Measurement of the rate of protein synthesis in muscle of postabsorptive young men by injection of a 'flooding dose' of [1-13C]leucine

1. The 'flooding dose' technique for measuring the rate of protein synthesis in tissues in vivo involves the injection of a large amount of unlabelled amino acid together with the tracer to minimize differences in isotopic enrichment of the free amino acid in plasma and tissue compartments. This approach has been investigated in human muscle by taking biopsies from postabsorptive male volunteers given [1-13C]leucine. 2. Intravenous injection of 4 g of unlabelled leucine resulted in a rapid rise in free leucine concentration of seven- to eleven-fold in plasma and five-fold in muscle. Values were still elevated by two-fold after 2 h. 3. Five minutes after injection of [1-13C]leucine (0.05 g/kg) the isotopic enrichment of plasma leucine was 82% that of the injected material, falling to 44% at 120 min. The enrichment of free leucine in sequential muscle biopsies was close to that in plasma and almost identical to that for plasma alpha-ketoisocaproate. 4. The rate of protein synthesis was determined from the increase in leucine enrichment in protein of muscle biopsies taken before and 90 min after injection of [1-13C]leucine (0.05 g/kg; 19 or 39 atom% excess) and the average plasma alpha-ketoisocaproate enrichment over this period (taken to represent muscle free leucine). The mean rate of muscle protein synthesis in 10 subjects was 1.95 (SEM 0.12) %/day. Rates of protein synthesis calculated from plasma leucine as precursor enrichment were only 5% lower than those calculated from plasma alpha-ketoisocaproate.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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

Utilization for protein synthesis of 2-ketoisocaproate relative to utilization of leucine, as estimated from exhalation of labelled CO2

1. We have previously shown that the ratio (RWBP) of incorporation of label from 2-ketoisocaproate (KIC) into the leucine of whole-body protein to the simultaneous incorporation of label from leucine itself into protein is a measure of the nutritional efficiency of KIC as a substitute for leucine. 2. In order to determine whether RWBP can be estimated indirectly from measurement of labelled CO2 excretion, rats were injected orally or intravenously with [4,5-3H]leucine and either [1-14C]leucine or [1-14C]KIC. Expired CO2 was collected for 6 h. 3. The results show that 9-14% of KIC underwent first-pass oxidation after oral administration. When isotopes were given intravenously, the mean rate of excretion of 14CO2 from KIC, after 20 min, remained 1.8 times the mean rate of excretion of 14CO2 from leucine. 4. Mean RWBP, measured in whole-body protein in rats given isotopes orally or intravenously along with small or large doses of carriers, was the same as mean RWBP estimated from mean cumulative CO2 excretion. 5. We conclude (1) that nutritional efficiency of KIC relative to leucine can be estimated from measurement of labelled CO2 excretion, and (2) that the relative inefficiency of KIC as a substitute for leucine in the rat is attributable to first-pass oxidation of 9-14% (when given orally) and 80% greater susceptibility to systemic oxidation than leucine.     

The effect of hemodialysis on protein metabolism. A leucine kinetic study.

To assess the effect of hemodialysis on protein metabolism, leucine flux was measured in seven patients before, during, and after high efficiency hemodialysis using cuprophane dialyzers and bicarbonate dialysate during a primed-constant infusion of L-[1-13C]leucine. The kinetics [mumol/kg per h, mean +/- SD] are as follows: leucine appearance into the plasma leucine pool was 86 +/- 28, 80 +/- 28, and 85 +/- 25, respectively, before, during, and after dialysis. Leucine appearance into the whole body leucine pool, derived from plasma [1-13C]alpha-ketoisocaproate enrichment, was 118 +/- 31, 118 +/- 31, and 114 +/- 28 before, during, and after dialysis, respectively. In the absence of leucine intake, appearance rate reflects protein degradation, which was clearly unaffected by dialysis. Leucine oxidation rate was 17.3 +/- 7.8 before, decreased to 13.8 +/- 7.8 during, and increased to 18.9 +/- 10.3 after dialysis (P = 0.027). Leucine protein incorporation was 101 +/- 26 before, was reduced to 89 +/- 23 during, and returned to 95 +/- 23 after dialysis (P = 0.13). Leucine net balance, the difference between leucine protein incorporation and leucine release from endogenous degradation, was -17.3 +/- 7.8 before, decreased to -28.5 +/- 11.0 during, and returned to -18.9 +/- 10.3 after dialysis (P < 0.0001). This markedly more negative leucine balance during dialysis was accountable by dialysate leucine loss, which was 14.4 +/- 6.2 mumol/kg per h. These data suggest that hemodialysis using a cuprophane membrane did not acutely induce protein degradation. It was, nevertheless, a net catabolic event because protein synthesis was reduced and amino acid was lost into the dialysate.     

