MCB Accepted Manuscript Posted Online 16 November 2015 Mol. Cell. Biol. doi:10.1128/MCB.00430-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
1
ADIPOCYTE-SPECIFIC
HIF2α
DEFICIENCY
EXACERBATES
OBESITY-
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INDUCED BROWN ADIPOSE TISSUE DYSFUNCTION AND METABOLIC
3
DYSREGULATION
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Running title: Garcia-Martin, Adipocyte HIF2α regulates WAT and BAT angiogenesis.
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Rubén García-Martín1,#, Vasileia I. Alexaki1, Nan Qin2, María F. Rubín de Celis3, Matina
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Economopoulou4, Athanasios Ziogas1, Bettina Gercken1, Klara Kotlabova1, Julia Phieler1,
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Monika Ehrhart-Bornstein3, Stefan R. Bornstein3, Graeme Eisenhofer2, Georg Breier5,
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Matthias Blüher8, Jochen Hampe6, Ali El-Armouche7, Antonios Chatzigeorgiou1,2,10,
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Kyoung-Jin Chung1 and Triantafyllos Chavakis1,2,9,10,#
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Department of Clinical Pathobiochemistry; 2Institute of Clinical Chemistry and Laboratory
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Medicine; 3Department of Medicine III; 4Department of Ophthalmology; 5Department of
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Psychiatry; 6Department of Medicine I; 7Department of Phamacology and Toxicology;
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Department of Endocrinology and Nephrology, University of Leipzig, Germany
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Center for Regenerative Therapies Dresden, Dresden Germany
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Medical Faculty, Technische Universität Dresden, Dresden, Germany
Paul-Langerhans Institute Dresden, Dresden Germany
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#Correspondence:
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[email protected];
[email protected]
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Word count for Material and Methods section: 1756
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Word count for Introduction, Results and Discussion: 4616
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ABSTRACT
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Angiogenesis is a central regulator for white (WAT) and brown adipose tissue (BAT)
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adaptation in the course of obesity. Here we show that deletion of hypoxia-inducible factor
28
2α (HIF2α) in adipocytes (by using Fabp4-Cre transgenic mice), but not in myeloid or
29
endothelial cells, negatively impacted on WAT angiogenesis and promoted WAT
30
inflammation, WAT dysfunction, hepatosteatosis and systemic insulin resistance in obesity.
31
Importantly, adipocyte HIF2α regulated vascular endothelial growth factor (VEGF)
32
expression and angiogenesis of the obese BAT as well as its thermogenic function.
33
Consistently, obese adipocyte-specific HIF2α-deficient mice displayed BAT dysregulation,
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associated with reduced levels of uncoupling protein-1 (UCP1) and a dysfunctional
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thermogenic response to cold exposure. VEGF administration reversed WAT and BAT
36
inflammation and BAT dysfunction in adipocyte HIF2α-deficient mice. Together,
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adipocyte HIF2α is protective against maladaptation to obesity and metabolic dysregulation
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by promoting angiogenesis in both WAT and BAT and by counteracting obesity-mediated
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BAT dysfunction.
40 41 42
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INTRODUCTION
44
The hypoxia response is mediated by the heterodimeric hypoxia-inducible factors (HIFs)
45
comprising an α-subunit (HIF1α or HIF2α) that is regulated by oxygen and an oxygen-
46
insensitive β-subunit. Hypoxia prevents hydroxylation of HIFα subunits by prolyl
47
hydroxylases (PHDs), thereby leading to inhibition of degradation of the HIFα subunits,
48
their stabilization and the subsequent HIF-dependent upregulation of genes essential for
49
cellular adaptation to and cell survival under hypoxic conditions (reviewed in (1)).
50 51
In obesity, excessive lipid storage and adipocyte hypertrophy are thought to result in
52
hypoxia in white adipose tissue (WAT) and brown adipose tissue (BAT) (2-6), although
53
there is some controversy concerning oxygen levels in obese AT (7). The presence of
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hypoxia in WAT may be associated with increased inflammation (2, 4, 5); hypoxia also
55
triggers an angiogenic response, including upregulation of the major angiogenic factor,
56
vascular endothelial growth factor (VEGF) in adipocytes (4). The angiogenic response is
57
crucial for the adaptation of the WAT in obesity. Previous studies have demonstrated that
58
defective angiogenesis in obese WAT may promote insulin resistance, inflammation and
59
adipocyte apoptosis (3, 8, 9). On the other hand, mice with adipocyte overexpression of
60
VEGF family member A (VEGF-A) are protected against the adverse effects of high-fat
61
diet (HFD) (8, 10, 11). In obese WAT, enhanced expression of both HIF1α and HIF2α is
62
observed (2, 6, 12). Previous studies have functionally implicated HIF1α (6, 12-14) and
63
different PHDs (15, 16) in the process of obesity. In contrast, less is known about the role
64
of HIF2α. HIF2α heterozygous-null mice were recently shown to have reduced insulin
65
sensitivity and enhanced AT inflammation upon HFD (17) and HIF2α was found to
3
66
regulate lipid metabolism in hepatocytes (18-20), whereas less data exist on the specific
67
role of HIF2α in adipocytes (6). This is particularly important given several findings
68
demonstrating that regulation of VEGF-A and angiogenesis in WAT is not dependent on
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HIF1α. Whereas adipocytes lacking HIF1β (the common and obligate partner for HIF1α
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and HIF2α) showed reduced VEGF expression (21), transgenic mice overexpressing
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HIF1α in adipocytes did not show any upregulation in VEGF-A expression or other
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proangiogenic factors (12). In addition, mice lacking adipocyte HIF1α showed no vascular
73
alterations, as compared to HIF1α-proficient mice (6). These observations point to a
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potential role of HIF2α in the WAT for the adaptive response to obesity that remains to be
75
established.
76 77
BAT is also a highly vascularized tissue; the grade of vascularization determines its ability
78
for lipid consumption and its thermogenic function (3, 22). Increased BAT activity results
79
in improved insulin sensitivity and glucose homeostasis (reviewed in (23)). Norepinephrine
80
derived from sympathetic nerves is a central player in inducing expression of both VEGF
81
and the major thermogenic factor, uncoupling protein-1 (UCP1) (24). Catecholamine-
82
mediated induction of UCP1 requires a signaling cascade involving cAMP, protein kinase
83
A and PGC1α (reviewed in (23)). VEGF resulting from beta adrenergic receptor
84
stimulation promotes BAT angiogenesis and functionality (3, 8, 10, 11, 25-27).
