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.     

Chronic paraplegia-induced muscle atrophy downregulates the mTOR/S6K1 signaling pathway.

Ribosomal S6 kinase 1 (S6K1) is a downstream component of the mammalian target of rapamycin (mTOR) signaling pathway and plays a regulatory role in translation initiation, protein synthesis, and muscle hypertrophy. AMP-activated protein kinase (AMPK) is a cellular energy sensor, a negative regulator of mTOR, and an inhibitor of protein synthesis. The purpose of this study was to determine whether the hypertrophy/cell growth-associated mTOR pathway was downregulated during muscle atrophy associated with chronic paraplegia. Soleus muscle was collected from male Sprague-Dawley rats 10 wk following complete T(4)-T(5) spinal cord transection (paraplegic) and from sham-operated (control) rats. We utilized immunoprecipitation and Western blotting techniques to measure upstream [AMPK, Akt/protein kinase B (PKB)] and downstream components of the mTOR signaling pathway [mTOR, S6K1, SKAR, 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor (eIF) 4G and 2alpha]. Paraplegia was associated with significant soleus muscle atrophy (174 +/- 8 vs. 240 +/- 13 mg; P < 0.05). There was a reduction in phosphorylation of mTOR, S6K1, and eIF4G (P < 0.05) with no change in Akt/PKB or 4E-BP1 (P > 0.05). Total protein abundance of mTOR, S6K1, eIF2alpha, and Akt/PKB was decreased, and increased for SKAR (P < 0.05), whereas 4E-BP1 and eIF4G did not change (P > 0.05). S6K1 activity was significantly reduced in the paraplegic group (P < 0.05); however, AMPKalpha2 activity was not altered (3.5 +/- 0.4 vs. 3.7 +/- 0.5 pmol x mg(-1) x min(-1), control vs. paraplegic rats). We conclude that paraplegia-induced muscle atrophy in rats is associated with a general downregulation of the mTOR signaling pathway. Therefore, in addition to upregulation of atrophy signaling during muscle wasting, downregulation of muscle cell growth/hypertrophy-associated signaling appears to be an important component of long-term muscle loss.     

Adrenalectomy enhances the insulin sensitivity of muscle protein synthesis

After confirming that adrenalectomy per se does not affect skeletal muscle protein synthesis rates, we examined whether endogenously produced glucocorticoids modulate the effect of physiological insulin concentrations on protein synthesis in overnight-fasted rats 4 days after either a bilateral adrenalectomy (ADX), ADX with dexamethasone treatment (ADX + DEX), or a sham operation (Sham; n = 6 each). Rats received a 3-h euglycemic insulin clamp (3 mU. min(-1). kg(-1)). Rectus muscle protein synthesis was measured at the end of the clamp, and the phosphorylation states of protein kinase B (Akt), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and ribosomal protein S6 kinase (p70(S6K)) were quantitated before and after the insulin clamp. The basal phosphorylation states of Akt, 4E-BP1, and p70(S6K) were similar between ADX and Sham rats. Insulin significantly enhanced the phosphorylation of Akt (P < 0.03), 4E-BP1 (P = 0.003), and p70(S6K) (P < 0.002) in ADX but not in Sham rats. Protein synthesis was significantly greater after insulin infusion in ADX than in Sham rats (P = 0.01). Glucocorticoid replacement blunted the effect of insulin on Akt, 4E-BP1, and p70(S6K) phosphorylation and protein synthesis. In conclusion, glucocorticoid deficiency enhances the insulin sensitivity of muscle protein synthesis, which is mediated by increased phosphorylation of translation initiation-regulatory proteins.     

Human soleus and vastus lateralis muscle protein metabolism with an amino acid infusion

