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The effects of thyroxine (T4) and triiodothyronine (T3) on growth, muscle protein degradation, and proteases activities in cultured chick muscle cells were studied. The cells were treated with a physiological level of T4 (60 ng/mL) or T3 (12 ng/mL) for 6 d. Calpain, cathepsins, and proteasome activities and N tau-methylhistidine release were measured as indexes of myofibrillar protein breakdown. Creatine kinase activity was also measured as an index of myotube formation. Calpain activity was increased by T4 and T3. Cathepsin D and proteasome activities and N tau-methylhistidine release were increased by T3, but not by T4. Neither were cathepsin B and B + L activities affected by T3 or T4. Creatine kinase activity was increased by T4 and T3. The results suggest that myotube formation is accelerated by T4 and T3, whereas myofibrillar protein degradation is accelerated by T3, but not by T4.

The objectives were to investigate the mechanisms by which glucocorticoids control proteolysis in muscle cells and the relationship between the calpain:calpastatin system and proteolysis in muscle. Female rabbits were treated with 1 mg dexamethasone (Dex)/kg body weight per day for 0, 1, 2 or 4 days after which animals were killed and muscle samples taken for analyses. Dex reduced urinary N tau-methylhistidine (NMH) 48% (day 4 versus day 1 of Dex treatment) and muscle NMH concentrations by 49% (day 1) to 40% (day 2) respectively, suggesting that protein degradation was reduced. To investigate whether the changes in apparent proteolysis were related to calpains, we examined the effects of Dex on muscle calpain and calpastatin activities. These were unaffected by Dex. This implies that Dex-dependent changes in degradation are not mediated by changes in muscle calpain or calpastatin activities. We studied the effects of Dex on calpain and calpastatin gene expression as a means of clarifying the relationships between proteinase gene expression and proteinase activities. mu-Calpain mRNA concentration was unaffected by Dex but m-calpain mRNA and calpastatin mRNA concentrations were reduced by 42-55% and 40% respectively. Dex had a similar effect on beta-actin mRNA. Although calpain and calpastatin genes behaved as house-keeping genes, changes in their expression mimicked apparent changes in proteolysis. The observation that calpain and calpastatin activities were unchanged indicates that additional regulation of the calpain:calpastatin system exists at other sites in muscle cells. To determine whether Dex-dependent changes in proteolysis were mediated indirectly, we assayed the effects of Dex on plasma thyroid hormone concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

In previous studies, interleukin-1 (IL-1) or tumor necrosis factor (TNF) have been demonstrated to augment skeletal muscle protein breakdown in a manner similar to that induced by bacterial endotoxin. This response to IL-1 or TNF was elicited only after they were administered to animals for various periods, as their addition in vitro to incubated muscles from normal untreated rats was without effect. This suggested that IL-1 and TNF may augment muscle proteolysis in an indirect fashion. Serum levels of IL-1, TNF as well as interleukin-6 (IL-6) are all elevated during infection induced by bacterial endotoxin. Both IL-1 and TNF can induce the synthesis of IL-6 by a variety of cells. Because of this, in the present report, the ability of IL-6 to stimulate skeletal muscle protein breakdown was examined. Muscle protein breakdown was evaluated by measuring the release of both tyrosine and 3-methylhistidine by incubated muscles. Pretreatment of rats with IL-6 for 6 hr induced fever and increased the release of both tyrosine and 3-methylhistidine by the extensor digitorum longus muscle. However, IL-6 did not augment muscle proteolysis when muscles from normal untreated rats were incubated in the presence of the cytokine. The data suggest that the acute treatment of animals with IL-6 can augment muscle proteolysis. Whether this response is due to a direct effect of IL-6 on the myocyte or whether it is due to the production of other mediators remains unclear.

Objective: The study clarified the effects of growth hormone treatment on forearm amino acid efflux in patients with full nutritional support after gastrointestinal surgery., Summary Background Data: Growth hormone attenuates net nitrogen loss after surgical trauma. An increase in net protein synthesis has been described, whereas the results regarding protein breakdown have been conflicting., Methods: Elective patients undergoing abdominal surgery were double blindly randomized to treatment with recombinant human growth hormone (GH, n = 9) 24 IU or placebo (PL, n = 10) the first 5 postoperative days. All received parenteral nutrition (nitrogen = 5.7 +/- .1 g/m2, energy = 1018 +/- 12 kcal/m2 (125 +/- .7% of BMR) and epidural analgesia. Amino acid plasma levels and forearm fluxes were measured., Results: The second postoperative day, growth hormone abolished forearm efflux of total amino acid nitrogen (GH: 170 +/- 117, PL: -785 +/- 192 nmol/100 mL/min, p = .0007) due to reduced losses of both essential and nonessential amino acids. Glutamine release was abolished (13 +/- 15 vs. -137 +/- 43 nmol/100 mL/min, p = .007) and alanine release attenuated (-61 +/- 17 vs. -211 +/- 51 nmol/100 mL/min, p = .01). 3-Methyl-histidine release was attenuated (-.20 +/- .11 vs. -.62 +/- .09 nmol/100 mL/min, p = .04). Growth hormone also induced decreased venous plasma amino acid levels., Conclusions: When given after gastrointestinal surgery in patients treated with total parenteral nutrition, growth hormone treatment abolished glutamine, 3-methylhistidine, and total amino acid nitrogen loss from forearm tissue. Alanine loss from forearm tissue was attenuated.

