Dvid J.Mtye ,Xun Qin ,Mohmmd Nzmul Hsn ,Lijie Gu ,Yung Di Clyton ,Feng Li ,c ,Tingng Li ,*
a Harold Hamm Diabetes Center,Department of Physiology,University of Oklahoma Health Sciences Center,Oklahoma City,OK,USA
b Department of Pathology and Immunology,Baylor College of Medicine,Houston,TX,USA
c NMR and Drug Metabolism Core,Baylor College of Medicine,Houston,TX,USA
Keywords:Bile acids Farnesoid X receptor (FXR)Fibroblast growth factor 15 (FGF15)Apical sodium-bile acid transporter (ASBT)Non-alcoholic steatohepatitis (NASH)Liver fibrosis
ABSTRACT Background and aims: Several bile acids-based monotherapies have been developed for non-alcoholic steatohepatitis (NASH) treatment but clinical trial findings suggest that they do not satisfactorily improve NASH and liver fibrosis in many patients.Recently,we have shown that combining a gutrestricted apical sodium-bile acid transporter (ASBT) inhibitor GSK2330672 (GSK) with adenoassociated virus (AAV)-mediated liver fibroblast growth factor 15 (FGF15) overexpression provides significantly improved efficacy than either single treatment against NASH and liver fibrosis in a high fat,cholesterol,and fructose (HFCFr) diet-induced NASH mouse model.The beneficial effects of the combined treatment can be attributed to the markedly reduced bile acid pool that reduces liver bile acid burden and intestinal lipid absorption.The aim of this study is to further investigate if combining GSK treatment with the orally bioavailable obeticholic acid (OCA),which induces endogenous FGF15 and inhibits hepatic bile acid synthesis,can achieve similar anti-NASH effect as the GSK+AAV-FGF15 cotreatment in HFCFr-diet-fed mice.Materials and methods: Male C57BL/6J mice were fed HFCFr diet to induce NASH and liver fibrosis.The effect of GSK,OCA,and GSK+OCA treatments on NASH development was compared and contrasted among all groups.Results: Findings from this study showed that the GSK+OCA co-treatment did not cause persistent reduction of obesity over a 12-week treatment period.Neither single treatment nor the GSK +OCA cotreatment reduce hepatic steatosis,but all three treatments reduced hepatic inflammatory cytokines and fibrosis by a similar magnitude.The GSK+OCA co-treatment caused a higher degree of total bile acid pool reduction (～55%) than either GSK or OCA treatment alone.However,such bile acid pool reduction was insufficient to cause increased fecal lipid loss.The GSK+OCA co-treatment prevented GSK-mediated induction of hepatic cholesterol 7alpha-hydroxylase but failed to induce ileal FGF15 expression.GSK did not reduce gallbladder OCA amount in the GSK+OCA group compared to the OCA group,suggesting that ASBT inhibition does not reduce hepatic OCA distribution.Conclusions: Unlike the GSK+AAV-FGF15 co-treatment,the GSK +OCA co-treatment does not provide improved efficacy against NASH and liver fibrosis than either single treatment in mice.The lack of synergistic effect may be partly attributed to the moderate reduction of total bile acid pool and the lack of high level of FGF15 exposure as seen in the GSK+AAV-FGF15 co-treatment.
