Vascular rarefaction mediates whitening of brown fat in obesity (original) (raw)
To assess the role of BAT vascularity in systemic metabolic dysfunction, a model of diet-induced obesity was established by imposing a high-fat, high-sucrose (HFHS) diet on mice for 8 weeks. Compared with normal chow (NC), HFHS diet in mice significantly increased body weight and induced systemic insulin resistance (Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/JCI71643DS1). Under these conditions, total BAT weight increased (Supplemental Figure 1C) in association with the accumulation of enlarged lipid droplets in BAT cells (Figure 1, A and B) and a 51% reduction in mitochondrial content per cell (Figure 1B), giving the appearance of BAT “whitening.” Since BAT consumes lipids stored in cytosol to generate heat through uncoupled respiration, these morphological changes led us to hypothesize that obesity induces BAT to whiten via mitochondrial dysfunction and loss. Consistent with this concept, the expression of the mitochondrial gene ND5 was reduced (Figure 1C), as was the expression of nuclear-encoded mitochondria marker genes including Ucp1, Ndufa, Atp5a, and Ppargc1a relative to β_-actin_ expression (Figure 1D). Accordingly, these changes were associated with an impaired thermogenic response in an acute cold–tolerance test (Figure 1E).
The whitening of BAT associated with capillary rarefaction in diet-induced obesity. (A) H&E staining of BAT from mice fed NC or HFHS diet. Scale bar: 50 μm. Right graph shows the number of large lipid droplets/field in BAT (×400, n = 4). (B) Electron micrographs of BAT from mice fed NC or HFHS diet. Right graph shows the number of mitochondria/cell (n = 3). Scale bar: 10 μm. (C and D) Real-time PCR expression of the mitochondrial-encoded transcript ND5 and the nucleus-encoded transcripts Ucp1, Ndufa, Atp5a, and Ppargc1a in BAT from mice fed NC or HFHS diet (n = 3–6). (E) Acute CTT for mice fed NC or HFHS (n = 5–7). (F) Immunofluorescent staining to detect blood vessels with Fluorescein Griffonia (Bandeiraea) Simplicifolia Lectin I (green) and adipocytes with Bodipy-TR (red) in BAT and WAT from mice fed NC or HFHS diet. Scale bars: 100 μm. (G and H) Pimonidazole staining (G) and positive area (H) in BAT and WAT of mice fed NC or HFHS diet determined by hypoxyprobe-1 staining (n = 4–6). Scale bars: 50 μm. (I) Oxygen levels in adipose tissues (AT pO2 [mmHg]) (n = 5–6). (J) Real-time PCR expression of Vegfa and Kdr in BAT and WAT of mice fed NC or HFHS diet (n = 4–10). Data were analyzed by 2-tailed Student’s t test (A–D and H) or ANOVA (E, I, and J). *P < 0.05; **P < 0.01. All values represent the mean ± SEM.
Diet-induced obesity led to marked reductions in the vascular network of both BAT and WAT (Figure 1F). An analysis of isolectin IB4–positive vascular structures in tissue sections revealed large reductions in capillary density per unit area and smaller, but statistically significant, reductions in vessel number per adipocyte in both fat depots (Supplemental Figure 1D). However, severe hypoxia, indicated by staining with pimonidazole, was predominantly observed in BAT (Figure 1, G and H). Pimonidazole labeling was also detected in WAT, but the magnitude of the labeling in BAT was much greater. Consistent with these observations, tissue oxygen levels declined markedly in BAT but modestly in WAT in response to dietary obesity, as determined by the measure of oxygen content using a fiber optic oxygen sensor (Figure 1I).
Previous studies have shown that VEGF-A has a crucial role in controlling vascular network formation in adult tissues including WAT and BAT (11, 23). Vegfa mRNA was expressed at substantially higher levels in BAT than in WAT (Figure 1J), consistent with the observation that BAT is more highly vascularized than WAT. Under the conditions of these assays, diet-induced obesity was associated with declines in VEGF-A transcript and protein expression in both adipose tissue depots (Figure 1J and Supplemental Figure 1, E–H). Transcript levels of the VEGF-A receptor Kdr also declined in parallel with tissue VEGF-A levels, consistent with the reductions in adipose tissue vascularity (Figure 1J).
