Abstract
β-Hydroxy-β-methylbutyrate (HMB) is a popular ergogenic aid used by human athletes and as a supplement to sport horses, because of its ability to aid muscle recovery, improve performance and body composition. Recent findings suggest that HMB may stimulate satellite cells and affect expressions of genes regulating skeletal muscle cell growth. Despite the scientific data showing benefits of HMB supplementation in horses, no previous study has explained the mechanism of action of HMB in this species. The aim of this study was to reveal the molecular background of HMB action on equine skeletal muscle by investigating the transcriptomic profile changes induced by HMB in equine satellite cells in vitro. Upon isolation from the semitendinosus muscle, equine satellite cells were cultured until the 2nd day of differentiation. Differentiating cells were incubated with HMB for 24 h. Total cellular RNA was isolated, amplified, labelled and hybridised to microarray slides. Microarray data validation was performed with real-time quantitative PCR. HMB induced differential expressions of 361 genes. Functional analysis revealed that the main biological processes influenced by HMB in equine satellite cells were related to muscle organ development, protein metabolism, energy homoeostasis and lipid metabolism. In conclusion, this study demonstrated for the first time that HMB has the potential to influence equine satellite cells by controlling global gene expression. Genes and biological processes targeted by HMB in equine satellite cells may support HMB utility in improving growth and regeneration of equine skeletal muscle; however, the overall role of HMB in horses remains equivocal and requires further proteomic, biochemical and pharmacokinetic studies.
Highlights
Abstract β-Hydroxy-β-methylbutyrate (HMB) is a popular ergogenic aid used by human athletes and as a supplement to sport horses, because of its ability to aid muscle recovery, improve performance and body composition
Gene expression determines most of the phenotype; the present study focused on revealing the molecular background of HMB action in equine skeletal muscle by investigating the impact of HMB on global gene expression in differentiating equine satellite cells (ESC) in vitro
The following materials were used during cell culture: the Ca salt of HMB (Ca-HMB) was purchased from Metabolic Technologies; Dulbecco’s Modified Eagle Medium (DMEM) (1×) with glutamax, fetal bovine serum (FBS), horse serum (HS) and antibiotics (AB) – penicillin– streptomycin and fungizone – were purchased from Gibco, Life Technologies; penicillium crystalicum (AB) was purchased from Polfa Tarchomin; PBS, protease from Streptomyces griseus and DMSO were purchased from Sigma Aldrich
Summary
Abstract β-Hydroxy-β-methylbutyrate (HMB) is a popular ergogenic aid used by human athletes and as a supplement to sport horses, because of its ability to aid muscle recovery, improve performance and body composition. The horse has become an extraordinary ‘athlete’, exercised for a broad range of sporting activities (racing, endurance rides, show jumping, dressage, 3-d eventing, heavy draught work, polo, reining, cutting and competitive driving, as well as pleasure riding)(1), which may be associated with serious muscle overloading and an increased risk of injuries. This concerns especially the top-level competitors that are exposed to maximal training loads to achieve even a tiny increase in performance; even this small edge over competitors may result in winning the competition[2]. Recent evidence has indicated additional benefits of HMB supplementation related to energy metabolism, including improved aerobic performance[11] as well as increased fat loss with exercise[12]; the underlying mechanisms are poorly understood
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