Jessic M.Ferrell ,Mtthew Dilts ,Zchry Sthl ,Shnnon Boehme ,Sit Pokhrel ,Xinwen Wng ,John Y.L.Ching
a Department of Integrative Medical Sciences,Northeast Ohio Medical University,Rootstown,OH,USA
b Department of Pharmaceutical Sciences,Northeast Ohio Medical University,Rootstown,OH,USA
Keywords:Non-alcoholic fatty liver disease (NAFLD)Liver fibrosis Takeda G protein-coupled receptor 5(TGR5)Serotonin (5-HT)High fat,high fructose,and high sucrose(HFS)
ABSTRACT Background and aims: Diet-induced obesity and metabolic syndrome can trigger the progression of fatty liver disease to non-alcoholic steatohepatitis and fibrosis,which is a major public health concern.Bile acids regulate metabolic homeostasis and inflammation in the liver and gut via the activation of nuclear farnesoid X receptor(Fxr)and the membrane receptor Takeda G protein-coupled receptor 5 (Tgr5).Tgr5 is highly expressed in the gut and skeletal muscle,and in cholangiocytes and Kupffer cells of the liver.Tgr5 is implicated in the mediation of liver and gut inflammation,as well as the maintenance of energy homeostasis.Here,we used a high fat,high fructose,and high sucrose (HFS) diet to determine how bile acid signaling through Tgr5 may regulate metabolism during the progression from fatty liver to nonalcoholic steatohepatitis and fibrosis.Materials and methods: Female C57BL/6J control wild type (WT) and Tgr5 knockout (Tgr5-/-) mice were fed HFS (high fat (40% kcal),high fructose,and 20% sucrose water) diet for 20 weeks.Metabolic phenotypes were characterized through examination of bile acid synthesis pathways,lipid and cholesterol metabolism pathways,and fibrosis and inflammation pathways.Results:Tgr5-/-mice were more glucose intolerant when fed HFS diet,despite gaining the same amount of weight as WT mice.Tgr5-/-mice accumulated significantly more hepatic cholesterol and triglycerides on HFS diet compared to WT mice,and gene expression of lipogenic genes was significantly upregulated.Hepatic cholesterol 7alpha-hydroxylase (Cyp7a1) gene expression was consistently elevated in Tgr5-/-mice,while oxysterol 7alpha-hydroxylase (Cyp7b1),sterol 27-hydroxylase (Cyp27a1), Fxr,and small heterodimer partner (Shp) were downregulated by HFS diet.Surprisingly,hepatic inflammation and fibrosis were also significantly reduced in Tgr5-/- mice fed HFS diet,which may be due to altered serotonin signaling in the liver.Conclusions: Tgr5-/-mice may be protected from high fat,high sugar-induced hepatic inflammation and injury due to altered serotonin metabolism.
Bile acids are physiological detergents that aid in lipid and nutrient absorption in the intestine.In hepatocytes,bile acid synthesis is initiated by the rate-limiting enzyme cholesterol 7alphahydroxylase (Cyp7a1),which uses cholesterol as its substrate.The branch-point enzyme sterol 12alpha-hydroxylase (Cyp8b1) determines the ratio of cholic acid (CA) and chenodeoxycholic acid(CDCA),the two main primary bile acids synthesized in humans.In addition to their roles in nutrient absorption,bile acids also mediate hepatic glucose,lipid and bile acid homeostasis by binding to receptors,including farnesoid X receptor (Fxr) and Takeda G protein-coupled receptor 5 (Tgr5).In the liver,bile acids bind Fxr and induce small heterodimer partner (Shp) to inhibit Cyp7a1 transcription and bile acid synthesis in a negative feedback loop.Fxr also induces bile salt export pump(Bsep)to promote export of bile acids from hepatocytes to the biliary system,protecting the liver from toxic bile acid accumulation.In enterocytes,bile acids again bind Fxr,which induces the release of fibroblast growth factor 15 (Fgf15,human ortholog FGF19) into portal circulation.Fgf15/19 activates the hepatic membrane FGF receptor 4 (Fgfr4)/beta (β)-Klotho complex to further inhibitCyp7a1gene transcription.
Bile acids also bind Tgr5,which is expressed widely throughout various tissues,but not in hepatocytes.In the ileum and colon,secondary bile acids produced by the gut microbiome bind Tgr5 to induce glucagon-like peptide 1 secretion from enterocytes,which in turn induces pancreatic insulin release.1Tgr5 induces gallbladder filling and adipose tissue browning,2,3and plays a role in mediating inflammation via inhibition of nuclear factor-kappaB (NF-kB) and induction of nitric oxide production.4,5Both Fxr and Tgr5 are drug targets for the treatment of non-alcoholic steatohepatitis (NASH)and cholestatic liver diseases.6
Non-alcoholic fatty liver disease(NAFLD)includes a spectrum of liver disorders from simple steatosis to inflammatory NASH,which can progress to liver fibrosis,cirrhosis,and hepatocellular carcinoma,the end stage of liver disease.NAFLD is estimated to occur in about 25%of the global population.7Metabolic syndrome presents a significant risk factor for the development of NAFLD,and patients with type 2 diabetes have a 2-fold increased risk for NAFLD over the general population.8The progression of NAFLD to NASH likely involves multiple hepatic insults,including insulin resistance and diabetes,increased hepatic lipid content and lipid oxidation,and oxidative stress.The severity of NASH is significantly associated with cardiovascular disease,chronic kidney disease,and extrahepatic cancer,9making NAFLD/NASH treatment a critical checkpoint for preventing further disease progression.Most studies of experimental NASH utilize male mice,though in recent years NAFLD prevalence has increased in women,10and NAFLD-related deaths have significantly increased in women.11For these reasons,we aimed to study bile acid signaling and the role of Tgr5 in the development of NAFLD and progression to NASH by utilizing a high fat,high fructose,and high sucrose (HFS) diet in female Tgr5 knockout(Tgr5-/-) mice.
