Draft · 2018. 8. 23. · Draft 1 ASSESSING THE MECHANISTIC TARGET OF RAPAMYCIN COMPLEX-1 PATHWAY IN RESPONSE TO RESISTANCE EXERCISE AND FEEDING IN HUMAN SKELETAL MUSCLE by MULTIPLEX

Draft

ASSESSING THE MECHANISTIC TARGET OF RAPAMYCIN

COMPLEX-1 PATHWAY IN RESPONSE TO RESISTANCE

EXERCISE AND FEEDING IN HUMAN SKELETAL MUSCLE by

MULTIPLEX ASSAY

Journal: Applied Physiology, Nutrition, and Metabolism

Manuscript ID apnm-2017-0852.R1

Manuscript Type: Article

Date Submitted by the Author: 20-Feb-2018

Complete List of Authors: McGlory, Chris ; McMaster University, Kinesiology Nunes, Everson; Federal University of Santa Catarina, Physiological Sciences Oikawa, Sara; McMaster University, Exercise Metabolism Research Group, Department of Kinesiology Tsakiridis, Evangelia; McMaster University Department of Kinesiology, Exercise Metabolism Research Group, Department of Kinesiology Phillips, Stuart; McMaster University,

Keyword: Resistance exercise, protein, anabolic signalling, multiplex technology

Is the invited manuscript for consideration in a Special

Issue? : N/A

https://mc06.manuscriptcentral.com/apnm-pubs

Applied Physiology, Nutrition, and Metabolism

Draft

1

ASSESSING THE MECHANISTIC TARGET OF RAPAMYCIN COMPLEX-1

PATHWAY IN RESPONSE TO RESISTANCE EXERCISE AND FEEDING IN HUMAN

SKELETAL MUSCLE by MULTIPLEX ASSAY

Chris McGlory1, Everson A. Nunes

2, Sara Y. Oikawa

1, Evangelia Tsakiridis

1, and Stuart M.

Phillips1*

.

1Department of Kinesiology, McMaster University, Hamilton, ON, Canada

2Department of Physiological Sciences, Federal University of Santa Catarina, Florianópolis, SC,

Brazil.

Corresponding Author:

Prof. Stuart M. Phillips, Ph.D.

Department of Kinesiology, McMaster University

1280 Main Street West

Hamilton, ON L8S 4K1 CANADA

Telephone: +1 905 525 9140 (ext. 24465)

Email: [email protected]

Page 1 of 19



Draft

2

ABSTRACT 1

Background: The mechanistic target of rapamycin complex-1 (mTORC-1) is a key nutrient and 2

contraction-sensitive protein that regulates a pathway leading to skeletal muscle growth. 3

Utilizing a multiplex assay, we aimed to examine the phosphorylation status of key mTORC-1-4

related signalling molecules in response to protein feeding and resistance exercise. 5

Methods: Eight healthy men (22.5 ± 3.1 yr, 80 ± 9 kg, 1-repetition maximum [1RM] leg 6

extension: 87 ± 5 kg) performed 4 sets of unilateral leg extensions until volitional failure. 7

Immediately following the final set, all participants consumed a protein-enriched beverage. A 8

single skeletal muscle biopsy was obtained from the vastus lateralis before (Pre) with further 9

bilateral biopsies at 1 h (1 h FEDEX and 1 h FED) and 3 h (3 h FEDEX and 3 h FED) post drink 10

ingestion 11

Results: Phosphorylated Akt Ser473

was significantly elevated from Pre at 1 h FEDEX. 12

Phosphorylated p70S6K1 Thr412

was significantly increased above Pre at 1 h FEDEX and 1 h 13

FED and was still significantly elevated at 3 h FEDEX but not 3 h FED. Phosphorylated rpS6 14

