Skeletal muscle and whole body protein turnover in thyroid disease

The effects of disturbances of thyroid hormone secretion on leg and whole body amino acid and protein metabolism have been investigated in seven patients with untreated thyrotoxicosis and eight patients with untreated hypothyroidism; the results were compared to those obtained in 11 normal control subjects. After treatment, the patients were restudied. Arterio-venous exchanges of tyrosine and 3-methylhistidine across leg tissue in the post-absorptive state were used as indices of net protein balance and myofibrillar protein breakdown, respectively. Whole body protein turnover was measured using stable isotope labelling techniques with 1-[1-13C] leucine. Efflux of tyrosine from leg tissues was six-fold greater in patients with untreated thyrotoxicosis than in normal control subjects (-19.39 +/- 2.21 vs. -4.20 +/- 0.31 nmol 100 g-1 leg tissue min-1, P less than 0.005, mean +/- SEM), but 3-methyl-histidine efflux was not significantly different (-0.11 +/- 0.03 nmol 100 g-1 leg tissue min-1 vs. 0.14 +/- 0.02 nmol 100 g-1 leg tissue min-1). After treatment, when the thyrotoxic patients became euthyroid, tyrosine efflux was normalized (at -4.94 +/- 0.84 nmol 100 g-1 leg tissue min-1) and 3-methylhistidine efflux was unchanged. In hypothyroid patients, neither tyrosine nor 3-methylhistidine effluxes were significantly different from those in normal subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small-intestinal mucosal protein synthesis and whole-body protein turnover in alcoholic liver disease

We used stable-isotope-labelled amino acids to measure the effects of alcoholic liver disease (ALD) on whole-body protein turnover and small-intestinal mucosal protein synthesis. Groups comprising eight patients with ALD and eight healthy control subjects were studied. They received primed, continuous intravenous infusions of L-[1-(13)C]leucine after an overnight fast; after 4 h, duodenal biopsies were obtained via endoscopy. Protein synthesis was calculated from protein labelling relative to intracellular leucine enrichment. Rates of duodenal mucosal protein synthesis were 2. 58+/-0.32%.h(-1) (mean+/-S.D.) in the normal subjects and 2.04+/-0. 18%.h(-1) in the ALD patients (P<0.003), despite the fact that the protein synthetic capacity (microgram of RNA/mg of protein) was higher in ALD patients (160+/-14 compared with 137+/-6 microgram/mg; P<0.003). The mucosal cell size (protein/DNA ratio) was lower in ALD patients (9.23+/-0.91 compared with 13+/-2.2 microgram/mg; P<0.002). Although the mean rates of whole-body protein turnover were not significantly different between the two groups (204+/-18 and 196+/-44 micromol leucine.h(-1).kg(-1) for ALD and control subjects respectively), there was, in the ALD patients, an inverse relationship between the rate of small-intestinal mucosal protein synthesis and the severity of ALD; furthermore, there was a direct relationship between the rate of whole-body protein turnover and the severity of ALD. Thus there was an inverse relationship between the rate of small-intestinal mucosal protein synthesis and the rate of whole-body protein turnover in ALD patients, which was not seen in the normal subjects.     

Influence of dietary protein intake on whole-body protein turnover in humans.

Methods for measuring rates of protein synthesis and degradation in the whole body of humans with isotopes of carbon and nitrogen are described and attention is drawn to their relative merits and drawbacks for studying the nutritional control of protein metabolism. A review of published work on dietary protein and protein metabolism leads to the conclusion that protein is the major dietary determinant of whole-body protein turnover rates, and that energy intake is comparatively unimportant. Dietary protein affects protein turnover at two levels: an immediate response to the intake of protein in meals and a longer-term adaptation after a change in protein intake. An increase in the level of dietary protein enhances the response to meals, which mainly consists of a decrease in the rate of protein degradation. The adaptation to higher protein intakes involves an increase in the basal (postabsorptive) rates of both synthesis and degradation. Suggestions for future investigation include more detailed studies of the acute and adaptive responses, to facilitate understanding of dietary protein requirements, and the effects of very-high-protein intakes with continued development of techniques for studying protein turnover in individual tissues in humans.     

