Increased Leucine Oxidation Research Articles

Heat Stress Reduces Metabolic Rate While Increasing Respiratory Exchange Ratio in Growing Pigs.

Simple SummaryCellular growth, particularly muscle hypertrophy, requires substantial energetics. In animals, proper substrate utilization is essential. Maximal growth is dependent on both carbohydrates and lipids being oxidized to provide as much energy as possible for protein syntheses, while amino acids must be spared from oxidation to provide an ample supply of proteogenic building blocks. However, maximal cellular growth is not a default metabolic state and is easily overridden by environmental factors such as heat stress (HS). HS has previously been hypothesized to increase metabolic rate, as is typical of a general stress response. Newer evidence, however, suggest that HS may limit energy production by inhibiting the use of lipids as a fuel source. HS metabolism instead depends on carbohydrates and even amino acids as energetic substrate, potentially limiting energy production. This study demonstrates that, contrary to current recommendations, HS reduces metabolic rate. Findings demonstrated a 38% reduction in relative energy expenditure (kcal/day/kg) due to HS. HS also caused a 33% increase in amino acid oxidation. A combination of decreased energy production and increased amino acid oxidation creates a metabolic state with severely limited growth potential which cannot be solved by simply increasing feed.Heat stress (HS) diminishes animal production, reducing muscle growth and increasing adiposity, especially in swine. Excess heat creates a metabolic phenotype with limited lipid oxidation that relies on aerobic and anaerobic glycolysis as a predominant means of energy production, potentially reducing metabolic rate. To evaluate the effects of HS on substrate utilization and energy expenditure, crossbred barrows (15.2 ± 2.4 kg) were acclimatized for 5 days (22 °C), then treated with 5 days of TN (thermal neutral, 22 °C, n = 8) or HS (35 °C, n = 8). Pigs were fed ad libitum and monitored for respiratory rate (RR) and rectal temperature. Daily energy expenditure (DEE) and respiratory exchange ratio (RER, CO2:O2) were evaluated fasted in an enclosed chamber through indirect calorimetry. Muscle biopsies were obtained from the longissimus dorsi pre/post. HS increased temperature (39.2 ± 0.1 vs. 39.6 ± 0.1 °C, p < 0.01) and RER (0.91 ± 0.02 vs. 1.02 ± 0.02 VCO2:VO2, p < 0.01), but decreased DEE/BW (68.8 ± 1.7 vs. 49.7 ± 4.8 kcal/day/kg, p < 0.01) relative to TN. Weight gain (p = 0.80) and feed intake (p = 0.84) did not differ between HS and TN groups. HS decreased muscle metabolic flexibility (~33%, p = 0.01), but increased leucine oxidation (~35%, p = 0.02) compared to baseline values. These data demonstrate that HS disrupts substrate regulation and energy expenditure in growing pigs.

Heat stress increases respiratory exchange ratio while reducing daily energy expenditure in growing pigs.