In this study we compared the effect of sepsis on muscle protein metabolism in infant (3 to 4 weeks) and adult (3 to 4 months) rats. Sepsis was induced by cecal ligation and puncture (CLP). Control animals underwent sham operation. Sixteen hours after CLP or sham operation, metabolic studies were performed in incubated intact extensor digitorum longus muscles from infant rats or in strips of the same muscle from adult rats. Protein synthesis rate was determined as incorporation of 3H-phenylalanine into protein; total and myofibrillar protein breakdown rates were determined as release of tyrosine and 3-methylhistidine, respectively. Mortality rate following CLP was similar in both age groups. Basal protein synthesis rate was 3 times higher, total protein breakdown rate was 50% higher, and myofibrillar protein breakdown rate was 3 times higher in infant than in adult animals. However, the relative changes in protein turnover rates induced by sepsis were similar in infant and adult rats: protein synthesis rate decreased by approximately 30%, total protein breakdown increased by 40% to 50%, and myofibrillar protein breakdown increased severalfold. The data suggest that despite prominent differences in basal protein turnover rates between infant and adult rats, the effect of sepsis on muscle protein metabolism is not age dependent.

This work reports on the muscle loss and recovery after 17 wk of continuous bed rest and 8 wk of reambulation in eight normal male volunteers. Muscle changes were assessed by urinary levels of 3-methylhistidine (3-MeH), nitrogen balance, dual-photon absorptiometry (DPA), magnetic resonance imaging (MRI), and isokinetic muscle performance. The total body lean tissue loss during bed rest calculated from nitrogen balance was 3.9 +/- 2.1 (SD) kg (P < 0.05). Although the total loss is minimal, DPA scans showed that nearly all of the lean tissue loss occurred in the lower limbs. Similarly, MRI muscle volume measurements showed greater percent loss in the limbs relative to the back muscles. MRI, DPA, and nitrogen balance suggest that muscle atrophy continued throughout bed rest with rapid recovery after reambulation. Isokinetic muscle strength decreased significantly (P < 0.05) in the thigh and calf with no loss in the arms and with rapid recovery during reambulation. We conclude that there is great variability in the degree and location of muscle loss in bed rest and that the lower limb muscles are primarily affected.

A sensitive and specific high performance liquid chromatographic method is described for measuring imidazole dipeptides and 3-methylhistidine in human muscle biopsies, serum and urine. Muscle extract, serum or urine was reacted with o-phthaldialdehyde and the derivatives were separated by reversed phase chromatography with column switching and fluorescence detection.

The goal of this study was to assess the effects of voluntary running activity in rats on various aspects of carbohydrate and protein metabolism. After 6 wk of exercise training, rats (ET) were rested for 24 h and their hindquarters, along with those from sedentary control (SC) and dietary control (DC) rats, were perfused with 0, 60, 250, or 6,000 microU/ml insulin. At 0 insulin, glucose clearance was similar for all groups, and it was increased with added insulin. However, the insulin effect was 20-40% greater for ET rats at all insulin concentrations (P less than 0.05). Muscle glycogen deposition also increased with added insulin but showed muscle-specific differences. Specifically, glycogen content of the plantaris muscle was significantly higher in ET compared with SC or DC rats, whereas this pattern was reversed in soleus muscle. In plantaris muscle, insulin stimulated glucose 6-phosphate (G-6-P)-independent (-G-6-P) glycogen synthase activity only in SC and DC rats and increased its affinity for G-6-P at 250 microU/ml in all groups. In contrast, the -G-6-P synthase activity was not increased in soleus muscle and was actually decreased in all groups at 6,000 microU/ml. Tyrosine release was suppressed by insulin in all groups, but this effect was significantly greater at insulin levels of 60 microU/ml (P less than 0.02) in hindquarters from ET rats compared with SC and DC rats. Neither insulin nor exercise training decreased 3-methylhistidine release from perfused hindquarters.(ABSTRACT TRUNCATED AT 250 WORDS)

