Skeletal Muscle Proteome Modifications following Antibiotic-Induced Microbial Disturbances in Cancer Cachexia.

Cancer cachexia is an involuntary loss of body weight, mostly of skeletal muscle. Previous research favors the existence of a microbiota-muscle crosstalk, so the aim of the study was to evaluate the impact of microbiota alterations induced by antibiotics on skeletal muscle proteins expression. Skeletal muscle proteome changes were investigated in control (CT) or C26 cachectic mice (C26) with or without antibiotic treatment (CT-ATB or C26-ATB, n = 8 per group). Muscle protein extracts were divided into a sarcoplasmic and myofibrillar fraction and then underwent label-free liquid chromatography separation, mass spectrometry analysis, Mascot protein identification, and METASCAPE platform data analysis. In C26 mice, the atrogen mafbx expression was 353% higher than CT mice and 42.3% higher than C26-ATB mice. No effect on the muscle protein synthesis was observed. Proteomic analyses revealed a strong effect of antibiotics on skeletal muscle proteome outside of cachexia, with adaptative processes involved in protein folding, growth, energy metabolism, and muscle contraction. In C26-ATB mice, proteome adaptations observed in CT-ATB mice were blunted. Differentially expressed proteins were involved in other processes like glucose metabolism, oxidative stress response, and proteolysis. This study confirms the existence of a microbiota-muscle axis, with a muscle response after antibiotics that varies depending on whether cachexia is present.

Cannabidiol protects C2C12 myotubes against cisplatin-induced atrophy by regulating oxidative stress.

Cancer and chemotherapy induce a severe loss of muscle mass (known as cachexia), which negatively impact cancer treatment and patient survival. The aim of the present study was to investigate whether cannabidiol (CBD) administration may potentially antagonize the effects of cisplatin in inducing muscle atrophy, using a model of myotubes in culture. Cisplatin treatment resulted in a reduction of myotube diameter (15.7 ± 0.3 vs. 22.2 ± 0.5 µm, P < 0.01) that was restored to control level with 5 µM CBD (20.1 ± 0.4 µM, P < 0.01). Protein homeostasis was severely altered with a ≈70% reduction in protein synthesis (P < 0.01) and a twofold increase in proteolysis (P < 0.05) in response to cisplatin. Both parameters were dose dependently restored by CBD cotreatment. Cisplatin treatment was associated with increased thiobarbituric acid reactive substances (TBARS) content (0.21 ± 0.03 to 0.48 ± 0.03 nmol/mg prot, P < 0.05), catalase activity (0.24 ± 0.01 vs. 0.13 ± 0.02 nmol/min/µg prot, P < 0.01), whereas CBD cotreatment normalized TBARS content to control values (0.22 ± 0.01 nmol/mg prot, P < 0.01) and reduced catalase activity (0.17 ± 0.01 nmol/min/µg prot, P < 0.05). These changes were associated with increased mRNA expression of GPX1, SOD1, SOD2, and CAT mRNA expression in response to cisplatin (P < 0.01), which was corrected by CBD cotreatment (P < 0.05). Finally, cisplatin treatment increased the mitochondrial protein content of NDUFB8, UQCRC2, COX4, and VDAC1 (involved in mitochondrial respiration and apoptosis), and CBD cotreatment restored their expression to control values. Altogether, our results demonstrated that CBD antagonize the cisplatin-induced C2C12 myotube atrophy and could be used as an adjuvant in the treatment of cancer cachexia to help maintain muscle mass and improve patient quality of life.NEW & NOTEWORTHY In an in vitro model, cisplatin treatment led to myotube atrophy associated with dysregulation of protein homeostasis and increased oxidative stress, resulting in increased apoptosis. Cotreatment with cannabidiol was able to prevent this phenotype by promoting protein homeostasis and reducing oxidative stress.

Associating Inulin with a Pea Protein Improves Fast-Twitch Skeletal Muscle Mass and Muscle Mitochondrial Activities in Old Rats.