The calf muscles, compared with the thigh, are less responsive to resistance exercise in ambulatory and bed-rested individuals, apparently due to muscle-specific differences in protein metabolism. We chose to evaluate the efficacy of using amino acids to elevate protein synthesis in the soleus, because amino acids have been shown to have a potent anabolic effect in the vastus lateralis. Mixed muscle protein synthesis in the soleus and vastus lateralis was measured before and after infusion of mixed amino acids in 10 individuals (28 +/- 1 yr). Phosphorylation of ribosomal protein p70 S6 kinase (p70S6K; Thr389) and eukaryotic initiation factor 4E-binding protein-1 (4E-BP1; Thr37/46) was also evaluated at rest and after 3 h of amino acid infusion. Basal protein synthesis was similar (P = 0.126), and amino acids stimulated protein synthesis to a similar extent (P = 0.004) in the vastus lateralis (0.043 +/- 0.011%/h) and soleus (0.032 +/- 0.017%/h). Phosphorylation of p70S6K (P = 0.443) and 4E-BP1 (P = 0.192) was not increased in either muscle; however, the soleus contained more total (P = 0.002) and phosphorylated (P = 0.013) 4E-BP1 than the vastus lateralis. These data support the need for further study of amino acid supplementation as a means to compensate for the reduced effectiveness of calf resistance exercise in ambulatory individuals and those exposed to extended periods of unloading. The greater 4E-BP1 in the soleus suggests that there is a muscle-specific distribution of general translational initiation machinery in human skeletal muscle.     

Eukaryotic initiation factors and protein synthesis after resistance exercise in rats.

Translational control of protein synthesis depends on numerous eukaryotic initiation factors (eIFs) and we have previously shown (Am. J. Physiol. Endocrinol. Metab. 276: E721-E727, 1999) that increases in one factor, eIF2B, are associated with increases in rates of protein synthesis after resistance exercise in rats. In the present study we investigated whether the eIF4E family of initiation factors is also involved with an anabolic response to exercise. Male Sprague-Dawley rats either remained sedentary (n = 6) or performed acute resistance exercise (n = 6), and rates of protein synthesis were assessed in vivo 16 h after the last session of resistance exercise. eIF4E complexed to eIF4G (eIF4E x eIF4G), eIF4E binding protein 1 (4E-BP1) complexed to eIF4E, and phosphorylation state of eIF4E and 4E-BP1 (gamma-form) were assessed in gastrocnemius. Rates of protein synthesis were higher in exercised rats compared with sedentary rats [205 +/- 8 (SE) vs. 164 +/- 5.5 nmol phenylalanine incorporated x g muscle(-1) x h(-1), respectively; P < 0.05]. Arterial plasma insulin concentrations were not different between the two groups. A trend (P = 0.09) for an increase in eIF4E x eIF4G with exercise was noted; however, no statistically significant differences were observed in any of the components of the eIF4E family in response to resistance exercise. These new data, along with our previous report on eIF2B, suggest that the regulation of peptide chain initiation after exercise is more dependent on eIF2B than on the eIF4E system.     

4E binding protein 1 expression is inversely correlated to the progression of gastrointestinal cancers

Several components of the eukaryotic protein synthesis apparatus have been associated with oncogenic transformation of cells. Overexpression of the initiation factor eIF4E occurs in a variety of human tumours. The aim of this study was to determine the level of expression and the phosphorylation state of eIF4E and 4E-binding protein 1 (4E-BP1) in gastrointestinal cancer, and to ascertain whether or not these factors can be used as diagnostic or prognostic markers within this type of cancer. The eIF4E levels were significantly higher in tumours compared with normal tissue (51. 5+/-4.4 vs 30.9+/-2.5 arbitrary units (A.U.)/mg of protein, p<0.001). However, phosphorylated eIF4E did not change in stomach cancers and decreased in colorectal cancers (67.1+/-1.2 vs 60.8+/-2.8%, p<0.05). 4E-BP1 expression increased in most of the gastrointestinal cancers studied. In addition, an inverse correlation between 4E-BP1 elevation and N and M stages was found, showing significant higher elevation of 4E-BP1 in Node-negative patients (11.21+/-5.74 vs 4. 03+/-2.36 n-fold, p<0.05) as well as in patients without distant metastasis (8.41+/-3.29 vs 0.97+/-0.35 n-fold, p<0.05). These results suggest that 4E-BP1 could function as a tumour suppressor. Moreover, the data show a significant dephosphorylation of 4E-BP1 in gastrointestinal tumours that correlated with an increase in the association of 4E-BP1 and eIF4E indicating a lower availability to eIF4E to recruit to the ribosomes. Our results support a possible role of 4E-BP1 as a prognostic factor in gastrointestinal carcinoma.     