Heat stress (HS) alters animal metabolism causing reduced performance (muscle growth) while increasing the incidence of disease and mortality. HS is particularly detrimental in the swine industry where the global economic burden of heat stress is in the billions of dollars annually. Excess environmental heat promotes a HS response increasing the expression of heat shock factors and heat shock proteins which coordinate a shift in metabolic substrate preference. The net result of these changes is a metabolic phenotype with limited lipid oxidation that relies on aerobic and anaerobic glycolysis as a predominant source of energy production. This study was designed to evaluate the effect of HS on substrate utilization and overall animal metabolic rate in growing pigs. Crossbred barrows (15.2±2.4 kg) were exposed to 5 days of TN (thermal neutral, 24 C°) or HS ( 35 C°) (n=8 per treatment), after a 5‐day acclimation period (24 C°). Pigs were fed ad libitum and monitored regularly for respiratory rate (RR) and rectal temperature (3x daily). A metabolic cart was used to assess daily energy expenditure (DEE), and respiratory exchange ratio (RER, CO2:O2), pigs were placed in an enclosed chamber and gas exchange was measured for 1 hour after a 30 min washout. Muscle biopsies were taken from the longissimus dorsi to evaluated palmitate, pyruvate, and leucine oxidation capacity and metabolic flexibility (metflex). Metabolic measures and muscle biopsies were taken after five days of acclimation (pre) and immediately following (post) the 5‐day environmental treatments. Initial DEE was positively correlated to weight (r2=0.55, p&lt;0.01) and feed intake (r2=0.40, p&lt;0.01), initial DEE relative to bodyweight (DEE/BW) correlated to metflex (r2=0.29, p=0.03). HS increased RR (94.3±4.5 vs 55.9±2 BPM, p&lt;0.01) and rectal temperature (39.6±0.1 vs 39.2±0.1 C°, p&lt;0.01) compared to TN controls. Weight gain and feed intake did not differ between HS and TN groups. Seven days of HS increased RER (0.91±0.02 vs 1.02±0.02 VCO2:VO2, p&lt;0.01) and decreased DEE/BW (68.8±1.7 vs 49.7±4.8 kcal/kg, p&lt;0.01) compared to baseline values. Heat stress also decreased muscle palmitate oxidation (−20.1%, p=0.04) and metflex (28.8±3.5 vs 19.2±3.7, P=0.01), while causing an increase in leucine oxidation (14.1±0.9 vs 19.0±2.2 nmol/mg‐pro/hr, p=0.02) compared to baseline values. Growth rates of HS animals were positively correlated to post DEE/BW (r2=0.75, p&lt;0.01), while negatively correlated to changes in rectal temperature (r2=0.54, p=0.04). Previous research has shown a J‐shaped relationship between environmental temperature and energy expenditure, with both cold and hot environments increasing energy expenditure. However, these data demonstrate that chronic HS may decrease energy expenditure at least relative to body weight in growing pigs. This reduction in energy expenditure appears to be mechanistically controlled through substrate regulation as HS increased RER and decreased lipid oxidation and metflex. To overcome limits in lipid oxidation, amino acid use for energy may be upregulated which can further limit potential for muscle growth.

Prolonged amino acid infusion into intrauterine growth-restricted fetal sheep increases leucine oxidation rates.

Overcoming impaired growth in an intrauterine growth-restricted (IUGR) fetus has potential to improve neonatal morbidity, long-term growth, and metabolic health outcomes. The extent to which fetal anabolic capacity persists as the IUGR condition progresses is not known. We subjected fetal sheep to chronic placental insufficiency and tested whether prolonged amino acid infusion would increase protein accretion in these IUGR fetuses. IUGR fetal sheep were infused for 10 days with either mixed amino acids providing ~2 g·kg-1·day-1 (IUGR-AA) or saline (IUGR-Sal) during late gestation. At the end of the infusion, fetal plasma leucine, isoleucine, lysine, methionine, and arginine concentrations were higher in the IUGR-AA than IUGR-Sal group ( P < 0.05). Fetal plasma glucose, oxygen, insulin, IGF-1, cortisol, and norepinephrine concentrations were similar between IUGR groups, but glucagon concentrations were fourfold higher in the IUGR-AA group ( P < 0.05). Net umbilical amino acid uptake rate did not differ between IUGR groups; thus the total amino acid delivery rate (net umbilical amino acid uptake + infusion rate) was higher in the IUGR-AA than IUGR-Sal group (30 ± 4 vs. 19 ± 1 μmol·kg-1·min-1, P < 0.05). Net umbilical glucose, lactate, and oxygen uptake rates were similar between IUGR groups. Fetal leucine oxidation rate, measured using a leucine tracer, was higher in the IUGR-AA than IUGR-Sal group (2.5 ± 0.3 vs. 1.7 ± 0.3 μmol·kg-1·min-1, P < 0.05). Fetal protein accretion rate was not statistically different between the IUGR groups (1.6 ± 0.4 and 0.8 ± 0.3 μmol·kg-1·min-1 in IUGR-AA and IUGR-Sal, respectively) due to variability in response to amino acids. Prolonged amino acid infusion into IUGR fetal sheep increased leucine oxidation rates with variable anabolic response.

Interaction between leucine and palmitate catabolism in 3T3-L1 adipocytes and primary adipocytes from control and obese rats

Metabolic profiling studies have highlighted increases in the plasma free fatty acid (FFA) and branched-chain amino acid (BCAA) concentrations, which are hallmarks of the obese and insulin-resistant phenotype. However, little is known about how the increase of the BCAA concentration modifies the metabolic fate of FFA, and vice versa, in adipocytes. Therefore, we incubated differentiated 3T3-L1 adipocytes or primary adipocytes from rats fed a control or a high-fat diet with: (1) 0, 250, 500 and 1000 μM of leucine and determined the oxidation and incorporation of [1-14C]-palmitate into lipids or proteins or (2) 0, 250, 500 or 1000 μM of palmitate and evaluated the oxidation and incorporation of [U-14C]-leucine into lipids or proteins. Leucine decreased palmitate oxidation and increased its incorporation into the lipid fraction in adipocytes; the latter was reduced in adipocytes from obese rats. However, palmitate increased leucine oxidation in adipocytes as well as reduced leucine incorporation into the protein and lipid fractions in adipocytes from obese rats. These results demonstrate that leucine modifies the metabolic fate of palmitate, and vice versa, in adipocytes and that the metabolic interaction between leucine and palmitate catabolism is altered in adipocytes from obese rats.

