Life Sciences 114 (2014) 36–44

Contents lists available at ScienceDirect

Life Sciences

j ourna l homepage: www.e lsev ie r .com/ locate / l i fesc ie

Insulin acutely triggers transcription of Slc2a4 gene: Participation of theAT-rich, E-box and NFKB-binding sites

Paulo Alexandre Moraes, Caio Yogi Yonamine, Danilo Correa Pinto Junior, João Victor DelConti Esteves,Ubiratan Fabres Machado, Rosana Cristina Mori ⁎Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, SP, Brazil

⁎ Corresponding author at: Department of PhysiologBiomedical Sciences, University of São Paulo, Av. Prof. Lin05508-900, Brazil. Tel.: +55 1130917494; fax: +55 1130

E-mail address: morirc-icb@hotmail.com (R.C. Mori).

http://dx.doi.org/10.1016/j.lfs.2014.07.0400024-3205/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 1 April 2014Accepted 31 July 2014Available online 11 August 2014

Keywords:GLUT4Skeletal musclePI3K/AKTMEF2HIF1AMYOD1

Aims: The insulin-sensitive glucose transporter protein GLUT4 (solute carrier family 2 member 4 (Slc2a4) gene)plays a key role in glycemic homeostasis. Decreased GLUT4 expression is a current feature in insulin resistantconditions such as diabetes, and the restoration of GLUT4 content improves glycemic control. This study investi-gated the effect of insulin upon Slc2a4/GLUT4 expression, focusing on the AT-rich element, E-box and nuclearfactor NF-kappa-B (NFKB) site.Main methods: Rat soleus muscles were incubated during 180 min with insulin, added or not with wortmannin(phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PI3K)-inhibitor), ML9(serine/threonine protein kinase (AKT) inhibitor) and tumor necrosis factor (TNF, GLUT4 repressor), andprocessed for analysis of GLUT4 protein (Western blotting); Slc2a4, myocyte enhancer factor 2a/d(Mef2a/d), hypoxia inducible factor 1a (Hif1a), myogenic differentiation 1 (Myod1) and nuclear factor ofkappa light polypeptide gene enhancer in B-cells 1 (Nfkb1) messenger ribonucleic acids (mRNAs)

(polymerase chain reaction (PCR)); and AT-rich- (myocyte-specific enhancer factor 2 (MEF2)-bindingsite), E-box- (hypoxia inducible factor 1 alpha (HIF1A)- and myoblast determination protein 1 (MYOD1)-binding site), and NFKB-binding activity (electrophoretic mobility assay).Key findings: Insulin increased Slc2a4mRNA expression (140%) and nuclear proteins binding to AT-rich andE-box elements (~90%), all effects were prevented by wortmannin and ML9. Insulin also increased Mef2a/dand Myod1 mRNA expression, suggesting the participation of these transcriptional factors in the Slc2a4enhancing effect. Conversely, insulin decreased Nfkb1 mRNA expression and protein binding to the NFKB-site (~50%). Furthermore, TNF-induced inhibition of GLUT4 expression (~40%) was prevented by insulinin an NFKB-binding repressing mechanism. GLUT4 protein paralleled the Slc2a4 mRNA regulations.Significance: Insulin enhances the Slc2a4/GLUT4 expression in the skeletal muscle by activating AT-rich andE-box elements, in a PI3K/AKT-dependent mechanism, and repressing NFKB-site activity as well. Theseresults unravel how post-prandial increase of insulin may guarantee GLUT4 expression, and how the insulinsignaling impairment can participate in insulin resistance-induced repression of GLUT4.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Glycemic homeostasis depends on thewhole-body glucose handling,with different tissues interacting to lead to the metabolic harmony(Herman and Kahn, 2006). In this process, the insulin- and contraction-sensitive glucose disposal in skeletal muscle is a fundamental phenome-non, in which the glucose transporter protein GLUT4, encoded by thesolute carrier family 2 member 4 (human) (SLC2A4) gene, plays a keyrole (Correa-Giannella and Machado, 2013). Decreased GLUT4 expres-sion is a current feature in insulin resistant conditions such as diabetes,

y and Biophysics, Institute ofeu Prestes, 1524, São Paulo, SP917285.

and the restoration of GLUT4 content improves glycemic homeostasis.In fact, transgenic mice overexpressing GLUT4 in skeletal muscle depictincreased insulin-inducedwhole-body glucose utilization and decreasedbasal glycemia (Tsao et al., 1996). Besides, exercise training in obese type2 diabetes mellitus (T2DM) patients increases GLUT4 content improvingglycemic control, independently of changes in the insulin signaling cas-cade (O'Gorman et al., 2006).

Since increasing GLUT4 expression in muscle efficiently treatsinsulin resistance/T2DM, SLC2A4 is a potential target gene forpharmacogenomics of insulin resistance (Correa-Giannella andMachado, 2013). Thus, a deep knowledge on the regulation of theSLC2A4 gene expression is of the utmost importance. Amazingly, al-though GLUT4 is widely known as the insulin-sensitive transporter,the insulin effects upon SLC2A4 gene are not clearly established.Studies in vitro and in vivo have shown that insulin may either

37P.A. Moraes et al. / Life Sciences 114 (2014) 36–44

increase or decrease solute carrier family 2 member 4 (murine)(Slc2a4) expression. Such apparently opposite effects are probablyrelated to the different concentrations and/or time of exposition to thehormone used in each study (Sivitz et al., 1989; Flores-Riveros et al.,1993; Yu et al., 2001; Silva et al., 2005), aswell as tometabolic, hormonal,neural and inflammatory signals that are known to influence theinsulin's effects (Alves-Wagner et al., 2009; Furuya et al., 2010;Zanquetta et al., 2014). This points out the complexity of the regula-tion, and depicts that the physiological effect of the dynamic changesin insulin concentration throughout the day is far from beingunderstood.

