Leucine Oxidation Research Articles

Abstract Tu108: Improved Skeletal Muscle Metabolism and Exercise Capacity Following Hydrogen Sulfide Therapy in Cardiometabolic HFpEF

Introduction: Exercise intolerance is the cardinal feature of cardiometabolic HFpEF that is largely driven by impairments in skeletal muscle metabolism and function. Hydrogen sulfide (H 2 S) is an essential regulator of mitochondrial function and metabolic homeostasis. We have previously demonstrated reductions in skeletal muscle enzymatic H 2 S production and H 2 S bioavailability in rodent models of HFpEF. The potential for H 2 S donor therapy to restore skeletal muscle metabolism and improve exercise capacity in HFpEF has not been explored. Hypothesis: We tested the hypothesis that oral H 2 S donor (SG-1002) therapy would restore critical aspects of skeletal muscle metabolism and exercise performance in cardiometabolic HFpEF. Methods: Five weeks after the onset of HFpEF (high-fat diet + L-NAME) male C57BL6/J mice (n=12 per group) were administered dietary SG-1002 (95 mg/kg/day) for 5 weeks and compared to HFD + L-NAME alone mice (i.e., control). Exercise performance was assessed using forced treadmill and voluntary wheel running (VWR) with continuous monitoring of indirect calorimetry. Skeletal muscle (soleus and gastroc) mitochondrial function was assessed by high-resolution respirometry. Palmitate (fatty acid oxidation, FAO) and leucine (BCAA) oxidation were determined by radiolabeled isotope ( 14 C) assays. A force grid meter was used to determine muscle grip force production. GC-chemiluminescence was used to measure H 2 S bioavailability. Results: SG-1002 therapy significantly (p < 0.001) increased circulating H 2 S bioavailability (0.57 ± 0.15 vs. 0.22 ± 0.08 µM) when compared to HFpEF control mice. Forced treadmill running (125 ± 87 vs 64 ± 33 meters), grip force production (9.6± 1.1 vs. 7.5 ± 1.3 g/g lean mass) were increased (p < 0.05 vs. control for both) following SG-1002 treatment. During VWR, whole-body fat oxidation was increased (p=0.0008, 0.3 ± .01 lower RER) in SG-1002-treated mice. At the skeletal muscle level, SG-1002 treatment reduced LEAK state respiration by 52% (p < 0.0001 vs. control) and increased complete FAO by 30% (p < 0.05) while reducing incomplete FAO by 33% (p < 0.05 between groups). Conclusion: We demonstrate that H 2 S donor therapy significantly improves exercise capacity and underlying skeletal muscle dysfunction in a mouse “two-hit” model of cardiometabolic HFpEF.

Abstract Tu104: Mitochondrial Hydrogen Sulfide Regulates Skeletal Muscle Dysfunction and Exercise Intolerance in Cardiometabolic HFpEF

