Yuzhong Xiao, Hao Liu, Junjie Yu, Zilong Zhao, Fei Xiao, Tingting Xia, Chunxia Wang,Kai Li, Jiali Deng, Yajie Guo, Shanghai Chen, Yan Chen, and Feifan Guo

Activation of ERK1/2 Ameliorates LiverSteatosis in Leptin Receptor–Deficient(db/db) Mice via StimulatingATG7-Dependent AutophagyDiabetes 2016;65:393–405 | DOI: 10.2337/db15-1024

Although numerous functions of extracellular signal–regulated kinase 1/2 (ERK1/2) are identified, a directeffect of ERK1/2 on liver steatosis has not been reported.Here, we show that ERK1/2 activity is compromised inlivers of leptin receptor–deficient (db/db) mice. Adenovirus-mediated activation of mitogen-activated proteinkinase kinase 1 (MEK1), the upstream regulator of ERK1/2,significantly ameliorated liver steatosis in db/db mice, in-creased expression of genes related to fatty acid b-oxidationand triglyceride (TG) export and increased serumb-hydroxybutyrate (3-HB) levels. Opposite effects wereobserved in adenovirus-mediated ERK1/2 knockdownC57/B6J wild-type mice. Furthermore, autophagy andautophagy-related protein 7 (ATG7) expression were de-creased or increased by ERK1/2 knockdown or activa-tion, respectively, in primary hepatocytes and liver.Blockade of autophagy by the autophagy inhibitor chlo-roquine or adenovirus-mediated ATG7 knockdown re-versed the ameliorated liver steatosis in recombinantadenoviruses construct expressing rat constitutivelyactive MEK1 Ad-CA MEK1 db/db mice, decreased ex-pression of genes related to fatty acid b-oxidation andTG export, and decreased serum 3-HB levels. Finally,ERK1/2 regulated ATG7 expression in a p38-dependentpathway. Taken together, these results identify a novelbeneficial role for ERK1/2 in liver steatosis via promotingATG7-dependent autophagy, which provides new insightsinto the mechanisms underlying liver steatosis and impor-tant hints for targeting ERK1/2 in treating liver steatosis.

Nonalcoholic fatty liver disease involves a serious patholog-ical change in liver (1). The initial stage of nonalcoholic fatty

liver disease is liver steatosis, characterized by the excessdeposition of triglyceride (TG) and/or cholesterol (TC) inliver (2). If uncontrolled, liver steatosis will progress tolife-threatening diseases, such as liver cirrhosis and dysfunc-tion (3). Abnormal hepatic lipid accumulation results fromincreased uptake of fatty acid/augmented de novo lipogen-esis and/or decreased b-oxidation/impaired TG export (4).

Autophagy, a cellular process that degrades intracellularorganelles and proteins (5), has recently been demon-strated to regulate lipid metabolism (6,7). Lipid dropletsare sequestered by autophagosome with the coordinationof autophagy-related genes (ATGs). Autophagosome isthen fused with lysosome (8) for the degradation of lipiddroplets into free fatty acids (FFAs). FFAs are then de-graded by mitochondrial b-oxidation to produce ATP orare reesterified into TG for storage (9). Impaired auto-phagy decreases hepatic fatty acid b-oxidation (FAO)and TG export and results in liver steatosis in mice(7,10), and fatty liver is ameliorated when hepatic auto-phagy is stimulated by certain compounds (11,12) orsome signaling pathways (13) in various animal models.

The mitogen-activated protein kinase–extracellular signal–regulated kinase (MEK-ERK) signaling pathway is in-volved in a wide variety of cellular processes (14–16).Several lines of evidence, however, have implied a link be-tween ERK1/2 and lipid metabolism (17–20). A direct effectof ERK1/2 on hepatic lipid metabolism, however, has notbeen reported. Based on the knowledge detailed above andthe fact that ERK1/2 is involved in autophagy (21–23), wehypothesized that ERK1/2 may play a role in liver steatosis

Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences,Shanghai Institute for Biological Sciences, Graduate School of the Chinese Acad-emy of Sciences, The Chinese Academy of Sciences, Shanghai, China

Corresponding author: Feifan Guo, ffguo@sibs.ac.cn.

Received 23 July 2015 and accepted 11 November 2015.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1024/-/DC1.

Y.X. and H.L. contributed equally to this study.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

Diabetes Volume 65, February 2016 393

METABOLISM

via affecting autophagy. The aim of current study was totest this hypothesis and elucidate underlying mechanisms.

RESEARCH DESIGN AND METHODS

Animals and TreatmentMale C57/B6J wild-type (WT) mice were purchased fromShanghai Laboratory Animal Co. Ltd. (Shanghai, China). Male10-week-old leptin receptor–deficient (db/db) mice werekindly provided by Xiang Gao, Nanjing University, Nanjing,China. Mice were maintained as previously described (24).A high-fat diet (HFD) was purchased from Research Diets(60% fat, D12492; Research Diets). For autophagy inhibitortreatment, chloroquine (C6628; Sigma-Aldrich) was dis-solved in PBS and injected at the dose of 60 mg/kg i.p. dailyin mice for 14 days (12,25). For VLDL secretion assay, micewere injected with poloxamer 407 (16758; Sigma-Aldrich) atthe dose of 1 g/kg i.p. and TG levels were measured in serumof tail vein blood taken at different time points (26). Theseexperiments were conducted in accordance with the guide-lines of the Institutional Animal Care and Use Committee ofthe Institute for Nutritional Sciences, Shanghai Institutes forBiological Sciences, Chinese Academy of Sciences.

Primary Hepatocyte Isolation, Cell Culture, andTreatmentsMouse primary hepatocytes were prepared by collagenaseperfusion and treated with SB203580 (S8307; Sigma-Aldrich) as previously described (24,27).

Generation and Administration of RecombinantAdenovirusesRecombinant adenoviral vector expressing rat constitu-tively active MEK1 (Ad-CA MEK1) was kindly provided byHaiyan Xu from Brown University. Adenoviruses express-ing (Ad-) scrambled (Ad-scrambled) and short-hairpin(sh)RNAs specific for ERK1/2 (Ad-shERK1/2) or ATG7(Ad-shATG7) were generated using the BLOCK-iT AdenoviralRNAi Expression System (K4941-00; Invitrogen) accord-ing to the manufacturer’s instructions. The scrambledsequence is 59-TTCTCCGAACGTGTCACGT-39, and theshRNA sequence for mouse ERK1/2 is 59-CACCGCAATGACCACATCTGCTACTCGAAAGTAGCAGATGTGGTCATTGC-39 and for mouse ATG7 is 59-CACCATGAGATCTGGGAAGCCATCGAAATGGCTTCCCAGATCTCAT-39. Double-stranded small interfering (si)RNA targeting mouse p38awas from Cell Signaling Technology (catalog no. 6417).The Ad-ATG7 was constructed using a human ATG7 ex-pression vector (Neuronbiotech Company, Shanghai,China). Recombinant adenoviruses were purified (24)and diluted in PBS and administrated at a dose of 1 3 107

plaque-forming units (pfu)/well in 12-well plates or via tailvein injection using 5 3 108 pfu/mouse (Ad-scrambled,Ad-shERK1/2, and Ad-shATG7) or 13 109 pfu/mouse(Ad–green fluorescent protein [Ad-GFP], Ad-ATG7,and Ad-CA MEK1).

