Branched-chain Amino Acid Metabolites Research Articles

Variant of the lactase LCT gene explains association between milk intake and incident type 2 diabetes.

Cow's milk is frequently included in the human diet, but the relationship between milk intake and type 2 diabetes (T2D) remains controversial. Here, using data from the Hispanic Community Health Study/Study of Latinos, we show that in both sexes, higher milk intake is associated with lower risk of T2D in lactase non-persistent (LNP) individuals (determined by a variant of the lactase LCT gene, single nucleotide polymorphism rs4988235 ) but not in lactase persistent individuals. We validate this finding in the UK Biobank. Further analyses reveal that among LNP individuals, higher milk intake is associated with alterations in gut microbiota (for example, enriched Bifidobacterium and reduced Prevotella) and circulating metabolites (for example, increased indolepropionate and reduced branched-chain amino acid metabolites). Many of these metabolites are related to the identified milk-associated bacteria and partially mediate the association between milk intake and T2D in LNP individuals. Our study demonstrates a protective association between milk intake and T2D among LNP individuals and a potential involvement of gut microbiota and blood metabolites in this association.

β-aminoisobutyrics acid, a metabolite of BCAA, activates the AMPK/Nrf-2 pathway to prevent ferroptosis and ameliorates lung ischemia-reperfusion injury

BackgroundLung ischemia-reperfusion (I/R) injury is a serious clinical problem without effective treatment. Enhancing branched-chain amino acids (BCAA) metabolism can protect against cardiac I/R injury, which may be related to bioactive molecules generated by BCAA metabolites. L-β-aminoisobutyric acid (L-BAIBA), a metabolite of BCAA, has multi-organ protective effects, but whether it protects against lung I/R injury is unclear.MethodsTo assess the protective effect of L-BAIBA against lung I/R injury, an animal model was generated by clamping the hilum of the left lung, followed by releasing the clamp in C57BL/6 mice. Mice with lung I/R injury were pre-treated or post-treated with L-BAIBA (150 mg/kg/day), given by gavage or intraperitoneal injection. Lung injury was assessed by measuring lung edema and analyzing blood gases. Inflammation was assessed by measuring proinflammatory cytokines in bronchoalveolar lavage fluid (BALF), and neutrophil infiltration of the lung was measured by myeloperoxidase activity. Molecular biological methods, including western blot and immunofluorescence, were used to detect potential signaling mechanisms in A549 and BEAS-2B cells.ResultsWe found that L-BAIBA can protect the lung from I/R injury by inhibiting ferroptosis, which depends on the up-regulation of the expressions of GPX4 and SLC7A11 in C57BL/6 mice. Additionally, we demonstrated that the Nrf-2 signaling pathway is key to the inhibitory effect of L-BAIBA on ferroptosis in A549 and BEAS-2B cells. L-BAIBA can induce the nuclear translocation of Nrf-2. Interfering with the expression of Nrf-2 eliminated the protective effect of L-BAIBA on ferroptosis. A screening of potential signaling pathways revealed that L-BAIBA can increase the phosphorylation of AMPK, and compound C can block the Nrf-2 nuclear translocation induced by L-BAIBA. The presence of compound C also blocked the protective effects of L-BAIBA on lung I/R injury in C57BL/6 mice.ConclusionsOur study showed that L-BAIBA protects against lung I/R injury via the AMPK/Nrf-2 signaling pathway, which could be a therapeutic target.

Abstract 14967: Branching Out: Disrupted Branched Chain Amino Acid Metabolism Exacerbates Pulmonary Arterial Hypertension

Introduction: Valine, leucine, and isoleucine are essential branched chain amino acids (BCAA), and recent epidemiological studies report elevated BCAAs predict worse outcomes in several cardiovascular diseases with metabolic origins such as diabetes, heart failure, and hypertension. However, the role of BCAA metabolism in pulmonary arterial hypertension pathophysiology is relatively unexplored. Hypothesis: BCAAs are elevated in human PAH and reduction of BCAAs via small molecule activation of BCAA catabolism and dietary interventions combats PAH severity in preclinical PAH. Methods: Literature search of human PAH metabolomics studies was conducted. For rodent studies, male Sprague Dawley rats were divided into three groups: control (saline injection), monocrotaline (MCT) vehicle (MCT-V), or MCT-BT2, a small molecule inducer of BCAA catabolism. BT2 was administered at 20mg/kg/day. A low BCAA dietary intervention was implemented, with male Sprague Dawley rats divided into two groups: MCT-standard chow diet and MCT-low BCAA diet. To assess exercise capacity, rodents underwent a maximal exercise testing protocol before echocardiographic and hemodynamic assessments of PAH. Proteomic analysis of lung mitochondrial extracts was performed to define the molecular effects of BT2. Results: Of 11 studies evaluating human PAH metabolomics, 7 studies reported elevated BCAAs or BCAA metabolites in PAH. BT2 treatment improved exercise performance, reduced PA pressures, and augmented RV function. Moreover, the MCT-BT2 group possessed a distinct lung proteomic signature with changes in inflammatory and metabolic pathways. Finally, the low BCAA diet intervention modestly combatted PAH severity. Conclusions: BCAA metabolism is disrupted in human PAH and interventions that reduce circulating BCAAs display therapeutic potential in rodent PAH.