Ser235/236 was also significantly increased above Pre at 1 h FEDEX and 1 h FED with 1 h FEDEX 15

greater than 1 h FED. 16

Conclusion: Our data highlight the utility of a multiplex assay to assess anabolic signaling 17

molecules in response to protein feeding and resistance exercise in humans. Importantly, these 18

changes are comparable to those as previously reported using standard immunoblotting and 19

protein activity assays. 20

21

Abstract word count: 241 22

23

Page 2 of 19



Draft

3

Key words: Resistance exercise, protein, anabolic signalling, multiplex technology 24

INTRODUCTION 25

The size of human skeletal muscle mass is largely influenced by fasted and fed rates of skeletal 26

muscle protein synthesis (MPS). For instance, protein ingestion results in suppression of rates of 27

muscle protein breakdown and a transient (~2-3h) increase in rates of MPS, an effect that is 28

potentiated by prior resistance exercise (Biolo et al. 1997; Witard et al. 2009). The regulation of 29

MPS in response to protein feeding and resistance exercise is underpinned by activation of the 30

mechanistic target rapamycin complex 1 (mTORC1) pathway (Drummond et al. 2009; Dickinson 31

et al. 2011). Thus, many studies measure the phosphorylation of proteins, as proxies of activity 32

of this pathway, in response to various nutritional and exercise stimuli to provide mechanistic 33

insight into the regulation of MPS. 34

Using immunohistochemistry and in vitro [γ-32

P] ATP kinase assays, we recently 35

demonstrated that protein feeding and resistance exercise activate the mTORC1 pathway as well 36

as the translocation of the catalytic subunit of mTORC1, mechanistic target of rapamycin 37

(mTOR), to the muscle cell membrane (Hodson et al. 2017). However, immunohistochemistry 38

and in vitro [γ-32

P] ATP assays do not provide information related to residue-specific 39

phosphorylation, a key posttranslational modification affecting kinase activity (Fischer and 40

Krebs 1955). Recent advancements in multiplex technology have enabled the simultaneous 41

measurement of the phosphorylation status of mTORC1-associated proteins complementing 42

existing, routine procedures such as immunoblotting (Bass et al. 2017). A significant advantage 43

of multiplex technology is that multiple analytes can be simultaneously measured using 44

fluorescence without the need for membrane separation by mass and subsequent transfer for 45

quantification. Unlike the wealth of information derived from immunoblotting, data obtained 46

from multiplex approaches characterizing changes in anabolic signaling molecule 47

Page 3 of 19



Draft

4

phosphorylation in response to protein feeding and exercise in humans are scarce (Gonzalez et 48

al. 2015a; Gonzalez et al. 2015b). Moreover, how such changes compare to those assessed by 49

gold-standard enzyme activity approaches such as in vitro [γ-32

P] ATP kinase assays as we have 50

previously reported (McGlory et al. 2014; Hodson et al. 2017), is unknown. 51

The aim of the present study was to utilize a multiplex assay to measure changes in the 52

phosphorylation of key mTORC1-related signalling molecules (Akt Ser473

, mTOR Ser2448

, 53

ribosomal protein S6 kinase 1 of 70 kDa [p70S6K1] Thr412

, and ribosomal protein S6 [rpS6] 54

Ser235/236) in response to protein feeding and when protein feeding was preceded by a bout of 55

resistance exercise. We also wished to compare these changes against those assessed utilizing 56

immunohistochemistry and in vitro [γ-32

P] ATP kinase assays. We hypothesized that 57

phosphorylation of mTORC1-related signaling molecules would be increased in response to 58

protein feeding and resistance exercise, and that these changes would be congruent to those seen 59

in our previous work (Hodson et al. 2017). 60

61

METHODS AND MATERIALS 62

Participants. Eight healthy, recreationally active men (age 22.5 ± 3.1 yr; body mass; 80 ± 9 kg, 63

1-repetition maximum [1 RM] leg extension; 87 ± 5 kg) participated in this investigation. The 64

study was approved by the Hamilton Integrated Research Ethics Board (REB 14-736) and 65

adhered to the ethical standards outlined by the Canadian Tri-Council policy statement regarding 66

the use of human participants in research as well as the principles of the Declaration of Helsinki, 67

as revised in 2008. 68

Experimental design. The experimental design has been fully detailed elsewhere (Hodson 69

et al. 2017). Briefly, following initial assessment for 1 RM leg extension strength, participants 70