Detection of mRNAs and ProteinsRelative quantification RT-PCR and Western blot analysiswere performed as previously described (24). Primary

antibodies (anti–phosphorylated [p]-ERK1/2 [Thr202/Tyr204] [9106], anti–total (t)-ERK1/2 [9102], anti–p-p38[Thr180/Tyr182] [9211], anti–t-p38 [8690], anti-MEK1[9124], anti-LC3 [2775], anti-CPT1a [12252], anti-proliferating cell nuclear antigen (anti-PCNA) [13110],anti–cyclin D1 [2926], anti-PARP [9542], anti-caspase3[9662], anti–t-AKT [9272], anti–p-AKT [Ser473] [9271],anti–t-GSK3b [9315], anti–p-GSK3b [Ser9] [9336], anti–t-HSL [4107], anti–p-HSL [Ser660] [4126], and anti-ATG7[2631]) (all from Cell Signaling Technology); anti-actin(A4700; Sigma-Aldrich); anti-tubulin (P8203; Sigma-Aldrich);and anti-SQSTM1 (ab56416; Abcam) were incubated over-night at 4°C, and specific proteins were visualized usingECL Plus (9589151; Amersham Biosciences).

Liver and Serum MeasurementsHepatic and cellular lipids were extracted with chloroformmethanol (2:1) as previously described (28). TG, TC, FFAs,alanine transaminase (ALT), aspartate aminotransferase(AST), and b-hydroxybutyrate (3-HB) were measuredwith a TG kit (290-63701; Wako), TC kit (294-65801;Wako), FFA kit (436-91995; Wako), ALT kit (700260;Cayman Chemical), AST kit (K735-100; Bio Vision), and3-HB colorimetric assay kit (700190; Cayman Chemical),respectively, according to the manufacturers’ instructions.

FAO AssaysFAO assays were conducted as previously described (29).

Immunofluorescence AssayImmunofluorescence assay was performed as previouslydescribed (30). Images were obtained using a Zeiss LSM510 confocal microscope (Carl Zeiss Imaging, Oberkochen,Germany).

Histological Analysis of TissuesFrozen sections of liver were stained with Oil red O.Paraformaldehyde-fixed, paraffin-embedded sections of liverwere stained with hematoxylin-eosin (H-E) for histology.

Blood Glucose, Serum Insulin, HOMA of InsulinResistance, Insulin Tolerance Test, and In Vivo InsulinSignaling AssayLevels of blood glucose and serum insulin were measuredusing a Glucometer Elite monitor and a MercodiaUltrasensitive Rat Insulin ELISA kit (03113-1; ALPCODiagnostic), respectively. The HOMA of insulin resistance(HOMA-IR) index was calculated according to the follow-ing formula: [fasting glucose levels (mmol/L)] 3 [fastingserum insulin (mU/mL)]/22.5. The insulin tolerance testwas performed by injection of 0.75 units/kg i.p. insulinafter 4 h of fasting. The in vivo insulin signaling assay wasperformed as previously described (24).

StatisticsAll data are expressed as means 6 SEM. Significant dif-ferences were assessed either by two-tailed Student t testor by one-way ANOVA followed by the Student-Newman-Keuls (SNK) test. P , 0.05 was considered statisticallysignificant.

394 ERK1/2 Regulates Liver Steatosis via Autophagy Diabetes Volume 65, February 2016

RESULTSERK1/2 Activation Ameliorates Liver Steatosis inLeptin Receptor–Deficient (db/db) MiceTo explore a role of ERK1/2 in liver steatosis, we examinedERK1/2 activity in liver of db/db mice, a classic animal modelfor liver steatosis (31). We found that ERK1/2 activity wascompromised in the livers of db/db mice compared with con-trol mice (Fig. 1A). We then injected db/db mice with Ad-CAMEK1, the specific upstream activator of ERK1/2 (17), or

control Ad-GFP. As predicted, overexpression of MEK1 in-creased hepatic ERK1/2 phosphorylation compared with con-trol mice but had no effect on body weight, liver weight, fatmass, or food intake (Fig. 1B–D). In contrast, the extensivelipid deposition manifested as macro- and microvesicularsteatosis (as examined by Oil red O and H-E staining)in the livers of db/db mice disappeared after overexpres-sion of ERK1/2 compared with control mice (Fig. 1B and E).Consistently, liver TG content was also decreased by ERK1/2

Figure 1—Activation of ERK1/2 by Ad-CA MEK1 ameliorates liver steatosis in leptin receptor–deficient (db/db) mice. p-ERK1/2 andt-ERK1/2 in the livers of C57/B6J WT and db/db mice (A). db/db mice were injected with Ad-GFP (- Ad-CA MEK1) or Ad-CA MEK1 (+ Ad-CAMEK1) via tail vein injection for 10 days, and livers were isolated. p-ERK1/2, t-ERK1/2, and MEK1 in the livers (B); body weight, liver weight, fatmass, and food intake of db/db mice injected with Ad-GFP or Ad-CA MEK1 (C and D); Oil red O and H-E staining of representative liversections (320) (E); liver and serum TG and TC and FFAs (F andG); mRNA levels of genes (H–K); CPT1a in the livers (L); and 3-HB and ALT/ASTin the serum (M–O) of mice under different treatments as indicated. Values are means6 SEM (n = 6–7/group) and were analyzed by two-tailedStudent t test. *P < 0.05.

diabetes.diabetesjournals.org Xiao and Associates 395

activation (Fig. 1F). However, serum TG levels were in-creased in Ad-CA MEK1 mice (Fig. 1G), suggesting thatVLDL secretion may be largely enhanced in these mice, asconfirmed by our VLDL secretion assay (Supplementary Fig.1). Liver TC levels were not changed, but serum TC levelswere increased by ERK1/2 activation (Fig. 1F and G). Nei-ther serum nor liver FFA levels were affected by ERK1/2activation (Fig. 1F and G).