Oxidation of leucine in human lymphocytes

Appropriate conditions for analysis of leucine oxidation in peripheral human lymphocytes were determined. Lymphocytes, isolated by Ficoll centrifugation, were washed in phosphate-buffered saline without additions, with 5 mmol/l ADP or with 5 mmol/l ADP plus 25 mmol/l NaF for determination of transaminase activity, and total and basal activity of branched chain keto acid dehydrogenase (BCKA-D), respectively. Cells were incubated for 20-60 min with [1-14C]-leucine, or [1-14C]-alpha-ketoisocaproic acid (KIC) in the presence of various concentrations of the respective unlabelled substrates. Pyridoxal phosphate (0.1 mmol/l) augmented transaminase activity, coenzyme A, NAD and thiaminepyrophosphate (0.5 mmol/l each) enhanced BCKA-D activity. Apparent Km values for transamination and for BCKA-D were 40 and 17 microM, respectively. Total capacity for leucine transamination was about five times greater than for oxidation of KIC. The mean activity state of BCKA-D was 30%. Oxidation of KIC declined when 5.6 mmol/l glucose was added to the medium. It is concluded that: (1) BCKA-D is rate-limiting for leucine catabolism in peripheral human lymphocytes, (2) the BCKA-D complex is normally only partially active, and (3) flux of leucine through BCKA-D is inhibited by physiological glucose concentrations.     

Inverse relationship of leucine flux and oxidation to free fatty acid availability in vivo.

To determine the effect of fatty acid availability on leucine metabolism, 14-h fasted dogs were infused with either glycerol or triglyceride plus heparin, and 46-h fasted dogs were infused with either nicotinic acid or nicotinic acid plus triglyceride and heparin. Leucine metabolism was assessed using a simultaneous infusion of L-[4,5-3H]leucine and alpha-[1-14C]ketoisocaproate. Leucine, alpha-ketoisocaproate (KIC), and totalleucine carbon (leucine plus KIC) flux and oxidation rates were calculated at steady state. In 14-h fasted animals, infusion of triglyceride and heparin increased plasma free fatty acids (FFA) by 0.7 mM (P less than 0.01) and decreased leucine (P less than 0.01), total leucine carbon flux (P less than 0.02), and oxidation (P less than 0.05). The estimated rate of leucine utilization not accounted for by oxidation and KIC flux decreased, but the changes were not significant. During glycerol infusion, leucine and KIC flux and oxidation did not change. In 46-h fasted dogs, nicotinic acid decreased FFA by 1.0 mM (P less than 0.01) and increased (P less than 0.05) the rate of leucine and total leucine carbon flux, but did not affect KIC flux. Leucine oxidation increased (P less than 0.01) by nearly threefold, whereas nonoxidized leucine utilization decreased. Infusion of triglyceride plus heparin together with nicotinic acid blunted some of the responses observed with nicotinic acid alone. In that changes in oxidation under steady state condition reflect changes in net leucine balance, these data suggest that FFA availability may positively affect the sparing of at least one essential amino acid and may influence whole body protein metabolism.     

Insulin-mediated reduction of whole body protein breakdown. Dose-response effects on leucine metabolism in postabsorptive men.

