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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. metabolicdisorders 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. Through two-way artificial selection breeding for treadmill running capacity, intrinsically high-capacity running (HCR) and low-capacity running (LCR) rats provide an excellent model system for studying the genetic factors mediating extremes in metabolic health. 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 (narrow-sense heritability, h2 = 0.47 ± 0.02 in HCRs and 0.43 ± 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). Thus, investigating the gene-regulatory mechanisms mediating these processes in a translational animal model system may provide new insight into the molecular basis controlling metabolic health. MicroRNAs (miRNAs) are short, noncoding RNAs that regulate gene expression by binding to mRNA, subsequently instigating degradation or repressing protein translation (2, 10). Altered miRNA expression has been implicated in the pathogenesis of several metabolic conditions, including obesity and Type 2 diabetes, through the regulation of key metabolic signaling networks involved in glucose and lipid handling, and mitochondrial metabolism (9, 13, 45). Additionally, divergent miRNA expression has recently been characterized in mice with inherently high or low physical activity levels, as well in human “high” and “low” responders to resistance exercise (5, 6). These findings suggest that miRNAs may contribute to the metabolic adaptation profile induced by physical exercise activity. Whether miRNAs contribute to the signaling pathways that mediate the intrinsic skeletal muscle metabolic phenotypes divergent between HCR and LCR rats is unknown. We aimed to determine the miRNA expression profile and interactions with predicted protein targets implicated in metabolic health in skeletal muscle from HCR and LCR rats. 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.


Mary MacKillop Institute for Health Research

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Journal Article

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