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The aims of this work were to establish whether glutamine infusion via alanyl-glutamine dipeptide provides effective therapy against muscle atrophy from glucorticoids and whether the glucocorticoid induction of glutamine synthetase (GS) is downregulated by dipeptide supplementation. Rats were given hydrocortisone 21-acetate or the dosing vehicle and were infused with alanine (AA) or alanyl-glutamine (AG) at the same concentrations and rates (1.15 mumol.min-1.100 g body wt-1, 0.75 ml/h) for 7 days. Compared with AA infusion in hormone-treated animals, AG infusion prevented total body and fast-twitch muscle mass losses by over 70%. Glucocorticoid treatment did not reduce muscle glutamine levels. Higher serum glutamine was found in the AG-infused (1.72 +/- 0.28 mumol/ml) compared with the AA-infused group (1.32 +/- 0.06 mumol/ml), but muscle glutamine concentrations were not elevated by AG infusion. Following glucocorticoid injections, GS enzyme activity was increased by two- to threefold in plantaris, fast-twitch white (superficial quadriceps), and fast-twitch red (deep quadriceps) muscle/fiber types of the AA group. Similarly, GS mRNA was elevated by 3.3- to 4.1-fold in these same muscles of hormone-treated, AA-infused rats. AG infusion diminished glucocorticoid effects on GS enzyme activity to 52-65% and on GS mRNA to 31-37% of the values with AA infusion. These results provide firsthand evidence of atrophy prevention from a catabolic state using glutamine in dipeptide form. Despite higher serum and muscle alanine levels with AA infusion than with AG infusion, alanine alone is not a sufficient stimulus to counteract muscle atrophy. The AG-induced muscle sparing is accompanied by diminished expression of a glucocorticoid-inducible gene in skeletal muscle. However, glutamine regulation of GS appears complex and may involve more regulators than muscle glutamine concentration alone.

Skeletal muscle atrophy from glucocorticoids is prevented by glutamine infusion. Because the gene-encoding glutamine synthetase (GS) is glucocorticoid inducible, it represented an appropriate model for resting whether glucocorticoids and glutamine exert opposing actions on the expression of specific genes related to atrophy in muscle tissue. Rats were administered hydrocortisone 21-acetate or the dosing vehicle (carboxymethyl cellulose) and were infused with saline (Sal) or glutamine (Gln, 240 mM, 0.75 ml/h) for 7 days. Hormone treatment did not significantly lower glutamine levels in fast-twitch white or red regions of the quadriceps. Despite higher serum glutamine concentrations with amino acid infusion [1.52 +/- 0.03 (Gln) vs. 1.20 +/- 0.04 (Sal) mumol/ml], muscle glutamine concentrations were not markedly increased in these fiber types. In saline-infused animals, glucocorticoid treatment produced 200-300% increases in plantaris, fast-twitch white, and fast-twitch red muscle GS enzyme activity and mRNA. Moreover, in all muscle types studied, glutamine infusion diminished glucocorticoid effects on GS enzyme activity to 131-159% and on GS mRNA to 110-200% of the values in saline-treated controls. These data demonstrate that glutamine infusion results in inhibiting GS expression, but the absence of changes in muscle glutamine concentration suggests the interplay of additional regulators of the GS gene.

Sepsis has been reported to cause mitochondrial dysfunction and inhibition of key enzymes that regulate the tricarboxylic acid (TCA) cycle. We investigated the effect of sepsis on high-energy phosphates, glycolytic and TCA cycle intermediates, and specific amino acids that are involved in regulating the size of the TCA cycle pool during changes in metabolic state of the heart. Sepsis was induced in 12 female rats by the cecal ligation and perforation technique under halothane anesthesia; seven control rats underwent cecal manipulation without ligation. At 36-42 h postsurgery, the rats were reanesthetized, the chest was opened, and the hearts were freeze-clamped. Perchloric acid extracts of the hearts were analyzed with fluorometric enzymatic methods and 31P nuclear magnetic resonance spectroscopy. There were no significant differences in the levels of the TCA cycle intermediates or high-energy phosphates between the septic and control rats. The major metabolic changes were the 28% decrease in alanine and the 31% decrease in glutamate in the septic hearts compared with control (P less than 0.05 and P less than 0.005, respectively). Phosphocholine, a component of membrane phospholipids, was increased by 91% in the septic hearts (P less than 0.01). We conclude that sepsis does not impair the TCA cycle or induce significant cellular ischemia in the heart. The increase in phosphocholine may represent significant cellular membrane disruption during sepsis.

Nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS or NOS2) has been implicated in the hypotension, organ failure, and death that complicate sepsis. To avoid the confounding effects and limitations of iNOS inhibitors, we used iNOS gene "knockout" mice to examine the effect of inducible NO production in a model of polymicrobial abdominal sepsis treated with antibiotics. We hypothesized that iNOS gene deficiency would significantly alter outcome.C57BL6 wild-type (control) and congenic iNOS knockout mice were studied concurrently. Under halothane anesthesia, the ceca were ligated with 4-0 silk suture and punctured twice with a 26-gauge needle (cecal ligation and puncture, CLP). Survival was followed for 7 days, after which necropsies were performed in surviving animals. In an accompanying study examining the acute effects of sepsis, organ injury at 18 hours after CLP as determined by histology and the degree of cell death by apoptosis were examined with the use of hematoxylin and eosin (HE) and TUNEL staining and two-channel fluorescence-activated cell sorter (FACS) analysis.Sham laparotomy produced no lethality in either knockout (n = 3) or wild-type (n = 3) animals. Compared with survival in controls (n = 20), survival after CLP in iNOS knockout mice (n = 21) was significantly decreased (P.01 at 2 days, P = .080 at 7 days, Mantel-Haenszel log-rank test). CLP-induced apoptotic cell death was significantly less in the thymus of iNOS knockout mice compared with wild-type mice.We conclude that iNOS gene function provides a survival benefit in septic mice and is associated with increased sepsis-induced thymocyte apoptosis. To our knowledge, this is the first survival study examining the effect of iNOS gene deficiency in a clinically relevant model of sepsis.