In vivo effects of insulin on plasma leucine and alanine kinetics were determined in healthy postabsorptive young men (n = 5) employing 360-min primed, constant infusions of L-[1-13C]leucine and L-[15N]alanine during separate single rate euglycemic insulin infusions. Serum insulin concentrations of 16.4 +/- 0.8, 29.1 +/- 2.7, 75.3 +/- 5.0, and 2,407 +/- 56 microU/ml were achieved. Changes in plasma 3-methyl-histidine (3-MeHis) were obtained as an independent qualitative indicator of insulin-mediated reduction in proteolysis. Hepatic glucose output was evaluated at the lowest insulin level using D-[6,6-2H2]glucose. The data demonstrate a dose-response effect of insulin to reduce leucine flux, from basal values of 77 +/- 1 to 70 +/- 2, 64 +/- 3, 57 +/- 3, and 52 +/- 4 mumol(kg X h)-1 at the 16, 29, 75, and 2,407 microU/ml insulin levels, respectively (P less than 0.01). A parallel, progressive reduction in 3-MeHis from 5.8 +/- 0.3 to 4.3 +/- 0.3 microM was revealed. Leucine oxidation estimated from the 13C-enrichment of expired CO2 and plasma leucine (12 +/- 1 mumol[kg X h]-1) and from the 13C-enrichment of CO2 and plasma alpha-ketoisocaproate (19 +/- 2 mumol[kg X h]-1) increased at the 16 microU/ml insulin level to 16 +/- 1 and 24 +/- 2 mumol(kg X h)-1, respectively (P less than 0.05 for each), but did not increase at higher insulin levels. Alanine flux (206 +/- 13 mumol(kg X h)-1) did not increase during the clamp, but alanine de novo synthesis increased in all studies from basal rates of 150 +/- 13 to 168 +/- 23, 185 +/- 21, 213 +/- 29, and 187 +/- 15 mumol(kg X h)-1 at 16, 29, 75, and 2,407 microU/ml insulin levels, respectively (P less than 0.05). These data indicate the presence of insulin-dependent suppression of leucine entry into the plasma compartment in man secondary to a reduction in proteolysis and the stimulation of alanine synthesis during euglycemic hyperinsulinemia.     

Measurement of tumour protein synthesis in vivo in human colorectal and breast cancer and its variability in separate biopsies from the same tumour.

1. A method is described for measuring the rates of protein synthesis in vivo in human colorectal and breast tumours by the intravenous injection of L-[1-13C]leucine as a 'flooding dose'. 2. The incorporation of isotope into colorectal tumour protein was measured in six patients, whose tumours were biopsied after the injection. Fractional rates of protein synthesis were calculated from the enrichment of leucine in protein and the average free leucine enrichment in plasma. The range of rates obtained was 17.2-33.9%/day, with a mean rate (+/- SEM) of 22.5 +/- 2.6%/day. 3. Tumour protein synthesis rates were also measured in 15 patients with breast cancer. The range of rates obtained was 5.3-15.9%/day, with a mean rate (+/- SEM) of 10.3 +/- 0.8%/day. These rates are significantly lower than those obtained with colorectal tumours (P less than 0.001). 4. In 9 of the breast cancer patients, protein synthesis was measured in multiple random biopsies taken from the same tumour. The mean (+/- SEM) difference between the highest and lowest rates in biopsies from the same tumour was only 1.1 +/- 0.3%/day. Only 13% of the variation in protein synthesis between separate tumours could be explained by sampling error because of variation within the tumour itself, the remainder being genuine variation between individual tumours.     

Differential effects of hyperinsulinemia and hyperaminoacidemia on leucine-carbon metabolism in vivo. Evidence for distinct mechanisms in regulation of net amino acid deposition.

