1

Myotubes from lean and severely obese subjects with and without type 2 diabetes respond 1

differently to an in vitro model of exercise 2

Yuan Z. Feng1, Nataša Nikolić

1, Siril S. Bakke

1, Eili T. Kase

1, Kari Guderud

1, Jøran 3

Hjelmesæth2,3

, Vigdis Aas4, Arild C. Rustan

1 and G. Hege Thoresen

1,5 4

5 1Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Norway 6

2The Morbid Obesity Center, Vestfold Hospital Trust, Tønsberg, Norway 7

3Department of Endocrinology, Morbid Obesity and Preventive Medicine, Institute of Clinical 8

Medicine, University of Oslo, Norway 9 4Faculty of Health, Oslo and Akershus University College of Applied Sciences, Oslo, Norway 10

5Department of Pharmacology, Institute of Clinical Medicine, Faculty of Medicine, University of 11

Oslo and Oslo University Hospital, Oslo, Norway 12

13

Corresponding address: 14

Yuan Zeng Feng 15

School of Pharmacy 16

P.O. Box 1068 Blindern 17

0316 Oslo 18

Norway 19

E-mail: y.z.feng@farmasi.uio.no 20

Phone: +47 22845675 21

Fax: +47 22854402 22

23

Running title; Myotubes from different donors respond differently to EPS 24

25

Word count; 249 words (abstract), 4193 words (main text), 1 table and 5 figures 26

27

Articles in PresS. Am J Physiol Cell Physiol (January 21, 2015). doi:10.1152/ajpcell.00314.2014

Copyright © 2015 by the American Physiological Society.

2

ABSTRACT 28

Exercise improves insulin sensitivity and oxidative capacity in skeletal muscles. However, the 29

effect of exercise on substrate oxidation is less clear in obese and type 2 diabetic subjects than in 30

lean subjects. We investigated glucose and lipid metabolism and gene expression after 48 h with 31

low-frequency electrical pulse stimulation (EPS), as an in vitro model of exercise, in cultured 32

myotubes established from lean non-diabetic subjects and severely obese subjects (BMI ≥ 40 33

kg/m2) with and without type 2 diabetes. EPS induced an increase in insulin sensitivity, but did 34

not improve lipid oxidation in myotubes from severely obese subjects. Thus, the EPS-induced 35

increases in insulin sensitivity and lipid oxidation were positively and negatively correlated to 36

BMI of the subjects, respectively. EPS enhanced oxidative capacity of glucose in myotubes from 37

all subjects. Furthermore, EPS reduced mRNA expression of slow fiber-type marker (MYH7) in 38

myotubes from diabetic subjects; however the protein expression of this marker was not 39

significantly affected by EPS in neither of the donor groups. On the contrary, mRNA levels of 40

interleukin-6 (IL-6) and IL-8 were unaffected by EPS in myotubes from diabetic subjects, while 41

IL-6 mRNA expression was increased in myotubes from non-diabetic subjects. EPS-stimulated 42

mRNA expression levels of MYH7, IL-6 and IL-8 correlated negatively with subjects’ HbA1c 43

and/or fasting plasma glucose, suggesting an effect linked to the diabetic phenotype. Taken 44

together, these data show that myotubes from different donor groups respond differently to EPS, 45

suggesting that this effect may reflect the in vivo characteristics of the donor groups. 46

47

48

49

3

INTRODUCTION 50

Physical activity plays an important role in both prevention and treatment of obesity and type 2 51

diabetes (T2D). Skeletal muscle is the largest insulin-sensitive organ in humans, accounting for 52

more than 80 % of insulin-stimulated glucose disposal (15). Physical training leads to extensive 53

adaptations in skeletal muscles (14, 46), but the molecular mechanisms underlying these 54

adaptations are still poorly understood, and there is increasing evidence that some individuals 55

may be inherently less responsive to exercise (20, 53). Regular physical activity not only 56

improves oxidative capacity, such as increased lipid and glucose oxidation (39), but is also 57

known to increase insulin-stimulated glucose uptake (45). The effect of exercise on substrate 58

oxidation is less clear in obese and type 2 diabetic subjects than in lean subjects (37). 59

Impairments have been noted in skeletal muscle glucose and fatty acid oxidation in obesity and 60

T2D in the resting state (6, 29, 30). It is therefore important to determine whether interventions, 61

either exercise or pharmacologically, can effectively reverse this impairment. 62