Page 4 of 19



Draft

5

reported to the laboratory at ~7:00 am following a 10-h overnight fast. On arrival, a catheter was 71

inserted into a forearm vein for repeated blood sampling and an initial skeletal muscle biopsy 72

was taken from the vastus lateralis (Pre). Participants then performed 4 sets of unilateral leg 73

extension (Atlantis, Laval, QC, Canada) at 70% 1 RM until volitional failure interspersed by 2 74

min recovery. Immediately following the final repetition of the final set, all participants 75

consumed a commercially available beverage (Gatorade Recover®) that provided 20 g, 44 g, and 76

1 g of protein, carbohydrate, and fat, respectively. Further bilateral skeletal muscle biopsies were 77

obtained from the vastus lateralis from both the exercised and non-exercised legs at 1 h (1 h 78

FEDEX and 1 h FED) and 3 h (3 h FEDEX and 3 h FED) after drink ingestion. 79

Plasma glucose, insulin, and amino acids. Plasma glucose concentrations were measured using 80

the glucose oxidase method (YSI 2300; Yellow Springs, OH, USA). Plasma insulin 81

concentrations were measured using the dual-site chemiluminescent method (Siemens Immulite 82

2000; Malvern, PA, USA). Plasma amino acid concentrations were determined using 83

Phenomenex EZ:fast amino acid analysis kit with gas chromatography-mass spectrometry (GC 84

Model 6890 Network, Agilent Technologies; MSD model 5973 Network, Agilent Technologies, 85

MA, USA). Intra-assay CV was < 5% for all blood analyses. 86

Muscle lysate preparation. Muscle biopsy samples were homogenized in lysis buffer 87

(MILLIPLEX® MAP Lysis Buffer catalog No. 43-040) with protease inhibitor (Complete 88

Protease Inhibitor Mini-Tablets, Roche, IN, USA- 1tablet/10mL of lysis buffer). Each sample 89

was prepared with 8-50 mg of muscle tissue in 500 µL of lysis buffer and homogenized. After 90

homogenization, samples were centrifuged at 15000 g for 10 min at 4°C. The supernatant was 91

removed, and protein concentrations were determined using a bicinchoninic acid protein assay 92

(Thermo Fisher Scientific Inc, MA, USA). 93

Page 5 of 19



Draft

6

Multiplex procedure. MILLIPLEX MAP assay kits (EMD Millipore, MA, USA) were 94

used to quantify phosphorylation status of Akt Ser473

(46-677MAG), mTOR Ser2448

(46-686MAG), 95

rpS6 Ser235/Ser236

(46-714MAG), and p70S6K1 Thr412

(46-629MAG) according to the 96

manufacturer's guidelines. Aliquots of protein homogenates (25 µL - 0.8 µg/µL) were combined 97

with Milliplex MAP Assay Buffer 2 (catalog No. 43-041) resulting in 20 µg total protein being 98

added per well. MCF7 cells lysates stimulated with IGF-1 (catalog No. 47-216) were used as a 99

positive control. Multiplex analysis was performed using the Luminex L200 instrument 100

(Luminex, Austin, TX, USA), and data were analyzed for mean fluorescence intensity (MFI) by 101

xPONENT software (Luminex, Austin, TX, USA). 102

Western blotting. Aliquots of the same samples used in the multiplex procedure were 103

prepared for immunoblotting. Working samples of equal concentrations (2.0 µg/µL) were 104

prepared in sample Laemmli buffer and all immunoblotting procedures were conducted as 105

previously described (McGlory et al. 2014). Primary antibody as follows: phospho-Akt Ser473

106

(1:1000, Cell Signaling Technology, #9271S), total Akt (1:1000, Cell Signalling Technology, 107

#4691S), and α-tubulin (1:2000, Cell Signalling Technology, #2125S). Secondary antibody 108

