Cooler temperatures inhibit mTORC2 activity in differentiated 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes, acclimated to 37 °C, were exposed to room temperature (24.5 °C) for 4 h, and whole-cell lysates were analyzed by western blot (A) to assess expression levels of Akt/pS473, AKT, p-S6RP/S240/244, and S6RP. Dashed line represents image splicing to remove irrelevant data in gel. B, bar graph representing average image quantification of Akt/pS473 normalized to total AKT from three separate experiments. Statistical difference between 37 °C and RT groups was calculated by unpaired two-tailed t-test. C, differentiated 3T3-L1 adipocytes were acclimated to 37 °C and then shifted to 39 °C, 37 °C, 31 °C, or RT (23 °C) for 5 or 9 h and whole-cell lysates were probed for Akt/pS473, AKT, p-S6RP/S240/244, and S6RP. Note progressive decrease in Akt/pS473 levels with decreasing temperature, in contrast to biphasic effect in mouse iBAT.