Skeletal muscle and whole body protein turnover in cardiac cachexia: influence of branched-chain amino acid administration

Muscle protein wasting commonly accompanies severe heart failure. The mechanism of this so-called cardiac cachexia has been investigated in eight patients with an average body weight decrement of 19%, whose results have been compared with those from 11 healthy control subjects. Exchanges of tyrosine and 3-methylhistidine across leg tissue were used as specific indicators of net protein balance and myofibrillar protein breakdown, respectively. Whole body protein turnover was measured using a stable isotope labelling technique with L-[1-13C]leucine as tracer. In patients with cardiac cachexia there were greater values, relative to those values in normal control subjects, of leg efflux of tyrosine (-8.1 +/- 0.6 nmol 100 ml leg tissue-1 min-1 vs. -4.2 +/- 0.3 nmol 100 ml-1 min-1 (P less than 0.01) and of 3-methylhistidine (-0.8 +/- 0.1 nmol 100 ml leg tissue-1 min-1 vs. -0.1 +/- 0.02 nmol 100 ml-1 min-1 (P less than 0.005), mean +/- SEM). The results suggest that in patients with cardiac cachexia the state of net negative protein balance across leg tissue is associated with an increased rate of myofibrillar protein breakdown. In cardiac cachexia, neither efflux of tyrosine (-8.4 +/- 0.7 nmol 100 ml leg tissue-1 min-1) nor of 3-methylhistidine (-1.0 +/- 0.2 nmol 100 ml leg tissue-1 min-1) were significantly altered by branched-chain amino acid (BCAA) infusion to plasma concentrations of 1300 +/- 14 mumol ml-1, i.e., four times normal plasma values (282 +/- 11 mumol ml-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Elevated protein requirements in cirrhosis of the liver investigated by whole body protein turnover studies

1. In patients with cirrhosis of the liver and in healthy control subjects, the rates of nitrogen flux, protein synthesis and protein breakdown were studied, using a single oral dose of 200 mg of [15N]glycine as a tracer. The nitrogen flux through the amino acid pool was measured separately with both urinary ammonia and urinary urea as end products; the average value was used for further calculations. 2. Subjects were studied in the fed state, both on an adequate and a protein-restricted diet, and also in the fasting state. 3. The rates of protein synthesis were markedly increased in the patients, not only in the fed but also in the fasting state. Protein breakdown rates were increased in the patients in the fed state. 4. The nitrogen balance in steady-state conditions in the fed state was more positive in the patients, while their nitrogen loss in the fasting state was no higher than that of control subjects. 5. A hypothesis is put forward that the high protein requirements of cirrhotic patients could be caused by small and inadequate liver glycogen stores; due to these small stores, gluconeogenesis from amino acids will take place and lead to an extra amino acid loss even during short-term fasting. This increased amino acid loss could explain the elevated protein requirements in cirrhotic patients.     

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.     

Leucine. A possible regulator of protein turnover in muscle.

Incorporation of radiolabeled precursors into muscle proteins was studied in isolated rat hemidiaphragms. A mixture of three branched-chain amino acids (0.3 mM each) added to media containing glucose stimulated the incorporation of [14C]lysine into proteins. When tested separately, valine was ineffective, isoleucine was inhibitory, but 0.5 mM leucine increased the specific activity of muscle proteins during incubation with [14C]lysine or [14C]acetate in hemidiaphragms from fed or fasted rats incubated with or without insulin. Preincubation with 0.5 mM leucine increased the specific activity of muscle proteins during a subsequent 30- or 60-min incubation with [14C]lysine or [14C]pyruvate without leucine. Preincubation with other amino acids (glutamate, histidine, methionine, phenylalanine, or tryptophan) did not exert this effect. When hemidiaphragms were incubated with a mixture of amino acids at concentrations found in rat serum and a [14C]lysine tracer, the specific activity of muscle proteins increased when leucine in the medium was raised from 0.1 to 0.5 mM. Experiments with actinomycin D and cycloheximide suggested that neither RNA synthesis nor protein synthesis are required for the initiation of the leucine effect. Leucine was not effective when added after 1 h preincubation without leucine. The concentration of lysine in the tissue water of diaphragms decreased during incubation with 0.5 mM leucine in the presence or absence of cycloheximide, suggesting that leucine inhibited protein degradation. During incubation with [3h]tyrosine (0.35 mM) the addition of 0.5 mM leucine increased the specific activity of muscle proteins, while the specific activity of intracellular tyrosine remained constant and its concentration decreased, suggesting that leucine also promoted protein synthesis. The concentration of leucine in muscle cells or a compartment thereof may play a role in regulating the turnover of muscle proteins and influence the transition to negative nitrogen balance during fasting, uncontrolled diabetes, and the posttraumatic state. Leucine may play a pivotal role in the protein-sparing effect of amino aicds.     