Aging is associated with a decline in muscle mass and function, leading to increased risk for mobility limitations and frailty. Dietary interventions incorporating specific nutrients, such as pea proteins or inulin, have shown promise in attenuating age-related muscle loss. This study aimed to investigate the effect of pea proteins given with inulin on skeletal muscle in old rats. Old male rats (20 months old) were randomly assigned to one of two diet groups for 16 weeks: a 'PEA' group receiving a pea-protein-based diet, or a 'PEA + INU' group receiving the same pea protein-based diet supplemented with inulin. Both groups showed significant postprandial stimulation of muscle p70 S6 kinase phosphorylation rate after consumption of pea proteins. However, the PEA + INU rats showed significant preservation of muscle mass with time together with decreased MuRF1 transcript levels. In addition, inulin specifically increased PGC1-α expression and key mitochondrial enzyme activities in the plantaris muscle of the old rats. These findings suggest that dietary supplementation with pea proteins in combination with inulin has the potential to attenuate age-related muscle loss. Further research is warranted to explore the underlying mechanisms and determine the optimal dosage and duration of intervention for potential translation to human studies.

Vitamin D status modulates mitochondrial oxidative capacities in skeletal muscle: role in sarcopenia.

Skeletal muscle mitochondrial function is the biggest component of whole-body energy output. Mitochondrial energy production during exercise is impaired in vitamin D-deficient subjects. In cultured myotubes, loss of vitamin D receptor (VDR) function decreases mitochondrial respiration rate and ATP production from oxidative phosphorylation. We aimed to examine the effects of vitamin D deficiency and supplementation on whole-body energy expenditure and muscle mitochondrial function in old rats, old mice, and human subjects. To gain further insight into the mechanisms involved, we used C2C12 and human muscle cells and transgenic mice with muscle-specific VDR tamoxifen-inducible deficiency. We observed that in vivo and in vitro vitamin D fluctuations changed mitochondrial biogenesis and oxidative activity in skeletal muscle. Vitamin D supplementation initiated in older people improved muscle mass and strength. We hypothesize that vitamin D supplementation is likely to help prevent not only sarcopenia but also sarcopenic obesity in vitamin D-deficient subjects.

Characterization of the Skeletal Muscle Proteome in Undernourished Old Rats.

Aging is associated with a progressive loss of skeletal muscle mass and function termed sarcopenia. Various metabolic alterations that occur with aging also increase the risk of undernutrition, which can worsen age-related sarcopenia. However, the impact of undernutrition on aged skeletal muscle remains largely under-researched. To build a deeper understanding of the cellular and molecular mechanisms underlying age-related sarcopenia, we characterized the undernutrition-induced changes in the skeletal muscle proteome in old rats. For this study, 20-month-old male rats were fed 50% or 100% of their spontaneous intake for 12 weeks, and proteomic analysis was performed on both slow- and fast-twitch muscles. Proteomic profiling of undernourished aged skeletal muscle revealed that undernutrition has profound effects on muscle proteome independently of its effect on muscle mass. Undernutrition-induced changes in muscle proteome appear to be muscle-type-specific: slow-twitch muscle showed a broad pattern of differential expression in proteins important for energy metabolism, whereas fast-twitch muscle mainly showed changes in protein turnover between undernourished and control rats. This first proteomic analysis of undernourished aged skeletal muscle provides new molecular-level insight to explain phenotypic changes in undernourished aged muscle. We anticipate this work as a starting point to define new biomarkers associated with undernutrition-induced muscle loss in the elderly.

Pea Proteins Have Anabolic Effects Comparable to Milk Proteins on Whole Body Protein Retention and Muscle Protein Metabolism in Old Rats.

Plant proteins are attracting rising interest due to their pro-health benefits and environmental sustainability. However, little is known about the nutritional value of pea proteins when consumed by older people. Herein, we evaluated the digestibility and nutritional efficiency of pea proteins compared to casein and whey proteins in old rats. Thirty 20-month-old male Wistar rats were assigned to an isoproteic and isocaloric diet containing either casein (CAS), soluble milk protein (WHEY) or Pisane™ pea protein isolate for 16 weeks. The three proteins had a similar effect on nitrogen balance, true digestibility and net protein utilization in old rats, which means that different protein sources did not alter body composition, tissue weight, skeletal muscle protein synthesis or degradation. Muscle mitochondrial activity, inflammation status and insulin resistance were similar between the three groups. In conclusion, old rats used pea protein with the same efficiency as casein or whey proteins, due to its high digestibility and amino acid composition. Using these plant-based proteins could help older people diversify their protein sources and more easily achieve nutritional intake recommendations.