(1:10000, GE Healthcare Life Science, #NA931) followed by detection with chemiluminescence 109

(Amersham Biosciences; Pierce Biotechnology, USA) and quantified by densitometry using 110

Image J software (National Institutes of Health). 111

Statistical analysis. All statistical analyses were performed using the IBM Statistical 112

Package for the Social Sciences, Version 22.0 (IBM Corp, NY, USA). Data were checked for 113

normality using the Shapiro-Wilk test and analyzed using a one-way repeated measures 114

ANOVA. A Fishers least significant difference test was used to evaluate significant time-115

dependent effects. Spearman correlation was applied to test for correlations between Akt or 116

Page 6 of 19



Draft

7

p70S6K1 activity data from (Hodson et al. 2017) and phosphorylation of Akt Ser473

and 117

p70S6K1Thr412

assayed by the multiplex procedure. For correlations, r > 0.7 and r <0.4 were 118

considered strong and weak correlations respectively. In all analyses, P < 0.05 was considered 119

statistically significant. 120

121

RESULTS 122

Plasma glucose, insulin, and amino acids. Plasma glucose was significantly (P < 0.05) increased 123

above Pre at 15 and 30 min post drink consumption then returned to Pre levels at 45 min (Figure 124

1A). Plasma insulin concentration was also significantly increased (P < 0.05) post drink 125

consumption between 15 and 90 min then returned to Pre levels (Figure 1B). The plasma 126

essential amino acid concentration was significantly (P < 0.05) elevated at 15 min post drink 127

ingestion and returned to Pre levels at 90 min (Figure 1C) whereas plasma leucine concentration 128

was elevated from Pre at 15 min and only returned to Pre levels at 120 min post drink 129

consumption (Figure 1D). 130

Protein phosphorylation. Phosphorylated Akt Ser473

was significantly elevated (~2.0-fold) 131

from Pre at 1 h FEDEX (P < 0.05), but not at 1 h FED. There was no significant difference in 132

phosphorylated Akt Ser473

from Pre at either 3 h FEDEX or 3 h FED (Figure 2A). Phosphorylated 133

Akt Ser473

as assessed by immunoblotting was significantly increased at 1 h FEDEX (~1.5-fold; P 134

< 0.05) and 1 h FED (~1.0-fold; P < 0.05). Besides showing a higher absolute value at 1 h 135

FEDEX, there was no statistical difference between 1 h FEDEX and 1 h FED (Figure 3). There 136

was no effect of exercise and feeding or feeding alone on the phosphorylation status of mTOR 137

Ser2448 (Figure 2B). Phosphorylated p70S6K1

Thr412 was significantly increased above Pre at 1 h 138

FEDEX (~3.0-fold) and 1 h FED (~1.0-fold; P < 0.05) and was still significantly elevated at 3 h 139

Page 7 of 19



Draft

8

FEDEX but not at 3 h FED (P < 0.05; Figure 2C). Phosphorylated rpS6 Ser235/236

was also 140

significantly increased above Pre at 1 h FEDEX (~3.0-fold; P < 0.05) and 1 h FED (~1.2-fold; P 141

< 0.05) with 1 h FEDEX greater than 1 h FED (P < 0.05). There was no significant difference in 142

phosphorylated rpS6 Ser235/236

at Pre and 3 h FEDEX or 3 h FED (Figure 2D). 143

Correlation analysis. Spearman correlation analysis of p70S6K1 Thr412

phosphorylation 144

vs. p70S6K1 activity obtained from (Hodson et al. 2017) demonstrated a strong degree (r = 0.80; 145

P < 0.001) of correlation between methods (Figure 4). However, comparable analysis between 146

Akt Ser473

using multiplex and panAkt activity demonstrated a weak degree of correlation (r = 147

