Abstract
Impairments in mitochondrial function and substrate metabolism are implicated in the etiology of obesity and Type 2 diabetes. MicroRNAs (miRNAs) can degrade mRNA or repress protein translation and have been implicated in the development of such disorders. We used a contrasting rat model system of selectively bred high- (HCR) or low- (LCR) intrinsic running capacity with established differences in metabolic health to investigate the molecular mechanisms through which miRNAs regulate target proteins mediating mitochondrial function and substrate oxidation processes. Quantification of select miRNAs using the rat miFinder miRNA PCR array revealed differential expression of 15 skeletal muscles (musculus tibialis anterior) miRNAs between HCR and LCR rats (14 with higher expression in LCR; P < 0.05). Ingenuity Pathway Analysis predicted these altered miRNAs to collectively target multiple proteins implicated in mitochondrial dysfunction and energy substrate metabolism. Total protein abundance of citrate synthase (CS; miR-19 target) and voltage-dependent anion channel 1 (miR-7a target) were higher in HCR compared with LCR cohorts (~57 and ~26%, respectively; P < 0.05). A negative correlation was observed for miR-19a-3p and CS (r = 0.32, P = 0.015) protein expression. To determine whether miR-19a-3p can regulate CS in vitro, we performed luciferase reporter and transfection assays in C2C12 myotubes. MiR-19a-3p binding to the CS untranslated region did not change luciferase reporter activity; however, miR-19a-3p transfection decreased CS protein expression (∼70%; P < 0.05). The differential miRNA expression targeting proteins implicated in mitochondrial dysfunction and energy substrate metabolism may contribute to the molecular basis, mediating the divergent metabolic health profiles of LCR and HCR rats.
Highlights
METABOLIC DISORDERS such as Type 2 diabetes and obesity are characterized by a loss of “metabolic plasticity,” in which skeletal muscle is unable to effectively transition between lipid- and carbohydrate-based oxidation in response to the prevailing hormonal milieu [17]
15 miRNAs with predicted mRNA targets involved in mitochondrial dysfunction and substrate oxidation were differentially expressed between high-capacity running (HCR) and low-capacity running (LCR) rats
We show the abundance of predicted protein targets citrate synthase (CS) and VDAC1 were altered between phenotypes in accordance with miRNA expression profile
Summary
METABOLIC DISORDERS such as Type 2 diabetes and obesity are characterized by a loss of “metabolic plasticity,” in which skeletal muscle is unable to effectively transition between lipid- and carbohydrate-based oxidation in response to the prevailing hormonal milieu [17] Development of these clinical conditions is determined by a complex interaction of environmental (lifestyle) and genetic (heritable) factors. The HCR rats present with more than eight-fold greater intrinsic aerobic running capacity at generation 28 compared with LCR rats, and over 40% of the variance of the running capacity phenotype is due to additive genetic variance (narrowsense heritability, h2 ϭ 0.47 Ϯ 0.02 in HCRs and 0.43 Ϯ 0.03 in LCRs) [33] This superior aerobic capacity and metabolic health profile of HCR rats have, in part, been attributed to an increased activity and/or expression of skeletal muscle proteins involved in mitochondrial function and substrate oxidation [15, 31, 34, 41] compared with the impaired mitochondrial function observed in LCR animals [36, 41]. We hypothesized that HCR and LCR rats would present divergent miRNA expression profiles in a nonexercise condition, with HCR rats displaying a miRNA profile that upregulates proteins promoting efficient substrate oxidation and enhanced mitochondrial function
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