Figure 4.

Pharmacologic inhibition of mTOR stimulates UCP1 expression in mouse iBAT and in differentiated 3T3-L1 and MEFs. Male mice (n = 5/vehicle group, for AZD8055 group n = 4) were acclimated to thermoneutrality for 2 weeks followed by treatment with either vehicle or AZD8055 (15 mg/kg/BW) for 20 h. Animals were sacrificed, and iBAT was isolated and assayed by western blot for thermogenic protein: UCP1, mTORC1, and two targets: S6RP/pS240/244 and Akt/pS473, S6RP and AKT (A). Bar graph represents image quantification of UCP1 immunoblot, Hsp90 was used to normalize UCP1. Difference between groups was calculated by unpaired two-tailed t-test (B). C, fully differentiated 3T3-L1 adipocytes were treated with AZD8055 (0.5 μM) for 4 h (n = 5) and total RNA was isolated and mRNA expression of UCP1 was analyzed by qPCR. TBP was used as normalizing control gene. Statistical difference between untreated and AZD8055 groups was calculated by unpaired two-tailed t-test. To test for the efficiency of AZD8055, whole-cell lysates were immunoblotted for Akt/pS473 and AKT (D). E and F, genetic deletion of mTORC2. WT and mSin1 knockout (mSIN1 KO) murine embryo fibroblasts (MEFs) were differentiated into adipocytes and assayed for UCP1 mRNA expression by qPCR (n = 6). Statistical difference between WT and mSIN1 KO groups was calculated by unpaired two-tailed t-test (E). Genetic deletion/loss of mTORC2 function was confirmed by probing whole-cell lysates for Akt/pS473 and AKT (F).