Non-alcoholic fatty liver disease (NAFLD) has become a major liver disease due to the increasing prevalence of obesity and type 2 diabetes worldwide.1Although most NAFLD patients with simple steatosis do not require treatment,some NAFLD patients progress to non-alcoholic steatohepatitis(NASH)that presents with hepatic inflammation,hepatocyte ballooning,and fibrosis that contribute to increased risk developing end-stage liver disease and liver cancers.Recent evidence suggests that NASH is a highly heterogenous disease with complex underlying causes.Unfortunately,there is still a lack of approved clinical treatment for NASH patients,and therefore an urgent need for developing safe and effective therapies to promote NASH resolution and reduce liver fibrosis.2
Bile acids are synthesized from cholesterol in hepatocytes and circulate between the liver and small intestine in a process called the enterohepatic circulation.3Bile acids are physiological detergents that facilitate dietary lipid absorption in the small intestine and signaling molecules that activate nuclear receptors and intracellular signaling pathways to regulate metabolic homeostasis.In recent years,therapeutic strategies targeting the bile acid transport and signaling have shown promise as potential NASH treatments.Bile acid homeostasis is maintained by a gut-liver bile acid feedback loop mediated by the nuclear receptor farnesoid X receptor (FXR)and endocrine hormone mouse fibroblast growth factor 15(FGF15)and its human ortholog FGF19.Bile acids activate hepatocyte FXR to inhibit the transcription of cholesterol 7alpha-hydroxylase(CYP7A1),the rate-limiting step inde novobile acid synthesis.4In addition,bile acids activate intestine FXR to induce FGF15/FGF19,which inhibits CYP7A1 via a gut-liver signaling axis.5FGF15 is not expressed in hepatocytes in mice,but human hepatocytes express and secrete FGF19 to inhibit CYP7A1 in an autocrine loop.3Studies have demonstrated that activation of FXR or FGF15/19 signaling attenuates obesity,hepatic steatosis,and insulin resistance in mouse models of NAFLD.6,7Based on these findings,FXR agonist,FGF19 analogue,and inhibitors of the apical sodium-bile acid transporter(ASBT)have been tested in clinical trials as therapeutics for treating NASH,but none of these monotherapies have been approved for clinical use due to either unsatisfactory efficacy or safety concerns.8-11
Emerging evidence suggests that human NASH is associated with elevated intrahepatic bile acids which contribute to hepatic inflammation and injury.12-16Human bile acid pool is more hydrophobic than mouse bile acid pool and can cause a higher degree of organelle stress,inflammatory infiltration,and stellate cell activation when accumulated in diseased livers.17,18Therefore,bile acid-based therapeutics that are associated with bile acid pool reduction may be beneficial in NASH.8,16However,inhibition of hepatic bile acid synthesis by FGF19 analogue or FXR agonist is associated with reduced fecal bile acid loss and therefore intestinal bile acid preservation,while inhibition of ASBT-mediated intestinal bile acid uptake causes induction of hepatic bile acid synthesis.These compensatory mechanisms limited the degree of bile acid pool reduction by therapeutic agents inhibiting hepatic bile acid synthesis or intestinal bile acid uptake.
Recently,we have shown that combining ASBT inhibitor with adeno-associated virus(AAV)-mediated FGF15 signaling activation caused a significantly higher degree of improved NASH and fibrosis than either monotherapy in high fat,cholesterol,and fructose(HFCFr) diet-fed mice.19This combination treatment caused a striking～90%reduction in total bile acid pool as a result of FGF15-mediated inhibition of compensatory induction of hepatic bile acid synthesis and higher fecal bile acid loss promoted by ASBT inhibitor,which reduced hepatic bile acid burden,intestinal cholesterol and fat absorption,and obesity.Given that FXR activation also inhibits hepatic bile acid synthesis and the FXR agonist obeticholic acid (OCA) is an approved orally bioavailable drug for treating human cholestasis,we further investigated the effects of combining ASBT inhibitor GSK2330672(GSK)and OCA on bile acid metabolism and NASH development in HFCFr diet-fed mice.
2.1.Ethical approval
All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Oklahoma Health Sciences Center.
2.2.Reagents
Aspartate aminotransferase(AST)and alanine aminotransferase(ALT) assay kits,total cholesterol assay kit,and triglyceride (TG)assay kit were purchased from Pointe Scientific (Canton,MI,USA).GSK and OCA were purchased from MedChemExpress LLC (Monmouth Junction,NJ,USA).Bile acid assay kit was purchased from Diazyme Laboratories(Poway,CA,USA).
2.3.Mice and treatments
Wild-type (WT) male C57BL/6J mice were purchased from The Jackson Laboratory(Bar Harbor,ME,USA).HFCFr diet contains 40%fat (20% w/w,mostly palm oil),2% cholesterol,and 20% fructose(D09100310,Research Diets,Inc.New Brunswick,NJ,USA).GSK and/or OCA were mixed with HFCFr diet(100 mg/kg diet)resulting in a～8 mg/kg body weight(BW)daily intake based on 4 g/day food intake in a 50 g mouse.Mice were housed in micro-isolator cages under 7 am-7 pm light cycle and 7 pm-7 am dark cycle.Average daily food intake was measured as previously described.19Mice were euthanized following a 6 h fast from 9 am to 3 pm.Animals received humane care according to the criteria outlined in the“Guide for the Care and Use of Laboratory Animals”.
