Highly active antiretroviral therapy dysregulates proliferation and differentiation of human pre-adipocytes.

AIM: To investigate the mechanism(s) by which potential effects of multi-drug highly-active antiretroviral therapy contributes to lipodystrophy syndrome. METHODS: Preadipocytes from healthy donors were assessed for proliferation and differentiation in the presence of nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) individually and in combination. Effects on proliferation were assessed with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and effects on differentiation were assessed from glycerol-3-phosphate dehydrogenase (GP DH) activity and quantitation of Oil Red O staining for intracellular lipid. Data were analyzed with a randomized block ANOVA with post-hoc Fisher's Least Significant Difference test. RESULTS: Preadipocyte proliferation was inhibited by a combination of NNRTI + NRTI (14% at 48 h, P < 0.001) and PI + NRTI (19% at 48 h, P < 0.001) with additional suppression when ritonavir (RTV) was added (26% at 48 h). The drug combination of atazanavir (ATV) + RTV + emtricitabine (FTC) + tenofovir (TDF) had the greatest inhibitory effect on proliferation at 48 h. Preadipocyte differentiation was most significantly reduced by the efavirenz + FTC + TDF assessed either by GPDH activity (64%) or lipid accumulation (39%), P < 0.001. Combining NRTIs with a PI (ATV + FTC + TDF) significantly suppressed differentiation (GPDH activity reduced 29%, lipid accumulation reduced by 19%, P < 0.01). This effect was slightly greater when a boosting amount of RTV was added (ATV + FTC + TDF + RTV, P < 0.001). CONCLUSION: Although combination antiretroviral therapy is clinically more efficacious than single drug regimens, it also has a much greater inhibitory effect on preadipocyte proliferation and differentiation.

Peripheral fat loss and decline in adipogenesis in older humans.

OBJECTIVE: Aging is associated with a redistribution of body fat including a relative loss of subcutaneous peripheral fat. These changes in body fat can have important clinical consequences since they are linked to increased risk of metabolic complications. The causes and mechanisms of loss of peripheral fat associated with aging are not clear. The aim of this study was to assess whether defects in adipogenesis contribute to fat loss in aging humans, as suggested from animal studies, and to evaluate the role of inflammation on pathogenesis of fat loss. MATERIALS/METHODS: Preadipocytes isolated from subcutaneous peripheral fat of healthy young and elderly subjects were compared in their ability to replicate and differentiate. RESULTS: The results show that both the rate of replication and differentiation of preadipocytes are reduced in older subjects. The reduction in adipogenesis is accompanied by a higher plasma level of the inflammatory marker, soluble tumor necrosis factor receptor 2, and greater release of tumor necrosis factor α from fat tissue. CONCLUSIONS: Thus, the gradual relative loss of peripheral fat in aging humans may in part result from a defect in adipogenesis, which may be linked to inflammation and increased release of proinflammatory cytokines from fat tissue.

New perspectives in the control of body protein metabolism.

Recent advances in elucidating the mechanisms that control body protein synthesis and degradation both expand and complicate our understanding of how these processes are regulated. This review presents an introduction to the multiple regulatory systems involved, emphasizing the number of potential controls. These include gene transcription, gene activation or suppression, activation or suppression of mRNA translation and activation or suppression of signaling pathways. The complexity of these interacting controls presents a challenge to our understanding of the overall coordinated regulation of protein synthesis and degradation and its response to any particular stimulus. Specific examples are used to illustrate regulatory mechanisms, including the ways in which protein metabolism is regulated by the amino acid leucine. In addition to regulation associated with gene expression and post-translational control, the expanding field of epigenetics adds another layer of complexity, including trans-generational responses to nutrient intake, highlighting the potential for long-term impact of nutritional experience on the metabolism of subsequent generations.

Rosiglitazone-Mediated Effects on Skeletal Muscle Gene Expression Correlate with Improvements in Insulin Sensitivity in Individuals with HIV-Insulin Resistance.