To explore the temporal relationship between reduced BAT vascularity and markers of mitochondrial dysfunction, WT mice fed a HFHS diet for shorter periods of time (1 and 4 weeks) were analyzed. Relative to mice fed NC, body weight and BAT weight were significantly elevated after 4 weeks on a HFHS diet, but not at 1 week (Supplemental Figure 2A). Correspondingly, lipid droplet enlargement was detected at 4 weeks, but not at 1 week (Supplemental Figure 2B). In contrast, reductions in VEGF-A protein and mRNA expression in BAT could be detected both at 1 and 4 weeks on the HFHS diet, and this corresponded to reductions in the transcript levels of Kdr and to a decrease in capillary density (Supplemental Figure 2, C–E). Staining for pimonidazole indicated the development of robust tissue hypoxia after 4 weeks of HFHS diet (Supplemental Figure 2F). Mitochondrial markers that were reduced by 8 weeks in the BAT of HFHS-fed mice (Figure 1, C and D) were not affected at 1 week. However, ND5, Ndufa, Atp5a, and Ppargc1a were downregulated at 4 weeks (Supplemental Figure 2G). These results suggest that capillary rarefaction precedes mitochondrial dysfunction in BAT.
To corroborate these findings, the ob/ob genetic model of obesity was examined. As expected, body weight and BAT weight were significantly increased in ob/ob relative to control mice (Supplemental Figure 3A), and these changes were associated with the development of the whitening phenotype in BAT and the appearance of enlarged lipid droplets (Supplemental Figure 3B). These changes were associated with marked capillary rarefaction and increased pimonidazole staining in BAT, but the WAT of ob/ob mice displayed relatively modest changes in these parameters (Supplemental Figure 3, C and D). VEGF-A protein levels were reduced in the BAT and WAT of ob/ob mice (Supplemental Figure 3, E and F), but transcript analyses revealed that Vegfa levels were considerably higher in BAT than WAT in the different experimental groups of mice (Supplemental Figure 3G). Transcript analyses also revealed that levels of the vascularity marker Kdr were elevated in BAT compared with WAT, and that these levels were reduced in the BAT and WAT of ob/ob mice relative to WT (Supplemental Figure 3G). The mitochondrial markers Ucp1, ND5, Ndufa, Atp5a, and Ppargc1a were also suppressed in the BAT of ob/ob mice, and these mice displayed a reduced thermogenic response in an acute cold–tolerance test (Supplemental Figure 3, H and I). These results further indicate that capillary rarefaction and reduced VEGF-A expression are associated with the development of the whitening phenotype in BAT.
VEGF-A ablation leads to BAT whitening. To test whether diminished BAT vascularity is causal for BAT whitening, we generated homozygous adipose tissue–specific _Vegfa_-KO (adipo–_Vegfa_-KO) mice by crossing Vegfaflox/flox with aP2-Cre+/– mice. No changes in body weight and food intake were observed between the KO mice and WT fed a standard chow diet (Supplemental Figure 4, A and B). The aP2-Cre recombinase activity is expressed in adipose tissues as well as in the central nervous system and macrophages (24, 25); however, there were no significant changes in Vegfa expression in bone marrow–derived macrophages, cerebrum, and cerebellum in the aP2-Cre+/– Vegfaflox/flox mice (data not shown). The genetic disruption of Vegfa by aP2-cre significantly reduced VEGF-A transcript and protein levels, by 48% and 63%, respectively, in BAT, and by 32% and 58%, respectively in WAT (Figure 2A and Supplemental Figure 4, C–F), approximately matching the declines in VEGF-A protein expression that are observed in the diet-induced obesity model (Supplemental Figure 1, E–H). Declines in both BAT and WAT vascularity were notable under these conditions (Figure 2B). Analysis of capillary density by immunostaining for isolectin IB4–positive endothelial cells in histological sections revealed a 42% reduction in BAT and a 51% reduction in WAT (Supplemental Figure 4G), which was similar to the declines in vascularity observed in the model of diet-induced obesity (Supplemental Figure 1D). Although Vegfa ablation led to a relatively small but detectable pimonidazole reactivity in WAT, the degree of hypoxyprobe signal in BAT was considerably greater (Figure 2C and Supplemental Figure 4H).