Decrease in human quadriceps muscle protein turnover consequent upon leg immobilization

Quadriceps muscle protein turnover was assessed in the post-absorptive state in six men immediately after the end of unilateral leg immobilization (37 +/- 4 days) in a plaster cast after tibial fracture. A primed-constant intravenous infusion of L-[1-13C]leucine was administered over 7 h. Quadriceps needle biopsies, taken bilaterally at the end of the infusion, were analysed for muscle protein leucine enrichment with 13C. Quadriceps muscle protein synthetic rate, calculated from the fractional incorporation of [13C]leucine into protein compared with the average enrichment of blood alpha-ketoisocaproate, was 0.046 +/- 0.012%/h in the uninjured leg, but was only 0.034 +/- 0.007%/h in the quadriceps of the previously fractured leg (P less than 0.05, means +/- SD). Muscle RNA activity (i.e. protein synthetic rate per RNA) fell from 0.27 +/- 0.08 microgram of protein synthesized h-1 microgram-1 of RNA in the control leg to 0.14 +/- 0.03 microgram of protein synthesized h-1 microgram-1 of RNA in the immobilized leg (P less than 0.02). Immobilization was associated with a significant atrophy of type I muscle fibres (mean diameter 69.5 +/- 21 microns immobilized, 81.1 +/- 18 microns control, P less than 0.05), but no significant change occurred in type II fibre diameter. Mean quadriceps fibre volume calculated from the values for fibre diameter and percentage of each fibre type, was smaller in the injured leg by 10.6%; this value was near to the calculated difference in muscle thigh volume (calculated from thigh circumference and skin-fold thickness) which was less by 8.3%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basal metabolic rate, body composition and whole-body protein turnover in Indian men with differing nutritional status.

1. Three groups of adult men were studied in Bangalore, India: two groups were controls who had been receiving an adequate diet. Of these, one group, designated 'normal weight controls', had a mean body mass index of 22; the other group, 'underweight controls', had a mean body mass index of 16.7. The third group consisted of poor labourers, whose daily food intake had been less than 10 MJ and whose mean body mass index was 16.6. Previous studies had shown that such men had a lower basal metabolic rate than well-nourished Indian control subjects. 2. The object of the present study was to find out whether a reduced rate of protein turnover, measured after a single dose of [15N]glycine, contributed to a lower basal metabolic rate. It was found, however, that after adjusting for body weight and fat-free mass by analysis of co-variance there was no significant difference in basal metabolic rate between the three groups. Adjusted rates of protein synthesis were higher in the underweight controls and the undernourished labourers than in the normal weight controls, but not significantly so. 3. Estimates based on creatinine excretion showed that within the fat-free mass the underweight groups had a higher proportion of non-muscle to muscle mass. This may explain the somewhat higher rates of protein turnover in these groups. 4. Nitrogen flux (Q) was determined from 15N abundance in two end products, urea (QU) and ammonia (QA). In the underweight and undernourished groups the ratio QU/QA was increased compared with the normal weight group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Skeletal muscle disease in alcoholism

Acute alcoholic myopathy, a syndrome of sudden muscle necrosis, occurs as a result of binge drinking, whereas chronic alcoholic myopathy is a more indolently evolving syndrome of proximal weakness and muscle atrophy that accompanies prolonged alcohol abuse. The characteristic features and management of these disorders are highlighted.     

Skeletal muscle damage and recovery

Muscular strength is essential for recovery after an acute illness. Disuse atrophy of muscle begins within 4 hours of the start of bed rest resulting in decreases in muscle mass, muscle cell diameter, and the number of muscle fibers. Strenuous exercise of atrophic muscle can lead to muscle damage including sarcolemmal disruption, distortion of the myofibrils' contractile components, and cytoskeletal damage. Assessment of skeletal muscle for disuse atrophy is done clinically at the bedside through strength assessment. Examination of the muscle itself can be conducted through the use of nuclear magnetic resonance imaging, whereas muscle strength can be quantified with a computerized dynamometer. Biochemical markers, including creatine kinase and troponin, also are available for the assessment of skeletal muscle damage. Activity management in the critical care environment focuses on an individualized plan, developed in cooperation with the recovering patient, with the goal of preserving and improving atrophic skeletal muscle.     

Comparison of 15N-labelled glycine, aspartate, valine and leucine for measurement of whole-body protein turnover.

1. Whole-body protein turnover was measured in rats by constant infusion of 15N-labelled glycine, aspartate, valine and leucine and measuring the enrichment of hepatic and renal urea and ammonia nitrogen. 2. The values obtained with [15N]glycine were comparable with values reported with methods based on different assumptions. 3. [15N]Aspartate gave rise to an increased enrichment of urea and ammonia and hence to lower protein-turnover rates. 4. [15N]Valine and [15N]leucine gave low enrichments of nitrogenous end products and hence to high protein-turnover rates. 5. All 15N-labelled amino acids are not equally suitable for measuring whole-body protein turnover by the end-product method. The relative amounts of 15N going to the end products can be predicted from the known individual metabolism of aspartate and the branched-chain amino acids.     

Effect of beta-hydroxybutyrate on whole-body leucine kinetics and fractional mixed skeletal muscle protein synthesis in humans.