Introduction: Cardiometabolic heart failure with preserved ejection fraction (HFpEF) results in severe exercise intolerance. Dysregulated skeletal muscle metabolism and mitochondrial function are key drivers of exercise intolerance in cardiometabolic HFpEF. Mitochondrial hydrogen sulfide (H 2 S) generated by 3-mercaptopyruvate sulfur transferase (3-MST) exerts potent bioenergetic properties. We have recently shown that H 2 S bioavailability is significantly attenuated in HFpEF. The role of H2S in skeletal muscle metabolism and exercise tolerance in HFpEF is currently unknown. Hypothesis: We tested the hypothesis that the loss of skeletal muscle H 2 S results in metabolic dysfunction in skeletal muscle and contributes to exercise intolerance in cardiometabolic HFpEF. Methods: Skeletal muscle (gastrocnemius) from L-NAME + High Fat diet (“two-hit”) treated, and chow-fed control male mice (n=10/group) were assessed for free H 2 S and 3-MST enzymatic activity using GC-chemiluminescence detection. Additionally, muscles were processed for RNA sequencing. Next, male global 3-MST knockout (KO) mice (n=12) and controls (n=10) were subjected to “two-hit” HFpEF for 10 weeks, and exercise tolerance was evaluated. Skeletal muscle (soleus and gastroc) was subjected to high-resolution respirometry and substrate oxidation of radiolabeled isotope ( 14 C) for leucine and palmitate. Results: Skeletal muscle H 2 S levels (0.73 ± 0.18 vs. 1.0 ± 0.24 nmol/g/tissue) and 3-MST activity (0.46 ± 0.01 vs 0.57 ± 0.01 µmol/g/tissue/min) were significantly (p < 0.05) reduced in “two-hit” HFpEF mice compared to control mice. At the transcriptome level, 3-MST was one of the top 50 differentially regulated genes (i.e., reduced expression) in “two-hit” mice (Adjusted p=0.01). Genetic deficiency of 3-MST KO mice exacerbated (p < 0.05) exercise intolerance as measured by treadmill running distance (53 ± 24 vs. 138 ± 104 meters) vs. “two-hit” HFpEF genotype controls. Mitochondrial Complex I (low ADP) respiration was reduced by 23% in 3-MST KO HFpEF mice (p < 0.05). Genetic deficiency of 3-MST also reduced skeletal muscle leucine oxidation (58 ± 19 vs 84 ± 8; p = 0.01) and dramatically increased incomplete palmitate oxidation by 2.5-fold (p < 0.001) without changing complete oxidation. Conclusion: We demonstrate that skeletal muscle mitochondrial H 2 S is a critical regulator of metabolic function and exercise capacity in cardiometabolic HFpEF.

Postexercise Dietary Leucine Retention for Whole-Body Anabolism Is Greater With Whey Protein Isolate and Fish-Derived Protein Hydrolysate Than Nonessential Amino Acids in Trained Young Men.

Marine-derived proteins, such as blue whiting-derived protein hydrolysates (BWPH), represent high-quality sources of dietary protein, but their ability to support postexercise anabolism is not established. The impact of BWPH on whole-body anabolism was compared with an isonitrogenous whey protein isolate (WPI) and nonessential amino acid (NEAA) control in 10 trained young males (31 ± 4years) who, on three separate visits, performed a session of whole-body resistance exercise and then consumed, in randomized crossover fashion, BWPH, WPI, or NEAA (0.33g/kg; 19, 33, and 0mg/kgleucine, respectively) with L-[1-13C]leucine. Breath, blood, and urine samples were collected for 6-hr postprandial to assess dietary leucine oxidation, amino acid (AA) concentrations, and 3-methylhistidine: creatinine ratio. Peak and area under the curve concentrations for leucine, branched-chain amino acids, and essential amino acids were greater in WPI compared with BWPH (all p < .05) but with no differences in time to peak concentration. Total oxidation reflected leucine intake (WPI > BWPH > NEAA; p < .01), whereas relative oxidation was greater (p < .01) in WPI (28.6 ± 3.6%) compared with NEAA (21.3 ± 4.2%), but not BWPH (28.6 ± 8.8%). Leucine retention, a proxy for whole-body protein synthesis, was greater in WPI (185.6 ± 9.5μmol/kg) compared with BWPH (109.3 ± 14.1 μmol/kg) and NEAA (5.74 ± 0.30μmol/kg; both p < .01), with BWPH being greater than NEAA (p < .01). Urinary 3-methylhistidine: creatinine ratio did not differ between conditions. Both WPI and BWPH produced essential aminoacidemia and supported whole-body anabolism after resistance exercise, but a higher intake of BWPH to better approximate the leucine and EAA content of WPI may be needed to produce an equivalent anabolic response.

Underpinning the Food Matrix Regulation of Postexercise Myofibrillar Protein Synthesis by Comparing Salmon Ingestion With the Sum of Its Isolated Nutrients in Healthy Young Adults