The whitening of BAT and impaired glucose metabolism in aP2-Cre+/– Vegfaflox/flox mice. (A) Real-time PCR expression of Vegfa and Kdr in BAT and WAT of aP2-Cre+/– Vegfaflox/flox (KO) and control Vegfaflox/flox mice (WT) (n = 4–19). (B) Immunofluorescent staining to detect vasculature with Fluorescein Griffonia (Bandeiraea) Simplicifolia Lectin I (green) and adipocytes with Bodipy-TR (red) in BAT and WAT from WT and KO mice. Scale bars: 100 μm. (C) Pimonidazole staining of BAT and WAT from WT and KO mice was performed by the hypoxyprobe-1 method. Scale bars: 50 μm. (D) H&E staining of BAT and WAT from WT and KO mice. Scale bars: 50 μm. (E) Real-time PCR detecting expression of ND5, Ucp1, Ndufa, Atp5a, and Ppargc1a in WAT and BAT of WT and KO mice (n = 3–6). (F and G) Electron micrographs of BAT of WT and KO mice (F) and the number of mitochondria/cell (G) (n = 3). Scale bar: 10 μm. (H) Acute CTT for mice prepared in A (n = 7). (I) GTT and ITT of mice prepared in A (n = 5–7). Data were analyzed by 2-tailed Student’s t test (E and G) or ANOVA (A, H, and I). *P < 0.05; **P < 0.01. All values represent the mean ± SEM.
An analysis of WAT revealed that Vegfa ablation led to an enlargement in average cell size and a modest increase in total tissue weight (Figure 2D and Supplemental Figure 4, I and J), although a statistically significant change in overall body weight was not observed. However, Vegfa ablation produced little or no changes in expression of inflammatory markers that contribute to systemic insulin resistance, such as EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1), TNF-α, and chemokine (C-C motif) ligand 2 (Ccl2/MCP-1) (data not shown), nor were there detectable changes in the expression of mitochondrial markers including ND5, Ucp1, Ndufa, Atp5a, and Ppargc1a in WAT (Figure 2E). In marked contrast to effects on WAT, Vegfa ablation led to a significant reduction in BAT mass (Supplemental Figure 4, K and L). Histological analysis of BAT revealed that VEGF-A deficiency led to a whitening phenotype that was accompanied by an increase in lipid droplet size and a reduction in mitochondrial number within BAT adipocytes (Figure 2, D, F, and G, and Supplemental Figure 4M). In contrast to WAT, the transcript expression levels of mitochondrial marker proteins were suppressed in BAT by adipose tissue Vegfa ablation (Figure 2E). These changes were associated with an impaired thermogenic response (Figure 2H), consistent with results of systemic Vegfr2 blockade (23). Vegfa ablation also led to modest impairment in glucose metabolism in mice fed the NC diet (Figure 2I). Collectively, these data suggest that the BAT phenotype resulting from Vegfa ablation is similar to that seen in conditions of diet-induced obesity in WT mice. These findings led us to hypothesize that capillary rarefaction in adipose tissue can lead to the selective development of a hypoxic state in BAT that promotes BAT dysfunction.