Myocardial Metabolomics of Human Heart Failure With Preserved Ejection Fraction.

The human heart primarily metabolizes fatty acids, and this decreases as alternative fuel use rises in heart failure with reduced ejection fraction (HFrEF). Patients with severe obesity and diabetes are thought to have increased myocardial fatty acid metabolism, but whether this is found in those who also have heart failure with preserved ejection fraction (HFpEF) is unknown. Plasma and endomyocardial biopsies were obtained from HFpEF (n=38), HFrEF (n=30), and nonfailing donor controls (n=20). Quantitative targeted metabolomics measured organic acids, amino acids, and acylcarnitines in myocardium (72 metabolites) and plasma (69 metabolites). The results were integrated with reported RNA sequencing data. Metabolomics were analyzed using agnostic clustering tools, Kruskal-Wallis test with Dunn test, and machine learning. Agnostic clustering of myocardial but not plasma metabolites separated disease groups. Despite more obesity and diabetes in HFpEF versus HFrEF (body mass index, 39.8 kg/m2 versus 26.1 kg/m2; diabetes, 70% versus 30%; both P<0.0001), medium- and long-chain acylcarnitines (mostly metabolites of fatty acid oxidation) were markedly lower in myocardium from both heart failure groups versus control. In contrast, plasma levels were no different or higher than control. Gene expression linked to fatty acid metabolism was generally lower in HFpEF versus control. Myocardial pyruvate was higher in HFpEF whereas the tricarboxylic acid cycle intermediates succinate and fumarate were lower, as were several genes controlling glucose metabolism. Non-branched-chain and branched-chain amino acids (BCAA) were highest in HFpEF myocardium, yet downstream BCAA metabolites and genes controlling BCAA metabolism were lower. Ketone levels were higher in myocardium and plasma of patients with HFrEF but not HFpEF. HFpEF metabolomic-derived subgroups were differentiated by only a few differences in BCAA metabolites. Despite marked obesity and diabetes, HFpEF myocardium exhibited lower fatty acid metabolites compared with HFrEF. Ketones and metabolites of the tricarboxylic acid cycle and BCAA were also lower in HFpEF, suggesting insufficient use of alternative fuels. These differences were not detectable in plasma and challenge conventional views of myocardial fuel use in HFpEF with marked diabetes and obesity and suggest substantial fuel inflexibility in this syndrome.

Abstract MP59: Host LCT Genotype Modifies the Relationship Between Milk Intake and Risk of Diabetes Partially Through the Gut Microbiome and Blood Metabolome: The Hispanic Community Health Study/Study of Latinos (HCHS/SOL)

Introduction: The relationship between milk intake and risk of diabetes is controversial with substantial ethnical difference. Whether such difference can be partially explained by LCT genotype that varies by ethnical background is unknown. Hypothesis: Higher milk intake is associated with lower risk of diabetes only in lactase non-persistent (LNP) individuals ( LCT rs4988235, GG) but not in LP individuals (AA/AG). Methods: We examined associations of milk intake with incident diabetes (n=7089, 768 incident cases over 6 years), 490 gut microbial species (n=1767) and 624 serum metabolites (n=3110), stratified by LCT genotype in US Hispanics from the HCHS/SOL. We related the identified microbial species and metabolites with metabolic traits and/or incident diabetes. Results: Higher milk intake was associated with lower risk of diabetes in LNP but not in LP individuals (Fig A). Milk intake was associated with different microbial species by LCT genotype, including 13 species specific to LNP individuals (Fig B), especially Bifidobacterium sp. (enriched), which were favorably associated with several metabolic traits (Fig C). In LNP individuals, milk intake was specifically associated with 20 metabolites (Fig D). Metabolites positively associated with milk, especially indolepropinate and β-cryptoxanthin, were favorably associated with metabolic traits and risk of diabetes (Fig E). Opposite patterns were found for metabolites negatively associated with milk, especially for several bile acids and branched chain amino acid metabolites (Fig E). Some of these metabolites are known microbiota related, were correlated with LNP specific milk-related bacteria, and partially mediated the milk-diabetes association in LNP individuals (Fig F). Such associations were not observed for LP specific milk-related bacteria or metabolites. Conclusion LCT genotype may modify the relationship between milk intake and diabetes, with a beneficial association in LNP individuals, partially explained by gut microbiota and serum metabolites.

