Advertisement

Mitochondrial protease ClpP supplementation ameliorates diet-induced NASH in mice

Published:April 11, 2022DOI:https://doi.org/10.1016/j.jhep.2022.03.034

      Highlights

      • Low ClpP protein levels were found in the livers of mice with non-alcoholic steatohepatitis.
      • ClpP deficiency induced steatohepatitis and augmented high-calorie diet-induced steatohepatitis in C57BL/6J mice.
      • ClpP overexpression reduced hepatic steatosis, inflammation, and fibrosis in high-calorie diet-fed C57BL/6J mice.
      • A chemical activator of ClpP ameliorated high-calorie diet-induced steatohepatitis in C57BL/6J mice.

      Background & Aims

      Mitochondrial dysfunction is considered a pathogenic linker in the development of non-alcoholic steatohepatitis (NASH). Inappropriate mitochondrial protein-quality control, possibly induced by insufficiency of the mitochondrial matrix caseinolytic protease P (ClpP), can potentially cause mitochondrial dysfunction. Herein, we aimed to investigate hepatic ClpP levels in a diet-induced model of NASH and determine whether supplementation of ClpP can ameliorate diet-induced NASH.

      Methods

      NASH was induced by a high-fat/high-fructose (HF/HFr) diet in C57BL/6J mice. Stress/inflammatory signals were induced in mouse primary hepatocytes (MPHs) by treatment with palmitate/oleate (PA/OA). ClpP levels in hepatocytes were reduced using the RNAi-mediated gene knockdown technique but increased through the viral transduction of ClpP. ClpP activation was induced by administering a chemical activator of ClpP.

      Results

      Hepatic ClpP protein levels in C57BL/6J mice fed a HF/HFr diet were lower than the levels in those fed a normal chow diet. PA/OA treatment also decreased the ClpP protein levels in MPHs. Overexpression or activation of ClpP reversed PA/OA-induced mitochondrial dysfunction and stress/inflammatory signal activation in MPHs, whereas ClpP knockdown induced mitochondrial dysfunction and stress/inflammatory signals in these cells. On the other hand, ClpP overexpression or activation improved HF/HFr-induced NASH characteristics such as hepatic steatosis, inflammation, fibrosis, and injury in the C57BL/6J mice, whereas ClpP knockdown further augmented steatohepatitis in mice fed a HF/HFr diet.

      Conclusions

      Reduced ClpP expression and subsequent mitochondrial dysfunction are key to the development of diet-induced NASH. ClpP supplementation through viral transduction or chemical activation represents a potential therapeutic strategy to prevent diet-induced NASH.

      Lay summary

      Western diets, containing high fat and high fructose, often induce non-alcoholic steatohepatitis (NASH). Mitochondrial dysfunction is considered pathogenically linked to diet-induced NASH. We observed that the mitochondrial protease ClpP decreased in the livers of mice fed a western diet and supplementation of ClpP ameliorated western diet-induced NASH.