In sepsis there is extensive apoptosis of lymphocytes, which may be beneficial by down-regulating the accompanying inflammation. Alternatively, apoptosis may be detrimental by impairing host defense. We studied whether Bcl-2, a potent antiapoptotic protein, could prevent lymphocyte apoptosis in a clinically relevant model of sepsis. Transgenic mice in which Bcl-2 was overexpressed in T cells had complete protection against sepsis-induced T lymphocyte apoptosis in thymus and spleen. Surprisingly, there was also a decrease in splenic B cell apoptosis in septic Bcl-2 overexpressors compared with septic HeJ and HeOuJ mice. There were marked increases in TNF-alpha, IL-1beta, and IL-10 in thymic tissue in sepsis in the three species of mice, and the increase in TNF-alpha and IL-10 in HeOuJ mice was greater than that in Bcl-2 mice. Mitotracker, a mitochondrial membrane potential indicator, demonstrated a sepsis-induced loss of membrane potential in T cells in HeJ and HeOuJ mice but not in Bcl-2 mice. Importantly, Bcl-2 overexpressors also had improved survival in sepsis. To investigate the potential impact of loss of lymphocytes on survival in sepsis, Rag-1-/- mice, which are totally deficient in mature T and B cells, were also studied. Rag-1-/- mice had decreased survival compared with immunologically normal mice with sepsis. We conclude that overexpression of Bcl-2 provides protection against cell death in sepsis. Lymphocyte death may be detrimental in sepsis by compromising host defense.

Apoptosis, a mechanism of cell death prominent in critical illnesses including disseminated inflammation and multiorgan dysfunction syndrome, is characterized by morphologic changes including cell shrinkage, condensation of organelles, blebbing, and chromatin fragmentation. These phenomena suggest substantial changes in cytoskeletal structure. We hypothesized that stress-induced apoptosis in endothelial cells is, in part, a consequence of a critical cytoskeletal rearrangement.Porcine aortic endothelial cells in culture, surrogates for the microvasculature in vivo, were exposed sequentially to Escherichia coli endotoxin (25 micrograms/mL; 18 hours) to induce the inflammatory response and then to sodium arsenite (160 mumol/L; 120 minutes) to induce the heat-shock response, a well-characterized model of stress-induced apoptosis. Laser confocal micrographs of fluorescein isothio-cyanate-labeled phalloidin-stained cells were analyzed to calculate the border fractal dimension of the cytoskeleton. Other cells were exposed to cytochalasin D, a fungal metabolite, which interferes with polymerization of actin from its globular to its filamentous form, and similarly were analyzed with respect to fractal dimension, viability (neutral red assay), and manner of death (annexin V fluorescence-activated cell scanning analysis).Induction of the inflammatory or heat-shock responses caused subtle and distinct rearrangement of the actin cytoskeleton. When these stimuli were applied in sequence, a synergistic interaction led to profound cytoskeletal collapse. Reversal of the sequence did not induce the cytoskeletal disruption. Cytochalasin D alone induced a dose-dependent cytoskeletal collapse indistinguishable from that caused by the acute phase-heat shock sequence that caused cell death by apoptosis. The effect of lower doses of Cytochalasin D could be potentiated by subsequent induction of the heat-shock response.Sequential stresses that mimic pathophysiologic "two-hit" stimuli induce a characteristic fractal rearrangement of the actin cytoskeleton. Because cytochalasin D-induced rearrangement of this cytoskeleton produced apoptosis indistinguishable from the stress-induced apoptosis, we conclude that the cytoskeletal rearrangement is likely a critical event in the pathway to apoptosis. This disruption of intracellular interconnections mirrors endotoxin-induced disruption in signals among organs and supports the mechanistic hypothesis that multiorgan dysfunction syndrome generally reflects disruption of signals and connections at several levels of biologic organization.

Calcium functions as a critical intracellular second messenger and regulates many cellular processes such as muscle contractility, glycogen and protein turnover, hormone secretion, and vascular smooth muscle tone which are markedly abnormal during sepsis/endotoxemia. There also is increasing recognition of the role of calcium in the production of a variety of cytokines such as tumor necrosis factor alpha and interleukin-1 beta, which are important mediators of sepsis. Our hypothesis is that disturbances in cellular calcium regulation are responsible for or contribute to many of the metabolic manifestations of sepsis/endotoxemia and may be the driving force behind the development of multiorgan failure. In this article, we focus on a) new insights into calcium's regulation of the inflammatory cascade, b) the controversy concerning whether free cytosolic calcium concentration ([Ca2+]i) is increased in the disorder, and c) the potential therapeutic uses of calcium antagonists. An important message is that there are fundamental differences in the pathophysiology of the endotoxin model versus the cecal ligation and perforation (CLP) model of sepsis. Although calcium antagonists improve survival in the endotoxin model, they increase mortality in the CLP model of sepsis. Possible reasons for the differences in the effect of the drugs in the two different models and insight into the mechanisms of cell injury in endotoxin versus sepsis are presented.

Calcium plays an important role in the toxic effects of endotoxin, and calcium antagonists also have been shown to improve survival in animals challenged with endotoxin. Calcium may be involved in regulating cytokine production. Therefore, the protective effect of calcium-antagonists in endotoxin may be due to decreased cytokine formation and/or systemic release. In a mouse model of endotoxin, dantrolene (10 mg/kg) and azumolene (20 mg/kg), drugs that decrease calcium release from intracellular stores, or diltiazem (20 mg/kg), a calcium channel blocker, decreased plasma tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and IL-1 beta (47.2, 63.2, and 62.4%, respectively, p.05) when the animals were injected intraperitoneally with endotoxin. Dantrolene and azumolene decreased IL-1 alpha by 56.6 and 65.4%, respectively, (p.05) and IL-1 beta by 51.7 and 69.7%, respectively (p.05). Diltiazem had no effect on IL-1 alpha or IL-1 beta. Dantrolene decreased TNF-alpha in lung (26.1%), liver (29.4%), and spleen (35.4%) (p.05) and IL-1 alpha in lung (30.0%) and liver (25.4%) (p.05). The present findings indicate that calcium-antagonists may be efficacious in treating cytokine mediated inflammatory disorders.

In addition to participating in protein synthesis in cells and tissues, L-arginine is essential for the synthesis of urea, creatine, creatinine, nitric oxide, and agmatine and influences hormonal release and the synthesis of pyrimidine bases. This places L-arginine, its precursors and its metabolites at the center of the interaction of different metabolic pathways and interorgan communication. Thus L-arginine participates in changing the internal environment in different but simultaneous ways, ranging from disposal of protein metabolic waste, muscle metabolism, vascular regulation, immune system function, and neurotransmission, to RNA synthesis and hormone-mediated regulation of the internal milieu. In normal rats, inhibition of the nitric oxide pathway results in systemic hypertension and decreased glomerular filtration rate and effective renal plasma flow. If the inhibition of this pathway is sustained, then glomerulosclerosis and death from uremia follow. Dietary intervention with L-arginine has resulted in amelioration of a number of experimental kidney diseases, such as those caused by subtotal nephrectomy, diabetic, nephropathy, cyclosporin A administration, salt-sensitive hypertension, ureteral obstruction, puromycin amino-nucleoside nephrosis, kidney hypertrophy due to high-protein feeding, and glomerular thrombosis due to administration of lipopolysaccharide. The present review addresses the current evidence for the beneficial effects of dietary intervention with L-arginine in a number of experimental renal diseases and describes the basis for the concept of L-arginine deficiency (absolute or relative) in certain settings in which supplementation of the diet with this amino acid may be beneficial.

