Thomas C. Vary

Effect of dietary protein on translation initiation in rat skeletal muscle and liver

The effect of dietary protein on the initiation of mRNA translation was examined in rats starved for 18 h and then fed isocaloric diets containing either 20% protein (20P) or no added protein (0P). Feeding the 20P diet, but not the 0P diet, stimulated protein synthesis in skeletal muscle and liver by 38 and 41%, respectively. The stimulation was associated with reduced binding of eukaryotic initiation factor (eIF) 4E to the translational repressor 4E-BP1, increased formation of the active eIF4E-eIF4G complex, and increased phosphorylation of 4E-BP1. In contrast, feeding a 0P diet had no effect on any of these parameters. Feeding a 20P diet resulted in partial dephosphorylation of eIF4E in both tissues. In liver, refeeding a 0P diet also resulted in partial eIF4E dephosphorylation, suggesting that the phosphorylation state of eIF4E is not important in the stimulation of protein synthesis under these conditions. Finally, plasma insulin concentrations were the same in rats fed either diet (14.8 +/- 4.9 vs. 15.5 +/- 4.5 microU/ml for 20P and 0P groups, respectively), suggesting that feeding-induced changes in plasma insulin are not sufficient to stimulate protein synthesis. Instead, a combination of dietary protein and insulin may be required to stimulate translation initiation.The effect of dietary protein on the initiation of mRNA translation was examined in rats starved for 18 h and then fed isocaloric diets containing either 20% protein (20P) or no added protein (0P). Feeding the 20P diet, but not the 0P diet, stimulated protein synthesis in skeletal muscle and liver by 38 and 41%, respectively. The stimulation was associated with reduced binding of eukaryotic initiation factor (eIF) 4E to the translational repressor 4E-BP1, increased formation of the active eIF4E-eIF4G complex, and increased phosphorylation of 4E-BP1. In contrast, feeding a 0P diet had no effect on any of these parameters. Feeding a 20P diet resulted in partial dephosphorylation of eIF4E in both tissues. In liver, refeeding a 0P diet also resulted in partial eIF4E dephosphorylation, suggesting that the phosphorylation state of eIF4E is not important in the stimulation of protein synthesis under these conditions. Finally, plasma insulin concentrations were the same in rats fed either diet (14.8 ± 4.9 vs. 15.5 ± 4.5 μU/ml for 20P and 0P groups, respectively), suggesting that feeding-induced changes in plasma insulin are not sufficient to stimulate protein synthesis. Instead, a combination of dietary protein and insulin may be required to stimulate translation initiation.

Regulation of amino acid–sensitive TOR signaling by leucine analogues in adipocytes

In adipocytes, amino acids stimulate the target of rapamycin (TOR) signaling pathway leading to phosphorylation of the translational repressor, eIF‐4E binding protein‐I (4E‐BP1), and ribosomal protein S6. L‐leucine is the primary mediator of these effects. The structure‐activity relationships of a putative L‐leucine recognition site in adipocytes (LeuRA) that regulates TOR activity were analyzed by examining the effects of leucine analogues on the rapamycin‐sensitive phosphorylation of the translational repressor, eIF‐4E binding protein‐I (4E‐BP1), an index of TOR activity. Several amino acids that are structurally related to leucine strongly stimulated 4E‐BP1 phosphorylation at concentrations greater than the EC50 value for leucine. The order of potency was leucine > norleucine > threo‐L‐β‐hydroxyleucine ≃ Ile > Met ≃ Val. Other structural analogues of leucine, such as H‐α‐methyl‐D/L‐leucine, S‐(−)‐2‐amino‐4‐pentenoic acid, and 3‐amino‐4‐methylpentanoic acid, possessed only weak agonist activity. However, other leucine‐related compounds that are known agonists, antagonists, or ligands of other leucine binding/recognition sites did not affect 4E‐BP1 phosphorylation. We conclude from the data that small lipophilic modifications of the leucine R group and α‐hydrogen may be tolerated for agonist activity; however, leucine analogues with a modified amino group, a modified carboxylic group, charged R groups, or bulkier aliphatic R groups do not seem to possess significant agonist activity. Furthermore, the leucine recognition site that regulates TOR signaling in adipocytes appears to be different from the following: (1) a leucine receptor that regulates macroautophagy in liver, (2) a leucine recognition site that regulates TOR signaling in H4IIE hepatocytes, (3) leucyl tRNA or leucyl tRNA synthetase, (4) the gabapentin‐sensitive leucine transaminase, or (5) the system L‐amino acid transporter. J. Cell. Biochem. 77:234–251, 2000.

