Myostatin signals through miR-34a to regulate Fndc5 expression and browning of white adipocytes

Myostatin (Mstn) has a pivotal role in glucose and lipid metabolism. Mstn deficiency leads to the increased browning of white adipose tissue (WAT), which results in the increased energy expenditure and protection against diet-induced obesity and insulin resistance. In this study, we investigated the molecular mechanism(s) through which Mstn regulates browning of white adipocytes.

Quantitative molecular analyses were performed to assess Mstn regulation of miR-34a and Fndc5 expression. miR-34a was overexpressed and repressed to investigate miR-34a regulation of Fndc5. Luciferase reporter analysis verified direct binding between miR-34a and the Fndc5 3'-untranslated region (UTR). The browning phenotype of Mstn^-/- adipocytes was assessed through the analysis of brown fat marker gene expression, mitochondrial function and infrared thermography. The role of miR-34a and Fndc5 in this browning phenotype was verified through antibody-mediated neutralization of FNDC5, knockdown of Fndc5 by small interfering RNA and through miR-34a gain-of-function and loss-of-function experiments.

Mstn treatment of myoblasts inhibited Fndc5 expression, whereas the loss of Mstn increased Fndc5 levels in muscles and in circulation. Mstn inhibition of Fndc5 is miR-34a dependent. Mstn treatment of C2C12 myoblasts upregulated miR-34a expression, whereas reduced miR-34a expression was noted in Mstn^-/- muscle and WAT. Subsequent overexpression of miR-34a inhibited Fndc5 expression, whereas blockade of miR-34a increased Fndc5 expression in myoblasts. Reporter analysis revealed that miR-34a directly suppresses Fndc5 expression through a miR-34a-specific binding site within the Fndc5 3'UTR. Importantly, Mstn-mediated inhibition of Fndc5 was blocked upon miR-34a inhibition. Mstn^-/- adipocytes showed reduced miR-34a, enhanced Fndc5 expression and increased thermogenic gene expression, which was reversed upon either neutralization of Fndc5 or Fndc5 knockdown. In agreement, Mstn^-/- adipocytes have increased mitochondria, improved mitochondrial function and increased heat production.

Mstn regulates Fndc5/Irisin expression and secretion through a novel miR-34a-dependent post-transcriptional mechanism. Loss of Mstn in mice leads to the increased Fndc5/Irisin expression, which contributes to the browning of white adipocytes.