The histone deacetylase inhibitor sodium butyrate improves insulin signalling in palmitate-induced insulin resistance in L6 rat muscle cells through epigenetically-mediated up-regulation of Irs1

Molecular and Cellular Endocrinology xxx (2016) 1e9

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The histone deacetylase inhibitor sodium butyrate improves insulin signalling in palmitate-induced insulin resistance in L6 rat muscle cells through epigenetically-mediated up-regulation of Irs1 Sabrina Chriett, Ouafa Zerzaihi, Hubert Vidal, Luciano Pirola* Carmen Laboratory, INSERM U1060, Lyon-1 University, South Lyon Medical Faculty, 69921 Oullins, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 April 2016 Received in revised form 7 September 2016 Accepted 8 September 2016 Available online xxx

Dietary administration of the histone deacetylase (HDAC) inhibitor butyric acid e a short chain fatty acid present in milk products and also bacterially produced in the intestine e has been shown to increase energy expenditure and favour insulin sensitivity in mice through induction of PGC1a (peroxisome proliferator-activated receptor gamma co-activator 1a) and AMPK (AMP-activated protein kinase) in skeletal muscle, and a consequential increase of mitochondrial fatty acid oxidation. Here, we investigate whether such physiological improvements are associated to epigenetic effects dependent on increased histone acetylation and whether butyrate exerts a direct action on skeletal muscle insulin signalling. We show that sodium butyrate (NaBut) ameliorates the insulin-resistant phenotype, induced in L6 myotubes by prolonged exposure to palmitate, by i) increasing the insulin-induced phosphorylation of both PKB (protein kinase B) and MAPK (mitogen activated protein kinase), the two branches of insulin signalling and ii) increasing histone H3 acetylation e even in the presence of palmitate - on chromatin in proximity of the Irs1 (insulin receptor substrate 1) transcriptional start site. Consequently, NaBut induced Irs1 mRNA and protein overexpression, which in turn relayed higher insulin-stimulated IRS1 tyrosine phosphorylation and PI 3-kinase (phosphoinositide 3-kinase) association, suggesting that the increased IRS1 expression may mediate the insulin-sensitizing effects of NaBut. Furthermore, downstream of PKB, NaBut induced GSK3b gene upregulation. Our observations indicate that NaBut e through its action as HDAC inhibitor e can promote insulin responsiveness in L6 myotubes under conditions of lipid-induced insulin resistance. © 2016 Elsevier Ireland Ltd. All rights reserved.

Keywords: Histone acetylation Insulin signalling HDACi Sodium butyrate L6 myotubes

1. Introduction The prospect of epigenetic therapies through modulation of DNA methylation and histone post-translational modifications (PTMs) via administration of small molecules, initially developed in the cancer field (Ellis and Pili, 2010; Spannhoff et al., 2009), is now being extended to potentially cover diabetes and metabolic diseases.

Abbreviations: FFAs, free fatty acids; HAT, histone acetyltransferase; HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor; HK2, hexokinase 2; NaBut, sodium butyrate; PI 3-kinase, phosphoinositide 3-kinase; PTMs, posttranslational modifications; TSA, trichostatin A. * Corresponding author. INSERM U1060, 165 Ch. du Grand Revoyet - BP12, 69921 Oullins, France. E-mail address: [email protected] (L. Pirola).

Among the many histone PTMs that shape the chromatin transcriptional landscape (Turner, 2002), pharmacological modulation of acetylation has been under intensive investigation. Activation of the class III NADþ-dependent deacetylase SIRT1 through the small natural molecule resveratrol has been shown to protect against metabolic disease in rodent models (Baur et al., 2006; Lagouge et al., 2006) and in small-cohort clinical trials (Timmers et al., 2011). Conversely, recent research suggests that inhibition of class I/II histone deacetylases (HDAC) is associated to improved metabolic responses. In liver, class IIa HDACs deacetylate and activate Forkhead box (FOXO), favouring the expression of gluconeogenic FOXO target genes. Thus, HDACs inhibition may provide a potential therapeutic approach for metabolic syndrome by decreasing gluconeogenesis (Mihaylova et al., 2011). The small dietary molecule NaBut inhibits class I and class II

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Please cite this article in press as: Chriett, S., et al., The histone deacetylase inhibitor sodium butyrate improves insulin signalling in palmitateinduced insulin resistance in L6 rat muscle cells through epigenetically-mediated up-regulation of Irs1, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.006

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Table 1 Sequences of primers used in this study for mRNA quantifications (upper table) and ChIP (lower table). Gene

Sequence

Amplicon size bp

Annealing temperature

Hprt1

AS: AGT-TGA-GAG-ATC-ATC-TCC-AC S: TTG-CTG-ACC-TGC-TGG-ATT-AC AS: AAG-ACG-TGA-GGT-CCT-GGT-TG S: ACC-AGA-GGA-CCG-TCA-ATA-GC

