TITLE 1

In vitro Contraction Protects Against Palmitate-Induced Insulin Resistance in C2C12 Myotubes 2

3

AUTHORS 4

Stephan Nieuwoudt 1, 2, Anny Mulya 2, Ciarán E. Fealy 2, Elizabeth Martelli 3, Srinivasan Dasarathy 2, 5

Sathyamangla V. Naga Prasad 3, John P. Kirwan 1, 2 6

7

AUTHOR AFFILIATIONS 8

1. Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio, USA 9

2. Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA 10

3. Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, USA 11

12

RUNNING HEAD 13

EPS Protects Against Palmitate-Induced Insulin Resistance 14

15

ADDRESS FOR CORRESPONDENCE 16

John P. Kirwan 17

kirwanj@ccf.org 18

Mail Code NE40 19

9500 Euclid Avenue 20

Cleveland, OH 44195, USA 21

22

Articles in PresS. Am J Physiol Cell Physiol (August 23, 2017). doi:10.1152/ajpcell.00123.2017

Copyright © 2017 by the American Physiological Society.

2

ABSTRACT 23

We are interested in understanding mechanisms that govern the protective role of exercise against lipid-24

induced insulin resistance, a key driver of type 2 diabetes. In this context, cell culture models provide a 25

level of abstraction that aid in our understanding of cellular physiology. Here we describe the 26

development of an in vitro myotube contraction system that provides this protective effect, and which 27

we have harnessed to investigate lipid-induced insulin resistance. C2C12 myocytes were differentiated 28

into contractile myotubes. A custom manufactured platinum electrode system and pulse stimulator, 29

with polarity switching, provided an electrical pulse stimulus (EPS) (1Hz, 6ms pulse width, 1.5 V/mm, 16 30

hours). Contractility was assessed by optical flow flied spot noise mapping and inhibited by application 31

of ammonium acetate. Following EPS, myotubes were challenged with 0.5 mM palmitate for 4 hours. 32

Cells were then treated with or without insulin for glucose uptake (30 mins), secondary insulin signaling 33

activation (10 mins), and phosphoinositide 3-kinase-α (PI3Kα) activity (5 mins). Prolonged EPS increased 34

non-insulin stimulated glucose uptake (83%, P=0.002), Akt (Thr308) phosphorylation (P=0.005), and IRS-35

1 associated PI3Kα activity (P=0.048). Palmitate reduced insulin specific action on glucose uptake (-49%, 36

P<0.001) and inhibited insulin stimulated Akt phosphorylation (P=0.049) and whole cell PI3Kα activity 37

(P=0.009). The inhibitory effects of palmitate were completely absent with EPS pretreatment at the 38

levels of glucose uptake, insulin responsiveness, Akt phosphorylation, and whole cell PI3Kα activity. This 39

model suggests that muscle contraction alone is a sufficient stimulus to protect against lipid-induced 40

insulin resistance as evidenced by changes in proximal canonical insulin-signaling pathway. 41

42

KEYWORDS 43

Exercise, insulin resistance, diabetes, muscle, lipid 44

3

INTRODUCTION 45

46

Cell culture based models are widely used to study cellular physiology. Such models are not beholden to 47

the methodological limitations of human and animal studies, allowing for higher throughput, while also 48

eliminating the need for tissue procurement. In vitro models have been applied to every facet of 49

physiological research, but their relevance hinges upon their reproducibility of observed phenomena 50

demonstrated in vivo and ex vivo. Several skeletal muscle contraction models have been published to 51

date, with the goal of improving our understanding of exercise physiology. These models have relied on 52

electrical pulse stimulation (EPS) of differentiated skeletal myotubes, and have faithfully reproduced 53

exercise induced hormone secretion (36, 38, 42, 45), substrate utilization and metabolic signaling (15, 54

41), protein synthesis (6, 17), and gene expression (7, 9). Despite their extensive application, many 55

exercise related phenomena still need cellular models to understand their beneficial roles with regards 56

to metabolism. 57

58

We (24) and others (46) have shown that physical exercise in adults can protect against lipid-induced 59

insulin resistance, a known driver of type 2 diabetes (12). This protective phenomenon has also been 60

demonstrated in a rat model of exercise (51), but is yet to be established in an in vitro cellular model 61

system. At the cellular level, insulin resistance manifests as a reduction in insulin stimulated glucose 62

uptake (11). Our earlier work indicated that the inflammatory cytokine tumor necrosis factor-α (TNFα) 63

reduces insulin stimulated glucose uptake and inhibits insulin signaling in C2C12 skeletal myotubes (13), 64

a murine cell line widely used to study muscle physiology (56). Subsequent experiments have shown 65

that the TNFα effect is mediated through saturated lipids (54), such as palmitate (16:0), which inhibit 66

insulin signaling in vitro through phosphoinositide-3-kinase (PI3K), Akt/PKB, and glucose uptake (16, 23, 67

4

49). Given the methodological limitations of clinical and animal studies, in depth investigation is needed 68

to better understand the protective role of exercise on elements of canonical insulin signaling pathway. 69

70

Our interest lies in determining the mechanisms that impart the beneficial effects of exercise, especially 71

in protection against insulin resistance. Although exercise is a systemic event resulting in various 72

metabolic and cardiovascular adaptations (32), the primary component of skeletal muscle contraction 73

could underlie the beneficial effects. Thus, we hypothesized that contraction in isolation is a sufficient 74

stimulus to protect muscle against lipid-induced insulin resistance by exploring the protective 75

phenomenon along the canonical insulin signaling pathway. We compare data from this in vitro muscle 76

contraction model with clinical observations, in order to contextualize the systemic and cell specific 77

responses to exercise. 78

79

EXPERIMENTAL PROCEDURES 80

81

Materials 82

All cell culture media (Dulbecco’s Modified Eagle’s Media, Phosphate Buffered Saline, and penicillin-83

streptomycin) were obtained from the Cleveland Clinic Lerner Research Institute Media Preparations 84

Core (Cleveland, OH, USA). Fetal Bovine Serum (FBS) and Horse Serum were obtained from Life 85