In the SLC2A4 promoter region, several SLC2A4 gene expressionenhancers and inhibitors bind to specific domains controlling the geneexpression (Santalucia et al., 2001; Im et al., 2007; Karnieli andArmoni, 2008), some of them playing special role in the skeletal muscle.Regarding contraction-stimulated Slc2a4 expression, two domains playup the enhancing effect: the AT-rich element and the E-box, controlledby themyocyte enhancer factors 2A and 2D (myocyte-specific enhancerfactor 2A (MEF2A) and myocyte-specific enhancer factor 2D (MEF2D))and by proteins from the basic helix loop helix family (bHLH), respec-tively (Mora and Pessin, 2000; Santalucia et al., 2001; Knight et al.,2003; Sparling et al., 2008; Lima et al., 2009). The bHLH family includesthe hypoxia inducible factor 1 alpha (HIF1A), which rapidly activates inresponse to reductions in the intracellularO2 tension (Lee et al., 2003), acurrent phenomenon during muscle contraction.

On the other hand, only a few SLC2A4 repressors have been proposed(Karnieli and Armoni, 2008). The most relevant one seems to be thenuclear factor NF-kappa-B (NFKB), a key mediator of some inflam-matory cytokines, pointing out that insulin resistance and T2DMare pro-inflammatory conditions (Hotamisligil et al., 1995). NFKB-mediated reduction in GLUT4 protein has long been reported(Ruan et al., 2002); however, only recently the NFKB-binding siteshave clearly been demonstrated in the Slc2a4 gene promoter region(Furuya et al., 2013).

Once several SLC2A4 gene enhancers and repressors are known, thetrue role of insulin must be elucidated, contributing to achieve a bettercontrol of the SLC2A4 expression. In this context, the present study in-vestigated, in skeletal muscle in vitro, the effect of insulin upon Slc2a4/GLUT4 expression, focusing on the AT-rich, E-box and NFKB-bindingsites. Our expectation is to contribute to understanding the physiologi-cal role of insulin upon SLC2A4 gene, as well as to determine potentialapproaches for prevention and/or treatment of insulin resistance.

Material and methods

Animals

Male Wistar rats obtained from the animal care facility of the Insti-tute of Biomedical Sciences, University of Sao Paulo were maintainedat 23 ± 2 °C, on a 12-h dark (18:00–06:00):12-h light (06:00–18:00)cycle, with free access to water and standard rodent chow diet (NuvilabCR-1, Nuvital, Curitiba, Brazil). The animals were studied at 2 months ofage, weighing 200–220 g and the experimental protocol (#054/02) wasapproved by the Ethical Committee for Animal Research (CEEA) of theInstitute of Biomedical Sciences, University of Sao Paulo.

In vitro incubation of soleus muscles

The left and right soleus muscles were gently dissected from anes-thetized rats (sodium pentobarbital, 50 mg/kg body weight, i.p.). Intacttendons of each muscle were attached to horizontal stainless steel sup-ports. All muscles were incubated for 180 min in 25 mL of oxygenatedKrebs–Henseleit bicarbonate buffer (pH 7.4) containing 0.1% bovineserum albumin and 8mMD-glucose at 37 °C. The 180min of incubationwas chosen based on two main reasons: 1) it is the time in which themaximum insulin effect on Slc2a4 expression is achieved (data not

shown); 2) it would provide time enough for theGLUT4 protein synthe-sis after the insulin stimulus. The buffer was continuously oxygenatedwith 95% O2–5% CO2. Control muscles were incubated in buffer alone(C), and experimental muscles were incubated with different concen-trations of insulin, as described in the figures, or with 15 nM insulin(I), 100 nM wortmannin (W), 50 μM 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML9) or 25 ng/mL tumornecrosis factor (TNF), either alone or in association with insulin(I + W; I + ML9; I + TNF). After every 45 min of incubation, thebuffer was replaced by fresh buffer in the same experimental con-dition. At the end of the 180 min incubation, the muscle sampleswere immediately frozen in liquid nitrogen for further analysis.Adequate oxygenation conditions of the muscles during 180 minincubation were confirmed based on the unchanged hypoxia in-ducible factor 1a messenger ribonucleic acid (mRNA) expressionobserved.

RT-PCR for mRNA analysis

Total RNA was extracted from the muscle samples using Trizol re-agent (Invitrogen, Carlsbad, CA, USA) and treated with RQ1 RNAasefree DNAase (Promega, Madison, WI, USA), according to themanufacturer's instructions. Complementary deoxyribonucleic acids(DNAs) were synthesized using InProm-IITM Reverse Transcriptase(Promega, Madison, WI, USA), oligo-dT primers and dNTP Set PCRGrade (Invitrogen, Carlsbad, CA, USA); and used for polymerase chainreaction (PCR) (GoTaq® Green Master Mix — Promega, Madison, WI,USA). Cycle number for each gene was defined after titration, and waswithin the logarithmic phase of amplification. The PCR cycling condi-tions and primers for beta actin (Actb), nuclear factor of kappa lightpolypeptide gene enhancer in B-cells (Nfkb1), hypoxia inducible fac-tor 1a (Hif1a), myocyte enhancer factor 2a (Mef2a) and myocyte en-hancer factor 2d Mef2d were according to Silva et al. (2005). ForSlc2a4 (NM_012751.1) and myogenic differentiation 1 (Myod1)(NM_176079.1), the primers were picked on Primer 3 Output (forward5′-CCCCTCCAGGGCAAAGGAT-3′; reverse 5′-TCCTGGAGGGGAACAAGAA-3′ and forward 5′-CGCTCCAACTGCTCTGATG-3′; reverse 5′-TAGTAGGCGTCGTAGCC-3′, respectively). The number of cycles was 31 for Actb, 38 forNf-κb, 36 for Hif1a, 33 for Mef2a, 30 for Mef2d, 40 for Slc2a4 and 31 forMyod1. Supplementary Table 1 provides the size of each amplicon. ThePCR products were separated on 1.5% EtBr-agarose gels and the band in-tensities were determined by digital scanning followed by quantificationusing Scion Image analysis software (Scion Corp., Frederick,MD). The re-sults were expressed as a ratio of target to Actb signals, and normal-ized considering the mean value of control samples as 100.

