Fig 2: mTORC2 drives lipogenic and Glut4 gene expression through ACLY in mature brown adipocytes.a Time-course western blots showing the indicated phospho-specific and total proteins in Rictor-iKOMBA or Acly-iKOMBA cells following acute and prolonged protein loss. b Direct acetyl-CoA measurements in Rictor-iKOMBA cells following acute Rictor loss (day 12) (n = 8 per group). c qRT-PCR analysis of the indicated genes following acute (day 12) and prolonged (day 14) Rictor loss (n = 6 per group). Rictor deletion was induced at day 8 during differentiation. d qRT-PCR analysis of the indicated genes Rictor-iKOMBA (n = 6 per group) stably expressing empty (EV), Myc-ACLY-WT, Myc-ACLY-S455A, or Myc-ACLY-455D constructs. Bar graphs represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, a represents ***p < 0.001 vs control (EV), and b represents ***p < 0.001 vs Rictor-iKOMBA (EV) cells. Statistical significance was calculated by using two-tailed unpaired Student’s t test (b) or two-way ANOVA with Sidak’s test multiple comparisons (c, d).

Fig 3: Divergent effects of mild and severe cold on BAT temperature and mTORC2 signaling.A, schematic design of the experiment. Male wild-type C57B6 mice (n = 28) were acclimated to thermoneutrality (30 °C) for 4 weeks followed by exposure to either RT/mild cold (n = 14) or 4 °C/severe cold (n = 14) for various time points. B and C, infrared images of mice exposed to RT (B) or 4 °C (C) for times shown. Images are displayed using the rainbow high contrast color palette in the FLiR Research IR program using a temperature linear display between 10 and 40 °C. D and E, compiled infrared temperature data for BAT (D) and tail (E). F, rectal temperature (direct measurement with rectal probe). G, male wild-type C57B6 mice (n = 4/group) were acclimated to thermoneutrality (30 °C) followed by exposure to RT or 4 °C for 1 h iBAT was collected and subjected to immunoblot for Akt/pS473, total AKT, ACLY/pS455, total ACLY, and Actin. H, bar graph representing image quantification of Akt/pS473 normalized to total AKT. I, bar graph representing image quantification of ACLY/pS455 normalized to total ACLY. J, iBAT lysates immunoblotted for p-S6RP/S240/244 (mTORC1 substrate), p-PKA substrates, and Actin. K, bar graph representing image quantification of p-S6RP/S240/244 normalized to actin. L, image quantification of p-PKA substrates normalized to actin. Difference between groups was calculated by unpaired two-tailed t-test, ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Fig 4: AKT1 HM phosphorylation is necessary for ACLY stimulation and differentiation, while ACLY-S455D is sufficient to rescue acetyl-CoA levels and differentiation upon Rictor loss.a Rictor-iKOPBAs cells were stably transfected with the indicated HA-tagged AKT constructs, which were tested for their ability to rescue ACLY-S455 phosphorylation by western blot. b, c Western blot (b) and ORO (c) of Akt1-iKOPBAs and Akt2-iKOPBAs cells. Scale bar represents 50 μm. d Direct acetyl-CoA measurements of Akt1-iKOPBAs cells (n = 6 per group). e, f Representative differentiation rescue experiments showing ORO staining (e) and pparγ expression (n = 6 per group) (f) in Akt1-iKO cells stably expressing empty (EV), Myc-ACLY-WT, Myc-ACLY-S455A, or Myc-ACLY-455D constructs. Scale bar represents 50 μm. g Direct acetyl-CoA measurements of Akt1-iKOPBA cells (n = 9) stably transfected with the indicated Myc-tagged ACLY constructs. h–j Western blot (h), pparγ expression (n = 7–8 per group) (i), and ORO staining (j) of Akt1-iKOPBA stably expressing empty (EV), HA-AKT1-WT, HA-AKT1-S473A, or HA-AKT1-473D constructs. Scale bar represents 50 μm. Bar graphs represent mean ± SEM. ***p < 0.001, a represents ***p < 0.001 vs control (EV), and b represents ***p < 0.001 vs Akt1-iKOPBA (EV). Statistical significance was calculated by using two-tailed unpaired Student’s t test (d) or two-way ANOVA with Sidak’s test multiple comparisons (f, g, i).

Fig 5: Schematic figure of the studied metabolic pathways. The expressions of glutaminolysis (GLS), glycolysis (GLUT1, GAPDH, PFKP LDHA, PDH), fatty acid metabolism (FASN, p-Acly, CPT1A), acetate consumption (ACSS2), mitochondrial oxidative phosphorylation (COXIV, ß-F1-ATPase), autophagy (LC3), ATP sensor protein (p-AMPK) and mTOR complex activity (p-mTOR, Raptor, Rictor, p-S6, p-Akt (Ser473)) related enzymes were studied by Western blot analyses, WES Simple. Further explanation can be found in the text