Amino acid-induced stimulation of translation initiation in rat skeletal muscle

Amino acids stimulate protein synthesis in skeletal muscle by accelerating translation initiation. In the two studies described herein, we examined mechanisms by which amino acids regulate translation initiation in perfused skeletal muscle hindlimb preparation of rats. In the first study, the effects of supraphysiological amino acid concentrations on eukaryotic initiation factors (eIF) 2B and 4E were compared with physiological concentrations of amino acids. Amino acid supplementation stimulated protein synthesis twofold. No changes were observed in eIF2B activity, in the amount of eIF4E associated with the eIF4E-binding protein (4E-BP1), or in the phosphorylation of 4E-BP1. The abundance of eIF4E bound to eIF4G and the extent of phosphorylation of eIF4E were increased by 800 and 20%, respectively. In the second study, we examined the effect of removing leucine on translation initiation when all other amino acids were maintained at supraphysiological concentrations. Removal of leucine from the perfusate decreased the rate of protein synthesis by 40%. The inhibition of protein synthesis was associated with a 40% decrease in eIF2B activity and an 80% fall in the abundance of eIF4E ⋅ eIF4G complex. The fall in eIF4G binding to eIF4E was associated with increased 4E-BP1 bound to eIF4E and a reduced phosphorylation of 4E-BP1. In contrast, the extent of phosphorylation of eIF4E was unaffected. We conclude that formation of the active eIF4E ⋅ eIF4G complex controls protein synthesis in skeletal muscle when the amino acid concentration is above the physiological range, whereas removal of leucine reduces protein synthesis through changes in both eIF2B and eIF4E.Amino acids stimulate protein synthesis in skeletal muscle by accelerating translation initiation. In the two studies described herein, we examined mechanisms by which amino acids regulate translation initiation in perfused skeletal muscle hindlimb preparation of rats. In the first study, the effects of supraphysiological amino acid concentrations on eukaryotic initiation factors (eIF) 2B and 4E were compared with physiological concentrations of amino acids. Amino acid supplementation stimulated protein synthesis twofold. No changes were observed in eIF2B activity, in the amount of eIF4E associated with the eIF4E-binding protein (4E-BP1), or in the phosphorylation of 4E-BP1. The abundance of eIF4E bound to eIF4G and the extent of phosphorylation of eIF4E were increased by 800 and 20%, respectively. In the second study, we examined the effect of removing leucine on translation initiation when all other amino acids were maintained at supraphysiological concentrations. Removal of leucine from the perfusate decreased the rate of protein synthesis by 40%. The inhibition of protein synthesis was associated with a 40% decrease in eIF2B activity and an 80% fall in the abundance of eIF4E. eIF4G complex. The fall in eIF4G binding to eIF4E was associated with increased 4E-BP1 bound to eIF4E and a reduced phosphorylation of 4E-BP1. In contrast, the extent of phosphorylation of eIF4E was unaffected. We conclude that formation of the active eIF4E. eIF4G complex controls protein synthesis in skeletal muscle when the amino acid concentration is above the physiological range, whereas removal of leucine reduces protein synthesis through changes in both eIF2B and eIF4E.

SEPSIS-INDUCED ALTERATIONS IN PYRUVATE DEHYDROGENASE COMPLEX ACTIVITY IN RAT SKELETAL MUSCLE: EFFECTS ON PLASMA LACTATE

ABSTRACT The pyruvate dehydrogenase (PDH) complex undergoes reversible phosphorylation catalyzed by a PDH kinase (inactivating) and a PDH phosphatase (activating). In skeletal muscle, a decreased proportion of PDH complex in the active, nonphosphorylated form (PDHa) limits glucose oxidation and promotes the conversion of pyruvate to lactate. Increased lactate formation with the accompanying hyperlactatemia is a frequent metabolic complication of sepsis. The time course for inactivation of the PDH complex in skeletal muscle during sepsis was contrasted with changes in PDHa during sterile inflammation 3, 7, or 14 days following the implantation of the foreign body nidus. Total PDH complex activity was not altered in any of the conditions examined. Sepsis, but not sterile inflammation, caused a reduction in the muscle PDHa measured 3 or 7 days following induction of sepsis. The inhibition of the muscle PDHa during sepsis was associated with a sustained hyperlactatemia. PDH kinase activity measured in extracts of mitochondria was enhanced twofold during this period. Fourteen days after induction of sepsis, there were no differences in the PDHa or plasma lactate concentrations in septic rats compared with either control or sterile inflammation. Furthermore, the PDH kinase activity was decreased to values observed in control values. The results are consistent with the hypothesis that a reduced PDHa in skeletal muscle during sepsis is responsible, in part, for the hyperlactatemia characteristic of septic hypermetabolism. Furthermore, the results provide evidence that the decrease in PDHa results from a stable stimulation of PDH kinase activity.