The effects of physiologic hyperinsulinemia and hyperaminoacidemia, alone or in combination, on leucine kinetics in vivo were studied in postabsorptive healthy subjects with primed-constant infusions of L-[4,5-3H]leucine and [1-14C]alpha-ketoisocaproate (KIC) under euglycemic conditions. Hyperinsulinemia (approximately 100 microU/ml) decreased (P less than 0.05 vs. baseline) steady state Leucine + KIC rates of appearance (Ra) from proteolysis, KIC (approximately leucine-carbon) oxidation, and nonoxidized leucine-carbon flux (leucine----protein). Hyperaminoacidemia (plasma leucine, 210 mumol/liter), with either basal hormone replacement or combined to hyperinsulinemia, resulted in comparable increases in leucine + KIC Ra, KIC oxidation, and leucine----protein (P less than 0.05 vs. baseline). However, endogenous leucine + KIC Ra was suppressed only with the combined infusion. Therefore, on the basis of leucine kinetic data, hyperinsulinemia and hyperaminoacidemia stimulated net protein anabolism in vivo by different mechanisms. Hyperinsulinemia decreased proteolysis but did not stimulate leucine----protein. Hyperaminoacidemia per se stimulated leucine----protein but did not suppress endogenous proteolysis. When combined, they had a cumulative effect on net leucine deposition into body protein.     

Tritium and 14C isotope effects using tracers of leucine and alpha-ketoisocaproate

Abstract. To test if different leucine tracers behave in an indistinguishable manner and, by implication, that their metabolism is identical to that of natural leucine, we measured whole body leucine turnover in dogs and humans and fibrinogen synthesis in dogs by simultaneously infusing either [1–¹⁴C]leucine or [4,5–³H]leucine or [I-¹⁴C]α-ketoisocaproate (KIC) and [4,5–³H]KIC. Whole body leucine fluxes calculated from the plasma specific activity of the transaminated product of the infused tracer (reciprocal pool model) were lower (dogs by 5.7%; humans by 6.4%, both P<0.02) when the plasma 'H specific activity compared to ¹⁴C specific activity were used with leucine tracers and were also lower (dogs by 4.4%, P<0.02; humans by 86%, P<0.06 ) using the KIC tracers. Using leucine or KIC tracers in dogs, the fractional rate of fibrinogen synthesis was 6.7% or 9.4% lower, respectively, (P<0.02) using the ³H versus the ¹⁴C tracer. The apparently lower incorporation of ³H into protein was only in part accounted for by detritiation (2.1%, P = 0.05) of [³H]leucine during acid hydrolysis of proteins. These results suggest that in vivo and/or in vitro differential isotope effects are small (˜5%), but should be considered when dual isotopes infusions are employed to partition amino acid metabolism.     

Increase in anterior tibialis muscle protein synthesis in healthy man during mixed amino acid infusion: studies of incorporation of [1-13C]leucine

1. Anterior tibial muscle protein synthesis in seven healthy postabsorptive men was determined from increases in muscle protein bound leucine enrichment during a primed continuous infusion of L-[1-13C]leucine. Biopsies were taken 30 min after the beginning of leucine infusion (when plasma 13C enrichment was steady), 240 min later during continued fasting and again after 240 min of infusion of a mixed amino acid solution which increased plasma total amino acid concentrations by 37%. The mean enrichment of 13C in plasma alpha-ketoisocaproate was used as an index of the enrichment of the precursor pool for leucine metabolism. 2. Anterior tibial muscle mixed protein synthetic rate during fasting was 0.055 (SD 0.008)%/h and this increased by an average of 35% during infusion of mixed amino acid to 0.074 (SD 0.021)%/h (P less than 0.05). 3. Whole-body protein breakdown (expressed as the rate of endogenous leucine appearance in plasma) was 121 (SD 8) mumol h-1 kg-1 during fasting and decreased (P less than 0.01) by an average of 12% during amino acid infusion. Leucine oxidation was 18 (SD 3) mumol h-1 kg-1 during fasting and increased (P less than 0.001) by 89% during amino acid infusion. Whole-body protein synthesis (non-oxidative leucine disappearance) was 104 (SD 6) mumol h-1 kg-1 during fasting and rose by 13% (P less than 0.001) during mixed amino acid infusion. 4. 13C enrichment of muscle free leucine was only 61 (SD 19)% of that in plasma alpha-ketoisocaproate and this increased to 74 (SD 16)% (P less than 0.02) during mixed amino acid infusion. 5. The results suggest that increased availability of amino acids reverses whole-body protein balance from negative to positive and a major component of this is the increase in muscle protein synthesis.     