Plasticity of skeletal muscle in response to regular exercise extends beyond the metabolic 63

changes, and PPARδ (peroxisome proliferator-activated receptor-δ) activation is one of many 64

pathways proposed to be involved in exercise adaptations (15, 35). PPARδ is an important 65

regulator of skeletal muscle metabolism, in particular lipid oxidation. It is well known that 66

endurance exercise increases mitochondrial content (46, 54), and that MHCI (myosin heavy chain 67

type I, slow-twitch oxidative) muscle fibers are associated with higher mitochondrial content 68

compared to MHCII (type II, fast-twitch glycolytic) fibers (32). On the other hand, it is debated 69

whether conversion of muscle type II to type I phenotype actually happens in adult humans (22). 70

4

Moreover, skeletal muscle has been identified as a secretory organ that releases a diversity of 71

biologically active proteins classified as myokines that can have autocrine, paracrine or endocrine 72

functions. Muscle contraction during exercise is a major stimulus of these endocrine functions, 73

and the myokines are thought to mediate beneficial effects of exercise and may have a role in the 74

protection against conditions associated with low-grade inflammation, such as T2D and 75

metabolic syndrome (38, 42, 56). The myokines, interleukin-6 (IL-6) and IL-8 are markedly 76

produced in contracting muscles and, at least for IL-6, released into plasma during the post-77

exercise period (2, 51) when the insulin sensitivity is enhanced (23). It seems that IL-6 works as 78

an energy sensor, and preserves fuel availability during exercise (40) by enhancing insulin-79

stimulated glucose disposal and fatty acid metabolism (10, 55). Interestingly, while IL-6 released 80

from skeletal muscle may promote insulin sensitivity (21), IL-6 secreted from adipose tissue may 81

induce insulin resistance in muscle (41). Thus, while it has become evident that contracting 82

skeletal muscle releases myokines that may influence metabolism and function of muscle tissue 83

and other tissues and organs, the secretion of myokines from obese and/or diabetic muscle is yet 84

to be fully clarified. 85

Previously, we have established an in vitro model of regular exercise of cultured human skeletal 86

muscle cells (myotubes) by applying chronic, low-frequency electrical pulse stimulation (EPS) 87

(36), and thereby enabling the study of exercise-induced cellular mechanisms under controlled 88

conditions. Human myotubes are considered a valid model for studying metabolic disorders as 89

obesity and T2D because perturbances evident in vivo, such as depressed lipid oxidation and 90

insulin resistance, are retained in myotubes in culture (1, 7, 19). After EPS, myotubes established 91

from lean non-diabetic subjects showed an increase in glucose and lipid metabolism as well as 92

indications of fast-twitch glycolytic muscle fibers transformation into slow-type oxidative fibers 93

5

(36), and enhanced expression and release of IL-6 and IL-8 (31, 44, 48). In the present study, we 94

wanted to explore whether there are differences in EPS response on insulin sensitivity, glucose 95

and lipid metabolism and gene expression in myotubes from three groups of donors; lean non-96

diabetic, severely obese non-diabetic and severely obese subjects with established T2D and 97

whether these differences reflect the in vivo characteristics of the donor groups. Moreover, 98

PPARδ agonist and exercise training are shown to act synergistically to increase the content of 99

oxidative muscle fibers, and PPARδ agonists have been proposed as exercise mimetics (35). 100

Therefore, we wanted to explore the combined effect of EPS together with the PPARδ agonist, 101

GW501516, on lipid metabolism as well. 102

103

6

MATERIALS AND METHODS 104

Human myotubes 105

Human myotubes grown from satellite cells were isolated as previously described (18) from the 106

M. obliquus internus abdominis from lean or severely obese subjects. The lean non-diabetic 107

donor biopsies were obtained from subjects donating a kidney at Oslo University Hospital, 108

Norway and the severely obese donor biopsies were obtained from subjects undergoing bariatric 109

surgery at The Morbid Obesity Center, Vestfold Hospital Trust, Norway. The isolation of satellite 110

cells from all biopsies was performed at the same location and by the same researchers. The 111

biopsies were obtained with informed consent and approved by the National Committee for 112

Research Ethics, Oslo, Norway (S-04133 and S-09078d). 113

Classification of the severely obese donors into the two groups; severely obese non-diabetic 114

subjects (SO-nD) and severely obese subjects with T2D (SO-T2D) was based on fasting blood 115

glucose values (≥ 7.0 mmol/L), HbA1c (glycosylated hemoglobin, ≥ 6.5 %) and/or use of one or 116

more antidiabetic drugs. The cells were cultured in DMEM-GlutamaxTM

(5.5 mmol/L glucose) 117

with supplements during proliferation and differentiation as previously described (25). Briefly, at 118

~ 80 % confluence, growth medium was replaced by differentiation medium to induce fusion of 119

myoblasts into multinucleated myotubes. Experiments were performed on cells from passage 120

number 2 to 5 and after 7 days of differentiation. There was no difference in mRNA expression 121

levels of muscle fiber type I marker between the myotubes from lean non-diabetic and severely 122

obese subjects, or between the myotubes from severely obese non-diabetic and diabetic subjects 123

