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. 2014 Sep 15;34(18):3450-60.
doi: 10.1128/MCB.00426-14. Epub 2014 Jul 7.

Insulin receptor substrates are essential for the bioenergetic and hypertrophic response of the heart to exercise training

Christian Riehle  1 , Adam R Wende  2 , Yi Zhu  3 , Karen J Oliveira  3 , Renata O Pereira  1 , Bharat P Jaishy  1 , Jack Bevins  3 , Steven Valdez  3 , Junghyun Noh  3 , Bum Jun Kim  3 , Annie Bello Moreira  3 , Eric T Weatherford  4 , Rajkumar Manivel  4 , Tenley A Rawlings  3 , Monika Rech  3 , Morris F White  5 , E Dale Abel  6
Affiliations

Insulin receptor substrates are essential for the bioenergetic and hypertrophic response of the heart to exercise training

Christian Riehle et al. Mol Cell Biol. .

Abstract

Insulin and insulin-like growth factor 1 (IGF-1) receptor signaling pathways differentially modulate cardiac growth under resting conditions and following exercise training. These effects are mediated by insulin receptor substrate 1 (IRS1) and IRS2, which also differentially regulate resting cardiac mass. To determine the role of IRS isoforms in mediating the hypertrophic and metabolic adaptations of the heart to exercise training, we subjected mice with cardiomyocyte-specific deletion of either IRS1 (CIRS1 knockout [CIRS1KO] mice) or IRS2 (CIRS2KO mice) to swim training. CIRS1KO hearts were reduced in size under basal conditions, whereas CIRS2KO hearts exhibited hypertrophy. Following exercise swim training in CIRS1KO and CIRS2KO hearts, the hypertrophic response was equivalently attenuated, phosphoinositol 3-kinase (PI3K) activation was blunted, and prohypertrophic signaling intermediates, such as Akt and glycogen synthase kinase 3β (GSK3β), were dephosphorylated potentially on the basis of reduced Janus kinase-mediated inhibition of protein phosphatase 2a (PP2A). Exercise training increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein content, mitochondrial capacity, fatty acid oxidation, and glycogen synthesis in wild-type (WT) controls but not in IRS1- and IRS2-deficient hearts. PGC-1α protein content remained unchanged in CIRS1KO but decreased in CIRS2KO hearts. These results indicate that although IRS isoforms play divergent roles in the developmental regulation of cardiac size, these isoforms exhibit nonredundant roles in mediating the hypertrophic and metabolic response of the heart to exercise.

