Margaret Brousseau, Zhong-Yi Wang, Peter ODonnell, Jonathan Turner, Alexandra Graff Meyer, Laura Bollepalli, Giovanni d’Ario, Guglielmo Roma, Walter Carbone, Vanessa Orsini, Stefano Annunziato, Michael Obrecht, Nicolau Beckmann, Chandra Saravanan, Arnaud Osmont, Philipp Tropberger, Shola Richards, Christel Genoud, Alexandra Aebi, Svenja Ley, Iwona Ksiazek, Florian Nigsch, Luigi Terraciano, Tewis Bouwmeester, Jan Tchorz, Heinz Ruffner
    bioRxiv 2021.11.22.469602; doi: https://doi.org/10.1101/2021.11.22.469602
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    Loss of hepatic Lgr4 and Lgr5 promotes nonalcoholic fatty liver disease
    Enrica Saponara, Carlos Penno, Meztli L. Matadamas Guzmán, Virginie Brun, Benoit Fischer, Margaret Brousseau, Zhong-Yi Wang, Peter ODonnell, Jonathan Turner, Alexandra Graff Meyer, Laura Bollepalli, Giovanni d’Ario, Guglielmo Roma, Walter Carbone, Vanessa Orsini, Stefano Annunziato, Michael Obrecht, Nicolau Beckmann, Chandra Saravanan, Arnaud Osmont, Philipp Tropberger, Shola Richards, Christel Genoud, Alexandra Aebi, Svenja Ley, Iwona Ksiazek, Florian Nigsch, Luigi Terraciano, Tewis Bouwmeester, Jan Tchorz, Heinz Ruffner
    bioRxiv 2021.11.22.469602; doi: https://doi.org/10.1101/2021.11.22.469602

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