ARTICLE

Role for sterol regulatory element binding protein-1c activationin mediating skeletal muscle insulin resistance via repressionof rat insulin receptor substrate-1 transcription

Yan Bi & Wenjun Wu & Junfeng Shi & Hua Liang & Wenwen Yin & Yingying Chen &

Sunyinyan Tang & Shu Cao & Mengyin Cai & Shanmei Shen & Qian Gao & Jianping Weng &

Dalong Zhu

Received: 31 August 2013 /Accepted: 15 November 2013 /Published online: 21 December 2013# Springer-Verlag Berlin Heidelberg 2013

AbstractAims/hypothesis Sterol regulatory element binding protein-1c(SREBP-1c) is a master regulator of fatty acid synthase andcontrols lipogenesis. IRS-1 is the key insulin signalling medi-ator in skeletal muscle. In the present study, we investigatedthe role of SREBP-1c in the regulation of IRS-1 in skeletalmuscle cells.Methods L6 muscle cells were treated with palmitic acid (PA)or metformin. Adenovirus vectors expressing Srebp-1c (alsoknown as Srebf1 ) and small interfering RNA (siRNA) againstSrebp-1c were transfected into the L6 cells. Protein–DNAinteractions were assessed by luciferase reporter analysis,electrophoretic mobility shift assay and chromatin immuno-precipitation assay.

Results We found that both gene and protein expression ofSREBP-1c was increased in contrast to IRS-1 expression inPA-treated L6 cells. SREBP-1c overproduction decreasedIrs-1 mRNA and IRS-1 protein expression in a dose-dependent manner, and suppressed the resultant insulin sig-nalling, whereas SERBP-1c knockdown by Serbp-1c siRNAblocked the downregulation of IRS-1 induced by PA. Protein–DNA interaction studies demonstrated that SREBP-1c wasable to bind to the rat Irs-1 promoter region, therebyrepressing its gene transcription. Of particular importance,we found that metformin treatment downregulated Srebp-1cpromoter activity, decreased the specific binding of SREBP-1c to Irs-1 promoter and upregulated Irs-1 promoter activityin PA-cultured L6 cells.

Yan Bi, Wenjun Wu, Junfeng Shi and Hua Liang contributed equally tothis study.

Electronic supplementary material The online version of this article(doi:10.1007/s00125-013-3136-1) contains peer-reviewed but uneditedsupplementary material, which is available to authorised users.

Y. Bi (*) :Y. Chen : S. Tang : S. Shen :D. ZhuDepartment of Endocrinology, Drum Tower hospitalaffiliated to Nanjing University Medical School,No321 Zhongshan Road, Nanjing( 210008,People’s Republic of Chinae-mail: biyan@nju.edu.cn

Y. Bi :W. Yin : S. Cao :D. Zhu (*)Department of Endocrinology, Drum Tower ClinicalMedical College of Nanjing Medical University, Nanjing( 210008,People’s Republic of Chinae-mail: zhudalong@nju.edu.cn

W. WuDepartment of Endocrinology, Wuxi People’s hospitalof Nanjing Medical University, Wuxi,People’s Republic of China

J. ShiDepartment of Oncology, the First Affiliated Hospital of NanjingMedical University, Nanjing, People’s Republic of China

H. Liang :M. Cai : J. Weng (*)Department of Endocrinology, the 3rd affiliated hospital of SunYat-sen University, No 600 Tianhe Road, Guangzhou( 510630,People’s Republic of Chinae-mail: wengjp@vip.163.com

J. WengKey Lab of Diabetology of Guangdong Province, Guangzhou,People’s Republic of China

Q. GaoNanjing University Medical School, Nanjing,People’s Republic of China

Diabetologia (2014) 57:592–602DOI 10.1007/s00125-013-3136-1

Conclusions/interpretation Our data indicate for the first timethat SREBP-1c activation participates in skeletal muscle insu-lin resistance through a direct effect of suppressing Irs-1transcription. These findings imply that SREBP-1c couldserve as an attractive therapeutic target for insulin resistance.

Keywords Insulin resistance . IRS-1 . Skeletal muscle .

SREBP-1c

Abbreviations2-NBDG 2-[N-(7-nitrobenz-2-oxa-1,

3-diazol-4-yl)-amino]-2-deoxy-D-glucoseAMPK AMP-activated protein kinasebHLH Basic helix-loop-helixChIP Chromatin immunoprecipitation assayEMSA Electrophoretic mobility shift assayFAS Fatty acid synthaseGAPDH Glyceraldehyde-3-phosphate dehydrogenaseGFP Green fluorescent proteinLXR Liver X receptorPA Palmitic acidPI3K Phosphatidlyinositol-3-kinaseSD rat Sprague-Dawley ratsiRNA Small interfering RNASRE Sterol regulatory elementSREBP-1c Sterol regulatory element binding protein-1c

Introduction

Lipotoxicity is the ectopic accumulation of lipid intermediatesin the non-adipose tissue of liver and muscle inducing patho-logical changes, and it is a well-accepted concept in theexplanation of obesity-associated insulin resistance [1–3].Among the known lipogenic regulators, the sterol regulatoryelement binding protein-1c (SREBP-1c) is a key transcriptionfactor that regulates cellular lipogenesis in liver, skeletal mus-cle and adipose tissue [4–7]. SREBP-1c is synthesised as aprecursor in the membranes of the endoplasmic reticulum.After sterol- or insulin-regulated cleavage, its amino-terminal basic helix-loop-helix (bHLH) leucine zipper do-mains are translocated to the nucleus where they induce theexpression of lipogenic genes, such as fatty acid synthase(FAS), and control lipogenesis [6, 7].