Western blotting

Total membrane protein samples were obtained, and used for im-munoblotting for GLUT4, as previously described (Barros et al., 2008;Alves-Wagner et al., 2009). Briefly, equal amounts of total membraneprotein (30 μg) were resolved by electrophoresis, electrotransferred toa nitrocellulose membrane and incubated with anti-GLUT4 antiserum(polyclonal rabbit raised anti-GLUT4, 07-1404/Millipore). Primary anti-bodywas detected by horseradish peroxidase-linked anti-rabbit immu-noglobulin (Amersham Biosciences, Buckinghamshire, UK), followed byenhanced chemiluminescence reagent Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione free acid, Sigma-Aldrich, PerkinElmer, Boston,MA). The intensity of chemiluminescence for each blot was quantifiedby densitometry (ImageScanner III, GE Healthcare, Uppsala, Sweden),and normalized by densitometric value of the respective lane in thepost-transferring Commassie-stained gel (Welinder and Ekblad, 2011)and/or in the Ponceau stained membrane. Results were then normalizedconsidering the mean of control samples in each gel as 100.

A

0

50

200

150

100

GLU

T4 p

rote

in(A

U)

C 1 10 50 100

***

*** *** ***## ## ##

Insulin (nM)

B

C 3.75 7.5 15

Slc2

a4m

RN

A(A

U)

0

50

200

150

100

250

***

***

*

&

&&&£££

Insulin (nM)

Slc2a4

Actb 510 bp

203 bp

GLUT4

Ponc

eau

stai

n

Fig. 1. Insulin effect on GLUT4 protein (panel A) and Slc2a4mRNA (panel B) expression insoleus muscle of rats. Muscles were incubated during 180min in control buffer (C) and inthe presence of different concentrations of insulin, as indicated. On top of panel A: repre-sentative blots of the GLUT4 protein and the corresponding lanes in the Ponceau stainedmembrane. Top of panel B: blots of the Slc2a4 and Actb amplicons. Data are expressed asmean ± SEM of 9 animals for GLUT4 and 5 animals for Slc2a4 mRNA; comparisons wereperformed using one-way ANOVA, followed by Student–Newman–Keuls; ***P b 0.001 vsC; ##P b 0.01 vs 1 nM; &P b 0.05 and &&&P b 0.001 vs 3.75 nM; £££P b 0.001 vs 7.5 nM.

38 P.A. Moraes et al. / Life Sciences 114 (2014) 36–44

Electrophoretic mobility shift assay (EMSA)

Nuclear protein extraction and binding to specific oligonucleotideswas performed as previously described (Lima et al., 2009; Campelloet al., 2012). Briefly, double-stranded oligonucleotides containing thesequences of the E-box (5′-GGGACCTGACATTTGGCGGA-3′-500/-480), AT-rich element (5′-CGTGGGAGCTAAAAATAGCCAT-3′-474/-453) (Lima et al., 2009) and κ-B site (5′-GGGTTGGGGGCGTGGCCTTTTGG-3′-166/-144) (Furuya et al., 2013), all from the ratSlc2a4 gene promoter, were end-labeled using T4 polymerase kinase(Invitrogen Life Technologies, Carlsbad, CA, USA) and [γ-32P]ATP(Amersham Pharmacia Biotech, Amersham, UK). Labeled oligonu-cleotides (30,000 cpm) were added to 15 μg protein from nuclearextract, at room temperature for 20 min, in binding buffer (20 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),pH 7.6, 50 mM potassium chloride (KCl), 10% glycerol, 0.2 mM eth-ylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT)and 2 μg of polydeoxyinosinicdeoxycytidylic acid (poly[dI-dC];Amersham Pharmacia Biotech)). The DNA–protein complexes wereelectrophoresed on 4% non-denaturing polyacrylamide gels at 4 °Cin 45 mM Tris, 45 mM borate and 1 mM EDTA buffer. The gels weredried and subjected to autoradiography and the blots were analyzedby scanner densitometry (ImageScanner III, GE Healthcare, Uppsala,Sweden). The results of the binding activity were normalized in eachgel considering the mean value of control samples as 100 and wereexpressed as arbitrary units.

Data analysis

All data were reported as mean ± SEM of 5 to 9 different animals.For each data set analyzed, themuscles incubatedwith Krebs–Henseleitbuffer constituted the control group, normalized to 100. The meanswere statistically analyzed by one-way ANOVA, with Student–Newman–Keuls post hoc testing.

Results

Insulin rapidly enhances Slc2a4/GLUT4 expression

We incubated soleusmuscles for 180min in the presence or absenceof insulin to check for the isolated effect of this hormone in the expres-sion of the Slc2a4 gene (Fig. 1). When growing doses of insulin werepresent in the incubation buffer, GLUT4 protein increased more than50% as from the lower dose of 1 nM (P b 0.001 vs C). With 10 nM, thisGLUT4 increasing effect reached the maximum response (~100%), asdemonstrated by the absence of further increase with 50 nM and100 nM (Fig. 1A). Since a significant difference was found between theeffects of 1 nM and 10 nM (P b 0.01) with no additional increase afterthis point, a 0–15 nM rangewas assumed as an interval in which insulineffect on Slc2a4/GLUT4 would be dose dependent. This was confirmedby the gradual increase in Slc2a4 mRNA expression within this range(Fig. 1B), in which the Pearson's correlation between insulin concentra-tion and Slc2a4 mRNA content was highly significant (r = 0.09992,P b 0.001).