151 pb

55  C

138 pb

60  C

AS: AGA-ATG-GAT-TCA-GAG-TCT-TCG S: TCG-ACT-TCT-TGT-CCC-ATC-AC AS: CCA-TGT-AGC-AGG-CGT-TGC-TG S: CTT-CTC-GTT-CCC-CTG-CCA-CC AS: CTG-GGT-TTC-ACC-TCC-TGC-TC S: GGG-TTT-CCA-GTA-TGT-TGC-GG AS: TCC-ATA-AAG-CAG-CCA-AAG-CC S: TCA-GAC-CCC-ATC-TTG-ACC-AC AS: AAC-ACC-ACT-GGA-AGC-TTG-TGC S: TTC-AGG-TGG-AGT-TGG-AAG-CTG-ATG-C

99 pb

55  C

202 pb

62  C

183 pb

60  C

260 pb

60  C

278 pb

62  C

Irs1 Irs2 Hk2 Glut4 GS Gsk3b Gene

Sequence

Annealing temperature

Irs1

AS: TGG TGG CGG CGG GGA CTG TT S: GGA GGC GGG CTG CCA AGT CC AS: GCG GTA CCA GCC CTC C S: AAG CAC AAG TAC CTG AT AS: GGG-GAG-TCT-GGG-AGT-GTC-AAC-CAG S: AGG-GCC-AAT-CTA-GGG-CCA-GGA-G AS: GCG-AGT-CGG-GGT-CGA-GTA-GAG-AAA-C S: CCT-GGT-TTC-AAA-GCG-GTC-GAA-CTG

58  C

Irs2 Hk2 (4339, upstream of the TSS) Hk2 (þ106, downstream of the TSS)

histone deacetylases in a competitive fashion (Sekhavat et al., 2007), and dietary administration of NaBut has been shown to improve systemic insulin sensitivity and increase energy expenditure in mice via up-regulation of mitochondrial function in skeletal muscle and brown fat, through peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a) induction and elevation of AMP-activated protein kinase (AMPK) activity (Gao et al., 2009). A further mechanisms accounting for the beneficial metabolic effects of NaBut is the induction of hepatocyte- and adipocyte-produced Fibroblast growth factor 21 (FGF21), a hormone promoting fatty acid oxidation in mice (Li et al., 2012). Increased FGF-21 production depends on acetylated and active peroxisome proliferator-activated receptors alpha (PPARa), and inhibition of the PPARaedeacetylating enzyme HDAC3 contributes to maintaining PPARa in its active state (Li et al., 2012). The condition of insulin resistance, that underpins the later development of diabetes and associated complications, can be elicited by various factors, including chronic hyperglycaemia, hyperinsulinemia, elevated circulating free fatty acids (FFAs), and proinflammatory cytokines. Circulating saturated FFAs cause skeletal muscle insulin resistance (Boden and Shulman, 2002), and exposure to palmitate is a widespread model to induce insulin resistance in cultured myotubes (Schmitz-Peiffer et al., 1999). These extracellular stimuli share a common mode of action in inducing an increase of intracellular reactive oxygen species (ROS) (Houstis et al., 2006). Post-translational covalent modifications, including acetylation, methylation and phosphorylation, of histones are a main mechanism of regulation of the chromatin structure, and consequently transcriptional regulation. In earlier work, we demonstrated that activation of gene transcription by insulin is mediated by changes in histone post-translational modifications. In particular, upregulation of hexokinase 2 (HK2) was associated to enhanced histone acetylation, on histone H3 and histone H2A.Z, in the promoter region of the gene (Zerzaihi et al., 2014). Based on these initial findings, we sought to investigate here whether positive modulation of histone acetylation via inhibition of HDACs through administration of NaBut might have a positive effect in a situation of insulin resistance.

55  C 60  C 58  C

Here, we show that NaBut alleviates the insulin resistant state induced by the saturated FFA palmitate in L6 myotubes, partially rescuing palmitate-induced downregulation of PKB and MAPK phosphorylation. NaBut administration increased total histone H3 acetylation as well as specific acetylation on a genomic locus at the transcriptional start site of the insulin signalling gene Irs1, resulting in increased Irs1 mRNA, and IRS1 protein expression, and insulininduced IRS1 tyrosine phosphorylation and association to PI 3kinase. Downstream of PKB, GSK3b mRNA levels, and to a lesser extent protein levels, were increased; while no regulation occurred on GLUT4 and glycogen synthase in our model. The beneficial

Fig. 1. Increased Histone H3 K9 and K14 acetylation during L6 myoblasts differentiation. (A) Histones were acid-extracted at the start of differentiation (day 0) and 2, 4, 7 and 8 days post-differentiation. Analysis with anti-H3K9Ac (T, antibodies from Temecula; M, antibodies from Millipore) and antiH3K9/K14Ac antibodies demonstrates a time-dependent increase of histone H3 acetylation. Total loading was assessed by immunoblotting to histone H3, and the time-dependent progression of differentiation was monitored by immunoblotting to HK2. (B) Quantification of H3K9 acetylation throughout differentiation. Acetylation signals are presented as chemiluminescence arbitrary units (a.u.) and are normalised to total histone H3. Error bars represent SEM. n  3, *p < 0.05 versus day 0 time-point as tested by one-way ANOVA test followed by a post-hoc Bonferroni post-hoc test.

