Sequential changes in in vivo muscle and liver protein synthesis and plasma and tissue glutamine levels in sepsis in the rat

We have investigated sequential changes in skeletal muscle and hepatic protein synthesis following sepsis, and their relationship to changes in circulating and tissue glutamine concentrations. Male Wistar rats underwent caecal ligation and puncture (CLP) or sham operation, with starvation, and were killed 24, 72 or 96 h later. A group of non-operated animals were killed at the time of surgery. Protein synthesis was determined using a flooding dose of L-[4-(3)H] phenylalanine, and glutamine concentrations were measured by an enzymic fluorimetric assay. Protein synthesis in gastrocnemius muscle fell in all groups. Gastrocnemius total protein content was reduced after CLP and at 72 and 96 h after sham operation. After CLP, protein synthesis was lower at 24 h, and total protein content was lower at 72 and 96 h, than in sham-operated animals. CLP was associated with increased liver protein synthesis at all time points, whereas there was no change after sham operation. Liver protein content did not change after CLP, but was lower at 72 and 96 h after sham operation than in non-operated animals. Plasma glutamine concentrations were reduced at 24 h after sham operation, and at 72 and 96 h after CLP. Muscle glutamine concentrations were reduced in all groups, with the decrease being greater following CLP than after sham operation. In the liver, glutamine concentrations were unchanged after CLP, but increased after sham operation. In rats with sepsis, decreases in muscle protein synthesis and content are associated with markedly reduced muscle glutamine concentrations. Plasma glutamine concentrations are initially maintained, but fall later. In liver, protein synthesis is increased, while glutamine concentrations are preserved. These results support a peripheral-to-splanchnic glutamine flux in sepsis.     

Platelet-activating factor (PAF)-induced decreases in whole-body and skeletal muscle protein synthesis

Platelet-activating factor (PAF) has been shown to reduce rat skeletal muscle amino acid uptake, which may restrict intracellular amino acid availability for protein synthesis and amino acid oxidation during endotoxemia. We investigated in rats the effect of PAF infusion on amino acid and protein metabolism by measuring (a) whole-body and tissue leucine kinetics; (b) plasma amino acid profile; and (c) muscle RNA activity (protein synthesis efficiency) and relative abundance of myofibrillar proteins. Fasted male Sprague-Dawley rats (250+/-20 g) were given a 4-h i.v. continuous infusion of L-(1-14C)-leucine to determine leucine kinetics during the infusion of PAF (2 microg/kg PAF as a priming i.v. bolus 1 h before a 4-h i.v. infusion of 2 microg/kg/h PAF) or vehicle. PAF infusion caused sustained hypotension, hyperglycemia, hematological alterations, and hyperlacticacidemia. Whole-body protein synthesis was decreased by 24% (P < 0.05) and leucine flux oxidized was increased by 23% (P < 0.05). Leucine flux was reduced, although not significantly (P = 0.07), in PAF-treated rats (n = 8) compared with controls (n = 8). PAF significantly decreased fractional protein synthesis in the rectus abdominus (33%), soleus (30%), and extensor digitorum longus (26%) muscles, but not in the liver. Plasma branched-chain amino acid levels decreased (approximately 30%, P < 0.05) in PAF-treated rats. Muscle RNA activity was 32% lower and myosin relative abundance declined whereas actin was unchanged in PAF-treated rats. PAF induced net protein catabolism as a result of elevated leucine oxidation at the expense of protein synthesis. PAF had the cumulative effects in the skeletal muscle of (a) attenuating amino acid uptake, (b) reducing protein synthesis efficiency, (c) decreasing fractional protein synthesis rate, and (d) decreasing myosin relative abundance. Thus, PAF may be an important mediator of decreased protein synthesis in skeletal muscle during endotoxic and septic shock.     

Vitamin D Deficiency, hypocalcemia, and increased skeletal muscle degradation in rats.