2.4.Histological analysis
Liver tissues were fixed in 4% paraformaldehyde and paraffin embedded.Hematoxylin and eosin (H&E) staining was performed with an automated stainer.Sirius Red staining was performed with Direct Red 80 solution (Sigma#365548,St.Louis,MO,USA).Sirius Red positive area was measured using ImageJ software.
2.5.TG,cholesterol,bile acid,and OCA analysis
Lipid extraction was performed using a mixture of chloroform:methanol (2:1;v:v),dried under nitrogen,and resuspended in isopropanol containing 1% Triton X-100.Total cholesterol and TG were measured with assay kit following the manufacturer"s instruction.Bile acids were extracted from liver,whole gallbladder bile,whole small intestine with content,and dried feces in 90%ethanol.Total bile acid amount was measured by assay kit.Bile acid pool was calculated as the total bile acids in liver,gallbladder,and small intestine.OCA in bile samples was measured by liquid chromatography-mass spectrometry (LC-MS) method.
2.6.Real-time polymerase chain reaction (PCR)
Total RNA from the liver and terminal ileum was purified with Trizol (Sigma-Aldrich,St.Louis,MO,USA).Reverse transcription was performed with Oligo dT primer and SuperScript III reverse transcriptase (Thermo Fisher Scientific,Grand Island,NY,USA).Real-time PCR was performed on a Bio-Rad CFX384 Real-time PCR system with iQ SYBR Green Supermix(Bio-Rad,Hercules,CA,USA).GAPDH or 18S was measured as housekeeping gene and used for normalization.The comparative CT (Ct) method was used to determine the relative mRNA expression with the control group arbitrarily set as “1”.
2.7.Statistical analysis
All results were expressed as mean±standard error of the mean(SEM).One-way ANOVA followed by Tukey post hoc test or Student"st-test was used to calculate theP-value.AP-value<0.05 was considered statistically significant.
3.1.The GSK and OCA combined treatment does not reduce obesity or hepatic steatosis in HFCFr diet-fed mice
Male C57BL/6J mice were first fed an HFCFr diet for 16 weeks.These mice were then divided into 4 groups that were fed the HFCFr diet or a HFCFr diet containing GSK,OCA,or GSK+OCA for additional 12 weeks.Although the GSK+OCA treatment resulted in significant weight reduction compared to the non-treated control group at a few early time points,such effect was lost at the end of the 12 weeks treatment period(Fig.1A).GSK or OCA alone did not cause weight reduction compared to the control group over the 12 weeks treatment period(Fig.1A).Average daily food intake during the 12th week was similar among all groups (Fig.1B).Further analysis revealed that either GSK or OCA alone caused a～20%reduction of liver weight (LW) to BW ratio and the GSK+OCA treatment resulted in further decrease of LW to BW ratio (Fig.1C).However,hepatic TGs were not significantly reduced by any single or combined treatment(Fig.1D).Hepatic cholesterol accumulation was significantly lower in the GSK group,the OCA group,and the GSK+OCA group(Fig.1E).Liver histology revealed that livers of the OCA group and the GSK+OCA group showed reduced large lipid droplets and more hepatocytes exhibiting micro-steatosis despite having similar amount of hepatic TG as the control group(Fig.1F).The underlying cause and pathological significance of such histological changes are currently unclear.We suspect that reduction of other neutral lipids such as cholesterol ester may contribute to the reduced LW to BW ratio and moderately reduced lipid droplets accumulation on H&E slides in the treated groups.
