Articles in PresS. Am J Physiol Endocrinol Metab (February 10, 2015). doi:10.1152/ajpendo.00618.2014

1

Mechanisms for independent and combined effects of calorie restriction and acute

2

exercise on insulin-stimulated glucose uptake by skeletal muscle of old rats

3 4

Naveen Sharma1,2,*, Haiyan Wang1,3,*, Edward B. Arias1, Carlos M. Castorena1 and Gregory D.

5

Cartee1,4,5

6 7

1

8

2

9

3

10

4

11

5

12

*Naveen Sharma and Haiyan Wang contributed equally to this manuscript.

Muscle Biology Laboratory, School of Kinesiology, University of Michigan, Ann Arbor, MI School of Health Sciences, Central Michigan University, Mount Pleasant, MI College of Physical Education and Health, East China Normal University, Shanghai, China Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI Institute of Gerontology, University of Michigan, Ann Arbor, MI

13 14

Running title: Calorie restriction, exercise and glucose uptake in old rats

15 16

Correspondence:

17

Gregory D. Cartee, Ph.D.

18

University of Michigan, School of Kinesiology, Room 4745F

19

401 Washtenaw Avenue, Ann Arbor, MI 48109-2214

20

Phone: (734) 615-3458

21

Fax: (734) 936-1925

22

email: [email protected]

23

Email Addresses: Naveen Sharma, [email protected]; Haiyan Wang, [email protected];

24

Edward B. Arias, [email protected]; Carlos M. Castorena,

25

[email protected]

26 1

Copyright © 2015 by the American Physiological Society.

27 28

ABSTRACT Either calorie restriction (CR; consuming 60-65% of ad libitum, AL, intake) or acute

29

exercise can independently improve insulin sensitivity in old age, but their combined effects on

30

muscle insulin signaling and glucose uptake were previously unknown. Accordingly, we

31

assessed the independent and combined effects of CR (beginning at 14 weeks-old) and acute

32

exercise (3-4 hours post-exercise) on insulin signaling and glucose uptake in insulin-stimulated

33

epitrochlearis muscles from 30 month-old rats. Either CR alone or exercise alone versus AL

34

sedentary controls induced greater insulin-stimulated glucose uptake. Combined CR and

35

exercise versus either treatment alone caused an additional increase in insulin-stimulated

36

glucose uptake. Either CR or exercise alone versus AL sedentary controls increased AktSer473

37

and AktThr308 phosphorylation. Combined CR and exercise further elevated Akt phosphorylation

38

on both sites. CR alone, but not exercise alone, versus AL sedentary controls significantly

39

increased Akt substrate of 160 kDa (AS160) Ser588 and Thr642 phosphorylation. Combined CR

40

and exercise did not further enhance AS160 phosphorylation. Exercise alone, but not CR

41

alone, modestly increased GLUT4 abundance. Combined CR and exercise did not further

42

elevate GLUT4 content. These results suggest that CR or acute exercise independently

43

increase insulin-stimulated glucose uptake via overlapping (greater Akt phosphorylation) and

44

distinct (greater AS160 phosphorylation for CR; greater GLUT4 for exercise) mechanisms. Our

45

working hypothesis is that greater insulin-stimulated glucose uptake in the combined CR and

46

exercise group versus CR or exercise alone relies on greater Akt activation, leading to greater

47

phosphorylation of one or more Akt substrate other than AS160.

48 49

Key Words: glucose transport; glucose transporter; insulin signaling; insulin resistance;

50

physical activity; aging

2

51

INTRODUCTION

52

Because multiple age-related diseases are linked to the development of whole body

53

insulin resistance (19), identifying and understanding interventions that can elevate insulin-

54

stimulated glucose disposal during old age has important implications for health. Skeletal

55

muscle, which accounts for the largest amount of insulin-stimulated blood glucose clearance

56

(18), is a prime target for interventions to enhance insulin sensitivity. Moderate calorie restriction

57

(CR; chronically consuming ~20-40% below ad libitum, AL, intake) and exercise can each

58

independently enhance insulin-stimulated glucose uptake by muscle in old rats (9, 11, 17, 40,

59

49). However, the combined effects of CR and acute exercise on muscle glucose uptake and

60

insulin signaling by skeletal muscle during old age are unknown.

61

Although the combined effects of CR and acute exercise on glucose uptake are

62

uncertain, earlier research has addressed potential mechanisms for enhanced insulin-mediated

63

glucose uptake caused by either CR or acute exercise alone. The complex insulin signaling

64

pathway leading to insulin-stimulated glucose transport (12, 46) begins when insulin binds its

65

receptor, inducing site-specific tyrosine phosphorylation of the receptor, which in turns, leads to

66

site-specific tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1). Tyrosine-

67

phosphorylated IRS-1 associates with phosphatidylinositol 3-kinase (PI3K), and increased PI3K

68

activity is required for insulin-stimulated glucose transport. Subsequently, the Ser/Thr protein

69

kinase Akt binds to membranes that are enriched in lipids that were phosphorylated by IRS-1-

70

PI3K, leading to greater Akt phosphorylation on Thr308 and Ser473. Akt catalyzes the

71

phosphorylation of a Rab GTPase activating protein (GAP) known as Akt Substrate of 160 kDa

72

(AS160; also called TBC1D4) on several sites, including Thr642 and Ser588, which are important

73

for insulin-stimulated glucose transport.