Mitochondrial pyruvate carrier inhibition initiates metabolic crosstalk to stimulate branched chain amino acid catabolism

ObjectiveThe mitochondrial pyruvate carrier (MPC) has emerged as a therapeutic target for treating insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). We evaluated whether MPC inhibitors (MPCi) might correct impairments in branched chain amino acid (BCAA) catabolism, which are predictive of developing diabetes and NASH. MethodsCirculating BCAA concentrations were measured in people with NASH and type 2 diabetes, who participated in a recent randomized, placebo-controlled Phase IIB clinical trial to test the efficacy and safety of the MPCi MSDC-0602K (EMMINENCE; NCT02784444). In this 52-week trial, patients were randomly assigned to placebo (n = 94) or 250 mg MSDC-0602K (n = 101). Human hepatoma cell lines and mouse primary hepatocytes were used to test the direct effects of various MPCi on BCAA catabolism in vitro. Lastly, we investigated how hepatocyte-specific deletion of MPC2 affects BCAA metabolism in the liver of obese mice and MSDC-0602K treatment of Zucker diabetic fatty (ZDF) rats. ResultsIn patients with NASH, MSDC-0602K treatment, which led to marked improvements in insulin sensitivity and diabetes, had decreased plasma concentrations of BCAAs compared to baseline while placebo had no effect. The rate-limiting enzyme in BCAA catabolism is the mitochondrial branched chain ketoacid dehydrogenase (BCKDH), which is deactivated by phosphorylation. In multiple human hepatoma cell lines, MPCi markedly reduced BCKDH phosphorylation and stimulated branched chain keto acid catabolism; an effect that required the BCKDH phosphatase PPM1K. Mechanistically, the effects of MPCi were linked to activation of the energy sensing AMP-dependent protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) kinase signaling cascades in vitro. BCKDH phosphorylation was reduced in liver of obese, hepatocyte-specific MPC2 knockout (LS-Mpc2−/−) mice compared to wild-type controls concomitant with activation of mTOR signaling in vivo. Finally, while MSDC-0602K treatment improved glucose homeostasis and increased the concentrations of some BCAA metabolites in ZDF rats, it did not lower plasma BCAA concentrations. ConclusionsThese data demonstrate novel cross talk between mitochondrial pyruvate and BCAA metabolism and suggest that MPC inhibition leads to lower plasma BCAA concentrations and BCKDH phosphorylation by activating the mTOR axis. However, the effects of MPCi on glucose homeostasis may be separable from its effects on BCAA concentrations.

Serum Branched-chain Amino Acid Metabolites Increase When Aerobic Exercise Is Initiated With Low Muscle Glycogen

PURPOSE: Initiating aerobic exercise with low muscle glycogen blunts anabolic signaling and myogenesis. The underlying mechanisms causing the reduction in post-exercise anabolic signaling is unclear. This study aimed to identify metabolic factors that might contribute to the apparent muscle anabolic resistance that occurs when aerobic exercise is initiated with low muscle glycogen using global metabolomics. METHODS: Using a randomized, crossover design, 11 men (mean ± SD, age: 21 ± 4 y; VO2peak: 44 ± 4 mL/kg/min) completed 2 cycle ergometry glycogen depletion trials, followed by 24 hours of isocaloric high fat (1.5 g/kg carbohydrate, 3.0 g/kg fat) or high carbohydrate (6.0 g/kg carbohydrate, 1.0 g/kg fat) refeeding to elicit low (LOW; 217 ± 103 mmol/kg dry wt) or adequate (AD; 396 ± 70 mmol/kg dry wt) muscle glycogen content. Participants then performed 80 min of cycle ergometry (64 ± 3% VO2peak) and ingested 146 g of carbohydrate. Serum samples for metabolomics analysis were collected before glycogen depletion under resting, fasted conditions (BASELINE), before (PRE) and immediately after (POST) exercise. Changes in metabolite profiles were calculated by subtracting BASELINE from PRE and POST within LOW and AD. RESULTS: Treatment or time-by-treatment interactions were observed in 309 metabolites (P < 0.05, Q < 0.10), with 78% of identified metabolites being related amino acid or fatty acid metabolism. There were greater decreases in 80% of diacylglycerol metabolites, while 69% of acyl-carnitine metabolites had greater increases in LOW compared to AD (P < 0.05, Q < 0.10). There were greater increases in 64% of branched-chain amino acids (BCAA) metabolites, as well as urea and 3-methylhistidine in LOW compared to AD (P < 0.05, Q < 0.10). CONCLUSION: Greater increases in BCAA and acyl-carnitine metabolites, as well as urea and 3-methylhistidine, suggest that when aerobic exercise is initiated low muscle glycogen, the reliance on BCAA catabolism for substrate oxidation is greater than when exercise is initiated with adequate glycogen stores. Findings may provide a metabolic explanation for why muscle is anabolically resistant to the mechanical stimulus of exercise, and suggest exogenous BCAA requirements to optimize muscle recovery are likely increased when glycogen stores are limited.