The myopathy associated with vitamin D deficiency was examined in vitamin D-deficient and vitamin D-supplemented rats. When compared with either vitamin D-supplemented ad lib. or pair-fed rats, weight gain and muscle mass were decreased in vitamin D-deficient hypocalcemic animals. With the exception of a modest decrease in muscle creatine phosphate levels, muscle composition was unchanged by vitamin D deficiency. Muscle protein turnover rates were determined in both in vivo and in vitro studies and demonstrated that myofibrillar protein degradation was increased in vitamin D deficiency. Normal growth rates could be maintained be feeding the rats vitamin D-deficient diets containing 1.6% calcium, which maintained plasma calcium within the normal range. In addition to its role in maintaining plasma calcium, vitamin D-supplemented rats had significantly higher levels of the anabolic hormone insulin. Vitamin D supplementation may affect muscle protein turnover by preventing hypocalcemia, as well as directly stimulating insulin secretion, rather than by a direct effect within skeletal muscle.     

Short-term amino acid infusion improves protein balance in critically ill patients

IntroductionEvidence behind the recommendations for protein feeding during critical illness is weak. Mechanistic studies are needed to elucidate the effects of amino acid and/or protein supplementation on protein metabolism before larger clinical trials with higher levels of protein feeding are initiated.MethodsWe studied the effects of parenteral amino acid supplementation (equivalent to 1 g/kg/day) over the course of 3 hours on whole-body protein turnover in critically ill patients in the intensive care unit (ICU) during the first week after admission. Patients were studied at baseline during ongoing nutrition and during extra amino acid supplementation. If the patient was still in the ICU 2 to 4 days later, these measurements were repeated. Protein kinetics were measured using continuous stable isotope-labeled phenylalanine and tyrosine infusions.ResultsThirteen patients were studied on the first study occasion only, and seven were studied twice. Parenteral amino acid supplementation significantly improved protein balance on both occasions, from a median of −4 to +7 μmol phenylalanine/kg/hr (P =0.001) on the first study day and from a median of 0 to +12 μmol phenylalanine/kg/hr (P =0.018) on the second study day. The more positive protein balance was attributed to an increased protein synthesis rate, which reached statistical significance during the first measurement (from 58 to 65 μmol phenylalanine/kg/hr; n =13; P =0.007), but not during the second measurement (from 58 to 69 μmol phenylalanine/kg/hr; n =7; P =0.09). Amino acid oxidation rates, estimated by phenylalanine hydroxylation, did not increase during the 3-hour amino acid infusion. A positive correlation (r =0.80; P <0.0001) was observed between total amino acids and/or protein given to the patient and whole-body protein balance.ConclusionExtra parenteral amino acids infused over a 3-hour period improved whole-body protein balance and did not increase amino acid oxidation rates in critically ill patients during the early phase (first week) of critical illness.

Electronic supplementary material

The online version of this article (doi:10.1186/s13054-015-0844-6) contains supplementary material, which is available to authorized users.     

Effect of growth hormone on muscle and liver protein synthesis in septic rats receiving glutamine-enriched parenteral nutrition

OBJECTIVE: Administration of recombinant human growth hormone (rhGH) to critically ill adults in an attempt to attenuate catabolism was associated with increased morbidity and mortality. Possible explanations included inhibition of glutamine release from skeletal muscle and consequent restriction of splanchnic glutamine supply. In this study, we examined the effects of rhGH on plasma glutamine levels and on muscle and liver glutamine concentrations and protein synthesis rates in sepsis. We investigated the possibility that administration of supplemental glutamine might ameliorate any adverse effects of rhGH. DESIGN: Prospective study in rats rendered septic by cecal ligation and puncture. SETTING: University hospital laboratory. SUBJECTS: A total of 78 male Wistar rats in six groups. INTERVENTIONS: Animals received 6-hr tail vein infusions, commencing 18 hrs after cecal ligation and puncture, of either (a) 0.9% sodium chloride, (b) a standard parenteral nutrition (PN) solution without glutamine, or (c) an isocaloric, isonitrogenous PN solution with glutamine. PN groups received 400 microg rhGH or equivolume 0.9% sodium chloride vehicle in a divided subcutaneous and intravenous dose at PN commencement. Sacrifice was at the end of the infusion period. A further group was unoperated and uninfused and killed at 24 hrs as baseline controls. MEASUREMENTS AND MAIN RESULTS: Glutamine concentrations were measured by fluorometry. Protein synthesis in muscle and liver was measured by a "flooding-dose" technique employing L-[4-H]phenylalanine. Plasma glutamine was increased after cecal ligation and puncture except in the saline and glutamine with rhGH animals. Muscle glutamine was reduced after cecal ligation and puncture and was significantly lower in animals receiving standard PN with rhGH vs. saline alone. Liver glutamine was increased in animals receiving saline and those receiving standard PN with rhGH. PN, with or without glutamine, increased muscle protein synthesis, and the administration of rhGH tended to further increase this effect. Neither PN, glutamine, nor rhGH had an effect on the increased liver protein synthesis characteristic of sepsis. CONCLUSIONS: In sepsis, increased muscle protein synthesis with PN and rhGH administration is not associated with increased muscle glutamine levels. Administration of rhGH does not result in reduced liver glutamine levels or rates of hepatic protein synthesis. PN containing glutamine was no more efficacious than standard PN at increasing muscle protein synthesis.     