74

Current knowledge about the independent effects of CR or acute exercise on the role of

75

insulin signaling in the regulation of muscle glucose uptake during old age is limited, and

76

apparently nothing has been published about the combined effects of CR and acute exercise on 3

77

insulin signaling in muscles, regardless of age. Accordingly, the current study was designed to

78

provide new insights into the mechanisms for both the independent and combined benefits of

79

CR and one exercise session on insulin signaling and glucose uptake by insulin-stimulated

80

muscle in old age. The specific aims were to determine in isolated epitrochlearis muscle from

81

30 month-old rats the effects of chronic CR (initiated at 14 week-old) and/or acute exercise on:

82

1) insulin-stimulated glucose uptake; 2) activation of key insulin signaling steps that regulate

83

glucose uptake (including IRS-1-PI3K activity, Akt Ser473 and Thr308 phosphorylation, and AS160

84

Ser588 and Thr642 phosphorylation); 3) Akt’s association with three protein binding partners that

85

can influence Akt phosphorylation (protein phosphatase 2A, PP2A; heat shock protein of 90

86

kDa, HSP90; and adaptor protein containing pleckstrin homology domain, phosphotyrosine

87

domain, and leucine zipper motif 1, Appl1); 4) abundance of GLUT4 and hexokinase II, proteins

88

responsible for muscle glucose transport and phosphorylation, respectively; and 5)

89

phosphorylation of AMP-activated protein kinase (AMPK).

90 91

EXPERIMENTAL PROCEDURES

92

Materials

93

Unless otherwise noted, all chemicals were purchased from Fisher Scientific (Hanover

94

Park, IL) or Sigma-Aldrich (St. Louis, MO). Reagents and apparatus for SDS-PAGE and

95

immunoblotting were from Bio-Rad Laboratories (Hercules, CA). Bicinchoninic acid protein

96

assay and Pierce MemCode Reversible Protein Stain Kit were purchased from Thermo Fisher

97

(Waltham, MA). Anti-phospho Akt Thr308 (pAktThr308; #9275), anti-phospho Akt Ser473 (pAktSer473;

98

#9272), anti-phospho AS160 Ser588 (pAS160Ser588; #8730), anti-phospho AMPKα Thr172

99

(pAMPKThr172; #2531), anti-phospho insulin receptor Tyr1146 (pIRTyr1146; #3021), anti-Akt (#4691),

100

anti-AMP-activated protein kinase-α (AMPK; #5831) , anti-hexokinase II (#2867) and anti-rabbit

101

IgG horseradish peroxidase conjugate (#7074) and ATP (#9804) were from Cell Signaling

102

Technology (Danvers, MA). Anti-Akt Substrate of 160 kDa (AS160; #ABS54) and anti-GLUT4 4

103

(#CBL243) were from EMD Millipore (Billerica, MA). Anti-Filamin-C (FLNc; #sc-48496), anti-

104

mouse IgG horseradish peroxidase conjugate (#sc-2060) and anti-goat IgG horseradish

105

peroxidase conjugate (#sc-2020) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-

106

phospho Filamin-C Ser2231 (pFLNcSer2231; #PB-131) was from Kinasource (Dundee, Scotland,

107

UK). Anti-phospho AS160 Thr642 (pAS160Thr642; #3028-P) was from Symansis (B-Bridge

108

International; Mountain View, CA). Anti-heat shock protein of 90 kDa (HSP90; #610419) and

109

anti-protein phosphatase 2A catalytic α (PP2A; #610556) were from BD Bioscience (San Jose,

110

CA). Anti-Appl1 (Appl1; #ab59592) was from Abcam (Cambridge, MA). Anti-AffiniPure Sheep

111

IgG horseradish peroxidase conjugate (#713-035-147) was from Jackson ImmunoResearch

112

Laboratories (West Grove, PA). 2-Deoxy-D-[3H]-glucose ([3H]-2-DG), [14C]-mannitol and

113

[32ATP]-ATP were from Perkin Elmer (Boston, MA).

114

Animal treatment

115

Procedures for animal care were approved by the University of Michigan Committee on

116

Use and Care of Animals. Male Fischer-344 x Brown Norway rats (both CR rats and their AL

117

controls) were obtained at ~29 months of age from National Institute of Aging (NIA) Calorie

118

Restricted Rodent Colony. Calorie restriction was initiated at 14 weeks of age in the CR group

119

by the NIA. Rats were housed at the University of Michigan for approximately one month prior

120

to experimentation. During this time the rats were housed individually in shoebox cages and

121

maintained on a 12-12 hour light-dark cycle (lights out at 17:00 h) in specific pathogen-free

122

conditions. Rats were provided chow (AL: NIH31 chow; CR: NIH31/NIA fortified chow) and

123

maintained on their respective feeding protocol (AL: free access to chow; CR: ~60-65% of AL

124

consumption). The muscle glucose uptake experiment was performed when the rats were ~30

125

months of age. Rats were fasted at ~19:00 on the night before the terminal experiment. The

126

following morning at ~07:00 h, exercised rats swam in a barrel filled with water (35oC; ~45 cm

127

depth; six rats swimming at time; 3 AL and 3 CR). The exercise protocol consisted of nine

128

bouts of swimming (10 minute duration per bout) with 10 minute rest intervals separating each 5

129

exercise bout. After 90 minutes of total exercise, exercising rats were dried and returned to

130

their cages without food, and epitrochlearis muscles were dissected from anesthetized time-

131

matched sedentary and exercised rats at 3-4 hours after the exercising group had completed

132

the protocol.

133

Muscle dissection and incubation

134

Muscle dissection and incubation procedures have been previously described (42). The

135

two longitudinal muscle strips prepared from each epitrochlearis were placed in vials containing

136

the appropriate media, shaken at 45 oscillations per minute, continuously gassed (95% O2/5%

137

CO2), and heated (35oC) in a reciprocating water bath. Muscles were initially incubated in vials

138

containing 2 ml Krebs Henseleit (KHB) supplemented with 0.1% bovine serum albumin (BSA), 2

139

mM sodium pyruvate, 6 mM mannitol, and either no insulin (basal) or a submaximally effective

140

concentration of insulin (0.6 nM) for 30 minutes. Muscles were then transferred to another vial

141

containing 2 ml KHB/BSA, the same concentration of insulin as the previous step, 0.1 mM 2-

142

DG; including a final specific activity of 2.25 mCi/mmol [3H]-2-DG), and 5.9 mM mannitol

143

(including a final specific activity of 0.022 mCi/mmol [14C]-mannitol) for 20 minutes. After this

144

step, muscles were blotted on filter paper moistened with ice-cold KHB, trimmed, freeze-

145

clamped using aluminum tongs cooled in liquid nitrogen, and stored at -80oC for later processing

146

and analysis.