Myofibrillar protein breakdown in skeletal muscle progresses through two distinct phases in response to chronic glucocorticoid administration in the rat, i.e., an early phase lasting 4-5 days, during which proteolysis increases followed by a later phase during which proteolysis decreases. The possible involvement of insulin and the iodothyronines in this phenomenon has now been examined. Diabetic, thyroidectomized, and normal rats were treated with corticosteroid for 10-11 days, and at timed intervals muscle proteolysis was evaluated by measuring the release of 3-methyl-L-histidine (3-MH) and tyrosine from the perfused hindquarter as well as the excretion of 3-MH in the urine. Corticosterone (CTC) administration to normal rats increased plasma insulin, whereas plasma 3,5,3'-triiodothyronine responded with an early rise followed by a fall after 4-5 days. However, the biphasic response of myofibrillar proteolysis to chronic glucocorticoid treatment was not abolished in CTC-treated diabetic or thyroidectomized rats. CTC treatment increased release of tyrosine by perfused muscle of diabetic rats but, unlike 3-MH release, did not diminish later. Thus the adaptation of myofibrillar proteolysis to chronic glucocorticoid treatment appears to be independent of insulin and thyroid hormones. However, insulin may play a role in curtailing glucocorticoid-induced breakdown of nonmyofibrillar proteins.

The present study characterized total and myofibrillar protein breakdown rates in a muscle preparation frequently used in vitro, i.e. incubated extensor digitorum longus (EDL) and soleus (SOL) muscles of young rats. Total and myofibrillar protein breakdown rates were assessed by determining net production by the incubated muscles of tyrosine and 3-methylhistidine (3-MH) respectively. Both amino acids were determined by h.p.l.c. Both total and myofibrillar protein breakdown rates were higher in SOL than in EDL muscles and were decreased by incubating the muscles maintained at resting length, rather than flaccid. After fasting for 72 h, total protein breakdown (i.e. tyrosine release) was increased by 73% and 138% in EDL muscles incubated flaccid and at resting length respectively. Net production of tyrosine by SOL muscle was not significantly altered by fasting. In contrast, myofibrillar protein degradation (i.e. 3-MH release) was markedly increased by fasting in both muscles. When tissue was incubated in the presence of 1 munit of insulin/ml, total protein breakdown rate was inhibited by 17-20%, and the response to the hormone was similar in muscles incubated flaccid or at resting length. In contrast, myofibrillar protein breakdown rate was not altered by insulin in any of the muscle preparations. The results support the concepts of individual regulation of myofibrillar and non-myofibrillar proteins and of different effects of various conditions on protein breakdown in different types of skeletal muscle. Thus determination of both tyrosine and 3-MH production in red and white muscle is important for a more complete understanding of protein regulation in skeletal muscle.

Elevated temperature has been proposed to contribute to accelerated muscle protein degradation during fever and sepsis. The present study examined the effect of increased temperature in vitro on protein turnover in skeletal muscles from septic and control rats. Sepsis was induced by cecal ligation and puncture (CLP); control rats were sham operated. After 16 h, the extensor digitorum longus (EDL) and soleus (SOL) muscles were incubated at 37 or 40 degrees C. Protein synthesis was determined by measuring incorporation of [14C]phenylalanine into protein. Total and myofibrillar protein breakdown was assessed from release of tyrosine and 3-methylhistidine (3-MH), respectively. Total protein breakdown was increased at 40 degrees C by 15% in EDL and by 29% in SOL from control rats, whereas 3-MH release was not affected. In muscles from septic rats, total and myofibrillar protein breakdown was increased by 22 and 30%, respectively, at 40 degrees C in EDL but was not altered in SOL. Protein synthesis was unaffected by high temperature both in septic and nonseptic muscles. The present results suggest that high temperature is not the primary mechanism of increased muscle protein breakdown in sepsis because the typical response to sepsis, i.e., a predominant increase in myofibrillar protein breakdown, was not induced by elevated temperature in normal muscle. It is possible, however, that increased temperature may potentiate protein breakdown that is already stimulated by sepsis because elevated temperature increased both total and myofibrillar protein breakdown in EDL from septic rats.

The 20-hr swimming increased the contants of protein, thyrosine and 3-methylhistidine (also per 1 g of protein) in skeletal muscle of rat within 2-24 hrs. A significant increase in 3-methylhistidine excretion followed this on the 2nd day of the recovery period. The revealed combination of changes suggests a simultaneous augmentation of both synthesis and disintegration of protein in muscles after their activity. Hence, the increased excretion of 3-methylhistidine during recovery period reflects an intensive renewal of the molecular content of proteins of actinomyosine complex. The 3-methylhistidine level in intestine increased only for a short time and seems not to contribute to the delayed increase in excretion of the amino acid.