Effects of a Ketogenic Diet and Chronic Aerobic Exercise on C57BL6 Mice

INTRODUCTIONLow carbohydrate ketogenic diets (LCKD) alter metabolic processes and have been proposed as a means to attenuate adiposity and reduce hyperglycemia. Aerobic exercise training promotes a suite of metabolic adaptations effective in reducing the incidence of metabolic syndrome. There is potential for combined LCKD and aerobic exercise training to alter substrate metabolism in a manner that could enhance body composition. To investigate whether a LCKD could favorably alter metabolic function and potentiate the effects of chronic aerobic exercise, mice were fed a LCKD for a total of 7 weeks, while during the final 3 weeks half of the mice concurrently engaged in daily treadmill exercise.METHODS40 C57BL6 mice were randomized into 4 groups (n=10/group): 2 groups were fed a low‐fat control diet (16% protein, 72% carbohydrate, 12% fat) with one group performing vigorous intensity (blood lactate &gt;4mM post‐exercise) daily treadmill exercise (CEX) and the other serving as sedentary controls (CSED). The remaining 2 groups were fed a LCKD (84% fat, 16% protein) with one exercise group (KEX) and a group of sedentary controls (KSED). Body composition (NMR) was measured weekly. At the end of the 4 week exercise training program, mice were euthanized and substrate oxidation was assessed using 14C labelled substrates, while blood was analyzed for lipids, glucose and β‐OHB.RESULTSThe LCKD resulted in a mild increase in circulating ketones; however, mice on this diet did not exhibit differences in serum fatty acids, triglycerides (TG), or glucose levels despite consuming a lipid‐enriched, carbohydrate‐deficient diet. Body weight and fat mass increased in response to the LCKD, but exercise training limited weight gain. A LCKD did not alter TG levels in liver or muscle; however, exercise training increased TG accrual in both tissues and this effect was more prominent in mice fed the control diet. In the liver, LCKD reduce carbohydrate oxidation, but increased leucine oxidation, incomplete palmitate oxidation, and peroxisomal oxidation. Alternatively, in skeletal muscle carbohydrate oxidation did not change in response to LCKD, whereas mitochondrial and peroxisomal fat oxidation both increased. Exercise training had minimal effect on hepatic substrate oxidation, but increased oxidation of leucine, palmitate, and lignocerate in skeletal muscle; however, no synergistic effects were detected between a ketogenic diet and exercise training.CONCLUSIONOur results indicate that mice fed an 84% fat, 16% protein diet demonstrate a shift in substrate utilization toward an increased reliance on fatty acid and branch‐chain amino acid catabolism in liver and skeletal muscle, while vigorous‐intensity exercise effects were more prominent in muscle. Despite the fact that both LCKD and exercise independently induced shifts in substrate metabolism, the effects do not appear to be additive or synergistic and mice fed an LCKD retained greater adiposity than mice fed a protein‐matched control dietSupport or Funding InformationThis work was supported by NIH R01 DK103860‐01 (R.C.N).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute effects of phenylbutyrate on glutamine, branched-chain amino acid and protein metabolism in skeletal muscles of rats.

Phenylbutyrate (PB) acts as chemical chaperone and histone deacetylase inhibitor, which is used to decrease ammonia in urea cycle disorders and has been investigated for use in the treatment of a number of lethal illnesses. We performed invivo and invitro experiments to examine the effects of PB on glutamine (GLN), branched-chain amino acid (BCAA; valine, leucine and isoleucine) and protein metabolism in rats. In the first study, animals were sacrificed one hour after three injections of PB (300mg/kg b.w.) or saline. In the second study, soleus (SOL, slow twitch) and extensor digitorum longus (EDL, fast twitch) muscles were incubated in a medium with or without PB (5mM). L-[1-14 C] leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. PB treatment decreased GLN, BCAA and branched-chain keto acids (BCKAs) in blood plasma, decreased BCAA and increased GLN concentrations in muscles, and increased GLN synthetase activities in muscles. Addition of PB to incubation medium increased leucine oxidation (55% in EDL, 29% in SOL), decreased BCKA and increased GLN in medium of both muscles, increased GLN in muscles, decreased protein synthesis in SOL and increased proteolysis in EDL. It is concluded that PB decreases BCAA, BCKA and GLN in blood plasma, activates BCAA catabolism and GLN synthesis in muscle and exerts adverse effects on protein metabolism. The results indicate that BCAA and GLN supplementation is needed when PB is used therapeutically and that PB may be a useful prospective agent which could be effective in management of maple syrup urine disease.

