Liver-adipose,tissue,crosstalk,in,alcohol-associated,liver,disease:,The,role,of,mTOR☆

Yssa Rodriguez,Jack Dunfield,Tyson Roderique,Hong-Min Ni

Department of Pharmacology,Toxicology and Therapeutics,University of Kansas Medical Center,Kansas City,KS,USA

Keywords:Alcohol-associated liver disease (ALD)Liver injury Mechanistic target of rapamycin (mTOR)Adipose atrophy Adipokine Liver-adipose tissue crosstalk

ABSTRACT Background: Alcohol-associated liver disease (ALD) is a major chronic liver disease around the world without successful treatment.Acute alcoholic hepatitis is one of the most severe forms of ALD with high mortality,which is often associated with binge drinking.Alcohol drinking dysregulates lipid metabolism,increases adipose tissue lipolysis,and induces liver steatosis and adipose tissue atrophy.Increasing evidence implicates that crosstalk of liver and adipose tissue in the pathogenesis of ALD.Mechanistic target of rapamycin (mTOR) is a phosphatidylinositol 3-kinase (PI3K)-like serine/threonine protein kinase that regulates lipid metabolism,cell proliferation and autophagy.However,the role of mTOR in regulating adipose-liver crosstalk in binge drinking-induced organ damage remains unclear.Methods: We generated liver-specific and adipocyte-specific regulatory-associated protein of mTOR(Rptor) knockout (RptorLKO and RptorAKO) as well as Mtor knockout (MtorLKO and MtorAKO) mice,by crossing Rptorflox and Mtorflox mice with albumin Cre or adiponectin Cre mice,respectively.In addition,we generated liver and adipocyte double deletion of Rptor or Mtor (MtorLAKO and RptorLAKO) mice.The knockout mice with their matched wild-type littermates (RptorWT and MtorWT) were subjected to acute gavage of 7 g/kg ethanol.Results: Mice with adipocyte deletion of Rptor or Mtor developed hepatomegaly and adipose tissue atrophy.Alcohol gavage increased liver injury,hepatic steatosis and inflammation in mouse livers as demonstrated by elevated serum alanine aminotransferase activities,increased hepatic levels of triglyceride and increased hepatic numbers of CD68 positive macrophages in mouse livers after alcohol gavage.Liver injury was further exacerbated by deletion of adipocyte Rptor or Mtor.Serum adipokine array analysis revealed that increased levels of pro-inflammatory cytokines IL-6 and TNFα as well as chemokine MCP-1 following acute alcohol gavage in wild-type mice,which were further increased in adipocyte-specific Mtor or Rptor knockout mice.Conversely,levels of anti-inflammatory cytokine IL-10 decreased in adipocyte-specific Mtor or Rptor knockout mice.The levels of circulating fibroblast growth factor 21 (FGF21) increased whereas levels of circulating adiponectin and fetuin A decreased in wild-type mice after alcohol gavage.Intriguingly,adipocyte-specific Mtor or Rptor knockout mice already had decreased basal level of FGF21 which increased by alcohol gavage.Moreover,adipocyte-specific Mtor or Rptor knockout mice already had increased basal level of adiponectin and decreased fetuin A which were not further changed by alcohol gavage.Conclusions: Adipocyte but not hepatocyte ablation of Mtor pathway contributes to acute alcoholinduced liver injury with increased inflammation.Our results demonstrate the critical role of adipocyte mTOR in regulating the adipose-liver crosstalk in ALD.

Alcohol-associated liver disease (ALD) is a major chronic liver disease around the world without successful treatment.The pathogenesis of ALD is characterized by simple steatosis,which can progress to more severe alcoholic hepatitis,fibrosis,cirrhosis,and eventually hepatocellular carcinoma.1-3Acute alcoholic hepatitis is one of the most severe forms of ALD with high mortality,which is often associated with binge drinking.1Alcohol consumption can lead to the damage of multiple organs,including the liver,pancreas,lung,kidney,adipose,and brain.4Chronic alcohol consumption dysregulates lipid metabolism,increases adipose tissue lipolysis,and induces liver steatosis and adipose tissue atrophy.This suggests a critical role of liver-adipose axis in the pathogenesis of ALD.5,6Moreover,chronic ethanol increases cytochrome P450 2E1 and aldehyde dehydrogenase(ALDH)activity in adipose tissues,which contributes to adipose tissue inflammation and the clearance of toxic metabolites in adipose tissue.7,8Binge drinking induces hepatic steatosis,however,studies on the effect of binge drinking on adipose tissue are scarce.9

