Skeletal and heart muscle protein turnover during long-term exposure to high environmental temperatures in young rats

A study was undertaken to determine the long-term effects of a hot environment on protein turnover in skeletal and cardiac muscles of young homeothermic animals. Three groups of 36 male 28 day old rats were housed at 35 degrees C (hot group), 25 degrees C (control group), or 25 degrees C but pair-fed to the intake of the hot group (pair-fed group). Rates of protein synthesis and degradation were measured in vivo on days 5, 10, 15, and 20. By day 20, soleus and gastrocnemius (skeletal muscle) protein masses were 7 and 14% lower in the hot group and 31 and 21% lower in the pair-fed group compared with the control group (P < 0.05). The fractional rate of protein synthesis (k(syn)) was on average 11% lower (P < 0.05) in the hot group compared with control rats and was not different from pair-fed rats. The fractional rate of skeletal muscle protein degradation (k(deg)) in hot rats was slightly lower than in control rats; k(deg) was on average 18% higher (P < 0.05) in the pair-fed group compared with the hot group and this difference appeared to be most prominent on day 5. In heart, by day 20, protein mass was 30% lower in the hot group and 40% lower in the pair-fed group compared with control rats (P < 0.05). k(syn) was on average 19% lower (P < 0.05) in the hot group compared with the control group, but not different from pair-fed rats. In the heart there were no differences in k(deg) among treatments. Plasma triiodothyronine (T3) concentration was lower in the hot group, but not in the pair-fed group, compared with controls. In conclusion, chronic exposure to hot environments was associated with lower skeletal and cardiac muscle mass and protein turnover; lower protein mass in this tissue was due to decreased k(syn); this is consistent with lower plasma T3 concentrations. In pair-fed rats, k(syn) was also reduced, but interestingly k(deg) was not, resulting in a greater loss of skeletal muscle mass. These results suggest that heat exposure invokes physiological adaptations to preserve skeletal muscle mass despite decreased food intake. In the heart, loss of protein was a result of decreased k(syn), which can be primarily ascribed to lower food intake.     

Antioxidant alpha-lipoic acid and protein turnover in insulin-resistant rat muscle

We have shown previously that the antioxidant alpha-lipoic acid (ALA) can stimulate glucose transport and can enhance the stimulation of this process by insulin in skeletal muscle from insulin-resistant obese Zucker rats. As insulin can also acutely activate general protein synthesis and inhibit net protein degradation in skeletal muscle, we hypothesized that ALA could directly affect protein turnover and also increase the effect of insulin on protein turnover in isolated skeletal muscle from developing obese Zucker rats. In epitrochlearis muscles isolated from obese Zucker rats, insulin (2 mU/ml) significantly (p < 0.05) increased in vitro protein synthesis (phenylalanine incorporation into protein) and decreased net protein degradation (tyrosine release), whereas a racemic mixture of ALA (2 mM) had no effect on either process. Interestingly, rates of protein synthesis in muscle from obese Zucker rats were substantially lower compared to those values observed in age-matched insulin-sensitive Wistar rats, whereas rates of protein degradation were comparable. Obese Zucker rats were also treated chronically with either vehicle or ALA (50 mg/kg/d for 10 d). Again, insulin significantly increased net protein synthesis and decreased net protein degradation in epitrochlearis muscles isolated from vehicle-treated obese Zucker rats; however, this stimulatory effect of insulin was not improved by prior in vivo ALA treatment. These results indicate that the previously described effect of the antioxidant ALA to increase insulin-stimulated glucose transport in skeletal muscle of obese, insulin-resistant rats does not apply to another important insulin-regulatable process, protein turnover. These findings imply that the cellular mode of action for ALA is restricted to signaling factors unique to the activation of glucose transport, and does not involve the pathway of stimulation of general protein synthesis and net protein degradation.     