In addition to the regulation of lipid synthesis, studiesreveal expanding roles for SREBP-1c in controlling pathwaysfor insulin resistance, in which the pathological process in-volves a series of cascades, including defective activation ofinsulin receptor substrates (IRS), phosphatidlyinositol-3-kinase (PI3K) and Akt [8–12]. Recent developments revealthat the core of such defects lies in alterations in IRS proteins,in particular the IRS-1 and IRS-2 isoforms [8]. IRS-1 is

required in glucose homeostasis in skeletal muscle, whereasIRS-2 is associated with lipid metabolism in liver and skeletalmuscle [11, 12]. In the liver, nuclear active SREBP-1c candirectly bind to the promoter region of Irs-2 , suppressingIrs-2 expression and the subsequent insulin signalling pathway,thereby leading to hepatic insulin resistance [13–15].

However, the link between SREBP-1c activation and IRS-1-associated insulin signalling in skeletal muscle is unclear.Intriguingly, we have previously shown that IRS-1 andSREBP-1c expression was consistently reciprocal in skeletalmuscles of high fat diet- and streptozotocin-induced Sprague-Dawley (SD) rat models of diabetes [16, 17]. This finding ledus to speculate that Irs-1 expression might be negativelyregulated by SREBP-1c in skeletal muscle, but this remainsto be determined.

The aim of the present study, therefore, was to assess therole of SREBP-1c in the regulation of IRS-1-associated insu-lin signalling in skeletal muscle. We found that SREBP-1coverproduction decreased Irs-1 gene expression in a dose-dependent manner and suppressed the resultant insulin signal-ling transduction. By contrast, SREBP-1c knockdown usingSrebp-1c (also known as Srebf1 ) small interfering RNA(siRNA) blocked the downregulation of IRS-1 caused bypalmitic acid (PA) in L6 muscle cells. Further studies demon-strated that the major mechanism of SREBP-1c in the sup-pression ofmuscular insulin signalling was to bind to the Irs-1promoter and repress its gene transcription. Specifically, wefound that metformin could downregulate Srebp-1c promoteractivity, decrease the specific binding of SREBP-1c to Irs-1promoter and upregulate Irs-1 promoter activity in PA-culturedL6 muscle cells. Our data support the theory that SREBP-1cplays a crucial role in skeletal muscle insulin resistance.

Methods

Antibodies Anti-SREBP-1c antibody was purchased fromSanta Cruz Biotechnology (Dallas, Texas, USA). Anti-IRS-1, anti-p-IRS-1 (Tyr608/612) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were obtain-ed from EMD Millipore (Billerica, MA, USA). Antibodiesagainst Akt, p-Akt (Ser473), p-Akt (Tyr308) and IRS-2 wereobtained from Cell Signaling Technology (Danvers, MA,USA). Anti-FAS antibody was obtained from BD Biosciences(Franklin Lakes, NJ, USA), and anti-p-IRS-2 (Ser731) anti-body was from AnaSpec (Fremont, CA, USA).

Adenovirus cDNA encoding the active amino terminalfragment of rat Srebp-1c (amino acids 1–430, a matureform) were integrated into the adenovirus vector (Invitrogen,Grand Island, NY, USA). Srebp-1c adenoviral vectors werepropagated in 293 cells and purified by caesium chloridedensity centrifugation.

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Cell culture and transfection L6 muscle cells were obtainedfrom the Chinese Academy of Sciences, cultured in DMEMsupplemented with 10% FBS (vol./vol.), and differentiatedinto myotubes in differentiation medium within 6 days, aspreviously described [18]. To study the effect of PA or liverX receptor (LXR) agonist (TO901317 from Sigma-Aldrich,Saint Louis,MO, USA), cells were incubated for 8 h in serum-free medium and treated with 0.5 mmol/l PA, 0.5 mmol/loleate or 5 mmol/l TO901317 for 24 h. After PA incubation,cells were incubated for a further 12 h with 100 nmol/l ofinsulin or 10 mmol/l of metformin if indicated. Additionally,to determine the time course effects of PA, L6 myobutes wereincubated with 0.5 mmol/l of PA for 0.5, 1, 3, 6, 12, 24 or 48 h.For transient transfection assays, L6 myotubes were addedwith adenovirus vectors expressing SREBP-1c or green fluo-rescent protein (GFP) and then transfected for 24 h. Themediawere renewed and cells were treated with or without100 nmol/l insulin for 12 h. The cells were then harvestedfor protein and mRNA immediately.

