Articles in PresS. Am J Physiol Endocrinol Metab (November 4, 2014). doi:10.1152/ajpendo.00115.2014 1 2 3 4 p38 MAPK activation upregulates pro-inflammatory pathways in skeletal muscle cells from insulin resistant type 2 diabetic patients Short title; p38 MAPK activation in type 2 diabetes 5 6 Audrey E Brown1, Jane Palsgaard2, Rehannah Borup3 Peter Avery4, David A Gunn5, Pierre 7 De Meyts2, Stephen J Yeaman1, and Mark Walker1, 8 1 Institute of Cellular Medicine. Newcastle University, Newcastle, UK 9 2 Receptor Systems Biology Laboratory, Hagedorn Research Institute, Novo Nordisk, 10 Denmark 11 3 Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Denmark 12 4 School of Mathematics and Statistics, Newcastle University, Newcastle, UK 13 5 Unilever Discover, Colworth Science Park, Sharnbrook, Bedford, England, MK44 1LQ 14 15 Corresponding author: 16 17 Mark Walker, 18 Institute of Cellular Medicine 19 Faculty of Medical Sciences 20 Framlington Place, 21 Newcastle University, Newcastle upon Tyne, NE2 4HH 22 Tel: +44(0191) 2464661 23 Email address: [email protected] 24 25 26 Copyright © 2014 by the American Physiological Society. 27 Abstract 28 Skeletal muscle is the key site of peripheral insulin resistance in type 2 diabetes. Insulin- 29 stimulated glucose uptake is decreased in differentiated diabetic cultured myotubes in 30 keeping with a retained genetic/epigenetic defect of insulin action. We investigated 31 differences in gene expression during differentiation between diabetic and control muscle cell 32 cultures. Microarray analysis was performed using skeletal muscle cell cultures established 33 from type 2 diabetic patients with a family history of type 2 diabetes and clinical evidence of 34 marked insulin resistance, and non-diabetic control subjects with no family history of 35 diabetes. Genes and pathways upregulated with differentiation in the diabetic cultures, as 36 compared to controls, were identified using Gene Spring and Gene Set Enrichment Analysis. 37 Gene sets upregulated in diabetic myotubes were associated predominantly with 38 inflammation. p38 MAPK was identified as a key regulator of the expression of these pro- 39 inflammatory gene sets, and p38 MAPK activation was found to be increased in the diabetic 40 vs control myotubes. While inhibition of p38 MAPK activity significantly decreased cytokine 41 gene expression and release from the cultured diabetic myotubes, it did not improve insulin- 42 stimulated glucose uptake. Increased cytokine expression driven by increased p38 MAPK 43 activation is a key feature of cultured myotubes derived from insulin resistant type 2 diabetic 44 patients. p38 MAPK inhibition decreased cytokine expression but did not affect the retained 45 defect of impaired insulin action in the diabetic muscle cells. 46 47 Keywords 48 Inflammation, cytokines, p38 MAPK, insulin resistance, human skeletal muscle cells 49 50 51 52 Abbreviations 53 β2-M; beta2-microglobulin, BST2; bone marrow stromal cell antigen 2, GSEA; gene set 54 enrichment analysis, IL6; interleukin 6, IL8; interleukin 8, MCP-1; monocyte 55 chemoattractant protein 1, MX1; myxovirus (influenza virus) resistance 1, interferon- 56 inducible protein p78. 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 Skeletal muscle is the key peripheral tissue site of the insulin resistance in type 2 diabetes, 78 manifested as decreased insulin-stimulated glucose uptake and storage (3). Evidence 79 supporting the role of genetic factors in the development of insulin resistance in type 2 80 diabetes includes the familial clustering of insulin resistance (13), the study of rare severe 81 phenotypes (18) and the retention of defects of insulin action in cultured human muscle cells 82 (8, 9, 14). We and others have shown defects of insulin action in cultured muscle cells from 83 patients with type 2 diabetes and non-diabetic 1st degree relatives of type 2 diabetic patients 84 (8, 9, 14). However, while recent genome-wide association studies (GWAS) of type 2 85 diabetes have identified well over 40 susceptibility loci, few appear to impact upon insulin 86 action (4). 87 We sought to optimise the chance of identifying genetic variants related to insulin resistance 88 and type 2 diabetes. We established skeletal muscle cell cultures from patients with both a 89 familial predisposition to type 2 diabetes and clinical evidence of marked insulin resistance, 90 and from non-diabetic control subjects with normal glucose tolerance and no family history 91 of diabetes. As previously reported, insulin action was normal in the diabetic myoblasts, but 92 upon differentiation to mature multinucleated myotubes there was both decreased insulin- 93 stimulated glucose uptake and glycogen synthesis (14). 