Adipocyte HIF2α functions as a thermostat via PKA Cα regulation in beige adipocytes

Abstract

Thermogenic adipocytes generate heat to maintain body temperature against hypothermia in response to cold. Although tight regulation of thermogenesis is required to prevent energy sources depletion, the molecular details that tune thermogenesis are not thoroughly understood. Here, we demonstrate that adipocyte hypoxia-inducible factor α (HIFα) plays a key role in calibrating thermogenic function upon cold and re-warming. In beige adipocytes, HIFα attenuates protein kinase A (PKA) activity, leading to suppression of thermogenic activity. Mechanistically, HIF2α suppresses PKA activity by inducing miR-3085-3p expression to downregulate PKA catalytic subunit α (PKA Cα). Ablation of adipocyte HIF2α stimulates retention of beige adipocytes, accompanied by increased PKA Cα during re-warming after cold stimuli. Moreover, administration of miR-3085-3p promotes beige-to-white transition via downregulation of PKA Cα and mitochondrial abundance in adipocyte HIF2α deficient mice. Collectively, these findings suggest that HIF2α-dependent PKA regulation plays an important role as a thermostat through dynamic remodeling of beige adipocytes.

Fig. 1. Cold exposure induces HIFα expression in thermogenic adipose tissues.

a, b Immunohistochemistry of (a) iWAT and (b) BAT sections upon TN or cold exposure using an anti-pimonidazole antibody and DAB staining. Scale bars, 50 μm. c, d Western blot analysis of HIFα and UCP1 (loaded 50 μg for iWAT and 10 μg for BAT) in c iWAT and d BAT upon TN (30 °C), RT (22 °C), or cold exposure (4–6 °C). e, f Immunofluorescence images of iWAT sections upon TN or cold exposure (3 days) using anti-PLIN1 (green) and e anti-HIF1α or f anti-HIF2α (red) antibodies with Hoechst (blue) staining. Scale bars, 50 μm. g, h Western blot analysis of HIFα and UCP1 in g iWAT and h BAT at TN upon daily CL administration (0.5 mg/kg, 4 days).

Fig. 2. Adipocyte-specific HIF2α KO mice exhibit increased thermogenic activities upon cold exposure.

a Changes in rectal temperature of WT (n = 9), HIF1α AKO (n = 5), HIF2α AKO (n = 6), and HIF1/2α DKO (n = 7) mice during cold exposure. b Infrared images of body surface temperature of WT, HIF1α AKO, HIF2α AKO, and HIF1/2α DKO mice upon cold exposure (4 h). c–e Representative images of hematoxylin and eosin (H&E) staining of iWAT from WT, c HIF1α AKO, d HIF2α AKO, and e HIF1/2α DKO mice upon TN or cold exposure (3 days). Scale bars, 50 μm. f mRNA levels in iWAT from WT (n = 7), HIF1α AKO (n = 6), HIF2α AKO (n = 6), and HIF1/2α DKO (n = 6) mice upon cold exposure (3 days). g Rectal temperature of vehicle- (n = 5) and YC-1- (30 mg/kg, n = 5) administered mice during cold exposure. YC-1 was injected i.p. 1 h prior to exposure to cold. h mRNA levels in iWAT from vehicle- (n = 3) and YC-1- (n = 4) administered mice upon cold exposure (3 days). i Representative images of H&E staining of iWAT from vehicle- and YC-1-administered mice upon cold exposure (3 days). Scale bars, 50 μm. j Rectal temperature of vehicle- (n = 6) and PT2385- (10 mg/kg, n = 6) administered mice during cold exposure. PT2385 was injected i.p. 1 h prior to exposure to cold. k mRNA levels in iWAT from vehicle- (n = 5) and PT2385- (n = 5) administered mice upon cold exposure (3 days). l Representative images of H&E staining of iWAT from vehicle- and PT2385-administered mice upon cold exposure (3 days). Scale bars, 50 μm. m Rectal temperature of vehicle- (n = 6) and DMOG- (40 mg/kg, n = 7) administered mice during cold exposure. DMOG was injected i.p. 1 h prior to exposure to cold. n mRNA levels in iWAT from vehicle- (n = 4) and DMOG- (n = 5) administered mice upon cold exposure (3 days). o Representative images of H&E staining of iWAT from vehicle- and DMOG-administered mice upon cold exposure (3 days). Scale bars, 50 μm. Data were expressed as the mean ± SEM by either two-tailed unpaired Student t-tests in (h, k, n), one-way ANOVA in (f), or two-way repeated-measures ANOVA in (a, g, j, m) followed by Holm–Sidak’s multiple comparisons test.