Defective suppression by insulin of leucine-carbon appearance and oxidation in type 1, insulin-dependent diabetes mellitus. Evidence for insulin resistance involving glucose and amino acid metabolism.

To determine whether a resistance to insulin in type 1, insulin-dependent diabetes mellitus (IDDM) is extended to both glucose and amino acid metabolism, six normal subjects and five patients with IDDM, maintained in euglycemia with intravenous insulin administration, were infused with L-[4,5-3H]leucine (Leu) and [1-14C]alpha ketoisocaproate (KIC). Steady-state rates of leucine-carbon appearance derived from protein breakdown (Leu + KIC Ra) and KIC (approximately leucine) oxidation were determined at basal and during sequential euglycemic, hyperinsulinemic (approximately 40, approximately 90 and approximately 1,300 microU/ml) clamps. In the euglycemic postabsorptive diabetic patients, despite basal hyperinsulinemia (24 +/- 6 microU/ml vs. 9 +/- 1 microU/ml in normals, P less than 0.05), Leu + KIC Ra (2.90 +/- 0.18 mumol/kg X min), and KIC oxidation (0.22 +/- 0.03 mumol/kg X min) were similar to normal values (Leu + KIC Ra = 2.74 +/- 0.25 mumol/kg X min) (oxidation = 0.20 +/- 0.02 mumol/kg X min). During stepwise hyperinsulinemia, Leu + KIC Ra in normals decreased to 2.08 +/- 0.19, to 2.00 +/- 0.17, and to 1.81 +/- 0.16 mumol/kg X min, but only to 2.77 +/- 0.16, to 2.63 +/- 0.16, and to 2.39 +/- 0.08 mumol/kg X min in the diabetic patients (P less than 0.05 or less vs. normals at each clamp step). KIC oxidation decreased in normal subjects to a larger extent than in the diabetic subjects. Glucose disposal was reduced at all insulin levels in the patients. In summary, in IDDM: (a) Peripheral hyperinsulinemia is required to normalize both fasting leucine metabolism and blood glucose concentrations. (b) At euglycemic hyperinsulinemic clamps, lower glucose disposal rates and a defective suppression of leucine-carbon appearance and oxidation were observed. We conclude that in type 1 diabetes a resistance to the metabolic effects of insulin on both glucose and amino acid metabolism is present.     

Pathway of free fatty acid oxidation in human subjects. Implications for tracer studies.

To determine the pathway of plasma FFA oxidation and the site(s) of label fixation observed during infusion of FFA tracers, [1-13C]palmitate and [1-14C]acetate were infused intravenously for 3 h in five volunteers. Breath 13CO2 enrichment and 14CO2 specific activity were followed for 6 h to determine the labeled CO2 decay rates. Acetate enters directly into the TCA cycle; hence, if palmitate transits a large lipid pool before oxidation, 13CO2 enrichment (from palmitate) should decay slower than 14CO2 specific activity (from acetate). Breath 13CO2 enrichment and 14CO2 specific activity decayed at a similar rate after stopping the tracer infusions (half-lives of 13CO2 and 14CO2 decay: mean [+/- SE] 106.6 +/- 8.9 min, and 96.9 +/- 6.0 min, respectively, P = NS), which suggests that palmitate enters the TCA cycle directly and that label fixation occurs after citrate synthesis. Significant label fixation was shown in plasma glutamate/glutamine and lactate/pyruvate during infusion of either [1,2-13C]acetate or [U-13C]palmitate, suggesting that TCA cycle exchange reactions are at least partly responsible for label fixation. This was consistent with our finding that the half-lives of 13CO2 enrichment and 14CO2 specific activity decreased significantly during exercise to 14.4 +/- 3 min and 16.8 +/- 1 min, respectively, since exercise significantly increases the rate of the TCA cycle in relation to that of the TCA cycle exchange reactions. We conclude that plasma FFA entering cells destined to be oxidized are directly oxidized and that tracer estimates of plasma FFA oxidation will underestimate the true value unless account is taken of the extent of label fixation.     

