3-Methyl-2-oxovaleric acid

Brown and beige adipose tissue regulate systemic metabolism through a metabolite interorgan signaling axis

Brown and beige adipose tissue are emerging as distinct endocrine organs. These tissues are functionally associated with skeletal muscle, adipose tissue metabolism and systemic energy expenditure, suggesting an interorgan signaling network. Using metabolomics, we identify 3-methyl-2-oxovaleric acid, 5-oxoproline, and β-hydroxyisobutyric acid as small molecule metabokines synthesized in browning adipocytes and secreted via monocarboxylate transporters. 3-methyl-2-oxovaleric acid, 5-oxoproline and β-hydroxyisobutyric acid induce a brown adipocyte-specific phenotype in white adipocytes and mitochondrial oxidative energy metabolism in skeletal myocytes both in vitro and in vivo. 3-methyl-2-oxovaleric acid and 5-oxoproline signal through cAMP-PKA-p38 MAPK and β-hydroxyisobutyric acid via mTOR. In humans, plasma and adipose tissue 3-methyl-2-oxovaleric acid, 5-oxoproline and β-hydroxyisobutyric acid concentrations correlate with markers of adipose browning and inversely associate with body mass index. These metabolites reduce adiposity, increase energy expenditure and improve glucose and insulin homeostasis in mouse models of obesity and diabetes. Our findings identify beige adipose-brown adipose-muscle physiological metabokine crosstalk.

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

Serum metabolomic signatures of gestational diabetes in South Asian and white European women

Introduction: This study aimed to identify serum metabolomic signatures associated with gestational diabetes mellitus (GDM), and to examine if ethnic-specific differences exist between South Asian and white European women.
Research design and methods: Prospective cohort study with a nested case-control analysis of 600 pregnant women from two Canadian birth cohorts; using an untargeted approach, 63 fasting serum metabolites were measured and analyzed using multisegment injection-capillary electrophoresis-mass spectrometry. Multivariate logistic regression modeling was conducted overall and by cohort.
Results: The proportion of women with GDM was higher in South Asians (27.1%) compared with white Europeans (17.9%). Several amino acid, carbohydrate, and lipid pathways related to GDM were common to South Asian and white European women. Elevated circulating concentrations of glutamic acid, propionylcarnitine, tryptophan, arginine, 2-hydroxybutyric acid, 3-hydroxybutyric acid, and 3-methyl-2-oxovaleric acid were associated with higher odds of GDM, while higher glutamine, ornithine, oxoproline, cystine, glycine with lower odds of GDM. Per SD increase in glucose concentration, the odds of GDM increased (OR=2.07, 95% CI 1.58 to 2.71), similarly for metabolite ratios: glucose to glutamine (OR=2.15, 95% CI 1.65 to 2.80), glucose to creatinine (OR=1.79, 95% CI 1.39 to 2.32), and glutamic acid to glutamine (OR=1.46, 95% CI 1.16 to 1.83). South Asians had higher circulating ratios of glucose to glutamine, glucose to creatinine, arginine to ornithine, and citrulline to ornithine, compared with white Europeans.
Conclusions: We identified a panel of serum metabolites implicated in GDM pathophysiology, consistent in South Asian and white European women. The metabolic alterations leading to larger ratios of glucose to glutamine, glucose to creatinine, arginine to ornithine, and citrulline to ornithine in South Asians likely reflect the greater burden of GDM among South Asians compared with white Europeans.

Metabolomic profiles to explore biomarkers of severe sarcopenia in older men: A pilot study

Background: The pathophysiology of sarcopenia is complex and multifactorial; however, it has not yet been fully elucidated. Identifying metabolomic profiles may help clarify the mechanisms underlying sarcopenia.
Objective: This pilot study explored potential noninvasive biomarkers of severe sarcopenia through metabolomic analysis in community-dwelling older men.
Methods: Twenty older men (mean age: 81.9 ± 2.8 years) were selected from the Korean Frailty and Aging Cohort Study. Participants with severe sarcopenia (n = 10) were compared with non-sarcopenic, age- and body mass index-matched controls (n = 10). Severe sarcopenia was defined as low muscle mass, low muscle strength, and low physical performance using the Asian Working Group for Sarcopenia 2019 criteria. Non-targeted metabolomic profiling of plasma metabolites was performed using capillary electrophoresis time-of-flight mass spectrometry and absolute quantification was performed in target metabolites.
Results: Among 191 plasma metabolic peaks, the concentrations of 10 metabolites significantly differed between severe sarcopenia group and non-sarcopenic controls. The plasma concentrations of L-alanine, homocitrulline, N-acetylserine, gluconic acid, N-acetylalanine, proline, and sulfotyrosine were higher, while those of 4-methyl-2-oxovaleric acid, 3-methyl-2-oxovaleric acid, and tryptophan were lower in participants with severe sarcopenia than in non-sarcopenic controls (all, p < 0.05). Among the 53 metabolites quantified as target metabolites, L-alanine (area under the receiver operating characteristic curve [AUC] = 0.760; p = 0.049), gluconic acid (AUC = 0.800; p = 0.023), proline (AUC = 0.785; p = 0.031), and tryptophan (AUC = 0.800; p = 0.023) determined the presence of severe sarcopenia.
Conclusions: Plasma metabolomic analysis demonstrated that L-alanine, gluconic acid, proline, and tryptophan may be potential biomarkers of severe sarcopenia. The identified metabolites can provide new insights into the underlying pathophysiology of severe sarcopenia and serve as the basis for preventive interventions.

Novel HILIC-ESI-MS method for urinary profiling of MSUD and methylmalonic aciduria biomarkers

Methyl malonic acid and branched-chain keto acids are important biomarkers for the diagnosis of cobalamin deficiencies and maple syrup urine disease. We report the development and validation of a HILIC-ESI-MS2 method for the quantification of these organic acids from neonatal urine. The samples were 100 times diluted and analyzed on a ZIC-HILIC column with 25-mM formic acid in water: 25-mM formic acid in acetonitrile (45:55) at a flow rate of 0.8 mL/min with a runtime of only 6 minutes. The method demonstrated a lower limit of detection of 10 ng/mL, Limit of Quantification (LOQ) of 50 ng/mL, linearity of r2 ≥ 0.990 and recoveries of 87-105% for all analytes. The intraday and interday precision CV's were <10% and 12%, respectively. Extensive stability studies demonstrated the analytes to be stable in stock and in matrix with a percent change within ±15%. The Bland-Altman analysis of the developed method with the gold standard GCMS method demonstrated a bias of 0.44, 0.11, 0.009 and -0.19 for methyl malonic acid, 3-methyl-2-oxovaleric acid, 2-hydroxy-3methylbutyric acid and 4-methyl-2-oxovaleric acid, respectively, proving the methods are comparable. The newly developed method involves no derivatization and has a simple sample preparation and a low runtime, enabling it to be easily automated with a high sample throughput in a cost-effective manner.