BackgroundProtein is most commonly consumed as whole foods as opposed to single nutrients. However, the food matrix regulation of the postprandial muscle protein synthetic response has received little attention. ObjectivesThe purpose of this study was to assess the effects of eating salmon (SAL) and of ingesting the same nutrients as an isolated mixture of crystalline amino acids and fish oil (ISO) on the stimulation of postexercise myofibrillar protein synthesis (MPS) and whole-body leucine oxidation rates in healthy young adults. MethodsTen recreationally active adults (24 ± 4 y; 5 men, 5 women) performed an acute bout of resistance exercise, followed by the ingestion of SAL or ISO in a crossover fashion. Blood, breath, and muscle biopsies were collected at rest and after exercise during primed continuous infusions of L-[ring-2H5]phenylalanine and L-[1-13C]leucine. All data are presented as means ± SD and/or mean differences (95% CIs). ResultsPostprandial essential amino acid (EAA) concentrations peaked earlier (P = 0.024) in the ISO group than those in the SAL group. Postprandial leucine oxidation rates increased over time (P < 0.001) and peaked earlier in the ISO group (1.239 ± 0.321 nmol/kg/min; 63 ± 25 min) than those in the SAL group (1.230 ± 0.561 nmol/kg/min; 105 ± 20 min; P = 0.003). MPS rates for SAL (0.056 ± 0.022 %/h; P = 0.001) and ISO (0.046 ± 0.025 %/h; P = 0.025) were greater than the basal rates (0.020 ± 0.011 %/h) during the 0- to 5-h recovery period, with no differences between conditions (P = 0.308). ConclusionWe showed that the postexercise ingestion of SAL or ISO stimulate postexercise MPS rates with no differences between the conditions. Thus, our results indicate that ingesting protein from SAL as a whole-food matrix is similarly anabolic to ISO in healthy young adults. This trial was registered at www.clinicaltrials.gov as NCT03870165.

Chronic Fetal Leucine Infusion Increases Rate of Leucine Oxidation but Not of Protein Synthesis in Late Gestation Fetal Sheep

BackgroundLeucine increases protein synthesis rates in postnatal animals and adults. Whether supplemental leucine has similar effects in the fetus has not been determined. ObjectiveTo determine the effect of a chronic leucine infusion on whole-body leucine oxidation and protein metabolic rates, muscle mass, and regulators of muscle protein synthesis in late gestation fetal sheep. MethodsCatheterized fetal sheep at ∼126 d of gestation (term = 147 d) received infusions of saline (CON, n = 11) or leucine (LEU; n = 9) adjusted to increase fetal plasma leucine concentrations by 50%–100% for 9 d. Umbilical substrate net uptake rates and protein metabolic rates were determined using a 1-13C leucine tracer. Myofiber myosin heavy chain (MHC) type and area, expression of amino acid transporters, and abundance of protein synthesis regulators were measured in fetal skeletal muscle. Groups were compared using unpaired t tests. ResultsPlasma leucine concentrations were 75% higher in LEU fetuses compared with CON by the end of the infusion period (P < 0.0001). Umbilical blood flow and uptake rates of most amino acids, lactate, and oxygen were similar between groups. Fetal whole-body leucine oxidation was 90% higher in LEU (P < 0.0005) but protein synthesis and breakdown rates were similar. Fetal and muscle weights and myofiber areas were similar between groups, however, there were fewer MHC type IIa fibers (P < 0.05), greater mRNA expression levels of amino acid transporters (P < 0.01), and a higher abundance of signaling proteins that regulate protein synthesis (P < 0.05) in muscle from LEU fetuses. ConclusionsA direct leucine infusion for 9 d in late gestation fetal sheep does not increase protein synthesis rates but results in higher leucine oxidation rates and fewer glycolytic myofibers. Increasing leucine concentrations in the fetus stimulates its own oxidation but also increases amino acid transporter expression and primes protein synthetic pathways in skeletal muscle.

A non-invasive 13CO2 breath test detects differences in anabolic sensitivity with feeding and heavy resistance exercise in healthy young males: a randomized control trial.