Chronic hypobaric hypoxia mediated skeletal muscle atrophy: role of ubiquitin–proteasome pathway and calpains

The most frequently reported symptom of exposure to high altitude is loss of body mass and decreased performance which has been attributed to altered protein metabolism affecting skeletal muscles mass. The present study explores the mechanism of chronic hypobaric hypoxia mediated skeletal muscle wasting by evaluating changes in protein turnover and various proteolytic pathways. Male Sprague-Dawley rats weighing about 200 g were exposed to hypobaric hypoxia (7,620 m) for different durations of exposure. Physical performance of rats was measured by treadmill running experiments. Protein synthesis, protein degradation rates were determined by (14)C-Leucine incorporation and tyrosine release, respectively. Chymotrypsin-like enzyme activity of the ubiquitin-proteasome pathway and calpains were studied fluorimetrically as well as using western blots. Declined physical performance by more than 20%, in terms of time taken in exhaustion on treadmill, following chronic hypobaric hypoxia was observed. Compared to 1.5-fold increase in protein synthesis, the increase in protein degradation was much higher (five-folds), which consequently resulted in skeletal muscle mass loss. Myofibrillar protein level declined from 46.79 ± 1.49 mg/g tissue at sea level to 37.36 ± 1.153 (P < 0.05) at high altitude. However, the reduction in sarcoplasmic proteins was less as compared to myofibrillar protein. Upregulation of Ub-proteasome pathway (five-fold over control) and calpains (three-fold) has been found to be important factors for the enhanced protein degradation rate. The study provided strong evidences suggesting that elevated protein turnover rate lead to skeletal muscle atrophy under chronic hypobaric hypoxia via ubiquitin-proteasome pathway and calpains.     

Higher skeletal muscle protein synthesis and lower breakdown after chemotherapy in cachectic mice

The influence of cancer cachexia and chemotherapy and subsequent recovery of skeletal muscle protein mass and turnover was investigated in mice. Cancer cachexia was induced using colon 26 adenocarcinoma, which is characteristic of the human condition, and can be cured with 100% efficacy using an experimental nitrosourea, cystemustine (C(6)H(12)CIN(3)O(4)S). Reduced food intake was not a factor in these studies. Three days after cachexia began, healthy and tumor-bearing mice were given a single intraperitoneal injection of cystemustine (20 mg/kg). Skeletal muscle mass in tumor-bearing mice was 41% lower (P < 0.05) than in healthy mice 2 wk after cachexia began. Skeletal muscle wasting was mediated initially by decreased protein synthesis (-38%; P < 0.05) and increased degradation (+131%; P < 0.05); later wasting resulted solely from decreased synthesis (~-54 to -69%; P < 0.05). Acute cytotoxicity of chemotherapy did not appear to have an important effect on skeletal muscle protein metabolism in either healthy or tumor-bearing mice. Recovery began 2 days after treatment; skeletal muscle mass was only 11% lower than in healthy mice 11 days after chemotherapy. Recovery of skeletal muscle mass was affected initially by decreased protein degradation (-80%; P < 0.05) and later by increased protein synthesis (+46 to +73%; P < 0.05) in cured compared with healthy mice. This study showed that skeletal muscle wasted from cancer cachexia and after chemotherapeutic treatment is able to generate a strong anabolic response by making powerful changes to protein synthesis and degradation.     

The ubiquitin-proteasome proteolytic pathway in heart vs skeletal muscle: effects of acute diabetes

The ubiquitin-proteasome system is thought to play a major role in normal muscle protein turnover and to contribute to diabetes-induced protein wasting in skeletal muscle. However, its importance in cardiac muscle is not clear. We measured heart muscle mRNA for ubiquitin and for the C2 and C8 proteasomal subunits, the amount of free ubiquitin and the proteasome chymotrypsin-like proteolytic activity in control and diabetic rats. Results were compared to those in skeletal muscle (rectus). Heart ubiquitin, C2 and C8 subunit mRNA and proteolytic activity were significantly greater than in skeletal muscle (P 相似文献   

Active involvement of PKC for insulin-mediated rates of muscle protein synthesis in Zucker rats