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gammaisoform (PI3K)/serine/threonine protein kinase (AKT) pathway mediatesthe insulin effect on Slc2a4/GLUT4 expression

Once we had observed the clear effect of insulin increasing theSlc2a4/GLUT4 expression in incubated soleus muscle, it was importantto demonstrate whether this effect would involve the classic insulin-activated PI3K/AKT pathway. Thus, we investigated the effect of PI3Kand AKT inhibition on the Slc2a4/GLUT4 content. In fact, the additionof either the PI3K inhibitor wortmannin or the AKT inhibitor ML9(Fig. 2A and B) decreased the insulin-stimulated GLUT4 increase

(P b 0.001 vs I). However, this inhibition was not complete, sincewortmannin plus insulin incubated muscles had 44% more GLUT4than control (P b 0.01 vs C) and ML9 plus insulin muscles were 50%richer in GLUT4 than control muscles (P b 0.001 vs C). This result sug-gests that GLUT4 increase in response to insulin may be stimulated, atleast in part, by mechanisms independent of the PI3K/AKT pathway.On the other hand, either wortmannin or ML9 completely abolishedthe insulin-stimulated Slc2a4 increase (Fig. 2C), suggesting the insulineffect on the gene expression, different from the effect on proteinexpression, depends exclusively on the PI3K/AKT pathway.

A

***

###**

C I+W I0

300

100

200

GLU

T4 (A

U) ***

***###

C I+ML9 I0

100

200

GLU

T4 (A

U)

B

C

***

### ###

Slc2

a4m

RN

A(A

U)

0

200

100

C I I+W I+ML9

Slc2a4

Actb 510 bp

203 bp

GLUT4

Ponc

eau

stai

n

GLUT4

Ponc

eau

stai

n

Fig. 2. PI3K/AKT pathway is involved in the insulin effect on GLUT4 protein (panels A and B) and Slc2a4 mRNA (panel C) expression in soleus muscle of rats. Muscles wereincubated during 180 min in control buffer (C) and in the presence of 15 nM insulin alone (I) or added with 100 nMwortmannin (I +W) or 50 μMML9 (I + ML9). Representativeblots of theGLUT4protein aswell as the corresponding lanes in the Ponceau stainedmembrane are shown on the top of panels A and B.On top of panel C: representative blots of the Slc2a4and Actb amplicons. Data are expressed as mean ± SEM of 5 animals; comparisons were performed using one-way ANOVA, followed by Student–Newman–Keuls; **P b 0.01 and***P b 0.001 vs C; ###P b 0.001 vs I.

39P.A. Moraes et al. / Life Sciences 114 (2014) 36–44

Insulin enhances the binding activity to the AT-rich element present in theSlc2a4 promoter and the PI3K/AKT pathway mediates this effect

Themyocyte enhancer factors (MEFs) are known to bind the AT-richelement, enhancing the Slc2a4 gene expression. To check whether theinsulin-stimulated Slc2a4 expression would relate to the activation ofthe AT-rich element, EMSA analysis was performed (Fig. 3A). Indeed,15 nM of insulin increased the nuclear protein binding to the AT-richelement in ~75% (Fig. 4A). Besides, insulin increased Mef2a and Mef2dmRNA by 70% (Fig. 3B) and 65% (Fig. 3C), respectively (P b 0.001 vsC). To confirm the PI3K/AKT pathway participation in this insulin effect,wortmannin or ML9was added to the incubation buffer and both drugscompletely abolished the insulin-stimulated AT-rich binding activity(Fig. 3A, P b 0.001 vs. I). It is interesting to notice that wortmanninand ML9 affected exclusively the binding of the MEFs to the AT-rich

element, having no effect on the Mef2a or Mef2d mRNA contents(Fig. 3B and C).

Insulin enhances the binding activity to the E-box element present in theSlc2a4 promoter and the PI3K/AKT pathway mediates this effect

HIF1A and myoblast determination protein (MYOD1) are transcrip-tional factors known to increase the Slc2a4 expression. Herein, we dem-onstrate that different doses of insulinwere unable to increase theHif1amRNA expression (Supplementary Fig. 2) and that inhibition of PI3K orAKTdid not affect theHif1a gene expression either (Fig. 4B). Interesting-ly, this unchanged Hif1a is a good indicator of the tissue integrity afterhandling and 180 min incubation, since this protein rapidly increaseswhenever the ATP/ADP rate decreases. Thus, the maintained Hif1aserved in this case, as a good quality control of the experimental

C I I+W I+ML9

***

######

200

0

100

Bin

ding

activ

ity(A

U)

AT-rich element /protein complex

C I I+W I+ML9

Mef

2am

RN

A(A

U)

200

0

100

*** *** ***

Mef2a

Actb 510 bp

436 bp

C I I+W I+ML9

200

0

100

Mef

2dm

RN

A(A

U) *** *** ***

Mef2d

Actb 510 bp

333 bp

A

B C

Fig. 3. Insulin enhances the binding activity in the AT-rich element located in the Slc2a4 promoter, in a PI3K/AKT-mediatedway. Muscles were incubated during 180min in control buffer(C) and in the presence of 15 nM insulin alone (I) or added with 100 nM wortmannin (I + W) or 50 μM ML9 (I + ML9). In A, electrophoretic mobility shift assay analysis showing therepresentative image of the AT-rich element/nuclear protein complex (left), and the binding activity analysis (right). In B and C, the myocyte enhancer factor 2 a (Mef2a) and Mef2dmRNAs expression, respectively; on the top of each panel, representative images of the Mef2a, Mef2d and Actb amplicons are shown, with base pair numbers indicated on the right.Data are expressed as mean ± SEM of 5 animals; comparisons were performed using one-way ANOVA, followed by Student–Newman–Keuls; ***P b 0.001 vs C; ###P b 0.001 vs I.

40 P.A. Moraes et al. / Life Sciences 114 (2014) 36–44

conditions. Differently from theHif1a expression,Myod1mRNA (Fig. 4C)increased in response to insulin (~20%, P b 0.05 vs C); but this effectwas not altered by inhibition of PI3K or AKT. Concerning the bindingactivity, insulin increased by 96% the protein binding to the E-boxsequence of Slc2a4 promoter (P b 0.001 vs C); wortmannin andML9 completely abolished this insulin-induced binding activity, re-vealing the involvement of PI3K/AKT pathway in the binding activity(Fig. 4A).