Please cite this article in press as: Chriett, S., et al., The histone deacetylase inhibitor sodium butyrate improves insulin signalling in palmitateinduced insulin resistance in L6 rat muscle cells through epigenetically-mediated up-regulation of Irs1, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.006

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Fig. 2. NaBut increases histone H3 acetylation and, in palmitate-induced insulin resistance, improves insulin-induced PKB and MAPK phosphorylations. (A) L6 myotubes were incubated 24 h in the presence of 500 nM TSA or 5 mM NaBut, and subsequently stimulated with 100 nM insulin for 20 min. Acid-extracted histones were immunoblotted with antibodies anti H3K9Ac, H3K9/14Ac and H3. (B) Immunoblot analysis of histone H3 acetylation with antibodies anti H3K9/14Ac in the presence of NaBut and palmitateinduced insulin resistance. Total H3 is visualised by immunoblotting with antibodies anti H3 and coomassie blue staining of acid-extracted histones. Quantifications of the acetylation signals normalised to total histone H3 are shown at the bottom. n  3 (C, D). Immunoblot analysis of pPKB S473 and total PKB and of pMAPK and total MAPK in L6 myotubes rendered insulin-resistant by palmitate treatment (500 mM, 24 h) and co-treated or not with NaBut (5 mM, 24 h) prior to medium replacement for 1 h and insulin stimulation (20 min, 100 nM). Quantification of the phosphorylation signals normalised to the total protein amounts are shown at the bottom. n  3, (except for the non-insulin stimulated condition of total PKB, n ¼ 2). Error bars represent SEM. *p < 0.05 among the indicated experimental conditions as tested by Students t tests.

effects of NaBut may thus in part be linked to its action on the epigenome at the level of the Irs1 gene. 2. Materials and methods 2.1. Materials and cell culture Sodium butyrate (NaBut), trichostatin A, palmitate, and recombinant human insulin were from Sigma-Aldrich. The following antibodies were used: anti-phospho-Ser-473 PKB, anti-PKB, antiphospho-MAP kinase1/2, anti MAP kinase 1/2, anti-p70S6K, antiphospho-Ser9-GSK3b and anti-GSK3b (Cell signalling technology); anti-GLUT4 H61 (Santa Cruz Biotechnology); anti-IRS1, anti-IRS2, anti-H3K9Ac (Millipore Upstate Biotechnology); anti-H3K9Ac (Temecula Upstate Biotechnology); anti-HK2, anti H3, anti-H3K9/ 14Ac (Santa Cruz Biotechnology); anti-tubulin (Sigma-Aldrich). HPR-conjugated anti-rabbit and anti-mouse secondary antibodies were from BioRad. L6 myoblasts were cultured in DMEM, containing 4,5 g/l glucose and 10% Fetal Calf Serum (FCS). Cells were differentiated for 7e8 days by switching to a differentiation medium (DMEM, containing 1 g/l glucose and 2% FCS), which was replaced every second day. Palmitate containing media was prepared by conjugating palmitate to delipidated BSA. Briefly, a 100 mM palmitate stock solution was

prepared in ethanol and then diluted in DMEM, containing 1 g/l glucose and 2% delipidated BSA to a final concentration of 3 mM palmitate. Control medium was prepared with the same amount of ethanol. Both preparations were heated, under agitation, for 10 min at 55  C. Solutions were purified using a 0.2 mm filter before use. Insulin-resistance was induced by a 24 h treatment with 500 mM palmitate in the presence or absence of 5 mM NaBut. After 24 h, palmitate/NaBut were removed for 1 h by medium replacement (DMEM, containing 1 g/l glucose, with no addition of FCS), followed by a 20-min stimulation with 100 nM insulin. In the Trichostatin A (TSA) treatment, TSA was used in the same conditions than NaBut, with a concentration of 500 nM. 2.2. Protein extraction and western blotting Cells lysates were prepared by extracting cells in a “standard lysis buffer” (SLB) composed of 20 mM Tris-HCl, 138 mM NaCl, 2.7 mM KCl, 5% (v/v), glycerol, 1 mM sodium-o-vanadate, 1% (v/v) Nonidet P-40, 5 mM EDTA, 20 mM NaF, 1:1000 proteases inhibitors cocktail (Sigma-Aldrich, P2714) pH 8.0; as described (Pirola et al., 2003). Residual pellets from cell lysates were incubated overnight at 4  C with 0.2 M HCl to solubilize and extract total histones. Solubilized histones were centrifuged (13,000 g, 15 min at 4  C) and supernatants collected and neutralized with 1 M Tris prior to

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Fig. 3. NaBut increases Gsk3b mRNA independently from palmitate treatment. (A) Quantification of the mRNA levels by RT qPCR for Glut4, GSK-3b and GS in L6 myotubes treated for 24 h with 5 mM NaBut (black bars), 500 mM Palmitate (light gray bars) or both agents (dark gray bars). (B) Amplification curves are normalised to the Hprt1 housekeeping gene. Data are expressed as fold enrichment over the control condition and represent the mean ± SEM from 3 independent experiments. (*p < 0.05, versus Ctrl group using two-way ANOVA and Bonferroni's post-hoc test). (B) Immunoblotting of protein expression levels for p70S6K, GLUT4, phospho-GSK3b and GSK3b. Blots shown are representative of three independent experiments.