A recent report from our group demonstrated that insulin facilitates muscle protein synthesis in obese Zucker rats. The purpose of this study was to determine whether PKC, a probable modulator of insulin signal transduction and/or mRNA translation, has a role in this insulin-mediated anabolic response. In the first portion of the study, gastrocnemius muscles of lean and obese Zucker rats (n = 5-7 for each phenotype) were bilaterally perfused with or without insulin to assess cytosolic and membrane PKC activity. Limbs perfused with insulin demonstrated greater PKC activity in both lean and obese Zucker rats (P < 0.05) compared with no insulin, but overall activity was greater in obese animals (by approximately 27% compared with lean, P < 0.05). To determine whether PKC plays a role in muscle protein synthesis, hindlimbs (n = 6-8 for each phenotype) were bilaterally perfused with or without insulin and/or GF-109203X (GF; a PKC inhibitor). The presence of GF did not influence the rates of insulin-mediated protein synthesis in gastrocnemius muscle of lean Zucker rats. However, when obese rats were perfused with GF (P < 0.05), the effect of insulin on elevating rates of protein synthesis was not observed. We also used phorbol 12-myristate 13-acetate (TPA, a PKC activator; n = 5-7 for each phenotype) with and without insulin to determine the effect of PKC activation on muscle protein synthesis. TPA alone did not elevate muscle protein synthesis in lean or obese rats. However, TPA plus insulin resulted in elevated rates of protein synthesis in both phenotypes that were similar to rates of insulin alone of obese rats. These results suggest that PKC is a modulator and is necessary, but not sufficient, for insulin-mediated protein anabolic responses in skeletal muscle.     

Mechanism of muscle glycogen autoregulation in humans

To examine the mechanism by which muscle glycogen limits its own synthesis, muscle glycogen and glucose 6-phosphate (G-6-P) concentrations were measured in seven healthy volunteers during a euglycemic ( approximately 5.5 mM)-hyperinsulinemic ( approximately 450 pM) clamp using (13)C/(31)P nuclear magnetic resonance spectroscopy before and after a muscle glycogen loading protocol. Rates of glycogen synthase (V(syn)) and phosphorylase (V(phos)) flux were estimated during a [1-(13)C]glucose (pulse)-unlabeled glucose (chase) infusion. The muscle glycogen loading protocol resulted in a 65% increase in muscle glycogen content that was associated with a twofold increase in fasting plasma lactate concentrations (P < 0.05 vs. basal) and an approximately 30% decrease in plasma free fatty acid concentrations (P < 0.001 vs. basal). Muscle glycogen loading resulted in an approximately 30% decrease in the insulin-stimulated rate of net muscle glycogen synthesis (P < 0.05 vs. basal), which was associated with a twofold increase in intramuscular G-6-P concentration (P < 0.05 vs. basal). Muscle glycogen loading also resulted in an approximately 30% increase in whole body glucose oxidation rates (P < 0.05 vs. basal), whereas there was no effect on insulin-stimulated rates of whole body glucose uptake ( approximately 10.5 mg. kg body wt(-1). min(-1) for both clamps) or glycogen turnover (V(syn)/V(phos) was approximately 23% for both clamps). In conclusion, these data are consistent with the hypothesis that glycogen limits its own synthesis through feedback inhibition of glycogen synthase activity, as reflected by an accumulation of intramuscular G-6-P, which is then shunted into aerobic and anaerobic glycolysis.     

Selected Contribution: IGF-I antibody prevents increases in protein synthesis in epitrochlearis muscles from refed, diabetic rats.

The purpose of this study was to examine whether immune neutralization of muscle-produced insulin-like growth factor I (IGF-I) would prevent an appropriate anabolic response to refeeding in diabetic rats. Male Sprague-Dawley rats were made diabetic by partial pancreatectomy and were randomly assigned to be either control-fed, fasted, or fasted-refed (n = 7-8 per group). Diabetes decreased rates of protein synthesis and increased rates of protein degradation in incubated epitrochlearis muscles (P < 0.05). In both groups of rats, fasting lowered protein synthesis and increased proteolysis and subsequent refeeding returned both parameters to near basal values (P < 0.05). Neutralization of muscle IGF-I by the addition of IGF-I antibody to the incubation medium reduced protein synthesis an average of 22% for all groups (P < 0.05). However, rates of protein degradation were not affected. In nondiabetic rats, refeeding increased protein synthesis in both control and antibody-treated muscles (P < 0.05). Refeeding also increased protein synthesis in the control muscles from diabetic rats (P < 0.01). In contrast, muscles from diabetic rats that were incubated with anti-IGF-I did not increase protein synthesis in response to refeeding. These data suggest that immune neutralization of muscle IGF-I in hypoinsulinemic rats negated the ability of endogenous IGF-I to promote protein synthesis and thereby prevented an appropriate anabolic response.     