Comparison of the effects of recombinant human insulin-like growth factor-I and insulin on glucose and leucine kinetics in humans.

To compare the metabolic effects of elevated plasma concentrations of IGF-I and insulin, overnight-fasted normal subjects were studied twice, once receiving IGF-I and once insulin at doses that resulted in identical increases in glucose uptake during 8-h euglycemic clamping. Recombinant human IGF-I or insulin were infused in one group at high doses (30 micrograms/kg per h IGF-I or 0.23 nmol/kg per h insulin) and in another group at low doses (5 micrograms/kg per h IGF-I or 0.04 nmol/kg per h insulin). Glucose rate of disappearance (measured by [6,6-D2]-glucose infusions) increased from baseline by 239 +/- 16% during high dose IGF-I vs 197 +/- 18% during insulin (P = 0.021 vs IGF-I). Hepatic glucose production decreased by 37 +/- 6% during high dose IGF-I vs 89 +/- 13% during insulin (P = 0.0028 vs IGF-I). IGF-I suppressed whole body leucine flux ([1-13C]-leucine infusion technique) more than insulin (42 +/- 4 vs 32 +/- 3% during high doses, P = 0.0082). Leucine oxidation rate decreased during high dose IGF-I more than during insulin (55 +/- 4 vs 32 +/- 6%, P = 0.0001). The decreases of plasma concentrations of free fatty acids, acetoacetate, and beta-hydroxybutyrate after 8 h of IGF-I and insulin administration were similar. Plasma C-peptide levels decreased by 57 +/- 4% during high doses of IGF-I vs 36 +/- 6% during insulin (P = 0.005 vs IGF-I). The present data demonstrate that, compared to insulin, an acute increase in plasma IGF-I levels results in preferential enhancement of peripheral glucose utilization, diminished suppression of hepatic glucose production, augmented decrease of whole body protein breakdown (leucine flux), and of irreversible leucine catabolism but in similar antilipolytic effects. The data suggest that insulin-like effects of IGF-I in humans are mediated in part via IGF-I receptors and in part via insulin receptors.     

Postabsorptive and insulin-stimulated energy homeostasis and leucine turnover in offspring of type 2 diabetic patients

OBJECTIVE: This study was performed to ascertain whether insulin resistance with respect to protein metabolism is an additional primary metabolic abnormality affecting insulin-resistant offspring of type 2 diabetic parents, along with insulin resistance with respect to glucose and lipid metabolism. RESEARCH DESIGN AND METHODS: We studied 18 young, nonobese offspring of type 2 diabetic parents and 27 healthy matched (by means of dual-energy X-ray absorption) individuals with the bolus plus continuous infusion of [6,6-(2)H(2)]glucose and [1-(13)C]leucine in combination with the insulin clamp (40 mU x m(-2) x min(-1)). RESULTS: Fasting plasma leucine, phenylalanine, alanine, and glutamine concentrations, as well as the glucose and leucine turnover (reciprocal pool model: 155 +/- 10 vs. 165 +/- 5 micromol x kg lean body mass(-1) x h(-1) in offspring of type 2 diabetic patients and healthy matched individuals, respectively), were also not different. During the clamp, glucose turnover rates were significantly reduced in offspring of type 2 diabetic patients (7.1 +/- 0.5) in comparison with healthy matched individuals (9.9 +/- 0.6 mg x kg lean body mass(-1) x min(-1); P < 0.01). Also, the suppression of leucine turnover was impaired in offspring of type 2 diabetic patients (12 +/- 1%) in comparison with healthy matched individuals (17 +/- 1%; P = 0.04) and correlated with the degree of the impairment of insulin-stimulated glucose metabolism (R(2) = 0.13; P = 0.02). CONCLUSIONS: Nonobese, nondiabetic, insulin-resistant offspring of type 2 diabetic patients were characterized by an impairment of insulin-dependent suppression of protein breakdown, which was proportional to the impairment of glucose metabolism. These results demonstrate that in humans, a primary in vivo impairment of insulin action affects glucose and fatty acid metabolism as previously shown and also protein/amino acid metabolism.     