Technologies (Carlsbad, CA, USA). Palmitate, Sodium pyruvate, D-mannitol, D-(+)-glucose, 2-deoxy-D-86

glucose, gelatin (from porcine skin), HEPES, potassium chloride, calcium chloride, magnesium chloride, 87

magnesium sulfate, and PMSF were purchased from Sigma Aldrich (St. Louis, MO, USA). Dimethyl 88

sulfoxide, glycine, sodium chloride, Tween 20, Tris base, and ScintiSafe Econo2 scintillation cocktail were 89

purchased from Fischer Scientific (Pittsburgh, PA, USA). Bovine serum albumin, and PhoSTOP Easy Pack 90

5

were purchased from Roche (Indianapolis, IN, USA). Sodium dodecyl sulfate was purchased from Pierce 91

(Rockford, IL, USA). Cell Extraction Buffer was purchased from Life Technologies (Carlsbad, CA, USA). 92

93

Cell Culture 94

C2C12 myoblasts were purchased from the American Type Culture Collection (Manassas, VA) and 95

proliferated in high glucose (25 mM) DMEM supplemented with 10% FBS and 1% penicillin-streptomycin 96

(100 U/ml) placed in a water-jacketed incubator set at 37 °C and 5% CO2. Prior to 80% confluence, cells 97

at passages 4 through 7 were plated onto gelatin coated twelve-well plates. Gelatin coating was 98

achieved by incubating wells with 0.1% gelatin (w/v in ddH2O) for two hours at 37 °C followed by PBS 99

washing. Upon confluence, media was switched to differentiation media comprised of DMEM high 100

glucose supplemented with 2% horse serum and 1% penicillin-streptomycin. Differentiation media was 101

replenished daily by removing 75% percent of cell media followed by the addition of fresh media. Cells 102

were deemed ready for stimulation after the C2C12 myoblasts fully differentiated into contractile 103

myotubes (4 to 5 days). Cells were then treated as shown in Figure 1B according to the techniques 104

described below. 105

106

Electrical Pulse Stimulation 107

Fully differentiated C2C12 myotubes were induced to contract via Electrical Pulse Stimulation (EPS), as 108

represented schematically in Figure 1A. An electric field was generated by submerged platinum (99.95%) 109

wire electrodes to promote membrane depolarization and action potential propagation as an artificial 110

excitation-contraction-coupling stimulus. Electrical current was provided at 1 Hz, 6 millisecond pulse 111

width (0.6% duty cycle), and 1.5 V/mm with a Grass S88 Dual Output Square Pulse Stimulator (Warwick, 112

RI, USA) set at Stimulus Isolation Unit mode. An AC-coupling capacitor (250 μF) was included to remove 113

the direct current component to attenuate reduction/oxidation at the electrodes. A Double Pull Double 114

6

Throw (DPDT) mechanical relay switched the electrical field polarity at 0.5 Hz in order to negate 115

electrophoretic effects. Differentiation medium was replaced prior to the 16 hour EPS bout for both 116

stimulated and non-stimulated control wells to ensure that nutrient deprivation did not blunt the 117

responses. 118

119

Contractility 120

Relative contractility of C2C12 myotubes was quantified using a modified adaptation of optical flow 121

measurements with the FlowJ plugin in ImageJ (NIH, MD, USA). FlowJ is generally used to detect 122

movement in video for object recognition, producing an optical flow field (a vector map) that represents 123

individual pixel movement (1). Video recordings of EPS (1 Hz, 6 ms pulse width, 3.3 V/mm) contracting 124

myotubes were captured with an Olympus IX81 Inverted Microscope at 10 frames per second. The 125

optical flow field was calculated using the Lucas and Kanade algorithm (all parameters set to 1). Shown 126

in Figure 2 are representative optical flow fields for two adjacent myotubes over the course of a single 127

contraction (Fig. 2A), or at rest (Fig. 2B). The direction and magnitude of each pixel vector is indicated by 128

the color wheel insert in Figure 2A. Spot noise maps were then created for each optical flow field, 129

combining both magnitude and direction into a single pixel color value (Fig. 2C-D). A mean histogram 130

density of spot noise flow field maps for each myotube was calculated to quantify contractility (53). 131

Since hyperammonemia impairs skeletal muscle contractile function in vivo, and modifies contractile 132

proteins in cell culture in vitro (37), the impact of 10 mM ammonium acetate on contractile function in 133

C2C12 mytoubes was quantified and compared with vehicle treated control cells. Decreases in 134

contractility are represented by decreases in mean histogram density relative to that of control 135

contractions prior to the addition of a final concentration of 10 mM ammonium acetate (Sigma Aldrich). 136

Videos were taken every 60 seconds for 5 seconds to quantify the average contractility for each 137

7

myotube (2-4) over five contractions. Relative contractility may be represented as a relative (to control) 138

mean of histogram density or as a percentage of maximal density (at baseline). 139

140

Palmitic Acid Challenge 141

Palmitate was dissolved in ethanol (200 Proof, Pharmaco-Aaper, Brookfield, CT, USA) at a concentration 142

of 100 mM. On the day of the challenge, the palmitic acid stock was conjugated with Free Fatty Acid-143

free BSA (10% w/v in ddH2O) via incubation at 60°C for 10 minutes with constant mild agitation. The 144

palmiate:BSA molar ratio during conjugation was 10:3, providing BSA binding sites for long-chain free 145

fatty acids in excess (48). The conjugated solution was diluted to a final concentration of palmitic acid at 146

0.5 mM and BSA at 1% with Krebs-Ringer-Hepes buffer (20 mM HEPES, 136 mM sodium chloride, 4.7 147

mM potassium chloride, 1.25 mM magnesium sulfate, 1.25 mM calcium chloride) supplemented 32 mM 148

D-mannitol, and 8 mM D-(+)-glucose. EPS was stopped, electrodes removed, and media aspirated prior 149

to the addition of the palmitic acid containing media. Control wells received the same media (including 150