Because intravenous infusion of beta-hydroxybutyrate (beta-OHB) has been reported to decrease urinary nitrogen excretion, we investigated in vivo metabolism of leucine, an essential amino acid, using L-[1-13C]leucine as a tracer during beta-OHB infusion. Leucine flux during beta-OHB infusion did not differ from leucine flux during normal saline infusion in nine normal subjects, whereas leucine oxidation decreased 18-41% (mean = 30%) from 18.1 +/- 1.1 mumol.kg-1.h-1 (P less than 0.01), and incorporation of leucine into skeletal muscle protein increased 5-17% (mean = 10%) from 0.048 + 0.003%/h (P less than 0.02). Since blood pH during beta-OHB infusion was higher than the pH during saline infusion, we performed separate experiments to study the effect of increased blood pH on leucine kinetics by infusing sodium bicarbonate intravenously. Blood pH during sodium bicarbonate infusion was similar to that observed during the beta-OHB infusion, but bicarbonate infusion had no effect on leucine flux or leucine oxidation. We conclude that beta-OHB decreases leucine oxidation and promotes protein synthesis in human beings.     

Effects in vivo of decreased plasma and intracellular muscle glutamine concentration on whole-body and hindquarter protein kinetics in rats.

Glutamine is considered to be a 'conditionally' essential amino acid. During situations of severe stress like sepsis or after trauma there is a fall in plasma glutamine levels, enhanced glutamine turnover and intracellular muscle glutamine depletion. Under these conditions, decreased intramuscular glutamine concentration correlates with reduced rates of protein synthesis. It has therefore been hypothesized that intracellular muscle glutamine levels have a regulatory role in muscle protein turnover rates. Administration of the glutamine synthetase inhibitor methionine sulphoximine (MSO) was used to decrease glutamine levels in male Wistar rats. Immediately after the MSO treatment (t=0 h), and at t=6 h and t=12 h, rats received intraperitoneal injections (10 ml/100 g body weight) with glutamine (200 mM) to test whether this attenuated the fall in plasma and intracellular muscle glutamine. Control animals received alanine and saline after MSO treatment, while saline was also given to a group of normal rats. At t=18 h rats received a primed constant infusion of L-[2,6-3H]phenylalanine. A three-pool compartment tracer model was used to measure whole-body protein turnover and muscle protein kinetics. Administration of MSO resulted in a 40% decrease in plasma glutamine and a 60% decrease in intracellular muscle glutamine, both of which were successfully attenuated by glutamine infusions. The decreased intracellular muscle glutamine levels had no effect on whole-body protein turnover or muscle protein kinetics. Also, glutamine supplementation did not alter these parameters. Alanine supplementation increased both hindquarter protein synthesis and breakdown but the net balance of phenylalanine remained unchanged. In conclusion, our results show that decreased plasma and muscle glutamine levels have no effect on whole-body protein turnover or muscle protein kinetics. Therefore, it is unlikely that, in vivo, the intracellular muscle concentration of glutamine is a major regulating factor in muscle protein kinetics.     

Effects of recombinant human growth hormone on muscle protein turnover in malnourished hemodialysis patients.

To assess the effect of recombinant human growth hormone (rhGH) on muscle protein metabolism in uremic patients with malnutrition, forearm [3H]phenylalanine kinetics were evaluated in six chronically wasted (body weight 79% of ideal weight) hemodialysis (HD) patients in a self-controlled, crossover study. Forearm protein dynamics were evaluated before, after a 6-wk course of rhGH (5 mg thrice weekly) and after a 6-wk washout period. After rhGH: (a) forearm phenylalanine net balance--the difference between phenylalanine incorporation into and phenylalanine release from muscle proteins--decreased by 46% (-8+/-2 vs. -15+/-2 nmol/min x 100 ml at the baseline and -11+/-2 after washout, P < 0.02); (b) phenylalanine rate of disposal, an index of protein synthesis, increased by 25% (25+/-5 vs. 20+/-5 at the baseline and 20+/-4 after washout, P < 0.03); (c) phenylalanine rate of appearance, an index of protein degradation, was unchanged (33+/-5 vs. 35+/-5 at the baseline and 31+/-4 after washout); (d) forearm potassium release declined (0.24+/-0.13 vs. 0.60+/-0.15 microeq/min at the baseline, and 0.42+/-0.20 microeq/min after washout P < 0.03); (e) changes in the insulin-like growth factor binding protein (IGFBP)-1 levels and insulin-like growth factor-I (IGF-I)/IGFBP-3 ratios accounted for 15.1% and 47.1% of the percent variations in forearm net phenylalanine balance, respectively. Together, these two factors accounted for 62.2% of variations in forearm net phenylalanine balance during and after rhGH administration. These data indicate: (a) that rhGH administration in malnourished hemodialysis patients is followed by an increase in muscle protein synthesis and by a decrease in the negative muscle protein balance observed in the postabsorptive state; and (b) that the reduction in net protein catabolism obtained with rhGH can be accounted for by the associated changes in circulating free, but not total, IGF-I levels.