Deficiency in peripheral glutamine production in pediatric patients with burns

Plasma glutamine levels decrease in association with severe injury, which suggests that the consumption of glutamine exceeds the production of glutamine or possibly represents a deficit in the release of glutamine from skeletal muscle. The goal of this study was to assess the peripheral glutamine kinetic response to prolonged stress in children with critical injuries. To accomplish this purpose, we quantitated peripheral glutamine kinetics in vivo with the use of 5N15 glutamine in 5 children with severe burns (total body surface area, 74%+/-14%; mean +/- SEM) and 3 children who underwent elective scar reconstruction. In the children with severe burns, leg blood flow was significantly elevated (16.2+/-2.1 vs 7.5 +/-0.3 mL/min/100 mL leg volume, P < .02) and the arterial concentration of glutamine was significantly reduced (0.31+/-0.04 vs 0.84+/-0.05 mmol/L, P < .001). The rate of glutamine turnover within the leg was significantly reduced in the patients with acute burns, whereas the net efflux of glutamine was similar between the 2 groups. These findings suggest that plasma glutamine concentrations decrease during severe stress as a result of a deficit in peripheral glutamine release in conjunction with an increased central consumption. This preliminary study supports the notion that exogenous glutamine supplementation in pediatric patients with severe injuries may be needed because of this inadequate skeletal muscle response.     

Effect of infused branched-chain amino acids on muscle and whole-body amino acid metabolism in man

1. Using the forearm balance method, together with systemic infusions of L-[ring-2,6-3H]phenylalanine and L-[1-14C]leucine, we examined the effects of infused branched-chain amino acids on whole-body and skeletal muscle amino acid kinetics in 10 postabsorptive normal subjects; 10 control subjects received only saline. 2. Infusion of branched-chain amino acids caused a four-fold rise in arterial branched-chain amino acid levels and a two-fold rise in branched-chain keto acids; significant declines were observed in circulating levels of most other amino acids, including phenylalanine, which fell by 34%. Plasma insulin levels were unchanged from basal levels (8 +/- 1 mu-units/ml). 3. Whole-body phenylalanine flux, an index of proteolysis, was significantly suppressed by branched-chain amino acid infusion (P less than 0.002), and forearm phenylalanine production was also inhibited (P less than 0.03). With branched-chain amino acid infusion total leucine flux rose, with marked increments in both oxidative and non-oxidative leucine disposal (P less than 0.001). Proteolysis, as measured by endogenous leucine production, showed a modest 12% decrease, although this was not significant when compared with saline controls. The net forearm balance of leucine and other branched-chain amino acids changed from a basal net output to a marked net uptake (P less than 0.001) during branched-chain amino acid infusion, with significant stimulation of local leucine disposal. Despite the rise in whole-body non-oxidative leucine disposal, and in forearm leucine uptake and disposal, forearm phenylalanine disposal, an index of muscle protein synthesis, was not stimulated by infusion of branched-chain amino acids. 4. The results suggest that in normal man branched-chain amino acid infusion suppresses skeletal muscle proteolysis independently of any rise of plasma insulin. Muscle branched-chain amino acid uptake rose dramatically in the absence of any apparent increase in muscle protein synthesis, as measured by phenylalanine disposal, or in branched-chain keto acid release. Thus, an increase in muscle branched-chain amino acid concentrations and/or local branched-chain amino acid oxidation must account for the increased disposal of branched-chain amino acids.     

