Intestinal SIRT1 Deficiency Protects Mice from Ethanol-Induced Liver Injury by Mitigating Ferroptosis
2019; Elsevier BV; Volume: 190; Issue: 1 Linguagem: Inglês
10.1016/j.ajpath.2019.09.012
ISSN1525-2191
AutoresZhou Zhou, Ting Jie Ye, Elizabeth DeCaro, Brian Buehler, Zachary Stahl, G Bonavita, Michael J. Daniels, Min You,
Tópico(s)Ferroptosis and cancer prognosis
ResumoAberrant liver sirtuin 1 (SIRT1), a mammalian NAD+-dependent protein deacetylase, is implicated in the pathogenesis of alcoholic liver disease (ALD). However, the role of intestinal SIRT1 in ALD is presently unknown. This study investigated the involvement of intestine-specific SIRT1 in ethanol-induced liver dysfunction in mice. Ethanol feeding studies were performed on knockout mice with intestinal-specific SIRT1 deletion [SIRT1i knockout (KO)] and flox control [wild-type (WT)] mice with a chronic–plus-binge ethanol feeding protocol. After ethanol administration, hepatic inflammation and liver injury were substantially attenuated in the SIRT1iKO mice compared with the WT mice, suggesting that intestinal SIRT1 played a detrimental role in the ethanol-induced liver injury. Mechanistically, the hepatic protective effect of intestinal SIRT1 deficiency was attributable to ameliorated dysfunctional iron metabolism, increased hepatic glutathione contents, and attenuated lipid peroxidation, along with inhibition of a panel of genes implicated in the ferroptosis process in the livers of ethanol-fed mice. This study demonstrates that ablation of intestinal SIRT1 protected mice from the ethanol-induced inflammation and liver damage. The protective effects of intestinal SIRT1 deficiency are mediated, at least partially, by mitigating hepatic ferroptosis. Targeting intestinal SIRT1 or dampening hepatic ferroptosis signaling may have therapeutic potential for ALD in humans. Aberrant liver sirtuin 1 (SIRT1), a mammalian NAD+-dependent protein deacetylase, is implicated in the pathogenesis of alcoholic liver disease (ALD). However, the role of intestinal SIRT1 in ALD is presently unknown. This study investigated the involvement of intestine-specific SIRT1 in ethanol-induced liver dysfunction in mice. Ethanol feeding studies were performed on knockout mice with intestinal-specific SIRT1 deletion [SIRT1i knockout (KO)] and flox control [wild-type (WT)] mice with a chronic–plus-binge ethanol feeding protocol. After ethanol administration, hepatic inflammation and liver injury were substantially attenuated in the SIRT1iKO mice compared with the WT mice, suggesting that intestinal SIRT1 played a detrimental role in the ethanol-induced liver injury. Mechanistically, the hepatic protective effect of intestinal SIRT1 deficiency was attributable to ameliorated dysfunctional iron metabolism, increased hepatic glutathione contents, and attenuated lipid peroxidation, along with inhibition of a panel of genes implicated in the ferroptosis process in the livers of ethanol-fed mice. This study demonstrates that ablation of intestinal SIRT1 protected mice from the ethanol-induced inflammation and liver damage. The protective effects of intestinal SIRT1 deficiency are mediated, at least partially, by mitigating hepatic ferroptosis. Targeting intestinal SIRT1 or dampening hepatic ferroptosis signaling may have therapeutic potential for ALD in humans. Alcoholic liver disease (ALD) is an alcohol-associated pathologic process characterized by a range of liver disorders from steatosis, steatohepatitis, hepatitis, fibrosis/cirrhosis, to hepatocellular carcinoma and liver failure.1Farooq M.O. Bataller R. Pathogenesis and management of alcoholic liver disease.Dig Dis. 2016; 34: 347-355Crossref PubMed Scopus (36) Google Scholar,2Gao B. Ahmad M.F. Nagy L.E. Tsukamoto H. Inflammatory pathways in alcoholic steatohepatitis.J Hepatol. 