There are limited tools to measure anabolic sensitivity non-invasively in response to acute physiological stimuli, which represents a challenge for research in free-living settings and vulnerable populations. We tested the ability of a stable isotope breath test to detect changes in leucine oxidation (OX) and leucine retention (intake-OX) across a range of anabolic sensitivities. Healthy males ingested a beverage containing 0.25 g·kg-1 protein and 0.75 g·kg-1 carbohydrate with the leucine content enriched to 5% with l-[1-13C]leucine at rest (FED) or after a bout of resistance exercise (EXFED), with a parallel group consuming only the tracer (FAST). Concurrent primed-constant infusions of l-[5,5,5-2H3]leucine revealed high peripheral bioavailability for FED (∼81%), EXFED (∼80%), and FAST (∼117%). After beverage ingestion, whole-body protein synthesis was greater in FED and EXFED than FAST. OX was greater in FED and EXFED than FAST, with OX lower in EXFED than FED. Leucine retention demonstrated expected physiological differences in anabolic sensitivity (EXFED>FED>FAST). We demonstrated that a non-invasive breath test based on an amino acid (leucine) that is preferentially metabolized in peripheral (muscle) tissues can detect differences in anabolic sensitivity. Future studies could examine this test within a variety of populations experiencing muscle growth or atrophy. This study was registered as a Clinical Trial at ClinicalTrials.gov (no. NCT04887727). Novelty: An oral l-[1-13C]leucine breath test can detect greater anabolic sensitivity after feeding and resistance exercise. This tool may be applied in growing (e.g., children) or wasting (e.g., aging) populations where invasive procedures are not possible.

SIRT4 is an early regulator of branched-chain amino acid catabolism that promotes adipogenesis.

Upon nutrient stimulation, pre-adipocytes undergo differentiation to transform into mature adipocytes capable of storing nutrients as fat. We profiled cellular metabolite consumption to identify early metabolic drivers of adipocyte differentiation. We find that adipocyte differentiation raises the uptake and consumption of numerous amino acids. In particular, branched-chain amino acid (BCAA) catabolism precedes and promotes peroxisome proliferator-activated receptor gamma (PPARγ), a key regulator of adipogenesis. In early adipogenesis, the mitochondrial sirtuin SIRT4 elevates BCAA catabolism through the activation of methylcrotonyl-coenzyme A (CoA) carboxylase (MCCC). MCCC supports leucine oxidation by catalyzing the carboxylation of 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA. Sirtuin 4 (SIRT4) expression is decreased in adipose tissue of numerous diabetic mouse models, and its expression is most correlated with BCAA enzymes, suggesting a potential role for SIRT4 in adipose pathology through the alteration of BCAA metabolism. In summary, this work provides a temporal analysis of adipocyte differentiation and uncovers early metabolic events that stimulate transcriptional reprogramming.

Regulation of cold-induced thermogenesis by the RNA binding protein FAM195A

Homeothermic vertebrates produce heat in cold environments through thermogenesis, in which brown adipose tissue (BAT) increases mitochondrial oxidation along with uncoupling of the electron transport chain and activation of uncoupling protein 1 (UCP1). Although the transcription factors regulating the expression of UCP1 and nutrient oxidation genes have been extensively studied, only a few other proteins essential for BAT function have been identified. We describe the discovery of FAM195A, a BAT-enriched RNA binding protein, which is required for cold-dependent thermogenesis in mice. FAM195A knockout (KO) mice display whitening of BAT and an inability to thermoregulate. In BAT of FAM195A KO mice, enzymes involved in branched-chain amino acid (BCAA) metabolism are down-regulated, impairing their response to cold. Knockdown of FAM195A in brown adipocytes in vitro also impairs expression of leucine oxidation enzymes, revealing FAM195A to be a regulator of BCAA metabolism and a potential target for metabolic disorders.

Leucine Is More Readily Oxidized When Ingested as an Isolated Nutrient versus Incorporated in Its Whole-Food Matrix