Fig.1.The GSK and OCA combined treatment does not reduce obesity or hepatic steatosis in HFCFr diet-fed mice.Male C57BL/6J mice at 10 weeks of age were fed HFCFr for 16 weeks.Mice were then treated with GSK(～8 mg/kg/day),OCA(～8 mg/kg/day),or GSK+OCA co-treatment.After 12 weeks,mice were fasted for 6 h(9 am-3 pm)and euthanized.(A) Body weight (BW)during the treatment period.(B) Food intake.(C) Liver weight (LW) to BW ratio.(D) Liver TG content.(E) Liver cholesterol content.(F) Representative H&E stain of liver sections.Scale bar=250 μm.All results are expressed as mean±SEM(n=6-14 per group).*P<0.05 vs.control(Con)group unless indicated otherwise.Abbreviations:GSK,GSK2 330672;H&E,hematoxylin and eosin;HFCFr,high fat,cholesterol,and fructose;OCA,obeticholic acid;SEM,standard error of the mean;TG,triglyceride.
3.2.The GSK + OCA combined treatment does not cause further reduction of hepatic inflammation and fibrosis compared to GSK or OCA monotherapy in HFCFr diet-fed mice
Serum transaminases were modestly but significantly lower in the GSK group but not the OCA group or the GSK+OCA group compared to the control group (Fig.2A).Further measurement of hepatic pro-inflammatory cytokine expression showed that all three treatments significantly reduced hepatic monocyte chemoattractant protein(MCP)1,interleukin(IL)6,and IL1β(Fig.2B).Sirius Red staining of liver fibrosis revealed that all three treatments decreased the Sirius Red positive area by～50% (Fig.2C and D),which was further supported by reduced hepatic mRNA expression of alpha smooth muscle actin (ACTA2) and collagen 1A1 (COL1A1)(Fig.2E).However,the GSK+OCA treatment did not cause further reduction of hepatic inflammatory cytokine expression or fibrosis compared to the GSK or OCA single treatment.
3.3.The GSK+OCA treatment-mediated reduction in total bile acid pool was insufficient to promote fecal lipid loss in HFCFr diet-fed mice
Fecal bile acid measurements revealed that all three treatments caused increased fecal bile acid loss to a similar degree (～2 fold)compared to the control group at 1 week after treatment initiation(Fig.3A).After 6 weeks,GSK-mediated fecal bile acid loss was maintained at～2-fold,but the effect of OCA treatment and the GSK+OCA treatment on fecal bile acid loss was significantly diminished compared to that during the 1st week of treatment(Fig.3A).In the GSK+OCA group,attenuated fecal bile acid loss in the presence of ASBT inhibition may indicate markedly reduced bile acid pool.However,at the 12th week,all three treatments increased fecal bile acid loss by～2-3 fold although the GSK+OCA group showed a relatively less fecal bile acid loss than the GSK group and the OCA group (Fig.3A).Bile acid pool measurement after the 12-week treatment period revealed that the GSK treatment reduced total bile acid pool by～20%,the OCA treatment reduced total bile acid pool by～40%,and the GSK+OCA treatment reduced total bile acid pool by～55% (Fig.3B).All three treatments reduced bile acid content in the small intestine and gallbladder,while liver bile acid content was only slightly reduced in the GSK+OCA group compared to the control group (Fig.3B).The highest degree of bile acid pool reduction in the GSK+OCA group was explained by abolished compensatory CYP7A1 induction by GSK presumably due to the presence of OCA (Fig.3C).OCA also increased hepatic mRNA of small heterodimer partner (SHP),an FXR target gene (Fig.3C).GSK treatment reduced the mRNA expression of ileal SHP and FGF15,presumably as a result of reduced bile acid uptake (Fig.3D).To our surprise,OCA alone did not inhibit hepatic CYP7A1,and only induced ileal SHP but did not increase ileal FGF15 expression (Fig.3D),nor did it significantly reduce ASBT mRNA expression (Fig.3D).These findings suggest that the OCA activation of FXR may be attenuated in the HFCFr diet fed obese mice or partially antagonized by the significantly reduced endogenous bile acids(Fig.3B).Analysis of fecal lipids revealed that only GSK treatment resulted in a trend of higher fecal TG and cholesterol loss,while the OCA group and the GSK+OCA group showed modestly but significantly reduced fecal TG and cholesterol content(Fig.3E and F).