N tau-Methylhistidine(3-methylhistidine) in urine of the rat is mainly derived from the degradation of actin and myosin in skeletal muscle, intestine and skin. The fractional degradation rates of the myosin-actin pools of these tissues were calculated from the time course of increase in the specific radioactivities of N tau-methylhistidine after daily administration of [methyl-14C]methionine to young adult rats under conditions of restricted food intake. The contributions to urinary excretion of N tau-methylhistidine from the three tissues were calculated from the fractional degradation rates and N tau-methylhistidine contents of the three tissues; 75.6% for skeletal muscle, 2.2% for intestine and 22.2% for skin. The results show that the skeletal muscle is the major source of urinary N-tau-methylhistidine output, but the contribution of skin is not negligible in rats. The specific radioactivity of N tau-methylhistidine in urine was much higher than that of skeletal muscle. The fractional degradation rates of myosin and actin in skeletal muscle had similar values. Although the specific radioactivities of N tau-methylhistidine in myosin and actin were very different, the mean value was similar to that in mixed skeletal muscle.

The content of 3-methylhistidine (3-MH) and 1-methylhistidine (1-MH) was measured in muscle biopsy specimens from 13 normal controls, 19 patients with Duchenne muscular dystrophy, 8 limb-girdle disease patients, and 23 disease controls with different forms of muscular pathology. 3-MH and 1-MH concentrations in normal human muscle did not appear to be influenced by sex, body weight, and age, at least for subjects in the 10--60 year age group examined. Skeletal muscle 1-MH levels did not significantly differ from mean control values in any of the pathologies investigated. In the patient population examined, the mean 3-MH level per unit of noncollagen protein (NCP) was significantly lower than normal in Duchenne dystrophy only, the reduction being related to disease severity. The significantly lower concentrations of 3-MH in muscle of Duchenne patients indicate the importance of measuring 3-MH in diseased muscle to obtain reliable estimates of the myofibrillar protein catabolic rate.

Myofibrillar protein breakdown during brief and prolonged starvation was assessed in perfused rat skeletal muscle from 8-week-old fat-fed rats that conserve skeletal muscle protein during starvation and survive for 12 to 15 days and age-matched chow-fed rats that do not conserve protein and survive only five to six days. Following the inhibition of protein synthesis with cycloheximide, myofibrillar proteolysis was assessed by measuring the release of 3-methylhistidine from the perfused rat hindquarter while simultaneous measurement of total protein breakdown was assessed by measuring tyrosine release. Myofibrillar proteolysis progressed through three distinct phases during starvation: an early phase occurring within 24 hours in which proteolysis increased in all rats, a middle phase, which took three to five days to develop and during which proteolysis decreased and was present only in fat-fed rats, and a late phase in which proteolysis again increased. Total protein breakdown (ie, tyrosine release) changed little in phase I, decreased in phase II, and increased in phase III. The release of 3-methylhistidine from the perfused hindquarter reflected changes in muscle and urine of intact rats suggesting that data obtained with the perfused hindquarter reflected the in vivo situation. Insulin, amino acids, high concentrations of glucose, indomethacin, or epinephrine as well as adrenalectomy failed to attenuate the increase in 3-methylhistidine release from the perfused hindquarter during brief and late starvation. Free fatty acids and ketone bodies were also without effect in vitro. Refeeding fasting rats for four hours decreased myofibrillar proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

The tissue origin of 3-methylhistidine (N tau-methylhistidine) was investigated in adult female rats. The decay of labelling of urinary 3-methylhistidine was compared with the labelling of protein-bound 3-methylhistidine in skeletal muscle and intestine after the injection of [methyl-14C]methionine. The decay curve for urinary 3-methylhistidine was much steeper than that in muscle or intestine, falling to values lower than those in either tissue after 30 days. The lack of decay of labelling in muscle during the first 30 days is shown to result from the persistence of label in the precursor S-adenosylmethionine. The relative labelling of urinary, skeletal-muscle and intestinal 3-methylhistidine cannot be explained in terms of skeletal muscle accounting for a major proportion of urinary 3-methylhistidine. Measurements were also made of the steady-state synthesis rate of protein-bound 3-methylhistidine in intestinal smooth muscle in vivo in adult female rats. This involved measurement of the overall rate of protein synthesis and measurement of the relative rates of synthesis of 3-methylhistidine and of mixed protein. The synthesis rate of 3-methylhistidine was 29.1%/day, compared with the overall rate of 77.1%/day for mixed, non-mucosal intestinal protein. Measurement of the amount of 3-methylhistidine in skeletal muscle (0.632 +/- 0.024 mumol/g) and in the whole body (0.332 +/- 0.013 mumol/g) indicate that, although the muscle pool is 86% of the total, because of its slow turnover rate of 1.1-1.6%/day, it only accounts for 38-52% of the observed excretion. Measurements of the mass of the intestine (9.95 g/250 g body wt.) and protein-bound 3-methylhistidine content (0.160 mumol/g of tissue) indicate a pool size of 1.59 mumol/250 micrograms rat. Thus 463 nmol of the urinary excretion/day would originate from the intestine, 22% of the total. The tissue source of the remaining urinary excretion is not identified, but other non-muscle sources constituting about 10% of the whole-body pool could account for this with turnover rates of only 6%/day, a much lower value than the turnover rate in the intestine.