94 This observation led us to the hypothesis that changes in gene expression during myotube 95 differentiation contributed to the impaired action of insulin in the diabetic muscle cultures. 96 The specific aim of this study, therefore, was to use microarray technology to compare gene 97 expression between the diabetic and control differentiated myotube cultures. 98 99 100 Methods Study Subjects 101 Muscle biopsies were obtained from six type 2 diabetic patients with clinical evidence of 102 marked insulin resistance. None of the patients had partial lipodystrophy. Specifically, we 103 recruited type 2 diabetic patients who were insulin treated requiring > 100 units per day in the 104 absence of marked obesity (BMI <32kg/m2), and who had at least one 1st degree relative with 105 type 2 diabetes. All patients had been treated with diet and oral hypoglycaemic agents for at 106 least 3 years after diagnosis before starting insulin treatment. Skeletal muscle was acquired 107 from six non-diabetic control subjects with no family history of type 2 diabetes. The control 108 and diabetic subjects were matched for age and BMI. All subjects gave written informed 109 consent, and the study was approved by the Newcastle and North Tyneside Joint Ethics 110 Committee. Clinical characteristics are summarised in Table 1. 111 General chemicals and reagents 112 Cell culture media was obtained from Lonza. Foetal bovine serum (FBS) and Trizol reagent 113 were obtained from Life Technologies (Paisley, UK). Chick embryo extract was purchased 114 from Sera Labs International (Sussex, UK) while antibodies were obtained from New 115 England Biolabs (Herts, UK). The Human U133 Plus 2.0 expression arrays were obtained 116 from Affymetrix. 2-Deoxy-D-[2,6-3H]glucose was purchased from NEN (Boston, MA, US). 117 Cytokine ELISA kits were from Qiagen (Sussex, UK). The p38 MAPK inhibitor SB203580 118 was purchased from Tocris Bioscience (Bristol, UK). 119 Cell culture 120 Muscle biopsies were obtained from the vastus lateralis and satellite cells isolated as 121 described previously (1). Cultures were purified as described previously (14) using a 122 magnetic bead system (Miltenyi Biotec). Briefly, cells were harvested and resuspended in 123 PBS containing 2 mM EDTA and 5% (v/v) FBS and incubated with anti-CD56 antibody 124 which recognizes a muscle-specific cell surface antigen. After washing and incubation with 125 microbead-conjugated secondary antibody, the cell suspension was applied to an MS column 126 within a magnetic field. The cells with microbeads attached were retained on the column, and 127 other cells passed through the column. The cells retained in the column were eluted and 128 returned to culture. Muscle cell origin was confirmed immunohistochemically using 129 antibodies to the muscle-specific protein desmin. Myoblasts were cultured in Ham’s F10 130 media supplemented with 20% (v/v) FBS and 2% (v/v) chick embryo extract. Differentiation 131 was induced by changing the media to minimal essential media supplemented with 2% (v/v) 132 FBS. All experiments were performed on day 7 differentiated myotubes between passages 5 133 and 8. 134 RNA isolation, cDNA synthesis and array hybridisation 135 RNA was extracted from myoblasts and myotubes differentiated for 7 days using Trizol, as 136 per the manufacturer’s instructions. Briefly, cells were lysed in Trizol, homogenised and 137 incubated at room temperature for 5mins. After the addition of 0.2 volumes of chloroform the 138 samples were mixed and centrifuged at 12000g for 15mins at 4oC. 0.5 volumes isopropanol 139 was added to the upper aqueous phase before centrifugation at 12000g for 10mins at 4oC. 140 The pellet was washed with 75% ethanol and centrifuged at 7500g for 5mins at 4oC before 141 resuspending in 20μl RNase-free water. cDNA synthesis was performed using Superscript II 142 (Life Technologies) and the cDNA cleaned up using the recommended protocol. Fragmented, 143 biotinylated cRNA was produced using recommended protocols (Affymetrix). Hybridisation 144 of the cRNA took place at 45oC for 16h in a GeneChip Hybridization Oven 640 (Affymetrix) 145 to Affymetrix Human Genome U133 Plus 2.0 Arrays. The arrays were subsequently washed 146 and stained in a GeneChip Fluidics Station 400. Finally, the arrays were scanned in a 147 GeneChip Scanner 3000. 