Testosterone increases the muscle protein synthesis rate but does not affect very-low-density lipoprotein metabolism in obese premenopausal women

Men and women with hyperandrogenemia have a more proatherogenic plasma lipid profile [e.g., greater triglyceride (TG) and total and low-density lipoprotein-cholesterol and lower high-density lipoprotein-cholesterol concentrations] than healthy premenopausal women. Furthermore, castration of male rats markedly reduces testosterone availability below normal and decreases plasma TG concentration, and testosterone replacement reverses this effect. Testosterone is, therefore, thought to be an important regulator of plasma lipid homeostasis. However, little is known about the effect of testosterone on plasma TG concentration and kinetics. Furthermore, testosterone is a potent skeletal muscle protein anabolic agent in men, but its effect on muscle protein turnover in women is unknown. We measured plasma lipid concentrations, hepatic very low density lipoprotein (VLDL)-TG and VLDL-apolipoprotein B-100 secretion rates, and the muscle protein fractional synthesis rate in 10 obese women before and after trandermal testosterone (1.25 g of 1% AndroGel daily) treatment for 3 wk. Serum total and free testosterone concentrations increased (P < 0.05) by approximately sevenfold in response to testosterone treatment, reaching concentrations that are comparable to those in women with hyperandrogenemia, but lower than the normal range for eugonadal men. Except for a small (～10%) decrease in plasma high-density lipoprotein particle and cholesterol concentrations (P < 0.04), testosterone therapy had no effect on plasma lipid concentrations, lipoprotein particle sizes, and hepatic VLDL-TG and VLDL-apolipoprotein B-100 secretion rates (all P > 0.05); the muscle protein fractional synthesis rate, however, increased by ～45% (P < 0.001). We conclude that testosterone is a potent skeletal muscle protein anabolic agent, but not an important regulator of plasma lipid homeostasis in obese women.     

Amino acids and insulin are both required to regulate assembly of the eIF4E. eIF4G complex in rat skeletal muscle

The respective roles of insulin and amino acids in regulation of skeletal muscle protein synthesis and degradation after feeding were examined in rats fasted for 17 h and refed over 1 h with either a 25 or a 0% amino acid/protein meal. In each nutritional condition, postprandial insulin secretion was either maintained (control groups: C(25) and C(0)) or blocked with diazoxide injections (diazoxide groups: DZ(25) and DZ(0)). Muscle protein metabolism was examined in vitro in epitrochlearis muscles. Only feeding the 25% amino acid/protein meal in the presence of increased plasma insulin concentration (C(25) group) stimulated protein synthesis and inhibited proteolysis in skeletal muscle compared with the postabsorptive state. The stimulation of protein synthesis was associated with increased phosphorylation of eukaryotic initiation factor (eIF)4E binding protein-1 (4E-BP1), reduced binding of eIF4E to 4E-BP1, and increased assembly of the active eIF4E. eIF4G complex. The p70 S6 kinase (p70(S6k)) was also hyperphosphorylated in response to the 25% amino acid/protein meal. Acute postprandial insulin deficiency induced by diazoxide injections totally abolished these effects. Feeding the 0% amino acid/protein meal with or without postprandial insulin deficiency did not stimulate muscle protein synthesis, reduce proteolysis, or regulate initiation factors and p70(S6k) compared with fasted rats. Taken together, our results suggest that both insulin and amino acids are required to stimulate protein synthesis, inhibit protein degradation, and regulate the interactions between eIF4E and 4E-BP1 or eIF4G in response to feeding.     

