Leucine Oxidation Research Articles

Epinephrine does not impair utilization of exogenous amino acids in humans

The effect of epinephrine on leucine and phenylalanine kinetics was measured by using the stable isotope amino acid tracers L-[1-(13)C]leucine and L-[phenyl-2H5]-phenylalanine in the postabsorptive state and during the intravenous administration of a standard amino acid solution with respect to the amino acid load. Infusion of epinephrine (plasma concentration: approximately 3600 pmol/L) decreased leucine and phenylalanine and increased ketoisocaproate plasma concentrations and increased the metabolic clearance rate of leucine and phenylalanine. Epinephrine neither influenced leucine or phenylalanine flux nor leucine oxidation or leucine net balance. Hyperaminoacidemia from amino acid infusion reduced endogenous leucine release and stimulated leucine oxidation and nonoxidative disposal of leucine, resulting in a dose-dependent increase in leucine net balance. Epinephrine did not influence any changes in amino acid kinetics during parenteral amino acid administration. Therefore, we conclude that epinephrine had no catabolic effects on amino acid metabolism and no negative effect on the utilization of a parenterally offered amino acid solution in healthy humans.

Mechanisms permitting nephrotic patients to achieve nitrogen equilibrium with a protein-restricted diet.

Clinical experience suggests nephrotic patients are at risk for malnutrition. To determine if nephrotic patients can adapt successfully to a protein-restricted diet, nephrotic (glomerular filtration rate, 52+/-15 ml/min; urinary protein [Uprot.], 7.2+/-2.2 grams/d) and control subjects completed a crossover comparison of diets providing 0.8 or 1.6 grams protein (plus 1 gram protein/gram Uprot.) and 35 kcal per kg per day. Nitrogen balance (BN) was determined and whole body protein turnover measured during fasting and feeding using intravenous -[1-13C]leucine and intragastric -[5,5, 5- 2H3]leucine. BN was positive in both nephrotic and control subjects consuming either diet and rates of whole-body protein synthesis, protein degradation, and leucine oxidation did not differ between groups. In both nephrotic and control subjects anabolism was due to a suppression of whole-body protein degradation and stimulation of protein synthesis during feeding. The principal compensatory response to dietary protein restriction was a decrease in amino acid oxidation and this response was the same in both groups. With the low protein diet leucine oxidation rates during feeding correlated inversely with Uprot. losses (r = -0.83; P < 0. 05). (a) a diet providing 0.8 gram protein (plus 1 gram protein/gram Uprot.) and 35 kcal per kg per day maintains BN in nephrotic patients; (b) nephrotic patients activate normal anabolic responses to dietary protein restriction (suppression of amino acid oxidation) and feeding (stimulation of protein synthesis and inhibition of protein degradation); (c) the inverse correlation between leucine oxidation and Uprot. losses suggests that proteinuria is a stimulus to conserve dietary essential amino acids.