Recently, investigators have reported that heat shock proteins (HSPs) can protect isolated cells from cytotoxicity induced by two important mediators of sepsis: interleukin-1 and tumor necrosis factor. The present study was undertaken to examine the hypothesis that transient whole body hyperthermia could decrease mortality from subsequent challenge with gram-negative endotoxin. We demonstrate that heat pretreatments improved long-term survival fivefold in a mouse endotoxin model and this was correlated with the production of HSPs. There was a marked difference in individual organ expression of the inducible 72-kDa heat shock protein (HSP72). Heat treatments caused significant HSP72 formation in lung, liver, kidney, and small intestine, but much lesser formation in heart, brain, and abdominal wall muscle. Additional experiments demonstrated that the protective effect of hyperthermic treatments against an endotoxin challenge occurred early, i.e., 1 and 2 h after heating, was maximal at 12 h, and had significantly diminished by 48 h. The formation and decay of HSP72 demonstrated a time course that paralleled the survival curve from endotoxin challenge, thus suggesting a possible role for HSP72 in the protective effect. Surprisingly, and in contrast to studies reported in incubated cells, endotoxin alone did not cause significant formation of HSP72 in vivo.

The effect(s) of L-arginine administration on the renal function of rats with untreated diabetes mellitus was examined. Rats received streptozotocin (N = 11) or vehicle (N = 12): Group 1 (normal rats, N = 6) drank tap water; Group 2 (normal rats, N = 6) drank tap water containing 1% L-arginine; Group 3 (diabetic rats, N = 5) drank tap water; and Group 4 (diabetic rats, N = 6) drank tap water with 1% L-arginine. Rats were fed a standard rat chow diet (22.8% protein, 142% L-arginine) with free access to food and water for 14 wk. Diabetic rats gained less weight, had significantly lower plasma levels of albumin and L-arginine, and had greater values for 24-h urine volumes and urine excretion of glucose, protein, urea, creatinine, nitrate, and nitrite than control rats. Diabetic rats given L-arginine (Group 4) had significantly lower protein and cGMP excretion in the urine than did rats of Group 3. The administration of L-arginine did not affect the plasma levels of glucose or L-arginine in Groups 2 or 4 compared with those of their respective controls. Group 3 had significantly higher values for GFR than did the other three groups of rats, but values for effective RPF, mean arterial pressure, hematocrit, and renal vascular resistance were not significantly different between Groups 3 and 4. There was no significant difference in glomerular morphology among the four groups of rats as determined by light microscopy, and both groups of diabetic rats exhibited the Armanni-Ebstein lesion in their tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Sepsis is frequently characterized by a number of metabolic abnormalities: increased plasma lactate concentration, metabolic acidosis, increased glycolysis, and an abnormal "delivery-dependent" oxygen consumption. Two hypotheses have been advanced to explain these metabolic abnormalities: (1) cellular hypoxia resulting from abnormal microcirculatory blood flow or (2) defect(s) in energy-producing metabolic pathways of cells. Results of our studies on rat muscle, liver, heart, brain, and plasma suggest that there is no evidence of bioenergetic failure in these septic tissues and that the increase in lactate production is not necessarily due to cellular hypoxia. The adequacy of cellular oxygenation and bioenergetics was verified using in vivo phosphorus 31 nuclear magnetic resonance spectroscopy, [18F]fluoromisonidazole, and microfluorometric enzymatic techniques. Findings from these studies as well as results from several clinical investigations indicate that neither hypothesis can adequately account for the metabolic features typical of sepsis and that the pathophysiology of sepsis awaits further clarification. These studies and important clinical implications are discussed.

Underlying cellular hypoxia, which may be difficult to detect, has been postulated to be a major cause of morbidity and mortality in sepsis. We employed the novel hypoxic marker [18F]fluoromisonidazole to determine whether cellular hypoxia was present in a peritonitis model of sepsis in the rat. A second group of septic and control rats had organ blood flow measurements determined by the radiolabeled microsphere technique to relate possible ischemia to decreased organ perfusion. No evidence of cellular hypoxia was detected in skeletal muscle, brain, liver, heart, or diaphragm in the septic rats. Ligation of the femoral artery caused a greater reduction in flow (55% decrease vs. 20% decrease, P less than 0.05) and an increased retention of [18F]fluoromisonidazole in skeletal muscle of the septic rats. We conclude that sepsis does not invariably result in systemic, i.e., multiorgan, cellular hypoxia and that underlying cellular hypoxia is not the major pathophysiological abnormality in sepsis. The greater reduction in muscle blood flow and the increased retention of [18F]fluoromisonidazole in the ischemic muscle of septic rats implies that they may be more vulnerable to hypoxia.

The effects of sepsis on intracellular Na+ concentration ([Na+]i) and glucose metabolism were examined in rat red blood cells (RBCs) by using 23Na- and 2H-nuclear magnetic resonance (NMR) spectroscopy. Sepsis was induced in 15 halothane-anesthetized female Sprague-Dawley rats by using the cecal ligation and perforation technique; 14 control rats underwent cecal manipulation without ligation. The animals were fasted for 36 h, but allowed free access to water. At 36 h postsurgery, RBCs were examined by 23Na-NMR by using dysprosium tripolyphosphate as a chemical shift reagent. Human RBCs from 17 critically ill nonseptic patients and from 7 patients who were diagnosed as septic were also examined for [Na+]i. Five rat RBC specimens had [Na+]i determined by both 23Na-NMR and inductively coupled plasma-atomic emission spectroscopy (ICP-AES). For glucose metabolism studies, RBCs from septic and control rats were suspended in modified Krebs-Henseleit buffer containing [6,6-2H2]glucose and examined by 2H-NMR. No significant differences in [Na+]i or glucose utilization were found in RBCs from control or septic rats. There were no differences in [Na+]i in the two groups of patients. The [Na+]i determined by NMR spectroscopy agreed closely with measurements using ICP-AES and establish that 100% of the [Na+]i of the RBC is visible by NMR. Glucose measurements determined by 2H-NMR correlated closely (correlation coefficient = 0.93) with enzymatic analysis. These studies showed no evidence that sepsis disturbed RBC membrane function or metabolism.