Effects of estradiol and progesterone on body composition, protein synthesis, and lipoprotein lipase in rats

Prior studies suggest that estradiol and progesterone regulate body composition in growing female rats. Because these studies did not consider the confounding effect of changes in food intake, it remains unclear whether ovarian hormones regulate body composition independently of their effects on food intake. We utilized a pair-feeding paradigm to examine the effects of these hormones on body composition. In addition, skeletal muscle protein fractional synthesis rate and adipose tissue lipoprotein lipase activity were measured to examine pathways of substrate deposition into fat and fat-free tissue. Female Sprague-Dawley rats [pubertal: 7-8 wk old; 190 +/- 0.5 (SE) g] were separated into four groups: 1) sham-operated (S; n = 8), 2) ovariectomized plus placebo (OVX; n = 8), 3) ovariectomized plus estradiol (OVX+E; n = 8), and 4) ovariectomized plus progesterone (OVX+P; n = 8). All ovariectomized groups were pair-fed to the S group. Body composition was measured using total body electrical conductivity. The relative increase in fat-free mass was greater (P < 0.01) in the OVX group (31 +/- 2%) than in the S (17 +/- 2%), OVX+E (18 +/- 2%), and OVX+P (22 +/- 2%) groups. The fractional synthetic rates of gastrocnemius muscle protein paralleled changes in fat-free mass: OVX had a higher (P < 0.05) synthesis rate (21 +/- 3%/day) than S (12 +/- 2%/day), OVX+E (11 +/- 2%/day), and OVX+P (8 +/- 1%/day) groups. Body fat increased in the S group (31 +/- 7%; P < 0.01), whereas the OVX groups lost fat (OVX: -10 +/- 7%; OVX+E: -15 +/- 7%; OVX+P: -13 +/- 7%). No differences in lipoprotein lipase were found. Our results suggest that estradiol and progesterone may regulate the growth of fat and fat-free tissues in female rats. Moreover, ovarian hormones may influence skeletal muscle growth through their effects on skeletal muscle protein synthesis.     

Protein kinetics in stable heart failure patients.

Heart failure (HF) is a slow progressive syndrome characterized by low cardiac output and peripheral metabolic, biochemical, and histological alterations. Protein loss and reduced protein turnover occur with aging, but the consequences of congestive HF (CHF) superimposed on the normal aging response are unknown. This study has two objectives: 1) to determine whether there was a difference between older age-matched controls and those with stable HF (i.e., ischemic pathology) in whole body protein turnover and 2) to determine whether protein metabolism in liver and skeletal muscle protein turnover is impacted by CHF. We measured the whole body protein synthesis rate with a U-(15)N-labeled algal protein hydrolysate in 10 patients with CHF and in 10 age-matched controls. Muscle fractional synthesis rate of lateral vastus muscle was determined with [U-(13)C]alanine on muscle biopsies obtained by a standard percutaneous needle biopsy technique. Fractional synthesis rates of five plasma proteins of hepatic origin (fibrinogen, complement C-3, ceruloplasmin, transferrin, and very low-density lipoprotein apoliprotein B-100) were determined by using (2)H(5)-labeled l-phenylalanine as tracer. Results showed that whole body protein synthesis rate was reduced in CHF patients (3.09 +/- 0.19 vs. 2.25 +/- 0.71 g protein x kg(-1) x day(-1), P < 0.05) as was muscle fractional synthesis rate (3.02 +/- 0.58 vs. 1.33 +/- 0.71%/day, P < 0.05) and very low-density lipoprotein apoliprotein B-100 (265 +/- 25 vs. 197 +/- 16%/day, P < 0.05). CHF patients were hyperinsulinemic (9.6 +/- 3.1 vs. 47.0 +/- 7.8 microU/ml, P < 0.01). The results were compared with those found with bed rest patients. In conclusion, protein turnover is depressed in CHF patients, and both skeletal muscle and liver are impacted. These results are similar to those found with bed rest, which suggests that inactivity is a factor in depressed protein metabolism.     