148 Array data analysis 149 The arrays were normalised in GeneSpring GX software (Agilent) by RMA and baseline 150 transformation to the median of all samples. Data were log transformed to obtain a normal 151 distribution and differences in expression between the controls and diabetic myotubes and 152 myoblasts determined. p values were calculated in GeneSpring using a Student’s t test 153 adjusted for false discovery rate correction using the Benjamini-Hochberg method. Pathway 154 analysis was performed using Gene Set Enrichment Analysis (GSEA) (20) using both 155 myoblast and myotube data. Curated gene sets (c2) in MSigDB were used in the analysis. 156 Gene set size filters filtered out 1053 gene sets leaving 3669 curated gene sets to take part in 157 the analysis with 2000 gene set permutations to obtain the nominal p values. The data have 158 been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series 159 accession 160 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE55650). 161 Quantitative real-time PCR 162 Quantitative real-time PCR was performed on a Lightcycler 480 (Roche) using either SYBR 163 green or Taqman primers and probes. Multiplex reactions were performed in a final volume 164 of 20μl using the Quantifast Multiplex master mix (Qiagen). Single colour reactions were 165 performed with Probes Master mix (Roche) using β2-microglobulin as a reference gene. IL6 166 (Hs00985639_m1) was obtained from Applied Biosystems as a predesigned Taqman primer- 167 probe mix. Other primers and probes used were: IL8 For; 168 GCAGAGCACACAAGCTTCTAGG, Rev: ATCAGGAAGGCTGCCAAGAGA, Probe; 169 TxRed-ACTTCCAAGCTGGCCGTGGC-BHQ2, MCP-1 For; 170 CTCAGCCAGATGCAATCAATG, Rev; AGATCTCCTTGGCCACAATGG, Probe; Cy5- 171 CAGTGCAGAGGCTCGCGAGC-BHQ2, β2-M For; GCCTGCCGTGTGAACCAT, Rev; 172 TTACATGTCTCGATCCCACTTACCTATC, Probe; FAM-TGACTTTGTCACAGCCCA- 173 TAMRA. SYBR green reactions were performed using LightCycler 480 SYBR green I 174 mastermix (Roche). Primers used were: MX1 For; GTTTCCGAAGTGGACATCGCA, Rev; 175 CTGCACAGGTTGTTCTCAGC, BST2 For; CACACTGTGATGGCCCTAATG, Rev; number GSE55650 176 GTCCGCGATTCTCACGCTT. Results were analysed using the standard curve method from 177 a six-point serially diluted standard curve. 178 Western blotting 179 Cells were lysed in extraction buffer (100mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 180 25mM KF, 1mM benzamidine, 0.5mM Na3VO4, 0.1% (v/v) Triton X-100), plus protease 181 inhibitor cocktail (Pierce) before sonicating for 10s. Protein concentrations were determined 182 spectrophotometrically at 595nm by a Coomassie binding method. 10μg samples were 183 prepared in Laemmli sample buffer (0.125M Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) 184 glycerol, 10% (v/v) 2-mercaptoethanol, and 0.004% (w/v) bromophenol blue) and boiled for 185 5min. After separation on 10% SDS-PAGE gels, proteins were transferred to PVDF 186 membranes using a mini-Hoeffer gel transfer system. After incubation with the appropriate 187 antibodies, detection took place using enhanced chemiluminescence. Densitometry was 188 performed using a Bio-RAD Molecular Imager GS-800 calibrated densitometer and Quantity 189 One software. 190 Cytokine ELISA 191 Secretion of specific molecules was determined by enzyme-linked immunosorbent assay 192 (ELISA) using the Single-Analyte ELISArray (Qiagen). Skeletal muscle cell cultures were 193 allowed to differentiate for 7 days. Cells were incubated in fresh media for the last 24h of 194 differentiation. After 24h, media was removed, centrifuged at 1000g for 10min and assayed 195 for secretion of specific cytokines according to the manufacturer’s protocol. A standard curve 196 was generated by serial dilution of the provided antigen standard and absorbance was read at 197 450nm. Background absorbance was subtracted from the values and the protein 198 concentrations of the samples calculated from the standard curve. The detection limit for each 199 ELISA was IL6 5.01pg/ml, IL8 64.16pg/ml and MCP-1 13.90pg/ml. The CVs for all ELISAs 200 were <15%. 201 Glucose uptake 202 Measurement of (2,6-3H) 2-deoxy-glucose uptake took place in 12 well cluster plates. 203 Diabetic myotubes were treated for the last 18h of differentiation with 10μM SB203580 204 before incubating in Krebs’ buffer (136mM NaCl, 4.7mM KCl, 1.25mM MgSO4, 1.2mM 205 CaCl2, 20mM HEPES, pH 7.4) with or without 100nM insulin or cytochalasin B (10μM) for 206 20min. 0.1mM 2-deoxy-glucose and 0.5μCi (2,6-3H) 2-deoxyglucose were added to each 207 well and incubated for a further 10min. The reaction was stopped by washing the plate 208 rapidly in ice cold PBS. Cells were lysed in 0.05% SDS before scintillation counting and 209 protein determination. 