Leucine metabolism in partially hepatectomized rats

We hypothesized that the decrease in plasma branched-chain amino acids (i.e. valine, leucine and isoleucine) and the increase in the oxidized leucine fraction demonstrated in cirrhotic rats in our previous study were caused by the reduced liver cell mass. In the present study we have evaluated the influence of the loss of a substantial amount of the hepatic tissue on changes in leucine metabolism. A two-thirds partial hepatectomy (PH) was performed in male Wistar rats, weighing 210-250 g. Sham-operated rats served as controls. Whole-body leucine kinetics and ketoisocaproate oxidation rates in the isolated perfused liver were investigated using continuous infusion of [1-14C]leucine and alpha-keto[1-14C]ketoisocaproate at 0 h, 24 h and 72 h after surgery. All groups were compared by analysis of variance, and differences were considered significant at the p < 0.05 level. A significant decrease in the sum of branched-chain amino acids in blood plasma was observed at 24 h after PH. The decrease in whole-body leucine utilization in protein synthesis observed at 24 h after PH was associated with a decrease in protein synthesis in the gastrocnemius muscle, in the small intestine and in the liver remnant (although protein synthesis per mg of liver protein was higher than in sham-treated animals). In contrast, the rate of whole-body leucine oxidation increased immediately after PH (PH: 4.5 +/- 0.7 vs. sham: 2.4 +/- 0.4; mumol .100 g b.w.-1.h-1). As a result of the opposite changes in protein synthesis and leucine oxidation, marked increases in oxidized leucine fraction were observed immediately (14.6 +/- 1.5%) and 24 h (15.1 +/- 1.6%) after PH in comparison to the sham-treated rats (7.1 +/- 0.8%). In isolated perfused livers of PH rats, an increase in ketoisocaproate oxidation per liver weight unit was observed at 24 h and 72 h in comparison to the sham group. The loss of liver capacity for ketoisocaproate oxidation was restored at 72 h after PH, although the liver weight did not reach the preoperative value. We conclude that the loss of hepatic tissue results in an increase in leucine oxidized fraction that is caused by both a decrease in protein synthesis and an increase in leucine oxidation. Both the liver remnant and the extrahepatic tissues are involved in this response.

TESTOSTERONE ATTENUATES THE ACCELERATED WHOLE BODY PROTEIN TURNOVER ASSOCIATED WITH PROLONGED BEDREST 1152

A decreased muscle protein synthesis, loss of body nitrogen, and reduction in lean body and muscle mass are all consequences of prolonged bedrest. Such physical inactivity has also resulted in structural and functional deficits in the musculoskeletal system. The purpose of this study was to determine whether testosterone administration during prolonged bedrest would attenuate the increased protein turnover and loss of body nitrogen. Fourteen healthy men were studied during 28d of strict bedrest at 6° headdown tilt. To further accelerate protein and bone turnover all subjects received a daily oral dose of triodothyronine (50 μg/d, T3). Six of the men were randomized to receive testosterone enanthate (T) at a dose 200 mg/wk. During the first three days of bedrest for the T treated subjects, a loading dose of 80 mg/d was given. T was administered by intramuscular injection and was sufficient to elevate serum testosterone to a high normal range (1200 - 1800 ng/dl) by the third week of bedrest. Prior to and on the 26th day of bedrest, fasted state whole body leucine kinetics were determined during a 3 hr primed constant infusion of L[1-13C]leucine. RMR and VCO2 during the test were determined by indirect calorimetry, expired breath 13CO2 was determined with IRMS, and 13C enrichment in α-ketoisocaproic acid was determined by GC/MS. Baseline leucine Ra for all subjects was 156.7 ± 15.5μmol/kg LBM/hr while baseline leucine oxidation was 28.8 ± 6.1μmol/kg LBM/hr. The combined bedrest and T3 treatment resulted in a 20 and 33% increase in leucine Ra and oxidation, respectively. In T treated subjects, leucine Ra increased by only 11% while leucine oxidationdecreased by 10%. The net result being a less net negative protein balance in T treated subjects (i.e., synthesis - breakdown = -28.3 ± 4.1 μmol/kg LBM/hr in T vs. -36.3 ± 5.4 in non-treated subjects; p< 0.05). We conclude that T treatment is sufficient to attenuate the protein catabolic consequences of prolonged bedrest.

Effects of diphenyliodonium chloride on proteolysis and leucine metabolism by rumen microorganisms