Protein metabolism in glucocorticoid excess: study in Cushing's syndrome and the effect of treatment

How protein metabolism is perturbed during chronic glucocorticoid excess is poorly understood. The aims were to investigate the impact of chronic glucocorticoid excess and restoration of eucortisolemia in Cushing's syndrome (CS) on whole body protein metabolism. Eighteen subjects with CS and 18 normal subjects (NS) underwent assessment of body composition using DEXA and whole body protein turnover with a 3-h constant infusion of l-[(13)C]leucine, allowing calculation of rates of leucine appearance (leucine R(a)), leucine oxidation (L(ox)), and leucine incorporation into protein (LIP). Ten subjects with CS were restudied after restoration of eucortisolemia. Percentage FM was greater (43.9 +/- 1.6 vs. 33.8 +/- 2.4%, P = 0.002) and LBM lower (52.7 +/- 1.6 vs. 62.1 +/- 2.3%, P = 0.002) in CS. LBM was significantly correlated (r(2) > 0.44, P < 0.005) to leuceine R(a), L(ox), and LIP in both groups. After correcting for LBM, leucine R(a) (133 +/- 5 vs. 116 +/- 5 micromol/min, P = 0.02) and L(ox) (29 +/- 1 vs. 24 +/- 1 micromol/min, P = 0.01) were greater in CS. FM significantly correlated (r(2) = 0.23, P < 0.05) with leucine R(a) and LIP, but not L(ox) in CS. In multiple regression, LBM was an independent determinant of all three indexes of leucine turnover, FM of leucine R(a), and LIP and CS of L(ox). Following restoration of eucortisolemia, L(ox) was reduced (Delta-7.5 +/- 2.6 micromol/min, P = 0.02) and LIP increased (Delta+15.2 +/- 6.2 micromol/min, P = 0.04). In summary, whole body protein metabolism in CS is influenced by changes in body composition and glucocorticoid excess per se, which increases protein oxidation. Enhanced protein oxidation is a likely explanation for the reduced protein mass in CS. Successful treatment of CS reduces protein oxidation and increases protein synthesis to prevent ongoing protein loss.     

Regulation of contraction-induced FA uptake and oxidation by AMPK and ERK1/2 is intensity dependent in rodent muscle

Muscle contraction activates AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK1/2), two signaling molecules involved in the regulation of muscle metabolism. The purpose of this study was to determine whether activation of AMPK and/or ERK1/2 contributes to the regulation of muscle fatty acid (FA) uptake and oxidation in contracting muscle. Rat hindquarters were perfused during rest (R) or electrical stimulation (E) of increasing intensity by manipulating train duration (E1 = 25 ms, E2 = 50 ms, E3 = 100 ms, E4 = 200 ms). For matched FA delivery, FA uptake was significantly greater than R during E1, E2, and E3 (7.8 +/- 0.7 vs. 14.4 +/- 0.3, 16.9 +/- 0.8, 15.2 +/- 0.5 nmol.min(-1).g(-1), respectively, P < 0.05), but not during E4 (8.3 +/- 0.3 nmol.min(-1).g(-1), P > 0.05). FA oxidation was significantly greater than R during E1 and E2 (1.5 +/- 0.1 vs. 2.3 +/- 0.2, 2.5 +/- 0.2 nmol.min(-1).g(-1), P < 0.05) before returning to resting levels for E3 and E4 (1.8 +/- 0.1 and 1.5 +/- 0.2 nmol.min(-1).g(-1), P > 0.05). A positive correlation was found between FA uptake and ERK1/2 phosphorylation from R to E3 (R(2) = 0.55, P < 0.05) and between FA oxidation and ERK1/2 phosphorylation from R to E2 (R(2) = 0.76, P < 0.05), correlations that were not maintained when the data for E4 and E3 and E4, respectively, were included in the analysis (R(2) = 0.04 and R(2) = 0.03, P > 0.05). A positive correlation was also found between FA uptake and FA oxidation and AMPK activity for all exercise intensities (R(2) = 0.57, R(2) = 0.65 respectively, P < 0.05). These results, in combination with previous data from our laboratory, suggest that ERK1/2 and AMPK are the predominant signaling molecules regulating FA uptake and oxidation during low- to moderate-intensity muscle contraction and during moderate- to high-intensity muscle contraction, respectively.     