Insulin represses the Slc2a4-NFKB-site activity and counterbalances the re-pressive effect of TNF on GLUT4 expression

NFKB activation has been demonstrated as an inhibitor of Slc2a4/GLUT4 expression. This is in agreement with our observation that insu-lin decreases by ~50% the binding activity to the Slc2a4 NFKB-site(Fig. 5A), as well as the nuclear factor of kappa light polypeptide geneenhancer in B-cells 1 Nfkb1 mRNA expression (Fig. 5B), independentlyof the dose. To confirm the insulin effect on the NFKB-site activity andthe GLUT4 protein expression, we investigated its effect on the TNF-induced hyperactivity of NFKB-site (Fig. 6). In fact, TNF increased theNFKB-site activity in 58% (P b 0.001) and decreased GLUT4 content in35%. (P b 0.01). Addition of insulin to TNF prevented the TNF-inducedincrease in the NFKB-site activity (TNF vs I + TNF, P b 0.01), and re-stored the GLUT4 content to a value higher than that observed in basal

control condition (C vs I+ TNF, P b 0.01). Altogether, these data confirmthat insulin counterbalances the TNF repressive effect on GLUT4expression (Fig. 6B).

Discussion

GLUT4 protein plays a key role in the glycemic homeostasis, and adeep knowledge of the transcriptional regulation of Slc2a4 can contrib-ute to establish potential approaches to control its expression. Thisstudy shows that insulin in vitro enhances Slc2a4mRNA andGLUT4 pro-tein expression in soleus muscle by stimulating AT-rich and E-box andby repressing κB binding domains in the Slc2a4 promoter region.

Seventy minutes of in vitro stimulation with insulin (16.7 nM) hasincreased Slc2a4mRNA in themuscle from 48 hour fasted rats, a condi-tion that decreases this mRNA (Silva et al., 2005). Now we show in themuscle from fed rats, that 180 min incubation with insulin increasesSlc2a4mRNA expression dose-dependently. Besides, the GLUT4 proteincontent also increased in the lowest 1 nM insulin concentration,suggesting this may occur during post-prandial periods, participatingin the physiological regulation of GLUT4, and in the maintenance ofglycemic control. Differently, in vivo 120 min infusion of insulin to 48-fasted rats has reduced the Slc2a4 mRNA expression (Zanquetta et al.,2014). However, two different experimental conditions in that studymust be considered: 1) the in vivo condition, in which fasting triggers

E-box element /protein complex

C I I+W I+ML9

***

### ###

200

0

100

Bin

ding

activ

ity(A

U)

Hif1a

Actb 510 bp

261 bp

C I I+W I+ML9

Hif1

am

RN

A(A

U)

200

0

100

A

B CActb 510 bp

Myod1 86 bp

C I I+W I+ML9

100

0

50

Myo

d1 m

RN

A(A

U)

* * *

Fig. 4. Insulin enhances the binding activity in the E-box region located in the Slc2a4 promoter, in a PI3K/AKT-mediated way. Muscles were incubated during 180 min in control buffer(C) and in the presence of 15 nM insulin alone (I) or added with 100 nM wortmannin (I + W) or 50 μM ML9 (I + ML9). In A, electrophoretic mobility shift assay analysis showing therepresentative image of the E-box element/nuclear protein complex (left), and the binding activity analysis (right). In B and C, the hypoxia inducible factor 1a (Hif1a) and the myogenicdifferentiation 1 (Myod1) mRNA expression, respectively; on the top of each panel, representative images of the Hif1a,Myod1 and Actb amplicons are shown, with base pair numbers in-dicated on the right. Data are expressed as mean ± SEM of 5 animals; comparisons were performed using one-way ANOVA, followed by Student–Newman–Keuls; *P b 0.05 and***P b 0.001 vs C; ###P b 0.001 vs I.

41P.A. Moraes et al. / Life Sciences 114 (2014) 36–44

modulators such as the Slc2a4 repressor NFKB (Silva et al., 2005), and 2)the lower 0.24 nM insulin concentration achieved by the infusion, ascompared to 1 nM concentration in the present study.

Interestingly, in the present study, the addition of wortmannin orML9 to insulin in the incubation buffer prevented the insulin-inducedenhancement of Slc2a4mRNA, highlighting the participation of the ca-nonical PI3K/AKT pathway in the insulin regulation of Slc2a4 gene. Ad-ditionally, PI3K/AKT inhibition strongly blocked the insulin-inducedGLUT4 protein increase, but this blocking effect was even more com-plete on the mRNA increase; a profile maybe related to the half-livesof the Slc2a4 mRNA/GLUT4 protein and/or changes in the translationefficiency.

The MEF2A/D heterodimer complex (Mora and Pessin, 2000), bybinding to the AT-rich element (Santalucia et al., 2001), activatesSlc2a4 gene expression in the skeletal muscle. MEF2A/D activity in-creases in response to muscle contraction as soon as 30 min aftercontractile stimulus (Silva et al., 2005; Lima et al., 2009). Thisstudy shows that insulin induced an important increase in the nu-clear protein binding activity into the AT-rich element, depicting,for the first time, the participation of this enhancer domain in the

insulin-induced regulation of Slc2a4 expression. This effect mustbe mediated by MEF2A/D activation, pointing out that insulin alsoincreased the MEF2A and MEF2D mRNA expression (SupplementaryFig. 1).

MEF2 activation in response to muscle contraction is a CaMK-(calcium/calmodulin-dependent protein kinase) and AMPK- (AMP-activated protein kinase) mediated effect (Smith et al., 2007; Holmeset al., 2005). Now, we demonstrate that the PI3K/AKT pathway alsoparticipates in the MEF2A/D activation, since wortmannin and ML9prevented the insulin-induced increase in the AT-rich elementbinding activity. Altered MEF2A/D activity has already been relatedto diabetes-induced reduction in GLUT4 (Mora and Pessin, 2000);therefore, we can propose that insulin resistance, by reducing PI3K/AKT pathway activity, decreases MEF2A/D activity, thus decreasingSlc2a4 expression as observed in diabetes.