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Figures

FIG 1
FIG 1
Preserved insulin-mediated signaling in CIRS1KO and CIRS2KO hearts. (a and b) Representative immunoblots for IRS1 and IRS2 in homogenates of various tissues from 8-week-old CIRS1KO and CIRS2KO mice and ventricle homogenates from mice with genotypes as indicated. (c to h) Representative immunoblots (c) and quantification of densitometry (d to h) in protein lysates obtained from isolated working hearts perfused for 1 h with Krebs-Henseleit buffer containing 5 mM glucose and 0.4 mM palmitate in the presence or absence of 1 nM insulin (n = 4). (i) Glucose uptake in isolated cardiomyocytes obtained from genotypes as indicated in the presence or absence of 1 nM insulin. Data are presented as fold changes relative to the same genotype, no insulin (n = 7 to 9). Two-way ANOVA was performed to analyze differences by insulin stimulation and genotype followed by Fisher's PLSD post hoc analysis. Significance: phosphorylation of Akt at Ser473, P < 0.05 for insulin (d) and Thr308, P < 0.05 for insulin, genotype, and their interaction (e); phosphorylation of AS160 at Thr642 (f), phosphorylation of p70 S6K at Thr389 (g), phosphorylation of S6 at Ser235/236 (h), and glucose uptake in isolated cardiomyocytes (i), P < 0.05 for insulin. *, P < 0.05 versus WT (same insulin concentration); †, P < 0.05 versus same genotype (0 nM insulin); ‡, P < 0.05 versus CIRS1KO (same insulin concentration).
FIG 2
FIG 2
IRS proteins modulate the hypertrophic response to exercise training. Two-way ANOVA was performed to analyze differences by swim training and genotype followed by Fisher's PLSD post hoc analysis. (a) Citrate synthase activity in mixed gastrocnemius muscle homogenates (n = 6; P < 0.05 for swim training); (b) heart weights (HW; P < 0.05 for genotype, P < 0.05 for the interaction between swim training and genotype); (c) heart weights normalized to tibia lengths (HW/TL) under sedentary conditions and following swim training (n = 10; P < 0.05 for swim training, genotype, and their interaction). (d and e) Representative pictures and quantification of WGA staining (n = 6 per group; scale bar, 20 μm; P < 0.05 for genotype, P < 0.05 for the interaction between swim training and genotype). The intensities of each panel were adjusted individually to allow visualization of cell size. (f to h) In vivo, left ventricular hemodynamic parameters (maximum [Max] or minimum [Min] rates of change of left ventricle [LV] contraction [dP/dt] and LV developed pressure [Dev P]) under sedentary conditions and following exercise training (n = 9 to 12; Max dP/dt and LV Dev P, P < 0.05 for genotype, respectively). *, P < 0.05 versus WT (same treatment); †, P < 0.05 versus sedentary (same genotype); ‡, P < 0.05 versus CIRS1KO (same treatment).
FIG 3
FIG 3
Attenuated PI3K activation and increased dephosphorylation of prohypertrophic signaling intermediates in CIRS1KO and CIRS2KO hearts following exercise training. Two-way ANOVA was performed to analyze differences by swim training and genotype followed by Fisher's PLSD post hoc analysis. (a) PI3 kinase enzymatic activity (P < 0.05 for genotype, P < 0.05 for the interaction between swim training and genotype); (b) densitometric quantification of PI3 kinase p85 subunit protein; (c) PI3 kinase enzymatic activity normalized to p85 protein (n = 6; P < 0.05 for the interaction between swim training and genotype). (d to k) Representative Western blots (d) and densitometric quantification of P AKT Thr308/Total AKT (P < 0.05 for genotype, P < 0.05 for the interaction between swim training and genotype) (e), P AKT Ser473/Total AKT (P < 0.05 for genotype, P < 0.05 for the interaction between swim training and genotype) (f), P GSK3b Ser9/Total GSK3b (P < 0.05 for swim training, P < 0.05 for the interaction between swim training and genotype) (g), 4E-BP1 γ/GAPDH (P < 0.05 for swim training, P < 0.05 for the interaction between swim training and genotype) (h), P PP2A Tyr307/Total PP2A (P < 0.05 for swim training, genotype, and their interaction) (i), P JAK2 Tyr221/Total JAK2 (P < 0.05 for swim training, P < 0.05 for the interaction between swim training and genotype) (j), and P STAT3 Tyr705/Total STAT3 (P < 0.05 for swim training, P < 0.05 for the interaction between swim training and genotype) (n = 8) (k). *, P < 0.05 versus WT (same treatment); †, P < 0.05 versus sedentary (same genotype); ‡, P < 0.05 versus CIRS1KO (same treatment). n.s., no significant difference observed.
FIG 4
FIG 4
IRS proteins mediate mitochondrial bioenergetics and the increase in PGC-1α protein in response to exercise training. Two-way ANOVA was performed to analyze differences by swim training and genotype followed by Fisher's PLSD post hoc analysis. (a to d) Mitochondrial capacity as measured by maximum ADP-stimulated mitochondrial respiration (VADP) and ATP synthesis in saponin-permeabilized cardiac fibers. VADP and ATP synthesis (P < 0.05 for swim training, genotype, and their interaction) with pyruvate (a and b) and VADP (P < 0.05 for swim training, genotype, and their interaction) (c) and ATP synthesis (P < 0.05 for swim training, P < 0.05 for genotype) (d) with palmitoyl-carnitine as the substrates each combined with malate as indicated (n = 4 to 6). (e and f) Citrate synthase (P < 0.05 for swim training, genotype, and their interaction) (e) and hydroxyacyl-CoA dehydrogenase (HADH) enzymatic activity (P < 0.05 for swim training, P < 0.05 for genotype) (f) (n = 8). (g to l) Representative Western blots (g) and densitometric analysis of CPT1b and CPT2 (P < 0.05 for swim training, P < 0.05 for the interaction between swim training and genotype) (h and i) and electron transport chains subunit complex I NDUFA9 (P < 0.05 for genotype) (j), complex II 30 kDa subunit (P < 0.05 for swim training, genotype, and their interaction) (k), and complex V subunit α (P < 0.05 for swim training, P < 0.05 for genotype) (l), each normalized to Coomassie blue stains. (m and n) Mitochondrial DNA content measured by RT-PCR (P < 0.05 for swim training, genotype, and their interaction) (m) and protein levels of the transcriptional coactivator PGC-1α normalized to GAPDH (P < 0.05 for genotype, P < 0.05 for the interaction between swim training and genotype; n = 3 to 8) (n). PGC-1α knockout heart homogenate was used as a negative control for PGC-1α immunoblotting (38). *, P < 0.05 versus WT (same treatment); †, P < 0.05 versus sedentary (same genotype); ‡, P < 0.05 versus CIRS1KO (same treatment).
FIG 5
FIG 5
IRS proteins mediate the metabolic response to exercise training. (a to c) Palmitate oxidation, glucose oxidation, and glycolysis rates in isolated working WT, CIRS1KO, and CIRS2KO hearts under sedentary conditions and following chronic exercise training (n = 6 to 10). Hearts were perfused with 5 mmol/liter glucose and 0.4 mmol/liter palmitate. (d) Cardiac glycogen content as indicated (n = 7 to 8). Data are presented as fold changes compared to sedentary controls (same genotype); †, P < 0.05 versus sedentary (same genotype).
FIG 6
FIG 6
Proposed model for attenuated hypertrophic and bioenergetic response in IRS-deficient hearts to exercise training. Changes in exercise-trained WT (a), CIRS1KO (b), and CIRS2KO (c) hearts relative to sedentary controls of the same genotype are shown. POX, palmitate oxidation; GLOX, glucose oxidation; Glycol, glycolysis.
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