Metabolic Fate of Branched-Chain Amino Acids During Adipogenesis, in Adipocytes From Obese Mice and C2C12 Myotubes.

Branched-chain amino acid (BCAA) catabolism is regulated by the branched-chain aminotransferase (BCAT2) and the branched-chain α-keto acid dehydrogenase complex (BCKDH). BCAT2 and BCKDH expression and activity are modified during adipogenesis and altered in adipose tissues of mice with genetic or diet-induced obesity. However, little is known about how these modifications and alterations affect the intracellular metabolic fate of BCAAs during adipogenesis, in adipocytes from mice fed a control or high-fat diet or in C2C12 myotubes. Here, we demonstrate that BCAAs are mainly incorporated into proteins during the early stages of adipocyte differentiation. However, they are oxidized and incorporated into lipids during the late days of differentiation. Conversely, 92% and 97% of BCAA were oxidized, 1.6% and 6% were used for protein synthesis and 1.2% and 1.5% were incorporated into lipids in adipocytes from epididymal and subcutaneous adipose tissue, respectively. All three pathways were decreased in adipocytes from mice fed a high-fat diet. In C2C12 myotubes, leucine is mainly used for protein synthesis and palmitate is incorporated into lipids. Interestingly, leucine decreased both palmitate oxidation and its incorporation to lipids and proteins; and palmitate increased leucine oxidation and decreased its incorporation to lipids and proteins in a dose-dependent manner. These results demonstrate that BCAA metabolic fate differs between the early and late stages of adipocyte differentiation and in adipocytes from mice fed a control or high-fat diet; and that leucine affects the metabolic fate of palmitate and vice versa in C2C12 myotubes. J. Cell. Biochem. 118: 808-818, 2017. © 2016 Wiley Periodicals, Inc.

Effect of palmitate on the metabolic fate of leucine and vice versa in primary adipocytes of a rat model of diet‐induced obsesity (575.7)

Metabolic profiling studies have spotlighted altered fatty acid (FA) or branched chain amino acid (BCAA) oxidation that marks the obese and insulin‐resistant phenotype. Interestingly, mRNA expression of Carnitine palmitoyltransferase I (CPT1) ‐the rate‐limiting enzyme of FA oxidation‐ and Isovaleryl‐CoA dehydrogenase (IVDH) ‐a mitochondrial enzyme involved in BCAA oxidation‐ are inversely correlated in the livers of 41 recombinant inbred mouse strains of the BxD genetic reference population (http://www.genenetwork.org/). The inverse correlation between CPT1 and IVDH suggest that a high rate of FA oxidation could decrease BCAA oxidation and vice versa. To test this hypothesis, we have evaluated the effect of increasing concentrations of palmitate or leucine in the metabolic fate of leucine or palmitate in primary adipocytes of a rat model of diet‐induced obsesity. We incubated primary adipocytes with: 1) 0, 250, 500 or 1000 µM of palmitate and evaluated the oxidation, incorporation to lipids or proteins of [U‐14C]‐leucine; or 2) 0, 250, 500 and 1000 µM of leucine and determined the oxidation, incorporation to lipids or proteins of [1‐14C]‐palmitic acid. Interstingly, palmitate increased leucine oxidation and decreased its incorporation to lipids and proteins in a dose‐dependent manner. Furthermore, leucine decreased both palmitate oxidation and its incorporation to lipids and proteins in a dose‐dependent manner. Overall, our results demonstrate that palmitate affects the metabolic fate of leucine and that leucine modifies the metabolic fate of palmitate in primary adipocytes of a rat model of diet‐induced obsesity.Grant Funding Source: CONACYT 155949

Acute supplementation of amino acids increases net protein accretion in IUGR fetal sheep