Inhibition of p38 MAPK and AMPK restores adenosine-induced cardioprotection in hearts stressed by antecedent ischemia by altering glucose utilization

p38 mitogen-activated protein kinase (MAPK) and 5'-AMP-activated protein kinase (AMPK) are activated by metabolic stresses and are implicated in the regulation of glucose utilization and ischemia-reperfusion (IR) injury. This study tested the hypothesis that inhibition of p38 MAPK restores the cardioprotective effects of adenosine in stressed hearts by preventing activation of AMPK and the uncoupling of glycolysis from glucose oxidation. Working rat hearts were perfused with Krebs solution (1.2 mM palmitate, 11 mM [(3)H/(14)C]glucose, and 100 mU/l insulin). Hearts were stressed by transient antecedent IR (2 x 10 min I/5 min R) before severe IR (30 min I/30 min R). Hearts were treated with vehicle, p38 MAPK inhibitor (SB-202190, 10 microM), adenosine (500 microM), or their combination before severe IR. After severe IR, the phosphorylation (arbitrary density units) of p38 MAPK and AMPK, rates of glucose metabolism (micromol x g dry wt(-1) x min(-1)), and recovery of left ventricular (LV) work (Joules) were similar in vehicle-, SB-202190- and adenosine-treated hearts. Treatment with SB-202190 + adenosine versus adenosine alone decreased p38 MAPK (0.03 +/- 0.01, n = 3 vs. 0.48 +/- 0.10, n = 3, P < 0.05) and AMPK (0.00 +/- 0.00, n = 3 vs. 0.26 +/- 0.08, n = 3 P < 0.05) phosphorylation. This was accompanied by attenuated rates of glycolysis (1.51 +/- 0.40, n = 7 vs. 3.95 +/- 0.65, n = 7, P < 0.05) and H(+) production (2.12 +/- 0.76, n = 7 vs. 6.96 +/- 1.48, n = 7, P < 0.05), and increased glycogen synthesis (1.91 +/- 0.25, n = 6 vs. 0.27 +/- 0.28, n = 6, P < 0.05) and improved recovery of LV work (0.81 +/- 0.08, n = 7 vs. 0.30 +/- 0.15, n = 8, P < 0.05). These data indicate that inhibition of p38 MAPK abolishes subsequent phosphorylation of AMPK and improves the coupling of glucose metabolism, thereby restoring adenosine-induced cardioprotection.     

Glucocorticoids modulate amino acid-induced translation initiation in human skeletal muscle

Amino acids are unique anabolic agents in that they nutritively signal to mRNA translation initiation and serve as substrates for protein synthesis in skeletal muscle. Glucocorticoid excess antagonizes the anabolic action of amino acids on protein synthesis in laboratory animals. To examine whether excessive glucocorticoids modulate mixed amino acid-signaled translation initiation in human skeletal muscle, we infused an amino acid mixture (10% Travasol) systemically to 16 young healthy male volunteers for 6 h in the absence (n = 8) or presence (n = 8) of glucocorticoid excess (dexamethasone 2 mg orally every 6 h for 3 days). Vastus lateralis muscles were biopsied before and after amino acid infusion, and the phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1), ribosomal protein S6 kinase (p70(S6K)), and eIF2alpha and the guanine nucleotide exchange activity of eIF2B were measured. Systemic infusion of mixed amino acids significantly stimulated the phosphorylation of 4E-BP1 (P < 0.04) and p70(S6K) (P < 0.001) and the dephosphorylation of eIF2alpha (P < 0.003) in the control group. Dexamethasone treatment did not alter the basal phosphorylation state of 4E-BP1, p70(S6K), or eIF2alpha; however, it abrogated the stimulatory effect of amino acid infusion on the phosphorylation of 4E-BP1 (P = 0.31) without affecting amino acid-induced phosphorylation of p70(S6K) (P = 0.002) or dephosphorylation of eIF2alpha (P = 0.003). Neither amino acid nor dexamethasone treatment altered the guanine nucleotide exchange activity of eIF2B. We conclude that changes of amino acid concentrations within the physiological range stimulate mRNA translation by enhancing the binding of mRNA to the 43S preinitiation complex, and the activity of p70(S6K) and glucocorticoid excess blocks the former action in vivo in human skeletal muscle.     

Reduced stroke volume during exercise in postural tachycardia syndrome.