BSA at 1% w/v) without palmitic acid. Cells were incubated for a total challenge time of 4 hours. Once 151

this treatment step was complete, media was carefully aspirated prior to the addition of insulin or non-152

insulin control media prior to final analysis. 153

154

Glucose Uptake 155

Insulin responsiveness was measured by an insulin stimulated glucose uptake assay adapted from Gao et 156

al. (21), whose methods ensured robust insulin stimulated glucose uptake. After EPS and palmitic acid 157

incubation, including control conditions, cells were stimulated with 1.0 µM insulin (Novolin R Regular 158

Human Insulin - Recombinant DNA Origin, Novo Nordisk, Plainsboro, NJ, USA) in KRH supplemented with 159

2 mM sodium pyruvate, 32 mM D-mannitol, and 0.1 % BSA for 30 minutes. Mannitol served as an 160

osmolyte, while sodium pyruvate served as a metabolic substrate during glucose-free incubation. Insulin 161

8

stimulation media was then aspirated and cells incubated for 10 minutes in KRH supplemented with 1 162

uCi/mL [3H]-2-deoxy-D-glucose (Perkin Elmer, Waltham, MA, USA) and 2 mM cold 2-deoxy-D-glucose. 163

Uptake of radiolabeled glucose was terminated by aspiration and rapid washing three times with ice 164

cold PBS. Cells were then lysed with 0.1 % SDS in ddH2O on a plate shaker for 30 minutes. Lysate was 165

used for both BCA protein quantification as an internal control and for scintillation counting following 166

the addition of ScintiSafe scinitillation cocktail. Glucose uptake rate was calculated as a percentage 167

increase over control uptake (without EPS, palmitic acid, or insulin treatment). Results from three 168

independent experiments, each in triplicate, were normalized to control non-insulin and insulin 169

stimulated glucose before compiling datasets. 170

171

Lipid Kinase Assay 172

Phosphoinositide-3-kinase-α (PI3Kα) activity was determined as previously described (39, 40). Briefly, 173

following EPS and palmitic acid challenge, cells were incubated with or without 1.0 µM insulin for 5 174

minutes. Insulin stimulation was stopped by aspiration and rapidly washed once with ice cold PBS. 175

Washed cells were placed on dry ice for immediate freezing. Plates were scraped and experimental 176

triplicates combined for a minimum of 300 µg, of which 150 µg was used for immunoprecipitation with 177

IRS-1 (Cell Signaling Technologies, Danvers, MA, USA) or pan-PI3Kα-P110 (Santa Cruz, Dallas, TX, USA) 178

antibodies. The same antibodies were also used for Western blotting. Lysates were then incubated with 179

phosphatidylinositol (PI) (Sigma Aldrich) and radiolabeled ATP [γ-32P] (Perkin Elmer) with continuous 180

agitation. Samples were then blotted onto thin layer chromatography plates for separation. Subsequent 181

radiographs were quantified by fixed area density calculations using ImageJ. 182

183

Western Blotting 184

9

Following treatment with or without EPS and palmitate, myotubes were incubated with or without 1.0 185

µM insulin for 10 minutes. Cells were then washed twice with ice cold PBS before the addition of a cell 186

extraction cocktail (5% Protease Inhibitor, 5% Phostop, 1% Sodium Orthovanadate, 0.35% PMSF, in Cell 187

Extraction Buffer). Plates were then stored at -80°C until ready for scraping and cell lysate preparation. 188

Collected crude cell lysates were spun down at 10,000 xg for 10 minutes to pellet any insoluble matter. 189

Supernatant was collected and quantified for total protein content (Pierce BCA Protein Assay Kit, 190

Thermo Scientific). Protein (25µg) lysate for each condition was prepared with Laemmli Buffer 191

containing β-mercaptoethanol, heated to 95°C for 30 minutes, and loaded onto a precast 4-12% Tris-192

glycine gel (Novex WedgeWell, Life Technologies). Gels were run until completion at fixed voltage (125V) 193

before setup for transfer onto PVDF membranes (Bio-Rad, Hercules, CA, USA) for 90 minutes at fixed 194

voltage (60V, Criterion Blotter, Bio-Rad). Membranes were blocked with 5% BSA in PBS-T (PBS, 0.15% 195

Tween-20), for at least 30 minutes before overnight incubation of primary antibodies (all at 1:1000 in 196

blocking buffer): Glut4 (G4048, Sigma Aldrich), phospho-Akt-Thr308 (9275S, CST), total Akt (9272S, CST), 197

total IRS-1 (2390S, CST), PI3Kα-P110 (sc-7189, Santa Cruz), and HSC70 (sc-7298, Santa Cruz) as an 198

internal loading control. Mouse and rat ECL secondary antibodies tagged to HRP (GE Healthcare, 199

Chicago, Illinois, USA) were used (2 hour secondary incubation), and chemiluminescent detection was 200

performed on a LAS 500 imager (GE Healthcare). Blots were quantified using ImageJ. 201

202

Statistical Analysis 203

All analyses were performed in GraphPad Prism 5 (La Jolla, CA, USA). Data were tested for normality 204

using the Kolmogorov-Smirnov test. For normally distributed data, a Student’s t-test was used to 205

determine statistically significant differences between treatments. For non-normally distributed data a 206

Mann-Whitney test was used instead. A Δ% between mean insulin and non-insulin stimulated glucose 207

uptake was calculated for the four treatment combinations (control, EPS, PA, EPS+PA) to represent 208

10

insulin specific action on glucose uptake (see Figure 3B). Standard deviations were determined from the 209

variance of the difference of the means as calculated using the Central Limit Theorem. Data from 210

independent experiments were normalized to the internal (non-insulin stimulated) control within each 211

experiment. The accepted P-value for significance was set at less than 0.05. All data are presented as 212

means and standard deviations, with replicate numbers indicated in figure legends. 213