85
Interestingly, UCP1-deficient mice display angiogenesis despite the absence of hypoxia in
86
their BAT (25, 28, 29); thus, hypoxia in BAT is not an absolute prerequisite for stimulation
87
of angiogenesis. Hypoxia may collaborate with norepinephrine in upregulating VEGF
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expression in brown adipocytes (24), whereas activation of HIF1α in these cells may occur
4
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even without hypoxia (28). However, HIF1α does not regulate expression of VEGF (3) or
90
UCP1 (6) in the BAT. On the other hand, although HIF2α expression is induced by cold
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exposure (25), its potential role in the adaptive response of the BAT to obesity and cold
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exposure, has not been addressed thus far.
93 94
To address the aforementioned questions pertinent to the role of HIF2α in both WAT and
95
BAT, we generated mice with adipocyte-specific HIF2α deletion. We found that lack of
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HIF2α in adipocytes resulted in systemic insulin resistance associated with reduced
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vascularization and a pro-inflammatory phenotype in both WAT and BAT in the course of
98
obesity. In contrast, myeloid or endothelial HIF2α did not affect obesity-related metabolic
99
dysregulation. In addition to reduced angiogenesis in the BAT, adipocyte HIF2α deficiency
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was associated with reduced expression of the major thermogenic factor UCP1 in the obese
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BAT. Treatment with VEGF reversed WAT and BAT inflammation and BAT dysfunction
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in obese mice lacking adipocyte HIF2α, suggesting that the metabolic dysregulation
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observed in adipocyte HIF2α deficiency, was, at least in part, mediated by diminished
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VEGF production. Thus, adipocyte HIF2α was identified as a factor contributing to the
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metabolic adaptation to diet-induced obesity in both WAT and BAT.
106 107 108
MATERIALS AND METHODS
109
Mice
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Mice carrying a floxed HIF2α (Epas1) allele (Jackson Laboratories, Bar Harbor, ME)
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(HIF2αfl/fl) were bred with mice carrying Cre recombinase under control of Fabp4 promoter
5
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(Jackson Laboratories) to generate adipocyte-specific HIF2α KO mice (AdHIF2KO)
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(Fabp4-Cre+ Epas1fl/fl). Similarly, Epas1fl/fl mice were crossed with LysM-Cre mice (30)
114
(Jackson Laboratories) to generate myeloid-specific HIF2α KO mice (MyeHIF2KO)
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(LysM-Cre+ Epas1fl/fl). Fabp4-Cre- Epas1fl/fl and LysM-Cre- Epas1fl/fl littermates were used
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as controls. For the study of endothelial HIF2α, we engaged a tamoxifen-mediated
117
inducible deletion by using CreERT, whose expression is driven by stem-cell leukemia
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promoter (Scl)-5’, which is specifically expressed in endothelial cells (31, 32). Scl-
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CreERT+ Epas1fl/fl (EndHIF2KO) and Scl-CreERT- Epas1fl/fl littermate control mice
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received i.p. tamoxifen (Sigma-Aldrich, Munich, Germany) (2 mg/mouse/day) at the age of
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8 weeks. Eight-ten week-old male mice were fed a normal diet (ND) or high-fat diet (HFD)
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with 10% Kcal from fat or 60% Kcal from fat, respectively (Research Diets, New
123
Brunswick, NJ) and feedings were conducted for up to 24 weeks. Animal experiments were
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approved by the Landesdirektion Sachsen, Germany.
125 126
In vivo metabolic analyses
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For glucose tolerance test, mice were fasted overnight before intraperitoneal injection of D-
128
(+)-glucose (Sigma-Aldrich, Munich, Germany) (1g/Kg body weight). At the indicated
129
times, blood was collected via tail vein for measuring glucose levels with an Accu-Chek
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glucose meter (Roche, Manheim, Germany). For insulin tolerance test, mice were fasted 6
131
hours before intraperitoneal injection of insulin (Lilly, Bad Homburg, Germany) (1 U/Kg
132
body weight) and blood glucose levels were measured at the indicated times. For glucose-
133
stimulated insulin secretion, mice were fasted overnight before intraperitoneal injection of
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D-(+)-glucose (Sigma-Aldrich, Munich, Germany) (1g/Kg body weight). At the indicated
6
135
times, blood was collected via tail vein for measuring plasma insulin with an ELISA kit
136
(Chrystal Chem, Cologne, Germany). Blood triglycerides and cholesterol were determined
137
using an Accutrend Plus system (Roche). For analysis of fasted plasma samples, mice were
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fasted overnight (16-18 hours), blood was collected via tail vein and plasma leptin,
139
adiponectin, insulin and FGF21 were determined using ELISA kits (R&D system,
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Wiesbaden-Nordenstadt, Germany; Chrystal Chem) following manufacturer’s instructions.
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For the lipid tolerance test, mice were fasted overnight before oral gavage of olive oil
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(Sigma-Aldrich) (5 μL/g of body weight) and blood triglycerides were determined as
143
described above. For the determination of free fatty acids, serum from 16-18 h fasted mice
144
was collected and a free fatty acid fluorometric assay kit was used (Cayman Chemical, Ann
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Arbor, MI). For in vivo insulin signalling pathway analysis, mice were fasted for 6 hours
146
before intraperitoneal injection of insulin (Lilly) (5 U/Kg); 8 min thereafter mice were
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euthanized and tissues were harvested and snap frozen for further analysis. Lean and fat
148
mass were measured in mice fed a HFD for 22 weeks by using computed tomography (CT)
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(33) (Skyscan 1178, Bruker, Rheinstetten, Germany). For cold exposure experiments, obese
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mice fed for at least 19 weeks with HFD were placed at 4ºC overnight. Body temperature
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was measured at the indicated times with a thermometer (Bioseb, Vitrolles, France).
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Thereafter, mice were sacrificed and tissues isolated and processed at 4ºC.