Glucose uptake assay L6 cells were treated with PA ortransfected with the expression plasmid pcDNA3.1-SREBP-1c. After 24 h of intervention, cells were incubated with100 nmol/l insulin for 12 h. The medium was then replacedwith low-glucose and serum-free DMEM containing100 μmol/l 2-[N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2-deoxy-D-glucose (2-NBDG) (Cayman, Ann Arbor,Michigan, USA). After incubation for 1 h, cells were collectedand the fluorescence intensity was measured at an excitationof 485 nm and emission of 520 nm using a FACSCalibur flowcytometer (BD Bioscience).

siRNA L6 myoblasts were transfected with siRNA directedagainst Srebp-1c (GenePharma, Shanghai, China) or controlsiRNA (siControl) via Lipofectamine 2000 (Invitrogen)according to the manufacturer’s instructions. The siRNAsequences for targeting Srebp-1c were as follows: 5′- GAGCAGAGAUGGCUCUAAUTT-3′. As a negative control, thefollowing siRNA sequencewas used: 5′-UUCUCCGAACGUGUCACGUdTdT-3. At 24 h after transfection, cells weretreated with 0.5 mmol/l of PA for an additional 24 h ifindicated.

Animal study SD rats (approximately 160 g, obtained fromShanghai Institutes for Biological Sciences, Shanghai, Peo-ple’s Republic of China) were housed at room temperaturewith a 12 h light/12 h dark cycle. Control rats (n =6) were feda normal diet (10% energy from fat, 64% energy from carbo-hydrate and 26% energy from protein). High-fat-fed rats(n =12) were fed a fat-enriched diet (45% energy from fat,37% energy from carbohydrate and 18% energy from protein)for 8 weeks [17], and were then randomised to receive met-formin (250 mg/kg) or not for 8 weeks. An intraperitoneal

glucose tolerance test was performed before and after the end ofthe experiment in a state of fasting for 16 h. Blood glucose wasmeasured using a potable blood glucose meter (LifeScan, John-son & Johnson, Shanghai, China). Insulin was measured with arat insulin ELISA kit (Linco Research, Saint Charles, MO,USA). Insulin sensitivity was evaluated as a glucose-insulinindex [17]. Gastrocnemius muscles were dissected and snapfrozen in liquid nitrogen. The animal protocol was reviewedand proved by the animal care committee of Drum TowerHospital affiliated to Nanjing University Medical School, Nan-jing, China (Characteristics of the experimental animals arepresented in electronic supplementary material [ESM] Table 1).

Quantitative real-time PCR Total RNAwas extracted using astandard Trizol RNA isolation method (Invitrogen). cDNAwas synthesised from 2 μg of total RNA using a PrimeScriptRT reagent Kit (Takara Bio, Otsu, Japan). Real-time PCRquantified amplifications of cDNAwith both sense and anti-sense primers (ESM Table 2), at a final volume of 20 μl usingthe SYBR Premix Ex Taq (Takara Bio) with an ABI PRISM7500 Sequence Detection System (Applied Biosystems,Grand Island, NY, USA) were performed, and the results werenormalised against Gapdh gene expression.

Western blotting L6 muscle cells were washed twice withcold phosphate-buffered saline and lysed in ice-cold cell lysisbuffer supplemented with Protease Inhibitor Cocktail (Roche,Basel, Switzerland). The protein concentrations were deter-mined by the bicinchoninic acid (BCA) method. The proteinlysates were boiled for 5 min and dissolved in Laemmli buffer.Proteins were then separated by SDS-PAGE, transferred topolyvinylidene difluoride membranes, blocked with 7.5%nonfat milk (wt/vol.), washed with Tri-Buffer saline andTween 20 (TBST; 10 mmol/l Tris HCl, 100 mmol/l NaCl,0.1% Tween 20) and incubated with appropriate primaryantibodies overnight at 4°C. Membranes were washed withTBST and incubated with an appropriate secondary antibody.Proteins were visualised by enhanced chemiluminescence(EMD Millipore) and quantified by densitometry (QuantityOne, Bio-Rad, Hercules, CA, USA).

Construction of promoter-reporter plasmids The expressionplasmid pcDNA3.1-Srebp-1c containing the coding ratSrebp-1c (amino acids 1–403) transcriptionally activeform or dominant-negative form (SREBP-1cdn, Tyr320Ala),which prevents DNA binding and sequestrates the endoge-nous wild-type form [19], was constructed by Invitrogen. Thereporter plasmid pGL3-Irs-1 1300 was constructed to containthe rat Irs-1 promoter region from −1,300 to +50 bp andcloned into theKpnI/HindIII sites of the pGL3-Basic luciferasevector (Promega, Madison WI, USA). Other pGL3-Irs-1reporter plasmids were produced by PCR using this constructas a DNA template, and the PCR products were inserted into

594 Diabetologia (2014) 57:592–602

the pGL3-Basic luciferase vector. The reporter plasmid pGL3-Srebp-1c was constructed to contain the rat Srebp-1c promoterregion from −1,000 to +100 bp. The primers used in promoteranalysis are listed in ESM Table 3.

Luciferase reporter assay L6 cells were cotransfected with aluciferase plasmid for rat Irs-1 promoter (500 ng), expressionplasmid (500 ng) and pRL-TK renilla plasmid (10 ng) usingLipofectamine 2000 (Invitrogen). After transfection for 36 h,luciferase activity was measured using the dual-luciferasereporter assay system (Promega) following the manufacturer’sinstructions. The level of activity was calculated as the ratio offirefly luciferase activity to renilla luciferase activity, and thiswas represented as the average of triplicate experiments. Thereporter plasmid pGL3- Srebp-1c (1,000 ng) and pRL-TKrenilla plasmid (20 ng) were co-transfected into L6 cells. After24 h of transfection, cells were switched to a medium supple-mented with 1% BSA, 0.5 mmol/l of PA or 10 mmol/l ofmetformin if indicated, incubated for an additional 12 h andthen assayed as described above.