Mechanistic target of rapamycin (mTOR) is a phosphatidylinositol 3-kinase (PI3K)-like serine/threonine protein kinase that regulates lipid metabolism,cell proliferation and autophagy.5,10,11mTOR is the catalytic core subunit of two distinct multiprotein complexes:mTOR complex 1(mTORC1)and mTORC2.mTORC1 and mTORC2 share several subunits: mTOR,DEP domain-containing mTOR-interacting protein (DEPTOR) and mammalian lethal with SEC13 protein 8 (mLST8).These two complexes also have distinct components: regulatory-associated protein of mTOR (RPTOR) and protein kinase B(AKT)/PKB substrate 40 kDa(PRAS40)in mTORC1 and Raptor-independent companion of mTOR (RICTOR),protein observed with Rictor-1 and Rictor-2(PROTOR 1/2)and mammalian stress-activated protein kinase-interacting protein (mSin1) in mTORC2.Pharmacological and genetic activation of mTOR promotes lipogenesis and suppresses lipolysis.10,11Chronic alcohol consumption altered mTOR signaling in adipose tissues and livers as well as lead to adipose atrophy and liver injury.5,12Adipocyte deletion ofRptorexacerbated adipose atrophy and liver injury in chronic alcohol-fed mice,suggesting adipocyte mTOR may play a critical role in liver-adipose tissue crosstalk in chronic alcoholinduced organ damage.5Our previous study showed that acute alcohol binge drinking also altered hepatic mTOR signaling.13However,the tissue-specific role of mTOR in regulating liveradipose tissue crosstalk in binge drinking-induced organ damage remains unclear.

In this study,we investigated the role of mTOR in regulating liver-adipose tissue crosstalk in an alcohol binge drinking model using mice with hepatic and/or adipocyte deletion ofRptororMtor.

2.1.Ethical approval

All procedures were approved by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center(21-05-175).

2.2.Animals

Mtorflox(#011009),Rptorflox(#013188),Albumin-cre (#003574)and Adipoq-cre (#010803) mice were purchased from the Jackson Laboratory (Bar Harbor,ME,USA).To generate liver-specificMtorknockout (KO) (MtorLKO) orRptorKO (RptorLKO) mice,MtorfloxorRptorfloxmice were crossed with Albumin-cre mice.To generate adipose-specificMtorKO (MtorAKO) orRptorKO (RptorAKO) mice,MtorfloxorRptorfloxmice were crossed with Adipoq-cre mice.To generate liver-and adipose-specificMtorDKO (MtorLAKO) orRptorDKO (RptorLAKO) mice,MtorLKOmice were crossed withMtorAKOmice orRptorLKOmice were crossed withRptorAKOmice.Cre negative mice were used as wild-type control(MtorWTandRptorWT).For the acute ALD model,8-10-week-old male mice were treated with 7 g/kg ethanol by oral gavage and control group mice were gavaged with maltose for 6 h.Mice were specific pathogen free and maintained in a barrier rodent facility under standard experimental conditions.

2.3.Hepatic triglyceride (TG) and cholesterol analysis

Hepatic lipid extraction was performed as described previously.14Frozen liver tissues(20-50 mg)were grinded into powder using a mortar and pestle followed by chloroform-methanol extraction.TG and cholesterol analysis of lipid extracts was performed using GPO-Triglyceride Reagent Set and Cholesterol liquid reagent (#T7532 and #C7510 respectively,Pointe Scientific)following the manufacturer"s instruction.

2.4.Serum biochemistry analyses

Blood samples were collected by cardiac puncture and sera were obtained by centrifugation.Serum alanine aminotransferase (ALT)activity (#A7526,Pointe Scientific),TG and cholesterol were measured by using commercially available kits following the manufacturer"s instruction as described previously.15

2.5.Histology and immunohistochemistry analyses

Livers and adipose tissues were fixed in 10% neutral formalin followed by paraffin embedding.Paraffin-embedded liver sections(5 μm) were stained with hematoxylin and eosin (H&E) for pathological evaluation.Immunostaining for F4/80 (#14-4801-82,Thermo Fisher) and CD68 (#137002,Biolegend) positive macrophages were performed as described previously.16

2.6.Protein extract and immunoblot analyses

Total liver proteins were extracted using radioimmunoprecipitation assay (RIPA) buffer (1% Igepal CA-630,0.5%sodium deoxycholate,0.1% sodium dodecyl (lauryl) sulfate (SDS)in phosphate buffered saline) supplemented with protease inhibitor cocktail(Bimake).Protein(30 μg)was separated on a SDSPAGE gel and transferred to a PVDF membrane.Membranes were probed with appropriate primary and secondary antibodies and visualized with SuperSignal plus chemiluminescent substrate(Thermo Fisher Scientific).Densitometry analysis was performed with Image J and normalized to β-actin.Primary antibodies anti-FGF21 (# ab64857,Abcam) and anti-β-actin (#A-5441,Sigma)were used in this study.