Initial Military Training Modulates The Serum Metabolome

PURPOSE: Initial military training (IMT) affects body composition by increasing lean mass and decreasing fat mass. The underlying metabolic adaptations associated with changes in body composition during IMT are unknown. Metabolomics analysis provides a comprehensive understanding of metabolic adaptations, which may yield mechanistic insight regarding these body composition changes. This study aimed to assess changes in body composition and the serum metabolome during IMT. METHODS: Data collection was conducted before (PRE) and after (POST) 22-week US Army IMT at Ft. Benning, GA. Fifty-four volunteers (mean±SD; 22 ± 3y; 24.6 ± 3.7 kg/m2) completed this longitudinal study. Body composition measurements (InBody 770, Cerritos, CA) and blood samples were collected under fasting, rested conditions PRE and POST IMT. Global metabolite profiling was performed to identify metabolites involved in energy, carbohydrate, lipid, and protein metabolism (Metabolon, Inc.). Paired t tests were used to determine differences between PRE and POST measures (P < 0.05). False discovery was controlled using the Benjamini Hochberg method. RESULTS: Body mass was maintained (mean difference ± SD, 0.4 ± 5.1, P > 0.05), fat mass was reduced (-1.7 ± 3.5 kg, P < 0.01), and lean mass was increased (2.1 ± 2.8 kg, P < 0.01) at POST compared to PRE. Of 682 identified metabolites, 367 differed at POST compared to PRE (P < 0.05, Q < 0.10). The majority of these metabolites were related to fatty acid (69%) and amino acid (23%) metabolism. Decreases were detected in 93% of long chain fatty acid metabolites, while 67% of primary bile acid metabolites increased. Increases were also detected in 52% of branched chain amino acid metabolites, 65% of histidine metabolites, and 40% of urea cycle metabolites. CONCLUSIONS: Changes in fatty acid metabolites indicate increased fat oxidation, consistent with reductions in fat mass observed during IMT. Increases in amino acid metabolites suggest higher rates of amino acid and nitrogen turnover, which may contribute to increases in fat free mass during IMT. Overall, changes in metabolomics profiles provide potential mechanistic insight into metabolic adaptions underlying changes in body composition during IMT.

Serum Metabolomics Reveals Distinct Profiles during Ischemia and Reperfusion in a Porcine Model of Myocardial Ischemia-Reperfusion.

Acute myocardial infarction (MI) is one of the leading causes of death worldwide. Early identification of ischemia and establishing reperfusion remain cornerstones in the treatment of MI, as mortality and morbidity can be significantly reduced by establishing reperfusion to the affected areas. The aim of the current study was to investigate the metabolomic changes in the serum in a swine model of MI induced by ischemia and reperfusion (I/R) injury, and to identify circulating metabolomic biomarkers for myocardial injury at different phases. Female Yucatan minipigs were subjected to 60 min of ischemia followed by reperfusion, and serum samples were collected at baseline, 60 min of ischemia, 4 h of reperfusion, and 24 h of reperfusion. Circulating metabolites were analyzed using an untargeted metabolomic approach. A bioinformatic approach revealed that serum metabolites show distinct profiles during ischemia and during early and late reperfusion. Some notable changes during ischemia include accumulation of metabolites that indicate impaired mitochondrial function and N-terminally modified amino acids. Changes in branched-chain amino-acid metabolites were noted during early reperfusion, while bile acid pathway derivatives and intermediates predominated in the late reperfusion phases. This indicates a potential for such an approach toward identification of the distinct phases of ischemia and reperfusion in clinical situations.