Fig. 3. HIF2α ablation in adipocytes potentiates energy expenditure upon β3-adrenergic activation.

a Representative images of H&E staining of iWAT from WT and HIF2α AKO mice upon daily CL administration (0.5 mg/kg, 4 days). Scale bars, 50 μm. b Western blot analysis of UCP1 in iWAT from WT and HIF2α AKO mice upon daily CL administration (0.5 mg/kg, 4 days). c mRNA levels in iWAT from WT (Vehicle; n = 5, CL; n = 4) and HIF2α AKO (n = 4) mice upon daily CL administration (0.5 mg/kg, 4 days). d–i d, e VO₂, f, g VCO₂, and h, i energy expenditure for WT and HIF2α AKO mice (n = 6). Data were expressed as the mean ± SEM by two-way ANOVA followed by Holm–Sidak’s multiple comparisons test.

Fig. 4. PKA signaling is activated by an increase in PKA Cα in HIF2α deficient adipocytes.

a Heatmap of upregulated DEGs in iWAT of HIF2α AKO mice upon cold exposure (3 days). b KEGG pathway and GO cellular component enrichment analysis upon cold exposure (3 days). c Scatter plot of thermogenesis-related DEGs upon cold exposure (3 days). d DEGs interaction network in iWAT of HIF2α AKO mice upon cold exposure (3 days). e mRNA levels in iWAT from WT (n = 7), HIF1α AKO (n = 6), HIF2α AKO (n = 6), and HIF1/2α DKO (n = 6) mice upon cold exposure (3 days). f, g Western blot analysis of PKA Cα in iWAT from WT and HIF2α AKO mice upon f cold exposure (3 days) or g daily CL administration (0.5 mg/kg, 4 days). h Western blot analysis of PKA signaling and UCP1 in iWAT from WT and HIF2α AKO mice upon TN or cold exposure (6 h). i Western blot analysis of PKA signaling in iWAT from WT and HIF2α AKO mice upon CL administration (0.5 mg/kg, 4 h). j Western blot analysis of PKA signaling and UCP1 in beige adipocytes from WT and HIF2α AKO mice without or with ISO (5 μM, 1 h). k Glycerol concentration in culture media of beige adipocytes from WT (n = 5) and HIF2α AKO (n = 5) mice without or with ISO (5 μM, 3 h). l Intracellular cAMP levels of beige adipocytes from WT (n = 4) and HIF2α AKO (n = 4) mice without or with ISO (1 μM, 15 min). m mRNA levels in beige adipocytes from WT (n = 3) and HIF2α AKO (n = 3) mice. n Western blot analysis of PKA Cα in beige adipocytes from WT and HIF2α AKO mice. o mRNA levels in beige adipocytes infected with Admock (n = 4) or AdHIF2α (n = 4). p Western blot analysis of PKA Cα beige adipocytes infected with Admock or AdHIF2α. Data were expressed as the mean ± SEM by either two-tailed unpaired Student t-tests in (m, o), one-way ANOVA in (e), or two-way ANOVA in (k, l) followed by Holm–Sidak’s multiple comparisons test. ISO isoproterenol.

Fig. 5. HIF2α deficiency upregulates mitochondrial OXPHOS in beige adipocytes.

a Relative mtDNA in iWAT from WT (TN; n = 4, Cold; n = 5) and HIF2α AKO (TN; n = 4, Cold; n = 5) mice upon TN or cold exposure (3 days). b mRNA levels in iWAT from WT (n = 5) and HIF2α AKO (TN; n = 4, Cold; n = 5) mice upon TN or cold exposure (3 days). c Western blot analysis of OXPHOS complexes in iWAT from WT and HIF2α AKO mice upon cold exposure (3 days). d Relative mtDNA in iWAT from WT (n = 4) and HIF2α AKO (n = 4) mice upon daily CL administration (0.5 mg/kg, 4 days). e mRNA levels in iWAT from WT (Vehicle; n = 5, CL; n = 4) and HIF2α AKO (n = 4) mice upon daily CL administration (0.5 mg/kg, 4 days). f Western blot analysis of OXPHOS complexes in iWAT from WT and HIF2α AKO mice upon daily CL administration (0.5 mg/kg, 4 days). g OCRs and quantification in beige adipocytes infected with Admock (n = 9) or AdHIF2α (n = 9). h OCRs and quantification in beige adipocytes from WT (n = 8) and HIF2α AKO (n = 8) mice. i OCRs and quantification in beige adipocytes from WT (n = 10) and HIF2α AKO (n = 10) mice without or with 1 h preincubation of H89 (50 μM). j Experimental scheme of siPrkaca injection. k Western blot analysis of PKA Cα, UCP1, and OXPHOS complexes in iWAT from WT and HIF2α AKO mice with siNC or siPrkaca injection upon cold exposure (3 days). l Representative images of H&E staining of iWAT from WT and HIF2α AKO mice with siNC or siPrkaca injection upon cold exposure (3 days). Scale bars, 50 μm. Data were expressed as the mean ± SEM by two-tailed unpaired Student t-tests in (g, h) or two-way ANOVA in (a, b, d, e, i) followed by Holm–Sidak’s multiple comparisons test. Oligo. oligomycin, ISO isoproterenol, Rot. rotenone, AA antimycin A.