All animal experiments were approved by the Institutional Animal Care and Use Committee of Northeast Ohio Medical University.
Female C57BL/6J wild type (WT;originally from The Jackson Laboratory,Bar Harbor,ME,USA)andTgr5-/-mice(originally from Merck,Rahway,NJ,USA)were bred in-house and were maintained on standard control diet(Ctl;LabDiet#5008,16%kcal from fat)and tap water or HFS (high fat,high fructose diet (Research Diets#D09100310,40% kcal from fat,20% kcal from fructose,2%cholesterol),and 20% sucrose in water) for 20 weeks.Glucose tolerance was determined using a OneTouch Ultra glucometer after an overnight fast followed by intraperitoneal (i.p.) injection of glucose (2 mg/kg).Indirect calorimetry was performed in mice maintained on HFS diet using a comprehensive lab animal monitoring system(CLAMS Oxymax,Columbus Instruments,Columbus,OH,USA).Food and water were availablead libitumand mice were housed in a temperature-controlled facility with a 12 h light and 12 h dark cycle.Mice were sacrificed in the fed state from approximately 9-11 am.
RNA was isolated from tissues using Trizol and cDNA was synthesized using 2 μg RNA.Taqman probes/primers (ThermoFisher,Waltham,MA,USA) were used to determine relative mRNA expression levels using the ddCt method against Gapdh as an internal control.
Protein was isolated from tissue using T-Per(ThermoFisher)and Western blotting was performed by loading 20 μg total protein or microsomal protein into 10%TGX gels(BioRad,Hercules,CA,USA).Blots were incubated with antibodies against phospho-forkhead box protein O1 (p-FoxO1;Cell Signaling Technology,Danvers,MA,USA,#9461S),FoxO1 (Cell Signaling Technology,#2880S),phospho-Akt (Ser473) (p-Akt;Cell Signaling Technology,#4060S)and Akt (Cell Signaling Technology,#4685) overnight and were imaged on a GBox Mini 6 Gel Documentation System (Syngene,Frederick,MD,USA).
The liver was fixed in 10% formalin overnight and was then paraffin processed and sectioned at 9 μm for Sirius red staining.Immunohistochemical staining for cytokeratin-19 (CK-19;Abcam,Waltham,MA,USA,#ab52625)and monoamine oxidase A(Mao-A;Abcam,#ab126751) was performed on 9 μm paraffin-embedded sections using citrate buffer antigen retrieval and DAB chromagen(SignalStain,Cell Signaling Technology).Sections were visualized using an Olympus IX73 inverted microscope and images were captured using Olympus cellSens Dimension software (scale bar=100 μm).Representative images are shown (n=3);percent area stained was quantified(n=6)for Sirius red,CK-19,and Mao-A using threshold detection in ImageJ (NIH).
To isolate lipids,liver and fecal samples were homogenized in PBS.Seven hundred microliters chloroform: isopropanol: NP40(7:11:0.1 v/v ratio) was added and samples were incubated in a sonicating water bath for 1 h.Samples were centrifuged at 8800×gand the organic layer was extracted and evaporated at 55°C.The remaining lipids were dissolved in 200 μL cholesterol assay buffer(BioVision,Waltham,MA,USA).Lipids were quantified in liver,feces and serum using commercially available kits (cholesterol &triglycerides: ThermoFisher #TR13421,#TR22421,free fatty acids:Fujifilm Wako,Richmond,VA,USA).Liver hydroxyproline (Bio-Vision),malondialdehyde(MDA;Cayman Chemical,Ann Arbor,MI,USA),and glutathione peroxidase (GPx;BioVision) activity were quantified using kits.
Bile acids were extracted by homogenizing 100 mg feces or frozen liver tissue,whole intestine,or whole gallbladder in ice-cold acetonitrile: methanol (1:1 v/v).Samples were incubated in a sonicating water bath for 1 h and bile acids were quantified using a kit (Diazyme,Poway,CA,USA).Bile acids were quantified directly from serum.Bile acid pool was determined by summing the backcalculation of total liver bile acids,total intestinal bile acids,and total gallbladder bile acids.