PI3K/AKT inhibition was not enough to reduce either MEF2A orMEF2D mRNA expression. Nevertheless, it is reasonable to supposethat the transcription factor binding activity is rapidly triggered, with-out changes in its mRNA and/or protein content. In fact, a single boutof exercise and CaMK activation both increase the binding activity of

Slc2a4-NFKB-site (-134) / protein complex

Insulin (nM)

C 3.75 7.5 15

C 3.75 7.5 15

***

150

0

50

Bin

ding

activ

ity(A

U)

100

*** ***

Insulin (nM)

Insulin (nM)

*** *** ***

A

B

Nfk

b1m

RN

A(A

U)

100

0

50

150

Nfkb1

Actb 510 bp

266 bp

Fig. 5. Insulin represses the nuclear factor NF-kappa B (NFKB) binding to the NFKB-site located in the Slc2a4 promoter. Muscles were incubated during 180min in control buff-er (C) and in the presence of different concentrations of insulin, as indicated. In A, electrophoretic mobility shift assay analysis showing the representative image of the NFKB-binding site/nuclear protein complex (left) and the binding activity analysis (right). In B, the Nfkb1mRNA expression; on the top, representative images of the Nfkb1 and Actbamplicons are shown, with base pair numbers indicated on the right. Data are expressed as mean ± SEM of 5 animals; comparisons were performed using one-way ANOVA,followed by Student–Newman–Keuls; ***P b 0.001 vs C.

42 P.A. Moraes et al. / Life Sciences 114 (2014) 36–44

MEF2A into the Slc2a4 promoter, without changes in the MEF2A pro-tein content (Smith et al., 2007). These findings indicate thatMEF2A/D activity is modulated independently of variations in theprotein content; and may involve MEF2 nuclear translocation(Holmes et al., 2005) and/or histone deacetylases-regulatedsumoylation and acetylation of MEF2 (Zhao et al., 2005), facilitatingthe MEF2/DNA binding of preexisting proteins.

E-box transcriptional activitywasfirstly reported as fundamental formyogenic cell differentiation, in which MYOD1 protein plays an impor-tant role (Winter et al., 1993). Later on, the HIF1A was found to bind tothe E-box, increasing GLUT4 expression in brain subjected to transienthypoxia (Royer et al., 2000). Furthermore, we have shown that thebinding of HIF1A to the E-box participates in the contraction-inducedSlc2a4/GLUT4 expression (Silva et al., 2005; Lima et al., 2009). Now,the results depict for the first time that insulin induces a 2-fold increasein the binding activity of nuclear proteins into the E-box of the Slc2a4promoter. Considering that in response to insulin the Hif1a mRNA

content remained unchanged, whereas the Myod mRNA increased, wecan propose that the E-box enhanced activity was mainly related toMYOD1.

Cyclic AMP dependent protein kinase (PKA) overactivity has beendescribed as a negative regulator of E-box transcriptional activity(Winter et al., 1993). The present study reveals that insulin increasesE-box binding activity in a PI3K/AKT-mediated pathway, since bothwortmannin and ML9 prevented the effect. However, this effect seemsto be specific to the protein binding activity, since neither wortmanninnorML9 altered the expression of bothHif1a andMyod1mRNAs. In fact,the observed modulations of Hif1a and Myod1mRNAs do not precludealterations in the protein content and binding to DNA, as discussedabove for MEF2. Interestingly, PI3K/AKT inhibition has recently been re-ported to repress HIF1A/DNA binding in childhood tumors (Kilic-Erenet al., 2013), revealing that PI3K/AKT participates in the regulation of E-box/HIF1A activity, and supporting the hypothesis that activation of thecanonical insulin pathway activates the E-box transcriptional activity.

C I+TNF I

***

###

200

0

100B

indi

ngac

tivity

(AU

)

TNF

**

&&

###

##

***&&###***

**

###

GLU

T4 (A

U)

100

0

200

C I+TNF ITNF

Slc2a4-NFKB-site (-134) /protein complex

A

BGLUT4

Ponc

eau

stai

n

Fig. 6. Insulin counterbalances the repressive effect of tumor necrosis factor (TNF) onSlc2a4/GLUT4 expression. Muscles were incubated during 180 min in control buffer(C) and in the presence of 15 nM insulin alone (I), 25 ng/mL TNF (TNF) or the associationof insulin plus TNF (I + TNF). In A, electrophoretic mobility shift assay analysis for thenuclear factor kappa B, showing the representative image of the NFKB-binding site/nuclear protein complex (left), and the binding activity analysis (right). In B, theGLUT4 protein analysis with representative image of the blots and the correspondinglanes in the Ponceau stained membrane on the top. Data are expressed as mean ±SEM of 5 animals; comparisons were performed using one-way ANOVA, followedby Student–Newman–Keuls; **P b 0.01 and ***P b 0.001 vs C; ##P b 0.01 and###P b 0.001 vs TNF; &&P b 0.01 vs I.

43P.A. Moraes et al. / Life Sciences 114 (2014) 36–44

NFKB inhibitory effect on GLUT4 expression was firstly described asmediating the repressor effect of TNF (Ruan et al., 2002). Insulin-induced inhibition of NFKB has been suggested in vivo, reducingintranuclear NFKB in mononuclear cells of obese humans (Dandonaet al., 2001), and in vitro, reducing NFKB-binding activity in human aor-tic endothelial cells (Aljada et al., 2001). However, clear evidences ofinsulin-inducedNFKB regulation of Slc2a4 gene have never been report-ed so far, since the NFKB-site in the Slc2a4 gene had not been identifieduntil recently (Furuya et al., 2013).