0.20; P > 0.05, data not shown). 148

149

DISCUSSION 150

Employing a multiplex assay, we show that protein feeding plus resistance exercise increased the 151

phosphorylation status of Akt Ser473

and p70S6K1 Thr412

. Additionally, we identified an increase in 152

the phosphorylation of rpS6 Ser235/236

, a downstream target of p70S6K1. These findings 153

complement our previous investigation that used a combination of immunohistochemistry and in 154

vitro [γ-32

P] ATP kinase assays, which demonstrated that protein feeding and resistance exercise 155

induced the translocation of mTOR to the muscle cell membrane, as well as activation of Akt, 156

and p70S6K1 in healthy, young men (Hodson et al. 2017). The findings of the present study 157

when taken together with our earlier work (Hodson et al. 2017), highlight the utility of multiplex 158

technology to assess changes in putative anabolic signalling molecules in response to protein 159

feeding and resistance exercise in human skeletal muscle. 160

Previous research utilizing multiplex approaches has failed to detect any change in Akt 161

Ser473 phosphorylation following resistance exercise and protein feeding in well trained men 162

Page 8 of 19



Draft

9

(Gonzalez et al. 2015a; Gonzalez et al. 2015b), which contrasts with our observation of an 163

increase in Akt Ser473

phosphorylation at 1 h FEDEX. To substantiate our finding, we probed for 164

Akt Ser473

phosphorylation utilising traditional immunoblotting and confirmed that the increase 165

was observable with both methods. To our knowledge, we are the first to make this comparison 166

in humans complementing existing data in rodents (Sharma et al. 2012). Interestingly, in contrast 167

to the multiplex approach, using immunoblotting we did detect a significant increase in Akt Ser473

168

phosphorylation at 1 h FED. We cannot fully explain the exact reason why we failed to detect 169

changes in Akt Ser473

phosphorylation at 1 h FED with the multiplex assay but it is reasonable to 170

assume that the small sample size, variability, and differences in antibody affinity between the 171

two methods may have played a role. 172

It is possible that methodological differences between studies may in part be responsible 173

for the discrepant findings between our study and that of (Gonzalez et al. 2015a; Gonzalez et al. 174

2015b). Indeed, Gonzalez and colleagues (Gonzalez et al. 2015a; Gonzalez et al. 2015b) 175

employed well-trained participants whereas the participants in our investigation were 176

recreationally active. Given that trained individuals may display a blunted anabolic response to 177

resistance exercise and protein feeding compared to their untrained counterparts (Coffey et al. 178

2006) it is conceivable that this difference in training status contributed to the disparate results. 179

Another, and we propose more likely factor, contributing to the differences was that the protein-180

enriched drink in our study contained 44 g of carbohydrate and in the study of Gonzalez et 181

al.(Gonzalez et al. 2015a) the carbohydrate content was only 6 g. The peak insulin concentration 182

in our study was therefore significantly higher than that of previous reports (Gonzalez et al. 183

2015a) (40 vs. 15 µIU.mL

-1). As Akt

Ser473 phosphorylation is sensitive to changes in insulin 184

concentration (Gonzalez et al. 2009), we propose that the detection of changes in Akt Ser473

185

Page 9 of 19



Draft

10

phosphorylation in our study is likely due to the substantially greater carbohydrate content of the 186

protein-enriched drink. It is also important to note that their investigation, Gonzalez et al. 187

(Gonzalez et al. 2015b) performed a muscle biopsy 2 h following the cessation of exercise and 188

not 1 h post-exercise as in the present investigation that may have been too late to detect a signal 189

response. 190

A distal target substrate of mTORC1 is p70S6K1, which is a kinase that plays important 191

roles in translation initiation, ribosomal biogenesis, and the upregulation of MPS in response to 192

exercise and feeding (Ma and Blenis 2009). In the present investigation, we demonstrated a 193

significant increase in phosphorylated p70S6K1 Thr412

at 1 h FEDEX, 1 h FED, and 3 h FEDEX. 194

This finding corroborates our previous study that also identified a significant increase in 195

p70S6K1 activity at 1 h FEDEX, 1 h FED, and 3 h FEDEX (Hodson et al. 2017). Furthermore, 196

in the present investigation, phosphorylation of the p70S6K1 substrate rpS6 Ser235/236

was 197

transiently increased 1 h FEDEX and 1 h FED. The fact that our changes in p70S6K1 Thr412

