Excessive Energy Intake Does Not Modify Fed‐state Tissue Protein Synthesis Rates in Adult Rats

The impact of chronic excessive energy intake on protein metabolism is still controversial. Male Wistar rats were fed ad libitum during 5 weeks with either a high‐fat high‐sucrose diet (HF: n = 9) containing 45% of total energy as lipids (protein 14%; carbohydrate 40% with 83.5% sucrose) or a standard diet (controls: n = 10). Energy intake and body weight were recorded. At the end of the experiment, we measured body composition, metabolic parameters (plasma amino acid, lipid, insulin, and glucose levels), inflammatory parameter (plasma α2‐macroglobulin), oxidative stress parameters (antioxidant enzyme activities, lipoperoxidation (LPO), protein carbonyl content in liver and muscle), and in vivo fed–state fractional protein synthesis rates (FSRs) in muscle and liver. Energy intake was significantly higher in HF compared with control rats (+28%). There were significant increases in body weight (+8%), body fat (+21%), renal (+41%), and epidydimal (+28%) fat pads in HF compared with control rats. No effect was observed in other tissue weights (liver, muscle, spleen, kidneys, intestine). Liver and muscle FSRs, plasma levels of lipids, glucose, insulin and α2‐macroglobulin, soleus and liver glutathione reductase and peroxidase acitivities, MnSOD activity, LPO, and protein carbonyl content were not altered by the HF diet. Only soleus muscle and liver Cu/ZnSOD activity and soleus muscle catalase activities were reduced in HF rats compared with control rats. Thus, chronic excessive energy intake and increased adiposity, in the absence of other metabolic alterations, do not stimulate fed‐state tissue protein synthesis rates.     

Presence of low-grade inflammation impaired postprandial stimulation of muscle protein synthesis in old rats

Aging is characterized by a decline in muscle mass that could be explained by a defect in the regulation of postprandial muscle protein metabolism. This study was undertaken to examine a possible link between the development of low-grade inflammation (LGI) in elderly and the resistance of muscle protein synthesis and degradation pathways to food intake. Fifty-five 20-month-old-rats were studied for 5 months; blood was withdrawn once a month to assess plasma fibrinogen and α2-macroglobulin. Animals were then separated into two groups at 25 months old according to their inflammation status: a control non-inflamed (NI, n=24) and a low-grade inflamed group (LGI, n=23). The day of the experiment, rats received no food or a meal. Muscle protein synthesis was assessed in vivo using the flooding dose method ([1-¹³C]phenylalanine) and muscle phosphorylation of protein S6 kinase, and protein S6 was measured in gastrocnemius muscle. Muscle proteolysis was assessed in vitro using the epitrochlearis muscle. Postabsorptive muscle protein synthesis and proteolysis were similar in NI and LGI. After food intake, muscle protein synthesis was significantly stimulated in NI but remained unresponsive in LGI. Muscle proteolysis was similar in both groups whatever the inflammation and/or the nutritional status. In conclusion, we showed that development of LGI during aging may be responsible, at least in part, for the defect in muscle protein synthesis stimulation induced by food intake in rats. Our results suggested that the control of LGI development in elderly improve meal effect on muscle protein synthesis and consequently slow down sarcopenia.     

Genomic and Proteomic Profiling Reveals Reduced Mitochondrial Function and Disruption of the Neuromuscular Junction Driving Rat Sarcopenia

Molecular mechanisms underlying sarcopenia, the age-related loss of skeletal muscle mass and function, remain unclear. To identify molecular changes that correlated best with sarcopenia and might contribute to its pathogenesis, we determined global gene expression profiles in muscles of rats aged 6, 12, 18, 21, 24, and 27 months. These rats exhibit sarcopenia beginning at 21 months. Correlation of the gene expression versus muscle mass or age changes, and functional annotation analysis identified gene signatures of sarcopenia distinct from gene signatures of aging. Specifically, mitochondrial energy metabolism (e.g., tricarboxylic acid cycle and oxidative phosphorylation) pathway genes were the most downregulated and most significantly correlated with sarcopenia. Also, perturbed were genes/pathways associated with neuromuscular junction patency (providing molecular evidence of sarcopenia-related functional denervation and neuromuscular junction remodeling), protein degradation, and inflammation. Proteomic analysis of samples at 6, 18, and 27 months confirmed the depletion of mitochondrial energy metabolism proteins and neuromuscular junction proteins. Together, these findings suggest that therapeutic approaches that simultaneously stimulate mitochondrogenesis and reduce muscle proteolysis and inflammation have potential for treating sarcopenia.     