Effects on skeletal muscle of intravenous glutamine supplementation to ICU patients

Objective: To evaluate the effect of four doses of intravenous glutamine supplementation on skeletal muscle metabolism. Design: A prospective, blinded, randomized study. Setting: The general Intensive Care Unit (ICU) of a university hospital. Patients: ICU patients with multiple organ failure (n=40), who were expected to stay in the unit for more than five days. Intervention: Patients received 0, 0.28, 0.57 or 0.86 g of glutamine per kg bodyweight per day intravenously for five days as part of an isocaloric, isonitrogenous and isovolumetric diet. Results: Plasma glutamine concentration responded to glutamine supplementation with normalization of plasma levels in a dose-dependent way, while free muscle glutamine concentration, as well as muscle protein synthesis and muscle protein content, did not change significantly. Conclusion: Intravenous glutamine supplementation to ICU patients for a period of five days resulted in normalization of plasma glutamine concentrations in a dose-dependent way whereas muscle glutamine concentrations were unaffected.     

Chronic alpha-tocopherol supplementation in rats does not ameliorate either chronic or acute alcohol-induced changes in muscle protein metabolism

Chronic alcohol muscle disease is characterized by reduced skeletal muscle mass precipitated by acute reduction in protein synthesis. The pathogenic mechanisms remain obscure, but several lines of evidence suggest that increased oxidative stress occurs in muscle in response to alcohol and this may be associated with impaired alpha-tocopherol status. Potentially, this implies a therapeutic role for alpha-tocopherol, especially as we have shown that supplemental alpha-tocopherol may increase the rate of protein synthesis in normal rats [Reilly, Patel, Peters and Preedy (2000) J. Nutr. 130, 3045-3049]. We investigated the therapeutic effect of alpha-tocopherol on plantaris muscle protein synthesis in rats treated either acutely, chronically or chronically+acutely with ethanol. Protein synthesis rates were measured with a flooding dose of L-[4-(3)H]phenylalanine. Protein, RNA and DNA contents were determined by standard laboratory methods. Ethanol caused defined metabolic changes in muscle, including decreased protein, RNA and DNA contents in chronically treated rats. In acute or chronic+acute studies, ethanol suppressed fractional rates of protein synthesis. alpha-Tocopherol supplementation did not ameliorate the effects of either acute, chronic or chronic+acute alcohol on plantaris muscle protein content or rates of protein synthesis. In control animals (not treated with alcohol), alpha-tocopherol supplementation decreased muscle protein content owing to increases in protein turnover (both synthesis and degradation). alpha-Tocopherol supplementation is not protective against the deleterious effects of alcohol on protein metabolism in skeletal muscle.     

Intracellular amino acid concentrations in the musculature of 3 species of animals under physiologic, pathophysiologic and infusion therapy conditions

In the first part of this paper methodological aspects of intracellular amino-acid analysis in three species of animals, i.e. in rats, rabbits, and cats are dealt with. The genetic homogeneity as well as the age of the animal groups, metabolic differences between carnivores and herbivores, the length of the intestine and, thus, the time elapsing before the postabsorptive state is reached, furthermore, the body size which, in studies with muscle biopsies, makes correlated samples either possible or impossible; all these factors are experimental variables potentially affecting the evidence of the results obtained. Amino acids being involved in ammonia detoxification or showing raised plasma levels in hepatic failure form the subject of the subsequent parts of this paper. In the muscle cells of the three above species investigated after an overnight fast, the concentration of glutamine was about 5.5 times and that of glutamate about 4.1 times higher than in human muscle. Regarding glutamine and glutamate, there were no marked differences between the three species. As to the other amino acids measured in the rats, rabbits, and cats, alanine, the branched-chain amino acid (BCAA) as well as methionine and phenylalanine had intracellular values similar to those observed in humans. To test the hypothesis that ammonia lowers the BCAA plasma levels by considerably increasing glutamine synthesis and decreasing the intracellular glutamate pool with consecutively intensified transamination of BCAA for partial replenishment of this pool, we infused ammonium salts and created portocaval shunts in animal experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin and insulin-like growth factor-I enhance human skeletal muscle protein anabolism during hyperaminoacidemia by different mechanisms.