Electrophoretic mobility shift assay Electrophoretic mobilityshift assay (EMSA) was performed using a LightShift Chemi-luminescent EMSA Kit (Thermo Scientific, Waltham, MA,USA). The wild-type and mutant probes were as follows: IRS-1 sterol regulatory element (SRE; wild type), CTGCGCCTCCCGAGGACC; IRS-1 SRE (mutant), CTGCTGTTAAATTAGACC. The wild-type probe was labelled at the 5′ end withBiotin (Invitrogen). The labelled oligos were annealed bymixing equal molar amounts of two single-stranded oligos,heating to 95°C for 5 min and slowly cooling to roomtemperature. Nuclear extracts (6 μg) were incubated in thereaction buffer for 30 min at room temperature before addingthe labelled DNA probe. For competition assays, a 200-foldexcess of unlabelled wild-type or mutant probe was added tothe preincubated binding mixture. The SREBP-1 antibody(Santa Cruz Biotechnology) was used to confirm the specificbinding. After adding the labelled DNA probe, all sampleswere incubated for a further 20 min at room temperature.Protein–DNA complexes were separated by electrophoresisin a 6.5% polyacrylamide gel (wt/vol.) and transferred to anylon membrane. The developed bands were visualised byexposing the membrane to x-ray film.

Chromatin immunoprecipitation assay L6 cells weretransfected with Srebp-1c adenovirus vectors for 48 h ortreated with PA for 24 h and cross-linked 1% formaldehyde(vol./vol.) for 10 min at room temperature. Chromatin solu-tions were sonicated and incubated with 4 μg of anti-SREBP-1c antibody (Santa Cruz Biotechnology) or with control IgG,and rotated overnight at 4°C. Immune complexes were col-lected with protein A Sepharose beads (EMD Millipore) for1 h at 4°C. To purify the immunoprecipitated DNA, the beads

were successively treated with DNase-free RNase A andproteinase K (EMD Millipore), followed by resuspension ofthe DNA in distilled water. To amplify the Irs-1 promoterregions containing SRE, the following primer sets were used:5′-ACCAGGTGACGATTGTCCAG-3′ and 5′- ACAATGTCAGCCCCAAACAT-3′. After amplification, PCR productswere analysed on a 2% agarose gel (wt/vol.). For quantifica-tion of the chromatin immunoprecipitation (ChIP) assay, inputgenomic DNA and immunoprecipitated DNAwere amplifiedby real-time PCR using SYBR Green (Takara Bio). All PCRsignals from immunoprecipitation samples were referred totheir respective input standard curve to normalise differencesin cell number and primer efficiency.

Statistical analysis Values are means ± SE. Differences be-tween groups were determined using two-way ANOVA.Fisher’s least significant difference post hoc analysis was usedto identify significant differences. A p value of less than 0.05was considered statistically significant.

Results

PA activates SREBP-1c and impairs the IRS-1/Akt pathway Wefound that Srebp-1c mRNA (Fig. 1a) and SREBP-1c protein(Fig. 1c) levels were increased, and Irs-1 gene (Fig. 1b) andIRS-1 protein (Fig. 1c) expression was decreased in PA-treated L6 myotubes compared with controls. Furthermore,PA treatment led to decreased expression of insulin-stimulatedp-IRS-1(Tyr608/612), p-Akt (Ser473) and p-Akt (Tyr308)proteins (Fig. 1c). However, protein levels of p-IRS-2(Ser731) and IRS-2 were not altered with PA treatment(Fig. 1c). In addition, 2-NBDG uptake under basal andinsulin-stimulated states was inhibited by PA treatment(Fig. 1d).

It has been reported that saturated PA promotes insulinresistance, whereas the monounsaturated oleate improves in-sulin sensitivity [20]. The current study compared the effectsof oleate and PA on SREBP-1c and IRS-1expression. Asshown in Fig. 1e, PA increased SREBP-1c expression anddecreased IRS-1 expression, but oleate had no effect on theexpression of either protein.

To determine the time course effects of PA treatment onSREBP-1c and IRS-1 expression, L6 myotubes were treatedwith PA for times ranging from 0.5, 1, 3, 6, 12, 24 to 48 h. Asshown in Fig. 1f, SREBP-1c protein expression increasedsignificantly after 1 h of exposure to PA, remaining at approx-imately this level thereafter. IRS-1 tended to decrease after 3 hof PA incubation and then decreased significantly to an unde-tectable level after 6 h of treatment. Taken together, our datasupport a close inverse relationship between SREBP-1c andIRS-1 expressions.

Diabetologia (2014) 57:592–602 595

LXR agonist promotes SREBP-1c and decreases the IRS-1/Aktpathway Srebp-1c gene expression has been reported to beupregulated by the nuclear receptor protein LXR [21]. Toinvestigate whether LXR-induced SREBP-1c activation cor-relates with IRS-1-associated insulin signalling, SREBP-1cand IRS-1 expression was compared in L6 myotubes follow-ing LXR agonist intervention. Similar to the observations

made with PA treatment, the expression of Srebp-1c mRNA,SREBP-1c protein and FAS protein was increased by LXRagonist treatment compared with controls (Fig. 2a, b). Bycontrast, protein expression of IRS-1, insulin-stimulatedp-IRS-1(Tyr608/612) and p-Akt (Ser473)/Akt was decreasedby LXR agonist treatment, compared with controls, furtherindicating a close relationship between SREBP-1c and IRS-1.