Association of Modifiable Lifestyle Factors with Plasma Branched-Chain Amino Acid Metabolites in Women

BackgroundCirculating branched-chain amino acids (BCAAs—isoleucine, leucine, and valine) are strongly associated with higher risk of incident type 2 diabetes (T2D); however, determinants of elevated fasting BCAA concentrations are largely unknown. ObjectivesWe aimed to characterize the modifiable lifestyle factors related to plasma BCAAs. MethodsWe performed a cross-sectional analysis among n = 18,897 women (mean ± SD age: 54.9 ± 7.2 y) in the Women’s Health Study, free of T2D and cardiovascular disease at baseline blood draw. Lifestyle factors, weight, and height were self-reported via questionnaire, including smoking status, alcohol, leisure-time physical activity (LTPA), diet quality scores [2010 Alternative Healthy Eating Index (without alcohol) (aHEI); alternate Mediterranean Diet (aMED)], and dietary sources of BCAAs. Plasma BCAAs were quantified via NMR spectroscopy. We calculated multivariable-adjusted percentage mean differences (95% CIs) and P values for linear trend of BCAAs stratified by categoric lifestyle factors. We estimated R2 from univariate cubic spline regression models to estimate the variability in BCAAs explained. ResultsCompared with women with BMI (in kg/m2) <25.0, BCAAs were 8.6% (95% CI: 8.0%, 9.3%), 15.3% (95% CI: 14.4%, 16.3%), and 21.0% (95% CI: 18.2%, 23.9%) higher for the BMI strata 25.0–29.9, 30.0–39.9, and ≥40.0, respectively (P-trend < 0.0001). Women with higher LTPA and higher alcohol intake compared with lower had modestly (∼1%) lower plasma BCAAs (P-trend = 0.014 and 0.0003, respectively). Differences in smoking status, aHEI, and aMED score were not related to plasma BCAAs. Women with higher dietary BCAAs had dose-response higher plasma BCAA concentrations, 3.4% (95% CI: 2.5%, 4.4%) higher when comparing the highest with the lowest quintile (P-trend < 0.0001). BMI explained 11.6% of the variability of BCAAs, whereas other factors explained between 0.1% and 1%. ConclusionsOur findings among a large cohort of US women indicate that BMI, but less so diet, physical activity, and other lifestyle factors, is related to plasma BCAAs.This trial was registered at clinicaltrials.gov as NCT00000479.

Characterization of a lactic acid bacteria using branched-chain amino acid transaminase and protease from Jinhua Ham and application in myofibrillar protein model

This study was to screen a strain with both branched-chain amino acid transaminase (BCAT) and protease activities for producing methyl-branched flavor compounds in a myofibrillar protein extract model. Lactic acid bacteria (LAB) was isolated from Jinhua ham and screened with BCAT activity by an electrochemical sensor and protease activity by the agar plate method. In the culture medium, strain L6 was selected with high utilization rate and characteristic metabolites content of branched-chain amino acids (BCAAs), and identified as Lactobacillus fermentum YZU-06 (L. fermentum). Compared with the previously reported L. plantarum, L. fermentum exhibited an excellent capacity of hydrolyzing myofibrillar protein with the higher contents of free amino acids, peptides and small molecular weight proteins. Moreover, L. fermentum group presented more BCAAs metabolites of 2-methylbutanal and 3-methylbutanal than that of L. plantarum group. In conclusion, L. fermentum YZU-06 is a promising starter culture to improve the flavor of fermented meat products.

Sex-Specific Catabolic Metabolism Alterations in the Critically Ill following High Dose Vitamin D.

Pharmacological interventions are essential for the treatment and management of critical illness. Although women comprise a large proportion of the critically ill, sex-specific pharmacological properties are poorly described in critical care. The sex-specific effects of vitamin D3 treatment in the critically ill are not known. Therefore, we performed a metabolomics cohort study with 1215 plasma samples from 428 patients from the VITdAL-ICU trial to study sex-specific differences in the metabolic response to critical illness following high-dose oral vitamin D3 intervention. In women, despite the dose of vitamin D3 being higher, pharmacokinetics demonstrated a lower extent of vitamin D3 absorption compared to men. Metabolic response to high-dose oral vitamin D3 is sex-specific. Sex-stratified individual metabolite associations with elevations in 25(OH)D following intervention showed female-specific positive associations in long-chain acylcarnitines and male-specific positive associations in free fatty acids. In subjects who responded to vitamin D3 intervention, significant negative associations were observed in short-chain acylcarnitines and branched chain amino acid metabolites in women as compared to men. Acylcarnitines and branched chain amino acids are reflective of fatty acid B oxidation, and bioenergesis may represent notable metabolic signatures of the sex-specific response to vitamin D. Demonstrating sex-specific pharmacometabolomics differences following intervention is an important movement towards the understanding of personalized medicine.

Association of Genetically Predicted BCAA Levels with Muscle Fiber Size in Athletes Consuming Protein.