3.4.Inhibition of intestinal ASBT by GSK treatment did not reduce OCA in the gallbladder bile of HFCFr diet-fed mice
After absorption in the small intestine,OCA is conjugated in the liver and re-secreted into bile.To determine if ASBT inhibition interferes with OCA absorption in the intestine to potentially explain some of the observed changes,we measured OCA in the gallbladder bile.We found that taurine conjugated OCA (T-OCA) was not detected in the bile samples from the control group or the GSK group,but was readily detected in the OCA group and the GSK+OCA group (Fig.4).T-OCA amount in the gallbladder was about 2-fold higher in the GSK+OCA group than the OCA group.Although this difference could be a result of altered hepatic secretion or gallbladder emptying dynamics,this result suggests that GSK treatment may not prevent intestinal OCA absorption.Unconjugated OCA was not detected in the bile samples from the 4 groups,suggesting that OCA was efficiently conjugated to taurine in mouse hepatocytes.The T-OCA amount is about～0.6% of total gallbladder bile acids in the OCA group and～2%of total gallbladder bile acids in the GSK+OCA group.This degree of biliary OCA enrichment is generally in line with a recent study conducted in WT mice treated with 10 mg/kg/day OCA for 4 weeks,and suggests that GSK inhibition of ileal ASBT does not reduce hepatic OCA distribution in mice.20
We have recently reported that combining GSK with AAVmediated FGF15 overexpression caused marked reduction in bile acid pool and significant improvement of obesity and NASH fibrosis than either single treatment in a pre-clinical NASH mouse model.19This beneficial effect of the combined GSK+AAV-FGF15 treatment may be significantly attributed to their synergistic action on hepatic bile acid synthesis and intestine bile acid re-uptake,which causes bile acid pool reduction,lipid malabsorption,and weight loss.In this follow-up study,we have further investigated if combining GSK with the potent FXR agonist OCA,which,unlike FGF19 analogue,can be administered via oral route and has been approved for clinical use in humans,may have similar effects on bile acid metabolism and NASH development in the HFCFr diet-induced NASH mouse model.Our findings showed that although GSK+OCA co-treatment resulted in further bile acid pool reduction than either single treatment,this co-treatment did not cause persistent reduction of obesity or hepatic steatosis.In addition,all three treatments caused similar reduction in hepatic cholesterol content,inflammatory cytokine expression,and liver fibrosis,and the GSK+OCA co-treatment did not provide additional benefits than either single treatment against NASH and liver fibrosis.We think that there are two possible reasons that the GSK+OCA cotreatment was not as effective as the GSK+AAV-FGF15 cotreatment in reversing obesity,NASH and liver fibrosis in the HFCFr diet-fed mice,and they are discussed next.
Fig.2.The GSK+OCA combined treatment does not cause further reduction of hepatic inflammation and fibrosis compared to GSK or OCA monotherapy in HFCFr diet-fed mice.Mice and experiments are described in Fig.1 legend.(A)Serum transaminases after 12 weeks of treatments.(B)Relative liver mRNA expression.(C)Representative Sirius Red stain of liver sections.Scale bar=600 μm.(D) Sirius Red positive area per field is quantified with ImageJ software.(E) Relative liver mRNA expression.Results are expressed as mean±SEM (n=5-10 per group).*P<0.05 vs. control (Con) group.Abbreviations: ACTA2,alpha smooth muscle actin;ALT,alanine aminotransferase;AST,aspartate aminotransferase;COL1A1,collagen 1A1;GSK,GSK2 330672;HFCFr,high fat,cholesterol,and fructose;IL,interleukin;MCP1,monocyte chemoattractant protein 1;OCA,obeticholic acid;SEM,standard error of the mean.
First,the GSK+OCA-mediated bile acid pool reduction was not as strong as that caused by GSK-AAV-FGF15 treatment.19The bile acid pool was reduced by～55%in the GSK+OCA group.Although this reduction was larger than that caused by either GSK or OCA alone,it was not associated with increased fecal cholesterol or fat loss.This suggests that such degree of bile acid pool reduction was not sufficient to cause intestinal lipid malabsorption.This is in contrast to our previous study where GSK+AAV-FGF15 cotreatment reduced total bile acid pool by～90%,leading to reduced intestinal cholesterol and lipid absorption that contributed to weight loss.19However,our previous study also showed that FGF15 overexpression alone reduced bile acid pool by～50% and also caused increased fecal lipid loss,which raises an intriguing question if FGF15 signaling limits intestinal lipid absorption independent of bile acid pool regulation.Furthermore,it was noticed that when the obese mice were treated with GSK +OCA,there was an initial weight loss which did not persist until the 12th week.The degree of fecal bile acid loss measured at 6 weeks was also reduced in the GSK+OCA group compared to that in the 1st week despite the presence of GSK that inhibited intestinal bile acid uptake,suggesting that reduced fecal bile acid loss at this time may reflect a high degree of overall reduction in bile acid pool that correlated with weight loss at these similar time points.However,the fecal bile acid content of the GSK +OCA group was restored to～2-fold during the 12th week of treatment.These data suggest that the mice may have adapted to the GSK +OCA co-treatment condition over time and largely reversed initial weight loss at the end of the treatment period.