protein quantifications (Shechter et al., 2007). Protein quantifications were performed with the Bradford reagent (BioRad). Protein lysates were separated by 7.5 or 10% SDS-PAGE, histones were separated by 15% SDS-PAGE. Standard immunoblotting procedures and ECL detection were employed. Chemiluminescence was detected on a BioRad Chemidoc™ XRS þ apparatus and images were processed using Image Lab 3.0 (BioRad). 2.3. RNA extraction, reverse transcription and real-time quantitative PCR Total RNA was isolated with Tri Isolation Reagent™ (Roche) according to the manufacturer's instructions. RNA was verified using optical density (OD) measurement on a Nanodrop 2000. OD 260/280 was >1.8, confirming RNA purity, OD 260/230 was >1.5, confirming the absence of residual solvents in the purified RNA. cDNA synthesis was performed using the Primescript™ RT reverse transcription kit (Takara) according to the manufacturer's instructions, using 1 mg of RNA as template. Quantitative PCR amplification was performed using a Rotor-Gene Real-Time PCR System. 5 ml of diluted cDNA template (obtained from a 60 dilution of the original cDNA), 5 pmoles of forward and reverse primers and 15 mL of ABsolute™ QPCR SYBR Green Mix (ABgene) were added in each reaction. Reactions were incubated at 95  C for 10 min, followed by 40 cycles of denaturation (95  C for 10 s), annealing (at gene-specific temperatures for 30 s, see Table 1 for primer sequences and annealing temperatures) and elongation (72  C for 30 s). As a quality control, qPCR amplicons were analyzed by melting curve and agarose gel. Analyzed genes were normalised against HPRT1. 2.4. Immunoprecipitation and chromatin immunoprecipitation (ChIP) For immunoprecipitation, cell lysates were incubated at 4  C for

30 min with rabbit polyclonal antibodies to IRS-1 (Santa Cruz, sc559) followed by addition of 20 mL of Protein A Magnetic Beads (Temecula Millipore) and further overnight incubation at 4  C. Immune complexes were washed twice with SLB, re-suspended in Laemmli sample buffer and denatured (7 min, 95  C) prior to immunoblotting analysis with antibodies to the p85a PI 3-kinase adapter subunit (Millipore 06e195), Phospho-Tyrosine (P-Tyr-99, Santa Cruz, sc-7020) and IRS1 (Upstate 06e248). ChIP was adapted from a previously published protocol (Zerzaihi et al., 2014). Briefly, cell culture medium was removed and cells were washed twice with ice-cold PBS before cross-linking for 10 min with 1% formaldehyde in PBS. Formaldehyde cross-linking was quenched by treatment with 0.125 M glycine, in PBS, for 10 min. Cells were then harvested and homogenized in 1% SDS cell lysis buffer. Chromatin was sheared by sonication on a Diagenode Bioruptor sonicator at maximal power for 30 min with 30 s pulses in a 4  C-cooled water bath. To assess shearing efficiency, a small aliquot of soluble sheared chromatin was reverse cross-linked and digested with proteinase K at 62  C for 2 h before DNA purification on minicolums (Nucleospin Extract II, Macherey Nagel) and analysis of the eluted DNA by agarose gel (Fig. 5A). Immunoprecipitation was performed on chromatin from approximately 1 million cells. Sheared chromatin was diluted to a final SDS concentration of 0.1% with ChIP dilution buffer, followed by addition of 5 mg of rabbit polyclonal antibody directed to acetylated H3K9/K14 (Santa Cruz, sc-8655) and 20 mL of Protein A Magnetic Beads (Temecula Millipore). Immune-complexes were captured on magnetic beads by overnight incubation at 4  C with rotation. Immune-complexes were washed for 5 min at 4  C sequentially with low salt buffer, high salt buffer, LiCl buffer and two final washes with TE buffer. Beads were transferred to a new tube for reverse cross-linking and purification of the immunopurified DNA. Immunoprecipitated DNA was quantified fluorometrically (Qubit 2.0, Invitrogen) using the Invitrogen reagent Quant-IT dsDNA. ChIP enrichments were

Please cite this article in press as: Chriett, S., et al., The histone deacetylase inhibitor sodium butyrate improves insulin signalling in palmitateinduced insulin resistance in L6 rat muscle cells through epigenetically-mediated up-regulation of Irs1, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.006