Insulin inhibits proteolysis in human muscle thereby increasing protein anabolism. In contrast, IGF-I promotes muscle protein anabolism principally by stimulating protein synthesis. As increases or decreases of plasma amino acids may affect protein turnover in muscle and also alter the muscle's response to insulin and/or IGF-I, this study was designed to examine the effects of insulin and IGF-I on human muscle protein turnover during hyperaminoacidemia. We measured phenylalanine balance and [3H]-phenylalanine kinetics in both forearms of 22 postabsorptive adults during a continuous [3H] phenylalanine infusion. Measurements were made basally and at 3 and 6 h after beginning a systemic infusion of a balanced amino acid mixture that raised arterial phenylalanine concentration about twofold. Throughout the 6 h, 10 subjects received insulin locally (0.035 mU/min per kg) into one brachial artery while 12 other subjects were given intraaterial IGF-I (100 ng/min per kg) to raise insulin or IGF-I concentrations, respectively, in the infused arm. The contralateral arm in each study served as a simultaneous control for the effects of amino acids (aa) alone. Glucose uptake and lactate release increased in the insulin- and IGF-I-infused forearms (P < 0.01) but did not change in the contralateral (aa alone) forearm in either study. In the aa alone arm in both studies, hyperaminoacidemia reversed the postabsorptive net phenylalanine release by muscle to a net uptake (P < 0.025, for each) due to a stimulation of muscle protein synthesis. In the hormone-infused arms, the addition of either insulin or IGF-I promoted greater positive shifts in phenylalanine balance than the aa alone arm (P < 0.01). With insulin, the enhanced anabolism was due to inhibition of protein degradation (P < 0.02), whereas IGF-I augmented anabolism by a further stimulation of protein synthesis above aa alone (P < 0.02). We conclude that: (a) hyperaminoacidemia specifically stimulates muscle protein synthesis; (b) insulin, even with hyperaminoacidemia, improves muscle protein balance solely by inhibiting proteolysis; and (c) hyperaminoacidemia combined with IGF-I enhances protein synthesis more than either alone.     

Amino acid and protein metabolism in critically ill patients

This study set out to investigate the alteration of amino acid (AA) and protein metabolism in patients with malnutrition, sepsis, acute pancreatitis and liver diseases. The results showed that in preoperative patients with malnutrition or protein catabolism (decreased levels of plasma proteins, increased urea production rate) the postoperative complications were significantly increased. An increased postoperative infusion of branched chain AA did not improve postoperative nitrogen retention nor plasma protein syntheses in patients with colon or rectum CA. Patients with sepsis or acute pancreatitis had drastically reduced levels of total muscular free AA, mainly due to a fall in muscle glutamine. In septic patients also the hepatic levels of free AA were decreased. These changes of AA metabolism found in clinical situation were not always reflected by results found in experimental rat models (sepsis, pancreatitis, burn injury). The parenteral administration of a synthetic dipeptide containing glutamine and alanine decreased the muscular decrease of glutamine and alanine and increased the hepatic uptake of these two AA in a catabolic dog model. In critically ill patients changes in amino acid and protein metabolism lead to a protein catabolic situation. Urea production rate and muscle glutamine levels seem to be closely related to the prognosis of catabolic patients.     

Glutamine attenuates post-traumatic glutathione depletion in human muscle

Glutathione is quantitatively the most important endogenous scavenger system. Glutathione depletion in skeletal muscle is pronounced following major trauma and sepsis in intensive care unit patients. Also, following elective surgery, glutathione depletion occurs in parallel with a progressive decline in muscle glutamine concentration. The present study was designed to test the hypothesis that glutamine supplementation may counteract glutathione depletion in a human trauma model. A homogeneous group of patients (n = 17) undergoing a standardized surgical procedure were prospectively randomly allocated to receive glutamine (0.56 g x day(-1) x kg(-1)) or placebo as part of isonitrogenous and isocaloric nutrition. Percutaneous muscle biopsies and blood samples were taken pre-operatively and at 24 and 72 h after surgery. The concentrations of muscle glutathione and related amino acids were determined in muscle tissue and plasma. In the control (unsupplemented) subjects, total muscle glutathione had decreased by 47+/-8% and 37+/-11% and reduced glutathione had decreased by 53+/-10% and 45+/-16% respectively at 24 and 72 h after surgery (P < 0.05). In contrast, in the glutamine-supplemented group, no significant post-operative decreases in total or reduced glutathione were seen following surgery. Muscle free glutamine had decreased at 72 h after surgery in both groups, by 41.4+/-14.8% (P < 0.05) in the glutamine-supplemented group and by 46.0+/-14.3% (P < 0.05) in the control group. In conclusion, the present study demonstrates that intravenous glutamine supplementation attenuates glutathione depletion in skeletal muscle in humans following standardized surgical trauma.     

