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

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 > 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 < 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

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 BCKA levels and C5-OH/C3-DC acylcarnitines (AC) in muscle from HFHS or HFD-fed mice, but did not worsen insulin resistance. A set of short and long-chain acyl CoAs were elevated by diet alone in muscle, liver and 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 two 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.

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

ABSTRACT Background 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. Objectives 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. Methods 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). Results 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). Conclusions 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

Context 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. Objective 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. Design and Setting An observational, community-based study was conducted. Participants Fifteen patients with T2DM, 13 FDR, and 17 CONs were included (age, 40-70 years; body mass index, 27-35 kg/m2). Main Outcome Measures 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. Results 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). Conclusions 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.

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.

Alterations in branched-chain amino acid kinetics in nonobese but insulin-resistant Asian men

ABSTRACT Background Branched-chain amino acids (BCAAs) are elevated in the insulin-resistant (IR) state. The reasons for this increase remain unclear, but it may be related to abnormalities in BCAA metabolism and free fatty acid (FFA) metabolism. Objective In this study, we quantified BCAA and FFA kinetics of IR and insulin-sensitive (IS) nonobese Asian men with the use of stable-isotope tracers. We hypothesized that in addition to greater substrate flux, the BCAA oxidative pathway is also impaired to account for the higher plasma BCAA concentration in the IR state. Design We recruited 12 IR and 14 IS nonobese and healthy Asian men. Oral-glucose-tolerance tests (OGTTs) were performed to quantify insulin sensitivity, and subjects underwent 2 stable-isotope infusion studies. [U-13C6]Leucine was infused to measure leucine flux and oxidation as indexes of BCAA metabolism, whereas [U-13C16]palmitate was infused to measure palmitate flux and oxidation to represent FFA metabolism, The 2H2O dilution method was used to estimate body composition. Results IR subjects had greater adiposity and significantly higher fasting and post-OGTT glucose and insulin concentrations compared with the IS group. However, none of the subjects were diabetic. Despite similar dietary protein intake, IR subjects had a significantly higher plasma BCAA concentration and greater leucine flux. Leucine oxidation was also greater in the IR group, but the relation between leucine oxidation and flux was significantly weaker in the IR group than in the IS group (r = 0.530 compared with 0.695, P < 0.0388 for differences between slope). FFA oxidation was, however, unaffected despite higher FFA flux in the IR group. Conclusion The higher plasma BCAA concentration in healthy nonobese individuals with IR is associated with a weaker relation between BCAA oxidation and BCAA flux and this occurs in the presence of accelerated FFA flux and oxidation.

The contribution of muscle, kidney, and splanchnic tissues to leucine transamination in humans

The first steps of leucine utilization are reversible deamination to α-ketoisocaproic acid (α-KIC) and irreversible oxidation. Recently, the regulatory role of leucine deamination over oxidation was underlined in rodents. Our aim was to measure leucine deamination and reamination in the whole body, in respect to previously determined rates across individual organs, in humans. By leucine and KIC isotope kinetics, we determined whole-body leucine deamination and reamination, and we compared these rates with those already reported across the sampled organs. As an in vivo counterpart of the “metabolon” concept, we analysed ratios between oxidation and either deamination or reamination. Leucine deamination to KIC was greater than KIC reamination to leucine in the whole body (p = 0.005), muscles (p = 0.005), and the splanchnic area (p = 0.025). These rates were not significantly different in the kidneys. Muscle accounted for ≈60% and ≈78%, the splanchnic bed for ≈15% and ≈15%, and the kidney for ≈12% and ≈18%, of whole-body leucine deamination and reamination rates, respectively. In the kidney, percent leucine oxidation over either deamination or reamination was >3-fold greater than muscle and the splanchnic bed. Skeletal muscle contributes by the largest fraction of leucine deamination, reamination, and oxidation. However, in relative terms, the kidney plays a key role in leucine oxidation.