2019; 70: 249-259Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar Studies in rodents and humans have demonstrated that excessive alcohol consumption induces pathophysiological conditions in multiple organs. Ethanol-induced liver dysfunctions are driven by organ crosstalk via intestine–liver, adipose–liver, or adipose–intestine-liver axis.1Farooq M.O. Bataller R. Pathogenesis and management of alcoholic liver disease.Dig Dis. 2016; 34: 347-355Crossref PubMed Scopus (36) Google Scholar, 2Gao B. Ahmad M.F. Nagy L.E. Tsukamoto H. Inflammatory pathways in alcoholic steatohepatitis.J Hepatol. 2019; 70: 249-259Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3You M. Zhou Z. Daniels M. Jogasuria A. Endocrine adiponectin-FGF15/19 axis in ethanol-induced inflammation and alcoholic liver injury.Gene Expr. 2018; 18: 103-113Crossref PubMed Scopus (13) Google Scholar Thus, identifying functional signaling molecules in the ethanol-mediated interorgan communications can lead to novel therapeutic strategies in treating human ALD. Gut is one of the major metabolic organs involved in the development and progression of ethanol-induced liver damage.1Farooq M.O. Bataller R. Pathogenesis and management of alcoholic liver disease.Dig Dis. 2016; 34: 347-355Crossref PubMed Scopus (36) Google Scholar, 2Gao B. Ahmad M.F. Nagy L.E. Tsukamoto H. Inflammatory pathways in alcoholic steatohepatitis.J Hepatol. 2019; 70: 249-259Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3You M. Zhou Z. Daniels M. Jogasuria A. Endocrine adiponectin-FGF15/19 axis in ethanol-induced inflammation and alcoholic liver injury.Gene Expr. 2018; 18: 103-113Crossref PubMed Scopus (13) Google Scholar Alcohol intake induces liver dysfunction by breaking barrier integrity and function of small intestine and by promoting growth of intestinal pathogenic bacteria and harmful metabolites (eg, lipopolysaccharides).1Farooq M.O. Bataller R. Pathogenesis and management of alcoholic liver disease.Dig Dis. 2016; 34: 347-355Crossref PubMed Scopus (36) Google Scholar,2Gao B. Ahmad M.F. Nagy L.E. Tsukamoto H. Inflammatory pathways in alcoholic steatohepatitis.J Hepatol. 2019; 70: 249-259Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar Ethanol exposure damages the liver by impairing fibroblast growth factor (FGF) 15/19, an ileum-derived hormone, and by disrupting FGF 15/19-mediated signaling.4Hartmann P. Hochrath K. Horvath A. Chen P. Seebauer C.T. Llorente C. Wang L. Alnouti Y. Fouts D.E. Stärkel P. Loomba R. Coulter S. Liddle C. Yu R.T. Ling L. Rossi S.J. DePaoli A.M. Downes M. Evans R.M. Brenner D.A. Schnabl B. Modulation of the intestinal bile acid/farnesoid X receptor/fibroblast growth factor 15 axis improves alcoholic liver disease in mice.Hepatology. 2018; 67: 2150-2166Crossref PubMed Scopus (143) Google Scholar, 5Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar, 6Wang J. Kim C. Jogasuria A. Han Y. Hu X. Wu J. Shen H. Chrast R. Finck B.N. You M. Myeloid cell-specific lipin-1 deficiency stimulates endocrine adiponectin-FGF15 axis and ameliorates ethanol-induced liver injury in mice.Sci Rep. 2016; 6: 34117Crossref PubMed Scopus (21) Google Scholar Altered intestinal microbiota and abnormal bile acid metabolism are also involved in the pathogenesis of ALD.4Hartmann P. Hochrath K. Horvath A. Chen P. Seebauer C.T. Llorente C. Wang L. Alnouti Y. Fouts D.E. Stärkel P. Loomba R. Coulter S. Liddle C. Yu R.T. Ling L. Rossi S.J. DePaoli A.M. Downes M. Evans R.M. Brenner D.A. Schnabl B. Modulation of the intestinal bile acid/farnesoid X receptor/fibroblast growth factor 15 axis improves alcoholic liver disease in mice.Hepatology. 