214

215

RESULTS 216

217

Contractility 218

A reliable model of exercise will most obviously produce contracting muscle cells, but more importantly 219

respond to contraction stimuli in a predictable manner. Supplemental Video 1 shows that EPS induced 220

contractions are directly dependent on stimulus frequency, with higher frequencies (>4 Hz) almost 221

inducing a tetanic state. Optical flow field spot noise mapping (Fig. 2A-D) calculations show that EPS 222

readily increases myotube contraction (Fig. 2E). The addition of 10 mM ammonium acetate instantly 223

inhibited contraction, despite continued EPS (Supp. Video 2). Within one minute, contractility is reduced 224

significantly (99.0 ± 3.5% vs. 64.0 ± 10.6%, P=0.008) (Fig. 2F). Mean contractility of ammonium acetate 225

treated myotubes remained significantly lower than control contractions throughout the 5-minute 226

observation period. 227

228

Glucose Uptake 229

Radiolabeled glucose uptake by C2C12 myotubes is presented here as a percentage increase over 230

control without insulin stimulation (Fig. 3A). In the absence of insulin, EPS significantly (P=0.002 vs. 231

control) increased the glucose uptake rate by 83%. Palmitate treatment alone acutely increased glucose 232

11

uptake (15%, P=0.01), to a much lower extent than EPS. EPS treatment followed by palmitate challenge 233

and EPS alone had similar glucose uptake rates in the absence of insulin (P=0.58) and was still 234

significantly greater than non-insulin stimulated control uptake rate (68%, P=0.007). Insulin stimulation, 235

as expected, resulted in an increase in glucose uptake (75%, P<0.001), with an effect size similar to that 236

of EPS alone (P=0.70) (Fig. 3A). Prior EPS stimulation, followed by a 4 hour “rest” period, produced a 237

higher insulin stimulated glucose uptake rate than insulin stimulation alone (138%, P=0.003). Prior 238

palmitate treatment reduced the insulin stimulated glucose uptake rate (54%, P=0.03, Fig. 3A), with an 239

effective reduction in insulin specific action on glucose uptake of 49% (75% vs. 39%, P<0.001, Fig. 3B). 240

EPS was able to rescue the inhibitory effect of palmitate on insulin stimulated glucose uptake and insulin 241

responsiveness (Fig. 3A and 3B). Total Glut4 protein expression remained unchanged (Fig. 3C and D). 242

243

Canonical Insulin Signaling Pathway 244

A key downstream signaling mediator of insulin stimulated glucose uptake is the serine and threonine 245

kinase Akt/PKB. This kinase is phosphorylated at the threonine-308 residue in response to insulin 246

stimulation (3). Insulin stimulation of muscle cells results in phosphorylation of Akt due to recruitment 247

of phosphoinositide-dependent kinase-1 (PDK1) and Akt to the plasma membrane through generation of 248

phosphatidylinositol (3,4,5)-trisphosphate (PIP3) due to activation of PI3Kα. PI3Kα is recruited to 249

activated insulin receptors through insulin receptor substrates, wherein PI3Kα converts 250

phosphatidylinositol 4,5-bisphosphate (PIP2) to PIP3, leading to subsequent recruitment of downstream 251

molecules (3). 252

253

EPS increased Akt phosphorylation in the absence of insulin (P=0.005), which was unaffected by 254

palmitate (Fig. 4B). As expected, insulin stimulation increased Akt phosphorylation by ten-fold (Fig. 4C). 255

Prior EPS stimulation did not affect this insulin-stimulated increase in phosphorylation. As with the 256

12

glucose uptake rate, palmitate significantly reduced insulin-stimulated phosphorylation of Akt to only 257

five-fold of non-insulin stimulated levels (P=0.048). This inhibitory effect was fully rescued by prior EPS 258

stimulation (Fig. 4C). Total protein expression of Akt was decreased by EPS stimulation, with a significant 259

reduction in total expression in the combination of EPS and palmitate treatment (P=0.013, Fig. 4D). 260

PI3Kα catalytic subunit expression was unaltered by either EPS or palmitate treatment (Fig. 4E). IRS-1 261

protein expression, however, was significantly reduced by about 50% following EPS treatment in the 262

presence (P=0.037) or absence (P=0.024) of the palmitate challenge (Fig. 4F). 263

264

We measured the activity of IRS-1 associated PI3K and PI3Kα, to determine the specificity of PI3Kα 265

activation in all treatment conditions (see Fig. 1B). IRS-1 associated PI3K activity was increased in 266

response to EPS treatment (P=0.048, Fig. 5C) similar to non-insulin stimulated Akt phosphorylation (Fig. 267

4B). While, insulin stimulated IRS-1 associated PI3K activity was not altered by EPS (Fig. 5E). However, 268

IRS-1 associated PI3K activity showed a slight but non-significant reduction with palmitate challenge (Fig. 269

5E). Interestingly, PI3Kα activity was not altered with either EPS or palmitate treatments in the absence 270

of insulin stimulation (Fig. 5D). Significantly, EPS protected against palmitate-induced reductions in 271

PI3Kα activity, despite the reduction in enzyme activity (Fig. 5F). These data match the insulin 272

responsiveness data shown in Figure 3B. 273

274

DISCUSSION 275

276

The objective of this study was to develop an in vitro contraction model that allows for elucidation of 277

the protective effects of exercise against lipid-induced insulin resistance. By taking a reductionist 278

approach, we were able to abstract the effects of contraction on muscle insulin signaling, independent 279

of the systemic effects of exercise. We demonstrate that the protective effect of exercise on peripheral 280

13

insulin sensitivity may be explained by muscle contraction alone. The protection is evident at the 281

proximal level of the insulin-signaling pathway, which is inhibited under insulin resistant states in type 2 282

diabetes (5). 283

284

Much of the difficulty in developing in vitro models lies in the optimization of cell culture methods. We 285

reviewed the literature of successfully implemented C2C12 contraction models (2, 4, 14, 18-20, 34, 35, 286