      Graphical abstract

      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 Journal of Hepatology
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Brunt E.M.
        • Wong V.W.S.
        • Nobili V.
        • Day C.P.
        • Sookoian S.
        • Maher J.J.
        • et al.
        Nonalcoholic fatty liver disease.
        Nat Rev Dis Primers. 2015; 1: 15080
        • Liu W.
        • Baker R.D.
        • Bhatia T.
        • Zhu L.
        • Baker S.S.
        Pathogenesis of nonalcoholic steatohepatitis.
        Cell Mol Life Sci. 2016; 73: 1969-1987
        • Somaya A.M.
        • Albhaisi S.A.M.
        • Sanyal A.J.
        New drugs for NASH.
        Liver Int. 2021; 41: 112-118
        • Ipsen D.H.
        • Lykkesfeldt J.
        • Tveden-Nyborg P.
        Molecular mechanisms of hepatic lipid accumulation in non-alcoholic fatty liver disease.
        Cell Mol Life Sci. 2018; 75: 3313-3327
        • Martinez-Lopez N.
        • Singh R.
        Autophagy and lipid droplets in the liver.
        Annu Rev Nutr. 2015; 35: 215-237
        • Musso G.
        • Cassader M.
        • Paschetta E.
        • Gambino R.
        Bioactive lipid species and metabolic pathways in progression and resolution of nonalcoholic steatohepatitis.
        Gastroenterology. 2018; 155: 282-302
        • Hirsova P.
        • Ibrahim S.H.
        • Gores G.J.
        • Malhi H.
        Lipotoxic lethal and sublethal stress signaling in hepatocytes: relevance to NASH pathogenesis.
        J Lipid Res. 2016; 57: 1758-1770
        • Farrell G.C.
        • van Rooyen D.
        • Gan L.
        • Chitturi S.
        NASH is an inflammatory disorder: pathogenic, prognostic and therapeutic implications.
        Gut Liver. 2012; 6: 149-171
        • Maiers J.L.
        • Malhi H.
        Endoplasmic reticulum stress in metabolic liver diseases and hepatic fibrosis.
        Semin Liver Dis. 2019; 39: 235-248
        • Khambu B.
        • Yan S.
        • Huda N.
        • Liu G.
        • Yin X.M.
        Autophagy in non-alcoholic fatty liver disease and alcoholic liver disease.
        Liver Res. 2018; 2: 112-119
        • Otoda T.
        • Takamura T.
        • Misu H.
        • Ota T.
        • Murata S.
        • Hayashi H.
        • et al.
        Proteasome dysfunction mediates obesity-induced endoplasmic reticulum stress and insulin resistance in the liver.
        Diabetes. 2013; 62: 811-824
        • Koliaki C.
        • Szendroedi J.
        • Kaul K.
        • Jelenik T.
        • Nowotny P.
        • Jankowiak F.
        • et al.
        Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis.
        Cell Metab. 2015; 21: 739-746
        • Sunny N.E.
        • Bril F.
        • Cusi K.
        Mitochondrial adaptation in nonalcoholic fatty liver disease: novel mechanisms and treatment strategies.
        Trends Endocrinol Metab. 2017; 28: 250-260
        • Simões I.C.M.
        • Fontes A.
        • Pinton P.
        • Zischka H.
        • Wieckowski M.R.
        Mitochondria in non-alcoholic fatty liver disease.
        Int J Biochem Cell Biol. 2018; 95: 93-99
        • Ciaula A.D.
        • Passarella S.
        • Shanmugam H.
        • Noviello M.
        • Bonfrate L.
        • Wang D.Q.
        • et al.
        Nonalcoholic fatty liver disease (NAFLD). Mitochondria as players and targets of therapies?.
        Int J Mol Sci. 2021; 20: 5375
        • Ahola S.
        • Langer T.
        • MacVicar T.
        Mitochondrial proteolysis and metabolic control.
        Cold Spring Harb Perspect Biol. 2019; 11: a033936
        • Deshwal S.
        • Fiedler K.U.
        • Langer T.
        Mitochondrial proteases: multifaceted regulators of mitochondrial plasticity.
        Annu Rev Biochem. 2020; 89: 501-528
        • Böttinger L.
        • Oeljeklaus S.
        • Guiard B.
        • Rospert S.
        • Warscheid B.
        • Becker T.
        Mitochondrial heat shock protein (Hsp) 70 and Hsp10 cooperate in the formation of Hsp60 complexes.
        J Biol Chem. 2015; 290: 11611-11622
        • Lebeau J.