Changes in the effluxes from the leg of 3-methylhistidine and tyrosine were studied in relation to alterations in the 24-hour excretion of 3-methylhistidine and total nitrogen in 11 patients before and after undergoing major surgical operation. On the first day after operation, efflux of 3-methylhistidine from the leg was significantly decreased by 40% compared to preoperative values. In contrast, tyrosine efflux was doubled at the same time as a transient 20% increase in oxygen uptake of the leg and a marked increase in catecholamine excretion were observed. These changes coincided with a 40% elevation in the excretion of both 3-methylhistidine and nitrogen. Leg metabolism returned to the preoperative pattern within a week. These results suggest that the loss of amino acids from the lean tissues of the leg is the result of a fall in protein synthesis accompanied by an adaptive fall in protein breakdown. Although the increase in nitrogen excretion in response to major surgical trauma reflects the negative amino acid balance of skeletal muscle, the changes in urinary 3-methylhistidine do not correlate with changes in efflux of 3-methylhistidine from the leg. These results suggest that the use of 3-methylhistidine excretion as a specific index of skeletal muscle protein breakdown in postoperative patients may be invalid. Tissues other than skeletal muscle appear to make a substantial contribution to the 3-methylhistidine excretion postoperatively.

Increased protein, tyrosine and 3-methylhistidine content has been observed in the skeletal muscles of rats 2-24 h after a 10-hour swimming period. This was accompanied by a significant rise in 3-methylhistidine excretion during the second day of the recovery period. Such combination of alterations suggests simultaneous augmentation of both protein synthesis and decomposition in the muscles after active work. The start of the alterations coincides with post-exercise increase of blood corticosterone level (2-6 h after work) and with the achievement of glycogen supercompensation in the liver and muscles.

1. Critical studies on the distribution of Ntau-methylhistidine (3-methylhistidine; Me-His) among organs and tissues in adult rats are reported. Adult rats contained 46-5+/-3-6 mg Me-His/kg body-weight. Almost 90% of the Me-His in the body was recovered from skeletal muscle. These results support the hypothesis that fractional catabolic rates of myosin and actin in skeletal muscle can be estimated by measuring urinary excretion of Me-His. 2. Dietary protein level did not affect the total amount of Me-His in the body. However, urinary excretion of Me-His increased as dietary protein intake was increased. 3. From these results it was concluded that fractional catabolic rates of myosin and actin increase as dietary protein intake increases.

We used the perfused hemicorpus preparation to measure individual rates of protein synthesis and degradation. Using fed animals, perfused either with or without insulin, muscle protein synthesis and hemicorpus protein degradation rates were similar, but myofibrillar protein degradation was clearly increased in the uremic preparations. When the animals were fasted, differences in the rates of skeletal muscle protein turnover were apparent. Uremic rats lost more wt at both 24 and 48 hr of fasting when compared to either ad libitum fed or pair-fed controls who started fasting at body wts equivalent to our uremic rats. The accelerated wt loss was accompanied by lower rates of protein synthesis, higher degradation rates, and greater net protein catabolism in our uremic rats. Alterations in body lipid content were present in uremia and correlated with the rate of protein degradation in both control and uremic rats. These data demonstrated that even in the fed state, uremia is associated with subtle alterations in skeletal muscle protein turnover. When stressed, these alterations become more pronounced. Insufficient stores of body lipids, either due to inadequate nutrition or altered metabolism, may contribute to the alterations in muscle protein turnover seen in chronic renal insufficiency.

The relative contributions of skeletal muscle, gastrointestinal tract, and skin to urinary N tau-methylhistidine (MH) excretion were estimated during in vitro studies using the rat hemicorpus preparation. After 0.5 h of perfusion, MH release into the perfusate was linear for 3 h and averaged 29.8 nmol . h-1 . 100 g hemicorpus-1. In vivo, 24-h urinary MH excretion averaged 37.3 nmol . h-1 . 100 g body wt-1. The ratio of soft tissue to skin weight is equal (3.2:1) in the whole rat and in the hemicorpus. The gastrointestinal tract released 16.0 nmol . h-1 . 100 g body wt-1 or approximately 41% of the total urinary MH excretion. Preparations perfused with or without skin showed modest differences in the rate of MH release that were not statistically significant. Skeletal muscle contains 89.8% of total body MH content, whereas gastrointestinal tract and skin contain 3.8 and 6.4%, respectively. Gastrointestinal tract actomyosin turns over rapidly with a fractional catabolic rate of 24%/day versus 1.4%/day for skeletal muscle actomyosin.