2.7.Serum adipokine array analysis

Equal amount of serum from each treatment group was pooled and 80 μL of pooled sera were subjected to adipokine array analysis using Proteome Profiler Mouse Adipokine Array Kit(#ARY013,R&D Systems) following the manufacture"s instruction.

2.8.Statistical analysis

All graphs and data were generated and analyzed using Graphpad Prism 8.All experimental data are expressed as the mean ± standard error of the mean (SEM) and subjected to oneway analysis of variance (ANOVA) with Turkey post hoc test(multi-group comparisons).P-value <0.05 was considered as significant.

3.1.Acute alcohol exposure leads to steatosis and liver injury and are exacerbated by adipocyte deletion of Mtor in mice

Our previous study showed that alcohol exposure altered mTOR signaling in the liver and adipose tissue.5,13To examine the effect of the mTOR pathway in the liver and adipose tissue of acute alcoholtreated mice,we establishedMtorwild-type(MtorWT),liver-specificMtorKO(MtorLKO),adipocyte-specificMtorKO(MtorAKO)as well as liver-and adipocyte-specificMtorKO (MtorLAKO) mice.The mice were treated with alcohol binge for 6 h.All of the mice developed steatosis as shown by H&E staining with increased hepatic triglyceride (TG),but not cholesterol in mouse livers after acute alcohol exposure (Fig.1A and B).Levels of hepatic TG inMtorLKOmice were comparable withMtorWTmice.Intriguingly,TG levels further increased in alcohol-treatedMtorAKOandMtorLAKOmouse livers but were similar inMtorAKOandMtorLAKOmice(Fig.1A and B).Moreover,MtorAKOandMtorLAKOmice developed hepatomegaly with or without alcohol gavage (Fig.1C).Similar to the steatosis data,acute alcohol exposure induced liver injury with increased serum alanine aminotransferase (ALT) activity inMtorWTandMtorLKOmice,which further increased inMtorAKOandMtorLAKOmice(Fig.1D).All these data suggested that acute alcohol exposure leads to steatosis and liver injury which are exacerbated by the deletion of adipocyte but not hepaticMtor.

Fig.1.Acute alcohol exposure leads to steatosis and liver injury and MtorAKO and MtorLAKO mice have exacerbated liver injury.Mtor wild-type(MtorWT),liver-specific Mtor KO(MtorLKO),adipocyte-specific Mtor KO(MtorAKO)as well as liver-and adipocyte-specific Mtor KO(MtorLAKO)mice were generated by crossing floxed Mtor mice with albumin Cre or adiponectin Cre mice.The mice were treated with ethanol 7 g/kg or equal amount water by oral gavage for 6 h.(A)Representative images of H&E staining of the livers.(B)Hepatic TG and cholesterol were quantified.(C) Liver/body weight ratio was quantified.(D) Serum ALT activities were measured.Data are expressed as means ± SEM (n=4 to 8) and subjected to one-way ANOVA with Turkey post hoc test.*P <0.05 EtOH vs. control;#P <0.05 vs. WT EtOH;^P <0.05 vs. LKO EtOH;&P <0.05 vs. WT control;$P <0.05 vs. LKO control.Original magnification,× 20.Abbreviations: ALT,alanine aminotransferase;EtOH,ethanol;H&E,hematoxylin and eosin;KO,knockout;mTOR,mechanistic target of rapamycin;TG,triglyceride;WT,wild-type.