210 Statistical analysis 211 For GSEA, a False Discovery Rate (FDR) <0.25 and Family Wise Error Rate (FWER) <0.05 212 was considered significant. Upstream regulator analysis in diabetic myotubes was performed 213 using Integrated Pathway Analysis (IPA) (Ingenuity, California). An overlap p value <0.01 214 and activation z-score greater than 2 (activating) and smaller than -2 (inhibiting) were 215 considered significant. 216 All results are expressed as mean±standard error of the mean (SEM). Student’s t-tests were 217 used to compare two groups and to test for changes after treatment with p<0.05 considered 218 significant. 219 220 Results 221 Microarray and Gene Set Enrichment Analyses 222 DNA microarray technology was used to compare the differences in gene expression in both 223 myoblasts and differentiated myotubes in type 2 diabetic skeletal muscle cell cultures and 224 age- and BMI-matched controls (Table 1). GeneSpring analysis showed that in both 225 myoblasts and myotubes, there were no genes significantly altered at an individual level in 226 the diabetic muscle cultures compared to controls after correction for multiple testing. 227 Gene Set Enrichment Analysis (GSEA) was therefore used to identify any coordinated 228 changes in gene expression within specific gene sets and pathways. As the previously 229 identified defects of insulin action in the diabetic muscle cell cultures were only apparent 230 with differentiation from myoblasts to myotubes (14), we focused on the analysis between 231 diabetic and control myotubes. 232 Applying the stringent cut off of FWER<0.05, 49 gene sets were significantly upregulated in 233 diabetic myotubes compared with control myotubes. The top 10 upregulated gene sets in the 234 diabetic myotubes are listed in Table 2. Most of the gene sets significantly upregulated are 235 related to inflammation. In particular, gene sets obtained by the incubation of cell lines with 236 interferons are particularly highly represented. Conversely, 13 gene sets were significantly 237 downregulated in the diabetic compared with the control myotubes (the top 10 are listed in 238 Table 3). It is worth noting that the inflammatory-related gene sets upregulated in the diabetic 239 myotubes (Table 2) were not upregulated in the corresponding diabetic myoblast cultures. 240 Upstream regulators of pro-inflammatory gene sets 241 Given the preponderance of inflammatory-related gene sets upregulated in the diabetic 242 myotubes, Ingenuity Pathway Analysis (IPA) was used to identify potential upstream 243 regulators of these pathways. Table 4 lists the top regulators and their predicted activation 244 state based on the direction of change in expression of genes in the diabetic myotubes 245 compared to controls. The top predicted activator was TNF. Consistent with the GSEA, 246 interferon gamma (IFNγ) was also predicted to activate the pathways upregulated in the 247 diabetic myotubes. Taking the GSEA and IPA results together, gene expression of the top 248 predicted regulators was measured by QPCR to determine whether expression of these 249 regulators was different between diabetic and control myotubes. TNF and interferon alpha 250 (IFNα) were expressed at very low levels in myoblasts with levels increasing slightly in 251 myotubes in both control and diabetic cells. However, there were no significant differences in 252 expression between the diabetic and control myotubes. Similarly, interferon beta (IFNβ) and 253 EPAS1 expression were also comparable between diabetic and control cells. Interferon 254 gamma (IFNγ) expression was undetectable in muscle cells thus suggesting that these 255 molecules are not the upstream regulators mediating the increased inflammatory profile 256 observed in the isolated diabetic muscle cells (data not shown). 257 p38 MAPK activation and inhibition in diabetic myotubes 258 Another predicted regulator was the p38 MAPK inhibitor SB203580 (Table 4). p38 MAPK is 259 a stress kinase that occupies a central point in the pathway regulating inflammatory 260 processes, and increased activation of this protein has been previously reported in skeletal 261 muscle from type 2 diabetic patients (11). Therefore, activation of p38 MAPK was examined 262 in the control and diabetic cultures in day 7 myotubes. Phosphorylation of p38 MAPK was 263 found to be significantly increased under both basal (p=0.02) and 30min insulin stimulation 264 (p=0.002) conditions in the diabetic myotubes compared to controls (Fig 1). Of all the key 265 predicted regulators identified using IPA and described above, p38 MAPK was the only one 266 examined which was significantly different between diabetic and control myotube cultures. 