Placental insufficiency decreases fetal amino acid uptake from the placenta, plasma insulin concentrations, and protein accretion, thus compromising normal fetal growth trajectory. We tested whether acute supplementation of amino acids or insulin into the fetus with intrauterine growth restriction (IUGR) would increase net fetal protein accretion rates. Late-gestation IUGR and control (CON) fetal sheep received acute, 3-h infusions of amino acids (with euinsulinemia), insulin (with euglycemia and euaminoacidemia), or saline. Fetal leucine metabolism was measured under steady-state conditions followed by a fetal muscle biopsy to quantify insulin signaling. In CON, increasing amino acid delivery rates to the fetus by 100% increased leucine oxidation rates by 100%. In IUGR, amino acid infusion completely suppressed fetal protein breakdown rates but increased leucine oxidation rate by only 25%, resulting in increased protein accretion rates by 150%. Acute insulin infusion, however, had very little effect on amino acid delivery rates, fetal leucine disposal rates, or fetal protein accretion rates in CON or IUGR fetuses despite robust signaling of the fetal skeletal muscle insulin-signaling cascade. These results indicate that, when amino acids are given directly into the fetal circulation independently of changes in insulin concentrations, IUGR fetal sheep have suppressed protein breakdown rates, thus increasing net fetal protein accretion.

Prolonged infusion of amino acids increases leucine oxidation in fetal sheep

Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n = 8) or saline [control (Con), n = 10] for ∼12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 ± 0.5 vs. 1.4 ± 0.6 μmol·min(-1)·kg(-1), P < 0.05). Fetal protein accretion (2.6 ± 0.5 and 2.2 ± 0.6 μmol·min(-1)·kg(-1) in AA and Con, respectively), synthesis (6.2 ± 0.8 and 7.0 ± 0.9 μmol·min(-1)·kg(-1) in AA and Con, respectively), and degradation (3.6 ± 0.6 and 4.5 ± 1.0 μmol·min(-1)·kg(-1) in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 ± 0.4 vs. 3.9 ± 0.1 mg·kg(-1)·min(-1), P < 0.05). The glucose-O(2) quotient also decreased over time in the AA group (P < 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 ± 24 vs. 59 ± 9 pg/ml, P < 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 ± 181 vs. 419 ± 76 pg/ml, P = 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon (r(2) = 0.38, P < 0.05), cortisol (r(2) = 0.31, P < 0.05), and NE (r(2) = 0.59, P < 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids.

A Protein–Leucine Supplement Increases Branched-Chain Amino Acid and Nitrogen Turnover But Not Performance

This study aimed to determine the effect of postexercise protein-leucine coingestion with CHO-lipid on subsequent high-intensity endurance performance and to investigate candidate mechanisms using stable isotope methods and metabolomics. In this double-blind, randomized, crossover study, 12 male cyclists ingested a leucine/protein/CHO/fat supplement (LEUPRO 7.5/20/89/22 g · h(-1), respectively) or isocaloric CHO/fat control (119/22 g · h(-1)) 1-3 h after exercise during a 6-d training block (intense intervals, recovery, repeated-sprint performance rides). Daily protein intake was clamped at 1.9 g · kg(-1) · d(-1) (LEUPRO) and 1.5 g · kg(-1) · d(-1) (control). Stable isotope infusions (1-(13)C-leucine and 6,6-(2)H2-glucose), mass spectrometry-based metabolomics, and nitrogen balance methods were used to determine the effects of LEUPRO on whole-body branched-chain amino acid (BCAA) and glucose metabolism and protein turnover. After exercise, LEUPRO increased BCAA levels in plasma (2.6-fold; 90% confidence limits = ×/÷ 1.1) and urine (2.8-fold; ×/÷ 1.2) and increased products of BCAA metabolism plasma acylcarnitine C5 (3.0-fold; ×/÷ 0.9) and urinary leucine (3.6-fold; ×/÷ 1.3) and β-aminoisobutyrate (3.4-fold; ×/÷ 1.4), indicating that ingesting ~10 g leucine per hour during recovery exceeds the capacity to metabolize BCAA. Furthermore, LEUPRO increased leucine oxidation (5.6-fold; ×/÷ 1.1) and nonoxidative disposal (4.8-fold; ×/÷ 1.1) and left leucine balance positive relative to control. With the exception of day 1 (LEUPRO = 17 ± 20 mg N · kg(-1), control = -90 ± 44 mg N · kg(-1)), subsequent (days 2-5) nitrogen balance was positive for both conditions (LEUPRO = 130 ± 110 mg N · kg(-1), control = 111 ± 86 mg N · kg(-1)). Compared with control feeding, LEUPRO lowered the serum creatine kinase concentration by 21%-25% (90% confidence limits = ± 14%), but the effect on sprint power was trivial (day 4 = 0.4% ± 1.0%, day 6 = -0.3% ± 1.0%). Postexercise protein-leucine supplementation saturates BCAA metabolism and attenuates tissue damage, but effects on subsequent intense endurance performance may be inconsequential under conditions of positive daily nitrogen balance.