Branched-chain amino acid (BCAA) levels are associated with skeletal muscle cross-sectional area (CSA). Serum BCAA levels are enhanced by whey protein supplementation (WPS), and evidence in clinical populations suggests an association of single nucleotide polymorphisms (SNPs) with BCAA metabolite levels. It is not known whether the same SNPs are associated with the ability to catabolise BCAAs from exogenous sources, such as WPS. The present study investigated whether possessing a higher number of alleles associated with increased BCAA metabolites correlates with muscle fiber CSA of m. vastus lateralis in physically active participants, and whether any relationship is enhanced by WPS. Endurance-trained participants (n = 75) were grouped by self-reported habitual WPS consumption and genotyped for five SNPs (PPM1K rs1440580, APOA5 rs2072560, CBLN1 rs1420601, DDX19B rs12325419, and TRMT61A rs58101275). Body mass, BMI, and fat percentage were significantly lower and muscle mass higher in the WPS group compared to Non-WPS. The number of BCAA-increasing alleles was correlated with fiber CSA in the WPS group (r = 0.75, p < 0.0001) and was stronger for fast-twitch fibers (p = 0.001) than slow-twitch fibers (p = 0.048). Similar results remained when corrected for multiple covariates (age, physical activity, and meat and dairy intake). No correlation was found in the Non-WPS group. This study presents novel evidence of a positive relationship between BCAA-increasing alleles and muscle fiber CSA in athletes habitually consuming WPS. We suggest that a high number of BCAA-increasing alleles improves the efficiency of WPS by stimulation of muscle protein synthesis, and contributes to greater fiber CSA.

Serum Branched-Chain Amino Acid Metabolites Increase in Males When Aerobic Exercise Is Initiated with Low Muscle Glycogen

This study used global metabolomics to identify metabolic factors that might contribute to muscle anabolic resistance, which develops when aerobic exercise is initiated with low muscle glycogen using global metabolomics. Eleven men completed this randomized, crossover study, completing two cycle ergometry glycogen depletion trials, followed by 24 h of isocaloric refeeding to elicit low (LOW; 1.5 g/kg carbohydrate, 3.0 g/kg fat) or adequate (AD; 6.0 g/kg carbohydrate 1.0 g/kg fat) glycogen. Participants then performed 80 min of cycling (64 ± 3% VO2 peak) while ingesting 146 g carbohydrate. Serum was collected before glycogen depletion under resting and fasted conditions (BASELINE), and before (PRE) and after (POST) exercise. Changes in metabolite profiles were calculated by subtracting BASELINE from PRE and POST within LOW and AD. There were greater increases (p < 0.05, Q < 0.10) in 64% of branched-chain amino acids (BCAA) metabolites and 69% of acyl-carnitine metabolites in LOW compared to AD. Urea and 3-methylhistidine had greater increases (p < 0.05, Q < 0.10) in LOW compared to AD. Changes in metabolomics profiles indicate a greater reliance on BCAA catabolism for substrate oxidation when exercise is initiated with low glycogen stores. These findings provide a mechanistic explanation for anabolic resistance associated with low muscle glycogen, and suggest that exogenous BCAA requirements to optimize muscle recovery are likely greater than current recommendations.

13-OR: Effects of Randomized Statin Therapy vs. Placebo on Plasma Branched-Chain Amino Acids (BCAAs) and Type 2 Diabetes (T2D) Risk: Secondary Analysis of the JUPITER Trial

Background: Statins increase T2D risk despite favorable effects on lipids, inflammation, and cardiovascular disease (CVD). Statins may modulate associations of T2D-related BCAAs with incident T2D. Methods: JUPITER (NCT00239681) randomized rosuvastatin vs. placebo for primary CVD prevention. Plasma BCAAs (isoleucine, leucine, valine) and alanine were quantified via NMR spectroscopy for n=12,441 at baseline and n=9,826 with repeat 12mo samples. We calculated % change in metabolites with mixed effects linear regression. We used Cox models to evaluate baseline and change (per SD) of metabolites with incident T2D (multivariable-adj HR [95%CI]) during 2y of follow-up (max. 5y). Results: Overall, statin vs. placebo increased T2D (HR 1.25 [95% CI: 1.05-1.49]). BCAA metabolites changed modestly at 12mo; only leucine differed with statin vs. placebo (+3.7% vs. +2.4%; p=0.03). Baseline BCAAs were associated with T2D. Each SD increase in isoleucine, leucine, valine, and alanine was associated with HR=1.3 (1.1-1.5), 1.1 (1.0, 1.3), 1.3 (1.1, 1.5), and 1.3 (1.1, 1.5) greater T2D risk (FIGURE). Associations did not differ with statin vs. placebo. Conclusion: High-intensity statin therapy had at most modest effects on BCAA metabolites. Baseline and change in BCAAs and alanine were associated with T2D, including during randomized statin therapy. Disclosure D. K. Tobias: None. P. Ridker: Consultant; Self; 360 Marketing Strategic Services, AstraZeneca, Corvidia Therapeutics, CSL Behring, DalCor Pharmaceuticals, Novartis AG, Omeicos, Flame, Uppton, Agepha, sIRNAomics, Research Support; Self; Amarin Corporation plc, Kowa Pharmaceuticals America, Inc., Novo Nordisk. R. Glynn: Research Support; Self; AstraZeneca, Kowa Research Institute, Inc., Novartis AG, Pfizer Inc. S. Mora: Consultant; Self; Pfizer Inc., Quest Diagnostics, Other Relationship; Self; LabCorp. Funding American Diabetes Association (1-19-JDF-115 to D.K.T.); Brigham and Women’s Hospital (to D.K.T.); National Institutes of Health (R01HL117861, R01HL134811, K24HL136852, R01DK112940 to S.M.); LabCorp

