Glycolytic fast-twitch muscle fiber restoration counters adverse age-related changes in body composition and metabolism

Abstract

Aging is associated with the development of insulin resistance, increased adiposity, and accumulation of ectopic lipid deposits in tissues and organs. Starting in mid-life there is a progressive decline in lean muscle mass associated with the preferential loss of glycolytic, fast-twitch myofibers. However, it is not known to what extent muscle loss and metabolic dysfunction are causally related or whether they are independent epiphenomena of the aging process. Here, we utilized a skeletal-muscle-specific, conditional transgenic mouse expressing a constitutively active form of Akt1 to examine the consequences of glycolytic, fast-twitch muscle growth in young vs. middle-aged animals fed standard low-fat chow diets. Activation of the Akt1 transgene led to selective skeletal muscle hypertrophy, reversing the loss of lean muscle mass observed upon aging. The Akt1-mediated increase in muscle mass led to reductions in fat mass and hepatic steatosis in older animals, and corrected age-associated impairments in glucose metabolism. These results indicate that the loss of lean muscle mass is a significant contributor to the development of age-related metabolic dysfunction and that interventions that preserve or restore fast/glycolytic muscle may delay the onset of metabolic disease.

Keywords: adipose tissue; diabetes; exercise; mTOR; sarcopenia; type IIb muscle.

Figure 1

Response to anabolic stimulation is blunted by aging. (A) Western blot analysis was performed to assess phosphorylation of insulin growth factor-1 (IGF-1) receptor beta at tyrosine 1135/1136, Akt at serine 473 and total Akt in gastrocnemius muscle of 3- or 12-month-old mice (n = 6/group). Vehicle group was injected with sterile water, and the IGF-1 group was treated with human insulin growth factor-1. (B) Phosphorylation or protein levels were determined relative to tubulin by Image J software (n = 6/group). Results are presented as mean ± SEM. *P < 0.05, **P < 0.01.

Figure 2

Akt1 activation increases lean mass and muscle weight in young and older mice. Body composition by quantitative magnetic resonance and gastrocnemius weight were measured in young and older control and double-transgenic (DTG) mice following 4 weeks of doxycycline administration. (A) Lean mass in experimental mice as a percentage of body weight (n = 7–10/group). (B) Gastrocnemius muscle of young and older mice expressed as a portion of total body weight. MCK-rtTA single transgenic mice were used as a control. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01; n.s., not significant. (C) Induction of Akt1 in skeletal muscle increased type IIb myofiber size. Representative gastrocnemius muscle sections stained with MHC type IIb (green), laminin (red) and the nuclear stain DAPI (blue) from 3- and 12-month-old control and DTG mice after 4 weeks of Akt1 activation in skeletal muscle. Scale bars = 50 μm. (D) Distribution of mean cross-sectional area (CSA) of muscle fibers in gastrocnemius muscle in the different experimental groups. (E) Mean CSA of muscle fibers (n = 4/group). Results are presented as mean ± SEM. *P < 0.05, **P < 0.01.

Figure 3

Transgene-mediated activation of Akt signaling in muscle. (A) Akt1 kinase activity in gastrocnemius muscle of 3- and 12-month-old control and double-transgenic mice after 4 weeks of doxycycline (DOX) treatment. MCK-rtTA single transgenic mice were used as controls. Top: representative GSK3α/β peptide phosphorylation. Bottom: histogram quantifying the data. n = 7/group. *P < 0.05, **P < 0.01. (B) Representative western blot analysis of Akt/PKB signaling. Phosphorylation of Akt at threonine 308 and serine 473, total Akt1 protein, Akt1 and transgene HA-tag was assessed in gastrocnemius muscle of the different experimental groups of mice. Representative blots are shown. HA, hemagglutinin. (C) Phosphorylation (p-Akt at Thr308 or Ser473) or protein levels (Akt1 or HA tag) were determined relative to GAPDH by Image J software analysis of autoradiography film (n = 7/group). Results are presented as mean ± SEM. *P < 0.05, **P < 0.01. (D) Regulation of downstream Akt effector molecules in muscle of young and older mice after 4 weeks of Akt transgene activation. Western blot analysis of downstream Akt targets mTOR and p70S6 kinase in gastrocnemius muscle. Representative blots of phosphorylation or total protein levels of mTOR and p70S6 kinase in control and double-transgenic (DTG) mice at 3 or 12 months of age. Phosphorylated levels of mTOR at Ser2448 or p70S6K at Thr389 were quantified relative to total mTOR or p70S6 kinase and then adjusted to GAPDH by Image J software analysis of autoradiography film (n = 7/group). (E) Relative mRNA expression levels of MAFbx, MURF, and TNF-α as measured relative to 36B4 by qRT-PCR in gastrocnemius muscle of control and DTG mice at 3 or 12 months of age (n = 7/group). Results are presented as mean ± SEM. *P < 0.05, **P < 0.01. (F) Myogenic activation of Akt signaling downregulates oxidative and upregulates glycolytic gene expression in muscle. Relative mRNA expression levels of PGC-1α, PPARα, PPARδ, pyruvate kinase isoenzyme M2 (PkM2), phosphofructokinase (Pfk), and LDHA as measured by qRT-PCR in gastrocnemius muscle of 3- or 12-month-old control and DTG mice after 4 weeks of DOX administration (n = 7/group). 36B4 was used as an internal control. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01.

