Jérôme Salles

Physiopathological Mechanism of Sarcopenia

The etiology of sarcopenia is multifactorial but still poorly understood, and the sequelae of this phenomenon represent a major public health issue. Age-related loss of muscle mass can be counteracted by adequate metabolic interventions including nutritional intake and exercise training. Other strategies including changes in daily protein pattern, the speed of protein digestion, or specific amino acid supplementation may be beneficial to improve short-term muscle anabolic response in elderly people. A multimodal approach combining nutrition, exercise, hormones, and specific anabolic drugs may be an innovative treatment for limiting the development of sarcopenia with aging.

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.

Paclitaxel therapy potentiates cold hyperalgesia in streptozotocin-induced diabetic rats through enhanced mitochondrial reactive oxygen species production and TRPA1 sensitization

Summary Paclitaxel potentiates cold hyperalgesia in streptozotocin‐induced diabetic rats through the combination of paclitaxel‐induced increase in mitochondrial reactive oxygen species and diabetes‐related overexpression of TRPA1. Abstract Diabetes comorbidities include disabling peripheral neuropathy (DPN) and an increased risk of developing cancer. Antimitotic drugs, such as paclitaxel, are well known to facilitate the occurrence of peripheral neuropathy. Practitioners frequently observe the development or co‐occurrence of enhanced DPN, especially cold sensitivity, in diabetic patients during chemotherapy. Preclinical studies showed that reactive oxygen species (ROS) and cold activate transient receptor potential ankyrin‐1 (TRPA1) cation channels, which are involved in cold‐evoked pain transduction signaling in DPN. Additionally, paclitaxel treatment has been associated with an accumulation of atypical mitochondria in the sensory nerves of rats. We hypothesized that paclitaxel might potentiate cold hyperalgesia by increasing mitochondrial injuries and TRPA1 activation. Thus, the kinetics of paclitaxel‐induced cold hyperalgesia, mitochondrial ROS production, and TRPA1 expression were evaluated in dorsal root ganglia of normoglycemic and streptozotocin‐induced diabetic rats. In diabetic rats, paclitaxel significantly enhanced cold hyperalgesia in comparison to normoglycemic paclitaxel‐treated control rats. These effects were prevented by N‐acetyl‐cysteine, a reducing agent, and by HC030031, an antagonist of TRPA1. In diabetic and control rats, paclitaxel treatment was associated with an accumulation of atypical mitochondria and a 2‐fold increase in mitochondrial ROS production. Moreover, mRNA levels of glutathione peroxidase 4 and glutathione‐S‐reductase were significantly lower in diabetic groups treated with paclitaxel. Finally, TRPA1 gene expression was enhanced by 45% in diabetic rats. Paclitaxel potentiation of cold hyperalgesia in diabetes may result from the combination of increased mitochondrial ROS production and poor radical detoxification induced by paclitaxel treatment and diabetes‐related overexpression of TRPA1.

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.

Chemokine Expression in Inflamed Adipose Tissue Is Mainly Mediated by NF-κB

Immune cell infiltration of expanding adipose tissue during obesity and its role in insulin resistance has been described and involves chemokines. However, studies so far have focused on a single chemokine or its receptor (especially CCL2 and CCL5) whereas redundant functions of chemokines have been described. The objective of this work was to explore the expression of chemokines in inflamed adipose tissue in obesity. Human and mouse adipocytes were analyzed for expression of chemokines in response to inflammatory signal (TNF-α) using microarrays and gene set enrichment analysis. Gene expression was verified by qRT-PCR. Chemokine protein was determined in culture medium with ELISA. Chemokine expression was investigated in human subcutaneous adipose tissue biopsies and mechanism of chemokine expression was investigated using chemical inhibitors and cellular and animal transgenic models. Chemokine encoding genes were the most responsive genes in TNF-α treated human and mouse adipocytes. mRNA and protein of 34 chemokine genes were induced in a dose-dependent manner in the culture system. Furthermore, expression of those chemokines was elevated in human obese adipose tissue. Finally, chemokine expression was reduced by NF-κB inactivation and elevated by NF-κB activation. Our data indicate that besides CCL2 and CCL5, numerous other chemokines such as CCL19 are expressed by adipocytes under obesity-associated chronic inflammation. Their expression is regulated predominantly by NF-κB. Those chemokines could be involved in the initiation of infiltration of leukocytes into obese adipose tissue.

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.

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.