153 154
VEGF administration
155
Controlled administration of VEGF was achieved via subcutaneous implantation of mini-
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osmotic pumps (Alzet, CA, USA). Pumps were filled with either recombinant murine
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VEGF (Peprotech, Hamburg, Germany) diluted in PBS with 0.1% BSA or with PBS with
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0.1% BSA as control. Delivery rate was set at 75 ng/hour. 5-week old mice were fed a HFD 7
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for 5 weeks before pump implantation; after pump implantation, mice were fed for
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additional 3 weeks prior to euthanasia and further analysis.
161 162
Protein detection
163
Adipose tissues (AT) were excised after euthanasia and proteins isolated as described
164
elsewhere (34). Briefly, AT were homogenized and digested in RIPA lysis buffer [1%
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TritonX-100; 0.5% sodium deoxycholate; 0.1% SDS; 50 mM Tris-HCL, pH 7.5; 150 mM
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NaCL; Mini Protease Inhibitor and Phosphatase Inhibitor Cocktail Tablet (Roche)],
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incubated on ice for 20 minutes and centrifuged to remove cellular debris and fat. Protein
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concentration was determined using BCA Protein Assay Kit (Thermo Scientific, Schwerte,
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Germany). Antibodies against UCP1 (Abcam, Cambridge, U.K.), phospho-Ser473 Akt,
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total Akt (Cell Signaling/New England Biolabs, Frankfurt am Main, Germany) and tubulin
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(Sigma-Aldrich) were used for immunoblotting. For blot quantification, densitometry was
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performed with ImageJ software (National Institute of Health, Bethesda, MD, USA);
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tubulin was used for UCP1 normalization, whereas total Akt was used for phospho-Ser473
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Akt normalization.
175 176
VEGF-A was determined in WAT and BAT lysates using an ELISA kit (R&D system).
177 178
Gene expression
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Total RNA from tissues or cells was isolated using TRIzol (Invitrogen, Darmstadt,
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Germany) following manufacturer’s instructions. After purification with DNase I treatment
181
(Thermo Scientific), 1 µg of RNA was reverse-transcribed using an iScript cDNA
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Synthesis kit (Bio-Rad, Munich, Germany), and real time PCR was performed with SsoFast 8
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EvaGreen Supermix (Bio-Rad) using a Bio-Rad CFX384 Touch Real Time PCR Detection
184
System (Bio-Rad). Calculation was based on ΔΔCt method (35) and normalization was
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performed to 18s RNA.
186 187
Ex vivo lipolysis assay in adipose tissue explants
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Subcutaneous and gonadal fat depots (scWAT and gonWAT, respectively) were surgically
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removed from 24-week HFD-fed control and AdHIF2KO mice and washed with ice-cold
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PBS. A piece of 100 mg was incubated for 2 hours at 37ºC in 250 μL DMEM containing
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2% fatty acid-free BSA (Sigma-Aldrich) and in the presence or absence of 10 μM
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isoprenaline (Sigma-Aldrich). Fatty acids released to the medium were quantified using a
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free fatty acid fluorometric assay kit (Cayman Chemical).
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Cell culture
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For the isolation of bone-marrow derived macrophages (BMDM), we followed our
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previously published protocol (36).
198 199
Histology
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Fresh tissues were excised, fixed in 4% paraformaldehyde, and paraffin embedded.
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Sections were stained with H&E. Histological scoring for liver NAFLD/NASH was read
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blinded to the experimental design using H&E staining. The degree of steatosis, ballooning
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and lobular inflammation was evaluated according to previously published criteria
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following the NASH-CRN Committee scoring system (37, 38). The NAFLD Activity Score
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(NAS) consists of the sum of steatosis, ballooning and lobular inflammation. A value of
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NAS>5 correlates with the presence of NASH (38). For immunohistochemistry, sections
207
were de-paraffinized and antigen retrieval was done by incubation with hot citrate buffer.
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Inhibition of intrinsic peroxidase activity was performed with 0.5 % H2O2 treatment. In
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addition, F4/80 staining required proteinase K (Sigma-Aldrich) treatment at 37°C, whereas
210
for UCP1 staining, sections were also incubated with pronase (Sigma-Aldrich) at 37°C.
211
Sections were permeabilized with 0.4% TritonX100 and blocked with serum from ABC kit
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(Vector Laboratories, Peterborough, U.K.). Sections were then incubated overnight with
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antibodies against UCP1 (Abcam), or against F4/80 (Novus Biologicals, Herford,
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Germany). The avidin–biotin complex was detected with an AEC Peroxidase Substrate Kit
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(Vector Laboratories). F4/80 quantification was performed by counting the number of
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F4/80 positive cells per adipocyte per field in at least 6 random low-magnification fields
217
per sample. TUNEL staining was performed using a Dead End Colorimetric TUNEL kit
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following manufacturer’s instructions (Promega, Mannheim, Germany). TUNEL
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quantification was performed by counting the number of TUNEL positive cells per
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adipocyte per field in at least 6 random low-magnification fields per sample. Pictures were
221
visualized by a computerized microscope (Zeiss, Oberkochen, Germany) and analysed with
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AxioVision Rel 4.8 software (Zeiss). For Oil Red O staining, 10 μm cryosections were
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prepared. Slides were fixed in 4% paraformaldehyde for 1 hour, rinsed in dH2O, and stained
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for 15 min in Oil Red O in 60% isopropanol. They were rinsed with 60% isopropanol and
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counterstained with Mayer’s hematoxylin. Quantification was performed as described (39).
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Fibrosis staining was performed by using a Masson trichrome staining kit (Sigma-Aldrich)
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following manufacturer's instructions. For hypoxia staining, mice were intraperitoneally
228
injected with 60 mg/kg of CCl-103F (Hypoxyprobe F6, Hypoxyprobe, Burlington, MA)
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and sacrificed 90 min after the injection. Tissues were fixed and processed as described 10
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above using Hypoxyprobe F6 kit (Hypoxyprobe) following manufacturer´s intructions;
231
quantification was performed by determining the percentage of the area staining positive
232
for hypoxia in 5 random low-magnification fields per sample. AT whole mount staining
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was performed following a previously described method (40) with some modifications.