The functional integrity of the gluconeogenic pathway was measured in nine term infants, four appropriate-for-gestational age (AGA), and five normoglycemic small-for-gestational age (SGA), by determination of 13C2 enrichment in blood glucose during the constant infusion of tracer [2,3]13C2]alanine between 4 and 8 h of postnatal age. Alanine flux, calculated from the steady-state blood [2,3-13C2]alanine enrichment was 16.6 +/- 1.3 (SE) (mumol.kg-1.min-1 in the AGA infants and not statistically different from the value of 15.3 +/- 0.7 mumol.kg-1.min-1 in the SGA infants. Alanine flux did not correlate with blood alanine level in either group. By 6 h of age, the earliest sampling time, there was 13C2 enrichment of blood glucose in every infant studied, indicating that the gluconeogenic pathway was functionally intact by that time and implying that it was operative sooner. At 8 h of age, 9.3 +/- 2.3% of blood glucose was derived from alanine in the AGA group and 12.9 +/- 2.4% in the SGA group, values not statistically different. These data indicate that the term human newborn has a functional gluconeogenic pathway very early in postnatal life and that intrauterine growth retardation per se does not impair maturation of the system. Furthermore, the plasma alanine level alone is a poor index of gluconeogenic carbon flow in these infants.

To determine whether the enhanced insulin-sensitivity of glucose metabolism in muscle after acute exercise also extends to protein metabolism, untrained and exercise-trained rats were subjected to an acute bout of exercise, and the responses of protein synthesis and degradation to insulin were measured in epitrochlearis muscles in vitro. Acute exercise of both untrained and trained rats decreased protein synthesis in muscle in the absence or presence of insulin, but protein degradation was not altered. Exercise training alone had no effect on protein synthesis or degradation in muscle in the absence or presence of insulin. Acute exercise or training alone enhanced the sensitivities of both protein synthesis and degradation to insulin, but the enhanced insulin-sensitivities from training alone were not additive to those after acute exercise. These results indicate that: a decrease in protein synthesis is the primary change in muscle protein turnover after acute exercise and is not altered by prior exercise training, and the enhanced insulin-sensitivities of metabolism of both glucose and protein after either acute exercise or training suggest post-binding receptor events.

To determine whether training reduces the acute catabolic stress of an exercise bout, control and azotemic rats, either exercise trained or untrained, were subjected to a bout of exercise or allowed to rest. Protein turnover was measured immediately following exercise in isolated muscles. Azotemia (no exercise) increased protein degradation; acute exercise (no azotemia) decreased protein synthesis. Protein catabolism was greatest in acutely exercised azotemic rats. Training reduced azotemia-induced and acute exercise-stimulated increase in protein degradation. To determine whether this improvement in protein metabolism by training was due to increased insulin sensitivity, the dose-response to insulin was determined. Resistance of protein synthesis or degradation to insulin was not observed in azotemic rats. Either acute exercise or training increased sensitivities of protein degradation and synthesis to insulin; training also increased responsiveness of protein degradation to insulin. Thus exercise training reduced the elevated muscle protein catabolism due to azotemia and/or acute exercise and enhanced the action of insulin on muscle.

The synthesis and release of alanine and glutamine were investigated with an intact rat epitrochlaris muscle preparation. This preparation will maintain on incubation for up to 6 hours, tissue levels of phosphocreatine, ATP, ADP, lactate, and pyruvate closely approximating those values observed in gastrocnemius muscles freeze-clamped in vivo. The epitrochlaris preparation releases amino acids in the same relative proportions and amounts as a perfused rat hindquarter preparation and human skeletal muscle. Since amino acids were released during incubation without observable changes in tissue amino acids levels, rates of alanine and glutamine release closely approximate net amino acid synthesis. Large increases in either glucose uptake or glycolysis in muscle were not accompanied by changes in either alanine or glutamine synthesis. Insulin increased muscle glucose uptake 4-fold, but was without effect on alanine and glutamine release. Inhibition of glycolysis by iodacetate did not decrease the rate of alanine synthesis. The rates of alanine and glutamine synthesis and release from muscle decreased significantly during prolonged incubation despite a constant rate of glucose uptake and pyruvate production. Alanine synthesis and release were decreased by aminooxyacetic acid, an inhibitor of alanine aminotransferase. This inhibition was accompanied by a compensatory increase in the release of other amino acids, such as aspartate, an amino acid which was not otherwise released in appreciable quantities by muscle. The release of alanine, pyruvate, glutamate, and glutamine were observed to be interrelated events, reflecting a probable near-equilibrium state of alanine aminotransferase in skeletal muscle. It is concluded that glucose metabolism and amino acid release are functionally independent processes in skeletal muscle. Alanine release reflects the de novo synthesis of the amino acid and does not arise from the selective proteolysis of an alanine-rich storage protein. It appears that the rate of alanine and glutamine synthesis in skeletal muscle is dependent upon the transformation and metabolism of amino acid precursors.

The synthesis and release of alanine and glutamine have been studied in the intact rat epitrochlaris skeletal muscle preparation. Aspartate, cysteine, leucine, valine, methionine, isoleucine, serine, theronine, and glycine increased significantly the formation and release of alanine from muscle. Cysteine, leucine, valine, methionine, isoleucine, tyrosine, lysine, and phenylalanine increased the rate of glutamine synthesis. Only ornithine, arginine, and tryptophan were without effect on the synthesis of either alanine or glutamine. Half-maximal stimulation of alanine and glutamine formation by added amino acids was observed with concentrations ranging between 0.5 and 1.0 mM. Increases in alanine and glutamine formation were not accompanied by changes in pyruvate production or glucose uptake. The progressive decline in alanine and glutamine synthesis noted on prolonged incubation was prevented by the addition of amino acids to the incubation medium. Stimulation of alanine synthesis by added amino acids was unaffected by inhibition of glycolysis with iodoacetate. Inhibition of alanine aminotransferase with aminooxyacetate significantly decreased alanine formation. Pyruvate and ammonium chloride did not increase further the rate of either alanine or glutamine formation above that produced by added amino acids. These data indicate that most amino acids are precursors for alanine and glutamine synthesis in skeletal muscle. A general mechanism is presented for the de novo formation of alanine from amino acids in skeletal muscle, and the importance of proteolysis for the supply of amino acid precursors for alanine and glutamine synthesis is discussed.

Effects of isometric contraction and catecholamines on glucose and glycogen metabolism in skeletal muscle were investigated with the in vitro rat epitrochlearis preparation. Mechanical performance and glycogenolysis exhibited two phases. During the initial 30 min, muscle work was 30% greater and glycogenolysis 8- to 10-fold faster than the steady-state values in the subsequent 3–4 h of contraction. Glucose uptake was increased by contraction and remained relatively constant during stimulation. Epinephrine (10(-9) to 10(-6) M) and norepinephrine (10(-7) to 10(-5) M) produced inotropic and glycogenolytic effects blocked by propranolol but not phentolamine. Chemical sympathectomy and propranolol blocked the initial glycogenolytic and inotropic effects produced by isometric contraction, suggesting that they were caused by the release of endogenous catecholamines. Net lactate production in resting muscles accounted for > 50% of total glucosyl units utilized. During contraction net lactate production accounted < 10–15% of total glycosyl flux indicating that rat fast-twitch pale muscle is capable of significant rates of aerobic glucose oxidation. Oleate and caprylate did not affect mechanical performance, glycogen, or glucose metabolism in resting or contracting muscles.

