Obesity-associated exosomal miRNAs modulate glucose and lipid metabolism in mice

Abstract

Obesity is frequently associated with metabolic disease. Here, we show that obesity changes the miRNA profile of plasma exosomes in mice, including increases in miR-122, miR-192, miR-27a-3p, and miR-27b-3p Importantly, treatment of lean mice with exosomes isolated from obese mice induces glucose intolerance and insulin resistance. Moreover, administration of control exosomes transfected with obesity-associated miRNA mimics strongly induces glucose intolerance in lean mice and results in central obesity and hepatic steatosis. Expression of the candidate target gene Ppara is decreased in white adipose tissue but not in the liver of mimic-treated (MIMIC) mice, and this is accompanied by increased circulating free fatty acids and hypertriglyceridemia. Treatment with a specific siRNA targeting Ppara transfected into exosomes recapitulates the phenotype induced by obesity-associated miRNAs. Importantly, simultaneously reducing free fatty acid plasma levels in MIMIC mice with either the lipolysis inhibitor acipimox or the PPARα agonist fenofibrate partially protects against these metabolic alterations. Overall, our data highlight the central role of obesity-associated exosomal miRNAs in the etiopathogeny of glucose intolerance and dyslipidemia.

Keywords: adiposity; dyslipidemia; exosome; glucose intolerance; miRNA.

Fig. 1.

Diet-induced central obesity changes the profile of circulating exosomal miRNAs. (A) IpGTT in C57B6J mice after 15 wk of HFD feeding. (B) Representative electron micrographs of exosomes isolated from mouse plasma. (C) Exosome size distribution determined by NTA in control and obese mice. (D) Western blot analysis of CD63 from equal volumes of plasma obtained from chow-fed and HFD mice and its quantification. (E) Number of exosomes from equal volumes of plasma estimated from esterase activity. (F) Volcano plot of real time RT-PCR profiling of the miRNA content of lean and obese plasma exosomes. Data are presented as mean ± SEM. n = 10 per group (A); n = 3 per group (B–E); n = 4 per group (F). *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, Student’s t test.

Fig. 2.

Exosomes from obese mice induce glucose intolerance in lean mice. (A) 3T3-L1 cells after 24 h incubation with unlabeled exosomes (Upper left), exosomes labeled with fluorescent marker PKH67 (Upper right), unlabeled exosomes transfected with a fluorescent miRNA mimic (Lower left), and 3T3-L1 cells transfected with the same fluorescent mimic (Lower right). (B) Liver (Upper) and eWAT (Lower) sections of control mice after 6 h injection with PBS (Left) or exosomes labeled with PKH67 (Right). (C) IpGTT in chow-fed mice after 4 wk of biweekly injections of obese exosomes. (D and E) IpGTT (D) and insulin tolerance test (0.35 U/kg) (E) in C57B6J mice after 8 wk of biweekly systemic injections of obese exosomes, with HFD feeding during the last 4 wk. Data are presented as mean ± SEM. n = 3 per condition (A and B); n = 10 per group (C); n = 5 per group except n = 4 CT-HFD (D and E). *P < 0.05, **P < 0.01, ****P < 0.001 with respect to control (CT) group, Student’s t test.

Fig. 3.

Exosomes transfected with obesity-associated miRNAs induce glucose intolerance dissociated from obesity. (A and B) Plasma TG (A) and FFA (B) concentrations from chow-fed mice after 4 wk of injections of exosomes loaded with mimics of four miRNAs enriched in obese exosomes. (C and D) IpGTT (C) and insulin tolerance test (0.175 U/kg) (D) in mice described in A and B. (E and F) Correlation between the glycemia AUC obtained from the IpGTT and either body weight (E) or percentage of eWAT (F). Data are presented as mean ± SEM. n = 5 per group (A–F). *P < 0.05, **P < 0.01, Student’s t test.

Fig. 4.

Mimic treatment induces eWAT inflammation and hepatic steatosis. (A) mRNA expression level of Ppar family members in 3T3-L1 cells after transfection with the four selected obesity-associated miRNA mimics. (B and G) Heat maps showing differential mRNA expression of candidate target genes involved in lipogenesis, FAO, and inflammation between either HFD or MIMIC mice and respective controls in the eWAT (B) and the liver (G). (C–E) mRNA expression level of Ppara (C), Pparg (D), and Ccl2 (E) in the eWAT of HFD mice, MIMIC mice, and respective controls. (F) Representative H&E staining of eWAT sections from control and MIMIC mice. (H and I) mRNA expression level of Ppara (H) and Pparg (I) in the liver of HFD mice, MIMIC mice, and respective controls. (J and K) Representative Oil Red staining of liver sections (J) and TG quantification in the liver of control and MIMIC mice (K). Data are presented as mean ± SEM. At least n = 7 per condition (A); n = 4 per group (B–E, G–I, and K); n = 2 per group (F and J). *P < 0.05, **P < 0.01, ***P < 0.005 with respect to either control (CT) or CHOW groups, Student’s t test.

Fig. 5.

Treatment with siPPARA-transfected exosomes recapitulates the central obesity phenotype of MIMIC mice. (A) mRNA expression level of Ppar family members in 3T3-L1 cells transfected with siPPARA siRNA. (B and C) mRNA expression of Ppara (B) and Ccl2 (C) in the eWAT of chow-fed mice after two injections of lean exosomes loaded with the siPPARA described in A. (D and E) mRNA expression of Ppara (D) and Pparg (E) in the liver of control and siPPARA mice. (F) Western blot analysis of mitochondrial complexes and housekeeping actin in the eWAT from MIMIC and siPPARA model mice. (G) FFA quantification in the liver of the mice described in B and C. (H) IpGTT in the mice described in B and C. (I and J) Correlation between the glycemia AUC obtained from the IpGTT and either body weight (I) or percentage of eWAT (J). Data are presented as mean ± SEM. n = 3 per condition (A); n = 4 per group (B–E); n = 2 per group (F); n = 8 per group (G); n = 5 per group (H); n = 9 per group (I); n = 8 control (CT) and 7 siPPARA (J). *P < 0.05, **P < 0.01, ***P < 0.005, Student’s t test.

Fig. 6.

Decreasing FFA plasma levels partially revert the pathologic phenotype. (A and B) Plasma FFA (A) and TG (B) concentrations from chow-fed mice after 4 wk of injections of exosomes loaded with mimics of four miRNAs enriched in obese exosomes and simultaneously administered acipimox (ACX) or fenofibrate (FF) orally. (C and D) IpGTT (C) and insulin tolerance test (0.5 U/kg) (D) in the mice described in A and B. (E) Proposed model: Injection of exosomes transfected with synthetic miRNAs simulating those enriched in obesity decreases Ppara expression and oxidative capacity in the eWAT. This is associated with increased FFA release to the bloodstream, which in turn induces adipose inflammation and hepatic steatosis. Treatment with the lipolysis inhibitor ACX or the PPARα agonist FF decreases plasma FFAs and partially reverts this phenotype. Data are presented as mean ± SEM. n = 5 per group (A–D). *P < 0.05, **P < 0.01, ****P < 0.001 with respect to the control (CT) group unless otherwise indicated, Student’s t test.