Aspects of protein metabolism after elective surgery in patients receiving constant nutritional support

1. The present study was designed in an attempt to resolve conflicting views currently in the literature relating to the effect of surgery on various aspects of protein metabolism. 2. Sequential post-operative (2, 4 and 6 days) changes in whole-body protein turnover, forearm arteriovenous difference of plasma amino acids, glucose, lactate and free fatty acids, muscle concentration of free amino acids, RNA and protein, urinary nitrogen and 3-methylhistidine, plasma concentrations of insulin, cortisol and growth hormone, and resting metabolic rate, were measured in six patients undergoing uncomplicated elective total abdominal hysterectomy. 3. All patients received a constant daily diet, either orally or intravenously, based on 0.1 g of nitrogen/kg and an energy content of 1.1 times the resting metabolic rate for 7 days before and 6 days after surgery. 4. Whole-body protein turnover, synthesis and breakdown increased significantly 2 days after surgery (P less than 0.05) and returned towards pre-operative levels thereafter. 5. Forearm release of branched-chain amino acids and alanine, and efflux of glucose and lactate, were enhanced 4 days after surgery (P less than 0.05). Muscle glutamine and alanine concentrations were decreased on the fourth and sixth days after surgery (P less than 0.05). The RNA/protein ratio (indicating the capacity for protein synthesis) was unaltered. 6. A significant increase in urinary nitrogen and 3-methylhistidine was observed on days 3 and 4 after surgery (P less than 0.05). Thereafter, these parameters remained elevated, although failing to reach statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of amino acid infusion on leg protein turnover assessed by L-[15N]phenylalanine and L-[1-13C]leucine exchange

A stable isotope technique depending on the use of [15N]phenylalanine and [1-13C]leucine to assess exchange was utilized to measure the components of protein turnover of the human leg and the effects of amino acid infusion. Eight healthy subjects (28.5 +/- 2.5 years) were studied when post-absorptive in the basal state and again during infusion of a mixed amino acid solution (55 g l-1, 1.52 ml kg-1 h-1). During the basal period leucine oxidation by the leg was 4.4 +/- 2.0 nmol 100 g-1 min-1 and this increased threefold during amino acid infusion (13.6 +/- 3.1 nmol 100 g-1 min-1, mean +/- SEM, P = 0.003). Amino acid infusion abolished the net negative balance between incorporation of leucine into, and release from, protein (basal, -31.8 +/- 5.8; during infusion, +3.1 +/- 7.1 nmol 100 g-1 P = 0.001). Phenylalanine exchange showed a similar pattern (basal, -13.7 +/- 1.8; during infusion, -0.8 +/- 3.0 nmol 100 g-1 min-1, P = 0.003). Basal entry of leucine into leg protein (i.e. protein synthesis) was 70.0 +/- 10.8 nmol 100 g-1 min-1 and this increased during amino acid infusion to 87.3 +/- 14.1 nmol 100 g-1 min-1 (P = 0.11). Phenylalanine entry to protein also increased with amino acid infusion (29.1 +/- 4.5 vs. 38.3 +/- 5.8 nmol 100 g-1 min-1, P = 0.09). Release from protein of leucine (101.8 +/- 9.1 vs. 84.2 +/- 9.1 nmol 100 g-1 min-1, P = 0.21) and of phenylalanine (42.8 +/- 4.2 vs. 39.1 +/- 4.2 nmol 100 g-1 min-1, P = 0.50) was unchanged by amino acid infusion. The results suggest that, in the post-absorptive state in man, infusion of mixed amino acids, without additional energy substrates; reverses negative amino acid balance by a mechanism which includes stimulation of muscle protein synthesis but which does not alter protein breakdown. Interpretation of the results obtained concurrently on whole-body protein turnover suggests that the increase in muscle protein synthesis contributes substantially to the whole-body increase, but the fall in whole-body breakdown with exogenous amino acids is independent of changes in muscle.     