The influence of contractile activity on protein degradation and amino acid metabolism in skeletal muscle was investigated by utilizing an in-vitro electrical stimulation model with the rat epitrochlearis muscle preparation. Graded decreases in contraction force and in the muscle content of ATP and PCr, and increases in lactate were recorded with different rates of stimulation (1 h) and with both isometric twitches and tetanic contractions. 3-Methylhistidine and phenylalanine were chosen as indicators of myofibrillar and total protein degradation, respectively. The release of 3-methylhistidine was significantly stimulated by contractile activity, but a significant increase in the total amount of this amino acid (released amount + tissue content) occurred only at the most intense contraction rates. The release rate, tissue content and total amount of phenylalanine were not influenced by the contractions. Glutamate formation was generally inhibited, but its release was increased. Alanine synthesis was increased in moderately and intensely stimulated muscles. Glutamine and glycine were not influenced by the contractions, however. Inhibition of protein synthesis did not significantly influence protein degradation or amino acid release. The data suggest that in the absence of anabolic factors in the medium, myofibrillar protein degradation is increased in heavily activated muscle. This takes place without total protein breakdown being affected.

Direct measurement of N tau-methylhistidine turnover in skeletal muscle, skin and gastrointestinal muscle indicates that these three tissues contribute only 24.9, 6.8 and 9.8% of the total urinary excretion. Measurement of the decay rate of radioactively labelled N tau-methylhistidine in urine indicates that skeletal muscle accounts for 74.5% of the urinary excretion and this is probably an overestimate. These results suggest that the common assumption, that N tau-methylhistidine in urine originates almost entirely from skeletal muscle, may be wrong.

1. The amount of 3-methylhistidine (3-MeH) has been measured in eighty-eight samples of tissue taken post-mortem from five adults. 2. The highest concentration (mumol/g fat-free dry weight) of 3-MeH was in skeletal muscle (3.31 +/- 0.05); intermediate values (2-3) were found in cardiac muscle and those tissues containing smooth muscle; and low values (less than 1) occurred in parenchymal tissues such as liver and kidney. 3. There was little variation between the mean 3-MeH content of striated muscles in different individuals, and no significant difference between the 3-MeH concentrations of striated muscles taken from six different sites. 4. The results suggest that it is justifiable to use values obtained from single muscles to calculate the rate of myofibrillar breakdown from urinary 3-MeH excretion.

We determined the effects of feeding, starvation, and glucose infusion after starvation in newborn guinea pigs. We determined the rate of 14C-leucine incorporation into skeletal muscle (KS) as a measure of muscle protein synthesis and the rate of excretion of 3-methylhistidine as a measure of muscle myofibrillar protein catabolism (Kc). Fed newborns, who were in positive nitrogen balance, had the highest Ks and lowest Kc, while starved newborns had the lowest Ks and highest Kc. Infusing glucose after starvation decreased net protein catabolism and Kc, but did not increase Ks. The magnitude of change of Kc in response to starvation and glucose infusion was much greater than Ks. Changes in catabolic rate may influence net muscle protein balance to a greater degree than changes in synthetic rate.

In this review paper various aspects of protein synthesis and breakdown in skeletal muscle will be surveyed as an introduction to the more specialized topics concerned with anabolic agents and the production of muscle protein. The quantitatively important role of skeletal muscle in total protein and amino acid metabolism will be evaluated by considering the size, composition (RNA and protein) and metabolic activity of this tissue in mammalian organism. This will provide an initial basis for understanding how internal and external environmental factors may affect the rate of growth and final size of the muscle mass in the intact organism. The steps in protein synthesis, involving aminoacid activation, initiation, peptide chain elongation and termination will be described, with particular reference to the synthesis of protein in fibers of skeletal muscle. This account will include a review of the specificity of myofibrillar protein synthesis and the relationships between the biochemical aspects of protein synthesis and the structural organization of the muscle cell. The role of messenger RNA, tRNA and soluble protein factors in the regulation of muscle protein synthesis in the growing animal will be reviewed, together with a short account of the ways in which hormones may modulate the rate of protein synthesis in muscle cells. However, the available data do not lead to definite conclusions concerning the rate limiting step(s) in muscle protein synthesis or the quantitative significance of factors which affect them. The mechanisms and regulation of protein degradation and the role of degradation in the regulation of muscle protein content will also be discussed.

The content of 3-methylhistidine (3MH) and creatine was measured in the heart and in nine skeletal muscles of control and dystrophic Re 129/J mice. The mean 3MH level per unit of non-collagen protein (NCP) was significantly lower than normal in all but the heart muscles of dystrophic mice.