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Fig. 1 PA activates SREBP-1c expression and impairs the IRS-1/Aktpathway in L6 cells. (a , b) mRNA expression of Srebp-1c (a) and Irs-1(b) measured using real-time PCR in control and PA-treated L6 musclecells. (c) Protein levels (normalised to GAPDH) measured using westernblotting in control and PA-treated L6 cells either in the absence of insulin(white bars) or in the presence of insulin (100 nmol/l, black bars). Graphsshow mean ± SE relative to control cells. (d) The uptake of 2-NBDG

performed in control and PA-treated L6 cells with or without insulin. (e)Protein expression of SREBP-1c and IRS-1 measured bywestern blottingin control, PA- and oleate-treated L6 cells. (f ) Time course effects ofPA treatment. Protein expression of SREBP-1c and IRS-1 wasmeasured by western blotting in L6 myotubes incubated with PA for0.5, 1, 3, 6, 12, 24 or 48 h. The graph shows the mean ± SE relative tocontrol cells; *p <0.05

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SREBP-1c overexpression decreases Irs-1 gene expressionand impairs IRS-1-Akt pathway To evaluate whetherSREBP-1c modulates Irs-1 gene expression, adenoviral vec-tors expressing Srebp-1c were transfected into L6 myotubes.Transduction of cultured L6myotubes with increasing titres ofthe Srebp-1c adenovirus resulted in dose-dependent expres-sion of Srebp-1c (Fig. 3a). The accumulation of SREBP-1cinduced gene expression of Fas mRNA (Fig. 3b), a well-established target gene for SREBP-1c, and decreased Irs-1mRNA levels in a dose-dependent manner (Fig. 3c).Furthermore, suppression of Irs-1 mRNA levels through over-production of SREBP-1c was accompanied by a reduction inIRS-1 protein levels in L6 myotubes (Fig. 3d).

To determine whether SREBP-1c-induced reduction ofIRS-1 protein levels affects insulin signalling, we measuredinsulin-mediated stimulation of p-IRS-1 (Tyr608/612) andp-Akt (Ser473) in L6 myotubes. As estimated by immuno-blotting assay in insulin-stimulated myotubes, SREBP-1c ledto a reduction of p-IRS-1 (Tyr608/612) and p-Akt (Ser473)levels in a dose-dependent manner, whereas Akt protein levelswere not affected (Fig. 3e). In addition, SREBP-1c overex-pression inhibited the uptake of 2-NBDG under basal andinsulin-stimulated states (Fig. 3f ). These data indicate thatSREBP-1c activation significantly suppresses insulin signal-ling in skeletal muscle cells.

SREBP-1c knockdown blocks the downregulation of IRS-1 byPA To further confirm the role of SREBP-1c in insulinsignalling in an independent setting, we adopted Srebp-1csiRNA to knockdown SREBP-1c in L6 cells (Fig. 4a). Asshown in Fig. 4b, compared with cells transfected withsiControl, SREBP-1c knockdown led to an upregulation ofIRS-1 levels as shown by western blotting assay, suggest-ing that endogenous SREBP-1c physiologically regulatesIRS-1 expression. In addition, we used western blotting toshow that knockdown of SREBP-1c blocked the downreg-ulation of PA-induced IRS-1 levels (Fig. 4c). Taken to-gether, the data suggest that SREBP-1c has a direct effecton the expression of Irs-1 .

SREBP-1c binds to Irs-1 promoter and represses itstranscription To examine the effect of SREBP-1c on Irs-1promoter activity, a fragment of Irs-1 promoter region be-tween −1,300 and +50 bp was cloned into the upstream of theluciferase coding gene. As shown in Fig. 5a, Irs-1 promoteractivity was repressed by SREBP-1c overexpression, whereasthe dominant negative form of SREBP-1c (a mutant ofTyr320Ala that lacks the ability to bind DNA) had no effect,indicating that SREBP-1c represses Irs-1 at the transcriptionallevel. Based on these results, a number of 5′ deletion promotersranging between −1,300 and −170 bp were constructed andsubjected to luciferase assay. We found that the serial deletionconstructs between −1,300 and −450 bp resulted in significantdecreases in promoter activity to similar degrees (approximately30–40%), whereas a further deletion of the sequences constructto position −210 bp resulted in markedly increased promoteractivity (Fig. 5b). These findings suggest a potential targetregion for SREBP-1c on the Irs-1 promoter vector.

Sequence analysis showed that the target region of the ratIrs-1 promoter gene contained a potential binding site (GCCTCCCGAG), the SRE variant located between −302 and−292 bp, which is conserved in promoter regions in the rat,mouse and human (ESM Fig. 1). To confirm that this putativesequence was functional, PCR site-directed mutagenesis(TGTTAAATTA) was generated and analysed using a lucif-erase assay (Fig. 5c). As shown in Fig. 5d, transcriptionalactivity of the wild-type Irs-1 promoter was dramaticallydownregulated by SREBP-1c, whereas transcriptional activitywas abolished in the promoter bearing the mutation ofSREBP-1c binding site.

