Translation Initiation In Muscle Research Articles

Leucine Supplementation Does Not Enhance Translation Initiation of Low Birth‐Weight Neonatal Pigs Despite Greater Sestrin2 Expression in Skeletal Muscle

Lean growth is impaired in low birth‐weight (LBWT) neonatal pigs and correlates with a reduction in protein synthesis rate in skeletal muscle. Translation initiation via mTOR signaling pathway is the rate limiting step in protein synthesis. Leucine binds and inactivates Sestrin2, an inhibitor of the mTORC1 complex. The objective of this study was to determine whether dietary branched‐chain amino acids (BCAA) stimulate translation initiation in skeletal muscle of LBWT pigs. LBWT and normal birth weight (NBWT) were defined when weight at birth was 2 SD below or within 0.5 of the corresponding litter average. Three day old LBWT and NBWT pigs (n=11‐13/group) were assigned to a Control or BCAA sow milk‐replacer formula in a 2×2 factorial arrangement. Formulas were isonitrogenous and contained either 1% BCAA (1:0.68:0.5 leucine:isoleucine:valine) or 0.7% alanine, and were offered at 250 mL·kg body weight−1·day−1 in 5 equal meals. Carotid and jugular catheters were surgically placed on day 21. On day 29, blood samples were taken over 4 h following a meal. Pigs were euthanized 1 h after a final meal. Data were analyzed using MIXED procedure of SAS version 9.4. BCAA supplementation increased BCAA concentration in plasma (P<0.0001) and in muscle free amino acid pool (P<0.001) compared to the Control diet, which similarly increased alanine concentration in plasma and muscle (P<0.0001). Although BCAA supplementation decreased circulating Pro, Ser, Asp, and Lys (P<0.001, 0.05, 0.05, 0.06, respectively), concentrations were not different in skeletal muscle. LBWT pigs expressed less BCAT2 mRNA in the longissimus dorsi than NBWT pigs, but expression was enhanced to normal levels with BCAA supplementation (P<0.05). BCKDHA expression was greater in the liver of LBWT compared to NBWT pigs irrespective of diet (P<0.05). Circulating branched‐chain α‐keto acids increased only in response to BCAA supplementation (P<0.0001). LBWT pigs expressed less Sestrin2 in skeletal muscle (P<0.01), suggesting enhanced sensitivity to leucine. However, despite lower expression of Sestrin2 and increased intramuscular BCAA concentrations, 4EBP1 and S6K1 phosphorylation were not affected by BCAA supplementation. These data suggest that branched‐chain amino acid supplementation did not enhance translation initiation in muscles of neonatal pigs regardless of birth weight.

Supplementation with leucine does not prevent development of obesity in rats fed a high fat diet (258.5)

Excess dietary fat consumption has been implicated in the development of obesity and diabetes. Obesity can be characterized by a disproportionate increase in fat mass. However, if muscle mass can be increased or maintained in obesity, this may facilitate weight loss. Historically, supplementation with the branched‐chain amino acid leucine has been shown to increase muscle protein synthesis via the protein kinase mTORC1. Recent studies suggest that supplementation with leucine also has the potential to reduce weight gain and fat deposition in high‐fat fed, obese mice. The objective of this study was to determine if long‐term dietary leucine supplementation prevents development of obesity in rats meal‐fed a high fat (60%) diet. Male, Sprague‐Dawley rats (n=30/dietary treatment) were meal‐fed (3 meals/day) either a control diet (C), control+leucine (CL), high‐fat (HF), or high‐fat+leucine (HFL) for 42 days. On day 42 rats were sacrificed at 0, 30 or 90 minutes postprandial. HF/HFL‐fed rats gained, 28 g more than rats fed C/CL diets (p<0.05). Insulin AUC was 35% higher in CL vs C and 13% higher in HFL vs HF. There was no difference in blood glucose values between treatment groups. Rats supplemented with leucine had significantly higher fat mass and significantly (p<0.05) lower muscle mass compared to their respective controls. However, preliminary data suggest that L and HFL activate translation initiation in muscle via 4E‐BP1 phosphorylation. Taken together, this study demonstrates that supplementation with leucine increases translation initiation, however this does not result in increased muscle mass. Supplementation with leucine does not prevent development of obesity in HF‐fed rats.Grant Funding Source: Supported by Arkansas Biosciences Institute.

Endotoxin Reduces Muscle Protein Synthesis and Restrains Translation Initiation by Decreasing eIF4G Phosphorylation in Neonatal and Young Pigs

Endotoxin (LPS) reduces muscle protein synthesis by restraining translation in the presence of fed levels of insulin and amino acids. In vivo effects of LPS on translation initiation in muscle during basal fasting insulin and amino acid conditions have not been determined in neonates, whose muscle protein synthesis rates are uniquely high. Overnight fasted 7‐ and 26‐d‐old pigs were infused for 8 h with LPS (0 and 10 μg·kg−1·hr−1) and fractional protein synthesis rates and translational control mechanisms in skeletal muscle were examined (n=5–7/group/age). LPS reduced the basal rate of muscle protein synthesis in 7‐ and 26‐day‐old pigs in the presence of fasting levels of insulin, glucose, and amino acids. LPS decreased eIF4G phosphorylation and the amount of the active eIF4G‐eIF4E complex in muscle of 7‐ and 26‐d‐old pigs, but had no effect on mTOR or eEF2 phosphorylation. Although muscle protein synthesis decreased and eEF2 phosphorylation increased with age, there was no effect of age on translation initiation factor activation in basal fasting conditions. The results suggest that the LPS‐induced decrease in eIF4G phosphorylation in muscle may be a limiting step in translation initiation which contributes to the sepsis‐associated decrease in basal protein synthesis rates in muscle during early postnatal life. NIH AR44474, NIH AR51563, NIH HD41648, and USDA 58‐6250‐6‐001.