Serum 7alpha-hydroxy-4-cholesten-3-one (C4) was quantified in mouse serum using an ultra performance liquid chromatography-mass spectrometry(UPLC-MS)/MS-based parallel reaction monitoring (PRM) assay.Briefly,20 μL mouse serum was mixed with 80 μL internal standard containing 50 ng/mL C4-d7 in methanol to precipitate proteins.The mixture was vortexed and centrifuged at 17,000×gfor 15 min at 4°C.Eighty microliters of the supernatant was transferred to a clean sample vial for UPLC-MS/MS analysis.UPLC-MS/MS assay was carried out on a Vanquish UPLC system coupled with a Q-Exactive Orbitrap mass spectrometry(ThermoFisher).UPLC separation was performed by an Accucore Vanquish C18 Column (50 mm × 2.1 mm,1.5 μm,ThermoFisher)using 10 μL sample.The mobile phases consisted of water containing 0.1% formic acid and 5 mM ammonium acetate as phase A and methanol containing 0.1% formic acid and 5 mM ammonium acetate as phase B.The mass spectrometer was operated on PRM mode to monitor all product ions of 401.3414 (C4) and 408.3853(C4-d7) at the collision energy of 60 eV.Representativem/ztransitions of C4(401.3414>97.0647)and C4-d7(408.3853>97.0647)were analyzed using Skyline software (University of Washington,Seattle,WA,USA),and serum C4 concentrations was determined with calibrators ranging from 3 ng/mL to 1 μg/mL.
Macrophages were derived from bone marrow of WT andTgr5-/-mice fed HFS diet for 20 weeks (n=3).Tibia and femur were collected,and bone marrow cells were harvested by flushing bones with cold RPMI media (supplemented with 10% FBS,5% nonessential amino acids,1% pen-strep,and 50 μM β-mercaptoethanol).Bone marrow cells were plated in the same media warmed to 37°C and were then differentiated into BMDMs using 10 ng/mL macrophage colony-stimulating factor (m-CSF).After 3 days,cells were replenished with fresh media containing 10 ng/mL m-CSF and cells were incubated for an additional 3 days.On day 6,mature BMDMs were stimulated with lipopolysaccharide (LPS;100 ng/mL,6 h) or interleukin-4 (IL-4;20 ng/mL,24 h) to induce M1 or M2 polarization,respectively.After stimulation,cells were harvested with Trizol for RNA isolation and quantitative real-time polymerase chain reaction(qPCR).
Statistical significance between two groups was determined using a two-tailed Student"st-test;statistical significance between multiple groups was determined using a two-way analysis of variance (ANOVA) followed by a Tukey multiple comparison post hoc test where appropriate.Data were analyzed using Prism(GraphPad Software,San Diego,CA,USA) and are presented as mean±standard error of the mean(SEM),withP<0.05 considered statistically significant.
FemaleTgr5-/-mice maintained on HFS diet for 20 weeks gained weight similarly to female WT mice (Fig.1A) and composition of fat and lean tissue mass did not differ between groups(Fig.1B),nor did average food intake (Fig.1C).Liver weight was significantly induced by HFS diet in both WT andTgr5-/-mice,though Tgr5-HFS livers were significantly smaller than WT livers in both the wet state and when normalized to body weight (Fig.1D).Mice maintained on control diet exhibited similar responses to glucose tolerance test(GTT;Fig.2A);however,Tgr5-HFS mice were more glucose intolerant compared to WT-HFS mice(Fig.2B).Serum insulin at the time of sacrifice was unchanged by genotype or diet(Fig.2C),despite significant reductions in mRNA expression of key genes involved in insulin metabolism including insulin receptor(Insr) and insulin-degrading enzyme (Ide) in Tgr5-HFS mice(Fig.2D).Protein analysis by Western blotting indicated significant reductions in the p-Akt:Akt ratio in Tgr5-Ctl mice,as well as significantly reduced p-FoxO1:FoxO1(Fig.2E).Additionally,hepatic genes involved in glucose and carbohydrate metabolism were investigated.Hepatic expression of glucose-6-phosphatase (G6pc)was significantly reduced in WT-HFS mice compared to WT-Ctl mice,while glucokinase (Gck) was significantly upregulated in WT-HFS mice compared to mice fed control diet(Fig.2F),consistent with the roles of these enzymes in gluconeogenesis and glycolysis,respectively.Tgr5-HFS mice did not exhibit alterations in either of these genes,consistent with the glucose intolerance observed in these mice.Expression of glucose transporter 2(Glut2)in the liver,ketohexokinase (Khk),responsible for the phosphorylation of fructose,and fructose-1,6-bisphosphatase 1(Fbp1)was unchanged across both genotypes and diets (Fig.2F).Expression of glycogen synthase 1 (Gys1) was significantly induced in WT-HFS but not in Tgr5-HFS mice,while glycogen phosphorylase (Pygl) and glycogen synthase kinase 3 beta (Gsk3b) were unchanged (Fig.2F).The luminal glucose transporterGlut5,which functions as a fructose transporter in the intestine,was unchanged,while expression of the ileal serosal glucose transporterGlut2trended increased in Tgr5-HFS mice but was significantly induced only in WT-HFS mice(Fig.2G).Lastly,skeletal muscle was examined for alterations in glucose metabolism.Genes involved in glucose phosphorylation(Hk1,Khk),gluconeogenesis(Pepck),and insulin-dependent glucose transport type 4 (Glut4) were unchanged by genotype or diet(Fig.2H),suggesting the glucose intolerance observed in Tgr5-HFS mice is independent of glycolysis and gluconeogenesis in myocytes.These data indicate thatTgr5-/-mice did not metabolically adapt to HFS diet,resulting in impaired glucose tolerance.