Rosiglitazone, an agonist of peroxisome proliferator activated receptor (PPARγ), improves insulin sensitivity by increasing insulin-stimulated glucose uptake into muscle tissue. This study was undertaken to assess changes in expression of PPAR-regulated genes in muscle tissue following treatment of HIV-associated insulin resistance with rosiglitazone. Muscle gene expression was assessed in twenty-two seronegative HIV subjects (control), 21 HIV-infected individuals with normal insulin sensitivity (HIV-IS) and 19 HIV-infected individuals with insulin resistance (HIV-IR). A subset of the HIV-IR group (N = 10) were re-evaluated 12 weeks after treatment with 8 mg/d of rosiglitazone. The HIV-IR group's rosiglitazone-mediated improvement in insulin sensitivity was highly correlated with increased expression of PPARγ and carnitine palmitoyl transferase-1 (CPT-1), (r = 0.87, P < .001) and (r = 0.95, P < .001), respectively. The changes in PPARγ expression were also correlated with the changes in CPT1 expression (r = 0.75, P = .009). The results suggest that rosiglitazone; may have a direct effect on muscle tissue to improve insulin sensitivity.

Effect of ritonavir and atazanavir on human subcutaneous preadipocyte proliferation and differentiation.

Protease inhibitors (PIs) have been implicated in the development of HIV-associated lipodystrophy through a reduction in the differentiation of preadipocytes. While atazanavir (ATV) is associated with fewer clinical metabolic abnormalities in the short-term, the effects of long-term exposure are not known. ATV effects on preadipocyte replication or differentiation would indicate the potential for long-term problems. This study compared ritonavir (RTV) and ATV effects on preadipocyte replication and differentiation in human primary cultures. Preadipocytes from subcutaneous fat were studied in the presence of therapeutic concentrations of RTV and ATV for replication, differentiation, and adipokine secretion. The effects of the drugs on the expression of PPARgamma and related genes during differentiation were also assessed by real-time quantitative PCR. RTV induced a significant inhibition of preadipocyte proliferation, differentiation and adiponectin secretion. ATV at concentrations within the range of therapeutic levels did not affect differentiation or adiponectin secretion, but did have inhibitory effects on preadipocyte proliferation. Inhibition of differentiation by PIs was associated with decreased expression of PPARgamma, C/EBPalpha, and aP2 genes. In summary, although ATV at therapeutic levels has a smaller impact on adipogenesis, alterations in preadipocyte proliferation suggest the potential for adverse effects with long-term use.

Effect of insulin on whole body protein metabolism in children with type 1 diabetes.

PURPOSE OF REVIEW: Untreated type 1 diabetes (T1D) is associated with abnormalities in protein metabolism, leading to protein loss. These alterations can be particularly detrimental in children, affecting both normal growth and development. A better understanding of the effects of insulin on protein metabolism in children with T1D is essential for optimizing therapy and minimizing consequences of the disease.The aim of the present review is to outline the effects of insulin on whole body protein metabolism in T1D, focusing particularly on studies in children with T1D. RECENT FINDINGS: Whole body protein degradation and amino acid oxidation are enhanced in children with T1D. Insulin reduces the rates at which body proteins are degraded. Whole body protein synthesis is either unaffected or reduced by insulin, even when insulin is administered together with amino acids to prevent insulin-dependent hypoaminoacidemia. Provision of insulin with oral nutrients improves protein balance by inhibiting whole body protein degradation, but does not affect protein synthesis. SUMMARY: In children with T1D the anticatabolic effects of insulin on whole body protein metabolism are mainly exerted through a reduction in rates at which body proteins are degraded. Nutritional factors enhancing the anabolic effect of insulin need to be further elucidated.

The effect of oral leucine on protein metabolism in adolescents with type 1 diabetes mellitus.

Lack of insulin results in a catabolic state in subjects with insulin-dependent diabetes mellitus which is reversed by insulin treatment. Amino acid supply, especially branched chain amino acids such as leucine, enhances protein synthesis in both animal and human studies. This small study was undertaken to assess the acute effect of supplemental leucine on protein metabolism in adolescents with type 1 diabetes. L-[1-(13)C] Leucine was used to assess whole-body protein metabolism in six adolescent females (16-18 yrs) with type 1 diabetes during consumption of a basal diet (containing 58 µmoles leucine/kg/h) and the basal diet with supplemental leucine (232 µmoles leucine/kg/h). Net leucine balance was significantly higher with supplemental leucine (56.33 ± 12.13 µmoles leucine/kg body weight/hr) than with the basal diet (-11.7 ± -5.91, P < .001) due to reduced protein degradation (49.54 ± 18.80 µmoles leucine/kg body weight/hr) compared to the basal diet (109 ± 13.05, P < .001).

Feeding acutely stimulates fibrinogen synthesis in healthy young and elderly adults.

