The differential role of Hif1β/Arnt and the hypoxic response in adipose function, fibrosis, and inflammation

Abstract

In obesity, adipocytes distant from vasculature become hypoxic and dysfunctional. This hypoxic response is mediated by hypoxia-inducible factors (Hif1α, Hif2α, and Hif3α) and their obligate partner, Hif1β (Arnt). We show that mice lacking Hif1β in fat (FH1βKO) are lean, exhibit reduced adipocyte size, and are protected from age- and diet-induced glucose intolerance. There is also reduced Vegf and vascular permeability in FH1βKO fat, but diet-induced inflammation and fibrosis is unchanged. Adipocytes from FH1βKO mice have reduced glucose uptake due to decreased Glut1 and Glut4, which is mirrored in 3T3-L1 adipocytes with Hif1β knockdown. Hif1β knockdown cells also fail to respond appropriately to hypoxia with reduced cellular respiration and reduced mitochondrial gene expression. Some, but not all, of these effects are reproduced by Hif1α knockdown. Thus, Hif1β/Arnt regulates glucose uptake, mitochondrial gene expression, and vascular permeability to control adipose mass and function, providing a target for obesity therapy.

Copyright © 2011 Elsevier Inc. All rights reserved.

Figures

Figure 1. aP2-Cre effectively ablates Hif1β in adipocytes

A) Expression level of Hif1β mRNA was compared using quantitative real-time PCR (qPCR) between adipocytes isolated from the subcutaneous fat (SCF) and perigonadal fat (PGF) and whole brown adipose tissue (BAT) in female control and FH1βKO mice. Mice were 6-8 weeks of age. Data are shown as mean ± SEM of five samples. Asterisks indicate p<0.05 in all panels. B) Western blot of Hif1β from isolated PGF adipocytes of control and FH1βKO mice at 12 weeks of age. Actin served as a loading control. Data are representative of four samples. C) Expression level of Hif1β mRNA by qPCR in liver, pancreas and heart of female control and FH1βKO mice. Mice were 6-8 weeks old. Data are shown as mean ± SEM of four samples. D) Expression level of Hif1β mRNA by qPCR of stromovascular fraction (SVF) isolated from subcutaneous or perigonadal in control and FH1βKO mice. Mice were 6-8 weeks of age. Data are shown as mean ± SEM of four samples. E) Expression level of Arnt2 mRNA was compared using qPCR in samples from

Figure 2. Ablation of Hif1β attenuates age and diet induced obesity

A) Body weight of female and male control and FH1βKO mice on chow diet. Data are shown as mean ± SEM of 5-6 animals/group. Asterisks indicate a significant difference (p20x. E) Median adipocyte size in adipocytes. Perigonadal and subcutaneous fat from 8 wk chow fed, 20 week chow fed, and 12 weeks of HFD (started at 6 weeks) mice (n = 4/group) were hematoxylin and eosin stained with five digital images (20x) from non-overlapping fields were taken from each slide (total 20 fields per group), and adipocyte diameters were calculated using Image J software. Values are median ± SEM of 4 animals.

Figure 3. FH1βKO adipocytes display defects in lipogenesis, glucose uptake, and glucose transporter expression

A) [14C] D-glucose incorporation into lipids in subcutaneous and perigonadal adipocytes isolated from control and FH1βKO animals at 12 weeks of age. Data are represented as 14C counts and normalized to lipid content. Data shown as mean ± SEM of 4 animals/group and are representative of two independent experiments. Asterisks in all panels indicate p<0.05, and double asterisks indicate p<0.01. B) [3H] 2-deoxyglucose uptake into subcutaneous and perigonadal adipocytes isolated from control and FH1βKO animals at 15 weeks of age. Data are represented as 3H counts and normalized to lipid content. Data are mean ± SEM of 4 animals/group. The entire experiment was repeated twice. Asterisks in all panels indicate p<0.05. C) Expression levels of Glut1 and Glut4 mRNA were compared using qPCR between subcutaneous and perigonadal in male control and FH1βKO mice. Mice were 6-8 weeks old. Data are mean ± SEM of four samples.

Figure 4. shRNA knockdown of Hif1β decreases glucose uptake

A) Expression level of Hif1β and Arnt2 mRNA was compared using qPCR between 3T3-L1 preadipocytes stably transfected with shHif1β-1 and shHif1β-2. Data are normalized by relative expression of Hif1β and Arnt2 mRNA in shGFP cells and are shown as mean ± SEM of triplicate samples, repeated five times. Asterisks indicate p<0.05 in all panels. B) Western blot of Hif1β from protein extracts from 3T3-L1 preadipocytes stably transfected with shHif1β-1, shHif1β-2, and shGFP cells. Actin was used as a loading control. C) Quantification of Oil Red-O staining in shHif1β-1, shHif1β-2, and shGFP adipocytes. Data are shown as mean ± SEM of triplicate samples repeated twice. Asterisks indicate a significant difference (p3H-labeled 2-deoxyglucose into shHif1β-1, shHif1β-2, and shGFP stably transfected adipocytes. Data are represented as 3H count normalized to protein content, and are shown as mean ± SEM of triplicate samples repeated three times. E) Expression of Glut1 and Glut4 as measured by qPCR. In cells with stable Knockdown of Hif1β-1, Hif1β-2 and shGFP. After eight days of differentiation, cells were subjected to normoxia, hypoxia (0.1% O2 for 16hrs), or 200 nM CoCl2 for 16 hrs. Data shown as mean ± SEM of triplicate samples and repeated three times. F) Western blot of Glut1 and Glut4 from protein extracts from shHif1β-2 and shGFP 3T3-L1 adipocytes after eight days of differentiation. Actin was used as a loading control.