Alanine and glutamine formation and release were studied using the intact epitrochlaris preparation of rat skeletal muscle. Alanine release from skeletal muscle was increased by fasting (65%), cortisone (145%), thyroxine (200%), and diabetes (185%). Glutamine release was decreased by cortisone (37%) and diabetes (23%) but not significantly affected by fasting or thyroxine. Tissue levels of alanine were unchanged but tissue glutamine levels were markedly reduced (30 to 60%) in all treatment groups. Insulin added in vitro did not affect amino acid release even with preparations obtained from diabetic animals. Inhibition of glycolysis with 0.2 mM iodoacetate had no effect on the rate of alanine and glutamine formation in any treatment group. Pyruvate generation was increased by all treatments even in the presence of the inhibitor. Total skeletal muscle alanine, aspartate, and branched chain aminotransferase, glutamate dehydrogenase, and malic enzyme activities were not significantly altered in any treatment groups. The addition of 10 mM aspartate, cysteine, branched chain amino acids, and serine significantly increased alanine formation, whereas the maximal rate of glutamine formation in the presence of stimulating amino acids was reduced in each treatment groups--the most marked effects were noted with cortisone and diabetic preparations. Although accelerated muscle proteolysis is an important factor regulating alanine formation in skeletal muscle, the redirection of carbon flow from glutamine toward alanine formation observed in fasting, cortisone, thyroxine-treated, and diabetic rats, indicates that factors other than proteolysis also participate in the control of amino acid release from muscle.

Insulin resistance has been demonstrated in chronic renal failure patients and may be improved by exercise training, but the mechanisms have not been identified. In this study, the response of glucose uptake, glycogen synthesis, and glucose utilization via glycolysis (glycolytic utilization) to stimulation by insulin and/or acute exercise were determined in isolated muscles from rats with moderate renal insufficiency that were exercise trained or remained sedentary. Moderate renal insufficiency had no effect on the basal rate, insulin sensitivity, or insulin responsiveness of glucose uptake, glycogen synthesis, or glycolytic utilization in muscle. The enhanced insulin responsiveness of both glycogen synthesis and glucose uptake following acute exercise, noted in control animals, was less in rats with moderate renal insufficiency, but the enhanced basal rate and insulin sensitivity after exercise were unaffected by moderate renal insufficiency. Exercise training increased the insulin sensitivity and responsiveness of muscle glucose uptake and glycolytic utilization in rats with moderate renal insufficiency and in controls. The effects of acute exercise and exercise training on insulin responsiveness of glucose uptake were additive in controls but not in animals with moderate renal insufficiency. These findings are compatible with the concept that moderate renal insufficiency is associated with a postreceptor defect in insulin's action in muscle, detectable only following maximal stimulation of glucose transport by insulin and exercise, and partially correctable by exercise training.

To characterize the action of growth hormone (GH) on gluconeogenesis in renal proximal tubule, glucose production was measured in suspensions of canine renal proximal tubular segments incubated with 1 mM L-alanine, 10 mM lactate, 1 mM succinate, and various concentrations (10(-11) to 10(-6) M) of recombinant bovine GH (bGH). Production of glucose increased as a function of time for 120 min. Bovine growth hormone (10(-6) M) increased glucose production at 120 min by 55 +/- 16%. Significant enhancement of glucose production occurred in suspensions of segments incubated with as little as 10(-10) M bGH. Half-maximal stimulation occurred at between 10(-9) and 10(-8) M. To ascertain whether these actions of bGH are mediated directly, we determined the effects of insulin-like growth factors (IGFs) I and II on glucose production. Addition of IGF-I to segments enhanced glucose production in a concentration-dependent manner. However, incubation with bGH did not induce measurable IGF-I production in the segments. In contrast to the action of IGF-I, IGF-II did not affect glucose production. We conclude that bGH acts directly on cells within proximal tubular segments to enhance gluconeogenesis. Stimulation of gluconeogenesis in vitro could reflect a counterregulatory action of GH exerted on renal proximal tubule in vivo.

Free fatty acid (FFA) transport was measured in 11 and glycerol turnover in 5 newborns with continuous tracer infusion of [1-13C]palmitate or [2-13C]glycerol, respectively. In addition, simultaneous determination of glucose production in the latter group with [6,6-2H2]glucose tracer and measurement of the appearance rate of [13C]glucose derived from [13C]glycerol allowed calculation of gluconeogenesis from glycerol. The average FFA inflow rate was 11.5±1.7 μmol kg−1min−1, 2.5-4.5 h after the last feeding, and 16.7±2.8 μmol kg−1min−1, 5-12 h after the last meal. These rates are comparable to those found in adults only after 8-16 h and ~72 h of fasting, respectively. FFA inflow in the newborn was directly correlated with time of fasting, plasma FFA level, and plasma glycerol level. Palmitate clearance and fractional removal were inversely related to palmitate level. Glycerol flux averaged 4.4±0.5 μmol kg−1min−1, a value three- to fourfold that of the postabsorptive adult. Approximately 75% of transported glycerol was converted to glucose and represented 5.0±0.6% of hepatic glucose production. Furthermore, there was a direct relationship between glycerol turnover and the fraction of glucose coming from glycerol. Despite the absolutely elevated neonatal FFA and glycerol transport rates, these were quantitatively similar to values found in adults with comparable elevated substrate levels. Furthermore, other similarities with the adult in the relationships between inflow transport and substrate values, and between transport and fractional removal suggest that the regulatory aspects of lipid transport in man are already well developed by the first day of life.

Alanine and glutamine formation and release were studied using the intact epitrochlaris preparation of rat skeletal muscle. Epinephrine reduced the release of alanine and glutamine in a concentration-dependent manner. Measurable inhibition was observed at 10(-9) M epinephrine, and maximal inhibition was obtained at 10(-5) M. Norepinephrine also reduced alanine and glutamine formation and release but the concentration required for maximal inhibition was approximately 100-fold greater than for epinephrine. Isoproterenol (beta agonist), but not phenylephrine (alpha agonist), reproduced the effects of epinephrine, and propranolol (beta antagonist), but not phentolamine (alpha antagonist), blocked the effect of the catecholamine. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate reproduced the effects of epinephrine and theophylline potentiated the effect of submaximal concentrations of the hormone. Glucagon and prostaglandin E2 had no observable effect on amino acid release. Insulin did not modify the inhibition of alanine and glutamine release produced by epinephrine. Alanine and glutamine formation from added precursor amino acids was unaffected by epinephrine or cyclic adenosine 3':5'-monophosphate. Epinephrine reduced alanine formation in muscles obtained from diabetic rats or animals treated with thyroxine or cortisone. These findings indicate that physiological levels of catecholamines reduce alanine and glutamine formation and release from skeletal muscle. This effect is mediated by a beta-adrenergic receptor and the adenylate cyclase system and can be accounted for by an inhibition of muscle protein degradation.

