Selective inhibition of hypoxia-inducible factor 1α ameliorates adipose tissue dysfunction

Abstract

Hypoxia-inducible factor 1α (HIF1α) induction in adipocytes is a critical component of the "fibrotic response," directly linked to metabolic dysfunction in adipose tissues under hypoxic conditions. We reasoned that inhibition of HIF1α may ameliorate the negative aspects of the obesity-associated fat pad expansion. We used the selective HIF1α inhibitor PX-478, whose effectiveness has previously been established in tumor models. We demonstrate that PX-478 treatment effectively suppresses the high-fat-diet (HFD)-induced HIF1α activation in adipose tissue. HIF1α inhibition causes a reduction of weight gain in mice on an HFD but not on a chow diet. Treatment increases energy expenditure and prompts resistance to HFD-mediated deterioration of metabolic parameters. Moreover, PX-478-treated mice have reduced fibrosis and fewer inflammatory infiltrates in their adipose tissues. We confirm the metabolic effects obtained with PX-478 treatment using an adipose tissue-specific, doxycycline-inducible dominant negative HIF1α mutant (dn-HIF1α). Consistent with the pharmacological results, genetic inhibition of endogenous HIF1α activity prompts similar metabolic improvements in HFD-fed mice. Collectively, our results demonstrate that HIF1α inhibition in the adipocyte leads to significant metabolic improvements, suggesting that selective HIF1α inhibition in adipose tissue may be an effective therapeutic avenue in the context of metabolic dysfunction.

Fig 1

Detection of hypoxia, HIF1α induction in EWAT of HFD-fed mice, and PX-478-mediated suppression of the HFD-induced HIF1α. (A) (Top) To monitor the adipose tissue oxygen levels, the animal was under isoflurane inhalation anesthesia (2%) and then placed with the EWAT exposed to the loop of a surface-coil resonator. Sonicated oxygen-sensing microcrystals of LiNc-BuO were then implanted in the fat tissue for oxygen detection. The probe particulates are black, indicated by an arrow. (Middle) H&E staining of EWAT. The arrow indicates the EPR probes (tiny black crystals) in fat tissue. (Bottom) Trichrome staining of the location where the probe particulates were implanted in EWAT. Note the absence of fibrosis around the particles. (B) Baseline body weight (before starting the HFD feeding) and body weight after HFD feeding for 10 weeks (n = 6). C57BL/6 wild-type mice at 13 to 14 weeks of age were used in this study. The statistical significance was assessed by a Student t test. **, P < 0.001. (C) pO₂ measurements for the mice before and after HFD feeding by EPR spectrometer in the EWAT. The peak-to-peak line width was used to calculate the pO₂ using the standard calibration curve. *, P < 0.05. (D) Immunohistochemical staining by anti-HIF1α in EWAT of mice fed regular chow or an HFD. The arrows in the bottom image indicate upregulated HIF1α induced by the HFD. (E) Western blot analysis for HIF1α in EWAT and BAT of mice after 3-week feeding with chow, an HFD, and an HFD plus PX-478. Five mouse tissue samples were pooled for each well. The bottom portion shows quantitative measurements of the band density by ImageJ software from the NIH. (F) qPCR analysis of HIF1α in EWAT (n = 5 for each group). The readings are normalized by HPRT. **, P < 0.001. (G) qPCR analysis of HIF1α direct target genes in EWAT. *, P < 0.05.

Fig 2

PX-478-treated mice gain less body weight, have smaller fat pads and smaller adipocytes, and exhibit reduced food intake under HFD challenge. (A) Body weight measurements in PX-478-treated and littermate control mice fed with regular chow (CHOW) or an HFD (n = 5 for each group). PX-478 treatment was started after HFD feeding for 11 days. *, P < 0.05; **, P < 0.001. (B) Comparison of sizes of different fat pads (EWAT, SWAT, and BAT) in PX-478-treated or control mice. (C) H&E staining of EWAT and BAT of mice fed regular chow, an HFD, and an HFD plus PX-478. The bars represent 50 μm. (D) Quantitative measurements of adipocyte sizes in EWAT of mice from the indicated groups. **, P < 0.001. (E) Accumulated food intake per day in PX-478-treated and littermate control mice (n = 5 for each group).

Fig 3

PX-478-treated mice exhibit improved glucose tolerance, increased energy expenditure, ameliorated circulating leptin levels, increased peripheral use of lipid, and hence decreased HFD-induced hepatic steatosis. (A) Circulating glucose levels measured during an OGTT in PX-478-treated or littermate control mice 6 weeks after HFD feeding (n = 5 for each group). The difference at each time point was determined by a Student t test. *, P < 0.05. (B) Circulating glucose levels in mice fed chow, an HFD, and an HFD plus PX-478 12 h after fasting. *, P < 0.05; **, P < 0.001 (n = 5 for each group). (C) Circulating glucose levels measured during an ITT in PX-478-treated mice or the littermate controls 2 weeks after PX-478 treatment. (D) Indirect calorimetry was performed in a TSE system by housing PX-478-treated mice or littermate controls after PX-478 treatment for 2 weeks. The mice were acclimated for 1 week in the metabolic chambers before the measurements were started. VO₂ and RER (VCO₂/VO₂) were analyzed in light (day) or dark (night) cycles. *, P < 0.05. The total calories in excreta were measured by collecting the stools from each cage for 3 days. (E) Serum cholesterol, triglyceride, and NEFA levels in mice fed chow, an HFD, and an HFD plus PX-478 (n = 5 for each group) after fasting for 12 h. *, P < 0.05; **, P < 0.001. (F) qPCR analysis for UCP-1 and PGC-1 in mice fed an HFD and an HFD plus PX-478 (n = 5 for each group). *, P < 0.05; **, P < 0.001. (G) H&E staining of the liver tissues from mice fed an HFD and an HFD plus PX-478. The bars represent 100 μm. (H) Leptin mRNA levels in EWAT (left) and circulating leptin (right) levels in mice fed chow, an HFD, and an HFD plus PX-478 (n = 5 for each group). **, P < 0.001.