267 This increase in p38 MAPK phosphorylation led to the question, would inhibition of p38 268 MAPK with SB203580 improve the inflammatory profile of the diabetic myotubes and 269 increase insulin-stimulated glucose uptake? We examined the expression and secretion of the 270 pro-inflammatory cytokines IL6, IL8 and MCP-1 and the expression of MX1 and BST2. 271 These were chosen because they satisfy one or more of the following criteria: (1) they are in 272 the list of top genes upregulated in the diabetic cells compared to controls (Table 5), (2) they 273 are molecules identified by GSEA as being involved in the enrichment score for the top gene 274 sets upregulated in the diabetic myotubes or (3) they are target molecules of the upstream 275 regulators identified by Ingenuity. Ingenuity analysis predicted that SB203580 would affect 276 31 target molecules, 17 of which were also present in the other upstream regulator groups. 277 Day 7 differentiated control and diabetic myotubes were treated for either the last 18 hours of 278 differentiation or for the 7 day duration of differentiation with 10μM SB203580. In keeping 279 with the gene set analysis data, there was a pattern of up-regulation of cytokine expression in 280 the diabetic myotubes (Figs 2 and 3). After 18h treatment, expression of IL6, IL8 and MCP- 281 1 decreased in both control and diabetic myotubes (Fig 2A). There were no significant 282 changes in MX1 or BST2 expression (Fig 2B). Similarly, after 7 day treatment, IL6, IL8 and 283 MCP-1 decreased in both the control and diabetic cultures (Figure 3A). There was no change 284 in MX1 or BST2 expression in control cultures, whilst BST2 was significantly decreased in 285 the diabetic cultures (Fig 3B). Release of IL6, IL8 and MCP-1 into the media was also 286 significantly decreased after SB203580 treatment (data not shown). However, SB203580 287 treatment did not improve insulin-stimulated glucose uptake in the diabetic myotube cultures 288 after either 18h (Fig 4A) or 7 day treatment (Fig 4B). 289 290 Discussion 291 The key finding of this work was the coordinated upregulation of inflammatory pathways in 292 differentiated diabetic myotubes identified using both GSEA and Ingenuity analyses. Of the 293 potential upstream regulators of these pathways, we found that p38 MAPK activation was 294 increased in the diabetic myotubes and selective p38 MAPK inhibition decreased the 295 inflammatory profile in these cultures. The implications of these findings are that skeletal 296 muscle contributes to the inflammatory process in type 2 diabetes, and involves activation of 297 the p38 MAPK pathway. 298 Increased activation of p38 MAPK has been demonstrated previously in both skeletal muscle 299 (11) and adipocytes (2) from type 2 diabetes patients. However, neither study explored 300 whether p38 MAPK inhibition directly affected insulin action in the tissues derived from the 301 diabetic patients. We are the first to show that p38 MAPK inhibition in diabetic skeletal 302 muscle cells did not improve the retained defect of insulin stimulated glucose uptake, despite 303 decreasing inflammatory cytokine expression. We also observed a decrease in cytokine 304 expression after p38 MAPK inhibition in the control cultures. This indicates that p38MAPK 305 regulates cytokine expression in non-diabetic muscle, but that the activation is increased in 306 muscle from diabetic subjects. Conflicting findings have been reported in relation to p38 307 MAPK activation and insulin action under other conditions. In a model of insulin resistance 308 in 3T3 L1 adipocytes, inhibition of p38 MAPK did not prevent insulin-induced loss of IRS-1 309 protein (2). Hepatic expression of a dominant-negative p38 MAPK in vivo lowered fasting 310 insulin levels while overexpression of wild-type p38 resulted in increased serine 311 phosphorylation on IRS-1 (7). Conversely, in the liver of ob/ob mice expressing 312 constitutively active MKK6, an upstream activator of p38 MAPK, increased p38 MAPK 313 activity was associated with improved glucose tolerance (12). 