Mild-to-Moderate Chronic Cholestatic Liver Disease Increases Leucine Oxidation in Children

Malnutrition is prevalent in children with chronic cholestatic liver disease. Using the noninvasive indicator amino acid oxidation (IAAO) technique, we recently determined that mild-to-moderate chronic cholestatic (MCC) liver disease increases the need for branched-chain amino acids (BCAA) in children. To examine the underlying mechanisms responsible for this increased need for BCAA in liver disease, we measured L-[1-(13C)]-leucine oxidation in the postabsorptive and fed states in 10 children with MCC liver disease (8.8 +/- 3.5 y) and in 11 healthy children (9.4 +/- 2.2 y). The oxidation of L-[1-(13C)]-leucine to 13CO2 [F13CO2 in micromol/(kg.h)] was determined after a primed, continuous oral administration of the tracer. Total BCAA in diet was provided at 300 mg/(kg.d) to ensure that leucine oxidation was measured when leucine intake was in excess of requirements. In the postabsorptive state, the rate of release of 13CO2 from 13C-leucine oxidation (F13CO2) and whole-body leucine oxidation were significantly higher in children with MCC liver disease (P < 0.05). However, F13CO2 and whole-body leucine oxidation did not differ in the fed state. We conclude that the increased need for dietary BCAA in MCC liver disease is mediated in part by increased leucine oxidation in the postabsorptive state.

Effect of increasing dietary protein content on muscle protein metabolism in healthy young and older people

Aging is associated with decreasing muscle mass and protein synthesis. Amino acid administration is reported to increase muscle protein synthesis and balance. We determined whether increasing dietary protein content from adequate (1.5g/kgFFM/d, AP) to high (3g/kgFFM/d, HP) had beneficial or adverse effects on whole body and muscle protein metabolism, renal function and insulin sensitivity in healthy young (24±1y) and older (70±2y) men and women. Subjects were randomized to a crossover design of 10 d of AP or HP protein diets, without changing caloric intake. Measurements of postabsoptive whole body leucine appearance rate (protein breakdown), oxidation, and non-oxidative leucine disposal (protein synthesis) rates were performed using L-[1-13C]-leucine and fractional synthesis rate (FSR) of mixed muscle proteins was done using [15N]-lysine as tracers. Glomerular filtration rate (GFR) and insulin sensitivity were determined by iodothalamate and intravenous glucose tolerance test, respectively. HP diet increased protein breakdown in both young and old (p<0.05) and increased leucine oxidation in both age groups with greater increase in young (p<0.005). WBPS, muscle protein FSR and insulin sensitivity were unaffected by diet. GFR was lower (p<0.01) in older than in young during both diets. HP diet increased GFR in young (p<0.01) but decreased GFR in older people (p<0.01). In conclusion, increasing dietary protein content had no detectable beneficial effects on muscle or whole body protein metabolism in healthy older people and may cause deleterious effects on renal function. Support: NIH R01AG09531.

Influence of glucagon on protein and leucine metabolism: A study in fasting man with induced insuiin resistance

The counter-regulatory hormones, including glucagon, may be involved in the generation of postoperative negative nitrogen balance. We examined the influence of glucagon on whole body and forearm muscle protein kinetics, determined by L-[1-13C, 15N]leucine, in two matched groups of healthy fasting subjects. In one study somatostatin alone was infused continuously (0.12 mg h-1) and in another with glucagon (0.04 mg h-1) to generate insulin resistance. Somatostatin infusion increased leucine oxidation (P less than 0.05) and reduced the negative protein balance (P less than 0.01) across the forearm; the 15 per cent decrease in protein breakdown was not significant. Whole body leucine kinetics showed increased flux (P less than 0.05) and synthesis (P less than 0.01) but reduced oxidation (P less than 0.05). Hyperglucagonaemia caused a threefold enhancement of leucine oxidation (P less than 0.02), while the negative protein balance further increased (P less than 0.05) across the forearm. Whole body leucine flux was unchanged; oxidation increased (P less than 0.01) and synthesis decreased (P less than 0.01). These studies confirm that physiological hyperglucagonaemia during insulin resistance is catabolic in the short-term and indicates, for the first time, that glucagon may influence muscle protein metabolism acutely in man. We suggest that therapeutic manoeuvres designed to reduce glucagon levels after surgery may ameliorate protein kinetic abnormalities.