Fig.1.Female WT and Tgr5-/- mice maintained on a high fat,high fructose,and high sucrose water (HFS) diet for 20 weeks.(A) Growth curve on HFS diet.(B) Body composition analysis.(C)Average food intake.(D)Wet liver weight and liver weight normalized to body weight(BW).n=8-10 per group.*indicates genotype effect(P<0.05);#indicates dietary effect (P <0.05).Abbreviations: Ctl,control diet;Tgr5,Takeda G protein-coupled receptor 5;WT,wild type.
Fig.2.Metabolic phenotyping of female WT and Tgr5-/-mice fed HFS diet.(A,B)Glucose tolerance tests(GTT).(C)Serum insulin.(D)Hepatic expression of insulin metabolism genes.(E)Hepatic expression of proteins involved in glucose and insulin metabolism.(F)Hepatic expression of carbohydrate metabolism genes.(G)Ileal expression of glucose and fructose transport genes.(H) Skeletal muscle expression of glucose metabolism genes. n=8-10 per group.* indicates P <0.05;for multiple group comparisons,* indicates genotype effect(P<0.05),#indicates dietary effect(P<0.05).Abbreviations:Ctl,control diet;Fbp1,fructose-1,6-bisphosphatase 1;Gck,glucokinase;Glut,glucose transporter;G6pc,glucose-6-phosphatase;Gsk3b,glycogen synthase kinase 3 beta;Gys1,glycogen synthase 1;HFS,high fat,high fructose,and high sucrose water;Hk1,hexokinase 1;Ide,insulindegrading enzyme;Insr,insulin receptor;Irs1,insulin receptor substrate 1;Khk,ketohexokinase;Pepck,phosphoenolpyruvate carboxykinase;p-FoxO1,phospho-forkhead box protein O1;Pygl,glycogen phosphorylase;Tgr5,Takeda G protein-coupled receptor 5;WT,wild type.
Indirect calorimetry experiments indicated that metabolic respiration was altered in Tgr5-HFS mice.Oxygen consumption(VO2) was significantly reduced in Tgr5-HFS mice (Fig.3A) while carbon dioxide production (VCO2) was unchanged (Fig.3B).Respiratory exchange ratio(RER),the ratio of VCO2/VO2,can be used as a marker of metabolic fuel preference,and trended increased in Tgr5-HFS mice (Fig.3C).An RER of approximately 0.7 indicates near-total fat usage,while an RER of 1.0 indicates total glucose usage.12RER was 0.73 in WT-HFS mice,while Tgr5-HFS mice had an RER of 0.77,indicating that Tgr5-HFS mice may be preferentially utilizing glucose over fat as fuel.Lastly,these effects in oxygen consumption and fuel usage were not due to differences in daily locomotor activity between WT-HFS and Tgr5-HFS mice (Fig.3D).
Fig.3.Metabolic respiration in female WT and Tgr5-/-mice fed HFS diet.(A)Oxygen consumption(VO2).(B)Carbon dioxide production(VCO2).(C)Respiratory exchange ratio(RER).(D)Locomotor activity.n=8-10 per group.*indicates P<0.05;black bar indicates the dark phase.Abbreviations:HFS,high fat,high fructose,and high sucrose water;Tgr5,Takeda G protein-coupled receptor 5;WT,wild type.
Liver cholesterol was significantly increased in Tgr5-HFS mice compared to WT-HFS and Tgr5-Ctl mice.Serum cholesterol was significantly increased by HFS diet in both WT andTgr5-/-mice,though this was reduced in Tgr5-HFS mice compared to WT-HFS mice (Fig.4A).Liver triglycerides were also significantly induced in Tgr5-HFS mice compared to WT-HFS mice,while serum triglycerides were elevated in Tgr5-Ctl mice and were then reduced after HFS feeding.Fecal cholesterol and triglyceride excretion were elevated in both WT andTgr5-/-mice fed HFS(Fig.4A and B).Hepatic free fatty acids were significantly elevated by HFS diet only inTgr5-/-mice,while serum free fatty acids were reduced inTgr5-/-mice fed HFS compared to WT mice fed the same diet.Overall,fecal free fatty acid excretion was significantly elevated in both groups fed HFS diet(Fig.4C).Expression of sterol regulatory element binding protein 1(Srebp1) was unchanged,while expression ofSrebp2,involved in cholesterol synthesis,was suppressed by HFS diet in WT mice only(Fig.4D).Consistent with high fructose feeding,13hepatic expressions of genes involved inde novolipogenesis were significantly upregulated in Tgr5-HFS mice,including acetyl-CoA carboxylase(Acc) and fatty acid synthase (Fasn) (Fig.4D).Expressions of genes involved in β-oxidation of fatty acids,including acyl-CoA dehydrogenase medium chain (Acadm) and long chain (Acadl),were significantly reduced in Tgr5-HFS,as was expression of fatty acid binding protein (Fabp1),while cluster of differentiation 36 (Cd36),also known as fatty acid translocase,was elevated in WT-HFS and reduced in Tgr5-HFS mice(Fig.4D).Fxr regulates hepatic lipogenesis and lipid metabolism.14Therefore,we investigated the expression of Fxr target genes after HFS feeding.Expressions of angiopoietin-like 3(Angptl3),scavenger receptor class B1(Srb1),microsomal triglyceride transfer protein (Mttp),and peroxisome proliferator-activated receptor alpha(Pparα)were unchanged by diet or lack ofTgr5(Fig.4E).These results may indicate that lipids are sequestered to the liver during HFS exposure inTgr5-/-mice,likely in an Fxr-independent fashion.
