Inhibition of miR-27b Regulates Lipid Metabolism in Skeletal Muscle of Obese Rats During Hypoxic Exercise by Increasing PPARγ Expression

Abstract

Hypoxic exercise may represent a novel therapeutic strategy to reduce and prevent obesity through the regulation of lipid metabolism. During hypoxic exercise, the targeting of peroxisome proliferator-activated receptor gamma (PPARγ) by miR-27b has been proposed to be one of the mechanisms involved in the modulation of lipid metabolism. We have previously shown that miR-27b can repress PPARγ and lipid metabolism-associated factors, thereby affecting lipid metabolism during hypoxic exercise in a rat model of obesity. In the current study, we aimed to confirm the role of miR-27b in the regulation of lipid metabolism. First, miR-27b expression was either upregulated or downregulated through the injection of adeno-associated virus (AAV) 9 containing a miR-27b expression cassette or miR-27b-3p inhibitor, respectively, into the right gastrocnemius muscle of obese rats. The rats were then subjected to a 4-week program of hypoxic exercise, and a series of parameters related to lipid metabolism were systematically evaluated, including body composition, blood lipid levels, miR-27b RNA levels, and mRNA and protein levels of PPARγ and those of its downstream lipid metabolism-associated factors. No significant differences were found in body composition between rats expressing different levels of miR-27b. However, regarding blood lipids, miR-27b overexpression led to increased concentrations of triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and free fatty acids (FFAs), while inhibition of miR-27b decreased the total cholesterol (TC) level and increased that of high-density lipoprotein cholesterol (HDL-C). At the mRNA level, miR-27b overexpression downregulated the expression of Pparγ, but upregulated that of lipid metabolism-associated factors such as heart-type fatty acid-binding protein (H-FABP), fatty acid transport protein 1 (FATP1), adipose triglyceride lipase (ATGL), and lipoprotein lipase (LPL), whereas miR-27b inhibition elicited the opposite effect; however, inhibition of miR-27b led to elevated cholesterol 7 alpha-hydroxylase (CYP7A1) and fatty acid translocase 36 (CD36) levels. Similarly, at the protein level, miR-27b overexpression promoted a decrease in the concentration of PPARγ, whereas miR-27b inhibition led to an increase in PPARγ levels, as well as those of CYP7A1, CD36, ATGL, and LPL. Overall, our results indicated that hypoxic exercise regulates lipid metabolism via the miR-27b/PPARγ pathway and modulates ATGL and LPL expression through inducing their post-transcriptional modifications.

Keywords: PPARγ; hypoxic exercise; lipid metabolism; miR-27b; skeletal muscle.

FIGURE 1

Flow chart describing the generation of a rat model of obesity and animal experiments.

FIGURE 2

The body weight of rats fed a high-fat diet (HFD; n = 100) and of those fed a normal diet (Control, n = 20) was measured weekly for 10 weeks. Data are presented as means ± SD.

FIGURE 3

Representative fluorescence images of the gastrocnemius muscle of rats injected with AAV9-ZsGreen-miR-27b (group O), AAV9-ZsGreen-miR-27b-3p inhibitor (group I), or AAV9-ZsGreen (group C), as well as of rats not injected with AAV9 (group H). The gastrocnemius muscle of rats in groups O, I, and C show ZsGreen fluorescence. Tissue sections of frozen gastrocnemius muscle were obtained using a freezing microtome and observed under a fluorescence microscope. Scale bar = 500 μm.

FIGURE 4

The expression levels of miR-27b relative to those of U6 in obese rats from groups O, I, C, and H measured by RT-qPCR. Results were compared by one-way ANOVA; all data are presented as means ± SD. **p < 0.01.

FIGURE 5

The expression of the lipid metabolism regulator PPARγ and that of its downstream effectors (CYP7A1, CD36, H-FABP, FATP1, ATGL, and LPL) were determined at the mRNA level by RT-qPCR (A) and at the protein level by western blot assays (B) in obese rats housed under hypoxic conditions, undergoing hypoxic training, and with regulated miR-27b expression. O: obese rats with overexpression of miR-27b and undergoing hypoxic training; I: obese rats with suppressed miR-27b expression and undergoing hypoxic training; C: obese rats undergoing hypoxic training only; and H: obese sedentary rats without regulation of miR-27b expression. Results were compared by one-way ANOVA; all data are presented as means ± SD. *p < 0.05, **p < 0.01.