234
Briefly, WAT and BAT were fixed with paraformaldehyde and cut in small pieces of 2 mm
235
x 2 mm. After blocking and permeabilization with BSA (1%) and Triton-X100 (0.5%),
236
mounts were stained with FITC-conjugated isolectin B4 (Sigma-Aldrich) at 4ºC. After
237
several washings with PBST, samples were visualized using a confocal microscope (Leica
238
TCS SP5, Leica, Wetzlar, Germany). Z-stacks of 5 µm depth were analysed and
239
quantification of fluorescence intensity was performed using LAS software (Leica).
240 241
Measurement of liver triglyceride content
242
Triglyceride content quantification in the liver was done using a commercially available kit
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(Abcam). Briefly, 100 mg of liver tissue was homogenized in 1 ml of 5% Triton-X100. The
244
samples were then heated to 95ºC and cooled to room temperature twice. Thereafter,
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samples were centrifuged, and triglyceride content in the supernatant was quantified using
246
enzymatic determination.
247 248
Flow cytometry
249
After sacrificing mice, scWAT and gonWAT were excised and the lymph nodes, immersed
250
in the fat depot, were removed. AT was then digested using collagenase type I (2 mg/ml per
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mg of tissue; Life Technologies) for 60 min at 37ºC. The suspension was resuspended in
252
DMEM containing 0.5% fatty acid–poor BSA (Sigma-Aldrich) and centrifuged to separate
253
the floating adipocyte fraction from the pelleted stromal vascular fraction (SVF). For FACS 11
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analysis, the following antibodies were used: Fc receptor–blocking Ab 2.4G2, CD45–Alexa
255
Fluor 488 (Biolegend, Fell, Germany); CD31-APC (eBioscience, Frankfurt, Germany).
256
FACS was carried out on a FACSCanto II (BD, Heidelberg, Germany) and analyzed with
257
FACSDiva Version 6.1.3 software.
258
Sorting of lung endothelial cells from endothelial-specific HIF2α KO mice was
259
performed as previously described (32). The solution was filtered and stained for CD31
260
(eBioscience) and sorting for CD31-positive and CD31-negative cells was performed using
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a sorter FACS Aria II (BD).
262 263
Metabolic cage analysis
264
Metabolic cage analysis was done with mice that were fed with HFD for 6 weeks. Mice
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were individually housed in metabolic cages (PhenoMaster, TSE Systems, Bad Homburg,
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Germany) with free access to water and food, maintaining a 12h:12h light-dark cycle. A
267
period of at least 16 hours acclimatization in the metabolic cages preceded initiation of the
268
experiment and data collection. Volume of oxygen consumption (VO2) and carbon dioxide
269
production (VCO2) were determined every 20 minutes. Respiratory exchange ratio (RER)
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was calculated as VCO2/VO2. Energy expenditure (EE) was calculated as 3.941xVO2 +
271
1.106xVCO2 (41). Food intake was also monitored. Data were normalized with respect to
272
body weight using ANCOVA analysis.
273 274
Statistical analyses
275
Data are expressed as mean ± s.e.m. and statistically analyzed by Student’s t-test, or Mann-
276
Whitney U as appropriate. Body temperature during cold exposure experiment was
277
analyzed by ANOVA. Significance was set at P < 0.05. 12
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RESULTS
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Adipocyte HIF2α deficiency promotes HFD-induced metabolic dysregulation.
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Mice with adipocyte-specific deletion of HIF2α were generated by crossing mice with a
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floxed Epas1 (HIF2α) allele with mice expressing Cre recombinase under the control of the
282
Fabp4 promoter. This resulted in Fabp4Cre+ Epas1fl/fl mice, in which HIF2α was deleted in
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white and brown adipocytes (AdHIF2KO) (Fig. 1A) (42), while Fabp4Cre- Epas1fl/fl mice
284
were used as littermate HIF2α-proficient mice (referred to as control mice). Efficient
285
deletion of HIF2α was observed in subcutaneous (sc) and visceral gonadal (gon) WAT, and
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in BAT, whereas HIF2α expression was unaffected in non-adipose tissues/cells such as the
287
liver, skeletal muscle, hypothalamus and bone marrow-derived macrophages (BMDM) (Fig.
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1A-1C). HIF1α expression was not affected in any of these tissues by adipocyte-specific
289
HIF2α deletion (Fig. 1A-1C).
290 291
AdHIF2KO and control mice were fed a HFD or ND. Mice lacking HIF2α in adipocytes
292
showed a significant increase in HFD-induced body weight gain (Fig. 2A), accompanied by
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increased weight of the scWAT, liver and BAT upon 16 and 24 weeks of HFD (Fig. 2B,
294
data from mice fed a HFD for 24 weeks; data from mice fed a HFD for 16 weeks not
295
shown). In contrast, the weight of gonWAT did not differ between HFD-fed AdHIF2KO
296
mice and HFD-fed control mice. The increase in the mass of scWAT, liver and BAT was
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not simply due to enhanced body weight, since a significant increase in the weight of these
298
tissues was also found when weights were plotted as percentage of total body weight (Fig.
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2B, data from mice fed a HFD for 24 weeks; data from mice fed a HFD for 16 weeks not
300
shown). In keeping with these data, fat body mass determined by computed tomography
13
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(CT) was elevated in AdHIF2KO mice, as compared to control mice, whereas no difference
302
was found in lean body mass between both genotypes (Fig. 2C).
303 304
AdHIF2KO mice on a HFD showed increased glucose intolerance and insulin resistance
305
compared to control mice (Fig. 2D-2E). Analysis of glucose-stimulated insulin secretion
306
(GSIS) demonstrated elevated insulin secretion upon glucose administration in AdHIF2KO
307
mice (Fig. 2F), reflecting the impaired insulin sensitivity. Furthermore, insulin signaling
308
was assessed, and BAT of AdHIF2KO mice exhibited reduced Akt phosphorylation in
309
response to insulin administration, as compared to control mice (Fig. 2G). In contrast,
310
insulin-induced Akt phosphorylation was not different in sc and gon WAT, liver and
311
muscle due to adipocyte HIF2α deficiency (data not shown). Fasting plasma insulin,
312
glucose, leptin and cholesterol levels were enhanced in obese AdHIF2KO mice, while
313
adiponectin levels were significantly lower in obese AdHIF2KO mice compared to control
314
mice (Fig. 2H-2L). In contrast, there was no significant difference in blood triglycerides or
315
FGF-21 (43) in AdHIF2KO mice (Fig. 2M and 2N). When fed a ND, AdHIF2KO mice
316
displayed no significant changes in body weight, fat mass and insulin sensitivity, as
317
compared to control mice (Fig. 3A-3C). These findings indicate that adipocyte HIF2α
318
contributes to the adaptive response of WAT to obesity and protects against HFD-induced
319
metabolic dysregulation.