2018; 67: 2150-2166Crossref PubMed Scopus (143) Google Scholar Sirtuin 1 (SIRT1), a class III histone deacetylase, plays critical and controversial roles in intestinal inflammation and development of colitis.7Lo Sasso G. Ryu D. Mouchiroud L. Fernando S.C. Anderson C.L. Katsyuba E. Piersigilli A. Hottiger M.O. Schoonjans K. Auwerx J. Loss of Sirt1 function improves intestinal anti-bacterial defense and protects from colitis-induced colorectal cancer.PLoS One. 2014; 9: e102495Crossref PubMed Scopus (33) Google Scholar, 8Wellman A.S. Metukuri M.R. Kazgan N. Xu X. Xu Q. Ren N.S.X. Czopik A. Shanahan M.T. Kang A. Chen W. Azcarate-Peril M.A. Gulati A.S. Fargo D.C. Guarente L. Li X. Intestinal epithelial Sirtuin 1 regulates intestinal inflammation during aging in mice by altering the intestinal microbiota.Gastroenterology. 2017; 153: 772-786Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 9Kazgan N. Metukuri M.R. Purushotham A. Lu J. Rao A. Lee S. Pratt-Hyatt M. Lickteig A. Csanaky I.L. Zhao Y. Dawson P.A. Li X. Intestine-specific deletion of SIRT1 in mice impairs DCoH2-HNF-1alpha-FXR signaling and alters systemic bile acid homeostasis.Gastroenterology. 2014; 146: 1006-1016Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 10Wang Y. Shi X. Qi J. Li X. Uray K. Guan X. SIRT1 inhibits the mouse intestinal motility and epithelial proliferation.Am J Physiol Gastrointest Liver Physiol. 2012; 302: G207-G217Crossref PubMed Scopus (16) Google Scholar Intestinal SIRT1 deficiency leads to abnormal gut microbiota, reduces inflammation, and protects mice from colitis.7Lo Sasso G. Ryu D. Mouchiroud L. Fernando S.C. Anderson C.L. Katsyuba E. Piersigilli A. Hottiger M.O. Schoonjans K. Auwerx J. Loss of Sirt1 function improves intestinal anti-bacterial defense and protects from colitis-induced colorectal cancer.PLoS One. 2014; 9: e102495Crossref PubMed Scopus (33) Google Scholar On the contrary, intestinal SIRT1 deficiency in mice increases concentrations of fecal bile acid, which in turn alter gut microbial composition, enhance intestinal inflammation, and increase susceptibility to colitis.8Wellman A.S. Metukuri M.R. Kazgan N. Xu X. Xu Q. Ren N.S.X. Czopik A. Shanahan M.T. Kang A. Chen W. Azcarate-Peril M.A. Gulati A.S. Fargo D.C. Guarente L. Li X. Intestinal epithelial Sirtuin 1 regulates intestinal inflammation during aging in mice by altering the intestinal microbiota.Gastroenterology. 2017; 153: 772-786Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,9Kazgan N. Metukuri M.R. Purushotham A. Lu J. Rao A. Lee S. Pratt-Hyatt M. Lickteig A. Csanaky I.L. Zhao Y. Dawson P.A. Li X. Intestine-specific deletion of SIRT1 in mice impairs DCoH2-HNF-1alpha-FXR signaling and alters systemic bile acid homeostasis.Gastroenterology. 2014; 146: 1006-1016Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar More important, intestine-specific SIRT1 regulates the homeostasis of extra-intestinal organs (eg, liver). For instance, intestine-specific ablation of SIRT1 alters systemic homeostasis of bile acid by increasing biosynthesis of hepatic bile acid and by attenuating hepatic accumulation of bile acids, and thus, protects mice from bile acid–induced liver damage.9Kazgan N. Metukuri M.R. Purushotham A. Lu J. Rao A. Lee S. Pratt-Hyatt M. Lickteig A. Csanaky I.L. Zhao Y. Dawson P.A. Li X. Intestine-specific deletion of SIRT1 in mice impairs DCoH2-HNF-1alpha-FXR signaling and alters systemic bile acid homeostasis.Gastroenterology. 