147

Muscle lysate preparation

148

Frozen muscles were weighed, homogenized in ice-cold lysis buffer (1 ml/muscle strip)

149

using a TissueLyser II homogenizer (Qiagen, Valencia, CA). For the samples analyzed for 2-

150

DG uptake and immunoblotting, the lysis buffer contained T-PER Tissue Protein Extraction

151

Reagent (#PI-78510; Thermo Scientific, Rockford, IL) supplemented with 1 mM EDTA, 1 mM

152

EGTA, 2.5 mM sodium pyrophosphate (NaPP), 1 mM sodium vanadate, 1 mM ß-

153

glycerophosphate, 1 µg/ml leupeptin, and 1 mM PMSF. For the samples analyzed for IRS-1-

154

PI3K activity and Akt co-immunoprecipitation, the lysis buffer contained 50 mM HEPES, pH 7.5, 6

155

150 mM sodium chloride, 1% octylphenoxy poly(ethyleneoxy)ethanol a (IGEPAL), 10% glycerol,

156

10 mM sodium fluoride, 2 mM EDTA, 10 mM NaPP, 1 mM magnesium chloride, 1 mM calcium

157

chloride, 1 mM PMSF, 5 µg/ml leupeptin, 1 tablet/10 ml PhosSTOP (#04906837001; Roche,

158

Indianapolis, IN) and 0.1 mM potassium bisperoxo(1,10-phenanthroline)oxovanadate

159

[bpV(phen)] (#203695, Millipore).

160

2-Deoxy-D-glucose uptake

161

The calculation of [3H]-2-deoxy-D-glucose (2-DG) uptake by skeletal muscle has been

162

previously described (8, 26). Briefly, [14C]-mannitol counts per minute, determined by liquid

163

scintillation counting of aliquots from muscle homogenates, were used to determine extracellular

164

space. The intracellular [3H]-2-DG of muscle was calculated as the difference between the total

165

[3H]-2-DG in muscle and the [3H]-2-DG in the extracellular space.

166

Immunoblotting

167

Western blotting procedures have been previously described (42). An equal amount of

168

protein of each sample was mixed with 6x Laemmli buffer, boiled for 5 minutes and separated

169

using SDS-PAGE (7% resolving gel), before being transferred to polyvinyl difluoride

170

membranes. The MemCode protein stain was used to confirm equal loading (3). Membranes

171

were blocked in 5% BSA in TBST (Tris-buffered saline, pH 7.5 plus 0.1% Tween-20) for 1 hour

172

at room temperature and transferred to 5% BSA-TBST with the appropriate primary antibody

173

overnight at 4°C. Membranes were washed 3 times for 5 minutes in TBST and incubated with

174

secondary antibody for 1 hour at room temperature. Blots were washed 3 times for 5 minutes in

175

TBST then washed 3 times for 5 minutes in TBS and then subjected to enhanced

176

chemiluminescence (Luminata Forte Western HRP Substrate; #WBLUF0100; Millipore) to

177

visualize protein bands. Immunoreactive proteins were quantified by densitometry (AlphaEase

178

FC; Alpha Innotech, San Leandro, CA). Values are expressed relative to the normalized

179

average of all the samples on each blot.

180

IRS-1-associated PI3K activity 7

181

Muscle IRS-1-PI3K activity was determined as previously described (42). After addition

182

of 2 µg of anti-IRS-1 antibody to 300 µg of supernatant protein from each muscle sample, the

183

immunocomplexes were allowed to form overnight at 4°C with slow rotation. Then 100 µl of

184

protein A-Sepharose beads (catalog no. 17-0469-01, GE Healthcare, Piscataway, NJ; 50%

185

slurry) were added to each aliquot, and samples were rotated for 2 hours at 4°C. Samples were

186

centrifuged at 3,000 g to pellet the protein A-Sepharose immunocomplex. Each immunopellet

187

was washed three times with buffer 1 (phosphate buffered saline, pH 7.5, containing 1%

188

IGEPAL and 100 µM sodium vanadate), three times with buffer 2 (100 mM Tris, pH 7.5, 500 mM

189

lithium chloride, and 100 µM sodium vanadate), and twice with buffer 3 (10 mM Tris, pH 7.5,

190

100 mM sodium chloride, 1 mM EDTA, and 100 µM sodium vanadate). After the immunopellet

191

was washed, all the buffer was removed, and the immunopellet was resuspended in 40 µl of the

192

10 mM Tris·1 mM EDTA, pH 7.5, buffer containing 10 µg of phosphatidylinositol (Avanti Polar

193

Lipids, Alabaster, AL) and 100 mM magnesium chloride. The reaction was initiated at room

194

temperature by addition of 5 µl of a phosphorylation mixture containing 880 µM ATP and 30 µCi

195

of γ-[32P] ATP. After 20 minutes with continuous rotation at 37°C, the reaction was stopped by

196

sequential addition of 20 µl of 8 N hydrochloric acid and 160 µl of chloroform-methanol (1:1).