3.2.Adipocyte deletion of Mtor but not acute alcohol binge leads to adipose atrophy in mice

We did not find significant changes in the serum levels of TG and cholesterol inMtorWTandMtorLKOmice with or without alcohol gavage.While no significant differences in serum levels of TG and cholesterol were found inMtorWTandMtorAKOorMtorLAKOmice gavaged with maltose,serum levels of cholesterol were significantly increased inMtorLAKOmice in alcohol gavaged mice compared with their respective maltose gavaged mice (Fig.2A).White adipose tissue (WAT) and brown adipose tissue (BAT) mass slightly increased but not significantly inMtorWTmice after alcohol gavage (Fig.2B).However,compared toMtorWTcontrol mice,MtorAKOandMtorLAKOcontrol mice had approximately 20-25%decreased epididymal WAT (eWAT),40-50% decreased inguinal WAT(iWAT),and 50-60%decreased interscapular BAT(iBAT)mass.iBAT further decreased by 32%inMtorAKOmice after alcohol gavage compared to their control mice.eWAT and iBAT decreased by 25%and 45% inMtorLAKOmice after alcohol gavage compared to their control mice(Fig.2B).The sizes of eWAT adipocytes were similar inMtorWTandMtorLKOmice with or without alcohol (Fig.2C and D).Although the adipose mass was smaller inMtorAKOandMtorLAKOmice,the sizes of eWAT adipocytes ofMtorAKOandMtorLAKOmice were similar to MtorWTandMtorLKOmice with or without alcohol gavage (Fig.2C and D).Together,these data indicate that loss of adipocyteMtorbut not acute alcohol consumption may lead to adipose atrophy in mice.

Fig.2.Adipocyte deletion of Mtor leads to adipose atrophy in mice.Mice were treated as in Fig.1.(A)TG and cholesterol levels were measured.(B)Adipose tissue/BW ratio was quantified.(C) Representative images of H&E staining of eWAT.(D) Distribution of adipocyte area of eWAT was analyzed.Around 1191-2294 adipocytes from 2 to 3 mice in each group were quantified.Data are expressed as means±SEM(n=4 to 8,A,B)and subjected to one-way ANOVA with Turkey post hoc test.#P<0.05 vs.WT EtOH;^P<0.05 vs.LKO EtOH;&P <0.05 vs. WT control;$P <0.05 vs. LKO control.Original magnification,× 20.Abbreviations: BW,body weight;EtOH,ethanol;eWAT,epididymal white adipose tissue;H&E,hematoxylin and eosin;iBAT,interscapular brown adipose tissue;iWAT,inguinal white adipose tissue;KO,knockout;mTOR,mechanistic target of rapamycin;TG,triglyceride;WT,wild-type.

3.3.The effects of adipocyte-deletion of Mtor on acute alcohol gavaged-induced inflammation and changes of adipokines and cytokines in mice

As we found that adipocyte but not hepatic deletion ofMtorexacerbated acute alcohol-induced steatosis and liver injury in mice,we next tried to determine the underlying mechanisms.As shown in Fig.3A and B,there was no significant induction of macrophage marker F4/80 inMtorWTandMtorLAKOmouse livers in response to alcohol.However,F4/80 increased inMtorLKOmouse livers and decreased inMtorAKOmouse livers after acute alcohol binge.In contrast to F4/80 staining data,alcohol increased CD68 staining in all groups (Fig.3C and D).

To further characterize the mechanism by which adipocyte deletion ofMtorcontributes to ALD,adipokine array was performed inMtorWTandMtorAKOmouse sera.Adiponectin,one of the major adipokines,regulates lipid and glucose metabolism,promotes insulin sensitivity,and plays a protective role in various metabolic scenarios.17MtorAKOmice had higher adiponectin compared toMtorWTmice after maltose gavage,but there were no significant changes after alcohol gavage in bothMtorAKOmice andMtorWTmice(Fig.3E).The levels of serum FGF21 were lower inMtorAKOmice in the control group but increased after alcohol gavage (Fig.3E).The levels of circulating fetuin A,another adipokine and hepatokine that inhibits adiponectin,were lower inMtorWTmice in response to alcohol (Fig.3E).The levels of circulating fetuin A inMtorAKOmice were lower compared withMtorWTmice regardless of alcohol gavage.On the other hand,alcohol gavage increased levels of proinflammatory cytokines and chemokine interleukin-6 (IL-6).It did not affect the levels of serum tumor necrosis factor alpha (TNFα)and monocyte chemoattractant protein-1 (MCP-1) but decreased anti-inflammatory cytokine interleukin-10 (IL-10) inMtorWTmice(Fig.3F).Adipocyte deletion ofMtorincreased levels of TNFα,MCP1 and IL-6 with or without alcohol gavage (Fig.3F).Hepatic FGF21 levels increased inMtorWTmice by alcohol(Fig.3G).Hepatic FGF21 levels were lower inMtorAKOcontrol mice and further decreased by alcohol (Fig.3G).Taken together,acute alcohol binge increases hepatic inflammation and the secretion of FGF21 but decreases the secretion of fetuin A in WT mice.Loss of adiposeMtordecreases the secretion of fetuin A but increases serum levels of TNFα and MCP-1,which are not affected by alcohol binge drinking.