EMSA was then performed to examine the binding ofSREBP-1c to the Irs-1 promoter region in vitro using probesto cover the conserved SRE variant (Fig. 5e). Compared withcontrols, the labelled wild-type probe successfully formed acomplex with nuclear proteins. The unlabelled wild-typeprobe abolished the complex formation, but the unlabelledmutant probe did not abolish the complex. The DNA–proteincomplex was partially abolished when anti-SREBP-1c anti-body was preincubated with the nuclear extract, with the

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Fig. 2 LXR agonist promotes SREBP-1c expression and decreasesthe IRS-1/Akt pathway in L6 cells. (a ) mRNA expression ofSrebp-1c , Irs-1 and Fas compared in LXR agonist-treated cells(black bars) and control L6 cells (white bars) using real-time PCR.

(b ) Protein levels (normalised to GAPDH) measured by westernblotting in LXR agonist-treated and control L6 cells. The graphshows mean ± SE relative to control cells; *p <0.05

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addition of normal rabbit IgG as the contrast. The data suggestthat the conserved SRE variant is responsible for the repres-sion of Irs-1 promoter by SREBP-1c.

ChIP assays were then performed to examine binding ofSREBP-1c to the Irs-1 promoter region in vivo. Figure 5fshows the primers spanning a sequence from −450 to −210 bpthat contained the SREBP-1c binding site in the Irs-1 genepromoter region. The results showed that SREBP-1c couldbind uniquely to the Irs-1 promoter when SREBP-1c wasoverexpressed or under PA conditions in L6 cells (Fig. 5g, h).Taken together, these findings provide evidence to show thatSREBP-1c can bind to the rat Irs-1 promoter region and repressits gene transcription.

Inhibition of SREBP-1c contributes to the beneficial effect ofmetformin on IRS-1 Metformin has been widely used in themanagement of insulin resistance and type 2 diabetes, but it isunknown whether SREBP-1c inhibition is required for the ben-eficial effect of metformin on IRS-1-associated insulin signalling

in muscle. We observed that in PA-cultured L6 cells metformindecreased Srebp-1c mRNA and protein expression (Fig. 6a, c),and increased Irs-1 mRNA and protein expression (Fig. 6b, c).In addition, metformin significantly increased the expression ofp-IRS-1(Tyr608/612) and p-Akt (Ser473) proteins. Similar find-ings were observed in skeletal muscle cells of SD rats fed with ahigh-fat diet and treated with metformin (ESM Fig. 2).

We performed luciferase reporter assays and ChIP as-says to further investigate whether increased IRS-1 expres-sion following metformin intervention is associated withthe inhibition of SREBP-1c activity. The results showedthat metformin significantly downregulated Srebp-1c pro-moter activity (Fig. 6d), upregulated the Irs-1 promoteractivity (Fig. 6e) and decreased the specific binding ofSREBP-1c to the Irs-1 promoter (Fig. 6f ) in PA-culturedL6 cells (Fig. 6f ). These results suggest that inhibition ofSREBP-1c expression contributes to the beneficial effectsof metformin on IRS-1-associated insulin signalling inmuscle.

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Fig. 3 SREBP-1coverexpression decreases Irs-1gene expression and impairs theIRS-1/Akt pathway in L6 musclecells. (a–c) mRNA expression ofSrebp-1c (a), Irs-1 (b) and Fas(c) in adenovirus-mediatedSREBP-1c-overexpressing L6cells. L6 cells were infected withGFP and SREBP-1c adenovirusat the indicated multiplicity ofinfection (MOI). The graphsshow mean ± SE relative to GFP-treated cells. (d) Protein levels(normalised to GAPDH) inSREBP-1c-overexpressing L6cells at the indicated MOImeasured by western blotting.The graphs show mean ± SErelative to GFP-treated cells. (e)Protein levels in SREBP-1c-overexpressing L6 cells at theindicated MOI in the absence orpresence of insulin. The graphshowsmean ± SE relative to GFP-treated cells under basal (whitebars) or insulin stimulated (blackbars) conditions. (f) 2-NBDGuptake performed in SREBP-1c-overexpressing L6 cells withinsulin (black bars) or withoutinsulin (white bars). The graphshowsmean ± SE relative to GFP-treated negative under basal orinsulin-stimulated conditions;*p <0.05

598 Diabetologia (2014) 57:592–602

Discussion

A number of prevailing hypotheses exist about the molecularmechanisms of lipotoxicity, such as inflammation, oxidativestress and endoplasmic reticulum stress [22–25]. By contrast,recent studies, suggest that activation of the inflammatorypathway in skeletal muscle does not play a causal role inPA-induced insulin resistance [26]. The initial mechanismsleading tomuscle insulin resistance remain to be clarified [27].In the present study, we report a new mechanism for insulinresistance of lipotoxicity. The accumulation of SREBP-1c, apivotal transcription factor regulating de novo lipogenesis, canresult in muscular insulin resistance by a direct effect ofsuppressing Irs-1 promoter transcription and gene expression.

