Figure 5. Diet-induced adipocyte precursor activation and adipogenesis requires Akt2 in adipocyte lineage cells.

(A) Western blot for AKT1 and AKT2 in lysates from SVF or FACS-sorted APs from the SWAT (S) or VWAT (V) depot pooled from 12 wild-type animals. Uncropped blots are shown in Supplementary Figure 7A–B. (B) Western blot of lysates from APs enriched from SVF via Sca-1 bead pull down (see methods) after 3 days of HFD or SD. Each lane represents pooled cells from 2 mice. Uncropped blots are shown in Supplementary Figure 7C–H. (C) Quantification of western blots in (B) showing normalization of pAKT1 and pAKT2 to total AKT1 and AKT2, respectively. (D) Quantification of BrdU incorporation into VWAT APs after 1 week of HFD or SD with daily injection of wortmannin (Wort), or vehicle (Veh) (n = 5 mice per group). (E) BrdU incorporation into VWAT APs of the indicated groups during the first week of HFD feeding compared to SD controls. (n = 7 for Akt2^fl/fl SD, n = 4 for PdgfRα-cre; Akt2^fl/fl SD, n = 14 for PdgfRα-cre; Akt2^fl/fl HFD, n = 8 for Akt2^fl/fl HFD, n = 5 for wild-type and PdgfRα-cre HFD) (F) Quantification of immunofluorescence staining for BrdU in adipocyte nuclei of VWAT from the indicated groups of mice after 1 week of BrdU treatment and 8 weeks on the indicated diet. (n = 8 mice for PdgfRα-cre; Akt2^fl/fl HFD, n = 5 mice for wild-type groups). Significance between the indicated groups in (C), (D), and (F) was calculated using a two-tailed student’s t-test. Significance in (E) was calculated using one-way ANOVA with Tukey’s test for multiple comparisons. Exact p-values are listed in Supplementary Table 1. Statistics source data for 5C can be found in Supplementary Table 2. Error bars represent mean ± s.e.m. * (P<0.05), ** (p<0.01), *** (P<0.001), **** (P<0.0001). SVF: stromal-vascular fraction, AP: adipocyte precursor, HFD: high-fat diet, SD: standard diet, BrdU: bromodeoxyuridine, Veh: vehicle, Wort: wortmannin.