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Fig. 4. NaBut specifically increases Irs1 mRNA and protein expression independently from palmitate treatment. (A) Quantification of the mRNA levels by RT qPCR for the insulin signalling molecules Irs1 and Irs2 and the metabolic enzyme Hk2 in L6 myotubes treated for 24 h with 5 mM NaBut (black bars), 500 mM Palmitate or both agents. Amplification curves are normalised to the Hprt1 housekeeping gene. (B,C) Quantification of Irs1 mRNA levels after dose-dependent and time-dependent NaBut treatment. (B) Myotubes were treated with NaBut for 24 h at 0, 0.5, 1 and 5 mM concentration. (C) Myotubes were treated with 5 mM NaBut for 0, 6, 12 and 24 h. Data are expressed as fold enrichment over the control condition and represent the mean ± SEM from 3 independent experiments. *p < 0.05, versus Ctrl group using two-way ANOVA and Bonferroni's posthoc test). (D) Immunoblotting analysis of protein expression levels for IRS1, IRS2 and HK2 and tubulin as loading control under the same experimental conditions of panel A. (E) The induction of IRS1 is also observed after a 24 h treatment of L6 myotubes with 500 nM trichostatin A (TSA).

quantified by real time qPCR relative to an input sample. Primers used to amplify specific genomic loci are reported in Table 1. Nonspecific immunoprecipitation with anti IgG antibodies raised in rabbit (DakoCytomation) consistently yielded negligible amplifications (data not shown) confirming the specificity of the immunopurification with anti-acetylated H3K9/K14 antibodies. 2.5. Statistical analysis Experiments were performed at least three times, or more when indicated. Values are expressed as means ± SEM. We determined significances using unpaired Student's t-test or ANOVA test, followed by Bonferroni's post-hoc pairwise comparisons. Differences were considered statistically significant when p < 0.05.

3. Results 3.1. Histone H3 acetylation increases during L6 cells differentiation We explored the time-dependent changes in histone acetylation during the differentiation process of rat L6 myoblast into myotubes. Immunoblotting analysis with three independent antibodies directed against acetylated histone H3 revealed a time-dependent increase in H3 acetylation, both at lysine 9 and lysine 14, throughout the differentiation process (Fig. 1A). Differentiation was confirmed visually, by the formation of multinucleated myotubes (data not shown), and biochemically by the upregulation of the metabolic enzyme HK2 (Fig. 1A). The observed increase of histone acetylation throughout

Please cite this article in press as: Chriett, S., et al., The histone deacetylase inhibitor sodium butyrate improves insulin signalling in palmitateinduced insulin resistance in L6 rat muscle cells through epigenetically-mediated up-regulation of Irs1, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.006

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Fig. 5. In L6 myotubes, NaBut increases histone H3 acetylation on the Irs1 gene. (A) Representative agarose gel showing the chromatin's shearing pattern from L6 myotubes after sonication and before realizing the ChIP experiment. ¼(B) Relative enrichments in acetylated H3 on lysine's 9/14 in the vicinity of the TSS of Irs1, Irs2 and Hk2 genes, and in a distal region of the Hk2 gene in L6 myotubes (error bars represent SD, n ¼ 3, with qPCR reactions performed in duplicate). The approximate position of the amplicons for each genomic locus is reported below the graph. C Modulation of histone acetylation by NaBut treatment around the Irs1 TSS. (Error bars represent SEM, n ¼ 4, *p < 0.05, versus Ctrl group using two-way ANOVA and Bonferroni's post-hoc test).

differentiation is a mark for active chromatin. Administration of TSA (500 nM, 24 h) or NaBut (5 mM, 24 h) to differentiated myotubes both induced histone H3 hyperacetylation as assessed with anti-H3K9/14Ac and anti-H3K9Ac antibodies (Fig. 2A). The promotion of histone acetylation by the two HDACi treatments suggests that they may change L6 myotubes physiology, including insulin sensitivity, in a similar manner. At the concentrations used, however, NaBut proved to be more effective in increasing H3 acetylation (Fig. 2A). Under standard cell culture conditions (i.e. without any palmitate addition), insulin stimulation (100 nM, 20 min) did not affect HDACi-induced hyperacetylation (Fig. 2A, compare lanes 2e3 and 5e6), nor the presence of TSA or NaBut affected insulin-induced PKB and MAPK phosphorylation (data not shown). 3.2. NaBut alleviates the insulin resistant state caused by exposure to palmitate To determine the effect of the HDACi NaBut on palmitate induced insulin-resistance, L6 myotubes were rendered insulin resistant by incubation with 500 mM palmitate for 24 h, in the presence or absence of 5 mM NaBut. The administration of NaBut induced histone H3 hyperacetylation, which was not altered by palmitate nor insulin stimulation (Fig. 2B). Insulin stimulation (100 nM, 20 min), revealed the establishment of an insulin resistant state in palmitate-treated cells, as monitored by a significant decrease in insulin-stimulated phosphorylation of PKB (Fig. 2C) and MAPK (Fig. 2D). However, the simultaneous administration of NaBut relieved this insulin-resistant condition, showing a significant improvement of PKB and MAPK phosphorylation (p < 0.05,