Diphenyliodonium chloride (DIC) at 0.3 and 0.8 mM inhibited hydrolysis of casein by a rumen microbial ecosystem unadapted to DIC by as much as 30%. Adaptive effects to DIC on leucine oxidation over 37 days were evaluated in a batch culture system repetitively diluted with culture media devoid of protein but containing carbon and nitrogen sources essential for growth. DIC concentrations up to 0.1 mM had no effect on concentrations of microbial protein during the 37 day period of semi-continuous culture. Concentrations of ammonia were depressed (P ≤ 0.05) by day 5 after initiating an influx of 0.1 mM DIC while other levels of DIC were without effect. Effects of DIC on oxidation of leucine were determined in aliquots of removed culture to which [1-14C]-leucine was added. Leucine oxidation was assessed as the 14CO2 evolved during a 1 h fermentation with [1-14C]-leucine and subsequent peroxide oxidation of the residual [1-14C]-α-ketoisocaporate (KIC). The proportion of 14CO2 recovered during fermentation and from KIC was essentially equal (regression coefficient of 1.0244 ± 0.04) and did not differ (P < 0.05) due to level of DIC or days of culture. The results suggests that leucine was deaminated via oxidative deamination to KIC followed by oxidative decarboxylation of KIC to isovalerate. These results illustrate that DIC can completely inhibit leucine degradation without detrimental effects on microbial growth in culture systems derived from rumen fluid that are allowed to adapt to DIC.

Effect of Quantity of Concomitant Energy (E) Intake of Low Birth Weight (LBW) Infants on Leucine Oxidation † 1388

We have shown that an E intake of 150 vs. 120 kcal/kg·d enhances protein utilization by LBW infants (Kashyap, et al., Pediatr Res 1994; 35:704). To determine the effect of quality of E intake on protein utilization, LBW infants (BW, 750-1600g were assigned randomly and blindly to receive one of 5 formulas differing only in quantity and quality of energy. Groups 1, 2 and 3 received formulas providing an E intake of 120 kcal/kg·d with 35, 50 and 65% of non-protein E, respectively, as carbohydrate (lactose + glucose polymers (CHO) and the remainder as fat; Groups 4 and 5 received an E intake of 150 kcal/kg·d with 35 and 65% of non-protein E, respectively, as CHO. Protein intake of all groups was 4g/kg·d. Leucine oxidation (i.e., the cumulative percent of a bolus dose of 1-13C-Leu recovered in breath from 0-8h) was determined in subsets of all groups (n=4-7) just prior to discharge. Leucine oxidation of Groups 1-5, respectively, was 24.2±5%a, 19.3±2.2%b, 14.8%c 13.7 ±2.9%c and 13.2±9,8%c of intake (values with different superscripts are statistically significant). Thus, both quantity and quality of concomitant E intake appear to affect protein utilization as measured by leucine oxidation. At E intakes of 120 kcal/kg·d, increasing the CHO content of non-protein E from 35% to 50% and from 50-65% increases protein utilization. A total E intake of 150 kcal/kg·d improves utilization relative to 120 kcal/kg·d with 35 and 50 but not 65% CHO. This suggests that increasing the CHO content of an E intake of 120 kcal/kg·d is as effective in promoting protein utilization as increasing E intake to 150 kcal/kg·d regardless of CHO content.

Correction of acidosis in hemodialysis decreases whole-body protein degradation.

Correction of acidosis in hemodialysis (HD) decreases protein degradation. The effect of the correction of chronic metabolic acidosis in chronic renal failure patients treated with HD was determined from the kinetics of infused L-[1-(13)C]leucine. Six HD patients were studied before (acid) and after (bicarbonate) correction of acidosis (pH: acid 7.36 +/- 0.01, bicarbonate 7.40 +/- 0.01, P < 0.005). Leucine appearance from body protein (PD) and leucine disappearance into body protein (PS) decreased significantly with correction of acidosis (PD: acid 180.6 +/- 7.3, bicarbonate 130.9 +/- 7.2 mumol.kg-1.h-1, P < 0.005; PS: acid 172.3 +/- 6.8, bicarbonate 122.0 +/- 6.8 mumol.kg-1.h-1, P < 0.005). There was no significant change in leucine oxidation or plasma amino acid concentrations. These results demonstrate that optimal correction of acidosis in HD is beneficial in terms of protein turnover and may improve long-term nutritional status in HD.