Abstract Objectives The ingestion of free amino acids, or isolated sources of protein, results in faster postprandial release of dietary amino acids into circulation, which stimulates muscle protein synthesis rates. However, indirect evidence suggests that this rapid release of dietary amino acids after the ingestion of free amino acids is coupled with higher amino acid oxidation rates when compared to a whole-food source. Whole food protein results in a reduced peak amplitude and prolonged postprandial aminoacidemia. This study aimed to assess the effects of eating a whole food source of protein on the stimulation of whole-body leucine oxidation rates versus eating these same nutrients in isolated form in healthy young adults. Methods In a crossover design, 10 recreationally active adults (24 ± 4 y; 5 M, 5 F) performed an acute bout of resistance exercise followed by the ingestion of salmon (SAL) (20.5 g protein and 7.5 g fat) or its matched constituents as crystalline amino acids and fish oil (ISO). Participants received priming doses of NaH13CO2 and L-[1–13C]leucine before initiating a constant L-[1–13C]leucine infusion. Blood and breath samples were collected at rest and after resistance exercise at regular intervals for the measurement of whole-body leucine oxidation rates and plasma leucine profiles. Data were tested using linear fixed effects models with time and group as fixed factors with Bonferroni's post hoc test. Results Postprandial plasma leucine concentrations did not differ between the SAL and ISO conditions (P &amp;gt; 0.05). Time to peak plasma leucine concentrations was faster in ISO (50 ± 27 min) vs. SAL (114 ± 64 min, P = 0.022) condition. Postprandial leucine oxidation rates were elevated from baseline at t = 30 min to t = 120 min in ISO and t = 60 min to t = 180 min in SAL (P &amp;lt; 0.001), with no total differences between group (P = 0.129). Time to peak leucine oxidation occurred sooner in ISO (66 ± 22 min; 1.358 ± 0.699 nmol · kg−1 · min−1) when compared to the SAL condition (105 ± 20 min; 1.067 ± 0.3076 nmol · kg−1 · min−1; P = 0.002). Conclusions We show that the ingestion of a whole-food source of protein resulted in a delayed stimulation of leucine oxidation when compared to free amino acid ingestion, but a similar net increase in oxidation during the 5 h postprandial period in healthy young adults. Funding Sources USDA National Institute of Food and Agriculture Hatch project 1017928

Emerging role of the mitochondrial branched-chain aminotransferase (BCATm) as an immunosuppressive enzyme during CD4+ T cell activation

Abstract The mitochondrial branched-chain aminotransferase (BCATm) catalyzes the transamination of leucine, an amino acid that is essential for the upregulation of complex 1 of the mammalian target of rapamycin (mTORC1) during T cell activation. Because BCATm is responsible for the mitochondrial degradation of leucine, we hypothesized that BCATm limits leucine availability for mTORC1 signaling and subsequently suppresses T cell activation. To test the hypothesis, we isolated CD4+T cells from the global BCATmKO mouse and measured the rates of leucine transamination/oxidation, the activity of the mTORC1 downstream targets S6 and 4EBP-1, and the release of IFNγ after 24–72h of T cell activation. To elucidate the role of BCATm in T cells in vivo, we used the Cre-LoxP strategy to create a T cell specific deletion of BCATm in C57BL/6 mice. We completed the initial characterization of the mice, which included a validation of absence of BCATm in T cells by using qRT-PCR and Western blotting. Results demonstrated that BCATm-deficient T cells experienced significant reductions in the transamination and oxidation of leucine, which led to increased intracellular leucine concentrations. This correlated with an increased phosphorylation of S6, but not 4EBP-1, suggesting that BCATm regulates mTORC1 signaling in S6 dependent manner. The T cells released more IFNγ in the absence of BCATm, which was highest after 72h of T cell activation. Thus, BCATm appears to play an immunosuppressive role by reducing CD4+ T cell activation. The newly designed T cell conditional BCATmKO mice, which do not express BCATm in CD4+ and CD8+ T cells will be engaged in preclinical studies aiming at deciphering the therapeutic potential of BCATm in T cell-driven immunological responses.

BCAA Supplementation in Mice with Diet-induced Obesity Alters the Metabolome Without Impairing Glucose Homeostasis.