The reversal effects of ERK1/2 activation on fatty liverin db/db mice are likely to reflect an effect on hepatic TGsynthesis, b-oxidation, uptake, and/or secretion of fattyacids. We first investigated whether genes underlying thesynthesis of triglycerides and uptake of fatty acids aredifferentially regulated in Ad-CA MEK1 or Ad-GFP db/dbmice. These genes included fatty acid synthase (Fas),acetyl CoA carboxylase 1 (Acc1), stearoyl CoA desatur-ase (Scd1), malic enzyme (Me), glycerol-3-phosphateacyltransferase (Gpat), and transcription factors includingperoxisome proliferator–activated receptor g (Pparg),sterol regulatory element–binding protein 1c (Srebp1c),and carbohydrate-responsive element-binding protein(Chrebp) (32). Genes related to fatty acid uptake includecluster of differentiation 36 (Cd36), fatty acid–bindingprotein (Fabp), and fatty acid–transporting protein (Fatp)(32). Interestingly, genes related to lipogenesis were notaffected or some even increased and fatty acid uptake genesshowed inconsistent changes in livers by Ad-CA MEK1 in-jection in db/db mice (Fig. 1H and I). Consistent with re-versal effects of ERK1/2 activation on liver steatosis, genesinvolved in TG secretion, including apolipoprotein B (ApoB)and apolipoprotein E (ApoE), and fatty acid oxidation, in-cluding peroxisome proliferator–activated receptor a (Ppara)and carnitine palmitoyltransferase 1a (Cpt1a), were sig-nificantly increased in the livers of Ad-CA MEK1 db/dbmice (Fig. 1J and K). Consistently, levels of hepatic CPT1aprotein and serum 3-HB were increased in Ad-CA MEK1db/dbmice (Fig. 1L andM). In addition, levels of serum ALTand AST were decreased by Ad-CA MEK1 (Fig. 1N and O).

ERK1/2 Inhibition Leads to Liver Steatosis in C57/B6JWT MiceTo further investigate the impact of ERK1/2 on liversteatosis in vivo, we injected WT mice with Ad-shERK1/2or Ad-scrambled. Ad-shERK1/2 significantly decreasedhepatic ERK1/2 protein levels, with no effects on bodyweight, liver weight, fat mass, or food intake, but causedliver steatosis as confirmed by Oil red O and H-E staining(Fig. 2A–D). Consistently, levels of hepatic TG, TC, andFFAs were also significantly increased in Ad-shERK1/2mice compared with control mice (Fig. 2E). In contrast,serum TC and FFA levels were decreased, but serum TGlevels were not affected, in Ad-shERK1/2 mice (Fig. 2F).Most genes related to lipogenesis (except for Pparg), FAO,and TG secretion were significantly decreased, but genesrelated to fatty acid uptake showed inconsistent changesin liver of Ad-shERK1/2 mice (Fig. 2G–J). Consistently,

levels of hepatic CPT1a protein and serum 3-HB were alsodecreased in Ad-shERK1/2 mice (Fig. 2K and L). In addi-tion, serum levels of ALT and AST were increased by Ad-shERK1/2 injection (Fig. 2M and N).

ERK1/2 Regulates Autophagy in Hepatocytes In Vivoand In VitroBecause autophagy accelerates FAO and TG export in mouselivers (7) and ERK1/2 is shown to regulate autophagy inHepG2 cells (22), we hypothesized that ERK1/2 might regu-late liver steatosis via autophagy. To test this possibility, weexamined expression of hepatic LC3-II (a positive autophagymarker) and SQSTM1/p62 (a negative autophagy marker)(33) in db/db and WT mice and found that autophagy wasattenuated (as demonstrated by decreased LC3-II and in-creased SQSTM1 expression) in db/db mice, as reported pre-viously (33), which was reversed by Ad-CAMEK1 (Fig. 3A andB). Opposite effects were observed in WT mice with ERK1/2knockdown (Fig. 3C). Similar effects were observed in primaryhepatocytes (Fig. 3D and E). Consistently, autophagic fluxwas enhanced by ERK1/2 activation or inhibited by ERK1/2knockdown, respectively, in primary hepatocytes transfectedor infected with RFP-LC3 plasmid or Ad-CA MEK1 or Ad-shERK1/2 (Fig. 3F). Because autophagy is a dynamic process,we further treated cells with chloroquine (CQ), a lysosomo-tropic weak base that blocks the fusion of autophagosomewith lysosome (12,25,33). In the presence of CQ, a greateramount of autophagasome was seen in control cells, but theincrease was much bigger in Ad-CA MEK1 cells or smaller inAd-shERK1/2 cells (Fig. 3F).

Inhibition of Autophagic Flux by CQ Reverses theAmeliorated Liver Steatosis in Ad-CA MEK1 db/dbMiceTo confirm a role for autophagy in mediating ERK1/2-ameliorated liver steatosis in db/db mice, we injected Ad-CAMEK1 db/db mice with PBS or CQ (12,25) for 14 days. p-ERK1/2 and MEK1 were increased by Ad-CA MEK1, in thepresence or absence of CQ treatment, in db/db mice (Supple-mentary Fig. 2). Autophagic flux inhibited by CQ (as con-firmed by Western blot) had no effects on body weight,liver weight, fat mass, or food intake but reversed the effectsof Ad-CA MEK1 on ameliorated liver steatosis (as confirmedby Oil red O and H-E staining and hepatic TG measurement)in db/db mice (Fig. 4A–E). CQ treatment had no effects onlevels of liver TC and FFAs and serum FFAs but decreasedserum TG and TC levels in Ad-CA MEK1 db/db mice (Fig. 4Eand F). In addition, CQ treatment reversed the effects of Ad-CA MEK1 on hepatic genes expression related to TG secretionand FAO, but had no effects on other genes, in db/db mice(Fig. 4G–J). Levels of hepatic CPT1a protein and serum 3-HBwere also reversed in Ad-CA MEK1 db/db mice by CQ treat-ment (Fig. 4K and L). Furthermore, the decreased serum levelsof ALT and AST of Ad-CA MEK1 db/db mice were also re-versed by CQ treatment (Fig. 4M and N). In addition, CQtreatment had similar effects in db/db mice injected with con-trol Ad-GFP (Supplementary Fig. 3).