198

phosphorylation using multiplex approaches mirrored those as detected using in vitro [γ-32

P] 199

ATP kinase assays (McGlory et al. 2014; Hodson et al. 2017) provides further, albeit indirect, 200

support for the utility of this methodology to detect changes in anabolic signaling molecules 201

following exercise and feeding in humans. 202

Although the present investigation provides novel and practically useful information for 203

researchers there are some limitations that we must acknowledge. Firstly, no direct measurement 204

of MPS was made and we therefore are unable to ascertain how feeding and exercise-induced 205

changes in these signalling molecules influenced rates of MPS. It is also important to note that 206

due to a lack of an available commercial antibody for the multiplex at the time of analysis the 207

phosphorylation of p70S6K1 in our investigation was indicative of the Thr412

residue and not the 208

Page 10 of 19



Draft

11

mTORC1-specific Thr389

residue. However, correlation analysis of p70S6K1 Thr412

and the 209

p70S6K1 activity data from (Hodson et al. 2017) demonstrated a strong degree of correlation 210

(Figure 4) similar to that as previously reported between p70S6K1 activity and p70S6K1 Thr389

211

(Apro et al. 2015). Nonetheless, we accept that corresponding immunoblotting p70S6K1 Thr389

as 212

well as our other molecular targets coupled with direct measurements of MPS would have 213

strengthened the findings of this study. 214

In conclusion, we demonstrate that a multiplex approach to assessing anabolic signaling 215

molecules in response to protein feeding and resistance exercise in humans results in comparable 216

changes in phosphorylation/activation as those detected using immunohistochemistry and in vitro 217

[γ-32

P] ATP kinase assays (McGlory et al. 2014; Hodson et al. 2017). Future work that combines 218

direct measures of MPS with multiplex technology would build on our findings. 219

220

CONFLICTS OF INTEREST 221

The authors declare no conflict of interest. 222

223

AUTHOR CONTIBUTIONS 224

C.M, E.N, and E.T performed sample analysis. C.M and E.N wrote the 1st draft of the manuscript 225

and all authors approved the final version prior to submission. 226

227

ACKNOWLEDGEMENTS 228

The authors thank Tracy Rerecich for her expert technical assistance. All authors played a role in 229

the design and conduct of the study. 230

231

Page 11 of 19



Draft

12

232

233

234

REFERENCES 235

Apro, W., Moberg, M., Hamilton, D.L., Ekblom, B., Rooyackers, O., Holmberg, H.C. et al. 236

2015. Leucine does not affect mechanistic target of rapamycin complex 1 assembly but is 237

required for maximal ribosomal protein s6 kinase 1 activity in human skeletal muscle following 238

resistance exercise. FASEB J. 29(10): 4358-73. DOI: 10.1096/fj.15-273474. 239

Bass, J.J., Wilkinson, D.J., Rankin, D., Phillips, B.E., Szewczyk, N.J., Smith, K., et al. 2017. An 240

overview of technical considerations for Western blotting applications to physiological research. 241

Scand. J. Med. Sci. Sports, 27(1): 4-25. DOI: 10.1111/sms.12702. 242

Biolo, G., Tipton, K.D., Klein, S., andWolfe, R.R. 1997. An abundant supply of amino acids 243

enhances the metabolic effect of exercise on muscle protein. Am. J. Physiol. 273(1 Pt 1): E122-244

E129. DOI: 10.1152/ajpendo.1997.273.1.E122 245

Coffey, V.G., Zhong, Z., Shield, A., Canny, B.J., Chibalin, A.V., Zierath, J.R., et al. 2006. Early 246

signaling responses to divergent exercise stimuli in skeletal muscle from well-trained humans. 247