(data not shown). 124

125

7

Electrical pulse stimulation of muscle cells 126

Human myotubes grown on Corning®

CellBIND®

6-well plates (Corning Life-Sciences, 127

Schiphol-Rijk, The Netherlands) were stimulated via carbon electrodes by applying chronic, low-128

frequency EPS (single, bipolar pulses of 2 ms, 30 V, 1 Hz) continuously for the last 48 h of the 129

differentiation period, as previously described (36). Culture media were changed every 12th

or 130

24th

h during EPS. Electrical pulses were generated by a muscle stimulator built at the Electronics 131

Lab, Institute of Chemistry, University of Oslo, Norway. Neither applying EPS for 48 h to the 132

myotubes nor changing the media every 12th

or 24th

h showed differences in floating cells 133

evaluated by microscopic inspection, or by measurement of cell protein content in the three donor 134

groups (data not shown). 135

Immunoblotting 136

After EPS, cells were harvested in Laemmli buffer (0.5 mol/L Tris-HCL, 10 % SDS, 20 % 137

glycerol 10 % β-mercaptoethanol and 5% bromphenyl blue). Total cell lysates were 138

electrophoretically separated on 4-20 % Mini-Protean®

TGX™ gels (BioRad, Copenhagen, 139

Denmark) with Tris/glycine buffer (pH 8.3) followed by blotting to nitrocellulose membrane and 140

incubation with antibodies against total Akt kinase, Akt phosphorylated at Ser473, β-actin, α-141

tubulin (all from Cell Signaling Technology, Beverly, MA), MHCI (Millipore, Billeria, MA), and 142

an OXPHOS antibody cocktail recognizing Complex I subunit NDUFB8, Complex II subunit, 143

Complex III subunit core 2, Complex IV subunit II and ATP synthase subunit alpha (Abcam, 144

Cambridge, UK). Immunoreactive bands were visualized with enhanced chemiluminescence 145

(Chemidoc XRS, BioRad, Denmark) and quantified with Image Lab (version 4.0) software. β-146

8

actin or α-tubulin were used to normalize the protein-antibody signals versus protein loading. 147

Glucose metabolism 148

After EPS, myotubes were exposed to serum-free DMEM supplemented with [14

C(U)]glucose 149

(0.5 μCi/mL, 1 mmol/L, PerkinElmer NEN®

, Boston, MA, US) and 1 mmol/L pyruvate in the 150

presence or absence of 100 nmol/L insulin for 3 h to study basal and insulin-stimulated glycogen 151

synthesis as previously described (16). Glucose oxidation was measured after 3 h incubation, 900 152

µL cell medium was transferred to airtight flasks, and 300 µL of phenyl ethylamine:methanol 153

(1:1, v/v) was added to a center well containing a folded filter paper. Subsequently, 200 µL of 1 154

mol/L perchloric acid was added to the cell medium through the stopper tops using a syringe, as 155

previously described (36). The flasks were placed at room temperature to trap labeled CO2 for 2 h, 156

and radioactivity was counted by liquid scintillation. All data were related to protein content. 157

Fatty acid oxidation 158

Myotubes were treated with 0.1 % DMSO (vehicle) or 10 nmol/L GW501516 for 96 h, and EPS 159

was applied to the cells for the last 48 h of the treatment period. After EPS, the myotubes were 160

exposed to 1 mL of DPBS supplemented with HEPES (10 mmol/L), NaHCO3 (44 mmol/L), 161

[1-14

C]oleic acid (1 µCi/mL, 100 µmol/L, PerkinElmer NEN®

, Boston, MA, US), 40 µmol/L 162

BSA and 1 mmol/L L-carnitine in a 5 % CO2-incubator at 37°C. After 2 h, CO2 and cell-163

associated radioactivity were measured as previously described (36). Briefly, 500 µL of cell 164

medium was transferred to airtight flasks, and 300 µL of phenyl ethylamine:methanol (1:1, v/v) 165

was added to a center well containing a folded filter paper. Subsequently, 100 µL of 1 mol/L 166

perchloric acid was added to the cell medium through the stopper tops using a syringe. The flasks 167

were placed at room temperature to trap labeled CO2 for 2 h. The cells were placed on ice and 168

9

washed twice with PBS, lysed with 0.1 mol/L NaOH, and cell-associated radioactivity was 169

counted by liquid scintillation to determine uptake of oleic acid. All data were related to protein 170

content. 171

Live imaging 172

After EPS, the myotubes were incubated at 37°C and 5 % CO2 with MitoTracker®

Red FM (100 173

nmol/L, Invitrogen, Carlsbad, CA) for 15 min to stain mitochondria, Bodipy 493/503 (2 µg/mL, 174

