Christophe Giraudet

Impaired anabolic response of muscle protein synthesis is associated with S6K1 dysregulation in elderly humans

Age‐related loss of muscle protein may involve a decreased response to anabolic factors of muscle protein synthesis through dysregulation of translation factors. To verify this hypothesis, we simultaneously investigated muscle protein synthesis and expression of some factors implicated in insulin signal transduction during hyperinsulinemia and hyperaminoacidemia in 6 young (25±1 year; mean±SEM) and 8 elderly subjects (72±2 year). Incorporation of L‐[1‐13C] leucine in muscle proteins (fractional synthesis rate, FSR) was measured in vastus lateralis, before and during a euglycemic hyperinsulinemic hyperaminoacidemic clamp, together with Western blot analysis of protein kinase B (PKB), mTOR, 4E‐BP1, and S6K1 phosphorylation. In basal state, muscle protein FSR was reduced in elderly in comparison with young subjects (0.061±0.004% per hour) vs 0.082±0.010% per hour, elderly vs. young, P<0.05). During clamp, muscle protein FSR was stimulated in young (0.119±0.006% per hour; P<0.05), but this response was significantly lower in elderly subjects (0.084±0.005% per hour, P<0.05 vs young subjects). Phosphorylation of PKB, mTOR, and 4E‐BP1 were similarly increased by insulin and amino acid in both groups, except for S6K1 phosphorylation, which was not stimulated in elderly subjects. In conclusion, 1) response of muscle protein synthesis to insulin and amino acid is impaired in elderly humans; 2) a defect in S6K1 pathway activation may be responsible for this alteration. This modification is a mechanistic basis of sarcopenia development during aging.

Leucine supplementation improves muscle protein synthesis in elderly men independently of hyperaminoacidaemia

The present study was designed to assess the effects of dietary leucine supplementation on muscle protein synthesis and whole body protein kinetics in elderly individuals. Twenty healthy male subjects (70 ± 1 years) were studied before and after continuous ingestion of a complete balanced diet supplemented or not with leucine. A primed (3.6 μmol kg−1) constant infusion (0.06 μmol kg−1 min−1) of l‐[1‐13C]phenylalanine was used to determine whole body phenylalanine kinetics as well as fractional synthesis rate (FSR) in the myofibrillar fraction of muscle proteins from vastus lateralis biopsies. Whole body protein kinetics were not affected by leucine supplementation. In contrast, muscle FSR, measured over the 5‐h period of feeding, was significantly greater in the volunteers given the leucine‐supplemented meals compared with the control group (0.083 ± 0.008 versus 0.053 ± 0.009% h−1, respectively, P < 0.05). This effect was due only to increased leucine availability because only plasma free leucine concentration significantly differed between the control and leucine‐supplemented groups. We conclude that leucine supplementation during feeding improves muscle protein synthesis in the elderly independently of an overall increase of other amino acids. Whether increasing leucine intake in old people may limit muscle protein loss during ageing remains to be determined.

Changes in Basal and Insulin and Amino Acid Response of Whole Body and Skeletal Muscle Proteins in Obese Men

CONTEXT Obesity-related insulin resistance of glucose and lipid metabolism may also affect protein kinetics, notably at the muscle level. OBJECTIVE We hypothesized that muscle protein response to insulin and amino acid is blunted during obesity. RESEARCH DESIGN AND METHODS Total (Tot) and mitochondrial (Mit) muscle proteins fractional synthesis rates (FSR) together with whole-body protein kinetics (WB) have been determined in postabsorptive state (PA) and during a hyperinsulinemic, hyperaminoacidemic, euglycemic clamp by using a continuous infusion of (13)C-leucine in six obese and eight nonobese subjects. RESULTS Responses of WB glucose disposal rate and protein breakdown to insulin and amino acid infusion were significantly lower in obese than in nonobese subjects (P < 0.05). In PA, Tot and Mit FSR were significantly lower (P < 0.05) in obese (Tot, 0.044 +/- 0.005% . h(-1); Mit, 0.064 +/- 0.008% . h(-1)) in comparison with nonobese subjects (Tot, 0.082 +/- 0.010% . h(-1); Mit, 0.140 +/- 0.006% . h(-1)). Tot FSR was similarly stimulated by insulin and amino acid in both groups (0.094 +/- 0.013 vs. 0.117 +/- 0.006% . h(-1), obese vs. nonobese; P < 0.05). Mit FSR was increased in nonobese subjects (0.179 +/- 0.007% . h(-1); P < 0.05) but not in obese subjects (0.078 +/- 0.012% . h(-1); P = not significant). CONCLUSIONS The obesity-related impairment of protein metabolism is characterized by 1) a reduced turnover rate of skeletal muscle proteins in PA; 2) a lack of stimulation of mitochondrial protein synthesis by insulin and amino acid; and 3) a lower inhibition of WB proteolysis by insulin and amino acid. Alterations of selective muscle protein kinetics may predispose obese subjects to muscle metabolic dysfunction leading to type 2 diabetes.