Deleterious Effect of High-Fat Diet on Skeletal Muscle Performance Is Prevented by High-Protein Intake in Adult Rats but Not in Old Rats.

The phenotype of sarcopenic obesity is frequently associated with impaired muscle strength and performance. Ectopic lipid deposition may interfere with muscle anabolic response especially during aging. Evidence is scarce concerning the potential interplay among aging and nutrient imbalance on skeletal muscle functionality. The objective of the present study was to investigate the impact of protein intake in the context of an obesogenic diet on skeletal muscle functional properties and intramuscular lipid infiltration. Two groups of forty-two adult and thirty-seven old male Wistar rats were randomly divided into four groups: isocaloric standard diet (12% protein, 14% lipid, as ST12); isocaloric standard (high-protein) diet (25% protein, 14% lipid, ST25); hypercaloric high-fat (normal-protein) diet (12% protein, 45% lipid, HF12); and hypercaloric high-fat (high-protein) diet (25% protein, 45% lipid, HF25). The nutritional intervention lasted 10 weeks. Total body composition was measured through Echo-MRI. Lipids were extracted from tibialis anterior muscle and analyzed by gas-liquid chromatography. The functional properties of the plantarflexor muscles were evaluated in vivo on an isokinetic dynamometer. Maximal torque was assessed from the torque-frequency relationship in isometric condition and maximal power was evaluated from the torque-velocity relationship in concentric condition. In adult rats high-protein intake combined with high-fat diet determined a lower decrease in relative isometric torque, normalized to either FFM or body weight, compared with adult rats fed a high-fat normal-protein diet. High-fat diet was also detrimental to relative muscle power, as normalized to body weight, that decreased to a larger extent in adult rats fed a high-fat normal-protein diet than their counterparts fed a normal-fat, high-protein diet. The effect of high-fat diet observed in adults, with the enhanced protein intake (25%) conferring some kind of protection against the negative effects of HFD, may be linked to the reduced intramuscular fat in this group, which may have contributed to preserve, at least partly, the contractile properties. A potential role for high-protein diet in preventing ectopic lipid deposition needs to be explored in future research. Detrimental effects of high- fat diet on skeletal muscle performance are mitigated by high- protein intake in adult rats but not in old rats.

Anabolic Properties of Mixed Wheat-Legume Pasta Products in Old Rats: Impact on Whole-Body Protein Retention and Skeletal Muscle Protein Synthesis.

The mechanisms that are responsible for sarcopenia are numerous, but the altered muscle protein anabolic response to food intake that appears with advancing age plays an important role. Dietary protein quality needs to be optimized to counter this phenomenon. Blending different plant proteins is expected to compensate for the lower anabolic capacity of plant-based when compared to animal-based protein sources. The objective of this work was to evaluate the nutritional value of pasta products that were made from a mix of wheat semolina and faba bean, lentil, or split pea flour, and to assess their effect on protein metabolism as compared to dietary milk proteins in old rats. Forty-three old rats have consumed for six weeks isoproteic and isocaloric diets containing wheat pasta enriched with 62% to 79% legume protein (depending on the type) or milk proteins, i.e., casein or soluble milk proteins (SMP). The protein digestibility of casein and SMP was 5% to 14% higher than legume-enriched pasta. The net protein utilization and skeletal muscle protein synthesis rate were equivalent either in rats fed legume-enriched pasta diets or those fed casein diet, but lower than in rats fed SMP diet. After legume-enriched pasta intake, muscle mass, and protein accretion were in the same range as in the casein and SMP groups. Mixed wheat-legume pasta could be a nutritional strategy for enhancing the protein content and improving the protein quality, i.e., amino acid profile, of this staple food that is more adequate for maintaining muscle mass, especially for older individuals.