Studies have established that SREBP-1c activation is in-volved in the development of insulin resistance mainlythrough the impairment of insulin signalling by promotingaccumulation of fatty acids [5–7]. Recently, however, expandingroles for SREBP-1c in controlling pathways for insulin resis-tance have been revealed [28]. Increased SREBP-1c activityresulting from hyperinsulinemia negatively correlates with theexpression of IRS-2, the major hepatic insulin signal mediator[13]. Consistent with this data, the available evidence in geneticand dietary rodent models of obesity suggest that SREBP-1ccan transcriptionally repress the expression of Irs-2 , inhibit thedownstream PI3K/Akt pathway and decrease glycogensynthesis [14, 15]. Therefore, these data suggest that SREBP-1ccontributes to liver insulin resistance.

Previous studies have not focused on the role of SREBP-1cactivation in skeletal muscle glucose metabolism, in whichIRS-1-associated signalling plays an important role. Althoughskeletal muscle is not thought to be as lipogenic as the liver,elevated skeletal muscle lipogenesis has been associated withinsulin resistance and the development of type 2 diabetes [29,30]. High amounts of muscular SREBP-1c were observed ininsulin-resistant states in animals and humans [16, 31, 32]. Inthe current work, we observed an inverse relationship betweenmuscular SREBP-1c and IRS-1 expression under fatty acidinduced insulin resistant states both in vivo and in vitro(Figs 1 and 6). Similar effects of LXR agonist, a stimulatorof SREBP-1c, on the expression of SREBP-1c and IRS-1were observed, further indicating a close relationship betweenthe two (Fig. 2). Through adenoviral overexpression experi-ments (Fig. 3) and knockdown studies using siRNA (Fig. 4),we demonstrated that overproduction of SREBP-1c decreasesIrs-1 gene expression in a dose-dependent manner andsuppresses the resultant insulin signalling pathway, leadingto impaired muscular insulin sensitivity; while knockdownof SREBP-1c blocks the downregulation of IRS-1 causedby PA in L6 cells. Taken together, our results are consis-tent with the hypothesis that SREBP-1c is as important forfatty acid-induced insulin resistance in skeletal muscle as it isin hepatocytes.

We further investigated the mechanism of SREBP-1c in theregulation of Irs-1 gene expression. Using luciferase, EMSAand ChIP assays (Fig. 5), we provide evidence to show thatSREBP-1c suppresses insulin signalling by binding to theIrs-1 promoter region and repressing its gene transcription.SREBP-1c is a bHLH transcription factor that can bind to theSRE sequence in the promoter regions of the target genes [33].Sequence analysis identified one SRE variant in the rat Irs-1promoter, conserved among species from rodents to humans(Fig. 5c, ESM Fig. 1), which mediates SREBP-1c-inducedtransinhibition and binding. Indeed, mutation of this SRE sitereversed the inhibition of the promoter activity of the wild-type in the rat Irs-1 promoter (Fig. 5d), whereas mutations ofother potential sites did not show any effect (data not shown).

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Fig. 4 SREBP-1c knockdown by Srebp-1c siRNA blocks the downreg-ulation of IRS-1 by PA. (a) The effects of Srebp-1c siRNA on the mRNAlevel of endogenous Srebp-1c were determined by real-time PCR. L6cells were transfected with the siRNA targeting Srebp-1c or an equalamount of control siRNA (siControl). (b) The effects of siRNA on theprotein levels of SREBP-1c and IRS-1 were determined by westernblotting. (c) The effects of siRNA on SREBP-1c and IRS-1 protein levelswere determined by western blotting with PA intervention. Graphs showmean ± SE relative to control; *p <0.05

Diabetologia (2014) 57:592–602 599

However, our data could not exclude the possibility thatSREBP-1c binds to the Irs-1 promoter by interfering withthe binding of other transcription factors in this region, as hasbeen observed in other promoters [15, 34]. Although addi-tional studies will be required to examine this hypothesis, theregion appears to be conserved in rodents and humans andtherefore could have important implications for the progres-sion of insulin resistance.

Metformin, a powerful activator of AMP-activated proteinkinase (AMPK), has been widely used in the management of

type 2 diabetes and insulin resistance by suppressing hepaticgluconeogenesis and activating insulin-stimulated peripheralglucose uptake [35, 36]. A recent study further indicated thatAMPK interacts with and directly phosphorylates SREBP-1c,suppresses SREBP-1c cleavage and nuclear translocation, andrepresses SREBP-1c target gene Fas expression in hepatocytesin response to metformin treatment, leading to reduced lipo-genesis [37]. In the current study we observed that muscleSREBP-1c expression was decreased and IRS-1 expressionwas increased in response to metformin under insulin resistant