3.4.Loss of adipocyte but not hepatic Rptor exacerbates acute alcohol gavage-induced liver injury

As mTOR exists in both mTORC1 and mTORC2,to determine whether the disruption of mTORC1 or mTORC2 would contribute to the detrimental effect on ALD,we establishedRptorwild-type(RptorWT),liver-specificRptorKO (RptorLKO),adipocyte-specificRptorKO (RptorAKO) as well as liver-and adipocyte-specificRptorKO (RptorLAKO) mice,which would only disrupt mTORC1 without affecting mTORC2.Similar to mTOR deletion mice,deletion of either adipocyte or liver Raptor did not affect hepatic steatosis after acute alcohol gavage as demonstrated by the accumulation of lipid droplets shown by H&E staining and hepatic TG but not cholesterol contents in mouse livers (Fig.4A and B).RptorAKOandRptorLAKOmice also developed hepatomegaly with or without alcohol gavage(Fig.4C).Acute alcohol increased liver injury with elevated serum ALT activity inRptorWTmice,which was further increased inRptorAKOandRptorLAKOmice but notRptorLKOmice (Fig.4D).

3.5.Adipocyte deletion of Rptor leads to adipose atrophy in mice

Acute alcohol binge slightly increased levels of serum TG inRptorWTmice (Fig.5A).However,RptorAKOandRptorLAKOmice had much higher serum levels of TG compared toRptorWTandRptorLKOmice after alcohol gavage(Fig.5A).RptorAKOandRptorLAKOmice had approximately 50% decreased eWAT,40-60% decreased iWAT and 40-50% decreased iBAT mass compared toRptorWTmice (Fig.5B).Alcohol gavage did not add more synergistic effects on adipose mass except that alcohol further decreased eWAT mass inRptorAKOandRptorLAKOmice compared toRptorWTmice(Fig.5B).The sizes of eWAT adipocytes and fat mass were similar inRptorWTandRptorLKOmice with or without alcohol gavage(Fig.5C and D).Interestingly,the average sizes of eWAT adipocytes were around 20% smaller inRptorAKOandRptorLAKOcontrol mice compared toRptorWTandRptorLKOmice.Notably,the sizes of adipocytes in eWAT ofRptorAKOandRptorLAKOmice were heterogeneous with increased number of smaller or larger adipocytes thanRptorWTadipocytes,which was different from the adipocyte deletion ofMtorin mice (Fig.5C and D).In addition to the differences of basal adipocyte size,alcohol gavage further increased the number of smaller or larger adipocytes compared to their control littermates gavaged with maltose(Fig.5C and D).These data suggested that adipocyte deletion ofRptorleads to epididymal adipose atrophy,which is further exacerbated by alcohol in mice.

3.6.The effects of adipocyte deletion of Rptor on acute alcohol gavage-induced inflammation and changes of serum adipokines and cytokines in mice

We found that the number of F4/80 positive cells decreased inRptorWTandRptorLAKOmice but did not change in alcohol gavagedRptorAKOandRptorLKOmice compared to maltose gavaged mice(Fig.6A and B).However,alcohol gavage significantly increased the number of CD68 positive cells in all the genotypes exceptRptorLAKOmice(Fig.6C and D).Notably,the number of CD68 positive cells was already significantly higher inRptorAKOandRptorLAKOmice only received maltose compared toRptorWTmice,suggesting possible increased basal level inflammation inRptorAKOandRptorLAKOmice.

Adipokine array analysis showed thatRptorAKOmice had higher levels of serum adiponectin but lower serum levels of FGF21 and fetuin A levels compared toRptorWTmice regardless of alcohol gavage (Fig.6E).Serum FGF21 further decreased inRptorAKOmice after alcohol gavage(Fig.6E).The serum levels of TNFα and IL-6 did not change with or without alcohol gavage inRptorWTandRptorAKOmice.Alcohol gavage decreased the levels of serum IL-10 in bothRptorWTandRptorAKOmice.The levels of serum MCP-1 were slightly higher inRptorAKOmice but were decreased by alcohol gavage compared toRptorWTwith or without alcohol gavage (Fig.6F).Together,these data indicates that acute alcohol consumption may increase hepatic inflammation and alter the circulating hepatokines and cytokines,which are partially affected by the deletion of adipocyteRptor.