Fig.4.Tgr5-/- mice have increased liver lipids after HFS diet feeding.(A) Cholesterol (Chol) quantified in liver,serum,and feces.(B) Triglycerides (TG) quantified in the liver,serum,and feces.(C) Free fatty acids (FFA) quantified in the liver,serum,and feces.(D) Hepatic expression of lipid synthesis and metabolism genes.(E) Hepatic expression of Fxr target genes involved in lipogenesis.n=8-10 per group.*indicates genotype effect (P<0.05),#indicates dietary effect(P<0.05).Abbreviations:Acadl,acyl-CoA dehydrogenase long chain;Acadm,acyl-CoA dehydrogenase medium chain;Acc,acetyl-CoA carboxylase;Angptl3,angiopoietin-like 3;Cd36,cluster of differentiation 36;Ctl,control diet;Fabp1,fatty acid binding protein 1;Fasn,fatty acid synthase;HFS,high fat,high fructose,and high sucrose water;Ldlr,low-density lipoprotein receptor;Mttp,microsomal triglyceride transfer protein;Pparα,peroxisome proliferator-activated receptor alpha;Srb1,scavenger receptor class B1;Srebp,sterol regulatory element binding protein;Tgr5,Takeda G protein-coupled receptor 5;WT,wild type.
Bile acids were quantified in tissues,serum,and feces of mice fed control or HFS diet.HFS diet elevated serum bile acids in WT mice,while serum bile acids were significantly reduced in Tgr5-HFS mice compared to WT-HFS mice (Fig.5A).Gallbladder bile acids were ubiquitously reduced inTgr5-/-mice,consistent with the role of Tgr5 in mediating gallbladder filling (Fig.5B).2Liver and intestinal bile acid content was unchanged regardless of genotype or diet (Fig.5C and D),and fecal bile acid excretion was significantly upregulated by HFS diet in both WT andTgr5-/-mice (Fig.5E).Overall,total bile acid pool size was unchanged (Fig.5F).Interestingly,mRNA expression ofCyp7a1,the rate-limiting enzyme for bile acid synthesis,was consistently elevated inTgr5-/-mice compared to WT mice (Fig.5G).Cyp8b1,responsible for CA synthesis,was unchanged,while oxysterol 7alpha-hydroxylase (Cyp7b1),sterol 27-hydroxylase (Cyp27a1),andFxrgene expressions were suppressed in both WT andTgr5-/-fed HFS diet.24-Hydroxycholesterol 7alpha-hydroxylase (Cyp39a1) was significantly reduced in Tgr5-HFS mice only.Shp,a negative regulator ofCyp7a1and a target gene of Fxr,was significantly suppressed in Tgr5-Ctl mice and by HFS diet in WT andTgr5-/-mice (Fig.5F),consistent with upregulatedCyp7a1gene expression inTgr5-/-mice.Serum C4,a biomarker of Cyp7a1 activity and bile acid synthesis,was significantly upregulated in Tgr5-Ctl mice(Fig.5H),also consistent with upregulatedCyp7a1expression,and remained elevated in Tgr5-HFS mice (Fig.5H).Overall,mRNA expression of Fxr targets did not change in the ileum.The negative regulators of Cyp7a1,ShpandFgf15,were not significantly increased,nor was ileal bile acid binding protein(Ibabp),while intestinal expression of the bile acid export protein organic solute transporter beta (Ostb)was significantly reduced only in WT-HFS mice(Fig.5I).These data indicate that HFS diet may induce a shift towards increased export or leakage of bile acids into serum,consistent with reports of elevated serum bile acids in patients with NASH,15,16whileTgr5-/-mice may be protected from this phenomenon.
Fig.5.Bile acid (BA) homeostasis is altered in Tgr5-HFS mice.BAs were quantified in (A) serum,(B) gallbladder,(C) liver,(D) intestine,and (E) feces.(F) BA pool.(G) Hepatic expression of genes involved in BA metabolism.(H)Serum 7alpha-hydroxy-4-cholesten-3-one(C4).(I)Ileal expression of BA metabolism and transport genes.n=8-10 per group.*indicates genotype effect (P <0.05),# indicates dietary effect (P <0.05).Abbreviations: Asbt,apical sodium dependent bile acid transporter;Ctl,control diet;Cyp7a1,cholesterol 7alpha-hydroxylase;Cyp7b1,oxysterol 7alpha-hydroxylase;Cyp8b1,sterol 12alpha-hydroxylase;Cyp27a1,sterol 27-hydroxylase;Cyp39a1,24-hydroxycholesterol 7alpha-hydroxylase;Fgf15,fibroblast growth factor 15;Fxr,farnesoid X receptor;HFS,high fat,high fructose,and high sucrose water;Ibabp,ileal bile acid binding protein;μM,μmol/L;Ost,organic solute transporter;Shp,small heterodimer partner;Tgr5,Takeda G protein-coupled receptor 5;WT,wild type.