Fig. 6. miR-3085-3p mediates HIF2α-dependent PKA Cα regulation in beige adipocytes.

a Prkaca 3′UTR sequence and predicted miR-3085-3p binding site. b Luciferase reporter activities of WT (n = 4) and mutant (n = 4) Prkaca 3′UTR upon con or miR-3085-3p transfection. c Upstream region of miR-3085-3p and hypoxia-response element (HRE) sites. d ChIP-qPCR analysis of BAC cells upon Admock (n = 5) or AdHIF2α (n = 5) infection. e miR-3085-3p level in iWAT from WT (TN; n = 5, Cold; n = 6) and HIF2α AKO (TN; n = 5, Cold; n = 6) mice upon TN or cold exposure (3 days). f miR-3085-3p level of iWAT from WT (n = 6), HIF1α AKO (n = 4), HIF2α AKO (n = 5), and HIF1/2α DKO (n = 5) mice upon cold exposure (3 days). g miR-3085-3p and h Prkaca levels in iWAT from vehicle- (n = 5) and PT2385- (n = 5) administered mice upon cold exposure (3 days). i mRNA levels in beige adipocytes from WT (n = 4) and HIF2α AKO (n = 4) mice upon con or miR-3085-3p mimic transfection with ISO (5 μM, 4 h). j OCRs and quantification in beige adipocytes from WT (n = 8) and HIF2α AKO (n = 8) mice upon con or miR-3085-3p mimic transfection. k mRNA levels in beige adipocytes infected with Admock (n = 4) or AdHIF2α (n = 4) upon con or miR-3085-3p inhibitor transfection with ISO (5 μM, 4 h). l OCRs and quantification in beige adipocytes infected with Admock (n = 10) or AdHIF2α (n = 10) upon con or miR-3085-3p inhibitor transfection. m Experimental scheme of miRNA mimic injection. n Representative images of H&E staining of iWAT from WT and HIF2α AKO mice with con or miR-3085-3p mimic injection upon cold exposure (3 days). Scale bars, 50 μm. o Western blot analysis of PKA Cα and UCP1 in iWAT from WT and HIF2α AKO mice with con or miR-3085-3p mimic injection upon cold exposure (3 days). Data were expressed as the mean ± SEM by two-tailed unpaired Student t-tests in (g, h), one-way ANOVA in (f), or two-way ANOVA in (b, d, e, i–l) followed by Holm–Sidak’s multiple comparisons test. Oligo. oligomycin, ISO isoproterenol, Rot. rotenone, AA antimycin A.

Fig. 7. During the re-warming process, adipocyte HIF2α deficiency fails to mitigate the mitochondrial activity.

a Experimental scheme (top) and western blot analysis of HIFα and UCP1 in iWAT of upon cold exposure or re-warming (bottom). b Experimental scheme of re-warming. c Representative images of H&E staining of iWAT from WT and HIF2α AKO mice upon cold exposure or re-warming (cold 2 weeks + TN 1 week). Scale bars, 100 μm. d miR-3085-3p level of iWAT from WT (n = 6) and HIF2α AKO (n = 5) mice upon cold and re-warming (cold 2 week + TN 1 week). e, f mRNA levels in iWAT from WT (n = 6) and HIF2α AKO (n = 5) mice upon cold exposure and re-warming (cold 2 weeks + TN 1 week). g Western blot analysis of OXPHOS complexes, PKA Cα, and UCP1 in iWAT from WT and HIF2α AKO mice upon cold exposure and re-warming (cold 2 weeks + TN 1 week). h Experimental scheme of re-warming with con or miR-3085-3p mimic injection. i Representative images of H&E staining of iWAT from WT and HIF2α AKO mice upon re-warming (cold 2 weeks + TN 1 week) with con or miR-3085-3p mimic injection. Scale bars, 50 μm. j Western blot analysis of OXPHOS complexes, PKA Cα, and UCP1 in iWAT from WT and HIF2α AKO mice upon re-warming (cold 2 weeks + TN 1 week) with con or miR-3085-3p mimic injection. Data were expressed as the mean ± SEM by two-tailed unpaired Student t-tests in (d) or two-way ANOVA in (e, f) followed by Holm–Sidak’s multiple comparisons test.

Fig. 8. Proposed model.

Upon cold exposure, an activated thermogenic program in beige adipocytes stimulates oxygen consumption, thereby resulting in the stabilization of HIFα. Among the HIFα isoforms, upregulated HIF2α fine-tunes thermogenic execution via miR-3085-3p-dependent Prkaca regulation. However, adipocyte HIF2α deficiency augments PKA signaling and potentiates thermogenic functions, leading to the retention of beige adipocytes. Our findings suggest that the HIF2α-miR-3085-3p-PKA Cα axis forms negative feedback for appropriate regulation of thermogenesis.