Whey and casein labeled with L-[1-13C]leucine and muscle protein synthesis: effect of resistance exercise and protein ingestion

Muscle protein turnover following resistance exercise and amino acid availability are relatively well described. By contrast, the beneficial effects of different sources of intact proteins in relation to exercise need further investigation. Our objective was to compare muscle anabolic responses to a single bolus intake of whey or casein after performance of heavy resistance exercise. Young male individuals were randomly assigned to participate in two protein trials (n = 9) or one control trial (n = 8). Infusion of l-[1-(13)C]leucine was carried out, and either whey, casein (0.3 g/kg lean body mass), or a noncaloric control drink was ingested immediately after exercise. l-[1-(13)C]leucine-labeled whey and casein were used while muscle protein synthesis (MPS) was assessed. Blood and muscle tissue samples were collected to measure systemic hormone and amino acid concentrations, tracer enrichments, and myofibrillar protein synthesis. Western blots were used to investigate the Akt signaling pathway. Plasma insulin and branched-chain amino acid concentrations increased to a greater extent after ingestion of whey compared with casein. Myofibrillar protein synthesis was equally increased 1-6 h postexercise after whey and casein intake, both of which were higher compared with control (P < 0.05). Phosphorylation of Akt and p70(S6K) was increased after exercise and protein intake (P < 0.05), but no differences were observed between the types of protein except for total 4E-BP1, which was higher after whey intake than after casein intake (P < 0.05). In conclusion, whey and casein intake immediately after resistance exercise results in an overall equal MPS response despite temporal differences in insulin and amino acid concentrations and 4E-BP1.     

The nature of the ingested protein has no effect on lean body mass during energy restriction in overweight rats

Severe energy restriction in obesity not only leads to fat mass loss but also to lean mass loss. The aim of this study was to compare the capacity of casein, a slowly digested protein, and milk soluble proteins (MSP; rapidly digested) to limit the loss of lean mass induced by energy restriction. Obesity was first induced in male Wistar rats by a 5-week feeding with a high-fat high-sucrose diet. The impact of energy restriction was then studied with high-protein (32%) diets containing either casein, MSP, or a 50/50 mixture of both proteins for 3 weeks (n = 10 per group). Food intake, body weight, nitrogen balance, creatinine, and 3-methyl-histidine excretion were measured during energy restriction. Then, tissue weights, plasma metabolic parameters (amino acids, glucose, insulin, cholesterol, triglycerides), and in vivo liver and extensor digitorum longus (EDL) muscle protein synthesis rates were measured in postabsorptive state at the end of the experimental period. Although significant differences relevant to protein metabolism were observed between groups (protein intake, plasma amino acid concentrations, fecal nitrogen excretion, muscle protein synthesis rates), week per week, there were no significant differences in nitrogen balance whatever the protein used. In conclusion, our results show that in young overweight energy restricted rats, using a high-protein diet, the nature of protein intake has no influence on body protein retention.     

A relative high dose of vitamin E does not attenuate unweighting-induced oxidative stress and ubiquitination in rat skeletal muscle