Because high calcium concentration in vitro stimulates muscle proteolysis, calcium has been implicated in the pathogenesis of increased muscle breakdown in different catabolic conditions. Protein breakdown in skeletal muscle is increased during sepsis, but the effect of sepsis on muscle calcium uptake and content is not known. In this study the influence of sepsis, induced in rats by cecal ligation and puncture, on muscle calcium uptake and content was studied. Sixteen hours after cecal ligation and puncture or sham operation, calcium content of the extensor digitorum longus (EDL) and soleus (SOL) muscles was determined with an atomic absorption spectrometer. Calcium uptake was measured in intact SOL muscles incubated in the presence of calcium 45 (45Ca) for between 1 and 120 minutes. Total and myofibrillar protein breakdown was determined in SOL muscles, incubated in the presence of different calcium concentrations (0; 2.5; 5.0 mmol/L), and measured as release into the incubation medium of tyrosine and 3-methylhistidine (3-MH), respectively. Calcium content was increased by 51% (p less than 0.001) during sepsis in SOL and by 10% (p less than 0.05) in EDL muscle. There was no difference in 45Ca uptake between control and septic muscles during the early phase (1 to 5 minutes) of incubation. During more extended incubation (30 to 120 minutes), muscles from septic rats took up significantly more 45Ca than control muscles (p less than 0.05). Tyrosine release by incubated SOL muscles from control and septic rats was increased when calcium was added to the incubation medium, and at a calcium concentration of 2.5 mmol/L, the increase in tyrosine release was greater in septic than in control muscle. Addition of calcium to the incubation medium did not affect 3-MH release in control or septic muscle. The results suggest that calcium uptake and content in skeletal muscle are increased during sepsis and that high calcium concentrations in vitro stimulate nonmyofibrillar protein breakdown. Muscles from septic animals may be more sensitive to the effect of calcium in vitro than muscles from nonseptic rats. Whether increased calcium uptake and content in skeletal muscle is partly responsible for accelerated muscle proteolysis during sepsis remains to be determined.

Actin and myosin, the contractile proteins of skeletal muscle, are methylated following peptide bond synthesis, with production of Ntau-methylhistidine (3-methylhistidine, 3-MeHis). During intracellular breakdown of these proteins, the 3-MeHis is released and excreted in the urine. Studies on tissue distribution of 3-MeHis and on its qunatitative excretion following administration to rats and to man show that urinary output of this amino acid provides a reliable index of the rate of myofibrillar protein breakdown in the musculature of intact rats and human subjects. Estimates of the fractional rate of muscle protein breakdown based on 3-MeHis data are consistent with rates computed by other techniques. By this technique, it has been shown that the fractional rate of muscle protein breakdown is not significantly different in the elderly as compared with young adults. However, since muscle mass is less in the elderly, it makes a smaller contribution to whole body protein breakdown with aging in humans. Output of 3-MeHis diminishes in growing rats and obese human subjects with protein or energy restriction, though the initial response of myofibrillar protein breakdown in growing rats to protein and protein-energy restriction differs. Measurement of 3-MeHis excretion has also proved useful in exploring the effects of physical and thermal trauma on the rate of muscle useful in exploring the effects of physical and thermal trauma on the rate of muscle protein breakdown.

The rate of myofibrillar protein degradation was measured by 3-methylhistidine (3MH) release in moderately uremic and sham-operated control rats. The rats were studied in the fed state or after 24 and 48 hours of fasting. When fed, both uremic and control rats gained weight at the same rate. During 48 hours of fasting, the uremic animals lost more weight (17.1%) than the shams (13.2%). The ratio of gastrocnemius muscle protein per gram of wet weight was not significantly different under any conditions. When fed and 48-hour fasted animals are compared, urinary excretion of 3MH rose from 0.81 to 1.32 moles per 24 hr per 100 g of initial body wt shams and from 0.90 to 1.30 moles per 24 hr per 100 g of initial body wt in the uremics. When ratio of 3MH to creatinine excretion in the urine was compared, there was no change in 3MH excretion between 0 and 24 hours, but there was an increase between 24 and 48 hours. Analysis of serum and muscle samples from fed and 48-hours fasted animals revealed that the concentrations of 3MH increased in both groups during fasting, with the uremic rats having a large increase in the total body 3MH pool. Myofibrillar degradation rates calculated from the sum of urinary excretion plus the changes in body 3MH pool size revealed that with moderate fasting, degradation rates increased in both sham and uremic rats with a larger increase being seen in the uremic group. After 48 hours of fasting, the increased amount of 3MH released from muscle of uremic rats was comparable to their larger percentage weight loss.

The daily flux of amino acids in the body is extensive. Protein synthesis is estimated to be 300 g daily in an adult man. This requires uptake and release of 150 g essential amino acids, yet the dietary requirement for essential amino acids in only 6 g. This indicates extensive and efficient recycling of essential amino acids released by protein breakdown. The catabolism of essential amino acids by the liver is sensitively regulated in relation to requirements. A study of availability of tryptophan to rats receiving various levels of tryptophan in the diet shows that plasma tryptophan increases only when intake exceeds requirements and at these higher levels of intake tryptophan oxygenase activity in the liver becomes increased shortly after meals. In addition, the carbohydrate content of the diet causes tryptophan to become deposited in the free amino acid pool of muscle through an insulin-dependent mechanism. Dietary carbohydrate also effects plasma tryptophan due to a fall in the plasma level of non-esterified fatty acids which compete with tryptophan for binding sites on serum albumin. Consequently, after carbohydrate the proportion of plasma tryptophan bound to serum albumin increases, so that there is less nonbound tryptophan in the plasma. The metabolic significance of this has yet to be demonstrated. Finally, protein metabolism in skeletal muscle exhibits considerable efficiency of reutilization of essential amino acids, since the main products passing into the blood are alanine and glutamine. It has been shown that 3-methylhistidine present in muscle protein in not reutilized for synthesis of protein and that its excretion in the urine can provide a useful index of muscle catabolism. In prolonged starvation of adults or protein deficiency in children, output of 3-methylhistidine is much reduced, suggesting an adaptive reduction in muscle protein catabolism. It is emphasized that, because of its function in monitoring dietary amino acid intake, liver protein metabolism responds rapidly to changes in protein intake and in consequence protein deficiency causes early depletion, whereas muscle protein undergoes depletion later and is subject to adaptive processes that restrict the loss.

