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. 2016 Oct 5:7:445.
doi: 10.3389/fphys.2016.00445. eCollection 2016.

Prolonged Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Intake and Associated microRNA Expression

Lee M Margolis  1 , Donato A Rivas  2 , Maria Berrone  2 , Yassine Ezzyat  2 , Andrew J Young  3 , James P McClung  3 , Roger A Fielding  2 , Stefan M Pasiakos  3
Affiliations

Prolonged Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Intake and Associated microRNA Expression

Lee M Margolis et al. Front Physiol. .

Abstract

Short-term (5-10 days) calorie restriction (CR) downregulates muscle protein synthesis, with consumption of a high protein-based diet attenuating this decline. Benefit of increase protein intake is believed to be due to maintenance of amino acid-mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1), however, there is limited evidence to support this contention. The purpose of this investigation was to determine the effects of prolonged CR and high protein diets on skeletal muscle mTORC1 signaling and expression of associated microRNA (miR). Twelve-week old male Sprague Dawley rats consumed ad libitum (AL) or calorie restricted (CR; 40%) adequate (10%, AIN-93M) or high (32%) protein milk-based diets for 16 weeks. Body composition was determined using dual energy X-ray absorptiometry and muscle protein content was calculated from muscle homogenate protein concentrations expressed relative to fat-free mass to estimate protein content. Western blot and RT-qPCR were used to determine mTORC1 signaling and mRNA and miR expression in fasted mixed gastrocnemius. Independent of dietary protein intake, muscle protein content was 38% lower (P < 0.05) in CR compared to AL. Phosphorylation and total Akt, mTOR, rpS6, and p70S6K were lower (P < 0.05) in CR vs. AL, and total rpS6 was associated with muscle protein content (r = 0.64, r2 = 0.36). Skeletal muscle miR expression was not altered by either energy or protein intake. This study provides evidence that chronic CR attenuates muscle protein content by downregulating mTORC1 signaling. This response is independent of skeletal muscle miR and dietary protein.

Keywords: energy deficit; miR-100; miR-99; muscle protein content; rpS6.

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Figures

Figure 1
Figure 1
Mean (± SEM) [n = 10 (AL 10% PRO; formula image), n = 10 (AL 32% PRO; formula image), n = 10 (CR 10% PRO; formula image), n = 10 (CR 32% PRO; □)] body mass (A), fat mass (B), fat-free mass (C), and muscle protein content (D). Data analyzed using a univariate ANOVA with Bonferroni correction to determine main effects of energy (AL vs. CR), protein (10 vs. 32%) and energy-by-time interactions. †Week 16 different from Baseline; P < 0.05. +Time-by-energy interaction, CR different from AL at week 16; P < 0.05. *CR different from AL; P < 0.05.
Figure 2
Figure 2
Mean (± SEM) [n = 10 (AL 10% PRO), n = 10 (AL 32% PRO), n = 10 (CR 10% PRO), n = 10 (CR 32% PRO)] phosphorylation (A) and totals (B) relative to GAPDH, and phosphorylation relative to total (C). Data analyzed using a univariate ANOVA with Bonferroni correction to determine main effects of energy (AL vs. CR), protein (10 vs. 32%) and energy-by-protein interactions. *CR body mass different from AL; P < 0.05.+Energy-by-protein interaction, CR 32% PRO different than AL 32% PRO; P < 0.05.
Figure 3
Figure 3
Association of total Akt (A) and rpS6 (B) to muscle protein content [n = 10 (AL 10% PRO ♦), n = 10 (AL 32% PRO formula image), n = 10 (CR 10% PRO formula image), n = 10 (CR 32% PRO ◦)]. Data analyzed using a spearman rho correlation coefficient. Significant associations were determined as P < 0.05.
Figure 4
Figure 4
Median (± SEM) [n = 10 (AL 10% PRO; formula image), n = 10 (AL 32% PRO; formula image), n = 10 (CR 10% PRO; formula image), n = 10 (CR 32% PRO; □)] mRNA (A) and miR (B) expression. Data analyzed using a univariate ANOVA with Bonferroni correction to determine main effects of energy (AL vs. CR), protein (10 vs. 32%) and energy-by-protein interactions. *CR body mass different from AL; P < 0.05. +32% PRO different than 10%; P < 0.05.
Figure 5
Figure 5
Median (± SEM) [n = 10 (AL 10% PRO; formula image), n = 10 (AL 32% PRO; formula image), n = 10 (CR 10% PRO; formula image), n = 10 (CR 32% PRO; □)] mRNA (A) and mean (± SEM) total AMPKα and PGC-1α (B) expression and citrate synthase activity (C). Data analyzed using a univariate ANOVA with Bonferroni correction to determine main effects of energy (AL vs. CR), protein (10 vs. 32%) and energy-by-protein interactions. *CR different from AL; P < 0.05.
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