320 321
We then analyzed the alterations in WAT induced by adipocyte HIF2α-deficiency. We
322
found that the mRNA expression of the adipocyte lipase Atgl was significantly reduced in
323
the gonWAT of obese AdHIF2KO mice; moreover, the expression of the lipase MgII was
14
324
lower (although not significantly), as compared to obese control mice (Fig. 4A). In the
325
gonWAT of AdHIF2KO mice, we also found decreased expression of genes involved in
326
peroxisomal (Acox, Crot) and mitochondrial lipid oxidation (Cpt1, Acsl1), of genes
327
involved in β-oxidation of long fatty acids (Vlcad, Lcad) and reduced expression of the
328
lipolysis regulator (Ppar-α) owing to adipocyte HIF2α-deficiency (Fig. 4A). In addition,
329
scWAT showed reduced expression of genes involved in lipolysis (MgII) and peroxisomal
330
lipid oxidation (Acox) (data not shown). Explants of scWAT from obese AdHIF2KO mice
331
showed reduced basal lipolysis, whereas gonWAT explants from obese AdHIF2KO mice
332
displayed diminished isoprenaline-stimulated lipolysis (Figure 4B), as compared to
333
respective explants from obese control mice. Furthermore, we found elevated RER in obese
334
AdHIF2KO mice indicating reduced lipid consumption, as compared to obese control mice,
335
while the food intake, oxygen consumption and energy expenditure (EE) were not affected
336
by adipocyte HIF2α−deficiency (Fig. 4C-4F). In addition, AdHIF2KO mice showed
337
elevated triglyceride levels upon an oral lipid tolerance test (Fig. 4G), suggesting defective
338
lipid metabolism due to adipocyte HIF2α deficiency.
339 340
Adipocyte HIF2α deficiency decreases WAT angiogenesis and enhances WAT
341
inflammation in obesity.
342
Given the strong link between insulin resistance and AT inflammation (44-46), we sought
343
to determine the inflammatory status of the obese WAT in HIF2α-deficiency.
344
Immunohistochemical staining for F4/80, a marker for macrophages, showed increased
345
abundance of macrophages in the gonWAT and scWAT of obese AdHIF2KO mice, as
346
compared to control mice (Fig. 5A). AT inflammation is associated with formation of
15
347
crown-like structures, found mostly in gonWAT, consisting of apoptotic adipocytes
348
surrounded by macrophages (47). Besides higher macrophage accumulation, we found
349
more apoptotic cells, as measured by TUNEL immunohistochemistry in the gonAT of
350
obese AdHIF2KO mice (Fig. 5B). Furthermore, WAT dysfunction is linked to WAT
351
fibrosis, which occurs by macrophage-mediated remodeling of the extracellular matrix (34,
352
48). By Masson´s trichrome staining, which is specific for collagen deposition, we found
353
increased fibrosis of the gonWAT in obese AdHIF2KO mice (Fig. 5C). Together, adipocyte
354
HIF2α protects against obesity-mediated WAT inflammation and fibrosis and thus, WAT
355
dysfunction.
356 357
Adipocyte hypertrophy in obesity leads to hypoxic areas in the WAT of men and mice (4,
358
5). AT hypoxia stimulates vascular growth in the AT (8). Interestingly, WAT of obese
359
AdHIF2KO mice displayed marked hypoxia compared to the WAT of obese control mice
360
(Fig. 5D and 5E). Limited vascularization of WAT is linked to elevated presence of
361
hypoxia, as well as insulin resistance, enhanced AT inflammation and adipocyte death (8).
362
On the other hand, enhanced vascularization of AT prevents obesity-related metabolic
363
dysregulation of AT (8, 11). HIF2α is a major regulator of proangiogenic responses in
364
several physiological and pathological situations (18, 49, 50). We found reduced WAT
365
vascularization in obese AdHIF2KO mice, as compared to control mice, which was
366
reflected by reduced numbers of endothelial cells in both WAT depots, as assessed by flow
367
cytometry (Fig. 5F). Moreover, vascularization was studied by staining with isolectin B4
368
and was found to be decreased in the scWAT and gonWAT of obese AdHIF2KO mice, as
369
compared to controls upon 16 and 24 weeks of HFD (Fig. 5G and 5H, data from mice fed a
16
370
HFD for 24 weeks; data from mice fed a HFD for 16 weeks not shown). A major HIF2α
371
target regulating angiogenesis is VEGF-A (18, 49, 50). Consistently, its expression was
372
reduced in the gonWAT of obese AdHIF2KO mice (Fig. 5I and 5J). Thus, the absence of
373
HIF2α resulted in insufficient angiogenic response in the obese WAT, thereby contributing
374
to enhanced WAT hypoxia, inflammation and cell death.
375 376
No role of HIF2α in myeloid or endothelial cells for WAT adaptation to obesity
377
Our findings so far indicate that adipocyte HIF2α deletion contributes to obese WAT
378
dysfunction, including reduced vascularization and enhanced inflammation in the obese
379
WAT, thereby resulting in insulin resistance. A major expression of HIF2α is found in
380
myeloid and endothelial cells, and HIF2α in these cells is capable of regulating angiogenic
381
responses (50, 51). As Fabp4-Cre transgenic mice have been previously reported to
382
potentially delete target genes in non-adipocytes, e.g. in macrophages (13, 52, 53), we
383
sought to clarify if myeloid HIF2α played any role for the WAT phenotype observed in
384
obese AdHIF2KO mice. To this end, we generated myeloid-specific HIF2α-deficient mice
385
(MyeHIF2KO) by engaging LysM-Cre transgenic mice. Compared to their littermate
386
controls, mice lacking HIF2α in myeloid cells did not show any metabolic changes in diet-
387
induced obesity, as assessed by analysis of body weight, fat mass or insulin tolerance test
388
(Fig. 6A-6D). These data suggest that myeloid HIF2α does not substantially contribute to
389
obesity-related WAT dysfunction.