The synthesis and release of alanine and glutamine were investigated with an intact rat epitrochlaris muscle preparation. This preparation will maintain on incubation for up to 6 hours, tissue levels of phosphocreatine, ATP, ADP, lactate, and pyruvate closely approximating those values observed in gastrocnemius muscles freeze-clamped in vivo. The epitrochlaris preparation releases amino acids in the same relative proportions and amounts as a perfused rat hindquarter preparation and human skeletal muscle. Since amino acids were released during incubation without observable changes in tissue amino acids levels, rates of alanine and glutamine release closely approximate net amino acid synthesis. Large increases in either glucose uptake or glycolysis in muscle were not accompanied by changes in either alanine or glutamine synthesis. Insulin increased muscle glucose uptake 4-fold, but was without effect on alanine and glutamine release. Inhibition of glycolysis by iodacetate did not decrease the rate of alanine synthesis. The rates of alanine and glutamine synthesis and release from muscle decreased significantly during prolonged incubation despite a constant rate of glucose uptake and pyruvate production. Alanine synthesis and release were decreased by aminooxyacetic acid, an inhibitor of alanine aminotransferase. This inhibition was accompanied by a compensatory increase in the release of other amino acids, such as aspartate, an amino acid which was not otherwise released in appreciable quantities by muscle. The release of alanine, pyruvate, glutamate, and glutamine were observed to be interrelated events, reflecting a probable near-equilibrium state of alanine aminotransferase in skeletal muscle. It is concluded that glucose metabolism and amino acid release are functionally independent processes in skeletal muscle. Alanine release reflects the de novo synthesis of the amino acid and does not arise from the selective proteolysis of an alanine-rich storage protein. It appears that the rate of alanine and glutamine synthesis in skeletal muscle is dependent upon the transformation and metabolism of amino acid precursors.

Acute and chronic uremia are associated with variable degrees of myopathy, a retardation in growth, and a reduction in lean body mass (1–3). With time, both the upper and lower extremities may demonstrate diffuse muscle atrophy and weakness (3). These data suggest that muscle protein catabolism may be increased, synthesis decreased, or both may coexist in uremia. Significant changes in plasma amino acids occur in both acute and chronic uremia. The plasma phenylalanine to tyrosine ratio is reduced and plasma non-essential amino acid levels are usually normal while the levels of essential amino acids including leucine, isoleucine, valine, and lysine are decreased (4, 5). These data suggest that significant abnormalities in protein metabolism do exist in uremia. Uremic rats fed identical diets exhibit significantly reduced growth rates compared to pair-fed control animals (6). Despite increased protein intake, growth was still impaired in the uremic animals. Thus, these data demonstrate that protein efficiency is reduced in the growing uremic animal. The mechanisms responsible for these observations have not been definitively elucidated.

To determine whether the differences in fasting glucose responses in men, women and children could be related to differences in glucoregulatory hormone secretion or availability of circulating gluconeogenic substrates, 20 adults (10 men and 10 women) were fasted for 86 hr and 15 children (6.1 +/- 0.8 yr, mean +/- SE) were fasted for 30 hr. Circulating concentrations of glucose, ketone bodies, potential gluconeogenic substrates and glucoregulatory hormones were determined at frequent intervals. In the postprandial state (1-14 hr fasting), substrate and hormone concentrations were similar in all groups with the exception of plasma glutamine concentrations which were higher in men (640 +/- 20 microM) than in women of children (490 +/- 20 microM and 480 +/- 50 microM, respectively, p less than 0.01 for both). Following 30 hr fasting children had the lowest glucose (53 +/- 3 mg/dl) and alanine (167 +/- 17 microM) concentrations and men had the highest (72 +/- 3 mg/dl and 279 +/- 16 microM, respectively) whereas those of women were intermediate (64 +/- 3 mg/dl and 226 +/- 19 microM. respectively). Following 30 hr of fasting betahydroxybutyrate concentrations were highest in children and lowest in men (the children, 3.7 +/- 0.4 mM; women, 1.7 +/- 0.2 mM and men 0.9 +/- 0.2 mM, p less than 0.02 between all groups). An inverse relationship (p less than 0.001 was observed between beta-hydroxybutyrate and glucose concentrations throughout the fast for each group (r greater than 0.92). Although plasma cortisol concentrations were higher in women and children when compared to those of the men, no differences in growth hormone, glucagon, or insulin concentrations were observed among the three groups studied. Differences in plasma substrate responses to fasting among the groups studied may reflect differences in glucose requirements among men, women and children, as well as the relative availability of both gluconeogenic substrate and ketone bodies.

Gluconeogenic competence of primary cultures of canine renal proximal tubular cells has been examined. Cells grown in 5 mM glucose media or in glucose-free media exhibited pyruvate-stimulated glucose production, as opposed to cells grown in 20 mM glucose media. By 72 h after the last media change, confluent cells grown in 5 mM glucose medium turn from a predominantly glycolytic to an oxidative type of metabolism. By this time, glucose production exhibited pH, 3-mercaptopicolinate, and insulin sensitivity. Parathyroid hormone, angiotensin II, and phenylephrine stimulated glucose production in a nonadditive fashion. Single-cell cystolic Ca2+ measurements using microspectrofluorometric techniques revealed that all three hormones elicited Ca2+ transients in proximal tubular cells. Ionomycin stimulated glucose production by proximal tubular cells, suggesting that Ca2+ transients could represent a sufficient stimulus for glucose production. When hormone-induced Ca2+ transients were curtailed by a pretreatment with the membrane-permeant Ca2+ chelator, Maptam, hormonal stimulation of glucose production was abolished, suggesting that Ca2+ transients represent not only a sufficient, but a necessary event in the stimulation of glucose production by these hormones.