Combined effects of hyperaminoacidemia and oxandrolone on skeletal muscle protein synthesis

We investigated whether the normal anabolic effects of acute hyperaminoacidemia were maintained after 5 days of oxandrolone (Oxandrin, Ox)-induced anabolism. Five healthy men [22 +/- 3 (SD) yr] were studied before and after 5 days of oral Ox (15 mg/day). In each study, a 5-h basal period was followed by a 3-h primed-continuous infusion of a commercial amino acid mixture (10% Travasol). Stable isotopic data from blood and muscle sampling were analyzed using a three-compartment model to calculate muscle protein synthesis and breakdown. Model-derived muscle protein synthesis increased after amino acid infusion in both the control [basal control (BC) vs. control + amino acids (C+AA); P < 0.001] and Ox study [basal Ox (BOx) vs. Ox + amino acids (Ox+AA); P < 0.01], whereas protein breakdown was unchanged. Fractional synthetic rates of muscle protein increased 94% (BC vs. C+AA; P = 0.01) and 53% (BOx vs. Ox+AA; P < 0.01), respectively. We conclude that the normal anabolic effects of acute hyperaminoacidemia are maintained in skeletal muscle undergoing oxandrolone-induced anabolism.     

Effects of ciliary neurotrophic factor (CNTF) on protein turnover in cultured muscle cells

The goal of the study was to evaluate the mechanism by which ciliary neurotrophic factor (CNTF) regulated protein metabolism in skeletal muscle. L8 myotubes were cultured and effects of various times and doses of CNTF on protein synthesis and degradation were evaluated. Effects of CNTF on turnover of specific pools of proteins (myofibrillar and non-myofibrillar) were also evaluated. Protein synthesis was assayed by incorporation of radioactive tyrosine into muscle proteins. Degradation was assessed by release of labelled tyrosine from pre-labelled myotubes. Effects of CNTF on protein turnover were found to be time- and dose-dependent. CNTF (1 and 10 ng/ml) increased myofibrillar protein synthesis after 12 h of exposure but had no effect on non-myofibrillar protein synthesis. Longer exposures of CNTF (24 h) reduced non-myofibrillar protein synthesis and had no effect on myofibrillar protein synthesis. High concentrations of CNTF (10 and 20 ng/ml) reduced myofibrillar protein degradation but had no effect on degradation of non-myofibrillar proteins. To evaluate the mechanism by which CNTF exerts control of protein turnover, we completed a Northern blot for CNTF receptor alpha-subunit (CNTFRalpha). This was non-detectable via conventional northern analysis. Use of RT-PCR, however, confirmed expression of CNTFRalpha, albeit at a low level compared to rat skeletal muscle. This low expression of the receptor in L8 myotubes may explain the limited effect of CNTF in vitro compared to the larger effects typically detected in vivo. CNTF regulated protein turnover through control of protein synthesis and degradation. Effects were dose and timedependent. These observations may explain ability of CNTF to exert both anabolic and catabolic actions in vivo.     

Influence of amino acid availability on protein turnover in perfused skeletal muscle

The effects of amino acids on protein turnover in skeletal muscle were determined in the perfused rat hemicorpus preparation. Perfusion of preparations from fasted young rats (81±2 g) with medium containing either a complete mixture of amino acids at five times (5×) their normal plasma levels, a mixture of leucine, isoleucine, and valine at 5× or 10× levels, or leucine alone (10×) resulted in a 25–50% increase in muscle protein synthesis and a 30% decrease in protein degradation compared to fasted controls perfused in the absence of exogenously added amino acids. When the branched-chain amino acids were omitted from the complete mixture, the remaining amino acids (5×) had no effect on protein turnover. The complete mixture at 1× levels was also ineffective. Comparison of the effects of amino acids with those of glucose and palmitate indicated that amino acids were not acting by providing substrates for energy metabolism. The stimulatory effect of amino acids on protein synthesis was associated with a facilitated rate of peptide-chain initiation as evidenced by a relative decrease in the level of ribosomal subunits. This response was not as great as that produced by insulin, and the amino acids did not augment the effect of insulin. Although protein synthesis in preparations from fed young rats (130±3 g) was stimulated by the addition of a mixture of the branched-chain amino acids (5×) to about the same extent as that observed in the fasted young rats, protein degradation was not affected. Furthermore, neither synthesis nor degradation were affected in preparations from fasted older rats (203±9 g) suggesting that the age and or nitritional state of the animal may influence the response of skeletal muscle to altered amino acid levels.     