AMINO ACIDS DO NOT SUPPRESS PROTEOLYSIS IN PRETERM NEONATES. • 1413

We have recently demonstrated dose dependent suppression of proteolysis with graded amino acid (aa) infusion in healthy term neonates (Am J Physiol, 1997). Rates of proteolysis are higher in preterm individuals, perhaps related to a greater need for aa supply for tissue remodeling and rapid growth. We hypothesized that preterm neonates would be resistant to suppression of proteolysis in response to aa infusion. To assess this, we measured the endogenous rates of appearance (Ra) of the essential aa's leucine (LEU) and phenylalanine (PHE) (reflecting proteolysis), LEU oxidation (LEU OX) and phenylalanine hydroxylation (PHE OH) (reflecting irreversible losses), and utilization of LEU and PHE for protein synthesis (PS) in 7 clinically stable preterm infants (32±0.5 wks gestation, 1.5±0.1 kg birth wt, 1.4±0.1 kg study wt, 6±1 days of age) during a basal glucose infusion (6mg/kg/min) and in response to a graded infusion of aa's (Aminosyn PF-1.2 & 2.5gm/kg/day).

Protein metabolism in children with edematous malnutrition and acute lower respiratory infection

This study tested the hypothesis that wholebody protein kinetics remain low in children with edematous malnutrition and acute infection. Thirteen children with edematous malnutrition and acute infection (subjects) were compared with 14 uninfected children with edematous malnutrition early in recovery (control children). Protein kinetics were determined by using a primed, constant intravenous infusion of [13C]leucine and [15N2]urea in the postabsorptive state. Calculations of rates of whole-body protein synthesis and breakdown were based on the rate of leucine appearance; the rate of leucine oxidation was estimated from the rate of urea appearance. Protein synthesis and breakdown rates were lower in subjects than in control children (97 +/- 30 compared with 153 +/- 67, P < 0.01, and 103 +/- 30 compared with 160 +/- 67 mumol leucine.kg-1.h-1, P < 0.01). No difference was found between the two groups in the rate of urea appearance, but this analysis only had a statistical power of 54%. The absence of the expected increase in the rate of protein turnover during acute infection in edematous malnutrition implies that acute phase proteins are made with a corresponding depletion of muscle, hepatic, and other body proteins such as albumin, and that there may also be a blunting of the acute phase response.

Amino acids suppress proteolysis independent of insulin throughout the neonatal period.

To determine how increased amino acid availability alters rates of whole body proteolysis and the irreversible catabolism of the essential amino acids leucine and phenylalanine throughout the neonatal period, leucine and phenylalanine kinetics were measured under basal conditions and in response to intravenous amino acids in two separate groups of healthy, full-term newborns (at 3 days and 3 wk of age). The endogenous rates of appearance of leucine and phenylalanine (reflecting proteolysis) were suppressed equally in both groups and in a dose-dependent fashion (by approximately 10% with 1.2 g x kg(-1) x day(-1) and by approximately 20% with 2.4 g x kg(-1) x day(-1)) in response to intravenous amino acid delivery. Insulin concentrations remained unchanged from basal values during amino acid administration. The irreversible catabolism of leucine and phenylalanine increased in a stepwise fashion in response to intravenous amino acids; again, no differences were observed between the two groups. This study clearly demonstrates that the capacity to acutely increase rates of leucine oxidation and phenylalanine hydroxylation is fully present early in the neonatal period in normal newborns. Furthermore, these data suggest that amino acid availability is a primary regulator of proteolysis in normal newborns throughout the neonatal period.

Metabolic effects of liver transplantation in cirrhotic patients.