Circulating branched chain amino acid (BCAA) levels are elevated in obese humans and genetically obese rodents. However, the relationship of BCAAs to insulin resistance in diet-induced obese mice, a commonly used model to study glucose homeostasis, is still ill-defined. Here we examined how high-fat high-sucrose (HFHS) or high-fat diet (HFD) feeding, with or without BCAA supplementation in water, alters the metabolome in serum/plasma and tissues in mice and whether raising circulating BCAA levels worsens insulin resistance and glucose intolerance. Neither HFHS nor HFD feeding raised circulating BCAA levels in insulin-resistant diet-induced obese mice. BCAA supplementation raised circulating BCAA and branched-chain α-keto acid levels and C5-OH/C3-DC acylcarnitines (AC) in muscle from mice fed an HFHS diet or HFD, but did not worsen insulin resistance. A set of short- and long-chain acyl CoAs were elevated by diet alone in muscle, liver, and white adipose tissue (WAT), but not increased further by BCAA supplementation. HFD feeding reduced valine and leucine oxidation in WAT but not in muscle. BCAA supplementation markedly increased valine oxidation in muscle from HFD-fed mice, while leucine oxidation was unaffected by diet or BCAA treatment. Here we establish an extensive metabolome database showing tissue-specific changes in mice on 2 different HFDs, with or without BCAA supplementation. We conclude that mildly elevating circulating BCAAs and a subset of ACs by BCAA supplementation does not worsen insulin resistance or glucose tolerance in mice. This work highlights major differences in the effects of BCAAs on glucose homeostasis in diet-induced obese mice versus data reported in obese rats and in humans.

Using breath δ13C analysis to determine the effects of dietary carbohydrate and protein on glucose and leucine oxidation at rest in the yellow-rumped warbler (Setophaga coronata)

Fat is the major fuel for migratory flight of birds, but protein is also catabolized. Flight range could be reduced if protein is used too quickly from muscles and organs, and it is important to understand factors that influence protein catabolism. Previous correlative studies suggested high protein diets may increase protein use in flight, although a wind tunnel study with yellow-rumped warblers (Setophaga coronata) did not support this relationship. We tested the hypothesis that diet composition affects nutrient oxidation in resting, fasted yellow-rumped warblers. For method development, we gavaged or subcutaneously injected warblers with 13C labelled glucose or leucine, and measured δ13C of breath CO2 in real time using infrared laser spectrometry. Regardless of route of administration, leucine had greater instantaneous and cumulative oxidation than glucose. Compared to subcutaneous injection, gavaged birds reached maximum oxidation rate faster for leucine and glucose, respectively, had a higher maximum oxidation rate, and reached final cumulative oxidation approximately faster for leucine or glucose, respectively, indicating immediate oxidation of the substrates by the digestive system. Warblers (N = 10 each) were fed isocaloric 60% carbohydrate or 60% protein diets for minimum 2 weeks, and subcutaneously injected with 13C labelled glucose or leucine. Diet composition had little effect on oxidation kinetics except that warblers fed high-carbohydrate reached final cumulative oxidation of leucine more quickly than those fed high-protein. The findings do not support the hypothesis that high protein diets increase the oxidation of protein during negative energy states in migratory birds, and provide methodology that could be applied to test it in flight.

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.

Branched-Chain Amino Acid Oxidation Is Elevated in Adults with Morbid Obesity and Decreases Significantly after Sleeve Gastrectomy

Plasma concentrations of branched-chain amino acids (BCAAs) are elevated in obese individuals with insulin resistance (IR) and decrease after bariatric surgery. However, the metabolic mechanisms are unclear. Our objectives are to compare leucine kinetics between morbidly obese and healthy-weight individuals cross-sectionally, and to prospectively evaluate changes in the morbidly obese after sleeve gastrectomy. We hypothesized that leucine oxidation is slower in obese individuals and increases after surgery. Ten morbidly obese [BMI (in kg/m2)≥32.5, age 21-50 y] and 10 healthy-weight participants (BMI <25), matched for age (median ∼30 y) but not gender, were infused with [U-13C6] leucine and [2H5] glycerol to quantify leucine and glycerol kinetics. Morbidly obese participants were studied again 6 mo postsurgery. Primary outcomes were kinetic parameters related to BCAA metabolism. Data were analyzed by nonparametric methods and presented as median (IQR). Participants with obesity had IR with an HOMA-IR (4.89; 4.36-8.76) greater than that of healthy-weight participants (1.32; 0.99-1.49; P <0.001) and had significantly faster leucine flux [218; 196-259 compared with 145; 138-149 μmol · kg fat-free mass (FFM)-1 · h-1], oxidation (24.0; 17.9-29.8 compared with 16.1; 14.3-18.5 μmol · kg FFM-1 · h-1), and nonoxidative disposal (204; 190-247 compared with 138; 129-140 μmol · kg FFM-1 · h-1) (P <0.017 for all). After surgery, the morbidly obese had a marked improvement in IR (3.54; 3.06-6.08; P=0.008) and significant reductions in BCAA concentrations (113; 95-157 μmol/L) and leucine oxidation (9.37; 6.85-15.2 μmol · kg FFM-1 · h-1) (P=0.017 for both). Further, leucine flux in this group correlated significantly with IR (r=0.78, P <0.001). BCAA oxidation is not impaired but elevated in individuals with morbid obesity. Plasma BCAA concentrations are lowered after surgery owing to slower breakdown of body proteins as insulin's ability to suppress proteolysis is restored. These findings suggest that IR is the underlying cause and not the consequence of elevated BCAAs in obesity.