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Fig. 5 SREBP-1c binds to the Irs-1 promoter and represses its genetranscription. (a) Effect of SREBP-1c on Irs-1 promoter activity. L6 cellswere transfected with pcDNA3.1, pcDNA3.1-Srebp-1c, or pcDNA3.1-Srebp-1c dominant-negative (dn) form (Tyr320Ala), and luciferase re-porter constructs containing the 5′-flanking region of the rat Irs-1 gene(pGL3/Irs-1 promoter luc). Data are mean ± SE relative to control cells.(b) Deletion studies in SREBP-1c repression of the Irs-1 promoter. Aseries of the 5′-deletion Irs-1 promoter reporter constructs were transientlytransfected into L6 cells and promoter activities of Irs-1 were measured. (c)The sequence, 5′-GCCTCCCGAG-3′ at −302 to −292 bp, was identified asa potential binding element. PCR site-directed mutagenesis was used togenerate mutation of TGTTAAATTA. (d) The intact and mutated con-structs were transfected into L6 cells and luciferase activities were mea-sured. (e) Interactions of SREBP-1c with the conserved SRE variant were

demonstrated by EMSA. Lane 1: the negative control without nuclearextract. Lane 2: DNA–protein complexes formed with the labelled probe.Lane 3: 200-fold molar excess of the unlabelled probe successfully com-peted with the labelled probe. Lane 4: 200-fold molar excess of theunlabelled mutant probe failed to compete with the labelled probe. Lane5: DNA–protein complex was unchanged with normal rabbit IgG. Lane 6:DNA–protein complex was weakened with anti-SREBP-1c antibody. (f)Diagram of the ChIP assay showing the primers spanning a sequence from−450 to −210 bp that contained SREBP-1c binding site in the rat Irs-1 genepromoter. (g) ChIP assay shows the binding of SREBP-1c to the Irs-1promoter region with SREBP-1c transfection or under PA treatment in L6cells. (h) The binding of SREBP-1c to the Irs-1 promoter was quantified byreal-time PCRwith Srebp-1c transfection or under PA treatment in L6 cells.Data are mean ± SE; *p<0.05

600 Diabetologia (2014) 57:592–602

states both in vivo and in vitro (Fig. 6 and ESM Fig. 2).Importantly, using luciferase reporter assays and ChIP analysis,we found that metformin significantly decreased Srebp-1c pro-moter activity and the specific binding of SREBP-1c to the Irs-1 promoter, and restored Irs-1 promoter activity that had beendecreased by PA. Therefore, our data suggest, for thefirst time to our knowledge, that inhibition of SREBP-1c contributes to the beneficial effect of metformin onIRS-1-associated insulin signalling in insulin resistantmuscle cells induced by fatty acid.

In conclusion, the current study indicates that a high levelof SREBP-1c results in impaired insulin signalling in skeletalmuscle cells via a direct effect of suppressing Irs-1 promotertranscription and gene expression. The data reveal a key rolefor SREBP-1c in the development of skeletal muscle insulinresistance correlated to abnormal fatty acid metabolism.Moreover, the amelioration of IRS-1-associated insulin sig-nalling with metformin is associated with its inhibitory effecton SREBP-1c, which implies that SREBP-1c could serve asan attractive therapeutic target for insulin resistance.

Acknowledgements We would like to thank Prof. Li Xiaoying fromShanghai Ruijin Hospital for his constructive discussion during manu-script preparation. Some of the data were presented as an abstract at the49th Annual European Association for the Study of Diabetes (EASD)Meeting in 2013 (number 2194).

Funding This work was sponsored by grants from the National NaturalScience Foundation of China Grant Award (81270906, 30800539), 973project (2012CB517506), National Science Fund for DistinguishedYoung Scholars (81025005), China postdoctoral Science Foundation(2012M521050), Jiangsu postdoctoral Science Foundation, JiangsuProvince’s Key Provincial Talents Program (RC2011011), JiangsuProvince’s Key Discipline of Medicine (XK201105), the Key Project ofNanjing Medical Science and Technology Development Foundation(ZKX11017), National Natural Science Foundation of China Grant Award(81000338, 81070636), New Drug Development, Construction andmanagement of Clinical Biobank forMajor Disease (2011ZX0907-001-08),the Project of National Key Clinical Division, Jiangsu Natural ScienceFoundation (KA037) and Guangdong Natural Science Foundation(10151008901000033).Contribution statement YB contributed to the study design, datainterpretation, drafting the article, and final approval of the version tobe published. WW contributed to the acquisition of data, drafting thearticle and approval of the final version. JS contributed to the studydesign, acquisition of data, drafting the article and approval of the finalversion. HL contributed the study design, data analysis, drafting thearticle and approval of the final version. WY, YC, ST, SC, MC, and SScontributed to acquisition of data, drafting the article and approval of thefinal version. QG contributed to the acquisition of data, drafting thearticle and approval of the final version. DZ contributed to the studydesign, acquisition of data, revision of the manuscript and final approvalof the version to be published. JW contributed to the study design,acquisition of data, revision of the manuscript and final approval of theversion to be published.

Duality of interest The authors declare that there is no duality ofinterest associated with this manuscript.

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Fig. 6 Inhibition of SREBP-1ccontributes to the beneficial effectof metformin on IRS-1. (a , b)Gene expression of Srebp-1c (a)and Irs-1 (b) in L6 cells culturedwith PA and treated withmetformin (10 mmol/l). (c)Protein expression (normalised toGAPDH) was measured in L6cells cultured with PA and treatedwith metformin. (d) Effects ofmetformin treatment on Srebp-1cpromoter activity in PA-treatedL6 cells measured by luciferasereporter assay. (e) Effects ofmetformin treatment on Irs-1promoter activity in PA-treatedL6 cells measured by luciferasereporter assay. (f) Effects ofmetformin treatment on thespecific binding of SREBP-1c tothe Irs-1 promoter measured byChIP analysis. Graphs showmean ± SE; *p<0.05

Diabetologia (2014) 57:592–602 601

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