To assess whether liver transplantation (LTx) can correct the metabolic alterations of chronic liver disease, 14 patients (LTx-5) were studied 5+/-1 mo after LTx, 9 patients (LTx-13) 13+/-1 mo after LTx, and 10 patients (LTx-26) 26+/-2 months after LTx. Subjects with chronic uveitis (CU) and healthy volunteers (CON) were also studied. Basal plasma leucine and branched-chain amino acids were reduced in LTx-5, LTx-13, and LTx-26 when compared with CU and CON (P < 0.01). The basal free fatty acids (FFA) were reduced in LTx-26 with respect to CON (P < 0.01). To assess protein metabolism, LTx-5, LTx-13, and LTx-26 were studied with the [1-14C]leucine turnover combined with a 40-mU/m2 per min insulin clamp. To relate changes in FFA metabolism to glucose metabolism, eight LTx-26 were studied with the [1-14C]palmitate and [3-3H]glucose turnovers combined with a two-step (8 and 40 mU/m2 per min) euglycemic insulin clamp. In the postabsorptive state, LTx-5 had lower endogenous leucine flux (ELF) (P < 0.005), lower leucine oxidation (LO) (P < 0.004), and lower non-oxidative leucine disposal (NOLD) (P < 0.03) with respect to CON (primary pool model). At 2 yr (LTx-26) both ELF (P < 0.001 vs. LTx-5) and NOLD (P < 0.01 vs. LTx-5) were normalized, but not LO (P < 0.001 vs. CON) (primary and reciprocal pool models). Suppression of ELF by insulin (delta-reduction) was impaired in LTx-5 and LTx-13 when compared with CU and CON (P < 0.01), but normalized in LTx-26 (P < 0.004 vs. LTx-5 and P = 0.3 vs. CON). The basal FFA turnover rate was decreased in LTx-26 (P < 0.01) and CU (P < 0.02) vs. CON. LTx-26 showed a lower FFA oxidation rate than CON (P < 0.02). Tissue glucose disposal was impaired in LTx-5 (P < 0.005) and LTx-13 (P < 0.03), but not in LTx-26 when compared to CON. LTx-26 had normal basal and insulin-modulated endogenous glucose production. In conclusion, LTx have impaired insulin-stimulated glucose, FFA, and protein metabolism 5 mo after surgery. Follow-up at 26 mo results in (a) normalization of insulin-dependent glucose metabolism, most likely related to the reduction of prednisone dose, and, (b) maintenance of some alterations in leucine and FFA metabolism, probably related to the functional denervation of the graft and to the immunosuppressive treatment.

Testosterone replacement increases fat-free mass and muscle size in hypogonadal men.

Testosterone-induced nitrogen retention in castrated male animals and sex-related differences in the size of the muscles in male and female animals have been cited as evidence that testosterone has anabolic effects. However, the effects of testosterone on body composition and muscle size have not been rigorously studied. The objective of this study was to determine the effects of replacement doses of testosterone on fat-free mass and muscle size in healthy hypogonadal men in the setting of controlled nutritional intake and exercise level. Seven hypogonadal men, 19-47 yr of age, after at least a 12-week washout from previous androgen therapy, were treated for 10 weeks with testosterone enanthate (100 mg/week) by im injections. Body weight, fat-free mass measured by underwater weighing and deuterated water dilution, and muscle size measured by magnetic resonance imaging were assessed before and after treatment. Energy and protein intake were standardized at 35 Cal/kg.day and 1.5 g/kg.day, respectively. Body weight increased significantly from 79.2 +/- 5.6 to 83.7 +/- 5.7 kg after 10 weeks of testosterone replacement therapy (weight gain, 4.5 +/- 0.6 kg; P = 0.0064). Fat-free mass, measured by underwater weighing, increased from 56.0 +/- 2.5 to 60.9 +/- 2.2 kg (change, +5.0 +/- 0.7 kg; P = 0.0004), but percent fat did not significantly change. Similar increases in fat-free mass were observed with the deuterated water method. The cross-sectional area of the triceps arm muscle increased from 2421 +/- 317 to 2721 +/- 239 mm2 (P = 0.045), and that of the quadriceps leg muscle increased from 7173 +/- 464 to 7720 +/- 454 mm2 (P = 0.0427), measured by magnetic resonance imaging. Muscle strength, assessed by one repetition maximum of weight-lifting exercises increased significantly after testosterone treatment. L-[1-13C]Leucine turnover, leucine oxidation, and nonoxidative disappearance of leucine did not significantly change after 10 weeks of treatment. There was no significant change in hemoglobin, hematocrit, creatinine, and transaminase levels. Replacement doses of testosterone increase fat-free mass and muscle size and strength in hypogonadal men. Whether androgen replacement in wasting states characterized by low testosterone levels will have similar anabolic effects remains to be studied.

