MiR-27b-3p Inhibition Enhances Browning of Epididymal Fat in High-Fat Diet Induced Obese Mice

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  Figure 1. Characteristics of high-fat diet induced obese mice. Mice were fed a high fat diet beginning at 3 weeks of age. Measurements were performed after 13-week feeding. Epididymal (eWAT), inguinal subcutaneous (iWAT) and retroperitoneal white adipose tissue (rWAT) was collected respectively. (A) Representative appearance of mice fed with high-fat diet (HFD) and with normal chow diet (NCD). (B) Total body weight (n = 10–12). (C) oGTT, performed after 12 weeks of HFD and mice were fasted for 16 h prior to obtain baseline blood glucose reading (n = 6). (D) ITT, performed after 13 weeks of HFD and mice were fasted for 6 h prior to obtain baseline blood glucose reading (n = 6). (E) Representative appearance of iWAT, eWAT, and rWAT. (F) Tissue weight (n = 10–12). (G) Ratio of tissue weight to body weight (n = 10–12). (H) Representative H&E staining of iWAT, eWAT, and rWAT (n = 6). Scale bars: 50 μm. (I) Mean adipocyte size was measured under microscope (n = 6). (J–L) qRT-PCR analysis of adipokines (Adipoq, Visfatin, Leptin, and Resistin) and inflammatory factors (MCP-1) expression of iWAT, eWAT, and rWAT (n = 10–12). Data are shown as mean ± SEM. *p < 0.05; **p < 0.01.

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  Figure 2. miR-27b-3p is predominantly expressed in eWAT compared with other white adipose tissues. (A) qRT-PCR analysis of miR-27b-3p expression in iWAT, eWAT, and rWAT from mice fed a NCD. (B) qRT-PCR analysis of miR-27b-3p expression in iWAT, eWAT, and rWAT after 13-week HFD or NCD feeding. (C) qRT-PCR analysis of miR-27b-3p expression in eWAT at the 1st, 2nd, 4th, 6th, 8th, and 10th week after HFD. Data are shown as mean ± SEM, n = 6. *p < 0.05; **p < 0.01, NCD vs. HFD; #p < 0.05; ##p < 0.01, 2nd, 4th, 6th, 8th, or 10th week vs. 1st week in the HFD group.

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  Figure 3. Impaired browning ability of eWAT in HFD induced obese mice. Whole body-energy metabolism analysis was measured after 13-week HFD feeding. (A) Respiratory Exchange Rate (RER). (B) Food intake. (C) Physical activity (n = 6). Mice were sacrificed after 13-week HFD feeding. (D) Representative immunohistochemical staining for Ucp1 in eWAT (n = 6). Scale bars: 50 μm. (E) Representative Ucp1 staining intensity designated by mean densitometry of the digital image (n = 6). (F) qRT-PCR analysis of Ucp1 expression and other brown adipose-selective genes in eWAT (n = 10–12). (G) qRT-PCR analysis of Ucp1 expression in eWAT at the 1st, 2nd, 4th, 6th, 8th, and 10th week after HFD. Data are shown as mean ± SEM, n = 6. *p < 0.05; **p < 0.01, NCD vs. HFD; #p < 0.05, 2nd, 4th, 6th, 8th, or 10th week vs. 1st week in the HFD group.

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  Figure 4. Inhibition of miR-27b-3p improves browning ability of eWAT. Mice were fed a HFD beginning at 3-week of age. Thirteen weeks later, mice received 2 injections of antimiR-27b-3p lentiviruses or scramble lentiviruses through the tail vein over 14 days. (A) Overview of anti-miR-27b-3p lentiviruses delivery. (B) qRT-PCR analysis of miR-27b-3p expression in iWAT, eWAT, and rWAT after mice were sacrificed (n = 12–13). (C–E) Energy expenditure of the mice during a 24-h period is reported as RER (C), food intake (D), and physical activity (E) were measured synchronously (n = 6, performed after two injections of anti-miR-27b-3p lentiviruses). (F) Representative Ucp1 staining in eWAT of the mice (n = 6). Scale bars: 50 μm. (G) Representative Ucp1 staining intensity designated by mean densitometry of the digital image (n = 6). (H) qRT-PCR analysis of Ucp1 and other brown adipose-selective genes in eWAT of the mice (n = 12–13). Data are shown as mean ± SEM. *p < 0.05; **p < 0.01, NCD+scr-miR vs. HFD+scr-miR; #p < 0.05; ##p < 0.01, HFD+scr-miR vs. HFD+antimiR-27b-3p.

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  Figure 5. Inhibition of miR-27b-3p improves metabolism of eWAT. Mice were fed a HFD beginning at 3-week of age. Thirteen weeks later, mice received 2 injections of antimiR-27b-3p lentiviruses or scramble lentiviruses through the tail vein over 14 days. (A) Total body weight of mice (n = 12–13). (B) Tissue weight of iWAT, eWAT, and rWAT (n = 12–13). (C) Ratio of tissue weight to body weight (n = 12–13). (D) oGTT, performed at 17th week and mice were fasted for 16 h prior to obtain baseline blood glucose reading (n = 6). (E) ITT, performed at 17th week and mice were fasted for 6 h prior to obtain baseline blood glucose reading (n = 6). (F) Representative H&E staining in eWAT of the mice (n = 6). Scale bars: 50 μm. (G) Mean adipocyte size was measured under microscope (n = 6). Data are shown as mean ± SEM. *p < 0.05; **p < 0.01, NCD+scr-miR vs. HFD+scr-miR; #p < 0.05; ##p < 0.01, HFD+scr-miR vs. HFD+antimiR-27b-3p.

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  Figure 6. miR-27b-3p inhibition enhances browning ability of adipocytes from BAT. (A–C) Mice were fed a HFD beginning at 3-week of age. Thirteen weeks later, mice received 2 injections of antimiR-27b-3p lentiviruses or scramble lentiviruses through the tail vein over 14 days. (A) Western blot analysis of Ucp1 expression in BAT of the mice. (B) Quantitative analysis of Ucp1 was performed with densitometric image analysis software (n = 6). (C) qRT-PCR analysis of Ucp1 and other brown adipose-selective genes expression in BAT of the mice. (D,E) SVF cells from BAT was isolated from 3-week-old male C57BL/6J mice. (D) qRT-PCR analysis of miR-27b-3p expression. (E) qRT-PCR analysis of Ucp1 and other brown adipose-selective genes: Prdm16, Cidea, Cox8b and Pgc1α. Data are presented as mean ± SEM; n = 3; *p < 0.05; **p < 0.01.