396 ERK1/2 Regulates Liver Steatosis via Autophagy Diabetes Volume 65, February 2016

ERK1/2 Regulates Autophagy via ATG7 in HepatocytesIn Vitro and In VivoTo investigate molecular mechanisms underlying ERK1/2control of autophagy, we examined mRNA expression ofautophagy regulators Atg4a, Atg5, Atg6 (Beclin-1 [Becn1]), andAtg7, all important for the production of autophagosome (34),in primary hepatocytes infected with Ad-CA MEK1 or controlAd-GFP. We found that mRNA levels of Atg4a, Atg5, and Atg7were increased by ERK1/2 activation (Fig. 5A). Opposite ef-fects were observed when ERK1/2 was knocked down by Ad-shERK1/2 (Fig. 5B). As observed in vitro, Ad-CA MEK1 alsoinduced Atg4a, Atg5, and Atg7 expression in the livers of db/db

mice, whereas Ad-shERK1/2 only inhibited Atg7, but notAtg4a and Atg5, expression in the livers of WT mice (Fig. 5Cand D). Hepatic Becn1 expression, however, was not affectedby Ad-CAMEK1 or Ad-shERK1/2 (Fig. 5A–D). Consistent withchanges in Atg7 mRNA, ATG7 protein levels were also in-creased or decreased by ERK1/2 activation or knockdown,respectively, in primary hepatocytes (Fig. 5E and F).Compared with WT mice, hepatic ATG7 protein levelswere decreased in db/db mice, which was further stimu-lated by ERK1/2 activation (Fig. 5G and H). Consistently,ERK1/2 knockdown decreased hepatic ATG7 protein lev-els in WT mice (Fig. 5I).

Figure 2—ERK1/2 inhibition induces liver steatosis in C57/B6J WT mice. WT mice were injected with Ad-scrambled (□) or Ad-shERK1/2(■) via tail vein injection for 10 days, and livers were isolated. t-ERK1/2 in the livers (A); body weight, liver weight, fat mass, and food intakeof mice injected with Ad-scrambled or Ad-shERK1/2 (B and C); Oil red O and H-E staining of representative liver sections (320) (D); liverand serum TG, TC, and FFAs (E and F ); mRNA levels of genes (G–J); CPT1a in the livers (K); and 3-HB and ALT/AST in the serum (L–N) ofmice under different treatments as indicated. Values are means 6 SEM (n = 6–7/group) and were analyzed by two-tailed Student t test.*P < 0.05.

diabetes.diabetesjournals.org Xiao and Associates 397

Inhibition of ATG7-Dependent Autophagy Reverses theAmeliorated Liver Steatosis in Ad-CA MEK1 db/db MiceTo investigate whether ATG7-dependent autophagy isresponsible for the ameliorated liver steatosis in Ad-CAMEK1 db/db mice, we injected db/db mice with Ad-CAMEK1 in combination with Ad-shATG7 or Ad-scrambled.ATG7 knockdown (as confirmed by Western blot) had no

effects on body weight, liver weight, fat mass, or foodintake but reversed the effects of Ad-CA MEK1 on im-proved autophagy (as measured by autophagy-relatedmarkers) and ameliorated liver steatosis (as confirmedby Oil red O and H-E staining and hepatic TG measure-ment) in db/db mice (Fig. 6A–D). ATG7 knockdown hadno effects on levels of liver TC and FFAs and serum FFAs

Figure 3—ERK1/2 regulates autophagy in vivo and in vitro. LC3-II and SQSTM1/p62 proteins in the livers of C57/B6J wild-type (WT) orleptin receptor–deficient (db/db) mice (A), in the livers of db/db mice injected with Ad-GFP (- Ad-CA MEK1) or Ad-CA MEK1 (+ Ad-CAMEK1) (B), or in the livers of WT mice or primary hepatocytes injected or infected with Ad-scrambled (- Ad-shERK1/2) or Ad-shERK1/2 (+Ad-shERK1/2) (C and E). LC3-II, SQSTM1, and MEK1 proteins in primary hepatocytes infected with Ad-GFP (- Ad-CA MEK1) or Ad-CAMEK1 (+ Ad-CA MEK1) (D). RFP-LC3 dots in primary hepatocytes infected with Ad-CA MEK1 or Ad-shERK1/2 and treated with CQ(10 mmol/L) or not for 6 h before fixed by 4% paraformaldehyde (363) (F ). Values are means 6 SEM (n = 6–7/group) of at least threeindependent in vitro experiments and were analyzed by two-tailed Student t test. *P < 0.05.

398 ERK1/2 Regulates Liver Steatosis via Autophagy Diabetes Volume 65, February 2016

but decreased serum TG and TC levels in Ad-CA MEK1db/db mice (Fig. 6E and F). In addition, ATG7 knockdownreversed the effects of Ad-CA MEK1 on hepatic gene ex-pression related to TG secretion and FAO, but not othergenes, in db/db mice (Fig. 6G–J). Consistently, levels ofhepatic CPT1a protein and serum 3-HB were reversed inAd-CA MEK1 db/db mice injected with Ad-shATG7 (Fig. 6Kand L). Furthermore, the decreased serum levels of ALTand AST of Ad-CA MEK1 db/db mice were also reversedby Ad-shATG7 injection (Fig. 6M and N). In addition,

Ad-shATG7 had similar effects in db/db mice injectedwith control Ad-GFP (Supplementary Fig. 4).

Restoration of ATG7-Dependent AutophagyAmeliorates Liver Steatosis in Ad-shERK1/2 MiceAtg7 triggers autophagy and ameliorates liver lipidstorage in leptin-deficient (ob/ob) mice (33). So, we in-jected Ad-shERK1/2 mice with Ad-ATG7 or Ad-GFP andexamined whether ATG7 overexpression could amelioratefatty liver in these mice. Ad-ATG7 overexpression (as

Figure 4—Inhibition of autophagic flux by CQ reverses the ameliorated liver steatosis in Ad-CA MEK1 leptin receptor–deficient (db/db)mice. db/db mice were injected with Ad-CA MEK1 (+ Ad-CA MEK1) via tail vein injection and PBS or CQ via intraperitoneal injection for14 days, and livers were isolated. LC3-II protein in the livers (A); body weight, liver weight, fat mass, and food intake of mice under differenttreatment as indicated (B and C); Oil red O and H-E staining of representative liver sections (320) (D); liver and serum TG, TC, and FFAs(E and F ); mRNA levels of genes (G–J); CPT1a in the livers (K); and 3-HB and ALT/AST in the serum (L–N ) of mice under different treatmentsas indicated. Values are means 6 SEM (n = 6–7/group) and were analyzed by two-tailed Student t test. *P < 0.05.

diabetes.diabetesjournals.org Xiao and Associates 399

confirmed by Western blot analysis) had no effects on bodyweight, liver weight, fat mass, or food intake but reversedthe suppressive effects of Ad-shERK1/2 on hepatic auto-phagy as demonstrated by the changes of autophagy-relatedmarkers and ameliorated ERK1/2 inhibition–induced liversteatosis as confirmed by H-E and Oil red O staining (Fig.7A–D). In addition, Ad-ATG7 reversed the increasing effectsof Ad-shERK1/2 on liver TG and FFAs, but not liver TC,compared with control mice (Fig. 7E). Ad-shERK1/2 de-creased serum TC but not TGs, and FFAs were increasedby Ad-ATG7 (Fig. 7F). Furthermore, Ad-ATG7 reversed thesuppressive effects of Ad-shERK1/2 on some of genes re-lated to lipogenesis and fatty acid uptake in liver (Fig. 7Gand H). Genes related to TG secretion and FAO, which weredecreased by Ad-shERK1/2 in liver, were also largely re-versed by Ad-ATG7 (Fig. 7I–J). Consistently, levels of hepaticCPT1a protein and serum 3-HB were reversed by Ad-ATG7in Ad-shERK1/2 mice (Fig. 7K and L). Furthermore, the in-creased serum levels of ALT and AST of Ad-shERK1/2 micewere also reversed by Ad-ATG7 (Fig. 7M and N).