390 391
Moreover, HIF2α in endothelial cells regulates angiogenesis-related functions (51). To
392
assess the role of endothelial HIF2α, we generated endothelial cell-specific HIF2KO mice
17
393
(EndHIF2KO) and subjected them to diet-induced obesity. In contrast to AdHIF2KO mice,
394
mice lacking HIF2α in endothelial cells did not develop any metabolic alterations, as
395
compared to littermate HIF2α-proficient mice (Fig. 6F-6I). Importantly, the number of
396
endothelial cells in the obese WAT did not change in both MyeHIF2KO and EndHIF2KO
397
mice (Fig. 6E and 6J), as compared to control littermate mice, thus, indicating that obese
398
WAT angiogenesis is regulated predominantly by adipocyte HIF2α.
399 400
Ectopic accumulation of fat in adipocyte-HIF2α deficient mice
401
Deficient lipolytic activity of WAT has been linked to ectopic accumulation of fat (e.g. in
402
the liver) along with reduced insulin sensitivity (10, 54). Livers of obese AdHIF2KO mice
403
showed not only increased size (Fig. 2B) but also increased lipid accumulation. As assessed
404
by histology analysis, we found enhanced abundance of lipid droplets in livers due to
405
adipocyte HIF2α deficiency in mice fed a HFD for 16 or 24 weeks (Fig. 7A-7B, data from
406
mice fed a HFD for 24 weeks; data from mice fed a HFD for 16 weeks not shown). This
407
finding was confirmed by measuring liver triglyceride content, which was higher in
408
AdHIF2KO mice (Fig. 7C). By histological assessment, AdHIF2KO mice displayed
409
enhanced steatosis and ballooning and an enhanced NAFLD Activity Score (NAS) (Fig.
410
7D); a NAS >5, as observed in AdHIF2KO mice, correlates with the presence of NASH
411
(38). Gene expression analysis showed unaltered expression of lipolytic markers, such as
412
Ppar-α, Mcad or Cpt1 (Fig. 7E), but significantly increased expression of lipogenic
413
markers, such as Ppar-γ and Scd1, as well as a tendency towards increased expression of
414
Srebp1c and Fas in livers from obese AdHIF2KO mice, as compared to littermate control
415
mice (Fig. 7F). These findings could explain the enhanced steatosis observed in adipocyte
18
416
HIF2α−deficiency under obese conditions. While the expression of the main hepatic
417
glucose transporter, Glut2, was unchanged, fatty acid transporter (Cd36) expression was
418
significantly increased in livers of obese AdHIF2KO mice, thereby suggesting enhanced
419
uptake of lipids by the liver (Fig. 7G). The enhanced hepatic lipid uptake was also in
420
keeping with reduced serum free fatty acid (FFA) levels in obese AdHIF2KO mice (Fig.
421
7H).
422 423
Deficiency of adipocyte HIF2α leads to BAT dysfunction in obesity.
424
We next analyzed the BAT of AdHIF2KO, as BAT is one of the main targets of the activity
425
of Cre recombinase in Fabp4-Cre-transgenic mice (Fig. 1A). The main function of BAT is
426
heat generation via lipid consumption in brown adipocytes. Recently, BAT has gained
427
attention in the context of obesity (reviewed in (23)); with the progression of obesity, BAT
428
gains weight, accumulates lipids and brown adipocytes become bigger, switching from the
429
typical brown appearance (several lipid droplets per cell) to a “white fat-like” shape
430
comprising a huge fat drop.
431 432
As described above (Fig. 2B), the BAT of mice lacking HIF2α in adipocytes displayed
433
higher mass. In addition to its enhanced mass, the BAT of obese AdHIF2KO displayed a
434
marked pale color, suggesting lipid accumulation (Fig. 8A). Microscopic analysis of the
435
BAT revealed that brown adipocytes from HFD-fed AdHIF2KO were larger than those in
436
littermate control mice (Fig. 8B and 8C, data from mice fed a HFD for 24 weeks; data from
437
mice fed a HFD for 16 weeks not shown). Interestingly, this was not observed under ND
438
conditions (data not shown). Accordingly, BAT from HFD-fed AdHIF2KO mice showed
19
439
enhanced expression of lipogenic markers (Fig. 8D), suggesting that excessive lipogenesis
440
could contribute to the elevated cell size of brown adipocytes in HIF2α−deficiency.
441 442
Consistent with recent reports with regards to induction of inflammation in the BAT during
443
obesity (55, 56), we found that dysfunction of the BAT in obese AdHIF2KO mice was
444
linked to higher BAT inflammation, as assessed by increased accumulation of F4/80-
445
positive macrophages (Fig. 8E, data from mice fed a HFD for 24 weeks; data from mice fed
446
a HFD for 16 weeks not shown). Moreover, gene expression of F4/80 and other
447
inflammatory factors, such as Tnf or Mcp-1 was upregulated in the BAT of obese
448
AdHI2KO mice, as compared to littermate control mice (Fig. 8F).
449 450
BAT function largely depends on angiogenesis (3, 57); by staining with fluorochrome-
451
labeled isolectin-B4, we found reduced vascularity in the BAT of obese AdHIF2KO mice
452
(Fig. 8G), as compared to littermate control mice, suggesting that HIF2α supports
453
angiogenic processes in the BAT in obesity. Consistently, VEGF-A levels were
454
downregulated in the BAT of obese AdHIF2KO (Fig. 8H). Together, these data
455
demonstrate that HIF2α is necessary for promoting angiogenesis not only in WAT, but also
456
in BAT in obesity, by regulating BAT VEGF-A levels.