197

The reaction mixture was vortexed for 5 minutes and then centrifuged at 3,000 g for 5 minutes;

198

50 µl of the organic phase containing the reaction products was spotted onto a thin layer

199

chromatography (TLC) plate (Whatman, Piscataway, NJ). The products were resolved in a

200

chloroform-methanol-water-ammonium hydroxide (60:47:11.3:2) solution and visualized by

201

autoradiography. The spots corresponding to the phosphatidylinositol phosphorylated product

202

were scraped from the TLC plate and counted in a scintillation counter.

203

Co-immunoprecipitation of HSP90, PP2A and Appl1 with Akt

204

For evaluation of Akt association with other proteins, 300 μg of protein from each

205

sample were combined with a 1:1,000 titer of Akt antibody and rotated overnight at 4°C. After

206

initial antibody incubation, 50 μl of protein G-magnetic beads (#10004D, Life Technologies, 8

207

Grand Island, NY) were added to the lysate-antibody mixture and rotated for 2 hours at 4°C.

208

The immunoprecipitation matrix (bead-antibody-antigen) for each sample was washed three

209

times with lysis buffer, with complete aspiration of buffer after the final wash, and 30 μl of 2×

210

Laemmli buffer was added. Samples were boiled for 5 minutes and centrifuged, and

211

supernatants were subjected to 10% SDS-PAGE and blotted for HSP90, PP2A and Appl1.

212

Statistical analysis

213

Two-way analysis of variance (ANOVA) was used to assess the main effects of diet (AL

214

or CR) and exercise (sedentary or 3 hours post-exercise) and the diet x exercise interaction

215

within each insulin level (minus or plus insulin), and the Tukey test was used for post-hoc

216

analysis to identify the source of significant variance (SigmaPlot version 11.0; Systat Software,

217

San Jose, CA). Data lacking normal distribution and/or equal variance were mathematically

218

transformed to achieve normality and equal variance prior to running two-way ANOVA. Kruskal-

219

Wallis one-way ANOVA on ranks was used if transformation failed to normalize the data, and

220

post-hoc analysis was performed by Dunn's method. The Spearman Rank Order Correlation

221

was used to evaluate associations between measured outcomes. Data are presented as mean

222

± SEM. A P value ≤ 0.05 was accepted as statistically significant.

223 224

RESULTS

225

2-Deoxy-D-glucose uptake

226

For 2-DG uptake in muscles incubated without insulin (Figure 1), the 3 hours post-

227

exercise and CR (3hPEX-CR) group exceeded the sedentary and AL (SED-AL) group (P
SED), as well as a significant

230

diet x exercise interaction (P < 0.05). Post-hoc analysis revealed that 2-DG uptake with insulin

231

in the SED-CR (P < 0.001) group and 3 hours post-exercise and AL (3hPEX-AL) group (P
CR; P < 0.01; data not

246

shown) and a diet x exercise interaction (P < 0.05) on total Akt abundance in the muscles

247

incubated without insulin, and post-hoc analysis indicated SED-AL values exceeded SED-CR

248

values (P < 0.001). For pAktThr308/Akt ratio in the absence of insulin, there were no significant

249

diet or exercise effects (Figure 3A). For pAktThr308/Akt ratio in the presence of insulin, there were

250

significant effects of diet (CR > AL; P < 0.001) and exercise (3hPEX > SED; P < 0.001), and a

251

significant diet x exercise interaction (P < 0.05; Figure 3A). Post-hoc analysis revealed that

252

both the SED-CR (P < 0.001) and 3hPEX-AL groups exceeded the SED-AL group (P < 0.05),

253

and the 3hPEX-CR group was greater than both the 3hPEX-AL and SED-CR groups (P
AL; P < 0.001; Figure 3B), and post-hoc analysis revealed that both the SED-CR

256

exceeded the SED-AL group (P < 0.01), and the 3hPEX-CR group was greater than the 3hPEX-

257

AL group (P < 0.01). In the presence of insulin, there were significant main effects of diet (CR > 10

258

AL; P < 0.001) and exercise (3hPEX > SED; P < 0.001) on the pAktSer473/Akt ratio (Figure 3B).

259

Post-hoc analysis demonstrated that the SED-CR group (P < 0.001) and the 3hPEX-AL group

260

(P < 0.05) were each greater than the SED-AL group, and the 3hPEX-CR group exceed both

261

the 3hPEX-AL (P < 0.001) and the SED-CR (P < 0.01) groups.

262

AS160

263

There were no significant effects of diet or exercise on AS160 total abundance (data not

264

shown). For the ratio of pAS160Ser588/AS160 in the absence of insulin, there were no significant

265

effects of diet or exercise (Figure 4A). For the ratio of pAS160Ser588/AS160 in the presence of

266

insulin, there was a significant effect of diet (CR > AL; P < 0.01; Figure 4A). Post-hoc analysis

267

revealed that SED-CR values exceeded SED-AL values (P < 0.05). For the ratio of

268

pAS160Thr642/AS160 in the absence of insulin, there were no significant effects of diet or

269

exercise (Figure 4B). For the ratio of pAS160Thr642/AS160 in the presence of insulin, there was a

270

significant effect of diet (CR > AL; P < 0.001; Figure 4B). Post-hoc analysis revealed that SED-

271

CR values exceeded SED-AL values (P < 0.01) and 3hPEX-CR values (P < 0.05) exceeded

272

3hPEX-AL values.