Fig.3.The GSK+OCA treatment-mediated reduction in total bile acid pool was insufficient to promote fecal lipid loss in HFCFr diet-fed mice.Mice were described in Fig.1 and the experimental methods.(A)Fecal bile acids during the 1st,6th,and 12th week of treatments.Four independent fecal samples were collected from each cage during a 1-week period.(B)Tissue bile acids and bile acid pool.Bile acid pool is the sum of bile acid in the liver,gallbladder,and small intestine with its content.(C)Relative liver mRNA expression.(D) Relative ileal mRNA expression.(E,F) Fecal TG and cholesterol during the 12th week of treatment.Results are expressed as mean±SEM (n=4-10 per group).*P<0.05 vs.control(Con)group unless indicated otherwise.Abbreviations:ASBT,apical sodium-bile acid transporter;CYP7A1,cholesterol 7alpha-hydroxylase;FGF15,fibroblast growth factor 15;GB,gallbladder;GSK,GSK2 330672;HFCFr,high fat,cholesterol,and fructose;OCA,obeticholic acid;SEM,standard error of the mean;SHP,small heterodimer partner;TG,triglyceride.
Second,it should be noted that reduced BW by the GSK+AAVFGF15 co-treatment may not be fully attributed to intestinal lipid malabsorption.19This is because FGF15/19 has been shown to improve hepatic lipid and glucose homeostasis.7,21In addition,FGF15/19 also targets extrahepatic tissues including the white and brown fat to increase overall energy expenditure and cause weight reduction.22However,this effect was mostly seen when FGF15/19 signaling was markedly activated at pharmacological level via direct administration or transgenic overexpression.6,16,23,24In contrast,GSK has a negative effect on ileal FGF15 expression due to reduced bile acid uptake and FGF15 was not induced in the GSK +OCA group.Furthermore,the effect elicited by induction of endogenous FGF15/19 by FXR activation is expected to be far less potent than the pharmacological effect of direct administration of FGF19 or transgenic FGF15/19 overexpression.These differences may also contribute to the lack of synergistic beneficial effects of the GSK +OCA co-treatment compared to the anti-NASH effect of the GSK+AAV-FGF15 co-treatment in the same mouse model.19It should also be noted that in mice FGF15 is mainly produced by the intestine,while in humans both the liver and intestine produce FGF19 which is induced by FXR agonist.In our previous study,we used AAV-FGF15 driven by a hepatocyte-specific thyroxine-binding globulin (TBG) promoter to overexpress FGF15,which presumably acts on hepatocytes via both autocrine and paracrine manners and remote target organs via an endocrine manner.19FGF15 produced from the intestine is known to act on the liver via an endocrine manner.However,it remains to be determined if liver-derived FGF15 via AAV-mediated expression and intestine-derived FGF15 induced by bile acids and FXR agonists may have differential metabolic impacts on NASH development and progression.
Fig.4.Inhibition of intestinal ASBT by GSK treatment did not reduce OCA in the gallbladder bile of HFCFr diet-fed mice.Mice and experiments are described in Fig.1 legend.Gallbladder bile was diluted in 1 mL 90% ethanol.A 50 μL aliquot from each sample was used for LC-MS measurement of taurine conjugated OCA(T-OCA)and free OCA.Free OCA was not detectable in any samples.Total T-OCA in each gallbladder bile was calculated.Results are expressed as mean±SEM (n=4-5 per group).Abbreviations: ASBT,apical sodium-bile acid transporter;GSK,GSK2 330672;HFCFr,high fat,cholesterol,and fructose;LC-MS,liquid chromatography-mass spectrometry;ND,not detected;OCA,obeticholic acid;SEM,standard error of the mean.