Figure 4

Age-associated accumulation of adipose tissue is reversed by restoration of glycolytic fast-twitch muscle. (A) Time course of body weight changes. Doxycycline (DOX) was administered to 3- or 12-month-old control and double-transgenic (DTG) mice to activate Akt1 in skeletal muscle. Body weight was measured weekly. MCK-rtTA single transgenic mice were used as controls. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01. vs. 12-month-old control mice. (B) Body weight of control and DTG mice at the 4 week time point of DOX administration. (C) Food intake of young or aged control and DTG mice. DOX was given to activate Akt1 transgene in skeletal muscle for 4 weeks, and food intake was monitored weekly in 3- or 12-month-old control and DTG mice (n = 7–9/group). (D) Whole body composition of fat mass was measured by quantitative magnetic resonance in the different experimental groups. DOX was administered for 4 weeks (n = 13–15/group). Subcutaneous fat (E) and inguinal fat (F) weight after 4 weeks of DOX administration in young and older mice. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01.

Figure 5

Myogenic Akt1 transgene activation corrects age-associated impairments in metabolism. (A) Glucose tolerance tests in 3- or 12-month-old control and double-transgenic (DTG) mice after 4 weeks doxycycline (DOX) treatment (n = 7–10/group). All mice were fasted overnight prior to measurements. Blood glucose was measured every 20 min for 2 h. *P < 0.05 vs. 12-month-old control mice. (B) Insulin tolerance tests in 3- or 12-month-old control and DTG mice after 4 weeks DOX treatment (n = 7–10/group). All mice were under 6-h fast condition. Blood glucose was measured every 15 min for 90 min. *P < 0.05 vs. 12-month-old control mice. Serum insulin (C) and serum leptin (D) levels in 3- or 12-month-old control and DTG mice after 4 weeks of DOX treatment (n = 8–10). Results are presented as mean ± SEM. *P < 0.05, **P < 0.01. (E) Akt1 activation increases whole body oxygen consumption. Young and older mice were monitored by Oxymax metabolic measuring system for 24 h under normal feeding conditions in control and DTG mice after 4 weeks administration of DOX (n = 6–7/group). Results are presented as mean ± SEM. *P < 0.05, **P < 0.01.

Figure 6

Myogenic activation of Akt1 signaling reduces hepatic steatosis. (A) Representative sections of liver stained with Oil red O in 3- or 12-month-old control and double-transgenic (DTG) mice after 4 weeks of doxycycline (DOX) administration. Scale bars = 100 μm. Quantitative analysis of total cholesterol (B) and triglyceride levels (C) in liver after 4 weeks DOX treatment in the different experimental groups (n = 8/group). Results are presented as mean ± SEM. *P < 0.05, **P < 0.01. (D) Myogenic activation of Akt1-signaling affects lipogenic gene expression. Relative mRNA expression levels of sterol regulatory element-binding protein (SREBP-1c), ACC1, FAS, and SCD1 were measured by qRT-PCR in liver of 3- or 12-month-old control and DTG mice after 4 weeks of DOX administration (n = 8/group). 36B4 was used as internal control. Results are presented as mean ± SEM. *P < 0.05, **P < 0.01.