ERK1/2 Regulates ATG7 and Autophagy in a p38-Dependent Pathway in Primary HepatocytesPrevious work has shown that ERK1/2 inhibits phosphory-lation of p38 (35), which is a negative regulator of autophagy

(23,36). We found that p38 phosphorylation was increasedin the livers of db/db mice, and the increased p38 phosphor-ylation was significantly decreased by Ad-CA MEK1 in thelivers of db/db mice or primary hepatocytes (Fig. 8A and B).Consistent with these changes, inhibition of ERK1/2 in-creased p38 phosphorylation in the livers of WT mice andprimary hepatocytes (Fig. 8C). To confirm a role of p38 inERK1/2 regulated autophagy, we infected or transfectedprimary hepatocytes with Ad-shERK1/2 or p38a siRNA for72 h. As predicted, p38 knockdown significantly increasedexpression of ATG7 and LC3-II and decreased expression ofSQSTM1 in primary hepatocytes infected with Ad-shERK1/2(Fig. 8D). Similar results were obtained with p38-specific in-hibitor SB203580 (Supplementary Fig. 5).

DISCUSSION

The research concerning the metabolic functions ofERK1/2 is primarily focused on nonhepatic tissues, suchas hypothalamus (37) and adipose tissue (14). In thisstudy, we found that 1) ERK1/2 activity is compromisedin the livers of db/db mice and activation of ERK1/2 up-stream regulator MEK1 is sufficient to reduce lipid accumula-tion in these mice, 2) knockdown of ERK1/2 in C57/B6J WTmice results in liver steatosis, and 3) manipulation of ERK1/2

Figure 5—ERK1/2 regulates autophagy via ATG7 in vitro and in vivo. Atg4a, Atg5, Becn1, and Atg7mRNA levels in primary hepatocytes orthe livers of leptin receptor–deficient (db/db) mice infected or injected with Ad-GFP (- Ad-CA MEK1) or Ad-CA MEK1 (+ Ad-CA MEK1)(A and C) or in primary hepatocytes or the livers of C57/B6J WT mice infected or injected with Ad-scrambled (- Ad-shERK1/2) or Ad-shERK1/2 (+ Ad-shERK1/2) (B and D). ATG7 protein in primary hepatocytes infected with Ad-GFP (- Ad-CA MEK1) or Ad-CA MEK1 (+ Ad-CA MEK1) (E), or Ad-scrambled (- Ad-shERK1/2) or Ad-shERK1/2 (+ Ad-shERK1/2) (F ), in the livers of WT and db/db mice (G), db/db miceinjected with Ad-GFP (- Ad-CA MEK1) or Ad-CA MEK1 (+ Ad-CA MEK1) (H), or WT mice injected with Ad-scrambled (- Ad-shERK1/2) or Ad-shERK1/2 (+ Ad-shERK1/2) (I). Values are means 6 SEM (n = 6–7/group) of at least three independent in vitro experiments and wereanalyzed by two-tailed Student t test. *P < 0.05.

400 ERK1/2 Regulates Liver Steatosis via Autophagy Diabetes Volume 65, February 2016

activity controls lipid accumulation in HepG2 cells and pri-mary hepatocytes (Supplementary Fig. 6). These resultsstrongly suggest that ERK1/2 serves as a potent regulatorof lipid accumulation both in vivo and in vitro. However,hepatic ERK1/2 activity is increased in ob/ob and HFD mice,as demonstrated by previous work (17). We speculate thatthe different ERK1/2 activity might be caused by the com-plicated mechanisms involved in the control of ERK1/2activity. In addition to well-known upstream cellular sur-face receptors and their downstream effectors, various

hormone levels (including insulin, glucagon, and leptin)and nutritional status (including HFD and high-carbohydratediet) and even self-signaling can influence ERK1/2 activ-ity (17,38,39).

Our results show that hepatic expression of genesrelated to FAO (Ppara, Cpt1a) and TG export (ApoB,ApoE) was increased by activation of ERK1/2 in db/dbmice or decreased by ERK1/2 knockdown in WT mice.The change in b-oxidation genes was accompanied bycorresponding changes in CPT1a protein expression,

Figure 6—Inhibition of ATG7-dependent autophagy reverses the ameliorated liver steatosis in Ad-CA MEK1 leptin receptor–deficient (db/db)mice. db/db mice were injected with Ad-CA MEK1 (+ Ad-CA MEK1), Ad-scrambled (- Ad-shATG7), or Ad-shATG7 (+ Ad-shATG7), as indicated,via tail vein injection for 10 days, and livers were isolated. LC3-II, SQSTM1, and ATG7 proteins in the livers (A); body weight, liver weight, fatmass, and food intake of mice under different treatment as indicated (B and C); Oil red O and H-E staining of representative liver sections (320)(D); liver and serum TG, TC, and FFAs (E and F); mRNA levels of genes (G–J); CPT1a in the livers (K); and 3-HB and ALT/AST in the serum (L–N)of mice under different treatments as indicated. Values are means 6 SEM (n = 6–7/group) and analyzed by two-tailed Student t test. *P < 0.05.

diabetes.diabetesjournals.org Xiao and Associates 401

serum 3-HB levels, and FAO rates measured under eachtreatment (Supplementary Fig. 7). In contrast, Jiao et al.(17) showed that the expression of these genes is de-creased when hepatic ERK1/2 is enhanced in WT mice,which might be explained by the difference in fat contentbetween WT and db/db mice. Because genes related to TGexport were also stimulated by ERK1/2 activation, thepossible contribution of increased TG export in ERK1/2ameliorating liver steatosis should not be ignored. Liverlipid levels can also be largely controlled by insulin

signaling in liver and lipolysis of fat tissue (40). Consis-tent with previous reports (16,17), we found that insulinsignaling is enhanced in Ad-shERK1/2 mouse livers, andmRNAs and proteins related to lipolysis in the fat tissueof Ad-shERK1/2 mice are inhibited (Supplementary Fig.8). These results suggest that insulin signaling and lipol-ysis in fat tissue may contribute to ERK1/2-regulated he-patic lipid storage in mice.