Fig.3.Acute alcohol exposure increases inflammation and altered adipokines in MtorAKO mice.Mice were treated as in Fig.1.(A) F4/80 was stained in the livers by immunohistochemistry staining and (B) F4/80 positive macrophages were quantified.(C) CD68 was stained in the livers by immunohistochemistry staining and (D) CD68 positive macrophages were quantified.(E,F) Equal amount of serum from the same group was pooled.Circulating adipokines (E) and inflammatory mediators (F) were analyzed by Proteome Profiler Mouse Adipokine Array Kit.Data are expressed as means ± SEM and subjected to one-way ANOVA with Turkey post hoc test. n=3 (A-D); n=5 to 8 (E,F).*P<0.05 EtOH vs.control.(G)Hepatic FGF21 levels in mice were measured by immunoblot analysis.Original magnification,×20.Abbreviations:EtOH,ethanol;FGF21,fibroblast growth factor 21;IL-6,interleukin-6;IL-10,interleukin-10;KO,knockout;MCP-1,monocyte chemoattractant protein-1;mTOR,mechanistic target of rapamycin;TNFα,tumor necrosis factor alpha;TG,triglyceride;WT,wild-type.

ALD is a burgeoning health problem worldwide without effective treatment.In this study,we characterized the effects of acute alcohol exposure on mTOR signaling in mouse livers and adipose tissues as well as liver-adipose tissue crosstalk in alcohol-induced steatosis and liver injury.Acute alcohol exposure induced hepatic steatosis and liver injury in mice as well as increased circulating FGF21,unchanged or slightly decreased adiponectin and decreased fetuin A.Hepatic deletion ofMtororRptordid not alter steatosis or liver injury.Adipocyte deletion ofMtororRptorsignificantly decreased adipose mass and further exacerbated alcohol-induced liver injury as well as decreased FGF21 and fetuin A but increased adiponectin.Double deletion ofMtororRptorin the liver and adipocyte had similar phenotype as single adipocyte deletion.

Fig.4.Acute alcohol exposure leads to steatosis and liver injury and RptorAKO and RptorLAKO mice have exacerbated liver injury.Rptor wild-type(RptorWT),liver-specific Rptor KO(RptorLKO),adipocyte-specific Rptor KO(RptorAKO)as well as liver-and adipocyte-specific Rptor KO(RptorLAKO)mice were generated by crossing floxed Rptor mice with albumin Cre or adiponectin Cre mice.The mice were treated with ethanol 7 g/kg or equal amount of water by oral gavage for 6 h.(A)Representative images of H&E staining of the livers.(B)Hepatic TG and cholesterol were quantified.(C)Liver/body weight ratio was quantified.(D)Serum ALT activities were measured.Data are expressed as means±SEM(n=4 to 7)and subjected to one-way ANOVA with Turkey post hoc test.*P <0.05 EtOH vs. control;#P <0.05 vs. WT EtOH;^P <0.05 vs. LKO EtOH;&P <0.05 vs. WT control;$P <0.05 vs. LKO control.Original magnification,×20.Abbreviations:ALT,alanine aminotransferase;EtOH,ethanol;H&E,hematoxylin and eosin;KO,knockout;Rptor,regulatory-associated protein of mTOR;TG,triglyceride;WT,wild-type.

Alcohol exposure including both acute and chronic induces hepatic steatosis.12,13Our present study confirmed that acute alcohol binge led to steatosis.mTOR is a nutrition sensor that regulates lipid metabolism,cell proliferation and autophagy.5,10,11Acute alcohol binge inhibits mTOR activation,activates autophagy and ameliorates alcohol-induced liver injury in the livers.13Inhibition of autophagy by the deletion of forkhead box O3 (FoxO3),a transcription factor which regulates autophagy,enhanced acute alcohol-induced steatosis and liver injury.9However,the role of mTOR in acute alcohol binge is still unclear.In our present study,we found that genetic deletion of mTOR or Rptor in the liver did not protect against alcoholinduced steatosis and liver injury.Our current data suggested that the inhibition of mTOR in the liver is not sufficient to ameliorate alcohol-induced liver injury in an acute alcohol binge model.In contrast to the acute alcohol binge model,chronic alcohol feeding activates mTOR signaling which contributes to ALD.12,18Chaoet al.12found that the activation of mTOR by alcohol-decreased transcription factor EB (TFEB) nuclear translocation and resulted in insufficient autophagy.Overexpression of TFEB or inhibition of mTOR by torin 1,an mTOR inhibitor,protected alcohol-induced steatosis and liver injury.On the other hand,Chenet al.18reported that the activation of mTOR by alcohol-decreased DEPTOR,an mTORC1 negative regulator,and increased transcriptional activity of the lipogenic transcription factor sterol regulatory element-binding protein-1(SREBP-1) and lipogenesis in the livers.Overexpression of DEPTOR ameliorated ALD.18The exact role of the mTOR pathway in ALD still needs to be further characterized.Interestingly,our study further demonstrated that mice with adipocyte deletion ofMtororRptorexacerbated acute alcohol-induced liver injury.Loss ofMtororRptorin the liver combined with adipocytes had similar liver injury to adipocyte deletion ofMtororRptor.All these results suggested adipocyte mTOR signaling but not hepatic mTOR contributes to liver injury in mice in response to acute alcohol exposure in mice.Future studies are needed to further investigate the different mTOR response to acute or chronic alcohol exposure.