Role of Insulin in the Regulation of Leucine Kinetics in the Conscious Dog

To study the effect of insulin on leucine kinetics, three groups of conscious dogs were studied after an overnight fast (16-18 h). One, saline-infused group (n = 5), served as control. The other two groups were infused with somatostatin and constant replacement amount of glucagon; one group (n = 6) received no insulin replacement, to produce acute insulin deficiency, and the other (n = 6) was constantly replaced with 600 muU/kg per min insulin, to produce twice basal hyperinsulinemia. Hepatic and extrahepatic splanchnic (gut) balance of leucine and alpha-ketoisocaproate (KIC) were calculated using the arteriovenous difference technique. l,4,5,[(3)H]Leucine was used to measure the rates (micromoles per kilogram per minute) of appearance (Ra) and disappearance (Rd), and clearance (Cl) of plasma leucine (milliliters per kilogram per minute).Saline infusion for 7 h resulted in isotopic steady state, where Ra and Rd were equal (3.2+/-0.2 mumol/kg per min). Acute insulin withdrawal of 4-h duration caused the plasma leucine to increase by 40% (P < 0.005). This change was caused by a decrease in the outflow of leucine (Cl) from the plasma, since Ra did not change. The net hepatic release of the amino acid (0.24+/-0.03 mumol/kg per min) did not change significantly; the arterio-deep femoral venous differences of leucine (-10+/-1 mumol/liter) and KIC (-12+/-2 mumol/liter) did not change significantly indicating net release of the amino and ketoacids across the hindlimb. Selective twice basal hyperinsulinemia resulted in a 36% drop in plasma leucine (from control levels of 128+/-8 to 82+/-7 mumol/liter, P < 0.005) within 4 h. This was accompanied by a 15% reduction in Ra and a 56% rise in clearance (P < 0.001, both). Net hepatic leucine production and net release of leucine and KIC across the hindlimb fell markedly. These studies indicate that physiologic changes in circulating insulin levels result in a differential dose-dependent effect on total body leucine metabolism in the intact animal. Acute insulin withdrawal exerts no effect on leucine rate of appearance, while at twice basal levels, insulin inhibited leucine rate of appearance and stimulated its rate of disappearance.     