Invitrogen, Carlsbad, CA) for 15 min to stain neutral lipids and Hoechst 33258 (2.5 µg/mL, 175

Invitrogen, Carlsbad, CA) for 15 min to stain nuclei and washed with PBS in between. Live 176

imaging was carried out as previously described (25). Images were randomly taken in 25-36 177

positions per well with a Scan^R platform (Olympus IX81 inverted fluorescence microscope) 178

equipped with a temperature incubator for long-term live imaging. After gating out aggregates 179

and dead cells, each parameter was determined from about 240 images per donor group (average 180

of 38 ± 4 nuclei per image). MitoTracker®

Red FM and Bodipy 493/503 intensity per image was 181

related to number of nuclei per image since for multinucleated myotubes the absolute number of 182

cells cannot be determined. This is an accepted method to account for differences in cell numbers 183

(13, 28, 34, 36). For the myotubes from L-nD subjects, the cells were also treated with 184

GW501516 for 96 h and EPS was applied to the cells for the last 48 h of the treatment period. 185

Gene expression analysis by qPCR 186

After EPS, total RNA was isolated from the myotubes, and qPCR was performed as previously 187

described (36). Briefly, total RNA from cells was isolated by Agilent Total RNA isolation kit 188

according to the supplier’s protocol. RNA was reverse-transcribed with oligo primers using a 189

PerkinElmer Thermal Cycler 9600 and qPCR was performed using an ABI PRISMT 7000 190

10

Detection System (Applied Biosystems, Warrington, UK). Transcription levels were normalized 191

to the housekeeping genes acidic ribosomal phosphoprotein P0 (36B4) and glyceraldehyde-3-192

phosphate dehydrogenase (GAPDH). The following forward and reverse primers (Molecular 193

Probes, Invitrogen, Carlsbad, CA) were used at concentration of 30 µmol/L, 36B4 (acc. no 194

M17885); GAPDH (acc. no NM_002046); IL-6 (acc.no. NM_000600), IL-8 195

(acc.no.NM_000584.2); MYH7 (acc.no.NM_000257.2). 196

Data presentation and statistics 197

Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 198

5.0 for Windows (GraphPad Software Inc., San Diego, CA, US). The value n represents the 199

number of different donors used, and each with at least duplicate observations. Two-tailed paired 200

Student’s t-tests were performed to determine the effects of treatments, and unpaired Student’s t-201

tests (independent samples) were performed to determine the difference in effects of EPS 202

between the donor groups. For correlation studies, Spearman correlation analysis was performed. 203

Linear mixed model analysis (LMM, SPSS 20.0.0.1, IBM SPSS Inc., Chicago, IL, US) was used 204

to determine the effect of EPS in OXPHOS experiments. The linear mixed model includes all 205

observations (complex I-V) in the statistical analyses and at the same time take into account that 206

not all observations are independent. P < 0.05 was considered statistically significant. 207

208

11

RESULTS 209

Clinical characteristics of the biopsy donors 210

Clinical characteristics of the donors are presented in Table 1. Fasting plasma glucose and HbA1c 211

were significantly higher in severely obese subjects with T2D (SO-T2D) than in non-diabetic 212

subjects with similar body mass index (SO-nD). Fasting plasma levels of insulin, TAG 213

(triacylglycerol) and HDL (high-density lipoprotein) were not different between the two obese 214

groups. As expected, clinical characteristics of lean non-diabetic subjects (L-nD) were 215

significantly different from severely obese subjects. The age was not significantly different 216

between the three donor groups. 217

Effect of EPS on insulin sensitivity in myotubes 218

Myotubes from severely obese subjects with T2D maintained their diabetic phenotype in culture 219

shown as absence of insulin-stimulated glucose uptake in myotubes from SO-T2D, and further, 220

phosphorylation of Akt tended to be lower in myotubes from SO-T2D compared to SO-nD (4). 221

To examine whether EPS could increase insulin sensitivity, insulin-stimulated phosphorylation of 222

Akt (Ser 473) and glycogen synthesis were measured in myotubes established from lean non-223

diabetic (L-nD), SO-nD and SO-T2D after EPS stimulation. In absence of insulin, 224

phosphorylation of Akt was not affected by EPS (Figure 1A), whereas insulin-stimulated 225

phosphorylation of Akt was significantly increased by EPS in myotubes from SO-T2D (Figure 226

1B). In absence of insulin, glycogen synthesis was not affected by EPS (Figure 1C), whereas 227

insulin-stimulated glycogen synthesis after EPS was increased in myotubes from both SO-nD and 228

SO-T2D subjects (Figure 1D). Further, EPS-induced effect on insulin-stimulated phosphorylation 229