Fig.5.Adipocyte deletion of Rptor leads to adipose atrophy and is further altered by alcohol in mice.Mice were treated as in Fig.4.(A)Serum triglyceride(TG)and cholesterol levels were measured.(B) Adipose tissue/BW ratio was quantified.(C) Representative images of H&E staining of eWAT.(D) Distribution of adipocyte area of eWAT was analyzed.Around 1783-3207 adipocytes from 2 to 4 mice in each group were quantified.Data are expressed as means±SEM(n=4 to 7,A,B)and subjected to one-way ANOVA with Turkey post hoc test.*P <0.05 EtOH vs. control;#P <0.05 vs. WT EtOH;^P <0.05 vs. LKO EtOH;&P <0.05 vs. WT control;$P <0.05 vs. LKO control.Original magnification,× 20.Abbreviations: BW,body weight;EtOH,ethanol;eWAT,epididymal white adipose tissue;H&E,hematoxylin and eosin;iBAT,interscapular brown adipose tissue;iWAT,inguinal white adipose tissue;KO,knockout;Rptor,regulatory-associated protein of mTOR;TG,triglyceride;WT,wild-type.

Fig.6.Acute alcohol exposure increases inflammation and altered adipokines in RptorAKO mice.Mice were treated as in Fig.4.(A) F4/80 was stained in the livers by immunohistochemistry staining and(B)F4/80 positive macrophages were quantified.(C)CD68 was stained in the livers by immunohistochemistry staining and(D)CD68 positive macrophages were quantified.(E,F) Equal amount of serum from the same group was pooled.Circulating adipokines (E) and inflammatory mediators (F) were analyzed by Proteome Profiler Mouse Adipokine Array Kit.Data are expressed as means ± SEM and subjected to one-way ANOVA with Turkey post hoc test. n=3 (A-D); n=4-7 (E-F).*P<0.05 EtOH vs.control;#P<0.05 vs.WT EtOH;^P<0.05 vs.LKO EtOH;&P<0.05 vs.WT control;$P<0.05 vs.LKO control.Original magnification,×20.Abbreviations:EtOH,ethanol;FGF21,fibroblast growth factor 21;IL-6,interleukin-6;IL-10,interleukin-10;KO,knockout;MCP-1,monocyte chemoattractant protein-1;Rptor,regulatory-associated protein of mTOR;TNFα,tumor necrosis factor alpha;TG,triglyceride;WT,wild-type.

Genetic deletion ofRptor/mTORC1 in adipose tissue has smaller adipose tissue mass.The heterogeneous adipocyte size which is either larger or smaller than normal is likely due to increased lipolysis and decreased lipogenesis.5,19,20Genetic deletion ofRictor/mTORC2 in adipose tissue has normal adipose tissue mass and distribution as well as normal adipocyte size.21Ablation of mTOR in adipose which loses both mTORC1 and mTORC2 has a similar adipose phenotype toRptoradipose deletion.22Consistent with these previous studies,our study also demonstrated that adipocyte deletion ofMtororRptordecreased adipose tissue mass at basal level.Adipocyte deletion ofRptorbut notMtoralso altered adipocyte sizes.Malnutrition is a major complication of ALD patients with clinical findings such as muscle wasting/skeletal muscle loss,lean body mass,and loss of subcutaneous fat.23,24Malnutrition is also associated with the severity of ALD.Adipocyte-deletion ofMtor/Rptormice had significantly decreased lean body mass and loss of adipose tissues which resembled some features with human ALD.Adipokines are important for adipose-liver and adiposemuscle crosstalk in ALD.However,whether loss of adipocyteMtor/Rptoraffects muscle strength needs to be further investigated.

