Skeletal Muscle β‐F1‐ATPase Translation is Inhibited by Hyperlipidemia‐Induced miR‐127‐5p Expression in Human Obesity

Abstract

We recently discovered persistent downregulation of the H+‐ATP synthase catalytic subunit (β‐F1‐ATPase) in skeletal muscle of obese individuals, which was linked to comorbid hyperlipidemia. β‐F1‐ATPase expression is primarily regulated post‐transcription, i.e. miRNAs, but the precise mechanism impeding expression are unclear. We tested the hypothesis that hyperlipidemia reduces β‐F1‐ATPase translational efficiency and is mediated by miR‐127‐5p, a miRNA known to control β‐F1‐ATPase translation. Biopsies isolated from the vastus lateralis of lean (BMI蠄25; n=7) and obese (BMI蠅30; n=6) subjects were evaluated for mRNA/miRNA expression via qRT‐PCR, protein by Western blot, and translation efficiency was calculated as the mRNA/protein ratio. Our results confirmed β‐F1‐ATPase translation was lower in obese individuals (P<0.01; 0.25±0.05%) compared to healthy weight controls (0.59±0.09%), and correlated with miR‐127‐5p expression (P<0.01; r=‐0.67). Bisulfite sequencing analysis of the miR‐127‐5p promoter revealed two hypomethylated sites in obese individuals, which may sustain an upregulation. Moreover, we found miR‐127‐5p present in muscle‐derived blood exosomes suggesting putative involvement in intracellular cross‐talk. In primary myotubes from a lean volunteer, prolonged fatty acid exposure increased miR‐127‐5p expression. Free Fatty Acid (mEq/L) Unsaturated/ Saturated mRNA (2‐DDCt) Protein (AU) Translation (%) CTRL 0.347 ‐ 1.70±0.113 0.216±0.062 0.130±0.040 Lo Fat 0.185 0.55 1.60±0.094 0.202±0.038 0.123±0.008 Hi Fat 0.251 2.29 1.35±0.133* 0.095±0.006* 0.073±0.010 Hi Fat 0.269 0.54 1.66±0.145 0.093±0.003* 0.050±0.004* *P<0.05; One‐way ANOVA, Tukey9s posttest Inhibiting miR‐127‐5p with locked nucleic acids dose‐dependently improved β‐F1‐ATPase translation. Inhibitor (nM) mRNA (2‐DDCt) Protein (AU) Translation (%) 0 1.84±0.314 0.22±0.062 0.647±0.120 10 1.93±0.387 1.24±0.135* 0.500±0.148 50 2.00±0.498 1.24±0.242** 1.483±0.233* 100 1.76±0.207 1.54±0.086* 0.892±0.096 *P<0.05; **P<0.01; ***P<0.001; 1‐way ANOVA, Tukey9s posttest Our results provide evidence implicating hyperlipidemia‐induced miR‐127‐5p expression in suppressing β‐F1‐ATPase translation in human obesity.Funding from NIH/NIDDK grant DK094062 to CSK