Protein metabolism in insulin-treated gestational diabetes.

OBJECTIVE: To test the hypothesis that protein metabolism is not totally normalized in insulin treated gestational diabetes mellitus (GDM) patients compared with normal, pregnant control subjects. RESEARCH DESIGN AND METHODS: Protein metabolism in eight Hispanic women with insulin-treated GDM and eight healthy Hispanic control women was studied in late gestation and at 6 weeks postpartum. Nitrogen flux was assessed from the disposal rate of [15N]-labeled urea over 12 h after a dose of [15N]-labeled leucine. Plasma amino acid concentrations were determined in fasting and 2-h postprandial samples using an amino acid analyzer. RESULTS: Protein turnover was normalized in insulin-treated GDM; however, fasting and postprandial plasma amino acids were elevated antepartum and postpartum. Nitrogen flux was significantly lower during pregnancy (P = 0.04-0.001) and did not differ between groups. Fasting and postprandial plasma amino acids were elevated in GDM antepartum and postpartum, despite satisfactory glycemic control. Fasting levels of taurine, hydroxyproline, glutamic acid, glutamine, cystine, tyrosine, phenylalanine, tryptophan, and histidine were higher in GDM antepartum and postpartum (P < 0.05). Postprandial concentrations of taurine, hydroxyproline, valine, cystine, isoleucine, leucine, tyrosine, phenylalanine, tryptophan, ornithine, lysine, histidine, and arginine were higher in GDM antepartum and postpartum (P < 0.05). With few exceptions, plasma amino acid concentrations were lower antepartum than postpartum (P < 0.05). CONCLUSIONS: Protein turnover was normalized in insulin-treated women with GDM; however, fasting and postprandial plasma concentrations of amino acids were elevated in the antepartum and postpartum periods, despite satisfactory maternal glycemic control.     

Intracellular free amino acids in muscle tissue of patients with chronic uraemia: effect of peritoneal dialysis and infusion of essential amino acids

1. Free amino acids were determined in the plasma and in the muscle tissue of 14 patients with chronic uraemia; eight were not on dialysis and six were having regular peritoneal dialysis. The concentration of each amino acid in muscle water was calculated with the chloride method. 2. In both groups of patients there were low intracellular concentrations of threonine, valine, tyrosine and carnosine, and high glycine/valine and phenylalanine/tyrosine ratios. Both groups of patients had increased amounts of 1- and 3-methyl-histidine in plasma and in muscle water. 3. The non-dialysed patients had low intracellular concentrations of lysine, and the dialysed patients had high intracellular concentrations of lysine, isoleucine, leucine and of some of the non-essential amino acids. 4. After peritoneal dialysis for 22 h, the plasma concentration of several amino acids decreased but the intracellular concentrations of most amino acids did not change significantly. 5. Intravenous administration of essential amino acids and histidine during the last 4 h of dialysis increased in muscle the total free amino acids, the ratio of essential to non-essential amino acids and the valine and phenylalanine concentrations. 6. The results demonstrated that the plasma and muscle concentrations of several amino acids are grossly abnormal in chronic uraemia. Non-dialysed and dialysed patients exhibit important differences, especially in the intracellular amino acid patterns. Infusion of essential amino acids may result in enhancement of protein synthesis.     

Efficiency of a cysteine-taurine-threonine-serine supplemented parenteral nutrition in an experimental model of acute inflammation