We previously reported that intragastric administration of cysteine could be beneficial to prevent unweighting-induced ubiquitination and degradation of muscle protein in association with redox regulation [Ikemoto et al., Biol. Chem., 383 (2002), 715-721]. In this study, we investigated whether vitamin E, another potent antioxidative nutrient, also had beneficial effects on the muscle protein catabolism. However, daily intragastric supplementation of 1.5 or 15 mg/rat of alpha-tocopherol did not prevent weight loss of hindlimb skeletal muscle in tail-suspended rats. To elucidate the reason for the non-effectiveness of vitamin E, we further examined concentrations of oxidative stress markers, ubiquitination of muscle proteins and fragmentation of myosin heavy chain in gastrocnemius muscle of rats daily treated with 15 mg of alpha-tocopherol. Unexpectedly, vitamin E increased concentrations of glutathione disulfide and thiobarbituric acid-reactive substance and decreased glutathione level in the muscle, compared with those of vehicle treatment, indicating that vitamin E enhanced unweighting-induced oxidative stress in skeletal muscle. The vitamin E supplementation did not suppress the ubiquitination of muscle proteins and fragmentation of myosin heavy chain caused by tail-suspension. Our results suggest that supplementation of a relative high dose of vitamin E could not inhibit ubiquitin-dependent degradation of muscle protein in tail-suspended rats possibly due to its prooxidant action.     

Effects of resistance training and protein plus amino acid supplementation on muscle anabolism,mass, and strength

Summary. This study examined 10 wks of resistance training and the ingestion of supplemental protein and amino acids on muscle performance and markers of muscle anabolism. Nineteen untrained males were randomly assigned to supplement groups containing either 20 g protein (14 g whey and casein protein, 6 g free amino acids) or 20 g dextrose placebo ingested 1 h before and after exercise for a total of 40 g/d. Participants exercised 4 times/wk using 3 sets of 6–8 repetitions at 85–90% of the one repetition maximum. Data were analyzed with two-way ANOVA (p < 0.05). The protein supplement resulted in greater increases in total body mass, fat-free mass, thigh mass, muscle strength, serum IGF-1, IGF-1 mRNA, MHC I and IIa expression, and myofibrillar protein. Ten-wks of resistance training with 20 g protein and amino acids ingested 1 h before and after exercise is more effective than carbohydrate placebo in up-regulating markers of muscle protein synthesis and anabolism along with subsequent improvements in muscle performance.     

Endurance training without weight loss lowers systemic, but not muscle, oxidative stress with no effect on inflammation in lean and obese women

Obesity is associated with oxidative stress. Endurance training (ET) in healthy individuals increases antioxidant enzyme activity and decreases oxidative stress, whereas its effects on oxidative status in obese humans have yet to be determined. We investigated the effects of obesity and ET on markers of oxidative stress, antioxidant defense, and inflammation. Obese (n=12) and lean (n=12) women underwent 12 weeks of ET with blood, 24-h urine, and muscle biopsies collected prior to and following training for determination of oxidative stress (urinary 8-hydroxy-2-deoxyguanosine and 8-isoprostanes, muscle protein carbonyls, and 4-hydroxynonenal), antioxidant enzyme protein content (muscle CuZnSOD, MnSOD, and catalase), and inflammation (C-reactive protein, leptin, adiponectin, interleukin-6). Obese women had elevated urinary 8-hydroxy-2-deoxyguanosine (P=0.03), muscle protein carbonyls (P=0.03), and 4-hydroxynonenal (P<0.001); serum C-reactive protein (P=0.01); and plasma leptin (P=0.0001) and interleukin-6 (P=0.03). ET decreased urinary 8-hydroxy-2-deoxyguanosine (P=0.006) and 8-isoprostanes (P=0.02) in all subjects and CuZnSOD protein content (P=0.04) in obese women, in the absence of changes in body weight or composition. ET without weight loss decreases systemic oxidative stress, but not markers of inflammation, in obese women.     

Activation of ubiquitin-proteasome pathway is involved in skeletal muscle wasting in a rat model with biliary cirrhosis: potential role of TNF-alpha