BAT-specific VEGF-A rescue improves BAT function and systemic glucose metabolism. To assess the functional significance of BAT hypoxia in obese or Adipo-_Vegfa_-KO mouse models, we performed gain-of-function experiments by injecting an adenoviral vector encoding Vegfa (ad-vegfa), or a control vector expressing β-gal directly into interscapular BAT (Supplemental Figure 5A). Transduction with a low dose of vector led to a doubling of VEGF-A transcript and protein levels in the BAT of NC-fed mice at 1 week after delivery, whereas the level of VEGF-A achieved in BAT of HFHS-fed mice approximately matched that of the control, NC-fed conditions (Figure 3A and Supplemental Figure 5B). Ad-vegfa delivery to BAT did not affect Vegfa or Kdr levels in epididymal WAT (Figure 3B). However, restoration of VEGF-A in the BAT of HFHS-fed mice reversed the decline in vascular density that was caused by dietary obesity as assessed by measures of vessel density and Kdr expression (Figure 3, A and C, and Supplemental Figure 5, C and D). Vegfa transduction of the BAT of obese mice suppressed the development of enlarged lipid droplets, and this was associated with the upregulation of ND5, Ucp1, Ndufa, and Ppargc1a transcripts that encode for mitochondria-associated proteins (Figure 3, D–F). Vegfa transduction of BAT normalized the thermogenic response to acute cold exposure in obese mice (Figure 3G). Vegfa delivery to the BAT also increased glucose uptake by BAT and improved systemic insulin sensitivity in obese mice (Figure 3, H and I).
BAT-specific Vegfa delivery induces the rebrowning of the whitened BAT in dietary obesity. (A and B) Real-time PCR analysis of Vegfa and Kdr expression in BAT (A) and WAT (B) of mice after injection of ad-vegfa or control vector into BAT of mice fed NC or HFHS diets. ad-lacZ was used as a control (Con) (n = 5–8). (C) Immunofluorescent staining with Fluorescein Griffonia (Bandeiraea) Simplicifolia Lectin I (green) to detect vasculature and with Bodipy-TR (red) to detect lipid in BAT from NC- or HFHS-fed mice after the injection of ad-vegfa of the control adenoviral vector. Scale bar: 100 μm. (D) H&E staining of BAT from NC- and HFHS-fed mice prepared in A after the injection of ad-vegfa or a control adenoviral vector. Scale bar: 50 μm. (E) Quantitative analysis of the number of large lipid droplets/field in BAT under the different experimental conditions (×400, n = 4). (F) Real-time PCR analysis of the expression of ND5, Ucp1, Ndufa, and Ppargc1a in BAT of mice described in A (n = 3–10). (G) Acute CTT of the different experimental groups of mice (n = 3–7). (H) Glucose uptake by BAT was evaluated by measuring 2DG uptake (n = 4–6). (I) GTT and ITT in the different experimental groups of mice receiving ad-vegfa or control adenovirus (n = 4–8). Data were analyzed by ANOVA (A, B, and E–I). *P < 0.05; **P < 0.01. All values represent the mean ± SEM.
At this low dose of Ad-Vegfa vector, vessel leakage in BAT was not detected following the injection of FITC-dextran (Supplemental Figure 6A), and there was no increase in markers of adipose tissue inflammation when the Ad-Vegfa vector was administered to the BAT of NC-fed mice (Supplemental Figure 6B). There were no detectable increase in serum VEGF-A levels determined by ELISA (Supplemental Figure 6C) and no detectable transgene expression or changes in vessel density in WAT (Supplemental Figure 6, D–F) or liver (data not shown). Ad-vegfa delivery to BAT did not lead to morphological changes in WAT adipocyte size and had no effect on the expression profiles of proinflammatory cytokines in the WAT of mice subjected to either NC or HFHS diets (Supplemental Figure 6, G and H), consistent with the lack of detectable transgene expression in WAT. Furthermore, the adenovirus treatment did not affect WAT weight, body weight, or food intake (Supplemental Figure 6, I–K). Ad-vegfa transduction of the BAT cell line did not change the expression profiles of mitochondria (ND5, Ucp1, Ndufa, Atp5a, Ppargc1a) or mitophagy (LC3 and Bnip3) markers (data not shown), suggesting that VEGF acts on BAT through its ability to restore vascularity. Finally Vegfa delivery to BAT of WT mice fed NC led to the overexpression of VEGF-A and increased BAT vessel density (Figure 3, A and C, and Supplemental Figure 5, B and C), but in contrast to findings in mice fed HFHS, this did not affect mitochondrial marker expression (Figure 3F), thermogenic response to acute cold exposure (Figure 3G), or metabolic responses to glucose or insulin under these conditions (Figure 3I).