41, 43, 50, 55, 57) and tested a variety of approaches. Pre-coating plates with gelatin, growth until 287

confluence, and daily partial changes in media resulted in full differentiation of C2C12 myocytes into 288

contractile myotubes within 5 days. These specific technical details were absent from previously 289

reported methods. Visually confirmed contraction was immediately evident upon EPS, without the need 290

for an acclimation period to align sarcomeres, as reported by others (18-20, 41). To confirm and quantify 291

myotube contractility, we used a novel application of a video analysis algorithm (Fig. 2A-E) (53). With 292

this technique, we are able to confirm the inhibitory effect of ammonium acetate on muscle 293

contraction, further demonstrating the ability of our model to replicate in vivo contractile dysfunction in 294

response to a specific intervention (Fig. 2F) (37). Traditionally, contractility measures are made via cell 295

shortening microscopy, force transducers or other engineered devices (22). This application of the 296

model with optical flow field mapping provides an accurate and higher throughput approach to study 297

the effects of various stimuli on contractility in vitro, which can be applied to simple bright field 298

microscopy videos. 299

300

We used a total of 16 hours of EPS in this model. An exercise bout of this length is not common in 301

humans or rodents. However, this duration, produced a persistent effect on potentiating glucose uptake 302

(Fig. 3A) and interleukin-6 secretion (data not shown), and are consistent with effects previously 303

observed after physical training (25). IRS-1 protein expression data gives an indication as to what type of 304

14

exercise this model mimics. Chibalin et al. have shown that 6 hours of daily swimming in rats reduces 305

muscle expression of IRS-1 only after 5 days (10), suggesting that our model is equivalent to several 306

consecutive days of aerobic training (Fig. 4F). IRS-1 associated PI3K activity as well as Akt 307

phosphorylation were also elevated following the 5 days of training in rats, consistent with our 308

observations (Fig. 4B and Fig. 5C). However, in contrast to their study, marked reduction in total Akt 309

protein expression was observed following EPS (Fig. 4D), suggesting a negative-feedback 310

downregulation as described by Chibalin et al. for the reduced IRS-1 expression (10). Despite protecting 311

against insulin resistance, EPS reduced expression of key elements in the canonical insulin-signaling 312

pathway, indicating a role for alternative non-canonical signaling pathways. Previous studies have 313

suggested that the insulin-receptor substrate-2 (IRS-2) may mediate the alternative pathways in 314

downstream signals (8, 10, 28, 29). Furthermore, insulin-stimulated PI3Kα activity in our model (Fig. 5F) 315

supports the idea that mechanisms independent of insulin receptor substrate may play a role in the 316

protective effect of contraction against palmitate-induced insulin resistance. 317

318

Shorter, 20 minute, palmitate incubations of L6 and C2C12 myotubes increases glucose uptake, in an 319

Akt/PKB and ERK1/2 dependent manner (44). Here we show that this acute effect of palmitate persists 320

for up to 4 hours (Fig. 3A). Palmitate does not appear to alter EPS induced increases in glucose uptake, 321

Akt phosphorylation, or PI3K activity in the absence of insulin. In the presence of insulin, the inhibitory 322

effects of palmitate are seen in our glucose uptake and Akt phosphorylation data, consistent with many 323

other observations (16, 49, 51, 52). We also show that EPS drives an increase in Akt phosphorylation 324

(Fig. 4B). Camera et al. reported that both endurance and resistance exercise can increase muscle Akt 325

phosphorylation in the absence of insulin stimulation for up to 60 minutes after exercise (8). Our data 326

here suggests that increased Akt phosphorylation is likely driven by increased IRS-1 associated PI3K 327

activity under non-insulin stimulated conditions (Fig. 5C), which persists in vitro for up to 4 hours (Fig. 328

15

4A). The effect of the combination of EPS and insulin stimuli on glucose uptake was not fully additive 329

(Fig. 3A-B), which may be due to a saturation in uptake rate with the dual stimuli. Alternatively, this 330

finding supports the notion of shared pools of recruitable glucose transporters (12). 331

332

Even one week of daily exercise results in improved peripheral insulin sensitivity in man (33). We have 333

also previously shown that individuals engaged in regular exercise training have greater insulin 334

stimulated activation of IRS-1 associated PI3K activity (31), which is diminished in muscle of T2D patients 335

(30). This in vitro model does not display a characteristic increase in insulin responsiveness at the levels 336

of glucose uptake (Fig. 3B) or IRS-1 associated PI3K activity (Fig. 5E) (26). Gao et al. have demonstrated 337

that a serum factor is necessary to produce contraction-induced increases in muscle insulin sensitivity 338

(21). Although a low concentration of horse serum is present during the EPS bout in our model, 339

subsequent treatments were performed in serum free medium to avoid the confounding effects of 340

additional fatty acids in serum. As a result, the necessary insulin sensitizing factor would not be present 341

during palmitate challenge or insulin stimulation. Furthermore, the horse serum and host serum (mouse 342

in this model) may be different in regards to its role in modulating insulin signaling. This observation, like 343

other aforementioned inconsistencies, suggest different systemic versus cell specific effects of exercise 344

that can only be identified through abstraction. 345

346

In this report, we have only used the model to examine the proximal insulin-signaling pathway. Our 347

clinical findings suggested that increased whole body lipid kinetics and skeletal muscle free fatty acid 348

oxidation play an important role in the reduction in lipid-induced insulin resistance after exercise 349

training (47). Schenk and Horowitz observed increased triglyceride synthesis, reduced ceramides, and 350

reduced pro-inflammatory signaling in human skeletal muscle, when protected from lipid-induced 351

insulin resistance by a single exercise bout (27, 46). Thrush et al. also observed the same increases in 352

16

lipid kinetics and fatty acid oxidation in their rat exercise model. Through ex vivo palmitate incubations 353

of excised muscle following treadmill running in rats, Thrush et al. also showed that in response to an 354

insulin stimulus, the protective effect was evident at the levels of skeletal muscle glucose uptake and 355

Akt phosphorylation (51). Within the proximal insulin-signaling pathway, these in vivo findings are 356

consistent with what we have observed here in vitro. Future applications of this model are ideally suited 357

to investigate lipid metabolism, as radioisotopic tracer methods, toxic inhibitors, and siRNA are readily 358

applied in cell culture. However, as with any model, there are limitations to in vitro interpretations as 359

they relate to the systemic effects of exercise. 360

361

We conclude that contraction alone may protect muscle from lipid-induced insulin resistance, evident at 362

the levels of glucose uptake and proximal insulin signaling. Our data support the presence of a non-363

canonical PI3Kα activating pathway, that could potentially be independent of insulin receptor substrate, 364

mediating the protective role of exercise. The robustness and validity of this “exercise in a petri dish” 365

system has been thoroughly tested here, and may be used as a high-throughput tool to investigate 366

exercise metabolism. 367

368

17

GRANTS 369

This study was funded by investigator initiated grants from the Metabolic Translational Research Center 370