12

of Akt and glycogen synthesis in the myotubes correlated positively with BMI of the subjects 230

(Figures 1E, F), i.e. the EPS effect increased with increasing BMI of the donors. 231

Effect of EPS on glucose and oleic acid metabolism 232

We wanted to explore whether there were differences in the EPS-induced response on oxidative 233

capacity in myotubes established from lean, non-diabetic and severely obese subjects with and 234

without T2D. Glucose oxidation after EPS was increased in myotubes from both lean and 235

severely obese subjects (Figure 2A), whereas EPS had effect on oleic acid oxidation in myotubes 236

from lean subjects only (Figure 2B). Furthermore, oleic acid oxidation after EPS was 237

significantly different in myotubes from lean subjects compared to myotubes from severely obese 238

subjects (Figure 2B), and the EPS-induced effect on oleic acid oxidation correlated also 239

negatively with the BMI of the subjects irrespective of donor group (Figure 2C). Mitochondrial 240

content shown as MitoTracker®

intensity related to nuclei was significantly increased by EPS in 241

myotubes from L-nD subjects (Figure 2D), but not in myotubes from severely obese subjects 242

(SO-nD, P = 0.076; SO-T2D, P = 0.32). Also the overall expression levels of proteins involved 243

in mitochondrial oxidative phosphorylation (OXPHOS proteins) were significantly increased by 244

EPS in myotubes from L-nD subjects (Figure 2E, F). Without EPS, lipid droplet number (Figure 245

2G) and neutral lipid content (data not shown) measured with Bodipy 493/503 were not different 246

between the myotubes from lean non-diabetic and severely obese subjects, or between myotubes 247

from severely obese non-diabetic and diabetic subjects. EPS revealed no further differences in 248

lipid droplet number between the groups (Figure 2G). In line with this, cellular uptake of oleic 249

acid, assessed as the sum of cell-associated and CO2-trapped radioactivity was not affected by 250

EPS in neither of the donor groups (Figure 2H). 251

252

13

Effect of EPS on oleic acid metabolism in the presence of PPARδ activation 253

To examine whether the PPARδ agonist GW501516 and exercise could act synergistically in our 254

cell model, myotubes were exposed to both EPS and GW501516. PPARδ activation increased 255

oleic acid oxidation in myotubes from both lean and severely obese subjects, whereas no further 256

increase with GW501516 and EPS in combination compared to GW501516 alone (Figure 3A) 257

was seen. Cellular uptake of oleic acid was increased after GW501516 treatment in myotubes 258

from all three donor groups, whereas there was no further increase with GW501516 and EPS in 259

combination (Figure 3B). Although only studied in myotubes established from lean non-diabetic 260

subjects, mitochondrial content measured as MitoTracker®

intensity was not further increased in 261

cells treated with both GW501516 and EPS compared to EPS alone (Figure 3C). 262

Effect of EPS on slow-oxidative (MHCI) fiber type 263

We wanted to investigate the effect of EPS on expressions of gene and protein for type I fibers 264

(MYH7, the gene that regulates protein expression of MHCI). After EPS, MYH7 gene expression 265

was reduced in myotubes from SO-T2D subjects (Figure 4A). Furthermore, the EPS effect on 266

gene expression of MYH7 in myotubes correlated negatively with fasting plasma glucose (Figure 267

4B) and HbA1c (Figure 4C) levels of the subjects. However, results obtained for protein 268

expression of MHCI (Figure 4D) showed no significant differences between the donor groups. 269

Effect of EPS on gene expression of IL-6 and IL-8 270

To investigate whether the EPS-induced gene regulations of myokines seen in myotubes from 271

lean non-diabetic subjects (48) also occurred in myotubes originating from severely obese 272

subjects, qPCR on selected genes was performed. Gene expression of IL-6 was increased after 273

EPS in myotubes established from L-nD and SO-nD subjects (Figure 5A). Furthermore, EPS-274

14

induced effect on IL-6 gene expression in myotubes correlated negatively with fasting plasma 275

glucose (Figure 5B) as well as HbA1c (r = -0.60, P < 0.05) levels of the subjects. Similar results 276

were also obtained for IL-8; although the EPS increased IL-8 expression was not significant 277

(Figure 5C). In line with this, effect of EPS on gene expression of IL-8 also correlated negatively 278

with fasting plasma glucose levels of the subjects (Figure 5D). 279

280

15

DISCUSSION 281

In this study, we show that insulin sensitivity was increased in myotubes established from 282

severely obese subjects after EPS. Furthermore, EPS enhanced oxidative capacity of glucose in 283

myotubes from all subjects, while oleic acid oxidation and mitochondrial content were only 284

improved in myotubes from lean subjects. Oleic acid oxidation was also increased after 285