Leucine supplementation of drinking water does not alter susceptibility to diet-induced obesity in mice.

Branched-chain amino acids (BCAA), Leu, and the signaling pathways they regulate have been reported to either improve or worsen adiposity and insulin sensitivity. Therefore, it is unclear whether dietary supplementation of Leu would be beneficial. To help address this question, we examined the effect of adding Leu (150 mmol/L; Expt. 1 and Expt. 2) or BCAA (109 mmol/L of each; Expt. 3) to the drinking water on diet-induced obesity (induced with a 60-kJ% fat diet) in singly housed C57BL6/J male mice for at least 14 wk. Liquid and solid food intakes were evaluated weekly along with body weight. During the last few weeks, several blood samples were taken at different times for plasma glucose, total cholesterol, or Leu measurements. Metabolic rate by indirect calorimetry, locomotor activity by light beam breaking, body composition by H1-NMR, and insulin tolerance were also determined. Compared with control, supplementation did not affect body weight, food intake, oxygen consumption, locomotor activity, body composition, insulin tolerance, or total cholesterol. In fed mice, this method of Leu supplementation only increased plasma Leu by 76% when the supplemented group was compared with control. On the other hand, after overnight food deprivation, the plasma Leu did not differ between these 2 groups, even though the mice in the supplemented group had continuous access to Leu-containing water during the solid food deprivation. Taken together, the results do not provide evidence that either Leu or BCAA supplementation of drinking water ameliorates diet-induced obesity in mice, although it may improve glycemia.

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 eIF2α or the amount of eIF2α in the phosphorylated form between alcohol-fed and control rats. Similarly, the relative amount of eIF2Bε 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 γ-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.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.

Alcohol myopathy: impairment of protein synthesis and translation initiation.

Alcohol consumption leads to numerous morphological, biochemical and functional changes in skeletal and cardiac muscle. One such change observed in both tissues after either acute alcohol intoxication or chronic alcohol consumption is a characteristic decrease in the rate of protein synthesis. A decrease in translation efficiency appears to be responsible for at least part of the reduction. This review highlights advances in determining the molecular mechanisms by which alcohol impairs protein synthesis and places these observations in context of earlier studies on alcoholic myopathy. Both acute and chronic alcohol administration impairs translational control by modulating various aspects of peptide-chain initiation. Moreover, this alcohol-induced impairment in initiation is associated with a decreased availability of eukaryotic initiation factor (eIF) 4E in striated muscle, as evidenced by an increase in the amount of the inactive eIF4E.4E-BP1 complex and decrease in the active eIF4E.eIF4G complex. In contrast, alcohol does not produce consistent alterations in the control of translation initiation by the eIF2 system. The etiology of these changes remain unresolved. However, defects in the availability or effectiveness of various anabolic hormones, particularly insulin-like growth factor-I, are consistent with the alcohol-induced decrease in protein synthesis and translation initiation.