4E-BP1 and 4E-BP2 double knockout mice are protected from aging-associated sarcopenia.

BACKGROUND: Sarcopenia is the loss of muscle mass/function that occurs during the aging process. The links between mechanistic target of rapamycin (mTOR) activity and muscle development are largely documented, but the role of its downstream targets in the development of sarcopenia is poorly understood. Eukaryotic initiation factor 4E-binding proteins (4E-BPs) are targets of mTOR that repress mRNA translation initiation and are involved in the control of several physiological processes. However, their role in skeletal muscle is still poorly understood. The goal of this study was to assess how loss of 4E-BP1 and 4E-BP2 expression impacts skeletal muscle function and homeostasis in aged mice and to characterize the associated metabolic changes by metabolomic and lipidomic profiling. METHODS: Twenty-four-month-old wild-type and whole body 4E-BP1/4E-BP2 double knockout (DKO) mice were used to measure muscle mass and function. Protein homeostasis was measured ex vivo in extensor digitorum longus by incorporation of l-[U-14 C]phenylalanine, and metabolomic and lipidomic profiling of skeletal muscle was performed by Metabolon, Inc. RESULTS: The 4E-BP1/2 DKO mice exhibited an increase in muscle mass that was associated with increased grip strength (P < 0.05). Protein synthesis was higher under both basal (+102%, P < 0.05) and stimulated conditions (+65%, P < 0.05) in DKO skeletal muscle. Metabolomic and complex lipid analysis of skeletal muscle revealed robust differences pertaining to amino acid homeostasis, carbohydrate abundance, and certain aspects of lipid metabolism. In particular, levels of most free amino acids were lower within the 4E-BP1/2 DKO muscle. Interestingly, although glucose levels were unchanged, differences were observed in the isobaric compound maltitol/lactitol (33-fold increase, P < 0.01) and in several additional carbohydrate compounds. 4E-BP1/2 depletion also resulted in accumulation of medium-chain acylcarnitines and a 20% lower C2/C0 acylcarnitine ratio (P < 0.01) indicative of reduced ß-oxidation. CONCLUSIONS: Taken together, these findings demonstrate that deletion of 4E-BPs is associated with perturbed energy metabolism in skeletal muscle and could have beneficial effects on skeletal muscle mass and function in aging mice. They also identify 4E-BPs as potential targets for the treatment of sarcopenia.

Nutritional evaluation of mixed wheat-faba bean pasta in growing rats: impact of protein source and drying temperature on protein digestibility and retention.

This study aimed to evaluate the nutritional value of pasta enriched with legume or wheat gluten proteins and dried at varying temperature. A total of four isonitrogenous experimental diets were produced using gluten powder/wheat semolina (6/94, g/g) pasta and faba bean flour/wheat semolina (35/65, g/g) pasta dried at either 55°C (GLT and FLT, respectively) or 90°C (FVHT and GVHT, respectively). Experimental diets were fed to ten 1-month-old Wistar rats (body weight=176 (sem 15) g) for 21 d. Growth and nutritional, metabolic and inflammatory markers were measured and compared with an isonitrogenous casein diet (CD). The enrichment with faba bean increased the lysine, threonine and branched amino acids by 97, 23 and 10 %, respectively. Protein utilisation also increased by 75 % (P<0·01) in FLT in comparison to GLT diet, without any effect on the corrected faecal digestibility (P>0·05). Faba bean pasta diets' corrected protein digestibility and utilisation was only 3·5 and 9 %, respectively, lower than the CD. Growth rate, blood composition and muscle weights were not generally different with faba bean pasta diets compared with CD. Corrected protein digestibility was 3 % lower in GVHT than GLT, which may be associated with greater carboxymethyllysine. This study in growing rats clearly indicates improvement in growth performance of rats fed legume-enriched pasta diet compared with rats fed gluten-wheat pasta diet, regardless of pasta drying temperature. This means faba bean flour can be used to improve the protein quality and quantity of pasta.