Direct effects of proteasome inhibitor AdaAhx3L3VS on protein and amino acids metabolism in rat skeletal muscle.

Proteasome inhibitors are novel potential drugs for therapy of many diseases, and their effects are not fully understood. We investigated direct effects of peptide vinylsulfone inhibitor AdaAhx3L3VS on protein and amino acids metabolism in rat skeletal muscle. Soleus and extensor digitorum longus muscles were incubated in a medium containing 30 micromol/l AdaAhx3L3VS or no inhibitors. Total proteolysis was determined according to the rates of tyrosine release into the medium during incubation. The rates of leucine oxidation and protein synthesis were evaluated during incubation in medium containing L-[1-14C]leucine. Amino acid concentrations in the medium were measured using HPLC. AdaAhx3L3VS decreased tyrosine release into the medium by 21 and 19 %, decreased leucine incorporation into proteins by 22 and 12 %, and increased leucine oxidation by 24 and 19 % in soleus and extensor digitorum longus muscles, respectively. The release of amino acids into the medium was reduced. We conclude that AdaAhx3L3VS significantly decreased proteolysis and protein synthesis and increased leucine oxidation.

Postoperative Protein Sparing With Epidural Analgesia and Hypocaloric Dextrose

We examined the hypothesis that epidural analgesia prevents the increase in amino acid oxidation after elective colorectal surgery in patients receiving hypocaloric infusion of dextrose. Increased oxidative protein loss after surgery may adversely affect postoperative outcome. We have previously shown that effective segmental pain relief by epidural analgesia improves postoperative substrate utilization, resulting in less protein catabolism. We randomly allocated 10 patients to receive general anesthesia combined with epidural analgesia using bupivacaine/fentanyl and 10 to receive general anesthesia followed by patient-controlled analgesia with intravenous morphine. All patients received a peripheral 72-hour infusion of dextrose 10% from the day before until the second day after surgery. The dextrose infusion rate was adjusted to provide 50% of the patients' resting energy expenditure. The primary end point was whole-body leucine oxidation as determined by a stable isotope tracer technique (l-[1-C]leucine). In the intravenous analgesia group, leucine oxidation increased from 19 +/- 4 to 28 +/- 6 micromol kg h after surgery. Epidural analgesia prevented this increase of leucine oxidation (preoperative 21 +/- 6 micromol kg h, postoperative 21 +/- 5 micromol kg h). This difference was statistically significant (P = 0.01; analysis of variance for repeated measures). Perioperative epidural analgesia and hypocaloric dextrose infusion suppress the postoperative increase in amino acid oxidation, thereby saving more than 100 g of lean body mass per day.

Gender differences in the regulation of amino acid metabolism.

Exercising men, compared with women, have a greater increase in leucine oxidation but not lysine rate of appearance. The cause for this sexual dimorphism is unknown; however, an inhibition of beta-adrenoreceptor activity has previously been shown to mediate amino acid metabolism (Lamont LS, McCullough AJ, and Kalhan SC. Am J Physiol Endocrinol Metab 268: E910-E916, 1995; Lamont LS, Patel DG, and Kalhan SC. J Appl Physiol 67: 221-225, 1989). This study was a gender comparison of leucine and lysine kinetics during a beta-adrenoreceptor blockade (beta1,beta2-blockade) and a placebo control by using a double-blind crossover protocol. Subjects exercised at 50% of their trial-specific maximal O2 consumption (1 h) after 7 days of dietary control. During exercise with beta-blockade, men had an increased nonprotein respiratory exchange ratio (P < 0.001), whereas women had an increased circulation of free fatty acids (P < 0.001). The genders also displayed distinct differences in exercise amino acid kinetics. The men, but not the women, increased leucine oxidation (P < 0.005) and lysine rate of appearance (P < 0.009) when exercising during beta-adrenergic blockade. This study indicates that during beta-blockade, exercising men increase their need for amino acids (and carbohydrate) to fuel energy needs, whereas women increase their mobilization of fat, thereby requiring less alternative fuels such as carbohydrate and amino acids. Gender-specific fuel preferences during exercise are regulated by beta-adrenergic-receptor activity. Substrate availability during exercise appears to modulate the amino acid oxidation differences between genders.