(MTRC 13-1563, Cleveland Clinic, Cleveland, Ohio, USA) and the NIH (R21 AR067477). 371

372

18

REFERENCES 373

1. Abramoff MD, Niessen WJ, and Viergever MA. Objective quantification of the motion of soft 374 tissues in the orbit. IEEE Trans Med Imaging 19: 986-995, 2000. 375 2. Akiyama Y, Furukawa Y, and Morishima K. Controllable bio-microactuator powered by muscle 376 cells. Conf Proc IEEE Eng Med Biol Soc Suppl: 6565-6568, 2006. 377 3. Andjelkovic M, Alessi DR, Meier R, Fernandez A, Lamb NJ, Frech M, Cron P, Cohen P, Lucocq 378 JM, and Hemmings BA. Role of translocation in the activation and function of protein kinase B. J Biol 379 Chem 272: 31515-31524, 1997. 380 4. Asano T, Ishizua T, and Yawo H. Optically controlled contraction of photosensitive skeletal 381 muscle cells. Biotechnol Bioeng 109: 199-204, 2012. 382 5. Bjornholm M, Kawano Y, Lehtihet M, and Zierath JR. Insulin receptor substrate-1 383 phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle from NIDDM subjects after 384 in vivo insulin stimulation. Diabetes 46: 524-527, 1997. 385 6. Brevet A, Pinto E, Peacock J, and Stockdale FE. Myosin synthesis increased by electrical 386 stimulation of skeletal muscle cell cultures. Science 193: 1152-1154, 1976. 387 7. Burch N, Arnold AS, Item F, Summermatter S, Brochmann Santana Santos G, Christe M, 388 Boutellier U, Toigo M, and Handschin C. Electric pulse stimulation of cultured murine muscle cells 389 reproduces gene expression changes of trained mouse muscle. PLoS One 5: e10970, 2010. 390 8. Camera DM, Edge J, Short MJ, Hawley JA, and Coffey VG. Early time course of Akt 391 phosphorylation after endurance and resistance exercise. Med Sci Sports Exerc 42: 1843-1852, 2010. 392 9. Chahine KG, Baracchini E, and Goldman D. Coupling muscle electrical activity to gene 393 expression via a cAMP-dependent second messenger system. J Biol Chem 268: 2893-2898, 1993. 394 10. Chibalin AV, Yu M, Ryder JW, Song XM, Galuska D, Krook A, Wallberg-Henriksson H, and 395 Zierath JR. Exercise-induced changes in expression and activity of proteins involved in insulin signal 396 transduction in skeletal muscle: differential effects on insulin-receptor substrates 1 and 2. Proc Natl 397 Acad Sci U S A 97: 38-43, 2000. 398 11. Ciaraldi TP, Abrams L, Nikoulina S, Mudaliar S, and Henry RR. Glucose transport in cultured 399 human skeletal muscle cells. Regulation by insulin and glucose in nondiabetic and non-insulin-400 dependent diabetes mellitus subjects. J Clin Invest 96: 2820-2827, 1995. 401 12. DeFronzo RA, and Tripathy D. Skeletal muscle insulin resistance is the primary defect in type 2 402 diabetes. Diabetes Care 32 Suppl 2: S157-163, 2009. 403 13. del Aguila LF, Claffey KP, and Kirwan JP. TNF-alpha impairs insulin signaling and insulin 404 stimulation of glucose uptake in C2C12 muscle cells. Am J Physiol 276: E849-855, 1999. 405 14. Dennis RG, and Kosnik PE, 2nd. Excitability and isometric contractile properties of mammalian 406 skeletal muscle constructs engineered in vitro. In Vitro Cell Dev Biol Anim 36: 327-335, 2000. 407 15. Evers-van Gogh IJ, Alex S, Stienstra R, Brenkman AB, Kersten S, and Kalkhoven E. Electric Pulse 408 Stimulation of Myotubes as an In Vitro Exercise Model: Cell-Mediated and Non-Cell-Mediated Effects. Sci 409 Rep 5: 10944, 2015. 410 16. Feng XT, Wang TZ, Leng J, Chen Y, Liu JB, Liu Y, and Wang WJ. Palmitate contributes to insulin 411 resistance through downregulation of the Src-mediated phosphorylation of Akt in C2C12 myotubes. 412 Biosci Biotechnol Biochem 76: 1356-1361, 2012. 413 17. Freud-Silverberg M, and Shainberg A. Electric stimulation regulates the level of Ca-channels in 414 chick muscle culture. Neurosci Lett 151: 104-106, 1993. 415 18. Fujita H, Endo A, Shimizu K, and Nagamori E. Evaluation of serum-free differentiation 416 conditions for C2C12 myoblast cells assessed as to active tension generation capability. Biotechnol 417 Bioeng 107: 894-901, 2010. 418