How does ablation of mTOR signaling in adipose tissue impact alcohol-induced liver injury in an acute alcohol binge model?There is growing evidence to suggest that adipose tissue is a key regulator of metabolism other than an inert energy storage organ.6It is well known that adipose tissue is an endocrine organ that actively synthesizes and secretes proteins (i.e.,adipokines) and other inflammatory mediators (i.e.,TNFα,IL-6) into the circulation to regulate the function of other tissues/organs in a paracrine manner.Adiponectin,one of the most abundant adipokines in adipose tissue,has an anti-steatotic effect on hepatocytes by enhancing free fatty acid oxidation,reducing free fatty acid influx andde novolipogenesis in ALD.17However,its response to alcohol exposure is still controversial.While most human studies demonstrated that adiponectin increased in human serum with alcohol consumption,many animal studies showed decreased adiponectin with alcohol consumption.25-28Consistent with most of the previous studies,alcohol decreased circulating adiponectin in WT mice.Adipocyte deletion ofMtororRptorincreased circulating adiponectin in mice.Fetuin A,a classic hepatokine,which has also recently become considered as an adipokine,is negatively correlated with adiponectin and promotes steatosis and fibrosis in metabolic liver diseases.29The role of fetuin A in ALD is not well studied.Only one study has shown that moderate alcohol consumption lead to a decreased in serum fetuin A among women.30Similar findings in alcohol binge mouse models showed that circulating fetuin A decreased.Our results suggested that increased adiponectin and decreased fetuin A are adaptive responses to alcohol.Circulating FGF21,another important adipokine and hepatokine and potential mediator of the liver-adipose tissue axis,increased in response to alcohol.The basal level of FGF21 was already low in mice with adipocyte deletion ofMtororRptorwhich further decreased in adipocyte deletion ofRptorby alcohol.These results were consistent with previous reported study that alcohol binge drinking increased circulating FGF21 in human and mice.31Previous studies demonstrated that FGF21 has hepatoprotective effect in liver injury.FGF21 null mice developed increased alcohol-induced liver injury with increased inflammation,steatosis and fibrosis when supplied alcohol in drinking water,and show higher mortality than their wild-type counterparts when fed with Lieber-DeCarli diet.31,32In addition to ALD model,FGF21 null mice exacerbated acetaminophen hepatoxicity and mortality.33Decreased circulating FGF21 in adipocyte deletion ofMtororRptormice may contribute to liver injury by alcohol binge.Previous study also demonstrated that FGF21 induces expression and secretion of adiponectin in adipocytes,and adiponectin is obligatory for FGF21 function in regulating glucose and lipid metabolism.34In this study,circulating FGF21 reduced but adiponectin increased inMtorAKOorRptorAKOcontrol mice (Figs.3E and 6E).Furthermore,alcohol-induced circulating FGF21 but not adiponectin (Figs.3E and 6E).All these data suggested that adiponectin secretion could be independent of FGF21.Future studies are needed to further dissect how alcohol alters adiponectin,fetuin A and FGF21 in either acute or chronic alcohol exposure and how altered basal levels of adiponectin,fetuin A and FGF21 affect the liver response to alcohol.

In human studies,it had been reported that women are enhanced vulnerability to develop alcohol-related diseases probably due to decreased gastric alcohol dehydrogenase activity which results in higher blood alcohol levels.35,36However,gender differences of ALD in mice were controversial.One study demonstrated that female mice were susceptible to alcohol-induce liver injury,the other one found that there were no gender differences in mouse model.37,38Overall,male mice were used in most ALD studies.However,gender differences in ALD mouse model need to be explored.

In conclusion,acute alcohol binge leads to steatosis and liver injury which is exacerbated by adipocyte deletion ofMtororRptorbut not hepatic deletion ofMtororRptor.Decreased circulating FGF21 in adipocyte-specificMtororRptorKO mice may contribute to elevated liver injury in response to acute alcohol.Targeting adipose mTOR signaling and adipocyte lipolysis could be a potential approach for improving ALD.

Authors’ contributions

Y.Rodriguez and J.Dunfield performed experiments,data analysis and interpretation and preparation of manuscript;T.Roderique participated in preparation of manuscript;H.-M.Ni participated in research design,performed experiments,data analysis and interpretation,and wrote the manuscript.All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

The research was supported in part by the USA NIDDK DK129234,NIAAA AA026904,and NIGMS P20GM144269 and P30GM118247 (to H.-M.Ni).

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