Arginine and nitric oxide metabolism in critically ill septic pediatric patients

OBJECTIVE: To investigate whole body, arginine metabolism and nitric oxide synthesis rates in septic, critically ill pediatric patients. DESIGN: Prospective study. SETTING: Pediatric intensive care unit at a general hospital. PATIENTS: Ten consecutive septic patients age 6-16 yrs. INTERVENTIONS: Septic patients received an 8-hr primed, constant intravenous tracer infusion of L-[guanidino-15N2]arginine, L-[1-13C]leucine, and [13C]urea. A 24-hr urine collection was obtained for determination of [15N]nitrate enrichment (15NO3(-)) and urinary nitrogen. The next day they received an infusion of L-[5-13C]arginine and L-[5-13C-ureido, 5,5, 2H2]citrulline. Blood samples were obtained for determination of plasma isotopic enrichment of the tracers given and of derived [15N]citrulline (nitric oxide synthesis), L-[13C-guanidino 5,5, 2H2]arginine (M+3 arg) (arginine synthesis), and [15N]urea (urea formation). Data are compared with historic controls from studies in healthy young adults. MEASUREMENTS AND MAIN RESULTS: Plasma arginine fluxes were 67 +/- 21 and 72 +/- 17 micromol x kg(-1) x hr(-1), respectively, for the [15N2 guanidino] and the [13C] arginine labels, which were not different from reported adult values. The rates of arginine oxidation were 22.9 +/- 10.8 micromol x kg(-1) x hr(-1) and were higher than arginine synthesis rates of 9.6 +/- 4.2 micromol x kg(-1) x hr(-1) (p <.01); therefore, these patients were in a negative arginine balance. The rates of nitric oxide synthesis as estimated by the [15N]citrulline method were 1.58 +/- 0.69 micromol x kg(-1) x hr(-1) for septic patients and higher (p <.05) than values of 0.96 +/- 0.1 micromol x kg(-1) x hr(-1) in healthy adults. Septic patients were in a negative protein (leucine) balance of about -1.00 +/- 0.40 g x kg(-1) x day(-1). CONCLUSIONS: Homeostasis of plasma arginine in septic patients was impaired compared with reported adult values. The rates of arginine oxidation were increased whereas net arginine synthesis was unchanged, leading to a negative arginine balance. The rates of nitric oxide synthesis and the fraction of plasma arginine used for nitric oxide and urea formation were increased. These findings suggest that under condition of sepsis, arginine becomes essential in critically ill children.     

Absorption of 13C-labeled stearic, oleic, and linoleic acids in humans: application to breath tests

Intestinal absorption of ingested [1-13C]stearic, [1-13C]oleic, and [1-13C]linoleic acid was compared in six healthy men. A bolus of each [1-13C]-labeled fatty acid was ingested in random order at 72-hour intervals with the breakfast meal. Subjects consumed fixed diets during a 9-day fecal collection period. Pooled 9-day fecal samples were homogenized and total fat extracted. Fat extracts were saponified and methylated, and individual fatty acids were quantitated by gas-liquid chromatography. Preparative high-performance liquid chromatography was used to obtain fractions containing stearic, oleic, and linoleic acid for combustion to CO2 and assay of 13C enrichment over background. Prelabel period 24-hour samples were treated similarly to measure background 13C abundance. Total fatty acid and stearic, oleic, and linoleic acid excretion (+/- SEM) in the six volunteers over the 9-day period was 41.5 +/- 7.3, 10.0 +/- 1.3, 8.8 +/- 2.9, and 0.8 +/- 0.1 mg/day/kg body weight, respectively. The absorption efficiency for [1-13C]stearic, [1-13C]oleic, and [1-13C]linoleic acid was 78.0% +/- 4.5%, 97.2% +/- 1.7%, and 99.9% +/- 0.1%, respectively. The reduced absorption of [1-13C]stearic acid observed emphasizes the importance of correcting breath test oxidation data for fecal loss of 13C substrate. The potential application of our method to other areas of intermediary metabolism is discussed.     

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)     

Effect of carbohydrate administration on leucine oxidation in newborn infants. Studies using the 13C leucine breath test

The branched-chain amino acid leucine plays an important role in the protein metabolism of human beings. It not only inhibits protein degradation but also stimulates protein synthesis. The oxidation rate of leucine and the influence which nutritional conditions have on this amino acid can be measured with the intravenous 13C-leucine breath test. In order the apply the breath test on newborn infants, the required dosage of L-(1-13C)-leucine and the reproducibility of the test had, firstly, to be determined. Following this, the extent to which the leucine oxidation rate was influenced by a simultaneous carbohydrate intake was investigated. An evident discrimination between the 13CO2-exhalation and the 13CO2-baseline exhalation is demonstrated after a bolus injection of 1 mg L-(1-13C)-leucine/kg B.W. We were able to measure reproducible values of the leucine oxidation rate in newborn infants with a tracer dosage of 4 mg L-(1-13C)-leucine/kg B.W. We found that a higher intake of carbohydrate given at the same time produced a lower rate of leucine oxidation, which indicates increased utilization of leucine for the benefit of protein synthesis.