FASEB J. 20(1): 190-192. DOI: 10.1096/fj.05-4809fje 248

Dickinson, J.M., Fry, C.S., Drummond, M.J., Gundermann, D.M., Walker, D.K., Glynn, E.L., et 249

al. 2011. Mammalian target of rapamycin complex 1 activation is required for the stimulation of 250

Page 12 of 19



Draft

13

human skeletal muscle protein synthesis by essential amino acids. J. Nutr. 141(5): 856-862. DOI: 251

10.3945/jn.111.139485. 252

Drummond, M.J., Fry, C.S., Glynn, E.L., Dreyer, H.C., Dhanani, S., Timmerman, K.L., et al. 253

2009. Rapamycin administration in humans blocks the contraction-induced increase in skeletal 254

muscle protein synthesis. J. Physiol. 587(Pt 7): 1535-1546. DOI: 10.1113/jphysiol.2008.163816. 255

Fischer, E.H. and Krebs, E.G. 1955. Conversion of phosphorylase b to phosphorylase a in 256

muscle extracts. J. Biol. Chem. 216(1): 121-32. PMID: 13252012 257

Gonzalez, A.M., Hoffman, J.R., Jajtner, A.R., Townsend, J.R., Boone, C.H., Beyer, K.S., et al. 258

2015a. Protein supplementation does not alter intramuscular anabolic signaling or endocrine 259

response after resistance exercise in trained men. Nutr. Res. 35(11): 990-1000. DOI: 260

10.1016/j.nutres.2015.09.006. 261

Gonzalez, A.M., Hoffman, J.R., Townsend, J.R., Jajtner, A.R., Wells, A.J., Beyer, K.S., et al. 262

2015b. Association between myosin heavy chain protein isoforms and intramuscular anabolic 263

signaling following resistance exercise in trained men. Physiol. Rep. 3(1). DOI: 264

10.14814/phy2.12268. 265

Gonzalez, E. and McGraw, T.E. 2009. Insulin-modulated Akt subcellular localization determines 266

Akt isoform-specific signaling. Proc. Natl. Acad. Sci. U S A, 106(17): 7004-9. DOI: 267

10.1073/pnas.0901933106. 268

269

Page 13 of 19



Draft

14

Hodson, N., McGlory, C., Oikawa, S.Y., Jeromson, S., Song, Z., Ruegg, M.A., et al. 2017. 270

Differential localisation and anabolic responsiveness of mTOR complexes in human skeletal 271

muscle in response to feeding and exercise. Am. J. Physiol. Cell Physiol. DOI: 272

10.1152/ajpcell.00176.2017. 273

Ma, X.M. and Blenis, J. 2009. Molecular mechanisms of mTOR-mediated translational control. 274

Nat. Rev. Mol. Cell Biol. 10(5): 307-18. DOI: 10.1038/nrm2672 275

McGlory, C., White, A., Treins, C., Drust, B., Close, G.L., Maclaren, D.P., et al. 2014. 276

Application of the [gamma-32P] ATP kinase assay to study anabolic signaling in human skeletal 277

muscle. J. Appl. Physiol. (1985). 116(5): 504-513. DOI: 10.1152/japplphysiol.01072.2013 278

Sharma, N., Sequea, D.A., Arias, E.B., andCartee, G.D. 2012. Greater insulin-mediated Akt 279

phosphorylation concomitant with heterogeneous effects on phosphorylation of Akt substrates in 280

soleus of calorie-restricted rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 303(12): R1261-7. 281

DOI: 10.1152/ajpregu.00457.2012 282

Witard, O.C., Tieland, M., Beelen, M., Tipton, K.D., van Loon, L.J., andKoopman, R. 2009. 283

Resistance exercise increases postprandial muscle protein synthesis in humans. Med. Sci. Sports 284

Exerc. 41(1): 144-54. DOI: 10.1249/MSS.0b013e3181844e79 285

286

Page 14 of 19



Draft

15

FIGURE LEGENDS

Fig. 1. Plasma glucose (A), insulin (B), essential amino acid (EAA) (C), and leucine (D)

concentrations prior (0 min) and following (15 -180 min) drink consumption. Data are presented

as mean ± SEM, n = 8. * denotes significantly different from Pre.