457 458
The main function of BAT is thermogenesis, which requires the action of UCP1. UCP1
459
increases the permeability of the inner mitochondrial membrane to reduce mitochondrial
460
membrane potential and thereby uncouple ATP generation from the respiratory chain
461
leading to heat generation (reviewed in (58)). Interestingly, we found reduced UCP1
20
462
protein and mRNA in the BAT of AdHIF2KO mice, as compared to littermate HIF2α-
463
proficient mice, under obese (Fig. 9A, 9B, 9F, data from mice fed a HFD for 24 weeks;
464
data from mice fed a HFD for 16 weeks not shown) but not lean conditions (data not
465
shown). These data suggested that HIF2α-mediated regulation of UCP1 is operative only
466
during HFD-induced metabolic deterioration of the BAT. In contrast, other BAT markers
467
were not changed between obese control and AdHIF2KO mice (Fig. 9C).
468 469
Because of the essential role of UCP1 in heat generation, we performed a cold resistance
470
test in HFD-fed AdHIF2KO and control littermate mice. Obese AdHIF2KO mice were
471
unable to maintain adequate body temperature, as compared to HFD-fed control mice (Fig.
472
9D and 9E). Thus, not only HIF2α expression is induced in mice subjected to cold
473
exposure (25), but adipocyte HIF2α contributes to cold adaptation. Furthermore, gene
474
expression analysis revealed a significant reduction of UCP1 expression in the BAT of
475
obese AdHIF2KO mice, as compared to littermate control mice under both cold and room
476
temperature conditions (Fig. 9F). Additionally, reduced expression of Vegf-a was observed
477
in the BAT of obese AdHIF2KO mice under both cold and room temperature conditions
478
(Fig. 9F), as compared to obese HIF2α-proficient mice. Together, these findings
479
demonstrate that adipocyte HIF2α deficiency promotes obesity-related BAT dysfunction.
480 481
BAT dysfunction and enhanced AT inflammation at early stages of obesity in
482
AdHIF2KO mice.
483
In order to understand the mechanisms underlying the metabolic alterations seen in
484
AdHIF2KO mice, we analyzed mice fed with a HFD for only 4 weeks, hence, before body
21
485
mass differences are established between AdHIF2KO and control mice. In contrast to
486
longer feedings (16-24 weeks; Fig. 2B and data not shown), AdHIF2KO fed for only 4
487
weeks did not show any difference in WAT weights while BAT weight was already
488
significantly elevated (Fig. 10A). Moreover, brown adipocytes were enlarged in
489
AdHIF2KO mice already at early stages of obesity (Fig. 10B). In addition, UCP1
490
expression was reduced and macrophages were more abundant in the BAT from
491
AdHIF2KO mice after only 4 weeks of diet (Fig. 10B-10C). Despite unchanged tissue mass,
492
sc and gon WAT from AdHIF2KO mice displayed enhanced accumulation of macrophages,
493
associated with reduced vascularization, indicating WAT dysfunction due to HIF2α
494
deficiency in adipocytes in early stages of obesity (Fig. 10D-10E). In contrast, lipid
495
accumulation in the liver, as well as markers for lipolysis, lipogenesis and FA uptake were
496
not altered in AdHIF2KO, as compared to control mice, upon 4 week HFD feeding (Fig.
497
10F-10I). Together, these data indicate that enhanced inflammation and dysfunction
498
especially of the BAT is already present at early stages of diet-induced obesity due to
499
adipocyte HIF2α deficiency and is likely the primary event leading to metabolic
500
dysregulation in these mice.
501 502
VEGF rescues BAT dysfunction in obese AdHIF2KO mice.
503
Mice lacking HIF2α in adipocytes showed BAT dysfunction already at early stages of
504
obesity (Fig. 10A-10C) accompanied by reduced VEGF expression in the BAT (Fig. 8H).
505
We therefore addressed next, whether the impaired levels of this major proangiogenic
506
factor could contribute to elevated BAT dysfunction and inflammation. To this end, obese
507
control and AdHIF2KO were subjected to VEGF administration via mini-osmotic pumps or
22
508
PBS as control treatment. VEGF treatment reversed the enhanced weight of obese BAT due
509
to adipocyte HIF2α deficiency (Fig. 11A). Consistently, VEGF treatment reversed the
510
HIF2α deficiency-associated reduced UCP1 expression of the BAT and elevated
511
macrophage accumulation in the BAT (Fig. 11B-11C). In other words the BAT dysfunction
512
in adipocyte HIF2α deficiency was reversed, at least partially, by VEGF administration.
513
Additionally, VEGF administration efficiently reversed the exacerbated macrophage
514
accumulation and the reduced vascularization in the WAT of obese AdHIF2KO mice (Fig.
515
11D-11E). Together, these data demonstrate that the reduced levels of VEGF resulting from
516
adipocyte HIF2α-deficiency contribute to BAT inflammation and dysfunction (reduced
517
UCP1 expression) in obese AdHIF2KO mice.
518 519
DISCUSSION
520
We identified here HIF2α in white and brown adipocytes as an important factor
521
counteracting the maladaptation of WAT and BAT to obesity. Especially, our present work
522
describes for the first time that adipocyte HIF2α contributes to the regulation of the
523
adaptation of BAT to obesity. Accordingly, obese AdHIF2KO mice displayed BAT
524
dysfunction, such as increased BAT mass, enlarged brown adipocytes and decreased UCP1
525
gene and protein expression.
526 527
Metabolic dysregulation of BAT in obese AdHIF2KO mice was linked to a dysfunctional
528
thermogenic response of these mice to cold exposure. In particular, obese AdHIF2KO,
529
exposed to cold, failed to sustain their body temperature. These data are in keeping with the
530
previous observation that cold exposure induced HIF2α expression (25). In fact, BAT
23
531
hypoxia is not only present in the course of obesity (3), but is also one of the earliest events
532
during cold challenge, likely resulting from the excessive demand of oxygen that is
533
required for UCP1 activity and heat generation (25).