Fibrinogen is a positive acute-phase protein and its hepatic synthesis is enhanced following inflammation and injury. However, it is not clear whether fibrinogen synthesis is also responsive to oral nutrients and whether the response to a meal may be affected by age. Our aim in this study was to investigate the acute effect of oral feeding on fibrinogen synthesis in both young and elderly men and women. Fibrinogen synthesis was determined in 3 separate occasions from the incorporation of l[(2)H(5)]phenylalanine (43 mg/kg body weight) in 8 young (21-35 y) and 8 elderly (>60 y) participants following the ingestion of water (control), a complete liquid meal (15% protein, 30% fat, and 55% carbohydrate), or only the protein component of the meal. The ingestion of the complete meal enhanced fibrinogen fractional synthesis rates (FSR) by 17 +/- 6% in the young and by 38 +/- 10% in the elderly participants compared with the water meal (P < 0.02). A comparable stimulation of FSR occurred with only the protein component of the meal in both young (29 +/- 7%) and elderly participants (41 +/- 9%) compared with the water meal (P < 0.005). Similar results were obtained when fibrinogen synthesis was expressed as absolute synthesis rates (i.e. mg.kg(-1).d(-1)). The results demonstrate that fibrinogen synthesis is acutely stimulated after ingestion of a meal and that this effect can be reproduced by the protein component of the meal alone, both in young and elderly adults.

Does increasing blood pH stimulate protein synthesis in dialysis patients?

BACKGROUND: Although the mechanism of muscle wasting in end-stage renal disease is not fully understood, there is increasing evidence that acidosis induces muscle protein degradation and could therefore contribute to the loss of muscle protein stores of patients on hemodialysis, a prototypical state of chronic metabolic acidosis (CMA). Because body protein mass is controlled by the balance between synthesis and degradation, protein loss can occur as result of either increased breakdown, impaired synthesis, or both. Correction of acidosis may therefore help to maintain muscle mass and improve the health of patients with CMA. We evaluated whether alkalizing patients on hemodialysis might have a positive effect on protein synthesis and on nutritional parameters. METHODS: Eight chronic hemodialysis patients were treated daily with oral sodium bicarbonate (NaHCO(3)) supplementation for 10-14 days, yielding a pre-dialytic plasma bicarbonate concentration of 28.6 +/-1.6 mmol/l. The fractional synthesis rates (FSR) of muscle protein and albumin were obtained by the L-[(2)H(5)ring]phenylalanine flooding technique. RESULTS: Oral NaHCO(3 )supplementation induced a significant increase in serum bicarbonate (21.5 +/- 3.4 vs. 28.6 +/- 1.6 mmol/l; p = 0.018) and blood pH (7.41 vs. 7.46; p = 0.041). The FSR of muscle protein and the FSR of albumin did not change significantly (muscle protein: 2.1 +/- 0.2 vs. 2.0 +/- 0.5% per day, p = 0.39; albumin: 8.3 +/- 2.2 vs. 8.6 +/- 2.5% per day, p = 0.31). Plasma concentrations of insulin-like growth factor 1 decreased significantly (33.4 +/- 21.3 vs. 25.4 +/- 12.3 nmol/l; p = 0.028), whereas thyroid-stimulating hormone, free thyroxin and free triiodothyronine did not change significantly and nutritional parameters showed no improvement. CONCLUSION: In contrast to other findings, raising the blood pH of dialysis patients was not associated with a positive effect on albumin and muscle protein synthesis, or nutritional and endocrinal parameters.

Importance of the high-molecular-mass isoform of adiponectin in improved insulin sensitivity with rosiglitazone treatment in HIV disease.

The present study was designed to investigate the relationship of isoforms of adiponectin to insulin sensitivity in subjects with HIV-associated insulin resistance in response to treatment with the thiazolidinedione, rosiglitazone. The two isoforms of adiponectin, HMW (high-molecular-mass) and LMW (low-molecular-mass), were separated by sucrose-gradient-density centrifugation. The amount of adiponectin in gradient fractions was determined by ELISA. Peripheral insulin sensitivity (Rd) was determined with hyperinsulinaemic-euglycaemic clamp, whereas hepatic sensitivity [HOMA (Homoeostasis Model Assessment) %S] was based on basal glucose and insulin values. Treatment with rosiglitazone for 3 months resulted in a significant improvement in the index of hepatic insulin sensitivity (86.4+/-15% compared with 139+/-23; P=0.007) as well as peripheral insulin sensitivity (4.04+/-0.23 compared with 6.17+/-0.66 mg of glucose/kg of lean body mass per min; P<0.001). Improvement in HOMA was associated with increased levels of HMW adiponectin (r=0.541, P=0.045), but not LMW adiponectin. The present study suggests that the HMW isoform of adiponectin is important in the regulation of rosiglitazone-mediated improvement in insulin sensitivity in individuals with HIV-associated insulin resistance, particularly in the liver.