Elevated Plasma Branched-Chain Amino Acid Levels Correlate With Type 2 Diabetes-Related Metabolic Disturbances.

Patients with type 2 diabetes mellitus (T2DM) have elevated plasma branched-chain amino acid (BCAA) levels. The underlying cause, however, is not known. Low mitochondrial oxidation of BCAA levels could contribute to higher plasma BCAA levels. We aimed to investigate ex vivo muscle mitochondrial oxidative capacity and in vivo BCAA oxidation measured by whole-body leucine oxidation rates in patients with T2DM, first-degree relatives (FDRs), and control participants (CONs) with overweight or obesity. An observational, community-based study was conducted. Fifteen patients with T2DM, 13 FDR, and 17 CONs were included (age, 40-70 years; body mass index, 27-35 kg/m2). High-resolution respirometry was used to examine ex vivo mitochondrial oxidative capacity in permeabilized muscle fibers. A subgroup of 5 T2DM patients and 5 CONs underwent hyperinsulinemic-euglycemic clamps combined with 1-13C leucine-infusion to determine whole-body leucine oxidation. Total BCAA levels were higher in patients with T2DM compared to CONs, but not in FDRs, and correlated negatively with muscle mitochondrial oxidative capacity (r = -0.44, P < .001). Consistently, whole-body leucine oxidation rate was lower in patients with T2DM vs CON under basal conditions (0.202 ± 0.049 vs 0.275 ± 0.043 μmol kg-1 min-1, P < .05) and tended to be lower during high insulin infusion (0.326 ± 0.024 vs 0.382 ± 0.013 μmol kg-1 min-1, P = .075). In patients with T2DM, a compromised whole-body leucine oxidation rate supports our hypothesis that higher plasma BCAA levels may originate at least partly from a low mitochondrial oxidative capacity.

Leucine acutely potentiates glucose-stimulated insulin secretion in fetal sheep.

A 9-day infusion of leucine into fetal sheep potentiates fetal glucose-stimulated insulin secretion (GSIS). However, there were accompanying pancreatic structural changes that included a larger proportion of β-cells and increased vascularity. Whether leucine can acutely potentiate fetal GSIS in vivo before these structural changes develop is unknown. The mechanisms by which leucine acutely potentiates GSIS in adult islets and insulin-secreting cell lines are well known. These mechanisms involve leucine metabolism, including leucine oxidation. However, it is not clear if leucine-stimulated metabolic pathways are active in fetal islets. We hypothesized that leucine would acutely potentiate GSIS in fetal sheep and that isolated fetal islets are capable of oxidizing leucine. We also hypothesized that leucine would stimulate other metabolic pathways associated with insulin secretion. In pregnant sheep we tested in vivo GSIS with and without an acute leucine infusion. In isolated fetal sheep islets, we measured leucine oxidation with a [1-14C] l-leucine tracer. We also measured concentrations of other amino acids, glucose, and analytes associated with cellular metabolism following incubation of fetal islets with leucine. In vivo, a leucine infusion resulted in glucose-stimulated insulin concentrations that were over 50% higher than controls (P < 0.05). Isolated fetal islets oxidized leucine. Leucine supplementation of isolated fetal islets also resulted in significant activation of metabolic pathways involving leucine and other amino acids. In summary, acute leucine supplementation potentiates fetal GSIS in vivo, likely through pathways related to the oxidation of leucine and catabolism of other amino acids.