When administered via oral route,OCA is conjugated and enters the enterohepatic circulation as other endogenous bile acids.One would expect that by combining GSK and OCA as a co-treatment,inhibition of intestinal bile acid uptake would diminish the bioavailability of OCA therefore limiting the effect of OCA in the cotreatment.Interestingly,we found that T-OCA was readily detected in the gallbladder bile of the GSK+OCA co-treated mice with no reduction than the OCA found in the gallbladder bile of the OCAtreated mice.One possibility is that unconjugated OCA may be absorbed via ASBT-independent mechanisms as unconjugated bile acids and subsequently conjugated in hepatocytes and secreted into bile.Therefore,the lack of synergistic NASH improvement by GSK+OCA co-treatment was not due to diminished OCA bioavailability.
In this and our previous study,we have found that the HFCFr diet-induced hepatic steatosis was not reversed by GSK,FGF15 overexpression,or OCA treatment.19This is in contrast to previously reported reduction of hepatic steatosis by these treatments in other obese mouse models.16,25Interestingly,we did observe reduced hepatic inflammatory cytokines and fibrosis in the GSK group,the OCA group,and the GSK+OCA group.We speculate that these may be partially attributed to reduction of hepatic cholesterol accumulation and bile acid burden,both of which are known to promote organelle dysfunction,hepatocyte injury,and inflammatory infiltration.16,17,26Bile acid synthesis is a major pathway for cholesterol elimination.Both OCA treatment and FGF19 analogue treatment in NASH humans inhibited bile acid synthesis and worsened hypercholesterolemia that was prevented with cholesterol lowering drug statins.27,28In a recent study conducted in mice fed a similar high fat high cholesterol diet with fructose drinking water,OCA failed to improve the atherogenic lipid profile,which was attributed to reduced cholesterol conversion to bile acids and fecal bile acid excretion.29In contrast,atrovstatin treatment was effective in improving fatty liver development,which was associated with increased bile acid synthesis.29This has raised the question if FXR antagonism that induces bile acid synthesis can be more beneficial than FXR agonism in fatty liver treatment.In this regard,we have shown that the GSK+AAV-FGF15 co-treatment repressed hepatic CYP7A1 and bile acid synthesis but still caused marked reduction in hepatic cholesterol accumulation,which could contribute to reduced hepatic organelle stress and inflammation in HFCFr dietfed mice.19Reduced hepatic cholesterol accumulation in the presence of bile acid synthesis repression in the GSK+AAV-FGF15 treated mice may be attributed to the predominant effect of reduced intestine cholesterol absorption,which is the major source of hepatic cholesterol in the HFCFr diet-fed mice.This may have contributed to the differential metabolic outcomes between OCA single treatment and GSK+AAV-FGF15 co-treatment,despite both treatments reduce hepatic bile acid synthesis.Given the highly complex and closely interconnected cholesterol and bile acid regulation in the liver and intestine,targeting different steps in the enterohepatic circulation with various therapeutic strategies could result in overlapping but also distinct metabolic changes that lead to differential modulation of disease development and progression.
In summary,we report that combining ASBT inhibitor GSK with FXR agonist OCA treatment does not provide additional therapeutic benefits than either single treatment against NASH or liver fibrosis in a preclinical mouse NASH model.This may be partly due to the insufficient reduction of total bile acid pool and the absence of high level of FGF15 exposure in the GSK +OCA co-treatment.Based on this and our previous report,combining GSK with FGF15/19 signaling activation may provide superior benefits than the GSK+OCA co-treatment against obesity,NASH,and liver fibrosis.19
Authors’ contributions
D.J.Matye,X.Qin,M.N.Hasan,L.Gu,Y.D.Clayton,and F.Li designed and performed the experiments.T.Li supervised the study and wrote the manuscript.
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgements
This study was supported in part by the USA National Institutes of Health(NIH)grants 1R01 DK117965-01A1(T.Li),1R01DK131064(T.Li) and R01DK121970(F.Li).
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