Supplementing Breakfast with a Vitamin D and Leucine-Enriched Whey Protein Medical Nutrition Drink Enhances Postprandial Muscle Protein Synthesis and Muscle Mass in Healthy Older Men.

Background: A promising strategy to help older adults preserve or build muscle mass is to optimize muscle anabolism through providing an adequate amount of high-quality protein at each meal.Objective: This "proof of principle" study investigated the acute effect of supplementing breakfast with a vitamin D and leucine-enriched whey protein medical nutrition drink on postprandial muscle protein synthesis and longer-term effect on muscle mass in healthy older adults.Methods: A randomized, placebo-controlled, double-blind study was conducted in 24 healthy older men [mean ± SD: age 71 ± 4 y; body mass index (in kg/m2) 24.7 ± 2.8] between September 2012 and October 2013 at the Unit of Human Nutrition, University of Auvergne, Clermont-Ferrand, France. Participants received a medical nutrition drink [test group; 21 g leucine-enriched whey protein, 9 g carbohydrates, 3 g fat, 800 IU cholecalciferol (vitamin D3), and 628 kJ] or a noncaloric placebo (control group) before breakfast for 6 wk. Mixed muscle protein fractional synthesis rate (FSR) was measured at week 0 in the basal and postprandial state, after study product intake with a standardized breakfast with the use of l-[2H5]-phenylalanine tracer methodology. The longer-term effect of the medical nutrition drink was evaluated by measurement of appendicular lean mass, representing skeletal muscle mass at weeks 0 and 6, by dual-energy X-ray absorptiometry.Results: Postprandial FSR (0-240 min) was higher in the test group than in the control group [estimate of difference (ED): 0.022%/h; 95% CI: 0.010%/h, 0.035%/h; ANCOVA, P = 0.001]. The test group gained more appendicular lean mass than the control group after 6 wk (ED: 0.37 kg; 95% CI: 0.03, 0.72 kg; ANCOVA, P = 0.035), predominantly as leg lean mass (ED: 0.30 kg; 95% CI: 0.03, 0.57 kg; ANCOVA, P = 0.034).Conclusions: Supplementing breakfast with a vitamin D and leucine-enriched whey protein medical nutrition drink stimulated postprandial muscle protein synthesis and increased muscle mass after 6 wk of intervention in healthy older adults and may therefore be a way to support muscle preservation in older people. This trial was registered at www.trialregister.nl as NTR3471.

Fast digestive, leucine-rich, soluble milk proteins improve muscle protein anabolism, and mitochondrial function in undernourished old rats.

SCOPE: One strategy to manage malnutrition in older patients is to increase protein and energy intake. Here, we evaluate the influence of protein quality during refeeding on improvement in muscle protein and energy metabolism. METHODS AND RESULTS: Twenty-month-old male rats (n = 40) were fed 50% of their spontaneous intake for 12 weeks to induce malnutrition, then refed ad libitum with a standard diet enriched with casein or soluble milk proteins (22%) for 4 weeks. A 13C-valine was infused to measure muscle protein synthesis and expression of MuRF1, and MAFbx was measured to evaluate muscle proteolysis. mTOR pathway activation and mitochondrial function were assessed in muscle. Malnutrition was associated with a decrease in body weight, fat mass, and lean mass, particularly muscle mass. Malnutrition decreased muscle mTOR pathway activation and protein FSR associated with increased MuRF1 mRNA levels, and decreased mitochondrial function. The refeeding period partially restored fat mass and lean mass. Unlike the casein diet, the soluble milk protein diet improved muscle protein metabolism and mitochondrial function in old malnourished rats. CONCLUSIONS: These results suggest that providing better-quality proteins during refeeding may improve efficacy of renutrition in malnourished older patients.

Muscle metabolic alterations induced by genetic ablation of 4E-BP1 and 4E-BP2 in response to diet-induced obesity.