273

Filamin C

274

Filamin C (FLNc) is an Akt substrate (21, 31). CR was recently found to result in greater

275

FLNcSer2231 phosphorylation in muscles from both 9 month-old and 24 month-old rats (41, 43),

276

but the effects of exercise alone or exercise combined with CR had not been previously

277

reported. For FLNc in the absence of insulin, there was a small (~6%), but significant diet effect

278

on total abundance (AL > CR; P < 0.005; data not shown), as well as a significant diet x

279

exercise interaction (P < 0.05) and post-hoc analysis indicated that SED-AL values were greater

280

than SED-CR values (P < 0.001) and 3hPEX-AL values (P < 0.01). For the FLNcSer2231/FLNc

281

ratio in the absence of insulin, there was a significant diet effect (CR > AL; P < 0.05; Figure 5A),

282

and post-hoc analysis revealed that the 3hPEX-CR values exceeded 3hPEX-AL values (P
11

284

AL; P = 0.002) and exercise (3hPEX > SED; P < 0.005) effects (Figure 5A). Post-hoc analysis

285

indicated that SED-CR and 3hPEX-AL groups were greater than the SED-AL group (P < 0.05),

286

and the 3hPEX-CR group was ~18% greater than the 3hPEX-AL and SED-CR groups (P


290

CR) either without insulin (P < 0.001) or with insulin (P < 0.005; data not shown). Post-hoc

291

analysis indicated that in the absence of insulin, SED-AL exceeded SED-CR (P < 0.001). For

292

pAMPKThr172/AMPK ratio in the absence of insulin, there were significant effects of diet (CR >

293

AL; P < 0.001) and exercise (3hPEX > SED; P < 0.01), and a significant diet x exercise

294

interaction (P < 0.01; Figure 6A). Post-hoc analysis indicated that SED-CR exceeded SED-AL

295

values (P < 0.01), and 3hPEX-CR exceeded both SED-CR and 3hPEX-AL values (P < 0.001).

296

For pAMPKThr172/AMPK ratio in the presence of insulin, there were significant effects of diet (CR

297

> AL; P < 0.001) and exercise (3hPEX > SED; P < 0.005), and a significant diet x exercise

298

interaction (P = 0.005; Figure 6A). Post-hoc analysis revealed that 3hPEX-CR values were

299

greater than 3hPEX-AL and SED-CR values (P < 0.001).

300

GLUT4 and hexokinase II

301

There was a moderate (~22%), but significant (P < 0.001) exercise effect on GLUT4

302

abundance (3hPEX > SED; Figure 7), and post-hoc analysis indicated that 3hPEX-AL exceeded

303

SED-AL (P < 0.01) and 3hPEX-CR exceeded SED-CR (P < 0.01). There was also a small

304

(~15%), but significant (P < 0.001) diet effect on hexokinase II abundance (AL > CR; Figure 8),

305

and post-hoc analysis indicated SED-AL exceeded SED-CR (P < 0.01), and 3hPEX-AL

306

exceeded 3hPEX-CR (P < 0.01).

307

HSP90, APPL1 and PP2A abundance and association with Akt

308

There were no significant effects of diet or exercise on Appl1 or PP2A abundance (data

309

not shown). There was a small (~9%), but significant main effect of diet (AL > CR; P < 0.01) on 12

310

HSP90 abundance, and post-hoc analysis indicated that SED-AL exceeded SED-CR (P < 0.05;

311

data not shown).

312

There was a significant main effect of diet (CR > AL; P < 0.05) for HSP90 associated

313

with Akt, and post-hoc analysis indicated that 3hPEX-CR exceeded 3hPEX-AL (P < 0.05; Figure

314

9). There was a significant main effect of exercise (SED > 3hPEX; P < 0.01) for PP2A

315

associated with Akt, and post-hoc analysis indicated that SED-AL exceeded 3hPEX-AL (P
SED-AL (P < 0.05). Post-hoc

503

analysis indicated for muscles with insulin: *SED-CR (P < 0.001) and 3hPEX-AL (P < 0.05) >

504

SED-AL, †3hPEX-CR > 3hPEX-AL and SED-CR, (P < 0.001). Values are means ± SE; n = 8-11

505

per treatment group.

506 507

Figure 2. IRS-1-associated PI3K activity in muscles from sedentary ad libitum (SED-AL),

508

sedentary calorie restricted (SED-CR), 3 hours post-exercise ad libitum (3hPEX-AL) and 3

509

hours post-exercise calorie restricted (3hPEX-CR) rats. Data were analyzed using two-way

510

ANOVA within each insulin level (without or with insulin). Values are means ± SE; n = 8-11 per

511

treatment group.

512

20

513

Figure 3. Phosphorylated AktThr308/Akt (A), and phosphorylated AktSer473/Akt (B), in muscles

514

from sedentary ad libitum (SED-AL), sedentary calorie restricted (SED-CR), 3 hours post-

515

exercise ad libitum (3hPEX-AL) and 3 hours post-exercise calorie restricted (3hPEX-CR) rats.

516

Representative western blots (C). Results for AktThr308/Akt without insulin were analyzed using

517

one-way ANOVA on ranks because these data were not normally distributed. All other data

518

were analyzed using two-way ANOVA within each insulin level (without or with insulin). Post-

519

hoc analysis indicated for muscles without insulin: **SED-CR > SED-AL, (P < 0.01); ‡3hPEX-

520

CR > 3hPEX-AL (P < 0.01). Post-hoc analysis indicated for muscles with insulin: *SED-CR (P

521

< 0.001) and 3hPEX-AL (P < 0.05) > SED-AL; †3hPEX-CR > 3hPEX-AL (P < 0.001) and SED-

522

CR, (P < 0.01). Values are means ± SE; n = 8-11 per treatment group.

523 524

Figure 4. Phosphorylated AS160Ser588/AS160 (A), and phosphorylated AS160Thr642/AS160 (B),

525

in muscles from sedentary ad libitum (SED-AL), sedentary calorie restricted (SED-CR), 3 hours

526

post-exercise ad libitum (3hPEX-AL) and 3 hours post-exercise calorie restricted (3hPEX-CR)

527

rats. Representative western blots (C). Data were analyzed using two-way ANOVA within each

528

insulin level (without or with insulin). Post-hoc analysis indicated for muscles with insulin:

529

*SED-CR > SED-AL, (P < 0.05 for AS160Ser588, P < 0.01 for AS160Thr642); †3hPEX-CR > 3hPEX-

530

AL, (P < 0.05). Values are means ± SE; n = 8-11 per treatment group.