Postural tachycardia syndrome (POTS) is characterized by excessive tachycardia without hypotension during orthostasis. Most POTS patients also report exercise intolerance. To assess cardiovascular regulation during exercise in POTS, patients (n = 13) and healthy controls (n = 10) performed graded cycle exercise at 25, 50, and 75 W in both supine and upright positions while arterial pressure (arterial catheter), heart rate (HR; measured by ECG), and cardiac output (open-circuit acetylene breathing) were measured. In both positions, mean arterial pressure, cardiac output, and total peripheral resistance at rest and during exercise were similar in patients and controls (P > 0.05). However, supine stroke volume (SV) tended to be lower in the patients than controls at rest (99 +/- 5 vs. 110 +/- 9 ml) and during 75-W exercise (97 +/- 5 vs. 111 +/- 7 ml) (P = 0.07), and HR was higher in the patients than controls at rest (76 +/- 3 vs. 62 +/- 4 beats/min) and during 75-W exercise (127 +/- 3 vs. 114 +/- 5 beats/min) (both P < 0.01). Upright SV was significantly lower in the patients than controls at rest (57 +/- 3 vs. 81 +/- 6 ml) and during 75-W exercise (70 +/- 4 vs. 94 +/- 6 ml) (both P < 0.01), and HR was much higher in the patients than controls at rest (103 +/- 3 vs. 81 +/- 4 beats/min) and during 75-W exercise (164 +/- 3 vs. 131 +/- 7 beats/min) (both P < 0.001). The change (upright - supine) in SV was inversely correlated with the change in HR for all participants at rest (R(2) = 0.32), at 25 W (R(2) = 0.49), 50 W (R(2) = 0.60), and 75 W (R(2) = 0.32) (P < 0.01). These results suggest that greater elevation in HR in POTS patients during exercise, especially while upright, was secondary to reduced SV and associated with exercise intolerance.     

Age-associated decrease in contraction-induced activation of downstream targets of Akt/mTor signaling in skeletal muscle

In this study, we investigated the effect of age on the association of eukaryotic initiation factor 4E (eIF4E) with eukaryotic initiation factor 4G (eIF4G), as well as the activity of its binding protein (4E-BP1) and the activity of glycogen synthase kinase-3 (GSK-3) after a single bout of rat hindlimb muscle contractile activity elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Tibialis anterior (TA) and plantaris (Pla) muscles from adult (Y; 6 mo old) and aged (O; 30 mo old) Fischer 344 x Brown Norway rats were collected immediately or 6 h after HFES. eIF4E-eIF4G association was elevated at 6 h of recovery in TA (1.9 +/- 0.2-fold, P < 0.05) and immediately and 6 h after exercise in Pla (2.1 +/- 0.3- and 2.1 +/- 0.7-fold, P < 0.05) in Y rats. No significant increase was observed in O rats. An increase in 4E-BP1 phosphorylation was observed only 6 h after HFES in TA (5.0 +/- 2.0-fold, P < 0.05) in Y rats. Phosphorylation of GSK-3alpha was increased immediately and 6 h after contraction in TA (1.6 +/- 0.3- and 4.1 +/- 0.8-fold, P < 0.05) and Pla (1.7 +/- 0.2- and 2.1 +/- 0.4-fold, P < 0.05) in Y rats and remained unaffected in O rats. Phosphorylation of GSK-3beta was observed only immediately after HFES in TA (1.5 +/- 0.2-fold, P < 0.05) in Y rats. Overall, eIF4E-eIF4G association and phosphorylation of 4E-BP1 and GSK-3 are increased after HFES in adult, but not in aged, animals. These observations suggest that the anabolic response to muscle stimulation is attenuated with aging and may contribute to the limited capacity of hypertrophy in aged animals.     

Postprandial body protein synthesis and amino acid catabolism measured with leucine and phenylalanine-tyrosine tracers

Whether phenylalanine-tyrosine (Phe-Tyr) tracers yield estimates of postprandial protein synthesis comparable to those of the widely used leucine (Leu) tracer is unclear. We measured Leu oxidation (Ox), Phe hydroxylation (Hy), and their disposal into whole body protein synthesis before and after the administration of a mixed meal (62 kJ/kg body wt, 22% of energy as protein), over 4 h in healthy subjects. Both plasma and intracellular precursor pools were used. The amino acid data were extrapolated to body protein by assuming a fixed ratio of Leu to Phe in the proteins. In the postabsorptive state, whole body protein synthesis (expressed as mg. kg(-1). min(-1)) was similar between Leu and Phe-Tyr tracers irrespective of the precursor pool used. After the meal, Leu Ox, Phe Hy, and body protein synthesis increased (P < or = 0.01 vs. basal). With the use of intracellular precursor pools, the increase of protein synthesis with Phe-Tyr (+0.51 +/-0.21 mg. kg(-1). min(-1)) and Leu tracers (+0.57 +/- 0.14) were similar (P = not significant). In contrast, with plasma pools the increase of protein synthesis was more than twofold greater with Phe-Tyr (+1.17 +/- 0.19 mg. kg(-1). min(-1)) than that with Leu (0.50 +/- 0.13 mg. kg(-1). min(-1), P < 0.01). Direct correlations were found between Leu and Ox [using both plasma and intracellular pools (r < or = 0.65, P < or = 0.01)] but not between Phe and either plasma or intracellular Hy. In conclusion, 1) Phe-Tyr and Leu tracers yield comparable estimates of body protein synthesis postprandially, provided that intracellular precursor pools are used; 2) both Leu Ox and Phe Hy are stimulated by a mixed meal; 3) Phe does not correlate with Hy, which might be better related to the (unknown) portal Phe.     