Influence of age on the association of retinol-binding protein 4 with metabolic syndrome.

OBJECTIVE: The relationships of retinol-binding protein 4 (RBP4) with insulin sensitivity and body fat distribution have been investigated in a few recent studies with conflicting results. This may have been due to differences in ages of the subjects in the different studies. The aim of this study was to investigate whether the association of RBP4 and insulin sensitivity and percent trunk fat are influenced by age. METHODS AND PROCEDURES: Cross-sectional analyses of 48 young subjects and 55 elderly subjects. Insulin sensitivity was determined by a hyperinsulinemic-euglycemic clamp. Body fat distribution was determined by a dual-energy X-ray absorptiometry (DXA). RESULTS: In the young subjects, RBP4 levels were associated with insulin sensitivity (r = -0.30, P = 0.04), percent trunk fat (r = 0.54, P < 0.001), triglycerides (r = 0.44, P = 0.003), low-density lipoprotein (r = 0.38, P = 0.01). In contrast, in the elderly subjects there was no correlation between RBP4 levels and insulin sensitivity (r = -0.18, P = 0.20), percent trunk fat (r = 0.00, P = 0.10), triglycerides (r = 0.25, P = 0.10), and low-density lipoprotein (r = -0.11, P = 0.47). DISCUSSION: The associations of RBP4 with insulin sensitivity, percent trunk fat, and lipid levels are influenced by age.

Endothelial adhesion molecules are associated with inflammation in subjects with HIV disease.

BACKGROUND: The presence of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and soluble vascular cell adhesion molecule-1 (VCAM-1) is associated with elevated risk of cardiovascular disease. Subjects with human immunodeficiency virus (HIV) disease have multiple risk factors for cardiovascular disease, including elevated serum lipid levels, insulin resistance, and elevated levels of ICAM-1 and VCAM-1. This study assessed the variables associated with elevated adhesion molecule levels in this patient population. METHODS: Serum levels of ICAM-1 and VCAM-1 were assessed in 31 subjects without HIV disease and 52 subjects with HIV disease. Pearson correlation indicated a significant relationship between ICAM concentration and other variables, including CD4+ cell count, HIV viral burden, insulin sensitivity, and serum lipid level. Multiple regression modeling was used to determine the strengths of association among the variables. RESULTS: Subjects with HIV disease had elevated levels of ICAM-1 and VCAM-1. Pearson correlation analysis revealed significant associations between ICAM-1 and VCAM-1 level and insulin sensitivity, plasma lipid level, and presence of type 2 soluble receptor for tumor necrosis factor-alpha (sTNFR2). With multiple regression modeling to control for interdependence, only sTNFR2, a marker of inflammation, was an independent predictor of ICAM-1 and VCAM-1 levels. CONCLUSIONS: The study suggests that many of the variables associated with ICAM-1 and VCAM-1 levels can be related to their impact on inflammation.

Altered protein metabolism following coronary artery bypass graft (CABG) surgery.

The aim of the present study was to investigate the acute effect of CABG (coronary artery bypass graft) surgery on the rates of synthesis of muscle protein, the positive acute-phase protein fibrinogen and the negative acute-phase protein albumin. Synthesis rates of muscle protein, fibrinogen and albumin were measured simultaneously before and 4 h after the end of surgery from the incorporation of L-[(2)H(5)]phenylalanine (given at 43 mg/kg of body weight) in 12 patients undergoing CABG surgery. Surgery was performed either with the use of extracorporeal circulation with cardiopulmonary bypass (on-pump; n=5) or with the beating heart procedure without cardiopulmonary bypass (off-pump; n=7). Post-surgical muscle protein fractional synthesis rates were decreased by 36+/-6.5% compared with pre-surgical values (1.59+/-0.10 compared with 0.97+/-0.08%/day respectively; P<0.001). In contrast, the synthesis rates of both fibrinogen (36+/-4 compared with 100+/-11 mg.day(-1).kg(-1) of body weight; P<0.0001) and albumin (123+/-12 compared with 178+/-19 mg.day(-1).kg(-1) of body weight; P<0.001) were both significantly increased after surgery. No significant differences were found between surgery performed with or without cardiopulmonary bypass. In conclusion, the results demonstrate that CABG surgery has a profound effect on protein metabolism, with a differential response of protein synthesis in muscle and liver.