Student's t-test) following insulin stimulation (Fig. 2C and D). 3.3. NaBut upregulates GSK3b gene and protein expression To extend our analysis on downstream components of insulin signalling, we measured gene expression of the main insulindependent glucose transporter Glut4, and the glycogen synthesis pathway genes glycogen synthase (Gs) and its major regulator glycogen synthase kinase 3b (Gsk3b). Neither NaBut nor palmitate induced gene or protein expression changes of Glut4 and Gs. However, Gsk3b gene expression was increased twofold by NaBut, both in the presence or absence of palmitate (Fig. 3A), and increased Gsk3b protein expression was also observed upon NaBut treatment, both in un-stimulated and insulin-treated cells (Fig. 3B). Insulin-stimulated GSK3b phosphorylation was also slightly increased in the presence of NaBut (Fig. 3B), consistent with the hormone's role to phosphorylate and therefore inactivate GSK3b0 s activity to allow glycogenosis by GS. Considering the effect of NaBut on Gsk3b gene and protein expression, we checked to see if this upregulation also happens at the protein level and on other PKB downstream targets. p70S6K phosphorylation also showed an upregulation by insulin (as observed by the band-shift of the protein), but no effect from palmitate or NaBut treatment on p70S6K protein expression or activation were observed. 3.4. NaBut induces IRS1 gene and protein upregulation As PKB and MAPK represent two biochemically independent branches of insulin signalling, we surmised that the action of NaBut on both kinases, and its prominent effects on MAPK, should be

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Fig. 6. NaBut increases insulin-induced IRS1 tyrosine phosphorylation and PI 3-kinase association. L6 myotubes were treated for 24 h with 0.5 mM palmitate and/or 5 mM NaBut as indicated. After 24 h, cells were exposed for 20 min to 100 nM insulin and cell lysates were immunoprecipitated with antibodies directed to IRS1. (A, left panel), upper gel: IRS1 associated tyrosine phosphorylation was detected in anti-IRS-1 immunoprecipitates (I.P.). Lower gel, total IRS1 was immunoblotted in total lysates as loading control. (A, right panel), quantification of IRS1 phosphotyrosine signals. (B, left panel), upper gel: IRS1-associated p85a PI3K adapter subunit was detected in anti-IRS1 immunoprecipitates (I.P.). Middle gel, total p85a was immunoblotted in total lysates as loading control. Lower gel, insulin-induced tyrosine phosphorylation of the insulin receptor was detected by antiphosphotyrosine immunoblotting. (B, right panel), quantification of IRS1-associated p85a was (upper graph) and tyrosine phosphorylation of the insulin receptor (lower graph). Blots shown are representative of at least three independent experiments. Error bars represent SEM. *p < 0.05, versus the corresponding treatment without insulin stimulation. #p < 0.05, versus the experimental condition of palmitate-treated insulin-stimulated cells as tested by Student's t tests.

regulated by an action of NaBut on an upstream component of the insulin signalling cascade. As inhibition of HDACs promotes histone acetylation, and thus a transcriptionally permissive form of the chromatic template, we evaluated the gene expression levels of the upstream signalling genes Irs1 and Irs2, as well as Hk2, by quantifying their mRNA levels by real time qPCR. As shown in Fig. 4A, NaBut treatments increased >3-fold the expression level of Irs1, while having no effect on Irs2 and Hk2. The effect of NaBut on Irs1 gene transcription was maintained in palmitate-treated cells (Fig. 4A). NaBut action on Irs1 transcription was concentrationdependent, with significant effects starting at 1 mM (Fig. 4B), and time dependent, with a 24 h incubation period being necessary to induce upregulation of Irs1 (Fig. 4C). Accordingly to the increased gene expression levels, we also observed increased IRS1 protein expression in NaBut treated cells, irrespectively of the simultaneous administration of palmitate (Fig. 4D). This observation indicates that IRS1 overexpression may be the biochemical event induced by NaBut that overrides the palmitate-induced insulin resistant state in L6 myotubes. The commonality of action of the two chemically unrelated HDACi, NaBut and TSA, was verified as both inhibitors increased IRS1 protein expression, while having no effect on IRS2 and HK2 protein expression (Fig. 4E). To search for a link between NaBut administration and increased IRS1 transcription and protein expression, we analyzed by chromatin immunoprecipitation the histone H3 acetylation levels in the vicinity of the Irs1 transcriptional start site (TSS). As histone acetylation is enriched around the TSSs of genes (Birney et al., 2007), we initially verified the preferential accumulation of acetylated H3K9/ 14 close to the TSS of Irs1, Irs2 and Hk2 as compared to a genomic

locus approximately 4 kbp upstream of the Hk2 TSS (Fig. 5B). As expected, H3K9/14 acetylation was higher on the TSS proximal loci of the insulin-responsive genes Irs1, Irs2 and Hk2 (Fig. 5B). Next, sheared chromatin from L6 myotubes treated with either or both NaBut and palmitate was immunoprecipitated with antibodies anti H3K9/14Ac, and quantitative real time PCR analysis revealed a NaBut-dependent increased acetylation of histone H3 in the vicinity of Irs1 TSS, both in the presence or absence of palmitate (Fig. 5C). Taken together, the ChIP and mRNA quantifications experiments suggest that NaBut exerts its insulin-sensitizing effects under an insulin resistant condition by upregulating Irs1 via an epigenetic mechanism implying the hyperacetylation of the chromatic template proximally to the Irs1 TSS. 3.5. NaBut-induces IRS1 upregulation sustains proximal insulin signalling NaBut-dependent upregulation of IRS1 gene and protein expression and improvement of PKB and MAPK phosphorylations in palmitate-dependent insulin resistance prompted us to investigate whether NaBut affects insulin-induced IRS1 tyrosine phosphorylation and IRS1 association to PI 3-kinase. Immunoprecipitated IRS1 displayed a higher insulin-induced tyrosine phosphorylation when myotubes were exposed to NaBut, irrespectively of the presence or not of palmitate (Fig. 6A). Such increased tyrosine phosphorylation, in turn, resulted in a higher insulin-induced PI 3-kinase p85a adapter subunit association (Fig. 6B, upper panel). The effect of NaBut on the increased insulindependent IRS1 phosphorylation and p85a association is likely