Ad-vegfa delivery to BAT was also assessed in adipo–_Vegfa_-KO and littermate control mice. VEGF-A rescue of the KO mice significantly increased the microvascular density of BAT (Supplemental Figure 7, A–C). Restoration of BAT vasculature promoted the “rebrowning” of BAT in the adipo–_Vegfa_-KO that was characterized by a reduction in the frequency of large lipid droplets and increased expression of ND5, Ndufa1, and Ppargc1a (Supplemental Figure 7, D and E). These changes were associated with the recovery of the thermogenic response in a cold-tolerance test and improved glucose metabolism in the lean KO mice (Supplemental Figure 7, F and G). However, delivery of Vegfa to BAT of adipo–_Vegfa_-KO mice had no detectable impact on WAT and body weight (Supplemental Figure 7, H and I) or vascularity in WAT (data not shown), suggesting that recovery of the vasculature in BAT leads to improved systemic metabolic function irrespective of the WAT phenotype.
Hypoxia promotes mitochondrial dysfunction and mitophagy in BAT. Mitochondria generate ROS during hypoxia (26, 27), contributing to mitochondrial damage that upregulates autophagic and apoptotic pathways (28). Thus, we hypothesized that capillary rarefaction in BAT would contribute to mitochondrial dysfunction and loss. Both diet-induced obesity and adipose tissue Vegfa ablation led to significantly elevated superoxide levels in mitochondria isolated from BAT, as detected by flow cytometric analysis of MitoSOX staining (Figure 4A and Supplemental Figure 8, A and B). These changes were associated with a downward shift in mitochondrial membrane potential determined by flow cytometric analysis of Mito Red staining (Figure 4B and Supplemental Figure 8, C and D). Treatment with CCCP, an uncoupler of oxidative phosphorylation, led to a near complete collapse of membrane potential in these assays (Figure 4B).
VEGF-A–mediated regulation of mitochondrial ROS production and autophagic responses in the BAT of obese mice. (A and B) FACS analysis for mitochondrial ROS (MitoSOX; A) and membrane potential (MitoRed with or without CCCP treatment; B) using isolated mitochondria extracted from BAT. (C) Western blot analysis of LC3A/B expression in BAT. The right graph indicates the quantification of LC3A/B-II expression relative to GAPDH-loading control (n = 3). (D and F) Immunofluorescent staining showing mitochondrial membrane protein Tom20 (green) colocalizing with autophagosomal membrane protein LC3 (red) in BAT of mice fed NC or HFHS diet with (F) or without (D) the injection of ad-vegfa or a control adenoviral vector (Con). Representative photomicrograph observed at ×3000 magnification. Scale bar: 3 μm. Merged areas are indicated by white arrows. The graph at right quantifies the number of puncta double stained with Tom20 and LC3 measured on 10 random fields and observed at ×3000 magnification (n = 3). (E and G) Real-time PCR expression of Bnip3 and Map1lc3b in BAT of mice (n = 3–6). (H–J) Western blot analysis of PINK1 (H), Parkin (I), and ubiquitin-conjugated protein (J) expression in isolated mitochondria extracted from BAT of mice. The graphs at right indicate the quantification relative to the expression of the Cox IV loading control (n = 3). In J, the level of ubiquitination is compared with the 25 kDa protein between the groups. Data were analyzed by 2-tailed Student’s t test (C–E and H–J) or ANOVA (F and G). *P < 0.05; **P < 0.01. All values represent the mean ± SEM.