Alanine and glutamine formation and release were studied using the intact epitrochlaris preparation of rat skeletal muscle. Alanine release from skeletal muscle was increased by fasting (65%), cortisone (145%), thyroxine (200%), and diabetes (185%). Glutamine release was decreased by cortisone (37%) and diabetes (23%) but not significantly affected by fasting or thyroxine. Tissue levels of alanine were unchanged but tissue glutamine levels were markedly reduced (30 to 60%) in all treatment groups. Insulin added in vitro did not affect amino acid release even with preparations obtained from diabetic animals. Inhibition of glycolysis with 0.2 mM iodoacetate had no effect on the rate of alanine and glutamine formation in any treatment group. Pyruvate generation was increased by all treatments even in the presence of the inhibitor. Total skeletal muscle alanine, aspartate, and branched chain aminotransferase, glutamate dehydrogenase, and malic enzyme activities were not significantly altered in any treatment groups. The addition of 10 mM aspartate, cysteine, branched chain amino acids, and serine significantly increased alanine formation, whereas the maximal rate of glutamine formation in the presence of stimulating amino acids was reduced in each treatment groups--the most marked effects were noted with cortisone and diabetic preparations. Although accelerated muscle proteolysis is an important factor regulating alanine formation in skeletal muscle, the redirection of carbon flow from glutamine toward alanine formation observed in fasting, cortisone, thyroxine-treated, and diabetic rats, indicates that factors other than proteolysis also participate in the control of amino acid release from muscle.

Injury is attended by accelerated skeletal muscle proteolysis. Accurate definition of this hypercatabolic response and its mediation is requisite for specific therapy. We measured protein dynamics in the incubated and intact epitrochlearis and soleus muscles excised from both forelimbs and both hindlimbs of rats 4 days after injury by either a single hind limb scald (90 degrees C water for 3 seconds; metabolic rate (MR) + 15%, urinary urea nitrogen (UUN) + 10%) or a 5% excision (dorsal skin removed to fascia; MR + 40%, UUN + 90%). Protein synthesis (3H phenylalanine incorporation) increased only in the injured soleus from the scalded hind limb (+100%). Actin and myosin breakdown (3-methylhistidine release) increased in all muscles tested and was consistently larger in epitrochlearis than in soleus muscles. Breakdown of the mixed protein pool (tyrosine release) increased but less so than 3-methylhistidine and did not reach significance in the uninjured soleus muscle of scalded rats. With respect to fiber type, white fiber epitrochlearis muscle demonstrated a more pronounced elevation of both measures of breakdown but at a lower metabolic rate than did red fiber soleus muscle. Increasing MR was associated with a linear increase in soleus proteolysis but no further change in epitrochlearis breakdown. We conclude that protein breakdown is increased in skeletal muscle distant from injury; however, even when metabolic stress is severe, synthesis is unchanged. Muscles of different fiber composition are not equally labile. Furthermore, myofibrillar protein is more labile than the mixed protein pool.

Long-term islet-transplanted rats were examined for their ability to secrete insulin and glucagon and maintain normal glucose homeostasis under various experimental conditions. Normal glucose tolerance and insulin secretion were noted in nonstressed transplanted animals after oral glucose loading, whereas the intravenous administration of glucose resulted in a prompt but reduced insulin release associated with a slower return to basal glucose concentrations. Both the insulin and glucagon responses of nonstressed transplanted rats to arginine were comparable to those seen in normal animals. In transplant rats, stress severely impaired glucose homeostasis, as indicated by a marked rise in plasma glucose and a significant decrease in plasma insulin under basal conditions. These abnormalities were prevented by either alpha-adrenergic blockade or adrenalectomy. Stress also severely impaired both oral and intravenous glucose tolerance tests in transplant animals and markedly inhibited insulin secretory responses to tolbutamide and theophylline. Control and transplant rats exhibited comparable plasma epinephrine and norepinephrine values under basal conditions and in response to stress. The stress-induced impairment in glucose homeostasis of transplant rats was attributed to an increased sensitivity of the transplanted A- and B-cells to catecholamines secondary to their denervated state. Transplanted islet cells exhibited appropriate adaptive responses to chronic modulating factors such as a 48-hour fast and the administration of cortisol. However, cortisol-treated rats were unable to sustain a sufficient secretion of insulin to maintain normal carbohydrate tolerance, and consequently there was a reappearance of the diabetic state.

Alanine and glutamine formation and release were studied using the intact epitrochlaris preparation of rat skeletal muscle. Epinephrine reduced the release of alanine and glutamine in a concentration-dependent manner. Measurable inhibition was observed at 10(-9) M epinephrine, and maximal inhibition was obtained at 10(-5) M. Norepinephrine also reduced alanine and glutamine formation and release but the concentration required for maximal inhibition was approximately 100-fold greater than for epinephrine. Isoproterenol (beta agonist), but not phenylephrine (alpha agonist), reproduced the effects of epinephrine, and propranolol (beta antagonist), but not phentolamine (alpha antagonist), blocked the effect of the catecholamine. N6,O2'-Dibutyryl adenosine 3':5'-monophosphate reproduced the effects of epinephrine and theophylline potentiated the effect of submaximal concentrations of the hormone. Glucagon and prostaglandin E2 had no observable effect on amino acid release. Insulin did not modify the inhibition of alanine and glutamine release produced by epinephrine. Alanine and glutamine formation from added precursor amino acids was unaffected by epinephrine or cyclic adenosine 3':5'-monophosphate. Epinephrine reduced alanine formation in muscles obtained from diabetic rats or animals treated with thyroxine or cortisone. These findings indicate that physiological levels of catecholamines reduce alanine and glutamine formation and release from skeletal muscle. This effect is mediated by a beta-adrenergic receptor and the adenylate cyclase system and can be accounted for by an inhibition of muscle protein degradation.

Effects of insulin on glycogen synthesis (GS), glycolytic utilization (GU), and glucose uptake (GT) were studied in isolated epitrochlearis muscles from exercise-trained or sedentary rats during recovery from acute exercise or at rest. During the 1st h after acute exercise, the enhanced basal and insulin-stimulated GT was directed mainly toward replenishment of glycogen but basal GU was also increased. During the second through third hours after exercise, basal GS decreased but remained greater than rest and basal GU and GT returned to normal. Insulin sensitivity of these parameters was enhanced. Training alone reduced basal GS but enhanced insulin sensitivity of GT and GU. Training reduced the acute exercise-stimulated increase in basal and insulin sensitivity of GS during recovery from acute exercise, probably due to elevated glycogen stores. Thus recovery from acute exercise or training, either alone or in combination, enhances insulin stimulated GT in muscle; however, the increased glucose is primarily channeled toward GS after acute exercise, which is reduced by prior training and is directed to GU in trained animals either at rest or after acute exercise.