Objective As is the case with glutamine, requirements for amino acids such as cysteine, taurine, and serine may be increased in stress situations. This study evaluated the potential usefulness of supplementation of total parenteral nutrition with a cysteine, taurine, threonine, and serine mixture (SEAS), with or without glutamine, in an experimental model of turpentine-induced acute inflammation.Design and setting Prospective, controlled animal study in male Sprague-Dawley rats.Interventions Twenty-seven rats received isonitrogenous, isocaloric total parenteral nutrition (260 kcal/kg, 2 gN/kg per day) for 5 days. They were divided into three groups according to the composition of the amino acid admixture: standard amino acids (control, n=9), standard amino acids partly substituted with SEAS (n=10) or with SEAS and glutamine (n=8). All rats received two subcutaneous turpentine injections (0.5 ml/100 g) 24 h (day 2) and 72 h (day 4) after the initiation of parenteral nutrition and were killed on day 5.Measurements and results Nitrogen balance was significantly increased (control 53±29, SEAS 153±21, SEAS+Gln 187±32 mg/24 h) and urinary 3-methylhistidine/creatinine ratio decreased (control 55±4, SEAS 43±4, SEAS+Gln 38±3 µmol/mmol) on day 5 in the two SEAS-treated groups. Hepatic and extensor digitorum longus muscle protein contents were significantly higher in the SEAS+Gln-treated group than in the other two groups. In addition to slight differences in liver amino acid content, other parameters including liver glutathione did not differ significantly between groups.Conclusions Improved nitrogen balance and reduction in urinary 3-methylhistidine suggest that SEAS supplementation improves nitrogen homeostasis in an experimental model of acute inflammation. Glutamine addition further improves protein status.This work was supported in part by a grant from Baxter S.A., Maurepas, France     

Measurement of whole-body protein turnover in insulin-dependent (type 1) diabetic patients during insulin withdrawal and infusion: comparison of [13C]leucine and [2H5]phenylalanine methodologies

1. The aims of this study were twofold: (i) to investigate the ability of a recently described [2H5]phenylalanine method for quantifying whole-body protein turnover during acute physiological perturbation; (ii) to determine specifically whether the previously observed increase in protein synthesis on insulin withdrawal in insulin-dependent (type 1) diabetic patients seen when employing the [13C]leucine technique could be corroborated by using [2H5]phenylalanine. 2. Whole-body protein turnover was measured by both the [2H5]phenylalanine and [13C]leucine primed continuous infusion methods applied simultaneously to six type I post-absorptive diabetic patients during insulin withdrawal and infusion. 3. Values were determined by the [13C]leucine method by measuring either [13C]leucine (primary pool) or alpha-[13C] ketoisocaproic acid (reciprocal pool) enrichment in plasma. 4. Values of whole-body protein breakdown during insulin withdrawal derived from the [2H5]phenylalanine and primary and reciprocal pool [13C]leucine models respectively were 3.54 +/- 0.43, 3.85 +/- 0.41 and 4.62 +/- 0.44 g day-1 kg-1 (means +/- SD). Insulin infusion resulted in a significant reduction (P less than 0.02) to 3.07 +/- 0.34, 3.05 +/- 0.26 and 3.82 +/- 0.4 g day-1 kg-1, respectively. Synthesis values fell significantly but by a smaller amount than breakdown, resulting in increased (P less than 0.05) net protein deposition, regardless of the model used. 5. These data demonstrate that the [2H5]phenylalanine and [13C]leucine methods generate similar results both in absolute and relative terms in response to short-term insulin infusion. 6. The confirmation of increased whole-body protein synthesis during insulin withdrawal by two independent methods supports the validity of this observation.     

Sepsis is associated with increased mRNAs of the ubiquitin-proteasome proteolytic pathway in human skeletal muscle.

Previous studies provided evidence that sepsis-induced muscle proteolysis in experimental animals is caused by increased ubiquitin-proteasome-dependent protein breakdown. It is not known if a similar mechanism accounts for muscle proteolysis in patients with sepsis. We determined mRNA levels for ubiquitin and the 20 S proteasome subunit HC3 by Northern blot analysis in muscle tissue from septic (n = 7) and non-septic (n = 11) patients. Plasma and muscle amino acid concentrations and concentrations in urine of 3-methylhistidine (3-MH), creatinine, and cortisol were measured at the time of surgery to assess the catabolic state of the patients. A three- to fourfold increase in mRNA levels for ubiquitin and HC3 was noted in muscle tissue from the septic patients concomitant with increased muscle levels of phenylalanine and 3-MH and reduced levels of glutamine. Total plasma amino acids were decreased by approximately 30% in the septic patients. The 3-MH/creatinine ratio in urine was almost doubled in septic patients. The cortisol levels in urine were higher in septic than in control patients but this difference did not reach statistical significance. The results suggest that sepsis is associated with increased mRNAs of the ubiquitin-proteasome pathway in human skeletal muscle.