GW501516 treatment in myotubes from all subjects, whereas combination of GW501516 286

treatment and EPS showed no additional effect on oleic acid oxidation. Of particular interest, the 287

impact of EPS on gene expressions of IL-6, IL-8 and was different in myotubes originating from 288

lean and severely obese non-diabetic subjects compared to myotubes from severely obese 289

diabetic subjects. EPS reduced mRNA expression of slow fiber-type marker (MYH7) in 290

myotubes from diabetic subjects, while protein expression of this marker was not affected by EPS 291

in neither of the donor groups. 292

Our findings suggest that human myotubes display the same phenotype as intact muscle in vivo, 293

in accordance to other studies (17, 19, 24). The precise mechanisms by which myotubes are able 294

to retain the in vivo characteristics are not known. However, a combination of genetic and 295

epigenetic mechanisms is probably involved, and this has been reviewed in a recent paper (1). 296

For example, epigenetic regulation of skeletal muscle stem cells and skeletal muscle 297

differentiation, exercise, diet and a family history of T2D, have all been described to influence 298

DNA methylation in human skeletal muscle, and these are traits that might follow the isolated 299

satellite cells into their corresponding cultured myotubes (1). 300

Exercise increases insulin sensitivity in vivo (23, 46), and in line with this, we have shown here 301

that EPS can increase insulin sensitivity in insulin-resistant myotubes from severely obese 302

16

diabetic subjects. The observed effect on insulin sensitivity was more evident in myotubes from 303

severely obese subjects, which is consistent with other studies reporting greater exercise-induced 304

changes in insulin sensitivity in subjects who were more insulin-resistant at baseline (5, 20), and 305

more evident in obese men compared to lean men (20). This suggests that interventions to 306

increase physical activity may be particularly effective at improving insulin sensitivity in 307

population groups who are more insulin resistant or have an increased predisposition to insulin 308

resistance. Lack of EPS-induced increase in insulin sensitivity in myotubes from lean non-309

diabetic subjects could be explained by maximal responsiveness to insulin in those cells per se 310

and limited capacity to respond further. In agreement with other studies reporting enhanced 311

carbohydrate oxidation after exercise in obese subjects (9), and in subjects with T2D (50), we 312

showed an increase in glucose oxidation after EPS in myotubes from all three donor groups. In 313

contrast, we have previously (36), and in the present study shown that mitochondrial content was 314

increased after EPS in myotubes from lean non-diabetic subjects, and this was followed by 315

increased rate of oleic acid oxidation. In this study, however, we showed that this is not the case 316

in myotubes from both groups of severely obese subjects. These data are in contrast to studies 317

reporting an increase in muscle ex vivo palmitate oxidation in obese subjects after in vivo exercise 318

(33). The discrepancy may to some extent be explained by differences between in vivo and in 319

vitro exercise models. Without EPS, there was no difference in basal lipid content between 320

myotubes from the three donor groups, excluding the possibility that the absence of EPS effect on 321

oleic acid oxidation was due to label dilution when using a labelled fatty acid as a measurement 322

of fatty acid oxidation. At resting state, the current consensus is that fatty acid oxidation is 323

reduced in obese versus lean muscle (6, 26). However, some studies have also reported similar 324

fatty acid oxidation in myotubes from lean and obese, diabetic subjects (49) and even higher 325

oxidation in obese than lean muscle (3). The reason behind this discrepancy is unclear, but may 326

17

be a result of different subgroups of obese populations studied. It is an ongoing debate whether 327

the impairment in oxidative capacity in obese and insulin-resistant subjects is due to lower 328

mitochondrial content or intrinsic mitochondrial dysfunction (54). Although it has been shown 329

that exercise improves both mitochondrial content and intrinsic mitochondrial function in obese 330

and insulin-resistant subjects (43), it has also been reported no changes in key regulators of 331

mitochondrial biogenesis in skeletal muscles in diabetic men after in vivo exercise (12). 332

Consistent with this, in our exercise model, myotubes from subjects with higher BMI were less 333

responsive to exercise-induced effects on fatty acid oxidation than myotubes from lean subjects. 334

It has been suggested that insulin resistance in obesity and T2D is associated with intracellular 335

accumulation of lipid in skeletal muscles (29). Consequently, much focus has been on the 336

possibility of increasing lipid utilization by exercise to avoid lipid accumulation. However, 337

evidence has emerged that increased lipid oxidation impair glucose uptake by metabolic 338

feedback, as suggested by the Randle cycle (reviewed in (29)). In line with this, we have shown 339

that EPS was able to improve insulin sensitivity and glucose oxidation in myotubes from both 340

groups of severely obese subjects without having impact on lipid oxidation. 341

Gene regulations involved in exercise adaptation are complex (14, 48), but genes regulated by 342