Integrated metabolomics analysis of the effect of PPARδ agonist GW501516 on catabolism of BCAAs and carboxylic acids in diabetic mice

The peroxisome proliferator-activated receptor (PPARδ) agonists are reported to improve insulin sensitivity, reduce glucose levels, and alleviate dysfunctional lipid metabolism in animal models of type 2 diabetes mellitus. However, the underlying mechanisms remain incompletely understood. Metabolism plays an essential role in the biological system. Monitoring of metabolic changes in response to disease conditions or drug treatment is critical for better understanding of the pathophysiological mechanisms. In this study, metabolic profiling analysis by gas chromatography-mass spectrometry integrated with targeted analysis by liquid chromatography-mass spectrometry was carried out in plasma samples of db/db diabetic mice after six-week treatment of PPARδ agonist GW501516. GW501516 treatment significantly altered levels of metabolites, such as branched-chain amino acids (BCAAs), BCAA metabolites (3-hydroxyisobutyric acid and 3-hydroxyisovaleric acid), long-chain fatty acids, uric acid and ketone bodies (3-hydroxybutyric acid and 2-hydroxybutyric acid) which are all associated with the impaired systemic insulin sensitivity. The present results indicate the beneficial effect of PPARδ agonist in alleviating insulin resistance of diabetic mice by favorably modulating metabolic profile, thus providing valuable information in understanding the therapeutic potential of PPARδ agonists in correcting metabolic dysfunction in diabetes.

Abstract 17285: Metabolite-Derived Network Reveals Cluster of Acylcholine Metabolites Associated With Better Diet Quality and Lower Prevalence of Type 2 Diabetes: Findings From the Boston Puerto Rican Health Study

Introduction: Puerto Rican (PR) adults living on the US mainland are at high risk for developing type 2 diabetes (T2D), and dietary factors may contribute to this increased risk. Network analysis is a data-reduction tool that can identify correlated clusters of co-regulated metabolites that reflect mechanisms underlying diet-T2D associations. Hypothesis: Diet quality will associate with T2D-associated metabolite clusters among PR adults. Methods: We used LC/MS to measure fasting plasma metabolites (&gt;700) among Boston PR Health Study participants, aged 45-75 years, with (n=258) and without (n=421) T2D. We applied an unsupervised correlation network-based method to identify metabolite clusters within a global metabolite network and calculated a score for each cluster using a weighted sum of metabolite concentrations. To estimate diet quality, we calculated a modified version of a previously validated American Heart Association diet score (AHA-DS). Logistic regression was used to assess cross-sectional associations between metabolite clusters and prevalent T2D, and linear regression was used to assess associations between the continuous AHA-DS and T2D-associated metabolite clusters among controls, adjusting for potential confounders and correcting for multiple testing. Results: We identified 7 metabolite clusters that were associated with prevalent T2D ( p &lt;0.05). For every 1-standard deviation (SD) increase in cluster score, the odds ratios for prevalent T2D and 95% confidence intervals were as the follows: acylcholines [0.40 (0.31, 0.50)], aromatic hydrocarbon derivatives [0.33 (0.22, 0.47)], sphingolipids [0.46 (0.33, 0.64)], tricarboxylic acid (TCA) cycle amino acids/peptides [0.39 (0.25, 0.62)], branched-chain amino acid metabolites [4.1 (2.9, 6.0)], acylcarnitines [1.8 (1.3, 2.5)], and TCA cycle/energy metabolites [2.0 (1.4, 3.0)]. The AHA-DS was only significantly associated with the acylcholine metabolites cluster [β (standard error) = 0.01 (0.004) SD increase in cluster score, p=0.02]. Conclusions: In individuals of PR descent, we identified a cluster of acylcholine metabolites where concentrations are higher among those with better diet quality and lower among those with prevalent T2D.

Effect of renal tubule-specific knockdown of the Na+/H+ exchanger NHE3 in Akita diabetic mice.