Figure 5. Hif1β ablation decreases vascular permeability and Vegf expression

A) Left panels: CD31 immunohistochemistry on whole mounts of subcutaneous fat from control and FH1βKO male mice at 8 weeks of age. The photograph was taken at 100x. Right panels: CD31 immunohistochemistry on whole mounts of subcutaneous fat from control and FH1βKO male mice after 12 weeks of exposure to high fat diet. B) Quantitation of capillary density in the perigonadal and subcutaneous fat pads after 12 weeks on HFD, starting at 6 weeks of age (n = 4/group). Slides were stained for GSL I - isolectin B4. Capillaries were counted from three digital images (20×) from non-overlapping fields were taken from each slide (total 12 fields per group) from control and FH1βKO males. Data are shown as mean ± SEM of three samples. Asterisks indicate pVegf mRNA were compared using qPCR between adipocytes isolated from the subcutaneous fat (SCF) and perigonadal fat (PGF). Mice were 6-8 weeks old. Data are shown as mean ± SEM of five samples. Asterisks indicate p<0.05. Western blot of Vegf from isolated PGF adipocytes of female control and FH1βKO− mice at 12 weeks of age. Western blot for actin is a loading control. Data are representative of four samples. E) mRNA was isolated from shHif1β-1, shHif1β-2, and shGFP stably transfected adipocytes after eight days of differentiation, and subjected to normoxia, hypoxia (0.1% O2 for 16hrs), or 200 nM CoCl2 for 16 hrs. Expression of Vegf was measured by qPCR. Data shown as mean ± SEM of triplicate samples and repeated three times. F) Western blot of Vegf from protein extracts from 3T3-L1 adipocytes stably transfected with shHif1β-1, shHif1β-2, and shGFP adipocytes, or media secreted by shHif1β-1, shHif1β-2, and shGFP adipocytes after eight days of differentiation. Actin was used as a loading control.

Figure 6. Hif1 β shRNA knockdown increases respiratory capacity during CoCl2 treatment

A) Bioenergetics profile, as measured by oxygen consumption rate with a Seahorse X24 extracellular flux analyzer, of shHif1β-1, shHif1β-2, and shGFP 3T3-L1 adipocytes under normoxic conditions. B) Bioenergetics profile, as measured by oxygen consumption rate with a Seahorse X24 extracellular flux analyzer, of shHif1β-1, shHif1β-2, and shGFP 3T3-L1 adipocytes after pre-treatment with CoCl2. C) Basal respiration and maximal respiratory capacity of shHif1β-1, shHif1β-2, and shGFP 3T3-L1 adipocytes was determined by calculating the area under the curve (AUC) in the basal phase and after uncoupling with FCCP. Values are means ± SEM of 6-7 replicates of three separate experiments. Asterisks indicate a significant difference compared to shGFP control in all panels (p<0.05). D) mRNA was isolated from shHif1β-1, shHif1β-2, and shGFP stably transfected adipocytes after eight days of differentiation, and subjected to normoxia, hypoxia (0.1% O2 for 16hrs), or 200 nM CoCl2 for 16 hrs. Expression of Cytc1 and Cox4.2 was measured by qPCR. Data shown as mean ± SEM of triplicate samples and repeated three times.

Figure 7. Ablation of Hif1α recapitulates a subset of the effects of Hif1β ablation

A) Triglyceride accumulation after 8 days of differentiation was visualized by Oil Red-O staining in shHif1α, shHif2α, shAhr, and shScr (control) adipocytes after 8 days of differentiation. B) 3H-labeled 2-deoxyglucose uptake into shHif1α, shHif2α, shAhr, and shScr stably transfected adipocytes after eight days of differentiation. Data are represented as 3H counts normalized to protein content. Data shown as mean ± SEM of four samples and repeated twice. Values are means ± SEM, and asterisks indicate a significant difference compared to shSCR control with the same treatment in all panels (p<0.05). C) mRNA was isolated from shHif1α, shHif2α, shAhr, and shScr stably transfected adipocytes after eight days of differentiation, and subjected to normoxia, hypoxia (0.1% O2 for 16h), or 200 nM CoCl2 for 16 hrs. Expression of Glut1, Glut4, Vegf, Cytc1 and Cox4.2 was measured by qPCR. Data shown as mean ± SEM of four samples and repeated twice.