Regulation of protein synthesis after acute resistance exercise in diabetic rats

These studies determined whether insulin-like growth factor-I (IGF-I) involvement in exercise-stimulated anabolic processes becomes more evident during hypoinsulinemia. Male Sprague-Dawley rats ( n = 6-12/group) were made diabetic (blood glucose ≅ 300 mg/dl) by partial pancreatectomy (PPX) or remained nondiabetic (glucose ≅ 144 mg/dl). Rats performed acute resistance exercise by repetitive standing on the hindlimbs with weighted backpacks (ex), or they remained sedentary (sed). Resistance exercise caused increases in rates of protein synthesis (nmol Phe incorporated ⋅ g muscle-1 ⋅ h-1, measured for gastrocnemius muscle in vivo 16 h after exercise) for both nondiabetic [sed = 154 ± 6 (SE) vs. ex = 189 ± 7] and diabetic rats (PPXsed = 152 ± 11 vs. PPXex = 202 ± 14, P < 0.05). Arterial plasma insulin concentrations in diabetic rats, ≅180 pM, were less than one-half those found in nondiabetic rats, ≅444 pM, ( P < 0.05). The activity of eukaryotic initiation factor 2B (eIF2B; pmol GDP exchanged/min) was higher ( P < 0.05) in ex rats (sed = 0.028 ± 0.006 vs. ex = 0.053 ± 0.015; PPXsed = 0.033 ± 0.013 vs. PPXex = 0.047 ± 0.009) regardless of diabetic status. Plasma IGF-I concentrations were higher in ex compared with sed diabetic rats ( P < 0.05). In contrast, plasma IGF-I was not different in nondiabetic ex or sed rats. Muscle IGF-I (ng/g wet wt) was similar in ex and sed nondiabetic rats, but in diabetic rats was 2- to 3-fold higher in ex ( P < 0.05) than in sed rats. In conclusion, moderate hypoinsulinemia that is sufficient to alter glucose homeostasis does not inhibit an increase in rates of protein synthesis after acute moderate-intensity resistance exercise. This preserved response may be due to a compensatory increase in muscle IGF-I content and a maintained ability to activate eIF2B.These studies determined whether insulin-like growth factor-I (IGF-I) involvement in exercise-stimulated anabolic processes becomes more evident during hypoinsulinemia. Male Sprague-Dawley rats (n = 6-12/group) were made diabetic (blood glucose congruent with 300 mg/dl) by partial pancreatectomy (PPX) or remained nondiabetic (glucose congruent with 144 mg/dl). Rats performed acute resistance exercise by repetitive standing on the hindlimbs with weighted backpacks (ex), or they remained sedentary (sed). Resistance exercise caused increases in rates of protein synthesis (nmol Phe incorporated. g muscle-1. h-1, measured for gastrocnemius muscle in vivo 16 h after exercise) for both nondiabetic [sed = 154 +/- 6 (SE) vs. ex = 189 +/- 7] and diabetic rats (PPXsed = 152 +/- 11 vs. PPXex = 202 +/- 14, P < 0.05). Arterial plasma insulin concentrations in diabetic rats, congruent with180 pM, were less than one-half those found in nondiabetic rats, congruent with444 pM, (P < 0.05). The activity of eukaryotic initiation factor 2B (eIF2B; pmol GDP exchanged/min) was higher (P < 0.05) in ex rats (sed = 0.028 +/- 0.006 vs. ex = 0.053 +/- 0.015; PPXsed = 0.033 +/- 0.013 vs. PPXex = 0.047 +/- 0.009) regardless of diabetic status. Plasma IGF-I concentrations were higher in ex compared with sed diabetic rats (P < 0.05). In contrast, plasma IGF-I was not different in nondiabetic ex or sed rats. Muscle IGF-I (ng/g wet wt) was similar in ex and sed nondiabetic rats, but in diabetic rats was 2- to 3-fold higher in ex (P < 0.05) than in sed rats. In conclusion, moderate hypoinsulinemia that is sufficient to alter glucose homeostasis does not inhibit an increase in rates of protein synthesis after acute moderate-intensity resistance exercise. This preserved response may be due to a compensatory increase in muscle IGF-I content and a maintained ability to activate eIF2B.

Analysis of physiological amino acids using dabsyl derivatization and reversed-phase liquid chromatography

A method is described for the measurement of the specific radioactivity of primary amino acids in physiological samples. The amino acids were dabsylated followed by separation using high-performance liquid chromatography. We measured the concentration of amino acids from rat plasma or liver samples. Chromatographic analyses resolved phenylalanine from a mixture of amino acids in plasma within 30 min. An extended chromatographic gradient program completely separated all physiological amino acids within 75 min. This method is as sensitive as any current method of amino acid analysis and offers several advantages including (1) simple pre-column derivatization and (2) stability of derivatized samples.

Mechanism of IL-1 induced inhibition of protein synthesis in skeletal muscle.

Chronic interleukin (IL)-1 administration is associated with negative nitrogen balance and the loss of lean body mass. To elucidate the molecular mechanism(s) by which IL-1 modulates protein metabolism in muscle, we investigated the effects of chronic (6 day) IL-1alpha infusion on protein synthesis in Individual muscles (gastrocnemius, soleus, heart) compared with saline-infused control rats. IL-1 significantly decreased muscle weight, protein content, and the rate of protein synthesis in gastrocnemius (fast-twitch muscle). IL-1 had no effect on these parameters in the heart, whereas only the rate of protein synthesis was reduced in soleus (slow-twitch muscle). The reduction in gastrocnemius protein synthesis was not the result of a decrease in total RNA content, but was associated with a diminished translational efficiency. The diminished translational efficiency correlated with a 40% reduction in the epsilon-subunit of eukaryotic initiation factor 2B (elF2Bepsilon) in gastrocnemius from IL-1 -treated animals. However, the content of the alpha-subunit of elF2 (elF2alpha) was unaffected. In contrast, the elF2alpha content in heart was increased by IL-1, although elF2Bepsilon levels were unchanged. Reductions in skeletal muscle protein synthesis were not associated with a concomitant reduction in circulating or tissue content of insulin-like growth factor I. In summary, the IL-1-induced decrease in gastrocnemius protein synthesis appears to be regulated at the level of RNA translation via a reduction in elF2Bepsilon. These findings support a regulatory role for IL-1 as a mediator of muscle protein synthesis and the alterations in body composition observed in catabolic states where this cytokine is overexpressed.