2014; 146: 1006-1016Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Hepatic SIRT1 has been identified as a protective player against alcoholic steatohepatitis in rodents and humans.11You M. Jogasuria A. Taylor C. Wu J. Sirtuin 1 signaling and alcoholic fatty liver disease.Hepatobiliary Surg Nutr. 2015; 4: 88-100PubMed Google Scholar,12Yin H. Hu M. Liang X. Ajmo J.M. Li X. Bataller R. Odena G. Stevens Jr., S.M. You M. Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.Gastroenterology. 2014; 146: 801-811Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Ablation of SIRT1 in hepatocytes exacerbated the development of steatosis, inflammation, and fibrosis in mice after ethanol consumption.12Yin H. Hu M. Liang X. Ajmo J.M. Li X. Bataller R. Odena G. Stevens Jr., S.M. You M. Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.Gastroenterology. 2014; 146: 801-811Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Treatment with a SIRT1 activator, resveratrol, impeded alcoholic fatty liver disease in mice experiments.13Ajmo J.M. Liang X. Rogers C.Q. Pennock B. You M. Resveratrol alleviates alcoholic fatty liver in mice.Am J Physiol Gastrointest Liver Physiol. 2008; 295: G833-G842Crossref PubMed Scopus (327) Google Scholar Nevertheless, it is still unknown whether intestinal SIRT1 acts as a coordinator in the gut–liver crosstalk and contributes to the development and progression of alcoholic steatohepatitis. In this study, with the use of an intestine-specific SIRT1 knockout mouse model (SIRT1iKO) and a chronic–plus-binge ethanol feeding protocol,14Bertola A. Mathews S. Ki S.H. Wang H. Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model).Nat Protoc. 2013; 8: 627-637Crossref PubMed Scopus (609) Google Scholar we investigated the roles of intestinal SIRT1 in ethanol-induced liver dysfunction. We demonstrate that intestinal deficiency of SIRT1 protects mice from the ethanol-induced liver damage by attenuating a novel form of iron-dependent cell death, ferroptosis. Antibodies of lipocalin 2 (LCN2) and serum amyloid A1 (SAA1) were purchased from Abcam (Cambridge, MA). SIRT1 antibody was purchased from MilliporeSigma (Cleveland, OH). Antibodies of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin were purchased from Cell Signal Technology (Beverly, MA). The intestinal-specific SIRT1 KO mice (SIRT1iKO) were kindly gifted by Dr. Xiaoling Li (National Institute of Environmental Health Sciences/NIH, Research Triangle Park, NC). These SIRT1iKO mice were produced by crossing mice carrying a SIRT1 exon 4 floxed allele (Sirt1flox/flox) with Villin-cre mice (The Jackson Laboratory, Bar Harbor, ME) in a C57BL/6 background.8Wellman A.S. Metukuri M.R. Kazgan N. Xu X. Xu Q. Ren N.S.X. Czopik A. Shanahan M.T. Kang A. Chen W. Azcarate-Peril M.A. Gulati A.S. Fargo D.C. Guarente L. Li X. Intestinal epithelial Sirtuin 1 regulates intestinal inflammation during aging in mice by altering the intestinal microbiota.Gastroenterology. 2017; 153: 772-786Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,9Kazgan N. Metukuri M.R. Purushotham A. Lu J. Rao A. Lee S. Pratt-Hyatt M. Lickteig A. Csanaky I.L. Zhao Y. Dawson P.A. Li X. Intestine-specific deletion of SIRT1 in mice impairs DCoH2-HNF-1alpha-FXR signaling and alters systemic bile acid homeostasis.Gastroenterology. 