The effect of diabetes on protein synthesis and the respiratory chain of rat skeletal muscle and kidney mitochondria

The effects of streptozotocin-induced diabetes mellitus upon mitochondria from rat skeletal muscle and kidney were examined. The rate of amino acid incorporation in vitro by isolated skeletal muscle mitochondria from diabetic animals was decreased by 50–60% from control values. Treatment of diabetic animals with insulin lowered blood glucose levels to control values and restored the rate of muscle mitochondrial protein synthesis in vitro to control levels. The rates of skeletal muscle mitochondrial protein synthesis were also decreased 23–27% by a 2-day fast. Comparison of the translation products synthesized by isolated muscle mitochondria from control and diabetic rats by dodecyl sulfate polyacrylamide-gel electrophoresis revealed a uniform decrease in the synthesis of all polypeptides. Aurintricarboxylic acid and pactamycin, inhibitors of chain initiation, blocked protein synthesis to a greater extent in muscle mitochondria from control as compared to diabetic animals suggesting that mitochondria from diabetics are unable to initiate protein synthesis at a rate comparable to control. Phenotypic changes observed in diabetic muscle mitochondria included a 36% decrease in the content of cytochromes aa₃ and a 27% decrease in cytochrome b, both established as containing mitochondrial translation products in lower eucaryotes. State 3 respiration with glutamate as substrate decreased by 27% and uncoupler-stimulated respiration decreased by 23% in the diabetic mitochondria. By contrast, the specific activities of NADH and succinate dehydrogenases, established as products of cytoplasmic protein synthesis in lower eucaryotes, were not decreased in skeletal muscle mitochondria from the diabetic animals. These results suggest that the considerable muscular atrophy observed in diabetics may involve decreases in both cytoplasmic and mitochondrial protein synthesis, the latter reflected in profound changes in the respiratory chain. By contrast, comparison of kidney mitochondria from control and diabetic rats revealed no differences in the rates of protein synthesis in vitro, nor in the mitochondrial translation products, which corresponded closely to liver and skeletal muscle translation products. Similarly, the mitochondrial content of cytochromes b, c + c₁, and aa₃, the specific activity of succinate dehydrogenase, the rate of state 3 respiration, and the recovery of mitochondria from kidney homogenates did not differ in control and diabetic animals. Kidney mitochondria are thus like liver mitochondria in being relatively unaffected by insulin deprivation.     

Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats

Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P < 0.05); refeeding reversed these changes (P < 0.05). Consistent with these effects being regulated by S6K1, activation of this kinase was suppressed by FD (-91%, P < 0.05) but was increased by refeeding. Gavaging rats subjected to FD with a mixture of amino acids partially restored muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P < 0.0001). Thus feeding stimulates fractional protein synthesis in skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.     

Combined ingestion of protein and free leucine with carbohydrate increases postexercise muscle protein synthesis in vivo in male subjects