Fig. 2. Phosphorylation status of Akt Ser473

(A), mTOR Ser2448

(B), p70S6K1 Thr412

(C), rpS6

Ser235/236 (D) at rest (Pre), 1 h post resistance exercise and feeding (1 h FEDEX), 1 h post feeding

(1 h FED), 3 h post resistance exercise and feeding (3 h FEDEX), and 3 h post feeding (3 h

FED). Boxes represent 25th

-75th

quartiles, whiskers represent maximum and minimum values,

horizontal line represents median, cross represents mean, n = 8. Means that do not share a letter

are significantly different. Median fluorescence intensity (MFI). Exercise cessation and

consumption of drink at 0 min.

Fig. 3. Phosphorylation status of Akt Ser473

assessed using immunoblotting at rest (Pre), 1 h post

resistance exercise and feeding (1 h FEDEX), 1 h post feeding (1 h FED), 3 h post resistance

exercise and feeding (3 h FEDEX), and 3 h post feeding (3 h FED). Boxes represent 25th

-75th

quartiles, whiskers represent maximum and minimum values, horizontal line represents median,

cross represents mean, n = 8. Means that do not share a letter are significantly different.

Fig. 4. Spearman correlation analysis of p70S6K1 Thr412

phosphorylation vs. p70S6K1 activity.

Median fluorescence intensity (MFI).

Page 15 of 19



Draft0 30 60 90 120 150 180

0

2

4

6

8

Time (min)

Plas

ma

gluc

ose

(mm

ol·L

-1)

A*

*

0 30 60 90 120 150 180

0

500

1000

1500

2000

TIme (min)

Plas

ma

EAA

(µm

ol. L

-1)

C

*

0 30 60 90 120 150 180

0

10

20

30

40

50

Time (min)

Plas

ma

insu

lin (µ

U. m

L-1)

B *

0 30 60 90 120 150 180

0

50

100

150

200

TIme (min)

Plas

ma

leuc

ine

(µm

ol. L

-1)

D *

Figure 1 Page 16 of 19



DraftPre

1 h FEDEX

1 h FED

3 h FEDEX

3 h FED

0.0

0.5

1.0

1.5

2.0

2.5

p-m

TOR

Ser

2448

B

Pre

1 h FEDEX

1 h FED

3 h FEDEX

3 h FED

0.0

1.0

2.0

3.0

4.0

5.0

6.0

p-p7

0S6k

Thr4

12

bc

c

C

a

a,c

Pre

1 h FEDEX

1 h FED

3 h FEDEX

3 h FED

0.0

2.0

4.0

6.0

8.0

10.0

p-rp

S6 S

er23

5/23

6

a

D

b

c a,ca

Pre

1 h FEDEX

1 h FED

3 h FEDEX

3 h FED

-1

0

1

2

3

4

p-Ak

t Ser

473 a

Ab a

Figure 2Page 17 of 19



Draft

Pre

1 h FEDEX

1 h FED

3 h FEDEX

3 h FED

0.0

1.0

2.0

3.0

4.0

p-Ak

t Ser

473 /

Tota

l Akt

56 kDa - p-Akt Ser 473

50 kDa - α-tubulin

56 kDa - Total Akt

50 kDa - α-tubulin

bb,c

a a,da,d

Figure 3 Page 18 of 19



Draft

0 10 20 30 40 50 600

25

50

75

100

125

150

p-p70S6k Thr412 (MFI)

p70S

6K1(

fmol

/min

/mg) r = 0.80; P = < 0.001

Figure 4Page 19 of 19



Documents

Draft · 2018. 8. 23. · Draft 1 ASSESSING THE MECHANISTIC TARGET OF RAPAMYCIN COMPLEX-1 PATHWAY IN RESPONSE TO RESISTANCE EXERCISE AND FEEDING IN HUMAN SKELETAL MUSCLE by MULTIPLEX