SCOPE: In recent years, several studies reported the role of eIF4E-binding proteins (4E-BPs) on the development of diet-induced obesity and insulin resistance. Our aim was to investigate the effect of 4E-BP protein deletion on lipid accumulation and metabolism in skeletal muscle in response to a high-fat diet induced obesity in 4E-BP1/2 DKO mice. METHODS AND RESULTS: Diet-induced obesity engendered increased ectopic accumulation of lipotoxic species in skeletal muscle of 4E-BP1 and 4E-BP2 double knockout mice (4E-BP1/2 DKO), namely diacylglycerols and ceramides. Increased lipid accumulation was associated with alterations in the expression of genes involved in fatty acid transport (FATP, CD36), diacylglycerol/triacylglycerol biosynthesis (GPAT1, AGPAT1, DGAT1), and ß-oxidation (CPT1b, MCAD). Diet-induced obesity resulted in increased lean mass and muscle in 4E-BP1/2 DKO mice despite the development of a more severe systemic insulin resistance. Since increased expression of genes of several proteolytic systems (MuRF1, atrogin/MAFbx, and cathepsin-l) in 4EBP1/2 DKO skeletal muscle was reported, the increase of skeletal muscle mass in 4E-BP1/2 DKO mice suggests that ablation of 4E-BPs compensate with activation of muscle anabolism. CONCLUSIONS: These findings indicate that 4E-BP proteins may prevent excess lipid accumulation in skeletal muscle and suggest that 4E-BPs are key regulators of muscle homeostasis regardless of insulin sensitivity.

Vitamin D supplementation restores the blunted muscle protein synthesis response in deficient old rats through an impact on ectopic fat deposition.

We investigated the impact of vitamin D deficiency and repletion on muscle anabolism in old rats. Animals were fed a control (1 IU vitamin D3/g, ctrl, n=20) or a vitamin D-depleted diet (VDD; 0 IU, n=30) for 6 months. A subset was thereafter sacrificed in the control (ctrl6) and depleted groups (VDD6). Remaining control animals were kept for 3 additional months on the same diet (ctrl9), while a part of VDD rats continued on a depleted diet (VDD9) and another part was supplemented with vitamin D (5 IU, VDS9). The ctr16 and VDD6 rats and the ctr19, VDD9 and VDS9 rats were 21 and 24 months old, respectively. Vitamin D status, body weight and composition, muscle strength, weight and lipid content were evaluated. Muscle protein synthesis rate (fractional synthesis rate; FSR) and the activation of controlling pathways were measured. VDD reduced plasma 25(OH)-vitamin D, reaching deficiency (<25 nM), while 25(OH)-vitamin D increased to 118 nM in the VDS group (P<.0001). VDD animals gained weight (P<.05) with no corresponding changes in lean mass or muscle strength. Weight gain was associated with an increase in fat mass (+63%, P<.05), intramyocellular lipids (+75%, P<.05) and a trend toward a decreased plantaris weight (-19%, P=.12). Muscle FSR decreased by 40% in the VDD group (P<.001), but was restored by vitamin D supplementation (+70%, P<.0001). Such changes were linked to an over-phosphorylation of eIF2α. In conclusion, vitamin D deficiency in old rats increases adiposity and leads to reduced muscle protein synthesis through activation of eIF2α. These disorders are restored by vitamin D supplementation.

Fast-digestive protein supplement for ten days overcomes muscle anabolic resistance in healthy elderly men.