2014; 146: 1006-1016Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Our study used 10- to 12-week–old female SIRT1iKO and female flox control [wild-type (WT)] mice. These female mice were subjected to a chronic plus a single binge ethanol feeding protocol [referred as the National Institute on Alcohol Abuse and Alcoholism (NIAAA) model].14Bertola A. Mathews S. Ki S.H. Wang H. Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model).Nat Protoc. 2013; 8: 627-637Crossref PubMed Scopus (609) Google Scholar Mice were divided into four dietary groups: i) WT control; ii) WT plus ethanol (identical to the control diet but with 5% v/w ethanol added); iii) SIRT1iKO control; iv) SIRT1iKO plus ethanol (SIRT1iKO + E). All female mice were first fed a liquid control diet (Lieber-DeCarli formulation; Bioserv, Flemington, NJ) for 5 days. Ethanol groups were then fed a liquid diet containing 5% v/w ethanol for 10 days, whereas control mice were pair-fed to their ethanol-fed counterparts for 10 days. At day 11, the ethanol groups were given a single oral gavage of ethanol (5 g/kg body weight, 31.25% ethanol), whereas WT or SIRT1iKO control mice were given an isocaloric gavage of dextrin maltose. No significant differences were found in food intake among the WT and SIRT1iKO mice groups fed with or without alcohol during the ethanol feeding period. Mice blood and tissues were collected after euthanasia. All animal experiments were approved by the Institutional Animal Care and Use Committee at Northeast Ohio Medical University. Serum analyses were performed with a SpectraMax i3x microplate reader (Molecular Devices, Sunnyvale, CA). Serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels were measured with ALT and AST assay kits (BioVision, Milpitas, CA). Serum lipocalin 2 (LCN2) levels were measured by using a Lipocalin-2 Mouse ELISA (enzyme-linked immunosorbent assay) Kit (R&D Systems, Minneapolis, MN). Serum amyloid A 1 (SAA1) levels were measured by using a SAA1 Mouse ELISA Kit (R&D Systems). Liver cholesterol and triglyceride levels were measured as described previously.5Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar,6Wang J. Kim C. Jogasuria A. Han Y. Hu X. Wu J. Shen H. Chrast R. Finck B.N. You M. Myeloid cell-specific lipin-1 deficiency stimulates endocrine adiponectin-FGF15 axis and ameliorates ethanol-induced liver injury in mice.Sci Rep. 2016; 6: 34117Crossref PubMed Scopus (21) Google Scholar,12Yin H. Hu M. Liang X. Ajmo J.M. Li X. Bataller R. Odena G. Stevens Jr., S.M. You M. Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.Gastroenterology. 2014; 146: 801-811Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Liver or intestine tissues were fixed in 10% formalin and embedded in paraffin. Sections of 4-μm thickness were prepared before staining.5Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar,6Wang J. Kim C. Jogasuria A. Han Y. Hu X. Wu J. Shen H. Chrast R. Finck B.N. You M. Myeloid cell-specific lipin-1 deficiency stimulates endocrine adiponectin-FGF15 axis and ameliorates ethanol-induced liver injury in mice.Sci Rep. 2016; 6: 34117Crossref PubMed Scopus (21) Google Scholar Hematoxylin and eosin staining and Prussian Blue staining were performed with a commercial staining kit (Sigma, St. Louis, MO).15Zhou Z. Ye T.J. Bonavita G. Daniels M. Kainrad N. Jogasuria A. You M. Adipose-specific lipin-1 overexpression renders hepatic ferroptosis and exacerbates alcoholic steatohepatitis in mice.Hepatol Commun. 