Kinetics and mechanistic study of oxidation of α-amino acid leucine by TCICA in aqueous acetic acid medium

Kinetics and mechanistic study of oxidation of α-amino acid leucine by TCICA in aqueous acetic acid medium

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.

Life-extending dietary restriction, but not dietary supplementation of branched-chain amino acids, can increase organismal oxidation rates of individual branched-chain amino acids by grasshoppers.

BACKGROUND:Life-extending dietary restriction increases energy demands. Branched-chain amino acids (BCAAs), at high levels, may be detrimental to healthspan by activating the mechanistic Target of Rapamycin (mTOR). Whether organismal oxidation of BCAAs increases upon dietary restriction is unknown.OBJECTIVE:Test whether dietary restriction (DR, which creates an energy deficit) or supplemental dietary BCAAs (superfluous BCAAs) increases oxidation of BCAAs, potentially reducing their levels to improve healthspan.METHODS:Grasshoppers were reared to middle-age on one of four diets, each a level of lettuce feeding and a force-fed solution: 1) ad libitum lettuce & buffer, 2) ad libitum lettuce & supplemental BCAAs, 3) DR lettuce & buffer, and 4) DR lettuce & supplemental BCAAs. On trial days, grasshoppers were force-fed one 13C-1-BCAA (isoleucine, leucine, or valine). Breath was collected and tested for 13CO2, which represents organismal oxidation of the amino acid. Additional trials re-tested oxidation of leucine (the most potent activator of mTOR) in both females and males on dietary restriction.RESULTS:Dietary restriction generally increased cumulative oxidation of each BCAA in females and hungry males over ∼8 hr. Results were consistent for isoleucine and valine, but less so for leucine. Supplementation of BCAAs, in combination with dietary restriction, increased isoleucine in hemolymph, with similar trends for leucine and valine. Despite this, supplementation of BCAAs did not alter oxidation of any BCAAs.CONCLUSIONS:Dietary restriction can increase oxidation of BCAAs, likely due to an energy deficit. The increased oxidation may decrease available BCAAs for activation of mTOR and improve healthspan.

251 Oxidation of energy substrates in tissues of Largemouth bass (Micropterus salmoides)

Abstract Because of growing interest in producing Largemouth bass (HMB) as a source of high-quality protein for human consumption worldwide, it is imperative to understand the metabolism of nutrients (including amino acids and carbohydrate) in this aquatic animal. The present study tested the hypothesis that amino acids are oxidized at a higher rate than carbohydrates (e.g., glucose) and fatty acids (e.g., palmitate) to provide ATP for tissues of LMB fed a 45%-crude protein diet. The liver, intestine, kidney, and skeletal muscle were isolated from juvenile LMB and incubated at 26 °C (the body temperature of LMB) for 2 h in 1 ml of oxygenated Krebs–Henseleit bicarbonate buffer (pH 7.4) containing a mixture of nutrients (2 mM glutamate, 2 mM glutamine, 2 mM aspartate, 2 mM alanine, 2 mM leucine, 5 mM glucose, and 2 mM palmitate). The rate of oxidation of each energy substrate was determined by using [U-14C]-labeled glutamate, glutamine, aspartate, alanine, leucine, glucose, or palmitate and collecting 14CO2 from each tracer. Results indicated that aspartate, glutamate and glutamine were extensively oxidized in all the four tissues and contributed to 67% of total ATP production. Glutamate contributed to more ATP than glutamine in the intestine, whereas similar amounts of ATP were produced from glutamate and glutamine in the liver, kidneys and skeletal muscle. In all the four tissues, rates of oxidation of alanine, leucine, palmitate and glucose were low and each of those nutrients contributed to &amp;lt; 10% of total ATP production. Together, the oxidation of aspartate, glutamate, glutamine, alanine plus leucine provided 82–85% of total ATP for the liver, intestine, kidney, and skeletal muscle. We conclude that amino acids, rather than glucose and long-chain fatty acids, are the primary energy substrates in the major tissues of Largemouth bass.