Hepatic cirrhosis is associated with negative nitrogen balance and loss of lean body mass. This study aimed to identify the specific proteolytic pathways activated in skeletal muscles of cirrhotic rats. TNF-alpha can stimulate muscle proteolysis; therefore, a potential relationship between TNF-alpha and muscle wasting in liver cirrhosis was also evaluated. Cirrhosis was induced by bile duct ligation (BDL) in male adult Sprague-Dawley rats. mRNA and protein levels of various targets were determined by RT-PCR and Western blotting, respectively. The proteolytic rate was measured ex vivo using isolated muscles. Compared with sham-operated controls, BDL rats had an increased degradation rate of muscle proteins and enhanced gene expression of ubiquitin, 14-kDa ubiquitin carrier protein E2, and the proteasome subunits C2 and C8 (P < 0.01). The muscle protein levels of free ubiquitin and conjugated ubiquitin levels were also elevated (P < 0.01). However, there was no difference between the two groups with regard to cathepsin and calpain mRNA levels. Cirrhotic muscle TNF-alpha levels were increased and correlated positively with free and conjugated ubiquitin (P < 0.01). We conclude that the ubiquitin-proteasome system is involved in muscle wasting of rats with BDL-induced cirrhosis. TNF-alpha might play a role in mediating activation of this proteolytic pathway, probably through a local mechanism.     

Dietary cysteine alleviates sucrose-induced oxidative stress and insulin resistance

Diets that promote oxidative stress favor impairment in glucose homeostasis. In this context, increasing the cysteine intake may be beneficial by maintaining glutathione status. We have investigated the effects of dietary cysteine on oxidative stress and glucose homeostasis in rats fed a high-sucrose (HS) diet. Rats were assigned for 6 weeks to a standard diet or to HS diets in which the protein source was either an alpha-lactalbumin-rich whey concentrate (a cysteine-rich protein) or the total milk proteins alone or supplemented with 5.8 or 20 g N-acetylcysteine per kilogram of food. Increasing the cysteine intake prevented HS-induced oxidative stress, as assessed by blood and tissue glutathione and carbonyl levels. At the same time, the HS-induced glucose intolerance, impaired postprandial glycemic control, and decrease in muscle and liver insulin-induced activation of insulin receptor substrate 1 and Akt were prevented by increasing the level of dietary cysteine, a major original finding. Of great interest was the observation that all beneficial effects of cysteine supplementation were duplicated by the consumption of a cysteine-rich protein. These data show that increasing the cysteine intake limits HS-induced impairment of glucose homeostasis and suggest that these effects are mediated by a reduction in oxidative stress.     

Amino acid absorption and subsequent muscle protein accretion following graded intakes of whey protein in elderly men

Whey protein ingestion has been shown to effectively stimulate postprandial muscle protein accretion in older adults. However, the impact of the amount of whey protein ingested on protein digestion and absorption kinetics, whole body protein balance, and postprandial muscle protein accretion remains to be established. We aimed to fill this gap by including 33 healthy, older men (73 ± 2 yr) who were randomly assigned to ingest 10, 20, or 35 g of intrinsically l-[1-13C]phenylalanine-labeled whey protein (n = 11/treatment). Ingestion of labeled whey protein was combined with continuous intravenous l-[ring-2H?]phenylalanine and l-[ring-2H?]tyrosine infusion to assess the metabolic fate of whey protein-derived amino acids. Dietary protein digestion and absorption rapidly increased following ingestion of 10, 20, and 35 g whey protein, with the lowest and highest (peak) values observed following 10 and 35 g, respectively (P < 0.05). Whole body net protein balance was positive in all groups (19 ± 1, 37 ± 2, and 58 ± 2 μmol/kg), with the lowest and highest values observed following ingestion of 10 and 35 g, respectively (P < 0.05). Postprandial muscle protein accretion, assessed by l-[1-13C]phenylalanine incorporation in muscle protein, was higher following ingestion of 35 g when compared with 10 (P < 0.01) or 20 (P < 0.05) g. We conclude that ingestion of 35 g whey protein results in greater amino acid absorption and subsequent stimulation of de novo muscle protein synthesis compared with the ingestion of 10 or 20 g whey protein in healthy, older men.     