314 Cytokine expression can be increased via a number of signalling pathways in skeletal muscle 315 including p38 MAPK, NFKB, JNK and the JAK-STAT pathway. However, the pattern of 316 inflammatory expression may differ. This is illustrated by the recent report of Green and 317 colleagues (6). They studied cultured skeletal muscle cells from obese diabetic patients and 318 found evidence of increased NFKB activation. This was associated with a trend to increased 319 TNFα and decreased IL6 expression. They found that suppression of NFKB activity via 320 AMPK activation normalised the cytokine response but did not improve insulin action. This 321 is in keeping with our own findings, and indicates that while upregulation of inflammatory- 322 related genes through the activation of different signalling pathways is a feature of cultured 323 diabetic cultured muscle cells, the accumulating evidence suggests that this pro-inflammatory 324 state does not directly contribute to the retained defects of insulin action. 325 A number of studies have been published describing microarray data from native skeletal 326 muscle (15, 16, 19), and identified altered expression of genes involved with metabolism in 327 diabetic muscle. However, this altered gene expression is likely to result from the 328 combination of retained primary genetic/epigenetic and secondary metabolic/lifestyle effects. 329 It is worth noting glycaemic control was generally poor in our diabetic patients despite high 330 dose insulin treatment. Hyperglycaemia per se can contribute to the insulin resistant state in 331 vivo, and so to limit any residual confounding effect we cultured the diabetic and control 332 muscle cells under standardised conditions out to passages 5-8 before conducting our 333 experiments. An earlier study of gene expression by microarray in cultured human diabetic 334 and control muscle cells found no robust differences between the 2 groups (5). However, it is 335 interesting to note that a pro-inflammatory interferon gamma pathway was in the top 10 gene 336 sets up-regulated in the diabetic cultures, although this was of nominal statistical significance. 337 This latter study was conducted on male participants. Although our study included males and 338 females, it has been shown previously that age, but not gender, influences gene expression 339 patterns in skeletal muscle (10). 340 It is widely accepted that low grade inflammation is a feature of type 2 diabetes. Our work 341 supports the growing body of evidence that skeletal muscle is involved in this pro- 342 inflammatory state. This could in turn contribute indirectly to the peripheral insulin resistance 343 in type 2 diabetes. Increased cytokine release, in particular MCP-1, would be predicted to 344 promote local inflammatory cell infiltration and amplification of the inflammatory process. 345 This is supported by the observation that CD163 macrophage-specific antigen concentration 346 and macrophage content is increased in skeletal muscle from type 2 diabetic patients (21) and 347 in murine models of obesity and insulin resistance (17), respectively. 348 In conclusion, we found an increased inflammatory profile and p38 MAPK activation in 349 differentiated myotubes from insulin resistant type 2 diabetic patients, and that inhibition of 350 p38 MAPK decreased cytokine expression but did not affect insulin-stimulated glucose 351 uptake. 352 Acknowledgements 353 Bioinformatics support was provided by the Bioinformatics Support Unit, Newcastle 354 University. We would like to acknowledge Liz McIntyre (Institute for Cellular Medicine, 355 Newcastle University) for establishing the cultures. No potential conflicts of interest relevant 356 to this article were reported. 357 This research was supported by Diabetes UK, Newcastle Hospitals NHS charity and by the 358 National Institute for Health Research (NIHR) Newcastle Biomedical Research Centre based 359 at Newcastle upon Tyne Hospitals NHS Foundation Trust and Newcastle University. The 360 views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or 361 the Department of Health. DAG is supported by Unilever plc. 362 Disclosure summary: The authors have nothing to disclose 363 364 365 References 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 1. Blau HM, and Webster C. Isolation and characterization of human muscle cells. Proceedings of the National Academy of Sciences of the United States of America 78: 5623-5627, 1981. 2. Carlson CJ, Koterski S, Sciotti RJ, Poccard GB, and Rondinone CM. 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Densitometry is presented as the mean±SEM from 6 separate cultures in each 451 group. – basal, + insulin stimulation. Open bars; basal, closed bars; insulin-stimulated. 452 *p=0.02, **p=0.002 453 454 Figure 2A QPCR analysis of cytokines after 18h SB203580 treatment. Control (left panel) 455 and diabetic cultures (right panel) were differentiated for 7 days and treated with 10μM 456 SB203580 for the last 18h of differentiation prior to RNA extraction. Data are normalised to 457 the reference gene β2-microglobulin and are expressed as the mean±SEM from 6 cultures 458 performed in triplicate. Closed bars: untreated, open bars: SB203580 treated. *p<0.05, 459 **p=0.003, ***p<0.005 vs untreated. 