BACKGROUND/OBJECTIVE: Adequate protein intake is crucial to maintain muscle protein content in elderly subjects, but quality of dietary proteins should be considered. The aim was to determine whether soluble milk protein offers an original strategy to increase muscle anabolism in elderly subjects via a synergistic effect of fast-digesting proteins together with a unique essential AA content. DESIGN: We investigated the effect of a 10-day adequate-protein (AP) or high-protein (HP) diet together with the protein source as caseins (CAS) or soluble milk proteins (PRO) on specific muscle protein fractional synthesis rates (FSRs) in healthy elderly men (71.8 ± 2.4 yr, n = 31). The isotopic study consisted of two periods of 4 h each: a post-absorptive and a postprandial period. The fed state was defined by consumption of either 15 g or 30 g of PRO or CAS, given fractionally every 20 min for 4 h. Soluble milk proteins are produced using a membrane process directly from pasteurized milk. MEASUREMENTS: Specific muscle protein FSRs were measured during both postabsorptive and postprandial period using a continuous infusion of l-[1-(13)C]leucine. RESULTS: FSR of sarcoplasmic muscle proteins and actin did not increase significantly in the postprandial state compared to postabsorptive state, whereas myosin FSR rate was increased by feeding whatever the protein source in HP groups (0.024 ± 0.005 vs 0.053 ± 0.011% h(-1), P < 0.05 and 0.026 ± 0.004 vs 0.050 ± 0.005% h(-1), P < 0.004 for PRO HP and CAS HP) but only with the PRO meal in the AP groups (0.031 ± 0.003 vs 0.062 ± 0.009% h(-1), P < 0.03 for PRO AP). Mitochondrial muscle protein FSR was also increased by feeding, irrespective of the protein quantity, but only in PRO meal groups (P < 0.02). CONCLUSION: Fast-digesting soluble milk proteins improved postprandial muscle protein synthesis, especially mitochondrial muscle proteins and myosin fractional synthesis rates, in elderly subjects.

Increased body fat mass and tissue lipotoxicity associated with ovariectomy or high-fat diet differentially affects bone and skeletal muscle metabolism in rats.

PURPOSE: The aim of this study was to evaluate and compare the musculoskeletal effects induced by ovariectomy-related fat mass deposition against the musculoskeletal effects caused by a high-fat diet. METHODS: A group of adult female rats was ovariectomized and fed a control diet. Two additional groups were sham-operated and fed a control or a high-fat diet for 19 weeks. Distal femur and serum bone parameters were measured to assess bone metabolism. Muscle protein metabolism, mitochondrial markers and triglyceride content were evaluated in tibialis anterior. Triglyceride content was evaluated in liver. Circulating inflammatory and metabolic markers were determined. RESULTS: The high-fat diet and ovariectomy led to similar increases in fat mass (+36.6-56.7%; p < 0.05) but had different impacts on bone and muscle tissues and inflammatory markers. Consumption of the high-fat diet led to decreased bone formation (-38.4%; p < 0.05), impaired muscle mitochondrial metabolism, muscle lipotoxicity and a 20.9% increase in tibialis anterior protein synthesis rate (p < 0.05). Ovariectomy was associated with higher bone turnover as bone formation increased +72.7% (p < 0.05) and bone resorption increased +76.4% (p < 0.05), leading to bone loss, a 17.9% decrease in muscle protein synthesis rate (p < 0.05) and liver lipotoxicity. CONCLUSIONS: In female rats, high-fat diet and ovariectomy triggered similar gains in fat mass but had different impacts on bone and muscle metabolism. The ovariectomy-induced mechanisms affecting the musculoskeletal system are mainly caused by estrogen depletion, which surpasses the potential-independent effect of adiposity.

Bee pollen improves muscle protein and energy metabolism in malnourished old rats through interfering with the Mtor signaling pathway and mitochondrial activity.

Although the management of malnutrition is a priority in older people, this population shows a resistance to refeeding. Fresh bee pollen contains nutritional substances of interest for malnourished people. The aim was to evaluate the effect of fresh bee pollen supplementation on refeeding efficiency in old malnourished rats. Male 22-month-old Wistar rats were undernourished by reducing food intake for 12 weeks. The animals were then renourished for three weeks with the same diet supplemented with 0%, 5% or 10% of fresh monofloral bee pollen. Due to changes in both lean mass and fat mass, body weight decreased during malnutrition and increased after refeeding with no between-group differences (p < 0.0001). Rats refed with the fresh bee pollen-enriched diets showed a significant increase in muscle mass compared to restricted rats (p < 0.05). The malnutrition period reduced the muscle protein synthesis rate and mTOR/p70S6kinase/4eBP1 activation, and only the 10%-pollen diet was able to restore these parameters. Mitochondrial activity was depressed with food restriction and was only improved by refeeding with the fresh bee pollen-containing diets. In conclusion, refeeding diets that contain fresh monofloral bee pollen improve muscle mass and metabolism in old, undernourished rats.