Protein oxidation and proteolysis during aging and oxidative stress

Previous studies in this laboratory have shown that glutamine synthetase (GS) and other key metabolic enzymes are inactivated by metal-catalyzed oxidation reactions in vitro. Oxidative inactivation renders these proteins highly susceptible to proteolysis, especially to a class of newly identified alkaline proteases which exhibit little or no activity against the native enzymes. These studies have suggested that oxidative inactivation may be an important marking step for intracellular protein degradation. Because many of the enzymes which have been shown to accumulate as inactive or less active forms during aging are readily inactivated by metal-catalyzed oxidation reactions in vitro, we have investigated the possible relationship between protein oxidation and proteolysis during aging and oxidative stress in vivo. Oxidized proteins accumulate in hepatocytes of rats exposed to 100% oxygen during the first 48 h of oxygen treatment. In the interval between 48 and 54 h the levels of oxidized proteins decline sharply. The specific activities of at least two liver enzymes, glutamine synthetase and glucose-6-phosphate dehydrogenase (G-6-PDH), decrease during the 54-h experiment. GS and G-6-PDH specific immunological cross-reactivity remains high during the first 48 h of oxygen treatment and then declines in the interval between 48 and 54 h. During this same interval the levels of alkaline proteases which degrade oxidized proteins increase, indicating that these activities are induced or activated in response to oxidative stress and subsequently degrade the proteins which have become oxidized during the initial phase of oxygen treatment. Oxidized proteins accumulate progressively during aging in hepatocytes from rats 3 to 26 months old, with the largest incremental increase between 20 and 26 months. The increase in protein oxidation is correlated with a loss of specific activity of GS and G-6-PDH without a concomitant loss of immunological cross-reactivity. The levels of alkaline proteases which degrade oxidized proteins in hepatocytes from 26-month-old rats is only 20% that of 3-month-old rats, suggesting that oxidized proteins accumulate in hepatocytes from old rats, in part, because the proteases which degrade them are deficient or defective. moreover, when old rats are subjected to treatment with 100% oxygen, the levels of oxidized proteins continue to increase and the alkaline protease activity remains low, indicating that these protease activities are not increased in response to oxidative stress in old rats.     

Proteomic analysis of altered protein expression in skeletal muscle of rats in a hypermetabolic state induced by burn sepsis

mRNA profiling has been extensively used to study muscle wasting. mRNA level changes may not reflect that of proteins, especially in catabolic muscle where there is decreased synthesis and increased degradation. As sepsis is often associated with burn injury, and burn superimposed by sepsis has been shown to result in significant loss of lean tissues, we characterized changes in the skeletal-muscle proteome of rats subjected to a cutaneous burn covering 20% of the total body surface area, followed 2 days later by sepsis induced by CLP (caecal ligation and puncture). EDL (extensor digitorum longus) muscles were dissected from Burn-CLP animals (n=4) and controls (sham-burned and sham-CLP-treated, n=4). Burn-CLP injury resulted in a rapid loss of EDL weight, increased ubiquitin-conjugated proteins and increased protein carbonyl groups. EDL protein profiles were obtained by two-dimensional gel electrophoresis using two immobilized pH gradient strips with overlapping pH range covering a pH 3-8 range. Seventeen spots were significantly altered in the Burn-CLP compared with the control group, representing 15 different proteins identified by peptide mass fingerprinting. The identities of three proteins including transferrin were further confirmed by liquid chromatography-tandem MS. The significant changes in transferrin and HSP27 (heat-shock protein 27) were verified by Western-blot analysis. HSP60, HSP27 and HSPbeta6 were down-regulated, along with HSP70, as detected by Western blotting. Six metabolic enzymes related to energy production were also down-regulated. A simultaneous decrease in chaperone proteins and metabolic enzymes could decrease protein synthesis. Furthermore, decreased HSPs could increase oxidative damage, thus accelerating protein degradation. Using cultured C2C12 myotubes, we showed that H2O2-induced protein degradation in vitro could be partially attenuated by prior heat-shock treatment, consistent with a protective role of HSP70 and/or other HSPs against proteolysis.     