460 Figure 2B QPCR analysis of MX1 and BST2 expression after 18h SB203580 treatment. 461 Control (left panel) and diabetic cultures (right panel) were differentiated for 7 days and 462 treated with 10μM SB203580 for the last 18h of differentiation prior to RNA extraction. Data 463 are normalised to the reference gene β2-microglobulin and are expressed as the mean±SEM 464 from 6 cultures performed in triplicate. Closed bars: untreated, open bars: SB203580 treated. 465 466 Figure 3A QPCR analysis of cytokines after 7 days SB203580 treatment. Control (left panel) 467 and diabetic cultures (right panel) were differentiated for 7 days and treated with 10μM 468 SB203580 for the duration of differentiation prior to RNA extraction. Data are normalised to 469 the reference gene β2-microglobulin and are expressed as the mean±SEM from 6 cultures 470 performed in triplicate. Closed bars: untreated, open bars: SB203580 treated. *p<0.05, 471 **p=0.002, ****p<0.0001 vs untreated. 472 Figure 3B QPCR analysis of MX1 and BST2 expression after 7 days SB203580 treatment. 473 Control (left panel) and diabetic cultures (right panel) were differentiated for 7 days and 474 treated with 10μM SB203580 for the duration of differentiation prior to RNA extraction. 475 Data are normalised to the reference gene β2-microglobulin and are expressed as the 476 mean±SEM from 6 cultures performed in triplicate. Closed bars: untreated, open bars: 477 SB203580 treated. *p<0.05 vs untreated. 478 479 480 Figure 4A Insulin-stimulated glucose uptake in diabetic muscle cultures after 18h SB203580 481 treatment. Day 7 myotubes were treated with p38 MAPK inhibitor for the last 18h of 482 differentiation before measuring glucose uptake. Data are presented as mean±SEM from 5 483 cultures. Open bars; basal, closed bars; insulin stimulated. ***p=0.0007 484 Figure 4B Insulin-stimulated glucose uptake in diabetic muscle cultures after 7 day 485 SB203580 treatment. Myotubes were treated for the duration of differentiation before 486 measuring glucose uptake. Data are presented as mean±SEM from 6 cultures. Open bars; 487 basal, closed bars; insulin stimulated. **p=0.001, ****p<0.0001. 488 489 490 491 492 493 494 495 496 Diabetic patients Controls Age (years) 59 ± 7 59 ± 11 Sex (M:F) 5:1 3:3 Time to insulin treatment (years) 10 ± 5 - Units of insulin/day 131.2 ± 9.6 - Fasting serum insulin (mU/l) - 7.1 ± 0.6 Fasting plasma glucose (mmol/l) - 5.4 ± 0.2 HbA1c (%) 9.0 ± 0.5 5.2 ± 0.1** BMI (kg/m2) 30 ± 0.7 28.5 ± 1.0 Waist:Hip ratio 1.0 ± 0.03 0.9 ± 0.02** Systolic BP (mmHg) 142.8 ± 6.2 131.7 ± 5.4 Diastolic BP (mmHg) 82.3 ± 3.0 76.7 ± 2.1 Total Cholesterol (mmol/l) 4.8 ± 0.2 5.9 ± 0.3** Triglycerides (mmol/l) 3.3 ± 0.5 1.6 ± 0.4* 497 498 Table 1 Metabolic and anthropometric characteristics of recruited subjects. Groups were 499 matched for age and BMI. The diabetics had significantly higher HbA1c, waist:hip ratio and 500 triglycerides. Controls had significantly higher total cholesterol attributed to statin therapy in 501 the diabetics. 502 Data are presented as mean±SEM, Diabetic vs control; ** p<0.01, * p<0.05 503 504 505 506 507 Gene set designation Description of gene set HECKER_IFNB1_TARGETS Genes transcriptionally modulated by interferon β in blood cells of patients with MS Genes upregulated in response to interferon α in hepatocytes Genes upregulated by treatment with decitabine in T24 cells Top 50 genes upregulated by interferon α in ovarian cancer progenitor cells Interferon, T and B lymphocyte genes clustered together across breast cancer samples. Top 50 genes downregulated in A549 cells expressing STAT3 Genes upregulated in primary fibroblasts after 6h treatment with interferon α. Genes upregulated in lung tissue after lipopolysaccharide aspiration and mechanical ventilation Interferon cluster genes upregulated in skin tumours treated with imiquimod Genes representing interferoninduced anti-viral module in sputum during asthma exacerbations RADAEVA_RESPONSE_TO_IFNA1_ UP LIANG_SILENCED_BY_METHYLAT ION_2 MOSERLE_IFNA_RESPONSE FARMER_BREAST_CANCER_CLUS TER_1 DAUER_STAT3_TARGETS_DN BROWNE_INTERFERON_RESPONSI VE_GENES ALTEMEIER_RESPONSE_TO_LPS_ WITH_MECHANICAL_VENTILATIO N UROSEVIC_RESPONSE_TO_IMIQUI MOD BOSCO_INTERFERON_INDUCED_A NTIVIRAL_MODULE Number of genes involved in enrichment score 50 (84) FWER 23 (51) <0.001 23 (51) <0.001 19 (28) <0.001 18 (37) <0.001 26 (47) <0.001 38 (63) <0.001 57 (117) <0.001 16 (22) <0.001 30 (68) <0.001 <0.001 508 509 Table 2 Top 10 gene sets significantly upregulated in diabetic myotubes compared to control 510 myotubes. Analysis was performed using GSEA and a FWER<0.05 was considered 511 significant. Numbers in brackets indicate the size of the gene set. 512 513 514 515 516 517 518 Gene set designation Description of gene set RICKMAN_HEAD_AND_NECK_CANCER _F Genes identifying an intrinsic group in head and neck squamous carcinoma Genes upregulated in myoblasts by insulin-like growth factor 1 vs plateletderived growth factor Genes downregulated in lobular carcinoma vs normal lobular breast cells Muscle development genes upregulated after knockdown of PAX3FOXO1 Genes involved in striated muscle contraction Genes upregulated in lobular carcinoma vs normal ductal breast cells Genes upregulated after PAX3-FOXO1 knockdown Genes involved in muscle contraction Genes in the mesenchymal transition signature common to all invasive cancer types Genes involved in collagen formation KUNINGER_IGF1_VS_PDGFB_TARGETS _UP TURASHVILI_BREAST_LOBULAR_CAR CINOMA_VS_LOBULAR_NORMAL_DN EBAUER_MYOGENIC_TARGETS_OF_PA X3_FOXO1_FUSION REACTOME_STRIATED_MUSCLE_CON TRACTION TURASHVILI_BREAST_LOBULAR_CAR CINOMA_VS_DUCTAL_NORMAL_UP EBAUER_TARGETS_OF_PAX3_FOXO1_F USION_UP REACTOME_MUSCLE_CONTRACTION ANASTASSIOU_CANCER_MESENCHYM AL_TRANSITION_SIGNATURE REACTOME_COLLAGEN_FORMATION Number of genes involved in enrichment score 31 (52) FWER 42 (72) <0.001 32 (66) <0.001 26 (49) <0.001 17 (27) <0.001 29 (61) <0.001 57 (184) <0.001 18 (44) <0.001 24 (60) 0.002 23 (56) 0.005 <0.001 519 520 Table 3 Top significantly down regulated gene sets in diabetic myotubes. Analysis was 521 performed using GSEA analysis and a FWER<0.05 was considered significant. Numbers in 522 brackets indicate the size of the gene set. 523 524 525 526 527 Upstream Regulator TNF EPAS1 IFNG IGF1R IFNA2 Poly rl:rC-RNA IKZF1 AHR IRF7 8-bromo-cAMP Sirolimus SB203580 Actinomycin D Staurosporine SREBF1 SREBPF2 Molecule Type Cytokine Transcription regulator Cytokine Transmembrane receptor Cytokine Chemical reagent Transcription regulator Ligand-dependent nuclear receptor Transcription regulator Chemical reagent Chemical drug Chemical - kinase inhibitor Chemical drug Chemical- kinase inhibitor Transcription regulator Transcription regulator Predicted Activation State Activated Activated Activated Activated Activated Activated Activated Activated Activated Activated Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Activation z-score 3.424 3.374 3.246 3.231 3.063 3.022 2.967 2.843 2.781 2.774 -2.943 -2.940 -2.917 -2.600 -2.598 -2.588 p-value of overlap 1.14E-22 2.72E-07 2.53E-10 1.32E-02 5.43E-04 3.79E-04 1.45E-03 1.50E-06 1.69E-02 1.00E-02 6.16E-06 2.10E-10 1.24E-05 1.80E-05 1.68E-03 4.21E-04 528 529 Table 4 Upstream regulator analysis using IPA. Upstream regulator analysis was used to 530 identify potential upstream factors involved in the increased inflammatory profile observed in 531 the diabetic myotubes compared to control myotubes. The top 10 predicted activators and the 532 6 predicted inhibitors are ranked based on activation score. 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 Probe set ID 202859_x_at 233533_at 202086_at 203001_s_at Gene symbol IL8 KRTAP1-5 MX1 STMN2 Fold Change 11.7 8.7 7.3 6.1 pvalue 0.03 0.01 0.04 0.04 202410_x_at 211356_x_at IGF2 LEPR 5.7 5.0 0.01 0.02 201641_at BST2 4.5 0.02 202803_s_at ITGB2 4.5 52837_at 204602_at KIAA1644 DKK1 4.2 4.2 219602_s_at PIEZO2 3.9 0.0009 Toll-like receptor signalling pathway 0.01 Unknown 0.04 Wnt receptor Yes signalling pathway 0.03 Ion transport 204415_at IFI6 3.9 0.02 201348_at GPX3 3.8 0.04 209869_at 242871_at ADRA2A PAQR5 GREM1 3.7 3.6 3.6 0.04 0.03 0.01 INHBB IFIT3 3.5 3.5 0.008 0.04 STC1 FRZB 3.4 3.4 0.01 0.02 218469_at 205258_at 229450_at 204597_x_at 203698_s_at GO Biological process Secreted? Inflammatory response Unknown Defence response Microtubule organisation MAPK cascade Cytokine-mediated signalling pathway Immune response Yes Yes Cytokine-mediated signalling pathway Glutathione metabolic Yes process Cytokine production Cell differentiation Cell differentiation Yes Defence response Yes Cytokine-mediated signalling pathway Ca2+ homeostasis Yes Wnt receptor Yes signalling pathway 551 552 Table 5 Top 20 probe sets and their corresponding gene ontology process, upregulated with 553 differentiation in the diabetic subjects compared to the controls, ranked by fold change. 554 Uncorrected P values were calculated by t test with equal variance. P<0.05 was considered 555 significant. 556 557
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