Time course evaluation of protein synthesis and glucose uptake after acute resistance exercise in rats.

The temporal pattern for changes in rates of protein synthesis and glucose uptake after resistance exercise, especially relative to each other, is not known. Male Sprague-Dawley rats performed acute resistance exercise (n = 7) or remained sedentary (n = 7 per group), and the following were assessed in vivo 1, 3, 6, 12 and 24 h later: rates of protein synthesis, rates of glucose uptake, phosphatidylinositol 3-kinase (PI3-kinase) activity, and p70(S6k) activity. Rates of protein synthesis in mixed gastrocnemius muscle did not increase until 12 h after exercise (e.g., at 12 h, sedentary = 138 +/- 4 vs. exercised = 178 +/- 6 nmol phenylalanine incorporated x g muscle(-1) x h(-1), mean +/- SE, P < 0.05), whereas at 6 h after exercise rates of glucose uptake were significantly elevated (sedentary = 0.18 +/- 0.020 vs. exercised = 0.38 +/- 0.024 micromol glucose 6-phosphate incorporated x kg muscle(-1) x min(-1), P < 0.05). At 24 h after exercise, rates of protein synthesis were still elevated, whereas glucose uptake had returned to basal levels. Arterial insulin concentrations were not different between groups at any time. Non-insulin-stimulated activities of PI3-kinase and p70(S6k) were higher at 6, 12, and 24 h after exercise (P < 0.05), and, generally, these occurred when rates of protein synthesis (12 and 24 h) and glucose uptake were elevated (6 and 12 but not 24 h) by exercise. These data suggest that regulators of protein synthesis and glucose uptake may respond to the same contraction-generated signals with different kinetics or that they respond to different intra- or extracellular signals that are generated by exercise.     

Reducing plasma HIV RNA improves muscle amino acid metabolism

We reported (Yarasheski KE, Zachwieja JJ, Gischler J, Crowley J, Horgan MM, and Powderly WG. Am J Physiol Endocrinol Metab 275: E577-E583, 1998) that AIDS muscle wasting was associated with an inappropriately low rate of muscle protein synthesis and an elevated glutamine rate of appearance (Ra Gln). We hypothesized that high plasma HIV RNA caused dysregulation of muscle amino acid metabolism. We determined whether a reduction in HIV RNA (> or =1 log) increased muscle protein synthesis rate and reduced R(a) Gln and muscle proteasome activity in 10 men and 1 woman (22-57 yr, 60-108 kg, 17-33 kg muscle) with advanced HIV (CD4 = 0-311 cells/microl; HIV RNA = 10-375 x 10(3) copies/ml). We utilized stable isotope tracer methodologies ([13C]Leu and [15N]Gln) to measure the fractional rate of mixed muscle protein synthesis and plasma Ra Gln in these subjects before and 4 mo after initiating their first or a salvage antiretroviral therapy regimen. After treatment, median CD4 increased (98 vs. 139 cells/microl, P = 0.009) and median HIV RNA was reduced (155,828 vs. 100 copies/ml, P = 0.003). Mixed muscle protein synthesis rate increased (0.062 +/- 0.005 vs. 0.078 +/- 0.006%/h, P = 0.01), Ra Gln decreased (387 +/- 33 vs. 323 +/- 15 micromol.kg fat-free mass(-1).h(-1), P = 0.04), and muscle proteasome chymotrypsin-like catalytic activity was reduced 14% (P = 0.03). Muscle mass was only modestly increased (1 kg, P = not significant). We estimated that, for each 10,000 copies/ml reduction in HIV RNA, approximately 3 g of additional muscle protein are synthesized per day. These findings suggest that reducing HIV RNA increases muscle protein synthesis and reduces muscle proteolysis, but muscle protein synthesis relative to whole body protein synthesis rate is not restored to normal, so muscle mass is not substantially increased.     