Free fatty acid (FFA) transport was measured in 11 and glycerol turnover in 5 newborns with continuous tracer infusion of [1-(13)C]palmitate or [2-(13)C]glycerol, respectively. In addition, simultaneous determination of glucose production in the latter group with [6,6-(2)H(2)]glucose tracer and measurement of the appearance rate of [(13)C]glucose derived from [(13)C]glycerol allowed calculation of gluconeogenesis from glycerol.The average FFA inflow rate was 11.5+/-1.7 mumol kg(-1)min(-1), 2.5-4.5 h after the last feeding, and 16.7+/-2.8 mumol kg(-1)min(-1), 5-12 h after the last meal. These rates are comparable to those found in adults only after 8-16 h and approximately 72 h of fasting, respectively. FFA inflow in the newborn was directly correlated with time of fasting, plasma FFA level, and plasma glycerol level. Palmitate clearance and fractional removal were inversely related to palmitate level. Glycerol flux averaged 4.4+/-0.5 mumol kg(-1)min(-1), a value three- to fourfold that of the postabsorptive adult. Approximately 75% of transported glycerol was converted to glucose and represented 5.0+/-0.6% of hepatic glucose production. Furthermore, there was a direct relationship between glycerol turnover and the fraction of glucose coming from glycerol. Despite the absolutely elevated neonatal FFA and glycerol transport rates, these were quantitatively similar to values found in adults with comparable elevated substrate levels. Furthermore, other similarities with the adult in the relationships between inflow transport and substrate values, and between transport and fractional removal suggest that the regulatory aspects of lipid transport in man are already well developed by the first day of life.

The synthesis and release of alanine and glutamine have been studied in the intact rat epitrochlaris skeletal muscle preparation. Aspartate, cysteine, leucine, valine, methionine, isoleucine, serine, theronine, and glycine increased significantly the formation and release of alanine from muscle. Cysteine, leucine, valine, methionine, isoleucine, tyrosine, lysine, and phenylalanine increased the rate of glutamine synthesis. Only ornithine, arginine, and tryptophan were without effect on the synthesis of either alanine or glutamine. Half-maximal stimulation of alanine and glutamine formation by added amino acids was observed with concentrations ranging between 0.5 and 1.0 mM. Increases in alanine and glutamine formation were not accompanied by changes in pyruvate production or glucose uptake. The progressive decline in alanine and glutamine synthesis noted on prolonged incubation was prevented by the addition of amino acids to the incubation medium. Stimulation of alanine synthesis by added amino acids was unaffected by inhibition of glycolysis with iodoacetate. Inhibition of alanine aminotransferase with aminooxyacetate significantly decreased alanine formation. Pyruvate and ammonium chloride did not increase further the rate of either alanine or glutamine formation above that produced by added amino acids. These data indicate that most amino acids are precursors for alanine and glutamine synthesis in skeletal muscle. A general mechanism is presented for the de novo formation of alanine from amino acids in skeletal muscle, and the importance of proteolysis for the supply of amino acid precursors for alanine and glutamine synthesis is discussed.

The effects of insulin and contraction on glucose transport and metabolism were investigated in rat epitrochlearis muscles in vitro. Insulin dose-response curves showed a threshold (approximately 50 microunits/ml) and saturation-type (approximately 1 mU/ml) kinetics, whereas isometric contraction activated glucose transport and metabolism in a linear fashion with no evidence of a threshold. Insulin and contraction increased the apparent maximal rate of uptake of the hexose transport system with minimal effect on its apparent Km. The stimulatory effects of insulin and contraction were additive; similar results were obtained with 2-deoxy-D-glucose. Contraction stimulated glucose transport in three different preparations of muscles depleted of insulin: 1) exhaustively washed for 2 h, 2) rats infused with anti-insulin serum, and 3) chronically (streptozotocin-induced) diabetic rats. Prostaglandin E2 augmented the effect of a submaximal concentration of insulin on glucose transport without exerting any effect by itself but had no effect on contraction-augmented glucose transport. It is concluded that insulin and contraction activate glucose transport and metabolism via independent mechanisms.

The effects of exercise training on muscle protein catabolism in uremia were studied in female rats. Rats made uremic by 3/4 nephrectomy were compared with sham-operated control female rats under conditions of exercise training by swimming or no exercise. The release of amino acids from epitrochlearis muscle in vitro was measured. Body weight epitrochlearis muscle weight, and epitrochlearis protein content were similar among groups. Uremia increased the release of phenylalanine and tyrosine 33% and alanine 50% from muscle of sedentary rats. Citrate synthase activity and glycogen content of muscle were increased twofold by exercise in both controls and uremics. Exercise increased the release of alanine (60%), glutamine (50%), and pyruvate (30%) from muscles of control rats, but it decreased to control levels the release of phenylalanine and tyrosine in uremic rats. Alanine release remained elevated. To determine if exercise training increases in vitro muscle sensitivity to insulin, we incubated muscle with and without 0.01 U/ml of insulin. Phenylalanine and tyrosine release was reduced minimally by insulin in both sedentary uremic and control groups. Glucose uptake was enhanced by 55% in both groups. But in the exercised uremic and control groups, insulin reduced phenylalanine and tyrosine release by 50% and increased glucose uptake by 75%. These results suggest that exercise training reduces muscle protein catabolism in uremia; and this reduction is associated with enhanced muscle response to pharmacologic doses of insulin in control and uremic rats.

An in vitro rat muscle preparation is described that can contract at rates of 12–240 twitches/min. Maximum dF/dt paralleled maximum twitch tension, their ratio being constant at approximately 8 ms for contraction rates of 12-120 twitches/min. Time to peak tension was 8–13 ms, time to peak dF/dt 5–8 ms, and half-relaxation time 4 ms. These parameters were unaffected by rate of contraction or duration of isometric work. Differential ATPase staining demonstrated that 60–65% of the fibers were fast-twitch white, 20% fast-twitch red, and 15% slow-twitch red. The preponderance of fast-twitch fibers correlated with the observed mechanical performance of the muscle. Muscles contracting for 60 min at rates up to 48 twitches/min maintained total adenine nucleotide content (ATP, ADP, AMP) at near resting levels. At higher twitch rates (72–240 twitches/min), total adenine nucleotide content decreased 40%, reflecting exclusively a fall in ATP in the presence of adequate phosphocreatine stores. Adequate oxygenation was reflected by lactate-to-pyruvate ratios in the range of 11–15 at all rates of contraction.

Chronic renal failure is associated with an enhanced catabolism of muscle protein. To determine the effect of exercise training and moderate renal insufficiency on net protein catabolism and protein synthesis in isolated epitrochlearis muscles, three-fourth nephrectomized and control rats were exercise trained or remained sedentary. Net muscle protein degradation was determined by measuring the rates of release of phenylalanine and tyrosine. Protein synthesis was determined by measuring the incorporation of [U-14C]phenylalanine into muscle protein. Exercise training reduced the elevated protein degradation of uremia to control levels. In control rats, exercise training had no effect on protein degradation. Exercise training increased alanine release in control rats but did not further increase the elevated alanine release of uremia. Protein synthesis was unaffected by both uremia and exercise training. Exercise training in control and uremic rats moderately increased the responsiveness of muscle to insulin by reducing net protein degradation but did not further enhance the insulin-stimulated increase in protein synthesis. Thus exercise training ameliorates the enhanced muscle protein degradation of moderate renal insufficiency.