Growth hormone modulates amino acid oxidation in the surgical patient: Leucine kinetics during the fasted and fed state using moderate nitrogenous and caloric diet and recombinant human growth hormone

Twelve patients (aged 70 ± 9 years) who were scheduled for resection of rectosigmoid colon adenocarcinoma but were otherwise healthy were randomly allocated after surgery to receive either peripheral parenteral nutrition alone ([PPN] n = 6) or in combination with recombinant human growth hormone (rGH) at a daily dose of 0.15 U · kg −1 · d −1 (PPN + rGH, n = 6). The daily nutritional regimen was 0.1 g nitrogen · kg −1 · d −1 and 20 kcal · kg −1 · d −1 (nonprotein energy was supplied as 60% lipid and 40% carbohydrate), and it was maintained for 6 days before and 6 days after surgery. Protein kinetics were studied in all 12 patients during the fasted and fed states before and 6 days after surgery using an 8-hour 13C-leucine tracer infusion. Daily urinary nitrogen, gaseous exchange, and plasma insulin, growth hormone, and insulin-like growth factor-I (IGF-I) were determined before and after surgery. Surgery was responsible for significant increases in postabsorptive whole-body protein flux and synthesis and leucine oxidation ( P < .01). Supplementation of PPN with rGH contributed to a significant attenuation of the postoperative increase in leucine oxidation ( P = .02), with a significant increase in whole-body protein synthesis ( P = .02) and no effect on protein breakdown ( P = .40). During the fed state, leucine oxidation increased significantly ( P =.005), with the greatest change occurring in the PPN group. Feeding was associated with a significant decrease in whole-body protein breakdown before and after surgery in both groups ( P = .001). Postoperative urinary nitrogen excretion was lower but was not statistically significant in the PPN + rGH group compared with the PPN group. There was a significant increase in oxygen consumption (Vo 2) and carbon dioxide production (Vco 2) as a result of feeding and surgery ( P < .01). Supplementation with rGH caused a decrease in the respiratory quotient (RQ) ( P = .04), particularly after surgery, indicating a direct effect of rGH on fatty acid oxidation. Circulating plasma insulin increased significantly in both groups with feeding and rGH supplementation ( P < .05). This was enhanced after surgery, particularly in the rGH group ( P < .05). Plasma growth hormone decreased after surgery in the PPN group ( P < .05), but did not change as a result of feeding. The circulating levels increased in the PPN + rGH group following subcutaneous administration before or after surgery. Plasma IGF-I decreased after surgery in the PPN group ( P < .05), and no changes occurred in the PPN + rGH group with feeding. The present findings suggest a distinct positive effect of rGH on protein synthesis in catabolic patients receiving a moderate intake of nitrogen and calories. This is achieved by modulation of amino acid oxidation. The acute effect of intravenous (IV) nutrients on protein metabolism during the catabolic phase of surgical stress caused a direct decrease in protein breakdown with no effect on protein synthesis.

Amino acid metabolism in rat hippocampus during the period of brain growth spurt.

We studied protein synthesis, lipid synthesis and CO2 production by oxidation of glycine, alanine and leucine by slices of rat hippocampus during the period of brain growth spurt. The metabolism of the three amino acids decreased with the age of the animals. A major reduction was observed in protein synthesis, which was 4 times higher at 7 days of age than at 21 days of age for all amino acids studied. Glycine oxidation to CO2 was twice as high as alanine oxidation and ten times higher than leucine oxidation. The major pathway of leucine utilization was incorporation into proteins. Glycine was the amino acid that had the highest metabolic rate.