        • Rainbolt T.K.
        • Wiseman R.L.
        Coordinating mitochondrial biology through the stress-responsive regulation of mitochondrial proteases.
        Int Rev Cell Mol Biol. 2018; 340: 79-128
        • Voos W.
        • Jaworek W.
        • Wilkening A.
        • Bruderek M.
        Protein quality control at the mitochondrion.
        Essays Biochem. 2016; 60: 213-225
        • Ngo J.K.
        • Pomatto L.C.
        • Davies K.J.
        Upregulation of the mitochondrial Lon protease allows adaptation to acute oxidative stress but dysregulation is associated with chronic stress, disease, and aging.
        Redox Biol. 2013; 1: 258-264
        • Cordova J.C.
        • Olivares A.O.
        • Shin Y.
        • Stinson B.M.
        • Calmat S.
        • Schmitz K.R.
        • et al.
        Stochastic but highly coordinated protein unfolding and translocation by the ClpXP proteolytic machine.
        Cell. 2014; 158: 647-658
        • Arnould T.
        • Michel S.
        • Renard P.
        Mitochondria retrograde signaling and the UPR mt: where are we in mammals?.
        Int J Mol Sci. 2015; 16: 18224-18251
        • Hill S.
        • Sataranatarajan K.
        • Remmen H.V.
        Role of signaling molecules in mitochondrial stress response.
        Front Genet. 2018; 9: 225
        • Fischer F.
        • Langer J.D.
        • Osiewacz H.D.
        Identification of potential mitochondrial CLPXP protease interactors and substrates suggests its central role in energy metabolism.
        Sci Rep. 2015; 5: 18375
        • Parthasarathy G.
        • Revelo X.
        • Malhi H.
        Pathogenesis of nonalcoholic steatohepatitis: an overview.
        Hepatol Commun. 2020; 4: 478-492
        • Luci C.
        • Bourinet M.
        • Leclère M.S.
        • Anty R.
        • Gual P.
        Chronic inflammation in non-alcoholic steatohepatitis: molecular mechanisms and therapeutic strategies.
        Front Endocrinol. 2020; 11: 597648
        • Begriche K.
        • Igoudjil A.
        • Pessayre D.
        • Fromenty B.
        Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it.
        Mitochondrion. 2006; 6: 1-28
        • Moreno-Cinos C.
        • Goossens K.
        • Salado I.G.
        • Van Der Veken P.
        • De Winter H.
        • Augustyns K.
        • Moreno-Cinos C.
        • et al.
        ClpP protease, a promising antimicrobial target.
        Int J Mol Sci. 2019; 20: 2232
        • Wong K.S.
        • Mabanglo M.F.
        • Seraphim T.V.
        • Mollica A.
        • Mao Y.Q.
        • Rizzolo K.
        Acyldepsipeptide analogs dysregulate human mitochondrial ClpP protease activity and cause apoptotic cell death.
        Cell Chem Biol. 2018; 25: 1017-1030
        • Grattagliano I.
        • Montezinho L.P.
        • Oliveira P.J.
        • Frühbeck G.
        • Gómez-Ambrosi J.
        • Montecucco F.
        • et al.
        Targeting mitochondria to oppose the progression of nonalcoholic fatty liver disease.
        Biochem Pharmacol. 2019; 160: 34-45
        • García-Ruiz C.
        • Baulies A.
        • Mari M.
        • García-Rovés P.M.
        • Fernandez-Checa J.C.
        Mitochondrial dysfunction in non-alcoholic fatty liver disease and insulin resistance: cause or consequence?.
        Free Radic Res. 2013; 47: 854-868
        • Longo M.
        • Meroni M.
        • Paolini E.
        • Macchi C.
        • Dongiovanni P.
        Mitochondrial dynamics and nonalcoholic fatty liver disease (NAFLD): new perspectives for a fairy-tale ending?.
        Metabolism. 2021; 117: 154708
        • Pintus F.
        • Floris G.
        • Rufini A.
        Nutrient availability links mitochondria, apoptosis, and obesity.
        Aging (Albany NY). 2012; 4: 734-741
        • Jovaisaite V.
        • Johan Auwerx J.
        The mitochondrial unfolded protein response-synchronizing Genomes.
        Curr Opin Cell Biol. 2015; 33: 74-81
        • Lin Y.F.
        • Haynes C.M.
        Metabolism and the UPR(mt).
        Mol Cell. 2016; 61: 677-682
        • Gat-Yablonski G.
        • Finka A.