Liver tissue was stained for collagen using Sirius red,and representative samples are shown in Fig.6.Tgr5-HFS mice have significantly reduced collagen staining compared to WT-HFS mice(quantified inn=6 mice).CK-19,a marker of cholangiocyte proliferation,was increased by HFS feeding in WT mouse liver,while Tgr5-HFS mice had significantly reduced CK-19 and cholangiocytes (quantified inn=6 mice;Fig.7).Liver hydroxyproline,the major amino acid component of collagen,was significantly elevated in both groups fed HFS diet,thoughTgr5-/-mice had significantly less hydroxyproline compared to WT mice (Fig.8A).Liver MDA,a product of lipid peroxidation,was significantly elevated in Tgr5-HFS mice (Fig.8B),consistent with elevated free fatty acid content in the liver.GPx,responsible for detoxifying hydrogen and lipid peroxides,was significantly elevated by HFS in both groups of mice (Fig.8C).Expression of genes involved in fibrosis and collagen deposition was then analyzed in the liver.Tgr5-HFS mice had reduced expression of tissue inhibitor of matrix metalloproteinase 1 (Timp1),matrix metalloproteinase 13 and 9(Mmp13andMmp9)and collagen type I alpha 1 and alpha 2(Col1a1andCol1a2)(Fig.8D).Tgr5-HFS mice also had significantly reduced expression of smooth muscle actin (Sma),transforming growth factor beta(Tgfb),andTimp2,while expression of arginase 1(Arg1),an M2 macrophage and antiinflammation marker,was significantly reduced in both WT-HFS and Tgr5-HFS mice (Fig.8D).Tgr5-HFS mice had significantly reduced mRNA expression of inflammatory mediators in liver tissue.IL-6,IL-1b,Toll-like receptor 2(Tlr2),Tlr4,lipocalin 2 (Lcn2),and tumor necrosis factor alpha (Tnfα) were significantly reduced inTgr5-/-mice fed HFS diet (Fig.8E).Expressions of platelet-derived growth factor(Pdgf),a pro-fibrogenic marker of stellate cell activation,17and lipoprotein lipase (Lpl),an enzyme found to be upregulated in patients with NASH and in NASH mouse models,18,19were both significantly upregulated in WT-HFS mice and significantly reduced in Tgr5-HFS mice(Fig.8E).
To further explore the phenomenon by which steatosis may uncouple from fibrosis pathogenesis in Tgr5-HFS mice,BMDMs were isolated from WT andTgr5-/-mice fed HFS diet for 20 weeks.After stimulation with IL-4 to induce M2 polarization,BMDM isolated fromTgr5-/-mice tended to express reducedTimp1andCol1a1and demonstrated significantly reduced expression ofSma(Fig.8F).When stimulated with LPS to induce M1 polarization,BMDM fromTgr5-/-mice expressed significantly increased levels ofIL-1bregardless of diet,while expression ofIL-6was significantly reduced in BMDM from Tgr5-HFS mice(Fig.8G).Overall,these data indicate that lack of Tgr5 may be protective against the development of high fat,high sugar-induced liver fibrosis,inflammation,and injury.
Fig.6.Reduced liver fibrosis in Tgr5-HFS mice. Sirius red staining in the liver shown in n=3 representative mice.Staining quantified in n=6 per group.Scale bar=100 μm.*indicates genotype effect(P<0.05),#indicates dietary effect(P<0.05).Abbreviations:Ctl,control diet;HFS,high fat,high fructose,and high sucrose water;Tgr5,Takeda G proteincoupled receptor 5;WT,wild type.
5-HT signaling has been implicated in mediating or exacerbating hepatic fibrosis,while inhibition of select 5-HT receptors may be protective.20Therefore,mediators of 5-HT signaling were investigated in HFS-fed mice to determine the mechanism by whichTgr5-/-mice may be protected against fibrotic progression.5-HT synthesis,initiated by the rate-limiting enzyme tryptophan hydroxylase(Tph),occurs primarily in the gut(mediated byTph1)while a small amount(10%) is synthesized in the brain byTph2.More recent studies indicate Tph1 is also expressed in the HepG2 cell line and in mouse liver.21GutTph1expression was not significantly changed by lack ofTgr5or by HFS diet (Fig.9A),indicating 5-HT synthesis may be unaltered in these mice.5-HT is metabolized primarily to 5-hydroxyindoleacetic acid byMaoa.Hepatic expression ofMaoawas significantly reduced in Tgr5-HFS compared to mice fed control diet and WT-HFS mice,whileMaobwas significantly reduced in both groups fed HFS diet (Fig.9B).Hepatic expression of the serotonin transporter(Sert)gene was unchanged.The expression of several 5-HT receptors implicated in the mediation of hepatic fibrosis were examined.Serotonin receptor 1b(Htr1b)was increased in Tgr5-HFS mice compared to WT-HFS mice.Expressions ofHtr2aandHtr2b,implicated in the development of hepatic fibrosis,were reduced in Tgr5-HFS mice(Fig.9C).Finally,liver sections were stained for Mao-A and content was quantified(Fig.10).These data indicate that altered 5-HT metabolism in the liver,including reduced processing by Mao-A and reduced signaling via fibrogenic 5-HT receptors,may mediate protection against diet-induced fibrosis inTgr5-/-mice.
