Muscle ectopic fat deposition contributes to anabolic resistance in obese sarcopenic old rats through eIF2α activation

Abstract

Obesity and aging are characterized by decreased insulin sensitivity (IS) and muscle protein synthesis. Intramuscular ceramide accumulation has been implicated in insulin resistance during obesity. We aimed to measure IS, muscle ceramide level, protein synthesis, and activation of intracellular signaling pathways involved in translation initiation in male Wistar young (YR, 6-month) and old (OR, 25-month) rats receiving a low- (LFD) or a high-fat diet (HFD) for 10 weeks. A corresponding cellular approach using C2C12 myotubes treated with palmitate to induce intracellular ceramide deposition was taken. A decreased ability of adipose tissue to store lipids together with a reduced adipocyte diameter and a development of fibrosis were observed in OR after the HFD. Consequently, OR fed the HFD were insulin resistant, showed a strong increase in intramuscular ceramide level and a decrease in muscle protein synthesis associated with increased eIF2α phosphorylation. The accumulation of intramuscular lipids placed a lipid burden on mitochondria and created a disconnect between metabolic and regulating pathways in skeletal muscles of OR. In C2C12 cells, palmitate-induced ceramide accumulation was associated with a decreased protein synthesis together with upregulated eIF2α phosphorylation. In conclusion, a reduced ability to expand adipose tissues was found in OR, reflecting a lower lipid buffering capacity. Muscle mitochondrial activity was affected in OR conferring a reduced ability to oxidize fatty acids entering the muscle cell. Hence, OR were more prone to ectopic muscle lipid accumulation than YR, leading to decreased muscle protein anabolism. This metabolic change is a potential therapeutic target to counter sarcopenic obesity.

Keywords: ceramide; eIF2α signaling; high-fat diet; muscle protein synthesis; obesity; sarcopenia.

Figure 1

Whole-body and skeletal muscle insulin sensitivity indices in young and old rats fed a low-fat or a high-fat diet for 10 weeks. (A) Index of whole-body insulin resistance measured by intraperitoneal glucose tolerance test. Insulin resistance was estimated by the product of the area under the curve (AUC) of the glucose and the AUC of insulin after intraperitoneal glucose infusion. (B) Akt phosphorylation state in tibialis anterior muscle. Phosphorylated Akt to total Akt protein content ratio was measured by Western blot. Values ± SEM for eight animals per group. *P < 0.05 age effect (same diet), ^$P < 0.05 diet effect (same age). LFD, low-fat diet; HFD, high-fat diet.

Figure 2

Triglyceride, diacyl-glycerol, and ceramide content in tibialis anterior muscle of young and old rats fed a low-fat or a high-fat diet for 10 weeks. Triglyceride (A), diacyl-glycerol (B), and ceramide (C) concentrations in tibialis anterior muscle were analyzed by gas-liquid chromatography after total lipid extraction. Ceramide, diacyl-glycerol (DAG) and triglyceride (TG) concentrations are expressed as nmoles/mg proteins. Values ± SEM for five animals per group. *P < 0.05 age effect (same diet), ^$P < 0.05 diet effect (same age). LFD, low-fat diet; HFD, high-fat diet.

Figure 3

Histological analysis of CD68+ macrophages and extracellular matrix staining, adipocyte diameters and transcript levels of inflammation and adipogenesis markers in abdominal adipose tissue of young and old rats fed a low-fat or a high-fat diet for 10 weeks. Immunohistochemical detection of CD68+ macrophages (A) was performed with the avidin–biotin peroxidase method. Slides of abdominal adipose tissue were stained with picrosirius red to detect areas of fibrosis (B). Adipocyte diameters were measured using perfectimage software (Claravision, France) (C). mRNA transcript levels of TNFα, IL1β MCP1, PPARgamma, SREBP1c, and acetyl-CoA carboxylase (ACC) in abdominal adipose tissue were measured by real-time Q-PCR (D->I). Data are expressed as a ratio of gene expression to GAPDH gene expression. Values ± SEM for eight animals per group. *P < 0.05 age effect (same diet), ^$P < 0.05 diet effect (same age). LFD, low-fat diet; HFD, high-fat diet.

Figure 4

Protein synthesis rate and activation of intracellular pathways related to the regulation of translational initiation in tibialis anterior muscle of young and old rats fed a low-fat or high-fat diet for 10 weeks. Total muscle protein (A) and muscle protein fractions, that is, myosin (B), actin (C), and mitochondrial proteins (D), were isolated, and the rate of protein synthesis was calculated as the ratio of the isotopic enrichment in proteins to the enrichment of the precursor pool of free amino acids. Protein synthesis rates in tibialis anterior muscle are expressed as %/d. The activation of specific intracellular pathways regulating translational initiation in tibialis anterior muscle was assessed in old rats by measuring the phosphorylation state of key intermediates (E). Ratio of phosphorylated protein to total protein content was measured by Western blot. Muscle protein synthesis rate was also measured after food intake in old rats (F). Postprandial protein synthesis rates in tibialis anterior muscle are expressed as % of change compared with postabsorptive values. Values ± SEM for eight animals per group. *P < 0.05 age effect (same diet), ^$P < 0.05 diet effect (same age). LFD, low-fat diet; HFD, high-fat diet.

Figure 5

Triglyceride, diacyl-glycerol, and ceramide content in C2C12 myotubes incubated in the presence or absence of fatty acids and of an inhibitor of ceramide synthesis. C2C12 myotubes were incubated in the presence or absence of palmitate (0.375 mm), oleate (0.375 mm), or fumonisin B1 (FB1, 50 μm) for 16 h before lipid extraction. Triglyceride (A), diacyl-glycerol (B), and ceramide (C) concentrations were analyzed by gas-liquid chromatography. Ceramide, diacyl-glycerol, and triglyceride concentrations are expressed as nmoles/mg proteins. Data are from six independent experiments. Values ± SEM. *P < 0.05 vs. control cells, ^$P < 0.05 vs. cells treated with palmitate.

Figure 6

Protein synthesis rate and activation of intracellular pathways related to the regulation of translational initiation in C2C12 myotubes incubated in the presence or absence of fatty acids and of pharmacological mediators of intracellular ceramide content. C2C12 myotubes were incubated in the presence or absence of palmitate (0.375 mm), oleate (0.375 mm), and fumonisin B1 (FB1, 50 μm) for 16 h or C2-ceramide (100 μm) for 30 min before insulin stimulation (100 nm). Proteins were isolated, and protein synthesis rate (A) was determined as in Fig.4. Protein synthesis rates in C2C12 myotubes are expressed as %/h. Data are from six independent experiments. The activation of specific intracellular pathways regulating translational initiation was assessed by measuring the phosphorylation state of key intermediates (B,C). Ratio of phosphorylated protein to total protein content was measured by Western blot. The implication of eIF2α in the regulation of protein translation in C2C12 myotubes treated with fatty acids was confirmed by measuring the mRNA expression of CHOP gene (D), one of the targets of eIF2α. All the data are from six independent experiments. Values ± SEM. *P < 0.05 vs. control cells, ^$P < 0.05 vs. cell treated with palmitate, and ^#P < 0.05 vs. cells treated with palmitate and FB1.