Na+/H+ exchanger isoform 3 (NHE3) contributes to Na+/bicarbonate reabsorption and ammonium secretion in early proximal tubules. To determine its role in the diabetic kidney, type 1 diabetic Akita mice with tubular NHE3 knockdown [Pax8-Cre; NHE3-knockout (KO) mice] were generated. NHE3-KO mice had higher urine pH, more bicarbonaturia, and compensating increases in renal mRNA expression for genes associated with generation of ammonium, bicarbonate, and glucose (phosphoenolpyruvate carboxykinase) in proximal tubules and H+ and ammonia secretion and glycolysis in distal tubules. This left blood pH and bicarbonate unaffected in nondiabetic and diabetic NHE3-KO versus wild-type mice but was associated with renal upregulation of proinflammatory markers. Higher renal phosphoenolpyruvate carboxykinase expression in NHE3-KO mice was associated with lower Na+-glucose cotransporter (SGLT)2 and higher SGLT1 expression, indicating a downward tubular shift in Na+ and glucose reabsorption. NHE3-KO was associated with lesser kidney weight and glomerular filtration rate (GFR) independent of diabetes and prevented diabetes-associated albuminuria. NHE3-KO, however, did not attenuate hyperglycemia or prevent diabetes from increasing kidney weight and GFR. Higher renal gluconeogenesis may explain similar hyperglycemia despite lower SGLT2 expression and higher glucosuria in diabetic NHE3-KO versus wild-type mice; stronger SGLT1 engagement could have affected kidney weight and GFR responses. Chronic kidney disease in humans is associated with reduced urinary excretion of metabolites of branched-chain amino acids and the tricarboxylic acid cycle, a pattern mimicked in diabetic wild-type mice. This pattern was reversed in nondiabetic NHE3-KO mice, possibly reflecting branched-chain amino acids use for ammoniagenesis and tricarboxylic acid cycle upregulation to support formation of ammonia, bicarbonate, and glucose in proximal tubule. NHE3-KO, however, did not prevent the diabetes-induced urinary downregulation in these metabolites.

Branched-chain amino acid metabolism in cancer.

Purpose of reviewThe current review aims to provide an update on the recent biomedical interest in oncogenic branched-chain amino acid (BCAA) metabolism, and discusses the advantages of using BCAAs and expression of BCAA-related enzymes in the treatment and diagnosis of cancers.Recent findingsAn accumulating body of evidence demonstrates that BCAAs are essential nutrients for cancer growth and are used by tumors in various biosynthetic pathways and as a source of energy. In addition, BCAA metabolic enzymes, such as the cytosolic branched-chain aminotransferase 1 (BCAT1) and mitochondrial branched-chain aminotransferase 2, have emerged as useful prognostic cancer markers. BCAT1 expression commonly correlates with more aggressive cancer growth and progression, and has attracted substantial scientific attention in the past few years. These studies have found the consequences of BCAT1 disruption to be heterogeneous; not all cancers share the same requirements for BCAA metabolites and the function of BCAT1 appears to vary between cancer types.SummaryBoth oncogenic mutations and cancer tissue-of-origin influence BCAA metabolism and expression of BCAA-associated metabolic enzymes. These new discoveries need to be taken into consideration during the development of new cancer therapies that target BCAA metabolism.

Military training elicits marked increases in plasma metabolomic signatures of energy metabolism, lipolysis, fatty acid oxidation, and ketogenesis.

Military training studies provide unique insight into metabolic responses to extreme physiologic stress induced by multiple stressor environments, and the impacts of nutrition in mediating these responses. Advances in metabolomics have provided new approaches for extending current understanding of factors modulating dynamic metabolic responses in these environments. In this study, whole‐body metabolic responses to strenuous military training were explored in relation to energy balance and macronutrient intake by performing nontargeted global metabolite profiling on plasma collected from 25 male soldiers before and after completing a 4‐day, 51‐km cross‐country ski march that produced high total daily energy expenditures (25.4 MJ/day [SD 2.3]) and severe energy deficits (13.6 MJ/day [SD 2.5]). Of 737 identified metabolites, 478 changed during the training. Increases in 88% of the free fatty acids and 91% of the acylcarnitines, and decreases in 88% of the mono‐ and diacylglycerols detected within lipid metabolism pathways were observed. Smaller increases in 75% of the tricarboxylic acid cycle intermediates, and 50% of the branched‐chain amino acid metabolites detected were also observed. Changes in multiple metabolites related to lipid metabolism were correlated with body mass loss and energy balance, but not with energy and macronutrient intakes or energy expenditure. These findings are consistent with an increase in energy metabolism, lipolysis, fatty acid oxidation, ketogenesis, and branched‐chain amino acid catabolism during strenuous military training. The magnitude of the energy deficit induced by undereating relative to high energy expenditure, rather than macronutrient intake, appeared to drive these changes, particularly within lipid metabolism pathways.