2019; 3: 656-669Crossref PubMed Scopus (26) Google Scholar Immunohistochemistry staining of myeloperoxidase (MPO) was performed with a primary MPO antibody (Biocare Medical, Pacheco, CA) and a horseradish peroxidase–based secondary detection kit (Vector Laboratories, Burlingame, CA). Liver MPO activity was determined by using assay kits (Biovision, Milpitas, CA) as described previously.5Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar,6Wang J. Kim C. Jogasuria A. Han Y. Hu X. Wu J. Shen H. Chrast R. Finck B.N. You M. Myeloid cell-specific lipin-1 deficiency stimulates endocrine adiponectin-FGF15 axis and ameliorates ethanol-induced liver injury in mice.Sci Rep. 2016; 6: 34117Crossref PubMed Scopus (21) Google Scholar Liver concentrations of malondialdehyde (MDA), one of the end products of lipid peroxidation, was measured with a lipid peroxidation assay kit (Cayman, Ann Arbor, MI).5Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar,6Wang J. Kim C. Jogasuria A. Han Y. Hu X. Wu J. Shen H. Chrast R. Finck B.N. You M. Myeloid cell-specific lipin-1 deficiency stimulates endocrine adiponectin-FGF15 axis and ameliorates ethanol-induced liver injury in mice.Sci Rep. 2016; 6: 34117Crossref PubMed Scopus (21) Google Scholar,12Yin H. Hu M. Liang X. Ajmo J.M. Li X. Bataller R. Odena G. Stevens Jr., S.M. You M. Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.Gastroenterology. 2014; 146: 801-811Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Iron concentrations were measured with an Iron Assay Kit (ab83366; Abcam) according to the manufacturer's instructions.5Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar,15Zhou Z. Ye T.J. Bonavita G. Daniels M. Kainrad N. Jogasuria A. You M. Adipose-specific lipin-1 overexpression renders hepatic ferroptosis and exacerbates alcoholic steatohepatitis in mice.Hepatol Commun. 2019; 3: 656-669Crossref PubMed Scopus (26) Google Scholar Hepatic glutathione (GSH) level was assessed with a GSH luminesce assay kit (Promega, Madison, WI) according to the manufacturer's instructions.15Zhou Z. Ye T.J. Bonavita G. Daniels M. Kainrad N. Jogasuria A. You M. Adipose-specific lipin-1 overexpression renders hepatic ferroptosis and exacerbates alcoholic steatohepatitis in mice.Hepatol Commun. 2019; 3: 656-669Crossref PubMed Scopus (26) Google Scholar Hepatic nicotinamide adenine dinucleotide phosphate (NADP)+ and NADPH concentrations were assessed with a NADPH colorimetric assay kit (Abcam). Liver glutathione peroxidase (GPX) enzyme activity was analyzed with a commercialized colorimetric assay kit (Biovision).15Zhou Z. Ye T.J. Bonavita G. Daniels M. Kainrad N. Jogasuria A. You M. Adipose-specific lipin-1 overexpression renders hepatic ferroptosis and exacerbates alcoholic steatohepatitis in mice.Hepatol Commun. 2019; 3: 656-669Crossref PubMed Scopus (26) Google Scholar Total RNA was purified from tissues with the use of TRIzol (Invitrogen, Waltham, MA).5Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar,6Wang J. Kim C. Jogasuria A. Han Y. Hu X. Wu J. Shen H. Chrast R. Finck B.N. You M. Myeloid cell-specific lipin-1 deficiency stimulates endocrine adiponectin-FGF15 axis and ameliorates ethanol-induced liver injury in mice.Sci Rep. 2016; 6: 34117Crossref PubMed Scopus (21) Google Scholar,12Yin H. Hu M. Liang X. Ajmo J.M. Li X. Bataller R. Odena G. Stevens Jr., S.M. You M. Deletion of SIRT1 from hepatocytes in mice disrupts lipin-1 signaling and aggravates alcoholic fatty liver.Gastroenterology. 