Participation of the NFKB in the insulin-induced Slc2a4 regulationwas suggested in skeletal muscle harvested from 48-hour fasted rats(Silva et al., 2005). However, that pioneering study investigated thepro-tein/DNA binding using an oligonucleotide containing a consensusNFKB-site, which actually does not exist in the Slc2a4 promoter. Nowwe have used the oligonucleotide corresponding to the exact sequenceof the recently characterized NFKB-site in the Slc2a4 promoter, flankedby6 bases in each 5′ and 3′ side, which is 74% homologous in the core, ascompared to the consensus binding site. Our data reveal that insulin de-creases the NFKB-site activity to 50% of the basal activity. Once the

repressor effect of this domain on Slc2a4 transcription is definitivelyconfirmed (Furuya et al., 2013), the present results depict that insulin-induced decrease in the NFKB-site activity participates in the stimula-tion of Slc2a4/GLUT4 expression.

In addition to the insulin-induced DNA binding of NFKB, this studyalso reveals that insulin rapidly decreases the Nfκb1 mRNA content.Nfκb1 encodes for the p105 protein, a precursor of the p50 protein.We have chosen the Nfκb1 gene because chromatin immunoprecipita-tion analysis has demonstrated that in vivo, there was much more p50than p60 bound into theDNA fragment (−151 to+73),which containsthe NFKB-site (Furuya et al., 2013).

In order to confirm the insulin effect on theNFKB-site activity, we in-vestigated whether insulin would affect the classic NFKB-mediatedTNF-induced repression of the Slc2a4 gene (Ruan et al., 2002). In fact,TNF induced an important increase in the NFKB-site binding activity;an effect prevented by insulin, confirming the repressor effect of insulinon the NFKB-binding activity. Besides, TNF also decreased GLUT4 pro-tein, as expected; however, when insulin and TNF were given together,the insulin enhancing effect on GLUT4 was only partially reduced; indi-cating the remaining stimulation must be ascribed to other enhancerssuch as those operating in the At-rich and E-box elements.

The NFKB-mediated effect of TNF on GLUT4 repression in adipocyteshas been related to a partial inhibition of AKT (Ruan et al., 2002). Thus, itis understandable that under insulin stimulus a remaining AKT activityensures at least a partial stimulation of the enhancer factors binding toAT-rich and E-box elements. These data highlight the importance ofinvestigating the TNF effect in the presence of insulin, as it must beoperating in inflammatory states in vivo. Besides, in vitro effects of TNFobserved in the absence of insulin should be revised, in order to betranslated to in vivo.

Conclusion

The present study demonstrates that insulin enhances the Slc2a4/GLUT4 expression in the skeletal muscle within a few hours. This effectis at least partially mediated by PI3K/AKT and involves increased bind-ing activity on the AT-rich and E-box elements of Slc2a4 gene promoter,and decreased binding activity on the NFKB-site as well. These resultsunravel how post-prandial insulin elevation may participate in themaintenance of GLUT4 expression and, consequently, of glycemic ho-meostasis, as well as how the insulin signaling impairment can par-ticipate in insulin resistance-induced repression of GLUT4,contributing to damage the glycemic control.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.lfs.2014.07.040.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

This research was supported by a Fundacao de Amparo a Pesquisado Estado de Sao Paulo (FAPESP) grant 2012/04831-1. P. A. Moraeswas recipient of a FAPESP Fellowship 2007/57873-5 and R.C. Moriis a postdoctoral fellow from CAPES, Brazil. The authors are indebtedto Kaio Vitzel for providing the primers used in the analysis ofmyod1mRNA expression.

References

Aljada A, Ghanim H, Saadeh R, Dandona P. Insulin inhibits NFkappaB and MCP-1 expres-sion in human aortic endothelial cells. J Clin Endocrinol Metab 2001;86:450–3.

Alves-Wagner AB, De Freitas HS, De Souza PB, Seraphim PM, Mori RC, Machado UF. Beta-adrenergic activity preserves GLUT4 protein in glycolytic fibers in fasting. MuscleNerve 2009;40:847–54.

Barros RP, Morani A, Moriscot A, Machado UF. Insulin resistance of pregnancy involvesestrogen-induced repression of muscle GLUT4. Mol Cell Endocrinol 2008;295:24–31.

44 P.A. Moraes et al. / Life Sciences 114 (2014) 36–44

Campello RS, Alves-Wagner AB, Abdulkader F, Mori RC, Machado UF. Carbohydrate- andlipid-enriched meals acutely disrupt glycemic homeostasis by inducing transientinsulin resistance in rats. Can J Physiol Pharmacol 2012;90:537–45.

Correa-GiannellaML,MachadoUF. SLC2A4gene: a promising target for pharmacogenomicsof insulin resistance. Pharmacogenomics 2013;14:847–50.

Dandona P, Aljada A, Mohanty P, Ghanim H, Hamouda W, Assian E, et al. Insulin inhibitsintranuclear nuclear factor kappaB and stimulates IkappaB in mononuclear cells inobese subjects: evidence for an anti-inflammatory effect? J Clin Endocrinol Metab2001;86:3257–65.

Flores-Riveros JR, McLenithan JC, Ezaki O, Lane MD. Insulin down-regulates expression ofthe insulin-responsive glucose transporter (GLUT4) gene: effects on transcriptionand mRNA turnover. Proc Natl Acad Sci U S A 1993;90:512–6.

Furuya DT, Poletto AC, Favaro RR, Martins JO, Zorn TM, Machado UF. Anti-inflammatoryeffect of atorvastatin ameliorates insulin resistance in monosodium glutamate-treated obese mice. Metabolism 2010;59:395–9.

Furuya DT, Neri EA, Poletto AC, Anhe GF, Freitas HS, Campello RS, et al. Identification ofnuclear factor-kappaB sites in the Slc2a4 gene promoter. Mol Cell Endocrinol 2013;370:87–95.

Herman MA, Kahn BB. Glucose transport and sensing in the maintenance of glucosehomeostasis and metabolic harmony. J Clin Invest 2006;116:1767–75.