alpha(1A) adrenergic receptor induces eukaryotic initiation factor 4E-binding protein 1 phosphorylation via a Ca(2+)-dependent pathway independent of phosphatidylinositol 3-kinase/Akt

Phosphorylation of the translation repressor eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is thought to be partly responsible for increased protein synthesis induced by growth factors. This study investigated the effect of a G(q)-coupled receptor on protein synthesis and the phosphorylation state and function of 4E-BP1 in Rat-1 fibroblasts expressing the human alpha(1A) adrenergic receptor. Treatment of cells with phenylephrine (PE), a specific alpha(1) adrenergic receptor agonist, increased protein synthesis and induced the phosphorylation of 4E-BP1 and its release from translation initiation factor 4E. Although the PE-induced phosphorylation of 4E-BP1 was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002, neither phosphatidylinositol 3-kinase nor Akt, its downstream effector, is activated in cells treated with PE (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z., J. Biol. Chem. 275, 4803-4809). The effect of PE on 4E-BP1 phosphorylation was also abolished in cells depleted of intracellular Ca(2+) and in cells pretreated with calmodulin antagonists. By contrast, phosphorylation of 4E-BP1 still occurred in cells in which the Ca(2+)- and diacylglycerol-dependent isoforms of protein kinase C were down-regulated by prolonged exposure to a phorbol ester. We conclude that activation of the alpha(1A) adrenergic receptor in Rat-1 fibroblasts leads to phosphorylation of 4E-BP1 via a pathway that is Ca(2+)- and calmodulin-dependent. Phosphatidylinositol 3-kinase, Akt, and phorbol ester-sensitive protein kinase C isoforms do not appear to be required in this signaling pathway.     

Role of eIF4E in stimulation of protein synthesis by IGF-I in perfused rat skeletal muscle

Insulin-like growth factor I (IGF-I) promotes anabolism by stimulating protein synthesis in skeletal muscle. In the present study, we have examined mechanisms by which IGF-I stimulates protein synthesis in skeletal muscle with a perfused rat hindlimb preparation. IGF-I (10 nM) stimulated protein synthesis over 2.7-fold. Total RNA content was unaffected, but translational efficiency was increased by IGF-I. We next examined the effect of IGF-I on eukaryotic initiation factor (eIF) 4E as a mechanism regulating translation initiation. IGF-I did not alter either the amount of eIF4E associated with the eIF4E binding protein 4E-BP1 or the phosphorylation state of 4E-BP1. Likewise, the phosphorylation state of eIF4E was unaltered by IGF-I. In contrast, the amount of eIF4E bound to eIF4G was increased threefold by IGF-I. We conclude that IGF-I regulates protein synthesis in skeletal muscle by enhancing formation of the active eIF4E x eIF4G complex.     

Translational regulation of ANG II type 1 receptors in proliferating vascular smooth muscle cells

The current study examined angiotensin receptor (ATR) regulation in proliferating rat aortic vascular smooth muscle cells (VSMCs) in culture. Radioligand competition analysis coupled with RNase protection assays (RPAs) revealed that angiotensin type 1a receptor (AT(1a)R) densities (B(max)) increased by 30% between 5 and 7 days in culture [B(max) (fmol/mg protein): day 5, 379 +/- 8.4 vs. day 7, 481 +/- 12, n = 3, P < 0.05] under conditions in which no significant changes in AT(1a)R mRNA expression occurred [in RPA arbitrary units (AU): day 5, 0.23 +/- 0.01 vs. day 7, 0.24 +/- 0.04, n = 4] or in mRNA synthesis determined by nuclear run-on assays [AU: day 5, 0.35 +/- 0.14 vs. day 7, 0.33 +/- 0.11, n = 5]. In contrast, polysome distribution analysis indicated that AT(1a)R mRNA was more efficiently translated in day 7 cells compared with day 5 [% of AT(1a)R mRNA in fraction 2 out of total AT1R mRNA recovered from the sucrose gradient: day 5, 20.9 +/- 9.9 vs. day 7, 56.8 +/- 5.6, n = 3, P < 0.001]. Accompanying the polysome shift was 50% less RNA-protein complex (RPC) formation between VSMC cytosolic RNA binding proteins in day 7 cells compared with 5-day cultures and the 5' leader sequence (5'LS) of the AT(1a)R [5'LS RPC (AU): day 5, 0.62 +/- 0.15 vs. day 7, 0.23 +/- 0.03; n = 4, P < 0.05] and also with exon 2 [Exon 2 RPC (AU): day 5, 35.0 +/- 5.7 vs. day 7, 17.2 +/- 3.6; n = 4, P < 0.05]. Taken together, these results suggest that AT(1a)R expression is regulated by translation during VSMC proliferation in part by RNA binding proteins that interact within exon 2 in the 5'LS of the AT(1a)R mRNA.     