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Fig. 7. Proposed model for the effect of NaBut in alleviating insulin resistance through acetylation-dependent transcriptional up-regulation of IRS1 mRNA and protein expression. Palmitate induced insulin-resistance is further exacerbated by simultaneous inhibition of PI3K, which removes the partially beneficial effects of PI3K on palmitate-induced insulin resistance.

dependent on the NaBut-induced IRS1 protein overexpression (Fig. 6A, lower panel; Fig. 4D,E), as insulin-dependent phosphorylation of the insulin receptor was not modified by NaBut treatment (Fig. 6B, lower panel). Preliminary observations indicate that NaBut does not affect the repressive IRS1 serine phosphorylation (Data not shown). Taken together, these data support the notion that the alleviation of palmitate-induced insulin resistance in L6 myotubes by NaBut is mainly mediated by its specific action on IRS1 transcription, protein upregulation and increased responsiveness to insulin. 4. Discussion The balance between HDACs inhibition and HATs activation regulates muscle cells differentiation. Here, we investigated whether differentiation of L6 myoblasts to myotubes is paralleled by increased histone H3 acetylation. Histone acetylation is a histone PTM leading to the establishment of a transcriptionally competent state of the chromatin template (Turner, 2002). The differentiation process from myoblasts to myotubes entails the overexpression of muscle structural genes (Myosin Heavy Chain) and of insulin signalling genes (Insulin receptor, GLUT4 glucose transporter), and the acetyltransferase CBP/p300 has been shown to be necessary for myogenic terminal differentiation (Polesskaya et al., 2001). Given that the relative balance of HDACs and HATs drives muscle cells differentiation, we monitored the extent of acetylation on the histone H3 throughout the L6 differentiation process, demonstrating increased acetylation on H3 lysines 9 and 14 (Fig. 1). Increased histone acetylation also occurred during differentiation of 3T3-L1 preadipocytes (Wellen et al., 2009; Yoo et al., 2006), suggesting that histone acetylation correlates with the insulin-

responsiveness of differentiated adipocytes and muscle cells. In this respect, a study has demonstrated increased insulin signalling in TSA-treated C2C12 myotubes (Sun and Zhou, 2008). Given the importance of the acetylation status in differentiated myotubes, and considering that the small dietary molecule - and HDAC inhibitor - NaBut positively regulate metabolism and counteract aging dependent muscle atrophy (Walsh et al., 2015), we assessed the capability of NaBut to alleviate the insulin-resistant state induced by palmitate treatment in L6 myotubes. Palmitate, and other long chain saturated fatty acids, induce insulin resistance via i) the induction of ceramide biosynthesis, which leads to Akt inhibition through the activation of protein phosphatase 2A and atypical PKCs (Glass and Olefsky, 2012) and ii) activation of JNK and classical PKCs which negatively modulate insulin action by phosphorylating IRS on serine residues (Pirola et al., 2004). The effects of butyrate seem to be multiple and occur at many levels to help maintain a healthy metabolism. Butyrate supplementation (5% in the diet) to animals submitted to a high fat diet prevented fat accumulation (Gao et al., 2009), and butyrate supplementation to ageing mice up to 26 months of age supported the maintenance of lean mass and prevented the development of muscle atrophy (Walsh et al., 2015). Butyrate, in several studies, showed a double way of action, as an HDACi but also by promoting mitochondrial activity. Increased histone acetylation levels were observed in multiple organs, with higher H3K9 acetylation found in liver and brain after butyrate supplementation (Walsh et al., 2015), consistent with our data where butyrate upregulated the acetylation levels of H3K9 and 14 in L6 myotubes. Co-administration of NaBut to palmitate-treated myotubes alleviated the establishment of the insulin resistant state, as demonstrated by a significant recovery in the insulin-induced phosphorylations of PKB and MAPK (Fig. 2). This in vitro result is consistent with what has been previously demonstrated concerning NaBut's ability to improve glucose tolerance and insulin sensitivity (Gao et al., 2009). Interestingly, no effect was seen downstream of PKB and MAPK in the insulin signalling pathway, except an increase in GSK3b gene expression. This particularity might be specific to our cell model and time of treatment. By gene expression analysis of the main insulin signalling genes, we discovered that NaBut treatment of myotubes is correlated to increased mRNA and protein expression of IRS1 (Fig. 4), which could mechanistically dependent on the acetylation status of chromatic histone H3 in the vicinity of the TSS of Irs1 gene (Fig. 5). IRS1 protein overexpression was also induced by TSA, further supporting the mechanism governing IRS1 overexpression through hyperacetylation of its genic region. Increased expression of IRS1 was linked to its increased tyrosine phosphorylation after insulin stimulation, and increased association to the PI 3-kinase p85a adapter subunit (Fig. 6), which can in turn explain the increased phosphorylation of the PI 3-kinase downstream targets PKB and GSK-3b. Overall, as depicted in Fig. 7, we propose a model whereby palmitate-induced insulin resistance is counteracted by NaBut via a molecular mechanism likely not directly affecting the palmitateinduced effects on the insulin signalling pathway, such as ceramide production or serine phosphorylation on IRS proteins, but rather via an independent mechanism which, through specific histone acetylation on the Irs1 gene, allows for increased Irs1 transcription, IRS1 protein overexpression and increased insulininduced IRS1 tyrosine phosphorylation and PI 3-kinase association. Together with previous research demonstrating the insulinsensitizing effects of the HDACi TSA in differentiated C2C12 myotubes (Sun and Zhou, 2008), our data extend this concept using NaBut, a microbiota-derived and also food-borne HDACi, which could thus be nutritionally supplemented (Chriett and Pirola, 2015). We show that the insulin-sensitizing effect of NaBut occurs in a

