Fig 2: β3-adrenergic stimulation is blunted in STAT5-deficient BAT and associates with diminished PKA activity. A, Basal and CL316243-stimulated lipolysis represented by NEFA release from BAT explants (n ≥ 9 explants from 3 mice/group). B, Heatmap of genes involved in β-adrenergic signalling that show significant (p < 0.05) changes in expression in BAT of Stat5Adipoq mice assessed by RNA-Seq. C, Western blot for pPKA substrate of lysates from BAT explants used in A. The signal for CL316243-induced pPKA substrate was quantified using Image J (n = 4/group). D, Representative Oil Red O staining of primary differentiated brown adipocytes at low magnification (upper panel, scale bar: 0.5 cm) and high magnification (lower panel, scale bar: 50 μm). Quantification (E) of Oil Red O content was performed by measuring the absorbance of the eluted staining (n = 5/genotype). F, Western blot analysis for adiponectin and STAT5 in lysates of primary differentiated brown adipocytes. HSC70 was used as loading control. Samples were loaded on the same gel but not consecutively as indicated. Quantification (G) was performed using ImageJ (n = 3/genotype). H, Bioenergetic profile of primary differentiated brown adipocytes of control and Stat5Adipoq mice shown as a representative curve. Baseline oxygen consumptions rates (OCR) and OCRs in the presence of oligomycin (Oligo), CL316243, FCCP and antimycin A (AA)/rotenone (Rot) were analysed using an XFe96 Seahorse Bioscience analyser (n = 16 wells/group). I, Respiratory capacities of primary brown adipocytes are shown from a representative experiment (n = 16 wells/group) from three experiments. J, Western blot for UCP1 in lysates of primary differentiated brown adipocytes. HSC70 serves as loading control. Samples were loaded on the same gel but not consecutively as indicated. Quantification (K) was performed using Image J (n = 6/genotype). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 3: OXGR1 is required for metabolic beneficial effects of resistance exercise ABody weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups receiving non-exercise or resistance exercise for 14 days (n = 8 per group).BExercise-induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of the non-exercise control group for each genotype (n = 8 per group).CExercise-induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice was subtracted by the average fat mass of the non-exercise control group for each genotype (n = 8 per group).DCumulative food intake of male WT littermates and OXGR1KO mice after 14-day resistance exercise (n = 8 per group).E, FWeight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).GBody composition of male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).HSerum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks old) fed with normal chow were receiving resistance exercise for 40 min (n = 8 per group). The serum AKG levels were tested before and immediately after exercise.ISerum E level in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).J–LThe mRNA expression (J) and protein expression of UCP1 (K) in the BAT or the mRNA expression of HSL and ATGL (L) in the gWAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).M–POxygen consumption (M, N) and RER (O, P) in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).Data information: Results are presented as mean ± SEM. In (A–D) *P = 0.05 and **P = 0.01 by two-way ANOVA followed by post hoc Bonferroni tests. In (E–L, N and P), different letters between bars indicate P = 0.05 by one-way ANOVA followed by post hoc Tukey's tests.

Fig 4: Schematic diagram showing adipocyte integrated responses to cold. Shown are responses to mild (left panel) versus severe (right panel) cold. Mild cold acts through the SNS to stimulate ß-adrenergic signaling, increase intracellular cAMP, and stimulate UCP1 expression. At the same time, cAMP stimulates mTORC2-dependent phosphorylation of Akt at S473, which stimulates lipogenesis via p-ACLY (S455), while blunting the stimulation of UCP1 expression. The net result is moderate stimulation of UCP1 together with enhanced DNL. In contrast, in addition to the canonical effect through the SNS, severe cold has a cell-autonomous PKA-independent effect to inhibit mTORC2 through an unknown cold receptor/sensor (referred to here as “?”/“X”). Stimulation of the thermogenic program is augmented as the inhibitory effect of mTORC2 is lifted. At the same time mTORC2 inhibition leads to a decrease in active Akt (pS473), and thus the SNS-dependent stimulation of lipogenesis through activation of Akt and p-ACLY (S455) is blocked. See text for further details.

Fig 5: TAZKD mice exhibit normal UCP1 and CL levels in BAT. (A) mRNA levels of PEMT and TAZ during differentiation of preadipocytes to brown adipocytes, n = 2. (B) Knockdown confirmation of PEMT and TAZ mRNA following silencing using the lentivirus system. shRNA against PEMT (shPEMT) or TAZ (shTAZ) administered to preadipocytes via infection with viral media for 48 h. Control cells received a scrambled virus (SC). After infection, preadipocytes were differentiated, n = 2. (C) Protein levels of UCP1, actin, and CS. (D) mRNA levels of UCP1 and other genes involved in adipogenesis, n = 2. (E) TAZ mRNA abundance in BAT, n = 4. (F) Body mass, n = 5–6. (G) Cold-tolerance test, n = 10. (H) Protein levels of UCP1 and CS. (I) Protein abundance of ETS complexes. (J) Mitochondrial phospholipids in BAT measured by TLC. (K) Mitochondrial phospholipids isolated from heart mitochondria. (L) mRNA levels of cardiolipin synthase (CRLS1) and acyl-CoA:lysocardiolipin acyltransferase-1 (ALCAT1), n = 3–5. (M) TAZ mRNA levels in heart, n = 4. Data are expressed as mean ± SEM, *p < 0.05.