Skeletal muscle protein synthesis and degradation exhibit sexual dimorphism after chronic alcohol consumption but not acute intoxication

Epidemiological evidence suggests alcoholic myopathy is more severe in females than males, but comparable animal studies are lacking that make elucidating the biochemical locus for this defect problematic. The present study determined whether skeletal muscle protein synthesis and markers of degradation exhibit a sexual dimorphic response to either chronic alcohol consumption or acute intoxication. Male and female rats were fed an alcohol-containing diet, pair-fed for 26 wk (chronic), or received an intraperitoneal injection of alcohol (acute). In males, chronic alcohol decreased gastrocnemius protein synthesis by 20%. This reduction was associated with a twofold increase in the inactive eukaryotic initiation factor (eIF) 4E.4E-binding protein 1 (4E-BP1) complex and a 60% reduction in the active eIF4E.eIF4G complex. This redistribution of eIF4E was associated with decreased phosphorylation of both 4E-BP1 and eIF4G (50-55%). The phosphorylation of ribosomal protein S6 was also reduced 60% in alcohol-consuming male rats. In contrast, neither rates of protein synthesis nor indexes of translation initiation in muscle were altered in alcohol-fed female rats despite blood alcohol levels comparable to males. Chronic alcohol ingestion did not alter atrogin-1 or muscle RING finger-1 mRNA content (biomarkers of muscle proteolysis) in males but increased their expression in females 50-100%. Acute alcohol intoxication produced a comparable decrease in muscle protein synthesis and translation initiation in both male and female rats. Our data demonstrate a sexual dimorphism for muscle protein synthesis, translation initiation, and proteolysis in response to chronic, but not acute, alcohol intoxication; however, they do not support evidence indicating females are more sensitive toward the development of alcoholic skeletal muscle myopathy.

Developmental changes in the feeding-induced stimulation of translation initiation in muscle of neonatal pigs.

The rapid gain in skeletal muscle mass in the neonate is associated with a marked elevation in skeletal muscle protein synthesis in response to feeding. The feeding-induced response decreases with development. To determine whether the response to feeding is regulated at the level of translation initiation, the expression, phosphorylation, and function of a number of eukaryotic initiation factors (eIF) were examined. Pigs at 7 and 26 days of age were either fasted overnight or fed porcine milk after an overnight fast. In muscle of 7-day-old pigs, the hyperphosphorylated form of the eIF4E repressor protein, 4E-binding protein 1 (4E-BP1), was undetectable in the fasting state but rose to 60% of total 4E-BP1 after feeding; eIF4E phosphorylation was unaffected by feeding status. The amount of eIF4E in the inactive 4E-BP1. eIF4E complex was reduced by 80%, and the amount of eIF4E in the active eIF4E. eIF4G complex was increased 14-fold in muscle of 7-day-old pigs after feeding. The amount of 70-kDa ribosomal protein S6 (p70(S6)) kinase in the hyperphosphorylated form rose 2.5-fold in muscle of 7-day-old pigs after feeding. Each of these feeding-induced responses was blunted in muscle of 26-day-old pigs. eIF2B activity in muscle was unaffected by feeding status but decreased with development. Feeding produced similar changes in eIF characteristics in liver and muscle; however, the developmental changes in liver were not as apparent as in skeletal muscle. Thus the results demonstrate that the developmental change in the acute stimulation of skeletal muscle protein synthesis by feeding is regulated by the availability of eIF4E for 48S ribosomal complex formation. The results further suggest that the overall developmental decline in skeletal muscle protein synthesis involves regulation by eIF2B.

Inhibition of muscle protein synthesis by alcohol is associated with modulation of eIF2B and eIF4E.

The present study examined potential mechanisms for the inhibition of protein synthesis in skeletal muscle after chronic alcohol consumption. Rats were maintained on an alcohol-containing diet for 14 wk; control animals were pair fed. Alcohol-induced myopathy was confirmed by a reduction in lean body mass as well as a decrease in the weight of the gastrocnemius and psoas muscles normalized for tibial length. No alcohol-induced decrease in total RNA content (an estimate of ribosomal RNA) was detected in any muscle examined, suggesting that alcohol reduced translational efficiency but not the capacity for protein synthesis. To identify mechanisms responsible for regulating translational efficiency, we analyzed several eukaryotic initiation factors (eIF). There was no difference in the muscle content of either total eIF2alpha or the amount of eIF2alpha in the phosphorylated form between alcohol-fed and control rats. Similarly, the relative amount of eIF2Bepsilon in muscle was also not different. In contrast, alcohol decreased eIF2B activity in psoas (fast-twitch) but not in soleus or heart (slow-twitch) muscles. Alcohol feeding also dramatically influenced the distribution of eIF4E in the gastrocnemius (fast-twitch) muscle. Compared with control values, muscle from alcohol-fed rats demonstrated 1) an increased binding of the translational repressor 4E-binding protein 1 (4E-BP1) with eIF4E, 2) a decrease in the phosphorylated gamma-form of 4E-BP1, and 3) a decrease in eIF4G associated with eIF4E. In summary, these data suggest that chronic alcohol consumption impairs translation initiation in muscle by altering multiple regulatory sites, including eIF2B activity and eIF4E availability.