Oxidative stress,neurodegeneration, and the balance of protein degradation and protein synthesis

Oxidative stress occurs in a variety of disease settings and is strongly linked to the development of neuron death and neuronal dysfunction. Cells are equipped with numerous pathways to prevent the genesis, as well as the consequences, of oxidative stress in the brain. In this review we discuss the various forms and sources of oxidative stress in the brain and briefly discuss some of the complexities in detecting the presence of oxidative stress. We then focus the review on the interplay between the diverse cellular proteolytic pathways and their roles in regulating oxidative stress in the brain. Additionally, we discuss the involvement of protein synthesis in regulating the downstream effects of oxidative stress. Together, these components of the review demonstrate that the removal of damaged proteins by effective proteolysis and the synthesis of new and protective proteins are vital in the preservation of brain homeostasis during periods of increased levels of reactive oxygen species. Last, studies from our laboratory and others have demonstrated that protein synthesis is intricately linked to the rates of protein degradation, with impairment of protein degradation sufficient to decrease the rates of protein synthesis, which has important implications for successfully responding to periods of oxidative stress. Specific neurodegenerative diseases, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and stroke, are discussed in this context. Taken together, these findings add to our understanding of how oxidative stress is effectively managed in the healthy brain and help elucidate how impairments in proteolysis and/or protein synthesis contribute to the development of neurodegeneration and neuronal dysfunction in a variety of clinical settings.     

低氧影响蛋白质合成和分解的平衡诱导骨骼肌萎缩

暴露在低氧环境下,可能会引起胃肠功能障碍和摄食量下降,打破骨骼肌蛋白质合成和分解平衡,造成骨骼肌萎缩。为探讨低氧环境下骨骼肌的萎缩是低氧环境引起的还是低氧诱发的摄食量减少所致,本研究检测大鼠腓肠肌中低氧时蛋白质合成与分解相关基因的蛋白质表达。将21只雄性SD大鼠,随机分为3组：常氧对照组、低氧组（氧浓度为12.4%,模拟海拔4 000 m高度）和配对组（大鼠的摄食量与低氧组前1 d的摄食量相同）,每组7只,每天记录大鼠体重和摄食量。4周后,HE染色法观察腓肠肌肌纤维形态,Western印迹测试相关蛋白质水平。低氧组和配对组摄食量在低氧干预初期,较常氧对照组有显著性下降(P<0.05),干预后期差异不明显;干预期间,低氧组大鼠体重平均增加量(102.10 g)、体重(341.20 ± 16.75 g)、肌肉总量（226.83 ± 8.33 g）和腓肠肌肌纤维横截面积(12.67 ± 1.83 mm)较常氧对照组（128.00 g;377.50 ± 20.75 g;260.50 ± 9.35 g;15.78 ± 2.38 mm）和配对组（119.40 g;375.86 ± 11.30 g;262.29 ± 7.90 g;15.71 ± 2.82 mm）均显著下降,配对组较常氧对照组无显著性差异;4周干预后,与常氧对照组相比,低氧组大鼠腓肠肌中与低氧相关的HIF1α显著增加(1.42 ± 0.19, P<0.05),Akt和p-Akt/Akt显著降低 (1.44 ± 0.13; 0.47 ± 0.08, P<0.05),配对组上述3种指标相对表达量均无显著性差异;在蛋白质合成方面,低氧组mTOR较常氧对照组显著下降(0.63 ± 0.18, P<0.05),配对组较常氧对照组差异不明显;低氧组腓肠肌中,4EBP1(1.14 ± 0.14)和p70S6K1(1.14 ± 0.11)较配对组显著下降(P<0.05)。在蛋白质分解方面,低氧组p-FoxO1和p-FoxO1/FoxO1比值较常氧对照组显著下降(0.71 ± 0.15; 0.78 ± 0.14, P<0.05);低氧组大鼠腓肠肌中,Atrogin1、MuRF1、Beclin1、LC3Ⅰ及LC3Ⅱ/Ⅰ比值均高于常氧对照组(1.35 ± 0.12; 1.30 ± 0.22; 1.17 ± 0.11; 1.03 ± 0.11; 1.35 ± 0.13, P<0.05);配对组与常氧对照组间无明显差异。低氧环境下骨骼肌中蛋白质合成相关基因表达减少,蛋白质分解相关基因表达增加,造成骨骼肌萎缩,体重下降,此变化与摄食量减少无关。     