Limb fat to trunk fat ratio in elderly persons is a strong determinant of insulin resistance and adiponectin levels.

BACKGROUND: Similar to lipodystrophy syndromes, aging results in increased visceral adiposity with loss of subcutaneous adipose tissue in the extremities. The hypothesis of this study is that the distribution of limb fat to trunk fat (LF/TF) ratio in elderly persons has a stronger correlation than trunk fat alone to insulin resistance and adiponectin levels. METHODS: Thirty-eight elderly participants were divided into an insulin-resistant (IR) group and an insulin-sensitive (IS) group. Limb fat and trunk fat were measured by dual-energy x-ray absorptiometry. Insulin resistance was measured by a hyperinsulinemic-euglycemic clamp. RESULTS: There was no significant difference between the IS and IR groups with respect to body mass index, body fat index, absolute amount of trunk fat, or percent body fat. However, the difference in LF/TF ratio between the IS (1.02 +/- 0.05) and the IR groups (0.77 +/- 0.05) was highly significantly different (p <.001). Insulin resistance had a stronger correlation to the LF/TF ratio (r = 0.61, p <.001) than to absolute trunk fat (r = -0.32, p =.051). Adiponectin levels had a strong association with the LF/TF ratio (r = 0.63, p <.001), but did not correlate to absolute trunk fat (r = -0.24, p =.18). CONCLUSIONS: The distribution of body fat (LF/TF ratio) in elderly persons is a stronger determinant of insulin resistance and adiponectin levels than is trunk fat alone. The LF/TF ratio can be a useful tool to assess insulin sensitivity in the elderly population.

Effect of coenzyme q10 on myopathic symptoms in patients treated with statins.

Treatment of hypercholesterolemia with statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) is effective in the primary and secondary prevention of cardiovascular disease. However, statin use is often associated with a variety of muscle-related symptoms or myopathies. Myopathy may be related in part to statin inhibition of the endogenous synthesis of coenzyme Q10, an essential cofactor for mitochondrial energy production. The aim of this study is to determine whether coenzyme Q10 supplementation would reduce the degree of muscle pain associated with statin treatment. Patients with myopathic symptoms were randomly assigned in a double-blinded protocol to treatment with coenzyme Q10 (100 mg/day, n = 18) or vitamin E (400 IU/day, n = 14) for 30 days. Muscle pain and pain interference with daily activities were assessed before and after treatment. After a 30-day intervention, pain severity decreased by 40% (p <0.001) and pain interference with daily activities decreased by 38% (p <0.02) in the group treated with coenzyme Q10. In contrast, no changes in pain severity (+9%, p = NS) or pain interference with daily activities (-11%, p = NS) was observed in the group treated with vitamin E. In conclusion, results suggest that coenzyme Q10 supplementation may decrease muscle pain associated with statin treatment. Thus, coenzyme Q10 supplementation may offer an alternative to stopping treatment with these vital drugs.

Response of albumin synthesis to oral nutrients in young and elderly subjects.