Please cite this article in press as: Chriett, S., et al., The histone deacetylase inhibitor sodium butyrate improves insulin signalling in palmitateinduced insulin resistance in L6 rat muscle cells through epigenetically-mediated up-regulation of Irs1, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.006

S. Chriett et al. / Molecular and Cellular Endocrinology xxx (2016) 1e9

specific insulin-resistant condition e i.e. induced by palmitate. Moreover, NaBut seems to ameliorate the cell status through an antioxidant action, and preventing oxidative damage on proteins that occur during aging (Walsh et al., 2015). A caveat in our study resides within the relatively high NaBut doses administered to L6 myotubes. Our experiments aiming at investigating the dose- and time-dependent exposure to butyrate show that a 1 mM concentration, and a 24-h administration are necessary to observe NaButinduced transcriptional effects. However, plasmatic concentrations of butyrate can at best attain transient low-millimolar levels (Clarke et al., 2008), while b-hydroxybutyrate, the main ketone body also described to possess an HDACi activity, may reach millimolar levels upon starving or prolonged physical effort (Shimazu et al., 2013). As studies in rodent models demonstrate the safety of in vivo administration of NaBut (Li et al., 2012), clinical studies are warranted to ascertain whether NaBut or perhaps the related molecule b-hydroxybutyrate could improve insulin sensitivity, and thus be a potentially valuable agent to target insulin resistance, in humans. Acknowledgements te  Francophone du This work was supported in part a SFD (Socie te)/Menarini Diagnostics research grant to LP. Diabe References Baur, J.A., Pearson, K.J., Price, N.L., Jamieson, H.A., Lerin, C., Kalra, A., et al., 2006. Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444 (7117), 337e342. Birney, E., Stamatoyannopoulos, J.A., Dutta, A., Guigo, R., Gingeras, T.R., Margulies, E.H., et al., 2007. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447 (7146), 799e816. Boden, G., Shulman, G.I., 2002. Free fatty acids in obesity and type 2 diabetes: defining their role in the development of insulin resistance and beta-cell dysfunction. Eur. J. Clin. Investig. 32 (Suppl. 3), 14e23. Chriett, S., Pirola, L., 2015. Essential roles of four-carbon backbone chemicals in the control of metabolism. World J. Biol. Chem. 6 (3), 223e230. Clarke, J.M., Topping, D.L., Bird, A.R., Young, G.P., Cobiac, L., 2008. Effects of highamylose maize starch and butyrylated high-amylose maize starch on azoxymethane-induced intestinal cancer in rats. Carcinogenesis 29, 2190e2194. Ellis, L., Pili, R., 2010. Histone deacetylase inhibitors: advancing therapeutic strategies in hematological and solid malignancies. Pharmaceuticals (Basel) 3 (8), 2411e2469. Gao, Z., Yin, J., Zhang, J., Ward, R.E., Martin, R.J., Lefevre, M., Cefalu, W.T., Ye, J., 2009. Butyrate improves insulin sensitivity and increases energy expenditure in mice. Diabetes 58 (7), 1509e1517. Glass, C.K., Olefsky, J.M., 2012. Inflammation and lipid signaling in the etiology of insulin resistance. Cell Metab. 15 (5), 635e645. Houstis, N., Rosen, E.D., Lander, E.S., 2006. Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature 440 (7086), 944e948. Lagouge, M., Argmann, C., Gerhart-Hines, Z., Meziane, H., Lerin, C., Daussin, F.,

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Please cite this article in press as: Chriett, S., et al., The histone deacetylase inhibitor sodium butyrate improves insulin signalling in palmitateinduced insulin resistance in L6 rat muscle cells through epigenetically-mediated up-regulation of Irs1, Molecular and Cellular Endocrinology (2016), http://dx.doi.org/10.1016/j.mce.2016.09.006