Oleate-enriched diet improves insulin sensitivity and restores muscle protein synthesis in old rats

BACKGROUND & AIMS Age-related inflammation and insulin resistance (IR) have been implicated in the inability of old muscles to properly respond to anabolic stimuli such as amino acids (AA) or insulin. Since fatty acids can modulate inflammation and IR in muscle cells, we investigated the effect of palmitate-enriched diet and oleate-enriched diet on inflammation, IR and muscle protein synthesis (MPS) rate in old rats. METHODS Twenty-four 25-month-old rats were fed either a control diet (OC), an oleate-enriched diet (HFO) or a palmitate-enriched diet (HFP) for 16 weeks. MPS using labeled amino acids and mTOR activation were assessed after AA and insulin anabolic stimulation to mimic postprandial state. RESULTS IR and systemic and adipose tissue inflammation (TNFα and IL1β) were improved in the HFO group. Muscle genes controlling mitochondrial β-oxidation (PPARs, MCAD and CPT-1b) were up-regulated in the HFO group. AA and insulin-stimulated MPS in the HFO group only, and this stimulation was related to activation of the Akt/mTOR pathway. CONCLUSIONS The age-related MPS response to anabolic signals was improved in rats fed an oleate-enriched diet. This effect was related to activation of muscle oxidative pathways, lower IR, and a decrease in inflammation.

Effects of trans MUFA from dairy and industrial sources on muscle mitochondrial function and insulin sensitivity.

Epidemiological studies suggest that chronic consumption of trans MUFA may alter muscle insulin sensitivity. The major sources of dietary trans MUFA (dairy fat vs. industrially hydrogenated oils) have different isomeric profiles and thus probably different metabolic consequences. These effects may involve alterations in muscle mitochondrial oxidative capacity, which may in turn promote insulin resistance if fatty acid oxidation is reduced. We report that in Wistar rats, an 8 week diet enriched (4% of energy intake) in either dairy, industrial, or control MUFA did not alter insulin and glucose responses to an intraperitoneal glucose tolerance test (1g/kg). In C2C12 myotubes, vaccenic and elaidic acids did not modify insulin sensitivity compared with oleic acid. Furthermore, the ex vivo total, mitochondrial and peroxisomal oxidation rates of [1-14C]oleic, vaccenic, and elaidic acids were similar in soleus and tibialis anterior rat muscle. Finally, an 8 week diet enriched in either dairy or industrial trans MUFA did not alter mitochondrial oxidative capacity in these two muscles compared with control MUFA but did induce a specific reduction in soleus mitochondrial ATP and superoxide anion production (P < 0.01 vs. control). In conclusion, dietary trans MUFA of dairy or industrial origin have similar effects and do not impair muscle mitochondrial capacity and insulin sensitivity.

Cumulative 3-nitrotyrosine in specific muscle proteins is associated with muscle loss during aging.

Post-translational oxidative protein modifications which are more marked during aging and/or high-calorie (HC) diets affect protein function and metabolism. Protein function and metabolism are different according to the type of muscle proteins. Oxidative muscle protein modifications may thus be associated with age-related sarcopenia, and HC may be implicated in the development of sarcopenia by emphasizing protein modifications. Understanding the role of protein modifications in the process of sarcopenia and metabolism associated with a high fat diet may be elucidated by investigations with skeletal muscle protein subfractionations. To study this hypothesis, carbonylated protein (CP) and 3-nitrotyrosine (3-NT) levels were measured in mixed, sarcoplasmic, myofibrillar and mitochondrial protein fractions of quadriceps in rats aged 6months (A) and 25months (O) fed a normal calorie (NC) or HC diet for 3months (AN, AH, ON, OH n=7-8). Muscle weight was lower in the older rats (AN: 0.79±0.03g, ON: 0.43±0.12g, P<0.05), but no HC effect was observed. CP did not differ between groups while 3-NT accumulated significantly in ON compared with AN, especially in mitochondria (2.4±0.5, 1.3±0.1, 1.9±0.4, 2.9±1.2 -fold in mixed, sarcoplasmic, myofibrillar and mitochondrial fractions respectively, P<0.05). 3-NT in mixed protein was negatively correlated with muscle mass (r(2)=-0.812). 3-NT accumulation during HC was observed only in specific proteins of mitochondria (100kDa) (1.0±0.6, 1.7±0.9, 3.3±1.4 and 7.0±2.5 -fold in AN, AH, ON and OH, respectively, P<0.05). Hence cumulative 3-NT in skeletal muscle protein appears associated with the development of age-related muscle loss. Mitochondrial proteins are more prone to nitration during aging and nutritional stress.