2014; 146: 801-811Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Levels of mRNA were determined by quantitative RT-PCR on a Bio-Rad thermocycler systems (Bio-Rad Laboratories, Hercules, CA). Relative mRNA levels were calculated with the comparative cycle threshold method and were normalized to the levels of GAPDH mRNA. Primer sets were either purchased or custom designed (Table 1).Table 1Primer Sequence for Real-Time PCRGeneDirectionSequenceNos2F5′-GTTCTCAGCCCAACAATACAAGA-3′R5′-GTGGACGGGTCGATGTCAC-3′GapdhF5′-TGACCTCAACTACATGGTCTACA-3′R5′-CTTCCCATTCTCGGCCTTG-3′Il1βF5′-TCGCTCAGGGTCACAAGAAA-3′R5′-CATCAGAGGCAAGGAGGAAAAC-3′Icam1F5′-CAATTTCTCATGCCGCACAG-3′R5′-AGCTGGAAGATCGAAAGTCCG-3′Cisd1F5′-GAGAGTAACGGCCTTTTTGTGA-3′R5′-GCTGTGCGAGTTGAGTGGAT-3′Cisd2F5′-GACAGCATCACCGGGTTCG-3′R5′-CTCATTCACCACCTTGGGATTTT-3′Lcn2F5′-TGGCCCTGAGTGTCATGTG-3′R5′-CTCTTGTAGCTCATAGATGGTGC-3′Ly6gF5′-TGCGTTGCTCTGGAGATAGA-3′R5′-CAGAGTAGTGGGGCAGATGG-3′Saa1F5′-TTAGCTCAGTAGGTTGTGCTGCTGG-3′R5′-ACAATGTTTCCCCAGAGAGCA-3′TnfaF5′-AGGCTGCCCCGACTACGT-3′R5′-GACTTTCTCCTGGTATGAGATAGCAAA-3′Vcam1F5′-TGAACCCAAACAGAGGCAGAGT-3′R5′-GGTATCCCATCACTTGAGCAGG-3′Acsl4F5′-CTCACCATTATATTGCTGCCTGT-3′R5′-TCTCTTTGCCATAGCGTTTTTCT-3′Ncoa4F5′-GAACCATCAGGACACATGGAAA-3′R5′-GGAGCCATAGCCTTGGGT-3′Dpp4F5′-TTTAAAGAGTACCTTTCGGGTCA-3′R5′-CAAGAGAACAAACAGTCGGTCA-3′Rpl8F5′-AAGGCGCGGGTTCTGTTTT-3′R5′-GCTCTGTCCGCTTCTTGAATC-3′CarsF5′-CATTCTGAGCATTAGTGACGAGG-3′R5′-CTGCCTGAACACGTCCACAT-3′Got1F5′-GCGCCTCCATCAGTCTTTG-3′R5′-ATTCATCTGTGCGGTACGCTC-3′Tp53F5′-GCGTAAACGCTTCGAGATGTT-3′R5′-TTTTTATGGCGGGAAGTAGACTG-3′Acsf2F5′-CTTCGGGAGGCTGTGTATCG-3′R5′-CACCATTCCAGAACTGAGAGC-3′Ireb2F5′-TTCTGCCTTACTCAATACGGGT-3′R5′-AGGGCACTTCAACATTGCTCT-3′Atp5G3F5′-CTGCATCAGTGTTATCTCGGC-3′R5′-CACCAGAACCAGCAACTCCTA-3′Chac1F5′-CTGTGGATTTTCGGGTACGG-3′R5′-CCCCTATGGAAGGTGTCTCC-3′Gls2F5′-GGTCTGCGCTATAACAAACTCT-3′R5′-CATGACACTGCCTGACTCACA-3′Slc1a5F5′-CATCAACGACTCTGTTGTAGACC-3′R5′-CGCTGGATACAGGATTGCGG-3′F, forward; R, reverse. Open table in a new tab F, forward; R, reverse. Proteins were extracted from mouse liver or intestine tissue samples. Protein concentrations were determined with a Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). Primary antibodies and secondary antibodies linked with horseradish peroxidase were used for detection as previously described.5Hu X. Jogasuria A. Wang J. Kim C. Han Y. Shen H. Wu J. You M. MitoNEET deficiency alleviates experimental alcoholic steatohepatitis in mice by stimulating endocrine adiponectin-Fgf15 axis.J Biol Chem. 2016; 291: 22482-22495Crossref PubMed Scopus (24) Google Scholar,6Wang J. Kim C. Jogasuria A. Han Y. Hu X. Wu J. Shen H. Chrast R. Finck B.N. You M. Myeloid cell-specific lipin-1 deficiency stimulates endocrine adiponectin-FGF15 axis and ameliorates ethanol-induced liver injury in mice.Sci Rep. 2016; 6: 34117Crossref PubMed Scopus (21) Google Scholar,15Zhou Z. Ye T.J. Bonavita G. Daniels M. Kainrad N. Jogasuria A. You M. Adipose-specific lipin-1 overexpression renders hepatic ferroptosis and exacerbates alcoholic steatohepatitis in mice.Hepatol Commun. 2019; 3: 656-669Crossref PubMed Scopus (26) Google Scholar Statistical significance between two groups was determined with a two-tailed t-test, and statistical significance between multiple groups was determined with one-way analysis of variance, followed by a Tukey post hoc test with the use of GraphPad Prism software version 8 (GraphPad Software Inc., San Diego, CA). Data were presented as means ± SEM; P < 0.05 was considered to be statistically significant. Consistent with published findings,8Wellman A.S. Metukuri M.R. Kazgan N. Xu X. Xu Q. Ren N.S.X. Czopik A. Shanahan M.T. Kang A. Chen W. Azcarate-Peril M.A. Gulati A.S. Fargo D.C. Guarente L. Li X. Intestinal epithelial Sirtuin 1 regulates intestinal inflammation during aging in mice by altering the intestinal microbiota.Gastroenterology. 