BACKGROUND: The synthesis of albumin after oral ingestion of nutrients provides a means of storing amino acids, which can be made available during periods of fasting. OBJECTIVE: This study was undertaken to see whether the response of albumin synthesis to the oral intake of nutrients is compromised in elderly subjects. DESIGN: Albumin synthesis was determined from the incorporation of 43 mg l-[(2)H(5)]phenylalanine/kg body wt. Eight elderly subjects (aged >60 y) and 8 young subjects (aged 21-35 y) were studied on 3 separate occasions: after the intake of water, a liquid meal (with 15% of energy from protein, 30% of energy from fat, and 55% of energy from carbohydrate), or an isonitrogenous but not isocaloric meal containing only protein. RESULTS: Mean (+/-SEM) albumin synthesis, expressed as an absolute rate (ie, the amount of albumin synthesized per day), was significantly lower in elderly subjects (108 +/- 7 mg . kg body wt(-1) . d(-1)) than in young subjects (141 +/- 7 mg . kg body wt(-1) . d(-1)). In response to the complete meal, albumin synthesis was significantly increased in both the elderly (144 +/- 7 mgkg body wt(-1) . d(-1)) and the young (187 +/- 11 mg . kg body wt(-1) . d(-1)) subjects. The protein component of the meal was sufficient to stimulate albumin synthesis in both the elderly (147 +/- 14 mg . kg body wt(-1) . d(-1)) and the young (182 +/- 6 mg . kg body wt(-1) . d(-1)) subjects. CONCLUSIONS: Elderly subjects have lower rates of albumin synthesis than do young subjects during fasting, but they stimulate albumin synthesis proportionately in response to the oral ingestion of protein. The intakes of additional fat and carbohydrate do not stimulate albumin synthesis further.

Retinol-binding protein 4 is associated with insulin resistance and body fat distribution in nonobese subjects without type 2 diabetes.

BACKGROUND: Adipose tissue is responsible for releasing various adipokines that have been related to insulin resistance. Understanding the relationship of these adipokines to insulin resistance may foster the development of new treatments for diabetes. OBJECTIVES: The primary objective of this study was to determine whether an association between retinol-binding protein 4 (RBP4) and insulin resistance exists in nonobese individuals without a family history or diagnosis of diabetes. The secondary objective was to determine by a dual energy x-ray absorptiometry scan which adipose tissue depot most closely relates to RBP4 levels. DESIGN: Cross-sectional analysis of 92 study participants ranging in age from 20 to 83 yr was performed. The range of body mass index (BMI) was from 18 to 30 kg/m(2). Exclusion criteria were a BMI greater than 30 kg/m(2), family history of diabetes, or a diagnosis of diabetes. Insulin sensitivity was determined by a hyperinsulinemic euglycemic clamp. Body fat was measured by dual energy x-ray absorptiometry scan. RESULTS: RBP4 values were lower in females (35.8 +/- 1.7 microg/ml) compared with males (39.9 +/- 1.4 microg/ml; P = 0.06). RBP4 levels were found to correlate negatively with insulin sensitivity (r = -0.32; P = 0.002) and positively with age (r = 0.38; P < 0.001). RBP4 levels did not correlate with BMI (r = -0.13; P = 0.22), trunk fat (r = 0.16; P = 0.22), or percent body fat (r = 0.07; P = 0.65). However, RBP4 levels did correlate with percent trunk fat (r = 0.36; P = 0.001). CONCLUSION: These findings indicate a relationship between RBP4, insulin sensitivity, and percent trunk fat in individuals who may not have features of insulin resistance.

Feeding meals containing soy or whey protein after exercise stimulates protein synthesis and translation initiation in the skeletal muscle of male rats.

The purpose of this investigation was to compare the early response of skeletal muscle protein synthesis and translation initiation following the ingestion of different protein sources after endurance exercise. Treadmill-acclimated rats were designated as either nonexercised controls (NEX) or treadmill exercised for 2 h at 26 m/min (approximately 75% VO2max) and then fed either carbohydrate only (EC), carbohydrate plus soy protein (ES), or carbohydrate plus whey protein (EW). One hour after exercise, serum insulin concentrations in EC, ES, and EW were greater than in NEX (P<0.05); the concentration in EW was greater than in EC, with that in ES intermediate. Serum concentrations of branched-chain amino acids in ES and EW were higher than in EC, but serum leucine and isoleucine in EW were higher than in ES (P<0.05). Nevertheless, both ES and EW promoted the fractional rate of skeletal muscle protein synthesis significantly more than EC. Likewise, compared with EC, both ES and EW increased formation of the mRNA cap binding complex eIF4F and stimulated phosphorylation of the translational repressor, 4E-BP1, the 70kD ribosomal protein S6 kinase (S6K1), and the mammalian target of rapamycin (mTOR) kinase at serine 2448. On the other hand, phosphorylation of S6K1 and mTOR was greater in EW than in ES (P<0.05). In conclusion, general protein synthesis and the mRNA cap binding step are promoted comparably by soy protein and whey protein in the skeletal muscle of exercised rats. Furthermore, the data suggest that mTOR signaling in skeletal muscle is acutely responsive to physiological variations in dietary amino acids.

Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase.

L-asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.