Effect of Intravenous Amino Acid Infusion on Leucine Oxidation Across the Mammary Gland of the Lactating Goat

Changes in the kinetics of leucine in the mammary gland were examined in four lactating goats (25, 38, 45, and 135 DIM) that were given an i.v. infusion of a mixture of 18 AA, not including leucine, to alter the availability of leucine to the gland relative to other AA. Arteriovenous monitoring of [1-13C]leucine kinetics across one-half of the mammary gland was conducted on the last day (d 6 or 7) of the saline (control) and the AA infusion periods. Although blood flow to the mammary gland and the arterial concentration of most AA other than leucine were increased by the AA infusion, milk and protein yields did not change. For goats in early lactation (n=3), arterial leucine concentrations fell considerably during AA infusion; however, the arteriovenous difference of leucine was maintained, resulting in uncommonly low leucine concentrations in venous plasma (8 μM). Whole body leucine flux (protein synthesis plus oxidation) was unaffected by AA infusion, but, because whole body leucine oxidation was reduced, whole body utilization of leucine for protein synthesis increased. The AA infusion reduced mammary oxidation of leucine to approximately one-third of control values. These results suggest that leucine oxidation can be reduced considerably without affecting milk protein output; thus, leucine oxidation may not be an irrevocable consequence of mammary metabolism. If catabolism of other AA either by the gland or in the whole body can be reduced, then the efficiency of milk yield can be improved.

The role of substrates in the regulation of protein metabolism

Substrates are powerful modulators of amino acid and protein turnover in vivo (Table 4). Intravenous infusions of amino acids exert a protein-anabolic effect, because they directly inhibit endogenous protein degradation and stimulate protein synthesis at the whole-body level. A stimulation of protein synthesis has been observed also at the forearm level. These changes resulted in an improvement of body and tissue protein balance, which is the ultimate goal of any nutritional intervention aimed at preserving body protein stores. In humans acute intravenous infusions of carbohydrates do not appear to affect either protein degradation or leucine oxidation. However, animal studies support the view that glucose availability spares essential amino acids at least in the fetus. The effects of hypercaloric refeeding with high-carbohydrate diets may, however, result in increased protein turnover. Lipids, in the form of long-chain fatty acids, inhibit endogenous protein breakdown and may suppress leucine oxidation in the whole body. They do not affect protein synthesis. In contrast, medium-chain fatty acids apparently increased leucine oxidation, and therefore increased net protein catabolism. Ketone bodies may be anabolic provided that fatty acid concentrations are not concurrently decreased.

Protein metabolism and liver disease

In health, the liver orchestrates the metabolism of proteins and amino acids. When the liver is diseased, the regulation of protein metabolism is frequently disturbed. The manifestations of disturbed protein metabolism in liver disease are varied and change with disease aetiology and severity. The hallmarks of protein and amino acid metabolism in liver disease are lowered concentrations of circulating branched-chain and increased concentrations of circulating aromatic amino acids with concomitantly altered amino acid kinetics. The changes in amino acid kinetics in liver disease are characterized by increased endogenous leucine flux, an indicator of protein breakdown, and leucine oxidation in the post-absorptive state (when calculated using a reciprocal-pool model and normalized for body cell mass). In addition, the increase in whole-body protein synthesis in response to an amino acid infusion may be attenuated in patients with cirrhosis. These changes are often accompanied by clinically apparent muscle wasting, manifest as protein-calorie malnutrition, and associated low levels of hepatically synthesized plasma proteins. While the pathogenesis of these changes in protein and amino acid metabolism has not been elucidated, altered levels of circulating hormones, known to affect protein metabolism, are probably important. Lowered levels of micronutrients and trace metals and elevated levels of cytokines may also play a role.