2017; 153: 772-786Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,9Kazgan N. Metukuri M.R. Purushotham A. Lu J. Rao A. Lee S. Pratt-Hyatt M. Lickteig A. Csanaky I.L. Zhao Y. Dawson P.A. Li X. Intestine-specific deletion of SIRT1 in mice impairs DCoH2-HNF-1alpha-FXR signaling and alters systemic bile acid homeostasis.Gastroenterology. 2014; 146: 1006-1016Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar the SIRT1iKO mice on the C57BL/6 background appeared healthy under a chow diet. Western blotting analyses confirmed the removal of intestine-specific SIRT1 protein in mice (Figure 1A and Supplemental Figure S1A). The protein levels of hepatic SIRT1 in SIRT1iKO mice was normal (Supplemental Figure S1, B and C). Hematoxylin and eosin histologic analysis revealed that removal of intestinal SIRT1 in mice did not alter intestinal morphologic structure (Supplemental Figure S1D).8Wellman A.S. Metukuri M.R. Kazgan N. Xu X. Xu Q. Ren N.S.X. Czopik A. Shanahan M.T. Kang A. Chen W. Azcarate-Peril M.A. Gulati A.S. Fargo D.C. Guarente L. Li X. Intestinal epithelial Sirtuin 1 regulates intestinal inflammation during aging in mice by altering the intestinal microbiota.Gastroenterology. 2017; 153: 772-786Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar,9Kazgan N. Metukuri M.R. Purushotham A. Lu J. Rao A. Lee S. Pratt-Hyatt M. Lickteig A. Csanaky I.L. Zhao Y. Dawson P.A. Li X. Intestine-specific deletion of SIRT1 in mice impairs DCoH2-HNF-1alpha-FXR signaling and alters systemic bile acid homeostasis.Gastroenterology. 2014; 146: 1006-1016Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar The involvement of intestinal SIRT1 in ethanol-induced liver damage was studied by challenging female SIRT1iKO and WT mice with ethanol.14Bertola A. Mathews S. Ki S.H. Wang H. Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model).Nat Protoc. 2013; 8: 627-637Crossref PubMed Scopus (609) Google Scholar In comparison with the WT controls, serum ALT levels were increased by approximately fivefold in the ethanol-fed SIRT1iKO mice (Figure 1B). However, the elevation of serum ALT associated with ethanol feeding in the SIRT1iKO mice was attenuated to nearly the levels of WT control mice (Figure 1B). Concordantly, the levels of serum AST in the ethanol-fed SIRT1iKO mice were reduced to lower than that in WT mice fed with or without ethanol (Figure 1C). Hepatic triglyceride concentrations were increased in ethanol-fed WT and ethanol-fed SIRT1iKO mice to the same extent as WT control mice (Figure 1D). Hepatic cholesterol concentrations were also similar in both ethanol-fed WT and ethanol-fed SIRT1iKO mice (Figure 1E). Liver histologic analysis confirmed similar steatosis in both WT and SIRT1iKO mice after ethanol consumption, suggesting that ethanol-induced hepatic lipid accumulation in mice may be independent of intestine-specific SIRT1 (Figure 1F). Taken together, these results demonstrated that intestinal SIRT1 deficiency ameliorated the ethanol-induced liver injury without significant alterations in steatosis in mice. SIRT1 is a master repressor of inflammation in multiple organs, including liver and small intestine.7Lo Sasso G. Ryu D. Mouchir
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