Holmes BF, Sparling DP, Olson AL, Winder WW, Dohm GL. Regulation of muscle GLUT4enhancer factor and myocyte enhancer factor 2 by AMP-activated protein kinase.Am J Physiol Endocrinol Metab 2005;289:E1071–6.

Hotamisligil GS, Arner P, Caro JF, Atkinson RL, Spiegelman BM. Increased adipose tissueexpression of tumor necrosis factor-alpha in human obesity and insulin resistance. JClin Invest 1995;95:2409–15.

Im SS, Kwon SK, Kim TH, Kim HI, Ahn YH. Regulation of glucose transporter type 4isoform gene expression in muscle and adipocytes. IUBMB Life 2007;59:134–45.

Karnieli E, Armoni M. Transcriptional regulation of the insulin-responsive glucose trans-porter GLUT4 gene: from physiology to pathology. Am J Physiol Endocrinol Metab2008;295:E38–45.

Kilic-Eren M, Boylu T, Tabor V. Targeting PI3K/AKT represses Hypoxia inducible factor-1alpha activation and sensitizes Rhabdomyosarcoma and Ewing's sarcoma cells forapoptosis. Cancer Cell Int 2013;13:36.

Knight JB, Eyster CA, Griesel BA, Olson AL. Regulation of the human GLUT4 gene promot-er: interaction between a transcriptional activator and myocyte enhancer factor 2A.Proc Natl Acad Sci U S A 2003;100:14725–30.

Lee M, Hwang JT, Lee HJ, Jung SN, Kang I, Chi SG, et al. AMP-activated protein kinase ac-tivity is critical for hypoxia-inducible factor-1 transcriptional activity and its targetgene expression under hypoxic conditions in DU145 cells. J Biol Chem 2003;278:39653–61.

Lima GA, Anhe GF, Giannocco G, Nunes MT, Correa-Giannella ML, Machado UF. Contrac-tile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle:involvement of MEF2D, HIF-1a, and TRalpha transcriptional factors. Am J PhysiolEndocrinol Metab 2009;296:E132–8.

Mora S, Pessin JE. The MEF2A isoform is required for striated muscle-specific expressionof the insulin-responsive GLUT4 glucose transporter. J Biol Chem 2000;275:16323–8.

O'Gorman DJ, Karlsson HK, McQuaid S, Yousif O, Rahman Y, Gasparro D, et al. Exercisetraining increases insulin-stimulated glucose disposal and GLUT4 (SLC2A4) proteincontent in patients with type 2 diabetes. Diabetologia 2006;49:2983–92.

Royer C, Lachuer J, Crouzoulon G, Roux J, Peyronnet J, Mamet J, et al. Effects of gestationalhypoxia on mRNA levels of Glut3 and Glut4 transporters, hypoxia inducible factor-1and thyroid hormone receptors in developing rat brain. Brain Res 2000;856:119–28.

Ruan H, Hacohen N, Golub TR, Van Parijs L, Lodish HF. Tumor necrosis factor-alpha sup-presses adipocyte-specific genes and activates expression of preadipocyte genes in3T3-L1 adipocytes: nuclear factor-kappaB activation by TNF-alpha is obligatory. Dia-betes 2002;51:1319–36.

Santalucia T, Moreno H, Palacin M, Yacoub MH, Brand NJ, Zorzano A. A novel functionalco-operation between MyoD, MEF2 and TRalpha1 is sufficient for the induction ofGLUT4 gene transcription. J Mol Biol 2001;314:195–204.

Silva JL, Giannocco G, Furuya DT, Lima GA, Moraes PA, Nachef S, et al. NF-kappaB, MEF2A,MEF2D and HIF1-a involvement on insulin- and contraction-induced regulation ofGLUT4 gene expression in soleus muscle. Mol Cell Endocrinol 2005;240:82–93.

Sivitz WI, DeSautel SL, Kayano T, Bell GI, Pessin JE. Regulation of glucose transportermessenger RNA in insulin-deficient states. Nature 1989;340:72–4.

Smith JA, Collins M, Grobler LA, Magee CJ, Ojuka EO. Exercise and CaMK activation bothincrease the binding of MEF2A to the Glut4 promoter in skeletal muscle in vivo.Am J Physiol Endocrinol Metab 2007;292:E413–20.

Sparling DP, Griesel BA, Weems J, Olson AL. GLUT4 enhancer factor (GEF) interacts withMEF2A and HDAC5 to regulate the GLUT4 promoter in adipocytes. J Biol Chem2008;283:7429–37.

Tsao TS, Burcelin R, Katz EB, Huang L, CharronMJ. Enhanced insulin action due to targetedGLUT4 overexpression exclusively in muscle. Diabetes 1996;45:28–36.

Welinder C, Ekblad L. Coomassie staining as loading control in Western blot analysis. JProteome Res 2011;10:1416–9.

Winter B, Braun T, Arnold HH. cAMP-dependent protein kinase represses myogenicdifferentiation and the activity of the muscle-specific helix–loop–helix transcriptionfactors Myf-5 and MyoD. J Biol Chem 1993;268:9869–78.

Yu ZW, Buren J, Enerback S, Nilsson E, Samuelsson L, Eriksson JW. Insulin can enhanceGLUT4 gene expression in 3T3–F442A cells and this effect is mimicked by vanadatebut counteracted by cAMP and high glucose—potential implications for insulin resis-tance. Biochim Biophys Acta 2001;1535:174–85.

ZanquettaMM, Alves-Wagner AB, Mori RC, Campello RS, Machado UF. Recovery of insulinsensitivity and Slc2a4 mRNA expression depend on T3 hormone during refeeding.Metabolism 2014;63:328–34.

Zhao X, Sternsdorf T, Bolger TA, Evans RM, Yao TP. Regulation of MEF2 by histonedeacetylase 4- and SIRT1 deacetylase-mediated lysine modifications. Mol Cell Biol2005;25:8456–64.