低氧影响蛋白质合成和分解的平衡诱导骨骼肌萎缩

暴露在低氧环境下,可能会引起胃肠功能障碍和摄食量下降,打破骨骼肌蛋白质合成和分解平衡,造成骨骼肌萎缩。为探讨低氧环境下骨骼肌的萎缩是低氧环境引起的还是低氧诱发的摄食量减少所致,本研究检测大鼠腓肠肌中低氧时蛋白质合成与分解相关基因的蛋白质表达。将21只雄性SD大鼠,随机分为3组：常氧对照组、低氧组（氧浓度为12.4%,模拟海拔4 000 m高度）和配对组（大鼠的摄食量与低氧组前1 d的摄食量相同）,每组7只,每天记录大鼠体重和摄食量。4周后,HE染色法观察腓肠肌肌纤维形态,Western印迹测试相关蛋白质水平。低氧组和配对组摄食量在低氧干预初期,较常氧对照组有显著性下降(P<0.05),干预后期差异不明显;干预期间,低氧组大鼠体重平均增加量(102.10 g)、体重(341.20 ± 16.75 g)、肌肉总量（226.83 ± 8.33 g）和腓肠肌肌纤维横截面积(12.67 ± 1.83 mm)较常氧对照组（128.00 g;377.50 ± 20.75 g;260.50 ± 9.35 g;15.78 ± 2.38 mm）和配对组（119.40 g;375.86 ± 11.30 g;262.29 ± 7.90 g;15.71 ± 2.82 mm）均显著下降,配对组较常氧对照组无显著性差异;4周干预后,与常氧对照组相比,低氧组大鼠腓肠肌中与低氧相关的HIF1α显著增加(1.42 ± 0.19, P<0.05),Akt和p-Akt/Akt显著降低 (1.44 ± 0.13; 0.47 ± 0.08, P<0.05),配对组上述3种指标相对表达量均无显著性差异;在蛋白质合成方面,低氧组mTOR较常氧对照组显著下降(0.63 ± 0.18, P<0.05),配对组较常氧对照组差异不明显;低氧组腓肠肌中,4EBP1(1.14 ± 0.14)和p70S6K1(1.14 ± 0.11)较配对组显著下降(P<0.05)。在蛋白质分解方面,低氧组p-FoxO1和p-FoxO1/FoxO1比值较常氧对照组显著下降(0.71 ± 0.15; 0.78 ± 0.14, P<0.05);低氧组大鼠腓肠肌中,Atrogin1、MuRF1、Beclin1、LC3Ⅰ及LC3Ⅱ/Ⅰ比值均高于常氧对照组(1.35 ± 0.12; 1.30 ± 0.22; 1.17 ± 0.11; 1.03 ± 0.11; 1.35 ± 0.13, P<0.05);配对组与常氧对照组间无明显差异。低氧环境下骨骼肌中蛋白质合成相关基因表达减少,蛋白质分解相关基因表达增加,造成骨骼肌萎缩,体重下降,此变化与摄食量减少无关。     

Skeletal and cardiac myopathy in HIV-1 transgenic rats

The mechanism by which human immunodeficiency virus (HIV)-1 infection in humans leads to the erosion of lean body mass is poorly defined. Therefore, the purpose of the present study was to determine whether transgenic (Tg) rats that constitutively overexpress HIV-1 viral proteins exhibit muscle wasting and to elucidate putative mechanisms. Over 7 mo, Tg rats gained less body weight than pair-fed controls exclusively as a result of a proportional reduction in lean, not fat, mass. Fast- and slow-twitch muscle atrophy in Tg rats did not result from a reduction in the in vivo-determined rate of protein synthesis. In contrast, urinary excretion of 3-methylhistidine, as well as the content of atrogin-1 and the 14-kDa actin fragment, was elevated in gastrocnemius of Tg rats, suggesting increased muscle proteolysis. Similarly, Tg rats had reduced cardiac mass, which was independent of a change in protein synthesis. This decreased cardiac mass was associated with a reduction in stroke volume, but cardiac output was maintained by a compensatory increase in heart rate. The HIV-induced muscle atrophy was associated with increased whole body energy expenditure, which was not due to an elevated body temperature or secondary bacterial infection. Furthermore, the atrophic response could not be attributed to the development of insulin resistance, decreased levels of circulating amino acids, or increased tissue cytokines. However, skeletal muscle and, to a lesser extent, circulating insulin-like growth factor I was reduced in Tg rats. Although hepatic injury was implicated by increased plasma levels of aspartate and alanine aminotransferases, hepatic protein synthesis was not different between control and Tg rats. Hence, HIV-1 Tg rats develop atrophy of cardiac and skeletal muscle, the latter of which results primarily from an increased protein degradation and may be related to the marked reduction in muscle insulin-like growth factor I.     