Fig 4

PX-478 treatment suppresses fibrosis and inflammation in EWAT induced by an HFD. (A) qPCR analysis for collagens (I and III) and their fiber linker enzyme LOX in mice fed chow, an HFD, and an HFD plus PX-478 (n = 5 for each group). **, P < 0.001. (B) Masson's trichrome staining in EWAT of mice fed chow, an HFD, and an HFD plus PX-478. (C) qPCR for IL-6 in EWAT of mice fed chow, an HFD, and an HFD plus PX-478 (n = 5 for each group). **, P < 0.001. (D) Immunohistochemical staining of F4/80 in EWAT in mice fed chow, an HFD, and an HFD plus PX-478. The arrows indicate the crown-like structures formed by macrophage aggregation in HFD-fed mice. (E) Quantitative measurements of the numbers of crown-like structures in mice fed chow, an HFD, and an HFD plus PX-478 (n = 5 for each group). **, P < 0.001. Bars, 50 μm.

Fig 5

dn-HIF1α induction exclusively in adipose tissue blocks its direct target genes in WAT. (A) Schematic representative of a mouse model for DOX-inducible adipose tissue-specific overexpression of dn-HIF1α. (B) qPCR analysis of dn-HIF1α overexpression in different fat pads (EWAT, SWAT, and BAT) in the double transgenic mice and their littermate controls 3 days after treatment with chow plus DOX. (C) qPCR analysis of direct HIF1α target genes in EWAT of the double transgenic mice and their littermate controls 3 days after treatment with chow plus DOX.

Fig 6

Overexpression of dn-HIF1α locally in adipose tissue ameliorates development of obesity, increases energy expenditure, and improves lipid metabolism under an HFD challenge. (A) Body weight gain in dn-HIF1α and their littermate controls during treatment with an HFD plus 600 mg/kg of DOX for 8 weeks (n = 5 for each groups). *, P < 0.05. (B) Circulating glucose levels during an OGTT and an ITT in dn-HIF1α and their littermate controls after treatment with an HFD plus DOX for 5 weeks (n = 5 for each group). *, P < 0.05. (C) H&E staining for EWAT and BAT of dn-HIF1α mice and their littermate controls 8 weeks after treatment with an HFD plus DOX. The bars represent 50 μm (left). The graph on the right shows quantification of the fat cells in EWAT in control and dnHIF1 transgenic groups. (D) Indirect calorimetry was performed in a TSE system for dn-HIF1α transgenic mice and their littermate controls after feeding with an HFD plus DOX for 6 weeks. VO₂, RER (VCO₂/VO₂), and core body heat were analyzed as the average results during a 24-h light-dark cycle since there are no differences between the light and dark cycles (n = 5 for each group). *, P < 0.05.

Fig 7

Overexpression of dn-HIF1α locally in adipose tissue increases peripheral use of lipid, decreases HFD-induced hepatic steatosis, and regulates circulating leptin levels. (A) Liver triglyceride (left) and cholesterol (right) in dn-HIF1α transgenic mice and their littermate controls after treatment with an HFD plus DOX for 8 weeks (n = 5 for each group). *, P < 0.05. (B) H&E staining for liver tissues in dn-HIF1α transgenic mice and their littermate controls. The bars represent 50 μm. (C) qPCR analysis of leptin in EWAT (left) and circulating leptin levels measured by ELISA (right) in dn-HIF1α transgenic mice and their littermate control mice after treatment with an HFD plus DOX for 8 weeks (n = 5 for each group). *, P < 0.05.

Fig 8

Overexpression of dn-HIF1α locally in adipose tissue suppresses fibrosis and inflammation induced by an HFD. (A) qPCR analysis for collagens (I, II, and VI) and LOX in dn-HIF1α transgenic mice and their littermate controls (n = 5 for each group). *, P < 0.05. (B) Masson's trichrome stain for EWAT of dn-HIF1α transgenic mice (left) and their littermate controls (right). The bars indicate 100 μm. (C) qPCR analysis for F4/80 and SAA3 for EWAT in dn-HIF1α transgenic mice and their littermate controls (n = 5 for each group, upper graphs) and circulating SAA3 levels in dn-HIF1α mice and their littermate controls (n = 5 for each group, lower graph). *, P < 0.05.