19

19. Fujita H, Nedachi T, and Kanzaki M. Accelerated de novo sarcomere assembly by electric pulse 419 stimulation in C2C12 myotubes. Exp Cell Res 313: 1853-1865, 2007. 420 20. Fujita H, Shimizu K, and Nagamori E. Novel method for measuring active tension generation by 421 C2C12 myotube using UV-crosslinked collagen film. Biotechnol Bioeng 106: 482-489, 2010. 422 21. Gao J, Gulve EA, and Holloszy JO. Contraction-induced increase in muscle insulin sensitivity: 423 requirement for a serum factor. Am J Physiol 266: E186-192, 1994. 424 22. Grosberg A, Nesmith AP, Goss JA, Brigham MD, McCain ML, and Parker KK. Muscle on a chip: 425 in vitro contractility assays for smooth and striated muscle. J Pharmacol Toxicol Methods 65: 126-135, 426 2012. 427 23. Haghani K, Pashaei S, Vakili S, Taheripak G, and Bakhtiyari S. TNF-alpha knockdown alleviates 428 palmitate-induced insulin resistance in C2C12 skeletal muscle cells. Biochem Biophys Res Commun 460: 429 977-982, 2015. 430 24. Haus JM, Solomon TP, Marchetti CM, Edmison JM, Gonzalez F, and Kirwan JP. Free fatty acid-431 induced hepatic insulin resistance is attenuated following lifestyle intervention in obese individuals with 432 impaired glucose tolerance. J Clin Endocrinol Metab 95: 323-327, 2010. 433 25. Helge JW, Stallknecht B, Pedersen BK, Galbo H, Kiens B, and Richter EA. The effect of graded 434 exercise on IL-6 release and glucose uptake in human skeletal muscle. J Physiol 546: 299-305, 2003. 435 26. Holloszy JO. Exercise-induced increase in muscle insulin sensitivity. J Appl Physiol (1985) 99: 338-436 343, 2005. 437 27. Horowitz JF. Exercise-induced alterations in muscle lipid metabolism improve insulin sensitivity. 438 Exerc Sport Sci Rev 35: 192-196, 2007. 439 28. Howlett KF, Sakamoto K, Hirshman MF, Aschenbach WG, Dow M, White MF, and Goodyear LJ. 440 Insulin signaling after exercise in insulin receptor substrate-2-deficient mice. Diabetes 51: 479-483, 441 2002. 442 29. Howlett KF, Sakamoto K, Yu H, Goodyear LJ, and Hargreaves M. Insulin-stimulated insulin 443 receptor substrate-2-associated phosphatidylinositol 3-kinase activity is enhanced in human skeletal 444 muscle after exercise. Metabolism 55: 1046-1052, 2006. 445 30. Kim YB, Nikoulina SE, Ciaraldi TP, Henry RR, and Kahn BB. Normal insulin-dependent activation 446 of Akt/protein kinase B, with diminished activation of phosphoinositide 3-kinase, in muscle in type 2 447 diabetes. J Clin Invest 104: 733-741, 1999. 448 31. Kirwan JP, del Aguila LF, Hernandez JM, Williamson DL, O'Gorman DJ, Lewis R, and Krishnan 449 RK. Regular exercise enhances insulin activation of IRS-1-associated PI3-kinase in human skeletal muscle. 450 J Appl Physiol (1985) 88: 797-803, 2000. 451 32. Kirwan JP, Sacks J, and Nieuwoudt S. The Essential Role of Exercise in the Management of Type 452 2 Diabetes. Cleveland Clinic Journal of Medicine 2017 (in press). 453 33. Kirwan JP, Solomon TP, Wojta DM, Staten MA, and Holloszy JO. Effects of 7 days of exercise 454 training on insulin sensitivity and responsiveness in type 2 diabetes mellitus. Am J Physiol Endocrinol 455 Metab 297: E151-156, 2009. 456 34. Manabe Y, Miyatake S, Takagi M, Nakamura M, Okeda A, Nakano T, Hirshman MF, Goodyear 457 LJ, and Fujii NL. Characterization of an acute muscle contraction model using cultured C2C12 myotubes. 458 PLoS One 7: e52592, 2012. 459 35. Marotta M, Bragos R, and Gomez-Foix AM. Design and performance of an electrical stimulator 460 for long-term contraction of cultured muscle cells. Biotechniques 36: 68-73, 2004. 461 36. Matthews VB, Astrom MB, Chan MH, Bruce CR, Krabbe KS, Prelovsek O, Akerstrom T, Yfanti C, 462 Broholm C, Mortensen OH, Penkowa M, Hojman P, Zankari A, Watt MJ, Bruunsgaard H, Pedersen BK, 463 and Febbraio MA. Brain-derived neurotrophic factor is produced by skeletal muscle cells in response to 464 contraction and enhances fat oxidation via activation of AMP-activated protein kinase. Diabetologia 52: 465 1409-1418, 2009. 466