Post-prandial protein metabolism

Current post-prandial studies of amino acid metabolism and utilization are consistent with a feeding mechanism mediated primarily by insulin and amino acids, with the balance between protein conservation and net deposition dependent on the amino acid supply [1-13C]leucine post-prandial kinetic tracer studies of leucine oxidation, non-oxidative disappearance and endogenous appearance allow study of the regulation of whole-body amino acid oxidation, protein synthesis and proteolysis. On the basis of these studies it appears that for leucine oxidation, the main determinant of the efficiency of protein utilization, the overriding regulatory influence is substrate availability rather than insulin. Such substrate sensitivity is manifest throughout the physiological range of insulin down to the lowest insulin levels observed suggesting that a basal insulin need is not an important part of regulation of this important catabolic pathway. The key protein turnover response is an inhibition of proteolysis sufficient to limit any increases in amino acid levels thus limiting any increase in amino acid oxidation. It appears that the influences of amino acids and insulin on proteolysis are separate and additive and may both be receptor mediated so that extracellular amino acid levels can regulate intracellular levels. It is likely that protein synthesis is regulated by intracellular amino acid levels but post-prandial stimulation through increases in amino acid levels appears to be unhelpful because of parallel increases in amino acid oxidation. Evidence for any influence of insulin on protein synthesis has yet to be unequivocally identified.

Diurnal pattern of the interrelationships among leucine oxidation, urea production, and hydrolysis in humans.

We investigated in six healthy adult men, who received an adequate intake of protein (1 g.kg-1.day-1), the relationship among urea production, excretion, and hydrolysis. At the end of a 6-day diet-adjustment period, subjects were studied using a 24-h continuous intravenous [1-13C]leucine and [15N,15N]urea tracer protocol (A. E. El-Khoury, N. K. Fukagawa, M. Sánchez, R. H. Tsay, R. E. Gleason, T. E. Chapman, and V. R. Young. Am. J. Clin. Nutr. 59: 1000-1011, 1994) to determine rates of irreversible protein nitrogen loss and urea kinetics. By combining leucine and urea kinetic data, we found a significant degree of urea hydrolysis over the 24-h period but no evidence to support the thesis that there is a net retention or "salvage" of the urea nitrogen liberated. Our measurements revealed little or no urea hydrolysis during the fed 12-h period of the 24-h tracer protocol but substantial hydrolysis during the 12-h fasting phase. Furthermore, a mass balance model and calculations (APPENDIX) indicated that nitrogen salvage, if any, is quantitatively indistinguishable from insensible nitrogen losses and aggregate estimation errors, accounting for no more than 5% of the nitrogen intake. We conclude that urea hydrolysis, via the intestinal microflora, although representing a component of the overall cycles of nitrogen flow within the body, does not contribute via a net retention of amino nitrogen to the maintenance of body nitrogen homeostasis in healthy adults consuming an adequate diet.

Growth hormone increases muscle mass and strength but does not rejuvenate myofibrillar protein synthesis in healthy subjects over 60 years old.

The rate of synthesis of myofibrillar proteins is slower in muscle of healthy subjects over 60 yr old than it is in young adults. Previous research suggests that reduced activity of the GH/insulin-like growth factor-I system could be a determinant of this slowing of protein synthesis. To test the hypothesis that GH could rejuvenate the rate of myofibrillar protein synthesis, we studied healthy subjects over 60 yr old, after a single injection (0.03 mg/kg.sc) of recombinant human GH (n = 6 males/2 females) or placebo (n = 6 males/2 females), or after 3 months of either GH (0.03 mg/kg, sc, 3 x /week, n = 5 males) or placebo (n = 5 males) treatment. Myofibrillar protein synthesis and whole-body protein metabolism were evaluated with the tracer L-[1-13C]leucine. GH reduced whole-body leucine oxidation by 36% (P < 0.01) in the single injection study. There was no effect of GH on whole-body protein breakdown or synthesis, or on myofibrillar protein synthesis in the quadriceps, either acutely or after 3 months of treatment. GH treatment for 3 months increased lean body mass (3.3 +/- 0.7 kg, P < 0.01, as evaluated by 40K counting), muscle mass (3.3 +/- 1.1 kg, P < 0.02, as evaluated by urinary creatinine excretion), and thigh strength (14 +/- 5%, P < 0.05, as evaluated by isokinetic dynamometry). We conclude that GH can increase muscle mass and strength in healthy men over 60 yr old, but does not restore a youthful rate of myofibrillar protein synthesis.