The present study was designed to determine postexercise muscle protein synthesis and whole body protein balance following the combined ingestion of carbohydrate with or without protein and/or free leucine. Eight male subjects were randomly assigned to three trials in which they consumed drinks containing either carbohydrate (CHO), carbohydrate and protein (CHO+PRO), or carbohydrate, protein, and free leucine (CHO+PRO+Leu) following 45 min of resistance exercise. A primed, continuous infusion of L-[ring-13C6]phenylalanine was applied, with blood samples and muscle biopsies collected to assess fractional synthetic rate (FSR) in the vastus lateralis muscle as well as whole body protein turnover during 6 h of postexercise recovery. Plasma insulin response was higher in the CHO+PRO+Leu compared with the CHO and CHO+PRO trials (+240 +/- 19% and +77 +/- 11%, respectively, P < 0.05). Whole body protein breakdown rates were lower, and whole body protein synthesis rates were higher, in the CHO+PRO and CHO+PRO+Leu trials compared with the CHO trial (P < 0.05). Addition of leucine in the CHO+PRO+Leu trial resulted in a lower protein oxidation rate compared with the CHO+PRO trial. Protein balance was negative during recovery in the CHO trial but positive in the CHO+PRO and CHO+PRO+Leu trials. In the CHO+PRO+Leu trial, whole body net protein balance was significantly greater compared with values observed in the CHO+PRO and CHO trials (P < 0.05). Mixed muscle FSR, measured over a 6-h period of postexercise recovery, was significantly greater in the CHO+PRO+Leu trial compared with the CHO trial (0.095 +/- 0.006 vs. 0.061 +/- 0.008%/h, respectively, P < 0.05), with intermediate values observed in the CHO+PRO trial (0.0820 +/- 0.0104%/h). We conclude that coingestion of protein and leucine stimulates muscle protein synthesis and optimizes whole body protein balance compared with the intake of carbohydrate only.     

Effect of diabetes on the rates of synthesis and degradation of ribosomes in rat muscle and liver in vivo

An important component of the decrease in protein synthesis in muscle of diabetic animals is a fall in the ribosome content. Therefore, we have investigated the turnover of ribosomes in skeletal muscle, heart, and liver of rats during the onset of diabetes. Synthesis rates were measured by incorporation of label into the protein moieties of the ribosomes, and a dual isotope technique was used to relate ribosome synthesis to that of total tissue protein. Degradation rates were calculated as the difference between the rates of synthesis and accumulation. The loss of ribosomes from gastrocnemius muscle and heart took place mainly between the 2nd and 4th days of insulin deficiency and was brought about largely by a very pronounced increase in the degradation rate, though synthesis also fell by a substantial amount. Rates of total tissue protein synthesis decreased markedly, but the degradation rates were only slightly elevated, if at all. Thus, the effect of diabetes on muscle ribosome breakdown was quite distinct from that on degradation of total tissue protein. In liver the response of protein synthesis to diabetes was much less pronounced than in muscle, and ribosome synthesis was not affected.     

Impact of protein coingestion on muscle protein synthesis during continuous endurance type exercise

This study investigates the impact of protein coingestion with carbohydrate on muscle protein synthesis during endurance type exercise. Twelve healthy male cyclists were studied during 2 h of fasted rest followed by 2 h of continuous cycling at 55% W(max). During exercise, subjects received either 1.0 g·kg(-1)·h(-1) carbohydrate (CHO) or 0.8 g·kg(-1)·h(-1) carbohydrate with 0.2 g·kg(-1)·h(-1) protein hydrolysate (CHO+PRO). Continuous intravenous infusions with l-[ring-(13)C(6)]phenylalanine and l-[ring-(2)H(2)]tyrosine were applied, and blood and muscle biopsies were collected to assess whole body protein turnover and muscle protein synthesis rates at rest and during exercise conditions. Protein coingestion stimulated whole body protein synthesis and oxidation rates during exercise by 22 ± 3 and 70 ± 17%, respectively (P < 0.01). Whole body protein breakdown rates did not differ between experiments. As a consequence, whole body net protein balance was slightly negative in CHO and positive in the CHO+PRO treatment (-4.9 ± 0.3 vs. 8.0 ± 0.3 μmol Phe·kg(-1)·h(-1), respectively, P < 0.01). Mixed muscle protein fractional synthetic rates (FSR) were higher during exercise compared with resting conditions (0.058 ± 0.006 vs. 0.035 ± 0.006%/h in CHO and 0.070 ± 0.011 vs. 0.038 ± 0.005%/h in the CHO+PRO treatment, respectively, P < 0.05). FSR during exercise did not differ between experiments (P = 0.46). We conclude that muscle protein synthesis is stimulated during continuous endurance type exercise activities when carbohydrate with or without protein is ingested. Protein coingestion does not further increase muscle protein synthesis rates during continuous endurance type exercise.