20

37. McDaniel J, Davuluri G, Hill EA, Moyer M, Runkana A, Prayson R, van Lunteren E, and 467 Dasarathy S. Hyperammonemia results in reduced muscle function independent of muscle mass. Am J 468 Physiol Gastrointest Liver Physiol 310: G163-170, 2016. 469 38. Miyatake S, Bilan PJ, Pillon NJ, and Klip A. Contracting C2C12 myotubes release CCL2 in an NF-470 kappaB-dependent manner to induce monocyte chemoattraction. Am J Physiol Endocrinol Metab 310: 471 E160-170, 2016. 472 39. Naga Prasad SV, Barak LS, Rapacciuolo A, Caron MG, and Rockman HA. Agonist-dependent 473 recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1. A role 474 in receptor sequestration. J Biol Chem 276: 18953-18959, 2001. 475 40. Naga Prasad SV, Laporte SA, Chamberlain D, Caron MG, Barak L, and Rockman HA. 476 Phosphoinositide 3-kinase regulates beta2-adrenergic receptor endocytosis by AP-2 recruitment to the 477 receptor/beta-arrestin complex. J Cell Biol 158: 563-575, 2002. 478 41. Nedachi T, Fujita H, and Kanzaki M. Contractile C2C12 myotube model for studying exercise-479 inducible responses in skeletal muscle. Am J Physiol Endocrinol Metab 295: E1191-1204, 2008. 480 42. Nedachi T, Hatakeyama H, Kono T, Sato M, and Kanzaki M. Characterization of contraction-481 inducible CXC chemokines and their roles in C2C12 myocytes. Am J Physiol Endocrinol Metab 297: E866-482 878, 2009. 483 43. Pan H, Xu X, Hao X, and Chen Y. Changes of myogenic reactive oxygen species and interleukin-6 484 in contracting skeletal muscle cells. Oxid Med Cell Longev 2012: 145418, 2012. 485 44. Pu J, Peng G, Li L, Na H, Liu Y, and Liu P. Palmitic acid acutely stimulates glucose uptake via 486 activation of Akt and ERK1/2 in skeletal muscle cells. J Lipid Res 52: 1319-1327, 2011. 487 45. Scheler M, Irmler M, Lehr S, Hartwig S, Staiger H, Al-Hasani H, Beckers J, de Angelis MH, Haring 488 HU, and Weigert C. Cytokine response of primary human myotubes in an in vitro exercise model. Am J 489 Physiol Cell Physiol 305: C877-886, 2013. 490 46. Schenk S, and Horowitz JF. Acute exercise increases triglyceride synthesis in skeletal muscle and 491 prevents fatty acid-induced insulin resistance. J Clin Invest 117: 1690-1698, 2007. 492 47. Solomon TP, Haus JM, Marchetti CM, Stanley WC, and Kirwan JP. Effects of exercise training 493 and diet on lipid kinetics during free fatty acid-induced insulin resistance in older obese humans with 494 impaired glucose tolerance. Am J Physiol Endocrinol Metab 297: E552-559, 2009. 495 48. Spector AA, John K, and Fletcher JE. Binding of long-chain fatty acids to bovine serum albumin. J 496 Lipid Res 10: 56-67, 1969. 497 49. Storz P, Doppler H, Wernig A, Pfizenmaier K, and Muller G. Cross-talk mechanisms in the 498 development of insulin resistance of skeletal muscle cells palmitate rather than tumour necrosis factor 499 inhibits insulin-dependent protein kinase B (PKB)/Akt stimulation and glucose uptake. Eur J Biochem 500 266: 17-25, 1999. 501 50. Thelen MH, Simonides WS, and van Hardeveld C. Electrical stimulation of C2C12 myotubes 502 induces contractions and represses thyroid-hormone-dependent transcription of the fast-type 503 sarcoplasmic-reticulum Ca2+-ATPase gene. Biochem J 321 ( Pt 3): 845-848, 1997. 504 51. Thrush AB, Harasim E, Chabowski A, Gulli R, Stefanyk L, and Dyck DJ. A single prior bout of 505 exercise protects against palmitate-induced insulin resistance despite an increase in total ceramide 506 content. Am J Physiol Regul Integr Comp Physiol 300: R1200-1208, 2011. 507 52. Thrush AB, Heigenhauser GJ, Mullen KL, Wright DC, and Dyck DJ. Palmitate acutely induces 508 insulin resistance in isolated muscle from obese but not lean humans. Am J Physiol Regul Integr Comp 509 Physiol 294: R1205-1212, 2008. 510 53. Vanwijk JJ. Spot Noise - Texture Synthesis for Data Visualization. Comp Graph 25: 309-318, 511 1991. 512 54. Varma V, Yao-Borengasser A, Rasouli N, Nolen GT, Phanavanh B, Starks T, Gurley C, Simpson P, 513 McGehee RE, Jr., Kern PA, and Peterson CA. Muscle inflammatory response and insulin resistance: 514

21

synergistic interaction between macrophages and fatty acids leads to impaired insulin action. Am J 515 Physiol Endocrinol Metab 296: E1300-1310, 2009. 516 55. Whitham M, Chan MH, Pal M, Matthews VB, Prelovsek O, Lunke S, El-Osta A, Broenneke H, 517 Alber J, Bruning JC, Wunderlich FT, Lancaster GI, and Febbraio MA. Contraction-induced interleukin-6 518 gene transcription in skeletal muscle is regulated by c-Jun terminal kinase/activator protein-1. J Biol 519 Chem 287: 10771-10779, 2012. 520 56. Yaffe D, and Saxel O. Serial passaging and differentiation of myogenic cells isolated from 521 dystrophic mouse muscle. Nature 270: 725-727, 1977. 522 57. Yamasaki K, Hayashi H, Nishiyama K, Kobayashi H, Uto S, Kondo H, Hashimoto S, and Fujisato 523 T. Control of myotube contraction using electrical pulse stimulation for bio-actuator. J Artif Organs 12: 524 131-137, 2009. 525

526

Figure 1. (A) Schematic of EPS system for inducing C2C12 myotube contraction. (B) Experimental designfor 4 different conditions (Control, EPS, PA, EPS PA) stimulated with or without insulin for 5, 10, or 30minutes for the indicated experimental outcomes.

A B

Figure 2. Quantification of reductions in in vitro C2C12 myotube contractility with ammonium acetate.(A) Directional 2D optical flow map of pixel movement from a single contraction over 10 frames with nocontraction control (B). Direction of and intensity of pixel movement is represented by the color wheelinsert(C). Spot noise map depicting total pixel movement for each point over 10 frames with a singlecontraction and no contraction control (D). (E) Average spotnoise histogram density for each myotube(n=4) relative to no contraction control. (F) Average spot noise histogram density (n=10) of controlmyotubes (circle) or 10mM ammonium acetate incubated myotubes (square). (*) P <0.01.

A B C D

E F

Figure 3. Glucose uptake rate and glucose transporter expression. (A) Relative glucose uptake rate ofcontrol, EPS, palmitate (PA), and combined stimulus (EPS+PA), under non insulin and insulin stimulatedconditions (n=3). (B) Delta % change in glucose uptake rate between non insulin and insulin stimulatedconditions (shown in panel A), representing insulin specific action on the background of non insulinmediated changes in glucose uptake. Representative Western blots (C) for Glut4 and (D) quantification(n=12) of total protein expression (both bands). (*) P < 0.05.

A

BC

D

Figure 4. Protein expression and phosphorylation for Akt (n=11), PI3K (n=9), and IRS 1 (n=9). (A)Representative Western blots. (B) Non insulin and (C) insulin stimulated phosphorylated Akt (Thr308)expression corrected to (D) total Akt protein (both corrected to HSC70 expression). Total expression of(E) PI3K and (F) IRS 1. (*) P < 0.05.

A B C

D E F

Figure 5. PI3K catalytic activity. Representative radiographs (A and B), arranged to match bar graphlayouts. Non insulin stimulated relative PI3K activity for (C) IRS 1 IP (n=6) and (D) pan PI3K IP (n=6) asquantified from PIP3 radiographs. Insulin stimulated relative PI3K activity for (E) IRS 1 IP (n=5) and (F)pan PI3K IP (n=6). (*) P < 0.05.

A B

C D

E F