Measurements have been made of protein turnover, RNA and DNA in thymus and skeletal muscle from rats fed on a zinc-deficient diet (ZD) for 10 and 17 d, in pair-fed controls (CI) and in muscle from rats fed on the ZD diet for 24 d and then fed on restricted amounts of the deficient diet with (RIZS) or without (RIZD) Zn supplementation, for 8 d. In thymus the ZD diet induced a loss of DNA and protein which was not observed with the CI rats. Accumulation of RNA was less affected but protein synthesis was reduced. In muscle the accumulation of DNA and protein was slowed by the ZD diet, particularly in glycolytic muscles compared with oxidative muscles, and Zn supplementation increased DNA and protein. Protein synthesis and RNA concentrations were reduced in the ZD rats compared with the CI rats, but Zn supplementation at constant restricted food intake did not increase protein synthesis. Muscle protein synthesis per unit RNA varied markedly in the ZD rats after 10 d when the characteristic cycling of the food intakes and body-weight was most pronounced, the highest values being observed in the anabolic phase of the cycle although these were less than values for well-fed controls. The variability was inversely correlated with the plasma Zn levels. The extent of the variability was much less after 17 d and was not apparent in the food-restricted ZD animals. Protein degradation in muscle, assessed as the difference between overall and net protein synthesis, was faster in the ZD rats compared with the CI rats and fluctuated considerably, partly accounting for the cyclic changes in muscle after 10 d, and was entirely responsible after 17 d. The concentration of muscle-free 3-methylhistidine and its urinary excretion rate indicated inconsistent results which could not be satisfactorily interpreted. Plasma insulin was reduced in the ZD rats compared with the CI rats and was insensitive to food intake in contrast to urinary corticosterone excretion which was inversely correlated with the cyclic changes in body-weight and food intake. Furthermore, adrenalectomized rats exhibited increased mortality and reduced cycling of body-weight and food intake. Thus Zn deficiency impairs growth by a combination of reduced food intake, a reduced anabolic response to food due to a reduced capacity for protein synthesis and reduced activation of protein synthesis, possibly reflecting impaired insulin secretion, and an increased catabolic response to the reduced intake in which corticosterone may play a role.

During four days of fasting in rats skeletal muscle protein synthesis fell progressively, whereas skeletal muscle protein breakdown was unchanged until the third and fourth days when it rose dramatically. In contrast, the synthetic rate of smooth muscle protein was unchanged during three days of fasting despite a loss of protein content, indicating an abrupt rise in protein breakdown in this tissue on the first day of fasting which was sustained thereafter. Urinary excretion of N tau-methylhistidine was significantly increased throughout fasting. The concentration of free N tau-methylhistidine in plasma and in muscle tissue was elevated throughout the period of fasting. This elevation was not caused by reduced renal clearance, but appears to have been mainly the result of increased breakdown of N tau-methylhistidine-containing proteins in tissues other than skeletal muscle.

Four groups of adult cockerels, consisting of a control group given adequate protein, a protein-depleted group, and two groups that had been given, respectively, a low and a higher nitrogen-containing diet following protein depletion, were killed after appropriate time intervals and histidine and histidine derivatives analyzed in breast muscle extract. The concentration of l-methylhistidine increased more than 12-fold during protein depletion and returned to near control levels during repletion. Carnosine decreased during depletion to less than 1/20 of the concentration in controls, yet increased more rapidly during repletion in the group fed a low-nitrogen diet free of histidine compared to the group fed a high-nitrogen diet containing histidine. Depleted cocks showed an elevation in the concentration of free histidine which decreased toward control levels after repletion. Levels of 3-methylhistidine were highest in the cocks fed diets containing histidine. Evidence for an accumulation in the breast muscle of adult cocks of 3-methylhistidine consisted of unusually high ratios of this derivative to muscle protein in all experimental groups. It was suggested that both 3-methylhistidine and 1-methylhistidine might serve as useful indices for assessing protein depletion in the adult cockerel, but that consideration should be given to the possibility of species differences in the metabolism of histidine derivatives during protein depletion.