531 532

Figure 5. Phosphorylated FLNcSer2231/FLNc (A) in muscles from sedentary ad libitum (SED-AL),

533

sedentary calorie restricted (SED-CR), 3 hours post-exercise ad libitum (3hPEX-AL) and 3

534

hours post-exercise calorie restricted (3hPEX-CR) rats. Representative western blots (B). Data

535

were analyzed using two-way ANOVA for samples with insulin. Post-hoc analysis indicated for

536

muscles without insulin: ‡3hPEX-CR > 3hPEX-AL, (P < 0.05). Post-hoc analysis indicated for

537

muscles with insulin: *SED-CR and 3hPEX-AL > SED-AL, (P < 0.05); †3hPEX-CR > 3hPEX-AL

538

and SED-CR, (P < 0.05). Values are means ± SE; n = 8-11 per treatment group. 21

539 540

Figure 6. Phosphorylated AMPKThr172/AMPK (A) in muscles from sedentary ad libitum (SED-

541

AL), sedentary calorie restricted (SED-CR), 3 hours post-exercise ad libitum (3hPEX-AL) and 3

542

hours post-exercise calorie restricted (3hPEX-CR) rats. Representative western blots (B). Data

543

were analyzed using two-way ANOVA within each insulin level (without or with insulin). Post-

544

hoc analysis indicated for muscles without insulin: *SED-CR > SED-AL, (P < 0.01); †3hPEX-CR

545

> SED-CR and 3hPEX-AL, (P < 0.001). Post-hoc analysis indicated for muscles with insulin:

546

‡

547

treatment group.

3hPEX-CR > 3hPEX-AL and SED-CR (P < 0.001). Values are means ± SE; n = 8-11 per

548 549

Figure 7. GLUT4 abundance in muscles from sedentary ad libitum (SED-AL), sedentary calorie

550

restricted (SED-CR), 3 hours post-exercise ad libitum (3hPEX-AL) and 3 hours post-exercise

551

calorie restricted (3hPEX-CR) rats. Data were analyzed using two-way ANOVA. Post-hoc

552

analysis indicated: *3hPEX-AL > SED-AL (P < 0.01) and †3hPEX-CR > SED-CR (P < 0.01).

553

Values are means ± SE; n = 8-11 per treatment group.

554 555

Figure 8. Hexokinase II abundance in muscles incubated from sedentary ad libitum (SED-AL),

556

sedentary calorie restricted (SED-CR), 3 hours post-exercise ad libitum (3hPEX-AL) and 3

557

hours post-exercise calorie restricted (3hPEX-CR) rats. Data were analyzed using two-way

558

ANOVA. Post-hoc analysis indicated: *SED-AL > SED-CR (P < 0.01) and †3hPEX-AL >

559

3hPEX-CR (P < 0.01). Values are means ± SE; n = 8-11 per treatment group.

560 561

Figure 9. HSP90 co-immunoprecipitated with Akt in muscles from sedentary ad libitum (SED-

562

AL), sedentary calorie restricted (SED-CR), 3 hours post-exercise ad libitum (3hPEX-AL) and 3

563

hours post-exercise calorie restricted (3hPEX-CR) rats. Data were analyzed using two-way

22

564

ANOVA. Post-hoc analysis indicated: *3hPEX-CR > 3hPEX-AL (P < 0.05). Values are means

565

± SE; n = 8-11 per treatment group.

566 567

Figure 10. PP2A co-immunoprecipitated with Akt muscles from sedentary ad libitum (SED-AL),

568

sedentary calorie restricted (SED-CR), 3 hours post-exercise ad libitum (3hPEX-AL) and 3

569

hours post-exercise calorie restricted (3hPEX-CR) rats. Data were analyzed using two-way

570

ANOVA. Post-hoc analysis indicated: *SED-AL > 3hPEX-AL (P < 0.05). Values are means ±

571

SE; n = 8-11 per treatment group.

572 573

Figure 11. Appl1 co-immunoprecipitated with Akt in muscles from sedentary ad libitum (SED-

574

AL), sedentary calorie restricted (SED-CR), 3 hours post-exercise ad libitum (3hPEX-AL) and 3

575

hours post-exercise calorie restricted (3hPEX-CR) rats. Data were analyzed using two-way

576

ANOVA. Values are means ± SE; n = 4-6 per treatment group.

577 578 579

23

580

REFERENCES

581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627

1.

2.

3. 4.

5.

6.

7. 8. 9. 10. 11.

12.

13.

14.

15.

16.

17.

