Comparative metabolomic analysis in horses and functional analysis of branched chain (alpha) keto acid dehydrogenase complex in equine myoblasts under exercise stress

Abstract

The integration of metabolomics and transcriptomics may elucidate the correlation between the genotypic and phenotypic patterns in organisms. In equine physiology, various metabolite levels vary during exercise, which may be correlated with a modified gene expression pattern of related genes. Integrated metabolomic and transcriptomic studies in horses have not been conducted to date. The objective of this study was to detect the effect of moderate exercise on the metabolomic and transcriptomic levels in horses. In this study, using nuclear magnetic resonance (NMR) spectroscopy, we analyzed the concentrations of metabolites in muscle and plasma; we also determined the gene expression patterns of branched chain (alpha) keto acid dehydrogenase kinase complex (BCKDK), which encodes the key regulatory enzymes in branched-chain amino acid (BCAA) catabolism, in two breeds of horses, Thoroughbred and Jeju, at different time intervals. The concentrations of metabolites in muscle and plasma were measured by ¹H NMR (nuclear magnetic resonance) spectroscopy, and the relative metabolite levels before and after exercise in the two samples were compared. Subsequently, multivariate data analysis based on the metabolic profiles was performed using orthogonal partial least square discriminant analysis (OPLS-DA), and variable important plots and t-test were used for basic statistical analysis. The stress-induced expression patterns of BCKDK genes in horse muscle-derived cells were examined using quantitative reverse transcription polymerase chain reaction (qPCR) to gain insight into the role of transcript in response to exercise stress. In this study, we found higher concentrations of aspartate, leucine, isoleucine, and lysine in the skeletal muscle of Jeju horses than in Thoroughbred horses. In plasma, compared with Jeju horses, Thoroughbred horses had higher levels of alanine and methionine before exercise; whereas post-exercise, lysine levels were increased. Gene expression analysis revealed a decreased expression level of BCKDK in the post-exercise period in Thoroughbred horses.

Keywords: Branched chain (alpha) keto acid dehydrogenase kinase complex (BCKDK) gene; Equine myoblast; Metabolite; Nuclear magnetic resonance (NMR) spectroscopy; mRNA expression.

Fig. 1.. Significant differences in plasma metabolite levels between Thoroughbred and Jeju horses (A) alanine (before exercise), (B) lysine (after exercise), (C) methionine (before exercise) and (D) taurine (before exercise).

*p < 0.05. All values are expressed in ppm as the mean ± SD. TH, Thoroughbred horse; JH, Jeju horse.

Fig. 2.. Significant differences in skeletal muscle metabolite levels between Thoroughbred and Jeju horses (A) leucine (before exercise), (B) isoleucine (before exercise), (C) lysine (before exercise), (D) aspartate (before exercise) and o-phosphocholine (after exercise).

*p < 0.05. All values are expressed in ppm as the mean ± SD. TH, Thoroughbred horse; JH, Jeju horse.

Fig. 3.. Analysis of amino acid sequences and phylogenetic tree of branched chain (alpha) keto acid dehydrogenase kinase complex (BCKDK) gene among various species.

(A) Gene structure of the branched chain (alpha) keto acid dehydrogenase kinase complex (BCKDK) gene in horses. Black boxes indicate exons, grey boxes indicate untranslated regions (UTR), and black lines indicate introns. (B) Phylogenetic tree of BCKDK. The phylogenetic tree was made with the full amino acid sequences of each species by Neighbor-Joining method after alignment by the MUSCLE method using GENEIOUS. Horse AXL was more similar to cow and dog than to frog and mouse. (C) ‘Alignments of histidine kinase-like ATPases’ domain of BCKDK from various species. The sequences were aligned by the MUSCLE method in GENEIOUS program.

Fig. 4.. BCKDK gene expression in skeletal muscle of Thoroughbred and Jeju horses.

(A) BCKDK in Thoroughbred horses. (B) BCKDK in Jeju horses. BCKDK gene expression significantly decreased after the exercise in Thoroughbred horses (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars indicate standard error). The relative expression for each gene was normalized to that of GAPDH and calculated with the 2−rr CT method (mean ± SD of triplicate experiments; two-tailed Student t-test). BCKDK, branched chain (alpha) keto acid dehydrogenase kinase complex.

Fig. 5.. Effects of exercise stress on BCKDK gene expression in horse muscle-derived cells and effects of methylsulfonylmethane (MSM) on stress reduction.

(A) Morphology of horse muscle-derived cells. (B) ATF4 gene, as exercise stress marker gene expression using qRT-PCR. white and grey bars represent gene expression in the presence and absence of cortisol treatment (30 ug/mL). (C) Expression of horse BCKDK analyzed using RT-PCR in horse muscle-derived cells after treatment with 30 μg/mL cortisol. Data are presented as one of three independent experiments. (D) Analysis of relative BCKDK gene expression using qRT-PCR under cortisol and MSM treatment. white and grey bars represent gene expression in the presence and absence of MSM treatment (100 mM), under exercise stress (30 μg/mL cortisol). The relative expression for each gene was normalized to that of GAPDH and calculated with the 2^−△△ CT method (mean ± SD of triplicate experiments; two-tailed Student t-test). BCKDK, branched chain (alpha) keto acid dehydrogenase kinase complex; qRT-PCR, quantitative real-time polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.