1,25(OH)2-vitamin D3 enhances the stimulating effect of leucine and insulin on protein synthesis rate through Akt/PKB and mTOR mediated pathways in murine C2C12 skeletal myotubes

SCOPE In recent years, there has been a growing body of evidence pointing to an effect of vitamin D on muscle mass and function. Our aim was to investigate the combined effect of 1,25(OH)2-vitamin D3 (1,25(OH)2D3) with anabolic factors insulin and leucine on protein fractional synthesis rate (FSR) and regulation in the mouse C2C12 myotube. METHODS AND RESULTS After differentiation, myotubes were cultured in 1,25(OH)2D3 solutions at 0, 1, or 10 nM for 72 h. Cells were treated by L-[1-(13) C]valine and puromycin in presence or not of leucine and insulin, and protein FSR was determined by measuring tracer enrichments and puromycin incorporation in proteins, respectively. Protein expression and phosphorylation state of insulin receptor (IR), Akt, GSK3, mTOR, p70 S6 kinase, rpS6, and 4EBP1 were measured by Western blot. Transcript levels of IR and 1,25(OH)2D3 receptor (VDR) were determined by qPCR. 1,25(OH)2D3 (10 nM) with leucine and insulin increased protein FSR in C2C12 myotubes (14-16%). IR and VDR mRNA expression was increased with 1,25(OH)2D3 treatment. The Akt/mTOR-dependent pathway was activated by insulin and leucine and further enhanced by 1,25(OH)2D3. CONCLUSION 1,25(OH)2D3 sensitizes the Akt/mTOR-dependant pathway to the stimulating effect of leucine and insulin, resulting in a further activation of protein synthesis in murine C2C12 skeletal myotubes.

Synergistic effects of caloric restriction with maintained protein intake on skeletal muscle performance in 21-month-old rats: a mitochondria-mediated pathway

Caloric restriction (CR) delays the onset of age‐related mitochondrial abnormalities but does not prevent the decline in ATP production needed to sustain muscle protein fractional synthesis rate (FSR) and contractile activity. We hypothesized that improving mitochondrial activity and FSR using a CR diet with maintained protein intakes could enhance myofibrillar protein FSR and consequently improve muscle strength in aging rats. Wistar rats (21 months old) were fed either an ad libitum (AL), 40% protein‐energy restricted (PER) or 40% AL‐isonitrogenous energy restricted (ER) diet for 5 months. ATP production, electron transport chain activity, reactive oxygen species (ROS) generation, protein carbonyl content and FSR were determined in both tibialis anterior (TA) and soleus muscle mitochondria. Myosin and actin FSR and grip force were also investigated. The ER diet led to improved mitochondrial activity and ATP production in the TA and soleus muscles in comparison with PER. Furthermore, mitochondrial FSR in the TA was enhanced under the ER diet but diminished under the PER. Mitochondrial protein carbonyl content was decreased by both the ER and PER diets. The ER diet was able to improve myosin and actin FSR and grip force. Therefore, the synergistic effects of CR with maintained protein intake may help to limit the progression of sarcopenia by optimizing the turnover rates and functions of major proteins in skeletal muscle.—Zangarelli, A., Chanseaume, E., Morio, B., Brugère, C., Mosoni, L., Rousset, P., Giraudet, C., Patrac, V., Gachon, P., Boirie, Y., Walrand, S. Synergistic effects of caloric restriction with maintained protein intake on skeletal muscle performance in 21‐month‐old rats: a mitochondria‐mediated pathway. FASEB J. 20, 2439–2450 (2006)

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

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.

Chronological Approach of Diet-induced Alterations in Muscle Mitochondrial Functions in Rats

Objective: Mitochondrial dysfunction might predispose individuals to develop insulin resistance. Our objective was to determine whether mitochondrial dysfunction or insulin resistance was the primary event during high‐fat (HF) diet.