Al-Regaiey KA, Masternak MM, Bonkowski MS, Panici JA, Kopchick JJ, Bartke A. Effects of caloric restriction and growth hormone resistance on insulin-related intermediates in the skeletal muscle. J Gerontol A Biol Sci Med Sci 62: 18-26, 2007. An D, Toyoda T, Taylor EB, Yu H, Fujii N, Hirshman MF, Goodyear LJ. TBC1D1 regulates insulin- and contraction-induced glucose transport in mouse skeletal muscle. Diabetes 59: 1358-1365, 2010. Antharavally BS, Carter B, Bell PA, Krishna Mallia A. A high-affinity reversible protein stain for Western blots. Anal Biochem 329: 276-280, 2004. Argentino DP, Dominici FP, Munoz MC, Al-Regaiey K, Bartke A, Turyn D. Effects of long-term caloric restriction on glucose homeostasis and on the first steps of the insulin signaling system in skeletal muscle of normal and Ames dwarf (Prop1df/Prop1df) mice. Exp Gerontol 40: 27-35, 2005. Arias EB, Kim J, Funai K, Cartee GD. Prior exercise increases phosphorylation of Akt substrate of 160 kDa (AS160) in rat skeletal muscle. Am J Physiol Endocrinol Metab 292: E1191-1200, 2007. Barger JL, Kayo T, Vann JM, Arias EB, Wang J, Hacker TA, Wang Y, Raederstorff D, Morrow JD, Leeuwenburgh C, Allison DB, Saupe KW, Cartee GD, Weindruch R, Prolla TA. A low dose of dietary resveratrol partially mimics caloric restriction and retards aging parameters in mice. PLoS ONE 3: e2264, 2008. Cartee GD. Roles of TBC1D1 and TBC1D4 in insulin- and exercise-stimulated glucose transport of skeletal muscle. Diabetologia 58: 19-30, 2015. Cartee GD, Bohn EE. Growth hormone reduces glucose transport but not GLUT-1 or GLUT-4 in adult and old rats. Am J Physiol 268: E902-909, 1995. Cartee GD, Briggs-Tung C, Kietzke EW. Persistent effects of exercise on skeletal muscle glucose transport across the life-span of rats. J Appl Physiol 75: 972-978, 1993. Cartee GD, Funai K. Exercise and insulin: Convergence or divergence at AS160 and TBC1D1? Exerc Sport Sci Rev 37: 188-195, 2009. Cartee GD, Kietzke EW, Briggs-Tung C. Adaptation of muscle glucose transport with caloric restriction in adult, middle-aged, and old rats. Am J Physiol 266: R1443-1447, 1994. Cartee GD, Wojtaszewski JF. Role of Akt substrate of 160 kDa in insulin-stimulated and contraction-stimulated glucose transport. Appl Physiol Nutr Metab 32: 557-566, 2007. Castorena CM, Arias EB, Sharma N, Bogan JS, Cartee GD. Fiber Type Effects on Contraction-stimulated Glucose Uptake and GLUT4 Abundance in Single Fibers from Rat Skeletal Muscle. Am J Physiol Endocrinol Metab: ajpendo 00466 02014, 2014. Castorena CM, Arias EB, Sharma N, Cartee GD. Postexercise Improvement in InsulinStimulated Glucose Uptake Occurs Concomitant With Greater AS160 Phosphorylation in Muscle From Normal and Insulin-Resistant Rats. Diabetes 63: 2297-2308, 2014. Cui M, Yu H, Wang J, Gao J, Li J. Chronic caloric restriction and exercise improve metabolic conditions of dietary-induced obese mice in autophagy correlated manner without involving AMPK. Journal of diabetes research 2013: 852754, 2013. Davidson RT, Arias EB, Cartee GD. Calorie restriction increases muscle insulin action but not IRS-1-, IRS-2-, or phosphotyrosine-PI 3-kinase. Am J Physiol Endocrinol Metab 282: E270-276, 2002. Dean DJ, Cartee GD. Brief dietary restriction increases skeletal muscle glucose transport in old Fischer 344 rats. J Gerontol A Biol Sci Med Sci 51: B208-213, 1996.

24

628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678

18.

19. 20.

21.

22.

23.

24.

25.

26.

27.

28. 29.

30.

31.

32. 33.

34.

DeFronzo RA, Jacot E, Jequier E, Maeder E, Wahren J, Felber JP. The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization. Diabetes 30: 1000-1007, 1981. Facchini FS, Hua N, Abbasi F, Reaven GM. Insulin resistance as a predictor of agerelated diseases. J Clin Endocrinol Metab 86: 3574-3578, 2001. Fisher JS, Gao J, Han DH, Holloszy JO, Nolte LA. Activation of AMP kinase enhances sensitivity of muscle glucose transport to insulin. Am J Physiol Endocrinol Metab 282: E18-23, 2002. Fujita M, Mitsuhashi H, Isogai S, Nakata T, Kawakami A, Nonaka I, Noguchi S, Hayashi YK, Nishino I, Kudo A. Filamin C plays an essential role in the maintenance of the structural integrity of cardiac and skeletal muscles, revealed by the medaka mutant zacro. Dev Biol 361: 79-89, 2012. Funai K, Cartee GD. Inhibition of contraction-stimulated AMP-activated protein kinase inhibits contraction-stimulated increases in PAS-TBC1D1 and glucose transport without altering PAS-AS160 in rat skeletal muscle. Diabetes 58: 1096-1104, 2009. Funai K, Schweitzer GG, Sharma N, Kanzaki M, Cartee GD. Increased AS160 phosphorylation, but not TBC1D1 phosphorylation, with increased postexercise insulin sensitivity in rat skeletal muscle. Am J Physiol Endocrinol Metab 297: E242-251, 2009. Gazdag AC, Sullivan S, Kemnitz JW, Cartee GD. Effect of long-term caloric restriction on GLUT4, phosphatidylinositol-3 kinase p85 subunit, and insulin receptor substrate-1 protein levels in rhesus monkey skeletal muscle. J Gerontol A Biol Sci Med Sci 55: B4446; discussion B47-48, 2000. Gonzalez AA, Kumar R, Mulligan JD, Davis AJ, Weindruch R, Saupe KW. Metabolic adaptations to fasting and chronic caloric restriction in heart, muscle, and liver do not include changes in AMPK activity. Am J Physiol Endocrinol Metab 287: E1032-1037, 2004. Hansen PA, Gulve EA, Holloszy JO. Suitability of 2-deoxyglucose for in vitro measurement of glucose transport activity in skeletal muscle. J Appl Physiol 76: 979985, 1994. Iwabe M, Kawamoto E, Koshinaka K, Kawanaka K. Increased postexercise insulin sensitivity is accompanied by increased AS160 phosphorylation in slow-twitch soleus muscle. Physiological reports 2, 2014. McCurdy CE, Cartee GD. Akt2 is essential for the full effect of calorie restriction on insulin-stimulated glucose uptake in skeletal muscle. Diabetes 54: 1349-1356, 2005. McCurdy CE, Davidson RT, Cartee GD. Brief calorie restriction increases Akt2 phosphorylation in insulin-stimulated rat skeletal muscle. Am J Physiol Endocrinol Metab 285: E693-700, 2003. McCurdy CE, Davidson RT, Cartee GD. Calorie restriction increases the ratio of phosphatidylinositol 3-kinase catalytic to regulatory subunits in rat skeletal muscle. Am J Physiol Endocrinol Metab 288: E996-E1001, 2005. Murray JT, Campbell DG, Peggie M, Mora A, Cohen P. Identification of filamin C as a new physiological substrate of PKBalpha using KESTREL. Biochem J 384: 489-494, 2004. O'Neill HM. AMPK and Exercise: Glucose Uptake and Insulin Sensitivity. Diabetes & metabolism journal 37: 1-21, 2013. Palacios OM, Carmona JJ, Michan S, Chen KY, Manabe Y, Ward JL, 3rd, Goodyear LJ, Tong Q. Diet and exercise signals regulate SIRT3 and activate AMPK and PGC1alpha in skeletal muscle. Aging (Albany NY) 1: 771-783, 2009. Pires RC, Souza EE, Vanzela EC, Ribeiro RA, Silva-Santos JC, Carneiro EM, Boschero AC, Amaral ME. Short-term calorie restriction improves glucose homeostasis in old rats: involvement of AMPK. Appl Physiol Nutr Metab 39: 895-901, 2014. 25