PPARδ may be involved (15). It has also been proposed that PPARδ agonists and exercise 343

training synergistically increase the proportion of type 1 fibers (MHCI) in adult mice (35). 344

However, selective biomarkers for fatty acid β-oxidation were no further induced with exercise 345

compared to GW501516 (35), which is consistent with our findings that oleic acid oxidation were 346

no further increased when myotubes were treated with a combination of GW501516 and EPS. 347

In response to acute exercise, an increase of IL-6 mRNA expression has been detected in muscle 348

during exercise and in the post-exercise period in both lean and obese subjects (11, 51). In 349

18

contrast to myotubes from lean and obese non-diabetic subjects, EPS did not increase mRNA 350

expression of IL-6 in myotubes from obese diabetic subjects. In support of our findings, other 351

studies have also reported no effect on IL-6 mRNA levels in muscle biopsies from obese diabetic 352

subjects (52) and in plasma from diabetic mice after in vivo exercise (8). This may reflect an 353

inherent abnormal IL-6 response in insulin resistant skeletal muscle (27, 47). It has previously 354

been shown that EPS increased gene expression of IL-8 in lean non-diabetic myotubes (48), and 355

our current data indicate that the impact of EPS on gene expression of IL-8 differs in myotubes 356

from severely obese non-diabetic subjects compared to diabetic subjects. Exercise-derived IL-8 is 357

postulated to stimulate angiogenesis in skeletal muscles (39), but the biological role of muscle-358

derived IL-8 remains to be clarified. Despite vast research on myokines the past decade, more 359

research is needed in order to clarify whether there is an altered myokine response in skeletal 360

muscles from subjects with disturbed metabolic status. 361

In summary, by applying chronic, low-frequency EPS on cultured myotubes established from 362

three different donor groups, lean non-diabetic subjects and severely obese subjects with and 363

without T2D, we observed an increase in insulin sensitivity with no improvement of lipid 364

oxidation after EPS in myotubes from both groups of severely obese subjects. These findings 365

were associated with BMI of the subjects suggesting an effect linked to obesity and indicating 366

that improving insulin sensitivity is not solely depended on combating lipid accumulation with 367

increased oxidative capacity of lipids. Furthermore, we observed a reduced or no effect of EPS on 368

gene expression of the myokines IL-6 and IL-8 in myotubes from severely obese diabetic 369

subjects. These findings were associated with fasting plasma glucose levels of the subjects 370

suggesting an effect linked to the diabetic phenotype. Taken together, these data show that 371

19

myotubes from different donor groups responded differently to EPS, suggesting that this effect 372

may reflect the in vivo characteristics of the donor groups. 373

374

ACKNOWLEDGEMENTS 375

The authors thank Terje Grønås (Electronics Lab, Institute of Chemistry, University of Oslo) for 376

support and technical assistance. We are also thankful to Gerbrand Koster and Oddmund Bakke 377

from the NORMIC-UiO imaging platform, Department of Molecular Biosciences, University of 378

Oslo, for support, use of equipment and for excellent technical assistance. 379

GRANTS 380

This work was founded by the University of Oslo, Diabetesforbundets forskningsfond and 381

Anders Jahres fond til vitenskapens fremme. 382

383

20

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Figure legends 553

554

555

Figure 1- EPS improved insulin sensitivity in myotubes derived from severely obese subjects. 556

EPS was applied to cultured myotubes for the last 48 h of the differentiation period. A, B: After 557

termination of EPS, myotubes were incubated for 15 min in presence or absence of 100 nmol/L 558

insulin, and immunoblotting analysis with antibodies against pAkt (Ser473) and total-Akt were 559

performed (n = 4-5). A: pAkt/total Akt without insulin. B: Insulin-stimulated pAkt/total Akt. C, D: 560

After termination of EPS, glycogen synthesis was measured as [14

C]glucose (1 mmol/L) 561

incorporation into glycogen in presence or absence of 100 nmol/L insulin for 3 h (n = 6-8). C: 562

Glycogen synthesis without insulin. D: Insulin-stimulated glycogen synthesis. EPS-induced 563

effect on insulin-stimulated pAkt/total Akt (E, n = 13) and glycogen synthesis (F, n = 21) in the 564

myotubes correlated positively with BMI of the donor subjects. *P ˂ 0.05 vs. control. 565

Figure 2- EPS improved oxidative capacity of human myotubes. The myotubes were electrically 566

stimulated for 48 h; glucose and oleic acid oxidation, mitochondrial staining and lipid droplets 567

were measured after termination of EPS. A: Glucose oxidation. Myotubes were exposed to 568