Dairy and industrial sources of trans fat do not impair peripheral insulin sensitivity in overweight women

BACKGROUND The 2 major dietary sources of trans fatty acids (TFAs) are partially hydrogenated oils and ruminant-derived products. Epidemiologic data suggest that chronic consumption of industrial sources of TFAs could be damaging to insulin sensitivity, but intervention studies on this issue have remained inconclusive. OBJECTIVE The trial was designed to compare the effects of dairy compared with industrial sources of TFAs on insulin sensitivity in overweight women. DESIGN Sixty-three healthy women with abdominal obesity [waist circumference >88 cm and a body mass index (in kg/m(2)) >28] were recruited. After a run-in period, the volunteers were randomly assigned to consume 1 of 3 four-week diets: 60 g low-TFA lipids/d (0.54 g/d; n = 21), ruminant TFA-rich lipids (4.86 g/d; n = 21), or industrial TFA-rich lipids (5.58 g/d; n = 21). Changes in peripheral insulin sensitivity were assessed by using hyperinsulinemic-euglycemic clamps. RESULTS After the intervention period, fasting glycemia and insulinemia and insulin sensitivity were not significantly modified in either group (P > 0.05). CONCLUSIONS These data indicate that consumption of dairy- and industrial-source TFAs for 4 wk at nutritional levels do not impair peripheral insulin sensitivity in insulin-resistant women. Our study may not preassess the effects of TFAs in normal insulin-sensitive individuals. This trial was registered at ClinicalTrials.gov as NCT00617435.

Time-course changes of muscle protein synthesis associated with obesity-induced lipotoxicity

•  Prolonged obesity leads to ectopic lipid accumulation in non‐adipose tissues, particularly in skeletal muscles, inducing metabolic dysfunctions (reduced glucose uptake, mitochondria dysfunction, lipotoxicity). •  Several studies in humans and rodents have shown that obesity induces a short‐term increase in fat‐free mass but a long‐term decrease in skeletal muscle mass. •  We investigated the mechanisms potentially involved in muscle loss by measuring simultaneously protein synthesis and lipid infiltration in different types of skeletal muscles, during the development of obesity. •  Our results show that protein synthesis rate in glycolytic muscles increased together with muscle mass during the early phase of obesity development, whereas it decreased later. Reduced protein synthesis rate was associated with a high lipid accumulation in glycolytic muscles. •  These results suggest that lipid accumulation in muscles during prolonged obesity is deleterious for amino acid incorporation in skeletal muscle proteins, and thus indirectly for muscle mass.

TNFα gene knockout differentially affects lipid deposition in liver and skeletal muscle of high-fat-diet mice

AIMS/HYPOTHESIS Inflammation and ectopic lipid deposition contribute to obesity-related insulin resistance (IR). Studies have shown that deficiency of the proinflammatory cytokine tumor necrosis factor-α (TNFα) protects against the IR induced by a high-fat diet (HFD). We aimed to evaluate the relationship between HFD-related inflammation and lipid deposition in skeletal muscle and liver. EXPERIMENTAL DESIGN Wild-type (WT) and TNFα-deficient (TNFα-KO) mice were subjected to an HFD for 12 weeks. A glucose tolerance test was performed to evaluate IR. Inflammatory status was assessed by measuring plasma and tissue transcript levels of cytokines. Lipid intermediate concentrations were measured in plasma, muscle and liver. The expression of genes involved in fatty acid transport, synthesis and oxidation was analyzed in adipose tissue, muscle and liver. RESULTS HFD induced a higher body weight gain in TNFα-KO mice than in WT mice. The weight of epididymal and abdominal adipose tissues was twofold lower in WT mice than in TNFα-KO mice, whereas liver weight was significantly heavier in WT mice. IR, systemic and adipose tissue inflammation, and plasma nonesterified fatty acid levels were reduced in TNFα-KO mice fed an HFD. TNFα deficiency improved fatty acid metabolism and had a protective effect against lipid deposition, inflammation and fibrosis associated with HFD in liver but had no impact on these markers in muscle. CONCLUSIONS Our data suggest that in an HFD context, TNFα deficiency reduced hepatic lipid accumulation through two mechanisms: an increase in adipose tissue storage capacity and a decrease in fatty acid uptake and synthesis in the liver.