679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725

35. 36. 37. 38.

39.

40.

41.

42.

43.

44.

45.

46. 47.

48.

49.

50.

Richter EA, Hargreaves M. Exercise, GLUT4, and skeletal muscle glucose uptake. Physiol Rev 93: 993-1017, 2013. Ruderman NB, Carling D, Prentki M, Cacicedo JM. AMPK, insulin resistance, and the metabolic syndrome. J Clin Invest 123: 2764-2772, 2013. Sato S, Fujita N, Tsuruo T. Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci U S A 97: 10832-10837, 2000. Schenk S, McCurdy CE, Philp A, Chen MZ, Holliday MJ, Bandyopadhyay GK, Osborn O, Baar K, Olefsky JM. Sirt1 enhances skeletal muscle insulin sensitivity in mice during caloric restriction. J Clin Invest 121: 4281-4288, 2011. Schweitzer GG, Arias EB, Cartee GD. Sustained postexercise increases in AS160 Thr642 and Ser588 phosphorylation in skeletal muscle without sustained increases in kinase phosphorylation. Journal of applied physiology 113: 1852-1861, 2012. Sequea DA, Sharma N, Arias EB, Cartee GD. Calorie restriction enhances insulinstimulated glucose uptake and Akt phosphorylation in both fast-twitch and slow-twitch skeletal muscle of 24-month-old rats. J Gerontol A Biol Sci Med Sci 67: 1279-1285, 2012. Sequea DA, Sharma N, Arias EB, Cartee GD. Greater filamin C, GSK3alpha, and GSK3beta serine phosphorylation in insulin-stimulated isolated skeletal muscles of calorie restricted 24 month-old rats. Mech Ageing Dev 134: 60-63, 2013. Sharma N, Arias EB, Bhat AD, Sequea DA, Ho S, Croff KK, Sajan MP, Farese RV, Cartee GD. Mechanisms for increased insulin-stimulated Akt phosphorylation and glucose uptake in fast- and slow-twitch skeletal muscles of calorie-restricted rats. Am J Physiol Endocrinol Metab 300: E966-978, 2011. Sharma N, Arias EB, Sequea DA, Cartee GD. Preventing the calorie restrictioninduced increase in insulin-stimulated Akt2 phosphorylation eliminates calorie restriction's effect on glucose uptake in skeletal muscle. Biochim Biophys Acta 1822: 1735-1740, 2012. Sharma N, Sequea DA, Castorena CM, Arias EB, Qi NR, Cartee GD. Heterogeneous effects of calorie restriction on in vivo glucose uptake and insulin signaling of individual rat skeletal muscles. PLoS One 8: e65118, 2014. Tanaka S, Hayashi T, Toyoda T, Hamada T, Shimizu Y, Hirata M, Ebihara K, Masuzaki H, Hosoda K, Fushiki T, Nakao K. High-fat diet impairs the effects of a single bout of endurance exercise on glucose transport and insulin sensitivity in rat skeletal muscle. Metabolism 56: 1719-1728, 2007. Thong FS, Dugani CB, Klip A. Turning signals on and off: GLUT4 traffic in the insulinsignaling highway. Physiology (Bethesda) 20: 271-284, 2005. Wang P, Zhang RY, Song J, Guan YF, Xu TY, Du H, Viollet B, Miao CY. Loss of AMP-activated protein kinase-alpha2 impairs the insulin-sensitizing effect of calorie restriction in skeletal muscle. Diabetes 61: 1051-1061, 2012. Wang ZQ, Floyd ZE, Qin J, Liu X, Yu Y, Zhang XH, Wagner JD, Cefalu WT. Modulation of skeletal muscle insulin signaling with chronic caloric restriction in cynomolgus monkeys. Diabetes 58: 1488-1498, 2009. Xiao Y, Sharma N, Arias EB, Castorena CM, Cartee GD. A persistent increase in insulin-stimulated glucose uptake by both fast-twitch and slow-twitch skeletal muscles after a single exercise session by old rats. Age (Dordr) 35: 573-582, 2013. Zaid H, Antonescu CN, Randhawa VK, Klip A. Insulin action on glucose transporters through molecular switches, tracks and tethers. Biochem J 413: 201-215, 2008.

726

26

A

B

C

A

B

C

A

B

A

B