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. Author manuscript; available in PMC: 2017 Oct 12.
Published in final edited form as: Cell Rep. 2017 May 9;19(6):1229–1246. doi: 10.1016/j.celrep.2017.04.035

Figure 6. BAT-Specific Overexpression of miR-32 during Cold Exposure Leads to Significantly Higher Core Body Temperature and Increased Energy Expenditure in Mice as the Result of Increased Thermogenic Response in BAT and Beige Cell Emergence in iWAT.

Figure 6

(A) miR-32 expression in BAT but not in liver or iWAT was increased in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7).

(B) miR-32-AAV-BS mice (n = 8) showed higher core body temperatures during cold exposure when compared to control-AAV-BS mice (n = 7).

(C) Total energy expenditure was significantly increased after 7 days’ cold stress in miR-32-AAV-BS mice (n = 6) as compared with control-AAV-BS mice (n = 6). Energy expenditure was normalized to lean body mass.

(D) Average total energy expenditure was significantly higher in miR-32-AAV-BS mice (n = 6).

(E) Percentage BAT mass was significantly higher in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7).

(F) In BAT, mRNA levels of Tob1 were lower in miR-32-AAV-BS mice, whereas expression of thermogenic genes was significantly higher in miR-32-AAV-BS mice.

(G) Protein levels of Tob1 were lower in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7). However, p-p38, p-ATF2, FGF21, UCP1, and PGC1α were higher in miR-32-AAV-BS mice (n = 8). Total p38 and total ATF2 expression were not significantly different between the two groups.

(H) Quantification of relative protein expression using ImageJ showed that protein level of Tob1 was reduced, but p-p38, p-ATF2, FGF21, UCP1, and PGC1α were significantly higher in BAT of miR-32-AAV-BS mice compared to control. Total p38 and total ATF2 expression were comparable between the two groups. Average intensities were normalized to that of Calnexin.

(I) Serum FGF21 levels were increased in miR-32-AAV-BS mice (n = 8) compared to control-AAV-BS mice (n = 7).

(J) mRNA levels of thermogenic genes in iWAT were significantly increased in miR-32-AAV-BS mice. Data were normalized to PPIA.

(K) Immunoblots showed that miR-32-AAV-BS mice (n = 8) had significantly increased UCP1 and PGC1α protein levels in iWAT compared to control-AAV-BS mice (n = 7).

(L) Quantification of relative UCP1 and PGC1α protein levels using ImageJ. Average intensities were normalized to that of Calnexin.

Data represent mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. See also Figure S6.