Autophagy is shown to stimulate lipid clearance(6,7,9), and manipulation of autophagic activity affects fat

Figure 7—ATG7 overexpression ameliorates liver steatosis in C57/B6J WT mice injected with Ad-shERK1/2. WT mice were injected withAd-scrambled (- Ad-shERK1/2) or Ad-shERK1/2 (+ Ad-shERK1/2), Ad-GFP (- Ad-ATG7), or Ad-ATG7 (+ Ad-ATG7), as indicated, via tail veininjection for 10 days, and livers were isolated. LC3-II, SQSTM1, t-ERK1/2, and ATG7 proteins in the livers (A); body weight, liver weight, fatmass, and food intake of mice under different treatment as indicated (B and C); Oil red O and H-E staining of representative liver sections(320) (D); liver and serum TG, TC, and FFAs (E and F ); mRNA levels of genes (G and J); CPT1a in the livers (K); and 3-HB and ALT/AST inthe serum (L–N) of mice under different treatments as indicated. Values are means 6 SEM (n = 6–7/group) and were analyzed by one-wayANOVA followed by the SNK test. *P < 0.05; #P < 0.05.

402 ERK1/2 Regulates Liver Steatosis via Autophagy Diabetes Volume 65, February 2016

accumulation in liver (11–13). In this study, we foundthat ERK1/2 positively regulates hepatic autophagy bothin vivo and in vitro. Furthermore, expression of ATG7, akey protein for autophagosome formation (34), is alsoregulated by ERK1/2. Although ATG7 protein can be post-translationally regulated via altered calpain activity (33),we did not observe any effects of ERK1/2 on calpainactivity (data not shown), suggesting that ERK1/2 regu-lation of ATG7 expression most likely occurs at transcrip-tional levels. The involvement of ATG7 in ERK1/2amelioration of liver steatosis was then confirmed bythe reversal effects of CQ treatment, and ATG7 knock-down on Ad-CA MEK1 ameliorated liver steatosis in db/dbmice. We also found that ATG7 overexpression signifi-cantly reversed liver steatosis in Ad-shERK1/2 mice, asshown previously (33). Again, ERK1/2-regulated expres-sion of hepatic genes related to FAO and TG export, aswell as serum 3-HB levels, was affected by CQ and ATG7under each different treatment.

Previous study has shown that ERK1/2 enhancesautophagy by positively regulating BECN1 (21). We didnot, however, observe any significant effects of ERK1/2on Becn1 expression in vitro or in vivo. Interestingly, wefound that p38 knockdown reversed the suppressive ef-fects of Ad-shERK1/2 on autophagy and ATG7 expressionin mouse primary hepatocytes. Consistent with our re-sults, a previous study has shown that activation of p38by osmotic stress inhibits autophagy in rat hepatocytes

(36). A possible role for p38 in ERK1/2 regulation of ATG7-dependent autophagy in vivo and underlying mechanisms,however, remains to be further explored in the future.

A change in liver fat content is normally associatedwith a change in liver weight, and ERK1/2 has beenshown to promote cell proliferation (15). Unexpectedly,we found that liver weight remains unchanged, thoughliver steatosis is ameliorated or induced by manipulationof hepatic ERK1/2 activity. We assume that the weight-lowering effects of the decreased liver TG on liver mightbe compensated by the enhanced cell proliferation asdemonstrated by the increased expression of proliferationmarkers PCNA and cyclin D1 after ERK1/2 activation(Supplementary Fig. 9). Reciprocally, a balanced liverweight is reached between the increased liver TG andthe decreased cell proliferation by ERK1/2 knockdown(Supplementary Fig. 9).

ERK1/2 is possibly activated in liver of young db/dband HFD mice, as insulin activates ERK1/2 (41) and se-rum insulin increases during this period (42). However,ERK1/2 may become inactivated in elder mice and resultin decreasing hepatocyte proliferation. Given the closerelationship between cell proliferation and lipid metabo-lism (43,44), changes in proliferation resulting from ma-nipulation of ERK1/2 activity may also influence the lipidaccumulation in db/db or HFD mice.

In addition to db/db mice, we also investigated therelevance of the role of ERK1/2 in the regulation of

Figure 8—ERK1/2 regulates ATG7 and autophagy in a p38-dependent pathway in primary hepatocytes. p-p38 and t-p38 proteins in thelivers of C57/B6J WT and leptin receptor–deficient (db/db) mice (A), in the livers of db/db mice or primary hepatocytes injected or infectedwith Ad-GFP (- Ad-CA MEK1) or Ad-CA MEK1 (+ Ad-CA MEK1) (B), or in the livers of WT mice or primary hepatocytes injected or infectedwith Ad-scrambled (- Ad-shERK1/2) or Ad-shERK1/2 (+ Ad-shERK1/2) (C ). ATG7, LC3-II, SQSTM1, t-p38a, and t-ERK1/2 proteins inprimary hepatocytes infected with adenovirus or transfected with p38a siRNA as indicated for 72 h (D). Working model (E). Values aremeans 6 SEM (n = 6–7/group) or at least three independent in vitro experiments and were analyzed by two-tailed Student t test. *P <0.05 in A–C, or one-way ANOVA followed by the SNK test. *P < 0.05; #P < 0.05 in D.

diabetes.diabetesjournals.org Xiao and Associates 403

autophagy and liver steatosis in mice maintained onan HFD for 12 weeks. Because hepatic ERK1/2 activityis increased in HFD mice (17), we injected HFD micewith Ad-shERK1/2 and found that ERK1/2 knockdownsignificantly decreased hepatic autophagy and exacerbatedliver steatosis in HFD mice (Supplementary Fig. 10). How-ever, another study shows that ERK1/2 knockdown hadno effect on hepatic TG in mice fed an HFD for 38 weeks(17). The lack of the response to ERK1/2 knockdown inthe latter case could be due to the much longer period forHFD feeding: mice maintained on an HFD for 38 weeksalready have much severe liver steatosis, which makes itdifficult to further increase liver steatosis by ERK1/2knockdown. A role for ERK1/2 in liver steatosis of ob/obmice is implied by a previous study showing that globalknockout of ERK1 reduces liver steatosis in ob/ob mice(45), but it is unclear whether it was due to a direct orindirect effect of ERK1 on liver steatosis. Therefore, a rolefor ERK1/2 in HFD and ob/ob mice remains to be inves-tigated in the future.