Fig.7.Reduced liver cytokeratin-19 (CK-19) in Tgr5-HFS mice. CK-19 staining in liver shown in n=3 representative mice.Staining quantified in n=6 per group.Scale bar=100 μm.*indicates genotype effect(P<0.05),#indicates dietary effect(P<0.05).Abbreviations:Ctl,control diet;HFS,high fat,high fructose,and high sucrose water;Tgr5,Takeda G protein-coupled receptor 5;WT,wild type.
NAFLD,and its progression to NASH/fibrosis,represents a global health concern for which there are no Food and Drug Administration (FDA)-approved treatments.NAFLD and NASH may involve sex-specific differences in presentation and outcome.Despite prevalence of NAFLD being lower in women compared to men,22one study demonstrated a 1.5-fold increase in all-cause mortality in female patients with NAFLD compared to male patients.23Here,we present data implicating Tgr5 signaling in the development of NAFLD and fibrosis in female mice.A high fat,high fructose,and high cholesterol diet induced hepatic inflammation and fibrosis in WT mice,whileTgr5-/-mice were protected.Tgr5-/-mice had increased liver cholesterol,triglycerides,and free fatty acids compared to WT control mice.Expressions ofFasnandAccwere also significantly upregulated inTgr5-/-mice,indicative of fructose-inducedde novolipogenesis.13Additionally,the fatty acid transporter Cd36 was significantly induced by HFS diet in control mice and was comparatively and significantly reduced in Tgr5-HFS mice.Both diet-induced and adenoviral-mediated increases in Cd36 were sufficient to exacerbate fatty liver disease,24indicating that reduced expression ofCd36in Tgr5-HFS mice may mediate some protection against fat-induced liver disease.Tgr5-HFS mice were also more glucose intolerant compared to WT-HFS mice.Tgr5-/-mice have reduced hepatic expression of Ide,a metalloproteinase that degrades insulin,glucagon,amylin,β-amylase,and other β-sheet-containing molecules.As was observed in Tgr5-HFS mice,liver-specific deletion ofIdein mice resulted in glucose intolerance,reduced expression of insulin receptor in the liver,and unaltered serum insulin,similar to control mice.25Liver-specificIdeknockout mice also had reduced expression of Akt and upregulation ofPepckandG6pc,which was not observed in Tgr5-HFS mice.The mechanistic origin of reduced Ide inTgr5-/-mice requires further investigation.
Our lab previously reported that maleTgr5-/-mice had reduced mRNA expression ofCyp7b1,whileCyp7a1was not different between WT andTgr5-/-mice.26Here,we demonstrate that femaleTgr5-/-mice have increasedCyp7a1expression while there was no change inCyp7b1expression between genotypes.Several cytochrome P450 enzymes exhibit sexually dimorphic expression,includingCyp7b1,which was reduced in embryonic and adult female mice compared to male mice.27,28Another study reported reduced expression ofCyp7a1inTgr5-/-mice,which was significantly induced by lithogenic diet.29Therefore,both sex and diet may be involved in the regulation of bile acid synthesis gene expression.Tgr5-/-mice also have reduced gallbladder volume and reduced bile flow.2Our data indicating reduced bile acid content in the gallbladders ofTgr5-/-mice confirm this finding,while increased liver cholesterol,increasedCyp7a1expression,and reducedShpexpression may be a compensatory response.Serum bile acids represent about 1%of the total bile acids present in mice under normal conditions,and may increase with cholestasis or other forms of liver injury.30Serum bile acids were significantly increased by HFS diet in WT mice only,indicatingTgr5-/-mice maybe protected from toxic circulating bile acids.Further analysis of bile acid composition,which may shift to a less hepatotoxic profile in the progression from NAFLD to NASH,31will lead to insight on the role of Tgr5 as a bile acid receptor in mediating the pathogenesis of NASH.
Fig.8.Reduced collagen and fibrogenic markers in Tgr5-HFS mice.(A)Liver hydroxyproline.(B)Liver malondialdehyde(MDA).(C)Liver glutathione peroxidase(GPx)activity.(D)Hepatic expression of genes involved in collagen synthesis and regulation of the extracellular matrix.(E) Hepatic gene expression of inflammatory and pro-fibrotic markers.(F)Expression of fibrotic genes in bone marrow-derived macrophage (BMDM) stimulated in an M2 state.(G) Expression of inflammatory genes in BMDM stimulated in an M1 state.(A-E)n=8-10 per group;(F,G)n=3 per group.*indicates genotype effect(P<0.05),#indicates dietary effect(P<0.05).Abbreviations:Arg1,arginase 1;Col1a1,collagen type I alpha 1;Ctl,control diet;HFS,high fat,high fructose,and high sucrose water;IL,interleukin;Lcn2,lipocalin 2;Lpl,lipoprotein lipase;Mmp,matrix metalloproteinase;Pdgf,plateletderived growth factor;Sma,smooth muscle actin;Tgfb,transforming growth factor beta;Tgr5,Takeda G protein-coupled receptor 5;Timp,tissue inhibitor of matrix metalloproteinase;Tlr,Toll-like receptor;Tnfα,tumor necrosis factor alpha;WT,wild type.