[14

C]glucose (1 mmol/L) for 3 h before CO2 assessment (n = 6-7). B: Oleic acid oxidation. 569

Myotubes were exposed to [14

C]oleic acid (100 µmol/L) for 2 h before CO2 assessment (n = 7-8). 570

C: EPS-induced effect on oleic acid oxidation correlated negatively with BMI of the subjects (n = 571

21). D: The myotubes were stained for mitochondrial content with MitoTracker®

Red FM, and 572

nuclei with Hoechst 33258. Data are shown as MitoTracker intensity related to nuclei (n = 5-6). E: 573

Expression of proteins involved in mitochondrial oxidative phosphorylation, OXPHOS (Complex 574

I subunit NDUFB8, Complex II subunit, Complex III subunit core 2, Complex IV subunit II and 575

ATP synthase subunit α). Expression levels were normalized to α-tubulin (n = 4-6). Linear mixed 576

model was performed for OXPHOS experiments. F: Representative immunoblots from one 577

25

experiment (L-nD). Ctr; control. G: The myotubes were stained for neutral lipids with Bodipy 578

493/503. Data are shown as lipid droplets related to nuclei. H: Cellular uptake of oleic acid was 579

assessed as the sum of cell-associated and CO2-trapped radioactivity (n = 7-8). *P < 0.05 vs. 580

control. #P < 0.05 vs. L-nD. 581

Figure 3- Effect of EPS combined with PPARδ activation on oleic acid metabolism. Myotubes 582

were treated with 10 nmol/L GW501516 for 96 h and EPS was applied to the cells for the last 48 583

h of the treatment period. A, B: After termination of EPS, myotubes were exposed to [14

C]oleic 584

acid (100 µmol/L) for 2 h. A: Oleic acid oxidation (n=5-10). B: Cellular uptake of oleic acid, 585

assessed as the sum of cell-associated and CO2-trapped radioactivity (n=5-7). C: After 586

termination of EPS, the myotubes were stained for mitochondrial content with MitoTracker®

Red 587

FM, and nuclei with Hoechst 33258. Data are shown as MitoTracker intensity related to nuclei 588

and normalized to unstimulated control cells (n = 5-6). *P < 0.05 vs. control. 589

Figure 4- Effect of EPS on gene and protein expression of MHCI. The myotubes were 590

electrically stimulated for 48 h. A: After termination of EPS, expression of MYH7 (the gene that 591

regulates protein expression of myosin heavy chain type I (MHCI)) was measured by qPCR and 592

compared relative to the housekeeping genes 36B4 and GAPDH (n =5-6). B, C: EPS effect on 593

gene expression of MYH7 correlated negatively with fasting plasma glucose (B) and HbA1c (C) 594

of the subjects (n =16). D: Immunoblotting analysis with antibodies against MHCI, related to β-595

actin, presented relative to unstimulated control.*P < 0.05 vs. control. 596

Figure 5- Effect of EPS on gene expression of IL-6 and IL-8. The myotubes were electrically 597

stimulated for 48 h and harvested after termination of EPS. Expression of IL-6 (A) and IL-8 (C) 598

were measured by qPCR and studied relative to the housekeeping genes 36B4 and GAPDH. 599

26

Fasting plasma glucose levels were inversely related to EPS effect on gene expressions of IL-6 (B, 600

n = 22) and IL-8 (D, n = 19). *P < 0.05 vs. control. 601

602

603

Table 1- Clinical characteristics of biopsy donors

L-nD (n = 14) SO-nD (n = 9) SO-T2D (n = 9)

Age (years) 49 ± 4 41 ± 2 48 ± 3

BMI (kg/m2) 24.3± 0.9 42.0 ± 1.7* 44.6 ± 2.2*

Fasting glucose (mmol/l) 5.6 ± 0.3 4.8 ± 0.1 8.2 ± 0.5†

HbA1c (%) - 5.5 ± 0.1 7.2 ± 0.6#

Insulin pmol/l - 89.4 ± 16 62.4 ± 18

TAG (mmol/l) 1.0 ± 0.1 1.6 ± 0.2* 2.0 ± 0.3*

HDL (mmol/l) 1.6 ± 0.1 1.1 ± 0.1* 1.0 ± 0.1*

Data are presented as mean ± SEM. L-nD; lean non-diabetic, SO-nD; severely obese non-diabetic,

SO-T2D; severely obese with type 2 diabetes. HbA1c and plasma insulin were not measured in the

lean non-diabetic cohort. *Significantly different from the lean group. †Significantly (P < 0.05)

different from both of the non-diabetic groups. #Significantly different from SO-nD. BMI; body

mass index, HbA1c; glycosylated hemoglobin, HDL; high-density lipoprotein, TAG;

triacylglycerol