We also explored the possible influence of ERK1/2 oninsulin resistance, as liver steatosis is shown to be linked toinsulin resistance. For example, it is shown that activation ofERK1/2 promotes insulin resistance and inhibition of ERK1/2 improves insulin sensitivity in WT mice and micemaintained on an HFD for 38 weeks (16,17), the effect ofwhich might be mediated by ERK1/2 stimulation of serinephosphorylation of insulin receptor substrate 1 (46). Onthe other hand, enhancement of autophagy is reported toameliorate insulin resistance and liver steatosis (33). Pos-sibly due to the combined effects of activation of insulinreceptor substrate 1 serine phosphorylation and stimula-tion of autophagy, we did not observe any significanteffects of ERK1/2 activation on fed/fasting blood glucose,fasting serum insulin, and HOMA-IR index in db/db mice,while the insulin tolerance test results indicated thatthe insulin resistance is exacerbated in Ad-CA MEK1db/db mice (Supplementary Fig. 11).

Amelioration of liver steatosis by inhibiting TG syn-thesis exacerbates liver damage (47). On the other hand,enhanced autophagy ameliorates liver injury and inhibitedautophagy exacerbates liver damage (12,25). Therefore, theincreased autophagy by ERK1/2 activation may accountfor the protective effects for liver damage in db/db mice,as demonstrated by the effects of ERK1/2 on serum ALTand AST under different treatment. Furthermore, lipidstorage in hepatocytes, autophagy, and ERK1/2 itselfare linked with apoptosis in a complex manner(15,48,49). However, we found that apoptosis is enhancedby ERK1/2 knockdown or inhibited by ERK1/2 activationin primary hepatocytes (Supplementary Fig. 12).

In summary, we have discovered a novel function ofERK1/2 in regulating hepatic lipid metabolism that ismediated by ATG7-dependent autophagy (Fig. 8E). Theseresults provide novel insights into a physiological role ofERK1/2 in liver and theoretical basis for activating ERK1/2as a potential treatment target for liver steatosis and its

associated diseases. However, in evaluation of the benefi-cial effects of ERK1/2 activation on liver steatosis, itspotential deleterious effects on insulin sensitivity andelevated serum TC, a risk factor for the developmentof heart attacks and strokes (50), should not be ignored.

Acknowledgments. The authors thank Haiyan Xu from Brown Universityfor kindly providing Ad-CA MEK1. The authors thank Zhixue Liu from the Institutefor Nutritional Sciences, Shanghai Institute for Biological Sciences, The ChineseAcademy of Sciences, for helpful discussion.Funding. This work was supported by grants from the National Natural ScienceFoundation (81130076, 81325005, 31271269, 81100615, and 81390350), theMinistry of Science and Technology of China (973 Program 2010CB912502), theBasic Research Project of Shanghai Science and Technology Commission(13JC1409000), and the International S&T Cooperation Program of China(Singapore 2014DFG32470) and by research supported by the The ChineseAcademy of Sciences/State Administration of Foreign Experts Affairs interna-tional partnership program for creative research teams. F.G. was also sup-ported by the One Hundred Talents Program of the Chinese Academy ofSciences.Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. Y.X. and H.L. researched data and wrote,reviewed, and edited the manuscript. J.Y., Z.Z., T.X., C.W., K.L., J.D., and Y.G.researched data. F.X. and Y.C. contributed to writing and helpful discussion. S.C.provided research material. F.G. directed the project, contributed to discussion,and wrote, reviewed, and edited the manuscript. F.G. is the guarantor of thiswork and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

References1. Liu W, Cao H, Yan J, Huang R, Ying H. ‘Micro-managers’ of hepatic lipidmetabolism and NAFLD. Wiley Interdiscip Rev RNA 2015;6:581–5932. Kerr TA, Davidson NO. Cholesterol and nonalcoholic fatty liver disease:renewed focus on an old villain. Hepatology 2012;56:1995–19983. Neuschwander-Tetri BA, Caldwell SH. Nonalcoholic steatohepatitis:summary of an AASLD Single Topic Conference. Hepatology 2003;37:1202–12194. Postic C, Girard J. Contribution of de novo fatty acid synthesis to hepaticsteatosis and insulin resistance: lessons from genetically engineered mice. J ClinInvest 2008;118:829–8385. Mizushima N. Physiological functions of autophagy. Curr Top MicrobiolImmunol 2009;335:71–846. Singh R, Cuervo AM: Lipophagy: connecting autophagy and lipid metabo-lism. Int J Cell Biol 2012;2012:2820417. Singh R, Kaushik S, Wang Y, et al. Autophagy regulates lipid metabolism.Nature 2009;458:1131–11358. Shen HM, Mizushima N. At the end of the autophagic road: an emergingunderstanding of lysosomal functions in autophagy. Trends Biochem Sci 2014;39:61–719. Dong H, Czaja MJ. Regulation of lipid droplets by autophagy. Trends En-docrinol Metab 2011;22:234–24010. Jaber N, Dou Z, Chen JS, et al. Class III PI3K Vps34 plays an essential rolein autophagy and in heart and liver function. Proc Natl Acad Sci U S A 2012;109:2003–200811. Sinha RA, Farah BL, Singh BK, et al. Caffeine stimulates hepatic lipidmetabolism by the autophagy-lysosomal pathway in mice. Hepatology 2014;59:1366–138012. Lin C-W, Zhang H, Li M, et al. Pharmacological promotion of autophagyalleviates steatosis and injury in alcoholic and non-alcoholic fatty liver conditionsin mice. J Hepatol 2013;58:993–999