Fig.9.Altered serotonin metabolism may protect Tgr5-HFS mice against liver fibrosis. (A) Ileal expression of serotonin synthesis gene tryptophan hydroxylase 1 (Tph1).(B)Hepatic expression of serotonin metabolism genes.(C)Hepatic expression of serotonin receptors implicated in mediating hepatic fibrosis.n=8-10 per group.*indicates genotype effect(P<0.05),#indicates dietary effect(P<0.05).Abbreviations:Ctl,control diet;HFS,high fat,high fructose,and high sucrose water;Htr,serotonin receptor;Maoa,monoamine oxidase A;Sert,serotonin transporter;Tgr5,Takeda G protein-coupled receptor 5;WT,wild type.
Fig.10.Reduced monoamine oxidase A (Mao-A) in Tgr5-HFS mice. Mao-A staining in liver shown in n=3 representative mice.Staining quantified in n=6 per group.Scale bar=100 μm.*indicates genotype effect(P<0.05),#indicates dietary effect(P<0.05).Abbreviations:Ctl,control diet;HFS,high fat,high fructose,and high sucrose water;Tgr5,Takeda G protein-coupled receptor 5;WT,wild type.
Development of hepatic fibrosis is mainly directed by hepatic stellate cells (HSCs),the resident mesenchymal cells normally responsible for formation and maintenance of basement membrane structures.32Under fibrogenic conditions,including in response to cytokines,reactive oxygen species,and lipid peroxidation,quiescent HSCs become activated and transition into proliferative and contractile myofibroblasts that secrete collagen and other components of the extracellular matrix.Under persistent activated conditions,HSCs will continue to deposit collagen and scar tissue that can ultimately impede liver function through reduced elasticity and impaired blood flow.33Tgr5-/-mice fed HFS diet had reduced liver collagen content,as detected by Sirius red staining and hydroxyproline quantification.Likewise,expressions of key genes involved in the formation of collagen and regulation of the ECM were significantly reduced inTgr5-/-fed HFS diet.Col1a1 may be the most abundant scar component,and knockdown ofCol1a1resulted in significant reduction in inflammatory responses as well as reduced gene expression of other collagen types.34Lastly,data from BMDMs,representative of macrophages recruited to the site of injury during hepatic fibrosis and inflammation,confirm that Tgr5-HFS mice have reduced expression of genes involved in collagen formation and fibrogenesis.Coupled with this,increased expression ofIL-1band reduced expression ofIL-6 in Tgr5-HFS mice may promote a cellular environment that favorably reduces fibrogenesis.Considering that macrophage polarization in either normal or disease states may be more plastic than static,35further studies should be conducted to determine how endogenous microenvironmental fluctuations regulate tissue remodeling homeostasis.
5-HT may play a mediating role in the development of fibrosis and in hepatic regeneration though activation of seven main receptor families (5-HT1-5-HT7),with considerable impact from the 5-HT2A/Bsubtypes.5-HT2Bantagonism improved cardiac and pulmonary fibrosis,36,37and silencing the 5-HT2Breceptor reducedCol1a1andSmagene and protein expressions in HSCs.38It was shown that 5-HT2Aantagonists reduced inflammation and HSC activation in fibrotic rats,39while the 5-HT2Breceptor subtype was identified as an HSC-specific cell marker in rat fibrotic liver.40Additionally,it was shown that 5-HT2Breceptors are required for the HSC-mediated inhibition of hepatic regeneration,and that 5-HT2Bantagonism reduced fibrogenesis and improved liver function in mice.41Another recent study demonstrated that 5-HT2A/B/Creceptors were upregulated in mouse models of cholangitis and in human patients with primary sclerosis cholangitis,and that antagonism of these receptors reduced fibrotic mRNA expression.42Here,we demonstrate that Tgr5-HFS mice have significantly reduced expression of bothMaoaand the 5-HT2Breceptor subtype,providing a possible mechanism for the reduced fibrogenic gene expression and collagen deposition in these mice.Our data also indicated that 5-HT synthesis may not be altered inTgr5-/-mice,thoughTph1expression was monitored only in ileal tissue,and studies indicateTph1is expressed widely throughout the enteroendocrine cells of the small intestine.Interestingly,Tgr5 was shown to regulate defecation via 5-HT release from enterochromaffin cells in the colon,and this response was blunted inTgr5-/-mice.43Understanding the mechanism by which Tgr5 influences serotonergic signaling with respect to the pathogenesis of liver fibrosis warrants study.
The bile acid receptor Tgr5 mediates inflammatory and metabolic signaling in the liver,gut,muscle,and brain.Here,we demonstrated that mice lacking Tgr5 were protected against the progression of NAFLD to NASH.Mechanistically,this may be due to altered 5-HT metabolism and signaling in the liver,which may protectTgr5-/-mice from diet-induced NASH-fibrosis.
Authors’ contributions
J.M.Ferrell designed experiments,performed experiments,analyzed data,and wrote the manuscript.M.Dilts performed experiments and analyzed data.Z.Stahl performed experiments and analyzed data.S.Boehme performed experiments and analyzed data.S.Pokhrel performed experiments and analyzed data.X.Wang performed experiments and analyzed data.J.Y.L.Chiang designed experiments and wrote the manuscript.
Declaration of competing interest
The authors declare that they have no conflicts of interest.
Acknowledgements
This work was supported by the USA National Institutes of Health (NIH) (AA015951,DK044442,and DK058379).
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