        • Pinto G.
        • Quadroni M.
        • Shtaif B.
        • Goloubinoff P.
        Quantitative proteomics of rat livers shows that unrestricted feeding is stressful for proteostasis with implications on life span.
        Aging (Albany NY). 2016; 8: 1735-1758
        • Mouchiroud L.
        • Houtkooper R.H.
        • Moullan N.
        • Katsyuba E.
        • Ryu D.
        • Cantó C.
        • et al.
        The NAD(+)/Sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling.
        Cell. 2013; 154: 430-441
        • Gariani K.
        • Menzies K.J.
        • Ryu D.
        • Wegner C.J.
        • Wang X.
        • Ropelle E.R.
        • et al.
        Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice.
        Hepatology. 2016; 63: 1190-1204
        • Deepa S.S.
        • Bhaskaran S.
        • Ranjit R.
        • Qaisar R.
        • Nair B.C.
        • Liu Y.
        • et al.
        Down-regulation of the mitochondrial matrix peptidase ClpP in muscle cells causes mitochondrial dysfunction and decreases cell proliferation.
        Free Radic Biol Med. 2016; 91: 281-292
        • Ferramosca A.
        • Di Giacomo M.
        • Zara V.
        Antioxidant dietary approach in treatment of fatty liver: new insights and updates.
        World J Gastroenterol. 2017; 23: 4146-4157
        • Tariq Z.
        • Green C.J.
        • Hodson L.
        Are oxidative stress mechanisms the common denominator in the progression from hepatic steatosis towards non-alcoholic steatohepatitis (NASH)?.
        Liver Int. 2014; 34: 180-190
        • Bai J.
        • Liu F.
        The cGAS-cGAMP-STING pathway: a molecular link between immunity and metabolism.
        Diabetes. 2019; 68: 1099-1108
        • Cho C.S.
        • Park H.W.
        • Ho A.
        • Semple I.A.
        • Kim B.
        • Jang I.
        • et al.
        Lipotoxicity induces hepatic protein inclusions through TANK binding kinase 1-mediated p62/sequestosome 1 phosphorylation.
        Hepatology. 2018; 68: 1331-1346
        • Murthy A.M.V.
        • Robinson N.
        • Kumar S.
        Crosstalk between cGAS-STING signaling and cell death.
        Cell Death Differ. 2020; 27: 2989
        • Xiao T.
        • Liang X.
        • Liu H.
        • Zhang F.
        • Meng W.
        • Hu F.
        Mitochondrial stress protein HSP60 regulates ER stress-induced hepatic lipogenesis.
        J Mol Endocrinol. 2020; 64: 67-75
        • Quirós P.M.
        • Langer T.
        • López-Otín C.
        New roles for mitochondrial proteases in health, ageing and disease.
        Nat Rev Mol Cell Biol. 2015; 16: 345-359
        • Gispert S.
        • Parganlija D.
        • Klinkenberg M.
        • Dröse S.
        • Wittig I.
        • Mittelbronn M.
        • et al.
        Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors.
        Hum Mol Genet. 2013; 22: 4871-4887
        • Bhaskaran S.
        • Unnikrishnan A.
        • Ranjit R.
        • Qaisar R.
        • Pharaoh G.
        • Maty S.
        • et al.
        A fish oil diet induces mitochondrial uncoupling and mitochondrial unfolded protein response in epididymal white adipose tissue of mice.
        Free Radic Biol Med. 2017; 108: 704-714
        • Hu D.
        • Sun X.
        • Liao X.
        • Zhang X.
        • Zarabi S.
        • Schimmer A.
        • et al.
        Alpha-synuclein suppresses mitochondrial protease ClpP to trigger mitochondrial oxidative damage and neurotoxicity.
        Acta Neuropathol. 2019; 137: 939-960
        • Bhaskaran S.
        • Pharaoh G.
        • Ranjit R.
        • Murphy A.
        • Matsuzaki S.
        • Nair B.C.
        • et al.
        Loss of mitochondrial protease ClpP protects mice from diet-induced obesity and insulin resistance.
        EMBO Rep. 2018; 19e45009
        • Becker C.
        • Kukat A.
        • Szczepanowska K.
        • Hermans S.
        • Senft K.
        • Brandscheid C.P.
        • et al.
        CLPP deficiency protects against metabolic syndrome but hinders adaptive thermogenesis.
        EMBO Rep. 2018; 19e45126