404 ERK1/2 Regulates Liver Steatosis via Autophagy Diabetes Volume 65, February 2016

13. Sharma S, Mells JE, Fu PP, Saxena NK, Anania FA. GLP-1 analogs reducehepatocyte steatosis and improve survival by enhancing the unfolded proteinresponse and promoting macroautophagy. PLoS One 2011;6:e2526914. Bost F, Aouadi M, Caron L, et al. The extracellular signal-regulated kinaseisoform ERK1 is specifically required for in vitro and in vivo adipogenesis.Diabetes 2005;54:402–41115. Wiesenauer CA, Yip-Schneider MT, Wang Y, Schmidt CM. Multiple anti-cancer effects of blocking MEK-ERK signaling in hepatocellular carcinoma. J AmColl Surg 2004;198:410–42116. Liu H, Yu J, Xia T, et al. Hepatic serum- and glucocorticoid-regulated proteinkinase 1 (SGK1) regulates insulin sensitivity in mice via extracellular-signal-regulated kinase 1/2 (ERK1/2). Biochem J 2014;464:281–28917. Jiao P, Feng B, Li Y, He Q, Xu H. Hepatic ERK activity plays a role in energymetabolism. Mol Cell Endocrinol 2013;375:157–16618. Tsai J, Qiu W, Kohen-Avramoglu R, Adeli K. MEK-ERK inhibition corrects thedefect in VLDL assembly in HepG2 cells: potential role of ERK in VLDL-ApoB100particle assembly. Arterioscler Thromb Vasc Biol 2007;27:211–21819. Mauvoisin D, Prévost M, Ducheix S, Arnaud MP, Mounier C. Key role of theERK1/2 MAPK pathway in the transcriptional regulation of the Stearoyl-CoADesaturase (SCD1) gene expression in response to leptin. Mol Cell Endocrinol2010;319:116–12820. Yousefi B, Darabi M, Baradaran B, et al. Inhibition of MEK/ERK1/2 signalingaffects the fatty acid composition of HepG2 human hepatic cell line. Bioimpacts2012;2:145–15021. Wang J, Whiteman MW, Lian H, et al. A non-canonical MEK/ERK signalingpathway regulates autophagy via regulating Beclin 1. J Biol Chem 2009;284:21412–2142422. Gong K, Chen C, Zhan Y, Chen Y, Huang Z, Li W. Autophagy-related gene 7(ATG7) and reactive oxygen species/extracellular signal-regulated kinase regulatetetrandrine-induced autophagy in human hepatocellular carcinoma. J Biol Chem2012;287:35576–3558823. Keil E, Höcker R, Schuster M, et al. Phosphorylation of Atg5 by the Gadd45b-MEKK4-p38 pathway inhibits autophagy. Cell Death Differ 2013;20:321–33224. Wang Q, Jiang L, Wang J, et al. Abrogation of hepatic ATP-citrate lyaseprotects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice. Hepatology 2009;49:1166–117525. Ding WX, Li M, Chen X, et al. Autophagy reduces acute ethanol-inducedhepatotoxicity and steatosis in mice. Gastroenterology 2010;139:1740–175226. Palmer WK, Emeson EE, Johnston TP. Poloxamer 407-induced athero-genesis in the C57BL/6 mouse. Atherosclerosis 1998;136:115–12327. Willaime S, Vanhoutte P, Caboche J, Lemaigre-Dubreuil Y, Mariani J, Brugg B.Ceramide-induced apoptosis in cortical neurons is mediated by an increase in p38phosphorylation and not by the decrease in ERK phosphorylation. Eur J Neurosci2001;13:2037–204628. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation andpurification of total lipides from animal tissues. J Biol Chem 1957;226:497–50929. Shao M, Shan B, Liu Y, et al. Hepatic IRE1a regulates fasting-inducedmetabolic adaptive programs through the XBP1s-PPARa axis signalling. NatCommun 2014;5:352830. Yuan L, Xiao Y, Zhou Q, et al. Proteomic analysis reveals that MAEL, acomponent of nuage, interacts with stress granule proteins in cancer cells. OncolRep 2014;31:342–35031. Anstee QM, Goldin RD. Mouse models in non-alcoholic fatty liver diseaseand steatohepatitis research. Int J Exp Pathol 2006;87:1–16

32. Horton JD, Shimomura I. Sterol regulatory element-binding proteins: activatorsof cholesterol and fatty acid biosynthesis. Curr Opin Lipidol 1999;10:143–15033. Yang L, Li P, Fu S, Calay ES, Hotamisligil GS. Defective hepatic autophagy inobesity promotes ER stress and causes insulin resistance. Cell Metab 2010;11:467–47834. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in auto-phagosome formation. Annu Rev Cell Dev Biol 2011;27:107–13235. Berra E, Diaz-Meco MT, Moscat J. The activation of p38 and apoptosis bythe inhibition of Erk is antagonized by the phosphoinositide 3-kinase/Akt path-way. J Biol Chem 1998;273:10792–1079736. Häussinger D, Schliess F, Dombrowski F, Vom Dahl S. Involvement ofp38MAPK in the regulation of proteolysis by liver cell hydration. Gastroenterology1999;116:921–93537. Rahmouni K, Sigmund CD, Haynes WG, Mark AL. Hypothalamic ERK me-diates the anorectic and thermogenic sympathetic effects of leptin. Diabetes2009;58:536–54238. Weng L-P, Smith WM, Brown JL, Eng C. PTEN inhibits insulin-stimulatedMEK/MAPK activation and cell growth by blocking IRS-1 phosphorylation and IRS-1/Grb-2/Sos complex formation in a breast cancer model. Hum Mol Genet 2001;10:605–61639. Li XC, Carretero OA, Shao Y, Zhuo JL. Glucagon receptor-mediated extra-cellular signal-regulated kinase 1/2 phosphorylation in rat mesangial cells: role ofprotein kinase A and phospholipase C. Hypertension 2006;47:580–58540. Gan L, Xiang W, Xie B, Yu L. Molecular mechanisms of fatty liver in obesity.Front Med 2015;9:275–28741. Feng B, Jiao P, Yang Z, Xu H. MEK/ERK pathway mediates insulin-promoteddegradation of MKP-3 protein in liver cells. Mol Cell Endocrinol 2012;361:116–12342. Wang B, Chandrasekera PC, Pippin JJ. Leptin- and leptin receptor-deficientrodent models: relevance for human type 2 diabetes. Curr Diabetes Rev 2014;10:131–14543. Sekiya S, Suzuki A. Glycogen synthase kinase 3 b-dependent Snail deg-radation directs hepatocyte proliferation in normal liver regeneration. Proc NatlAcad Sci U S A 2011;108:11175–1118044. Liu Y, Shao M, Wu Y, et al. Role for the endoplasmic reticulum stress sensorIRE1a in liver regenerative responses. J Hepatol 2015;62:590–59845. Jager J, Corcelle V, Grémeaux T, et al. Deficiency in the extracellular signal-regulated kinase 1 (ERK1) protects leptin-deficient mice from insulin resistancewithout affecting obesity. Diabetologia 2011;54:180–18946. Bouzakri K, Roques M, Gual P, et al. Reduced activation of phosphatidyli-nositol-3 kinase and increased serine 636 phosphorylation of insulin receptorsubstrate-1 in primary culture of skeletal muscle cells from patients with type 2diabetes. Diabetes 2003;52:1319–132547. Yamaguchi K, Yang L, McCall S, et al. Inhibiting triglyceride synthesis im-proves hepatic steatosis but exacerbates liver damage and fibrosis in obese micewith nonalcoholic steatohepatitis. Hepatology 2007;45:1366–137448. Li ZZ, Berk M, McIntyre TM, Feldstein AE. Hepatic lipid partitioning and liverdamage in nonalcoholic fatty liver disease: role of stearoyl-CoA desaturase. J BiolChem 2009;284:5637–564449. Wu M, Lao Y, Xu N, et al. Guttiferone K induces autophagy and sensitizescancer cells to nutrient stress-induced cell death. Phytomedicine 2015;22:902–91050. Verschuren WM, Jacobs DR, Bloemberg BP, et al. Serum total cholesteroland long-term coronary heart disease mortality in different cultures. Twenty-five-year follow-up of the seven countries study. JAMA 1995;274:131–136

diabetes.diabetesjournals.org Xiao and Associates 405