Whole body leucine flux in HIV-infected patients treated with or without protease inhibitors

The present study was carried out to assess the effects of protease inhibitor (PI) therapy on basal whole body protein metabolism and its response to acute amino acid-glucose infusion in 14 human immunodeficiency virus (HIV)-infected patients. Patients treated with PIs (PI+, 7 patients) or without PIs (PI-, 7 patients) were studied after an overnight fast during a 180-min basal period followed by a 140-min period of amino acid-glucose infusion. Protein metabolism was investigated by a primed constant infusion of l-[1-(13)C]leucine. Dual-energy X-ray absorptiometry for determination of fat-free mass (FFM) and body fat mass measured body composition. In the postabsorptive state, whole body leucine balance was 2.5 times (P < 0.05) less negative in the PI+ than in the PI- group. In HIV-infected patients treated with PIs, the oxidative leucine disposal during an acute amino acid-glucose infusion was lower (0.58 +/- 0.09 vs. 0.81 +/- 0.07 micromol x kg FFM(-1) x min(-1) using plasma [(13)C]leucine enrichment, P = 0.06; or 0.70 +/- 0.10 vs. 0.99 +/- 0.08 micromol x kg FFM(-1) x min(-1) using plasma [(13)C]ketoisocaproic acid enrichment, P = 0.04 in PI+ and PI- groups, respectively) than in patients treated without PIs. Consequently, whole body nonoxidative leucine disposal (an index of protein synthesis) and leucine balance (0.50 +/- 0.10 vs. 0.18 +/- 0.06 micromol x kg FFM x (-1) x min(-1) in PI+ and PI- groups respectively, P < 0.05) were significantly improved during amino acid-glucose infusion in patients treated with PIs. However, whereas the response of whole body protein anabolism to an amino acid-glucose infusion was increased in HIV-infected patients treated with PIs, any improvement in lean body mass was detected.     

Insulin action on protein metabolism in acromegalic patients

Insulin resistance in acromegaly causes glucose intolerance and diabetes, but it is unknown whether it involves protein metabolism, since both insulin and growth hormone promote protein accretion. The effects of acromegaly and of its surgical cure on the insulin sensitivity of glucose and amino acid/protein metabolism were evaluated by infusing [6,6-(2)H(2)]glucose, [1-(13)C]leucine, and [2-(15)N]glutamine during a euglycemic insulin (1 mU x kg(-1) x min(-1)) clamp in 12 acromegalic patients, six studied again 6 mo after successful adenomectomy, and eight healthy controls. Acromegalic patients, compared with postsurgical and control subjects, had higher postabsorptive glucose concentration (5.5 +/- 0.3 vs. 4.9 +/- 0.2 micromol/l, P < 0.05, and 5.1 +/- 0.1 micromol/l) and flux (2.7 +/- 0.1 vs. 2.0 +/- 0.2 micromol x kg(-1) x min(-1), P < 0.01, and 2.2 +/- 0.1 micromol x kg(-1) x min(-1), P < 0.05) and reduced insulin-stimulated glucose disposal (+15 +/- 9 vs. +151 +/- 18%, P < 0.01, and 219 +/- 58%, P < 0.001 from basal). Postabsorptive leucine metabolism was similar among groups. In acromegalic and postsurgical subjects, insulin suppressed less than in controls the endogenous leucine flux (-9 +/- 1 and -12 +/- 2 vs. -18 +/- 2%, P < 0.001 and P < 0.05), the nonoxidative leucine disposal (-4 +/- 3 and -1 +/- 3 vs. -18 +/- 2%, P < 0.01 and P < 0.05), respectively, indexes of proteolysis and protein synthesis, and leucine oxidation (-17 +/- 6% in postsurgical patients vs. -26 +/- 6% in controls, P < 0.05). Within 6 mo, surgery reverses insulin resistance for glucose but not for protein metabolism. After adenomectomy, more leucine is oxidized during hyperinsulinemia.