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.     

Leucine and Protein Metabolism in Obese Zucker Rats

Branched-chain amino acids (BCAAs) are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1-¹⁴C]-leucine metabolism, tissue-specific protein synthesis and branched-chain keto-acid (BCKA) dehydrogenase complex (BCKDC) activities. Male obese Zucker rats (11-weeks old) had increased body weight (BW, 53%), liver (107%) and fat (∼300%), but lower plantaris and gastrocnemius masses (−21–24%). Plasma BCAAs and BCKAs were elevated 45–69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%), leucine (Leu) turnover and proteolysis [35% per g fat free mass (FFM), urinary markers of proteolysis: 3-methylhistidine (183%) and 4-hydroxyproline (766%)] and decreased BCKDC per g kidney, heart, gastrocnemius and liver (−47–66%). A process disposing of circulating BCAAs, protein synthesis, was increased 23–29% by obesity in whole-body (FFM corrected), gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193–418%) than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and protein turnover along with impaired BCKDC activity. Elevated BCAAs/BCKAs may contribute to observed elevations in protein synthesis and BCAA oxidation.     

Fed-state clamp stimulates cellular mechanisms of muscle protein anabolism and modulates glucose disposal in normal men

Since maximum anabolism occurs postprandially, we developed a simulated fed state with clamped hyperinsulinemia, physiological hyperglycemia, and hyperaminoacidemia (Hyper-3) and explored muscle cellular mechanisms. Whole body [1-(13)C]leucine and [3-(3)H]glucose kinetics in healthy men were compared between hyperinsulinemic, euglycemic, isoaminoacidemic (Hyper-1, n = 10) and Hyper-3 (n = 9) clamps. In Hyper-3 vs. Hyper-1, nonoxidative leucine R(d) [rate of disappearance (synthesis)] was stimulated more (45 +/- 4 vs. 24 +/- 4 micromol/min, P < 0.01) and endogenous R(a) [rate of appearance (breakdown)] was inhibited similarly; hence net balance increased more (86 +/- 6 vs. 49 +/- 2 micromol/min, P < 0.001). Glucose R(d) was similar; thus Hyper-3 metabolic clearance rate (331 +/- 23 vs. 557 +/- 41 ml/min, P < 0.0005) and R(d)/insulin (M, 0.65 +/- 0.10 vs. 1.25 +/- 0.10 mg.min(-1).pmol(-1).l, P < 0.001) were less, despite higher insulin (798 +/- 74 vs. 450 +/- 24 pmol/l, P < 0.005). In vastus lateralis muscle biopsies, phosphorylation of Akt (P = 0.025), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70(S6K1); P = 0.008), S6 (P = 0.049), and 4E-binding protein 1 (4E-BP1; P = 0.001) increased. With decreased eukaryotic initiation factor-4E (eIF4E).4E-BP1 complex (P = 0.01), these are consistent with increased mTOR complex 1 (mTORC1) signaling and translation initiation of protein synthesis. Although mRNA expression of ubiquitin, MAFbx 1, and MuRF-1 was unchanged, total ubiquitinated proteins decreased 20% (P < 0.01), consistent with proteolysis suppression. The Hyper-3 clamp increases whole body protein synthesis, net anabolism, and muscle protein translation initiation pathways and decreases protein ubiquitination. The main contribution of hyperaminoacidemia is stimulation of synthesis rather than inhibition of proteolysis, and it attenuates the expected increment of glucose disposal.