Diet-induced obesity led to a significant increase in the expression of the autophagosomal protein LC3A/B-II in BAT (Figure 4C). Histological analysis of tissue sections revealed that LC3 formed puncta that colocalized with the mitochondrial marker Tom20 (Figure 4D). Similarly, Vegfa ablation in BAT led to the coalescence of LC3 into puncta that colocalized with mitochondria (Supplemental Figure 8E). Consistent with these observations, levels of the autophagy markers Bnip3 and Map1lc3b were increased in the BAT of dietary obese and VEGF-A–deficient mice (Figure 4E and Supplemental Figure 8F). The LC3-positive puncta phenotype was reversed by ad-vegfa delivery into BAT (Figure 4F and Supplemental Figure 8G), and this treatment also led to reductions in Bnip3 and Map1lc3b expression in both models (Figure 4G and Supplemental Figure 8H). PTEN-induced putative kinase protein 1 (PINK1) promotes mitophagy via the Parkin-dependent ubiquitination of mitochondria (29). Consistent with an increased mitophagic response in the BAT of the dietary obese model, there were significant increases in the expression of both PINK1 and Parkin, and there was a marked increase in the ubiquitination of the mitochondrial proteins (Figure 4, H–J). Collectively, these results suggest that obesity leads to hypoxic stress in BAT, promoting the activation of a mitophagic program via the PINK1-Parkin system.
To test whether hypoxia per se is sufficient to induce mitochondrial dysfunction and autophagy, BAT from WT mice was cultured ex vivo under normoxic and hypoxic conditions. Hypoxia significantly increased mitochondrial ROS production, which was associated with a reduction in mitochondrial membrane potential (Supplemental Figure 9, A and B). This condition also led to increased LC3A/B-II protein expression and elevations in Bnip3 and Map1lc3b levels, whereas the expression levels of mitochondrial marker genes were reduced (Supplemental Figure 9, C–E). The effects of hypoxia on a brown adipocyte cell line were also evaluated. Hypoxic conditions increased mitochondrial ROS and led to a reduction in mitochondrial membrane potential (Supplemental Figure 9, F–H). Hypoxia led to an upregulation of LC3A/B-II protein expression, and the LC3 signal was associated with mitochondria, indicative of an autophagic response (Supplemental Figure 9, I–K). Hypoxia also promoted Bnip3 and Map1lc3b expression (Supplemental Figure 9L). Conversely, levels of the mitochondrial markers, such as ND5, Ndufa, and Ppargc1a, were reduced under conditions of hypoxia (Supplemental Figure 9M), consistent with mitochondrial dysfunction and loss in the brown adipocyte cell line.
Hypoxic conditions during WAT expansion lead to the induction of Hif1α (13, 15, 16). However, in this context, Hif1α is not proangiogenic and its overexpression fails to induce Vegfa in adipose tissue (19, 30). In our study, dietary obesity led to an increase in Hif1α levels both in BAT and WAT, but the magnitude of the induction was much greater in BAT (Supplemental Figure 10A). Hypoxia also increased Hif1α protein levels in the brown adipocyte cell line (Supplemental Figure 10B). Hif1α is reported to induce autophagy via the induction of Bnip3 (31), but a potential role of Hif1α in mitochondrial clearance in adipose tissues has not been explored previously. Transduction of the cultured brown adipocyte line under normoxic conditions with an adenoviral vector expressing Hif1a (ad-Hif1a) did not upregulate Vegfa expression (Supplemental Figure 10, C and D), but increased Bnip3 and reduced ND5 levels (Supplemental Figure 10, E and F), which is consistent with the activation of a mitophagic response. Similarly, the injection of ad-Hif1a into the interscapular BAT of mice did not increase Vegfa levels (Supplemental Figure 10, G and H). However, delivery of ad-Hif1a to BAT increased Bnip3 expression and reduced ND5 (Supplemental Figure 10, I and J). Transduction of Hif1a into BAT also increased other Hif1a target genes involved in fibrotic processes such as elastin (Eln) and lysyl oxidase (Lox) (Supplemental Figure 10K); however, other target genes such as Glut1, hexokinase-2 (HK2) and pyruvate kinase PKM (Pkm2) showed no significant change (data not shown). Eln and Lox expression were also increased in the BAT of dietary obese mice (Supplemental Figure 10L). These results suggest that Hif1α is not proangiogenic in BAT, but that it may be involved in the induction of mitophagy or fibrotic processes triggered by hypoxia.