Vitamin D deficiency down-regulates Notch pathway contributing to skeletal muscle atrophy in old wistar rats.

BACKGROUND: The diminished ability of aged muscle to self-repair is a factor behind sarcopenia and contributes to muscle atrophy. Muscle repair depends on satellite cells whose pool size is diminished with aging. A reduction in Notch pathway activity may explain the age-related decrease in satellite cell proliferation, as this pathway has been implicated in satellite cell self-renewal. Skeletal muscle is a target of vitamin D which modulates muscle cell proliferation and differentiation in vitro and stimulates muscle regeneration in vivo. Vitamin D status is positively correlated to muscle strength/function, and elderly populations develop a vitamin D deficiency. The aim of this study was to evaluate how vitamin D deficiency induces skeletal muscle atrophy in old rats through a reduction in Notch pathway activity and proliferation potential in muscle. METHODS: 15-month-old male rats were vitamin D-depleted or not (control) for 9 months (n = 10 per group). Rats were 24-month-old at the end of the experiment. Gene and/or protein expression of markers of proliferation, or modulating proliferation, and of Notch signalling pathway were studied in the tibialis anterior muscle by qPCR and western blot. An unpaired student's t-test was performed to test the effect of the experimental conditions. RESULTS: Vitamin D depletion led to a drop in concentrations of plasma 25-hydroxyvitamin D in depleted rats compared to controls (-74%, p < 0.01). Tibialis anterior weight was decreased in D-depleted rats (-25%, p < 0.05). The D-depleted group showed -39%, -31% drops in expression of two markers known to modulate proliferation (Bmp4, Fgf-2 mRNA levels) and -56% drop in one marker of cell proliferation (PCNA protein expression) compared to controls (p < 0.05). Notch pathway activity was blunted in tibialis anterior of D-depleted rats compared to controls, seen as a down-regulation of cleaved Notch (-53%, p < 0.05) and its target Hes1 (-35%, p < 0.05). CONCLUSIONS: A 9-month vitamin D depletion induced vitamin D deficiency in old rats. Vitamin D depletion induces skeletal muscle atrophy in old rats through a reduction in Notch pathway activity and proliferation potential. Vitamin D deficiency could aggravate the age-related decrease in muscle regeneration capacity.

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.

Mitochondrial protein synthesis is increased in oxidative skeletal muscles of rats with cardiac cachexia.

Since cardiac cachexia could be associated with alterations in muscular mitochondrial metabolism, we hypothesized that the expected alterations in the activities of mitochondrial oxidative enzymes could be associated with changes in mitochondrial protein synthesis in oxidative skeletal muscles. Cardiac cachexia was provoked in male rats by the ligation of the left coronary artery. Six cachectic and 6 control rats were age-paired, and their food intake was observed. The synthesis of mitochondrial proteins was measured by [1-13C]-valine infusion in soleus, tibilais, myocardium, and liver. Muscles (soleus, gastrocnemius, and tibialis anterior), heart, kidneys, liver, and visceral adipose tissue were weighed. Mitochondrial cytochrome c oxydase IV as well as citrate synthase and myosin ATPase activities were measured. As expected, decreased food intake was observed in the cachectic group. Heart, kidney, and liver weights were higher in the cachectic group, while the visceral adipose tissue weight was lower (P < .01). No changes in muscle weights were observed. Soleus mitochondrial proteins fractional synthesis rate was higher in the cachectic group (P = .054). Cytochrome c oxydase IV activity was reduced (P = .009) and increased (P = .038) in the soleus and liver of the cachectic rats, respectively. No change in citrate synthase activity was observed. Myosin ATPase activity was reduced in the gastrocnemius of the cachectic group (P < .01). Mitochondrial protein synthesis is increased in the soleus of rats with cardiac cachexia, suggesting a compensatory mechanism of the impaired oxidative mitochondrial function. Further work should assess whether the mitochondrial protein synthesis is altered in chronic heart failure patients with cardiac cachexia, and whether this is the cause or the consequence of cachexia.