Efficiency of utilization of wheat and milk protein in healthy adults and apparent lysine requirements determined by a single-meal [1-13C]leucine balance protocol

We recently reported better wheat-protein utilization and a higher apparent lysine requirement than would be predicted, because of adaptive mechanisms of lysine conservation. However, such findings may be subject to the feeding protocol of frequent small meals. We used a [1-13C]leucine balance, large single-meal protocol to estimate the utilization of wheat and the consequent lysine requirements. Wheat and milk utilization were compared in 5 adults infused for 9 h with L-[1-13C]leucine, in the postabsorptive (0-3 h) and postprandial (3-9 h) states after ingestion of a single meal of either milk (30.4 kJ/kg; 32% of energy as protein) or a mixture of wheat gluten and whole wheat (29.2 kJ; 26.7% of energy as protein). Premeal nitrogen balance was predicted from [1-13C]leucine oxidation and postmeal balance predicted from cumulative increased leucine oxidation, enabling evaluation of the metabolic demand for protein, the efficiency of postprandial protein utilization (PPU), and the requirements for wheat protein and lysine. Mean (+/-SD) PPU was 0.61 +/- 0.03 and 0.93 +/- 0.02 for wheat and milk (P < or = 0.001), respectively, and the estimated average wheat-protein requirement (0.6 g.kg(-1).d(-1)/PPU) was 0.98 +/- 0.05 g.kg(-1).d(-1), indicating a lysine requirement of 18.3 +/- 1.0 mg. kg(-1).d(-1). Measured wheat utilization efficiency at 0.61 was considerably higher than the value predicted from wheat lysine intake and milk protein lysine deposition (ie, 0.222 +/- 0.004). These results confirm our previous finding that lysine conservation allows wheat protein to be utilized more efficiently than expected and is consistent with a lysine requirement in fully adapted individuals of 19 mg.kg(-1).d(-1), as indicated by recalculated nitrogen balance data.

Variations in Mammary Protein Metabolism During the Natural Filling of the Udder with Milk over a 12-h Period Between Two Milkings: Leucine Kinetics

To define the temporal variations of whole body and mammary leucine kinetics over a 12-h period between two milkings, we used two groups of four Holstein cows, one in their second and the other in their third or fourth lactation. Cows were infused with L-[1-13C]leucine during the 12-h interval between two milkings. Blood was sampled every 30min during that period from arterial and mammary sources. Time after milking did not affect whole body irreversible loss rate of leucine but affected whole body leucine oxidation, which broadly followed variations in arterial plasma leucine concentration. Similarly, mammary leucine irreversible loss rate and leucine used for protein synthesis were not affected by time after milking. Leucine oxidation by the mammary gland was, however, affected by time after milking. It increased by 15% from the first 2-h period to the following 4-h period and then decreased by 13% over the following 2-h period. A 21% increase in leucine oxidation was observed from 8 to 10h after milking, and then it decreased by 26% over the last 2-h period. Protein degradation expressed as percentage of mammary leucine flux followed a similar temporal pattern. Leucine used for protein synthesis by the mammary gland was unaltered over time after milking, suggesting that the increased availability of leucine resulting from mammary protein breakdown would increase intracellular concentrations of leucine, which would have favored its catabolism. Overall, these results confirm the high metabolic activity of the mammary gland, as protein synthesis by the mammary gland averaged 43% of whole body protein synthesis.

Gender differences in leucine, but not lysine, kinetics.

There is a controversy in the literature as to the effects of gender on leucine kinetics. Two research groups found that men oxidize more leucine during exercise, whereas another group showed no gender effects. The purpose of our study was to examine the effects of gender on leucine and, for comparison purposes, lysine kinetics. Our subjects (n = 14) were seven matched pairs of men and women selected for their exercise habits and age. After 1 wk of a standardized diet, they exercised at 50% of maximal O(2) uptake for 1 h. There was an effect of exercise in both genders: an increased leucine oxidation and an attenuation in nonoxidative leucine disposal compared with rest (P < 0.05). Furthermore, our study confirms that there are gender differences in leucine, but not lysine, kinetics. Men had a higher rate of leucine oxidation and a lower rate of nonoxidative leucine disposal during exercise (P < 0.05). For women, a larger proportion of their exercise energy needs came from fat; for men, a greater fraction came from carbohydrate (P < 0.05). We conclude that female exercisers rely to a greater extent on fat as an energy source, thereby using less carbohydrate, amino acid, and protein as a fuel source.