Hypoxia inhibits β-adrenergic signaling in BAT. β-Adrenergic signaling activates Vegfa expression in BAT via activation of cAMP-dependent PKA (32). Whereas sympathetic overactivity is generally associated with obesity (33), catecholamine sensitivity is decreased in the BAT of obese organisms (34) due at least in part to diminished β-adrenergic receptor expression (35, 36). In the model of diet-induced obesity employed in this study, significant reductions in β1- and β3-adrenergic receptor expression in BAT were observed after imposing the HFHS diet (Figure 5A). BAT hypoxia, imposed by VEGF deficiency, also led to similar reductions in β1- and β3-adrenergic receptor expression in BAT (Figure 5B). Whereas levels of the Adrb3 transcript were present at a higher copy number than Adrb1 in BAT, obesity and hypoxia led to a similar reduction in the expression of these receptor transcripts. These changes in receptor expression caused by dietary obesity or genetic Vegfa ablation led to accompanying reductions in PKA signaling (Figure 5, C and D). These data suggest that intracellular lipid droplet enlargement (whitening) is due in part to the downregulation in β-adrenergic signaling.
Hypoxia inhibits β-adrenergic signaling. (A) Relative copy number of Adrb1 and Adrb3 transcript expression in BAT under these experimental conditions (n = 4–5). (B) Relative copy number of Adrb1 and Adrb3 transcript expression in BAT of the KO and WT mice (n = 3–8). (C and D) Western blot analysis of phosphorylated PKA (pPKA Thr197) and total PKA in BAT from mice fed NC or HFHS diet or BAT from aP2-Cre+/– Vegfaflox/flox (KO) and control Vegfaflox/flox mice (WT). Right graphs indicate quantification relative to PKA (for pPKA) and GAPDH-loading control (for PKA) (n = 3). (E) Graphical illustration of the downregulation of adrenergic signaling under conditions of obesity and the proposed positive feedback loop caused by hypoxic conditions. Data were analyzed by 2-tailed Student’s t test (C and D) or ANOVA (A and B). *P < 0.05; **P < 0.01. All values represent the mean ± SEM.
To define aspects of the mechanisms by which metabolic dysfunction contributes to hypoxia-mediated BAT dysfunction, FFA levels were determined in the different experimental groups of mice. As expected, HFHS diet led to elevation in FFA levels in BAT at 8 weeks (Supplemental Figure 11A), and this increase in FFA levels in BAT was detected as early as 1 week after initiating the feeding regimen (Supplemental Figure 11B). In the brown adipocyte cell line, treatment with palmitic acid led to a reduction in VEGF-A protein and transcript expression (Supplemental Figure 11, C and D), as would be expected from its ability to downregulate PKA-targeted gene expression (37). In contrast, palmitic acid did not downregulate Adrb1 or Adrb3 expression (Supplemental Figure 11E). However, exposure to hypoxia for 24 hours was sufficient to downregulate receptor expression in the brown adipocyte cell line (Supplemental Figure 11, F and G), and this corresponded to reductions in receptor density (Supplemental Figure 11, H and I), PKA phosphorylation (Supplemental Figure 11J), and cAMP levels (Supplemental Figure 11K). Since it is established that VEGF is regulated by β-adrenergic signaling in BAT (32, 38, 39), these data suggest that multiple regulatory inputs contribute to the downregulation of VEGF in BAT under conditions of obesity (Figure 5E).