534 535
Noteworthy, we found that the expression of the major thermogenic factor UCP1 was
536
altered in adipocyte HIF2α deficiency. In contrast, a recent report did not show alterations
537
in UCP1 expression in the BAT of adipocyte-specific HIF1α-deficient mice compared to
538
adipocyte HIF1α-proficient mice in obesity (6). Although the underlying mechanisms of
539
UCP1 regulation by adipocyte HIF2α are not known and merit future investigations, our
540
findings are consistent with previous work showing that mice with adipocyte-specific
541
deficiency of PHD2, which is a negative regulator of HIF2α, showed enhanced UCP1
542
expression in obese BAT (15). VEGF has been previously shown to promote UCP1
543
expression (3, 11, 57). The fact that VEGF treatment rescued the reduced UCP1 expression
544
in HIF2α-deficient brown adipocytes suggests that the regulation of UCP1 by HIF2α may
545
involve VEGF. Moreover, UCP1 expression was not affected in lean AdHIF2KO mice, as
546
compared to control mice, indicating that the HIF2α-mediated regulation on UCP1 may be
547
operative in adaptive rather than constitutive BAT responses, such as under chronic
548
excessive lipid consumption characteristic of obesity or under acute cold exposure. Taken
549
together, our data demonstrate that adipocyte HIF2α is one of the factors that contribute to
550
BAT adaptation to obesity.
551 552
Furthermore, deletion of adipocyte HIF2α resulted in enhanced WAT dysfunction only in
553
the obese but not in the lean state, as indicated by reduced WAT angiogenesis, enhanced
24
554
WAT inflammation, increased WAT fibrosis and adipocyte death. Consistent with the
555
reduced expression of lipases and of genes involved in lipid oxidation, with the reduced ex
556
vivo lipolysis and the reduced lipid tolerance in adipocyte HIF2α-deficiency, we found
557
enhanced blood cholesterol levels, reduced serum free fatty acids and ectopic fat
558
accumulation in the liver (54). AdHIF2KO mice subjected to a short HFD feeding (4 weeks)
559
displayed BAT dysfunction and enhanced AT inflammation, as compared to control mice,
560
whereas liver lipid accumulation was not affected at the early stage of obesity. A recent
561
study briefly described that lack of HIF2α in adipocytes enhanced body weight and
562
gonWAT inflammation and promoted insulin resistance (6) without addressing WAT
563
angiogenesis or assessing further metabolic organs apart from the gonWAT, such as the
564
scWAT and the liver, and more importantly without reporting the role of adipocyte
565
HIF2α in BAT, as we have thoroughly done here. We provide here the crucial detailed
566
insights with regards to WAT and BAT dysfunction in obese AdHIF2KO mice.
567 568
A recent report has shown that heterozygous HIF2α+/- mice are prone to insulin resistance
569
and AT inflammation in obesity (17); these authors attributed their findings to the absence
570
of HIF2α from macrophages because clodronate-mediated macrophage depletion improved
571
glucose intolerance in HIF2α+/- mice. However, the more specific strategy employed here
572
(myeloid-specific HIF2α-deficiency) did not reveal a role of macrophage HIF2α in obesity-
573
related WAT inflammation and metabolic dysregulation. It is likely that the phenotype of
574
heterozygous HIF2α+/- mice in obesity resulted from the partial deficiency of adipocyte
575
HIF2α. Macrophage-mediated WAT inflammation is a common downstream event of
576
several pathways leading to WAT dysfunction and several examples exist that amelioration
25
577
of WAT inflammation improves obesity-related insulin resistance (59). Thus, it is quite
578
possible that obesity-related metabolic dysregulation initiated by WAT dysfunction due to
579
adipocyte-specific HIF2α-deficiency could be improved by manipulating a downstream
580
effector, e.g. by macrophage ablation. Together, our data clearly demonstrate that adipocyte
581
but not endothelial or myeloid HIF2α orchestrates the intimate crosstalk between
582
adipocytes, macrophages and the endothelium within the WAT.
583 584
Although Fabp4-Cre transgenic mice have been extensively used to achieve adipocyte -
585
specific deletion, some degree of recombination has been observed in other cells/tissues,
586
such as macrophages or endothelial cells, has been reported (53). In the present work we
587
did not find significant HIF2α deletion in the liver, skeletal muscle, heart, hypothalamus or
588
bone marrow macrophages of AdHIF2KO mice. This finding together with the absence of
589
any phenotype in the myeloid- and endothelium-specific HIF2α deficient mice in diet-
590
induced obesity, allow us to conclude that the observed phenotypes in AdHIF2KO mice
591
essentially derive from the deletion of HIF2α in adipocytes. Together, adipocyte but not
592
myeloid or endothelial HIF2α is responsible for regulating the angiogenic response within
593
the WAT in obesity and for reducing obesity-related WAT inflammation and metabolic
594
dysregulation.
595 596
Our findings support and extend a previous report demonstrating that vascular rarefaction
597
by adipocyte-specific deletion of VEGF promotes BAT dysfunction and whitening (3).
598
Although previous reports have shown that hypoxia in obesity induces HIFs (2, 6, 12) and
599
have illustrated the importance of proangiogenic responses in WAT and BAT adaptation to
26
600
obesity (3, 8, 9, 11, 57), the involvement of HIF1α or HIF2α in this process was not
601
clarified so far. In fact, several lines of evidence have suggested that HIF1α does not
602
participate in regulating Vegf-a expression and in inducing angiogenesis in both WAT and
603
BAT. On the other hand, adipocytes lacking HIF1β, and thereby HIF1α and HIF2α
604
signaling,
605
overexpression of HIF1α in adipocytes did not enhance VEGF-A expression (12) and
606
adipocyte-specific HIF1α-deleted mice had no alterations in endothelial cell numbers, as
607
compared to HIF1α-proficient mice (6). Similarly, HIF1α overexpression in brown
608
adipocytes did not enhance Vegf-a expression (3). These findings on the role of HIF1β and
609
HIF1α, together with our present findings of reduced VEGF-A levels, endothelial cell
610
numbers and vascularity of WAT and BAT in obese adipocyte-specific HIF2α-deficient
611
mice unequivocally underline the primacy of adipocyte HIF2α as the major HIF isoform
612
orchestrating the angiogenic response in the WAT and BAT in obesity. The clarification of
613
the distinct actions of HIF1α and HIF2α in adipocytes adds yet another example to the
614
variable, non-redundant and often opposite functions these two transcription factors have in
615
several biological processes (1).
showed
reduced
VEGF-A
expression
(21).
Additionally,
transgenic
616 617
In conclusion, we demonstrate that adipocyte HIF2α regulates angiogenesis in the obese
618
WAT and BAT. Furthermore, adipocyte HIF2α is integral to the thermogenic response of
619
the BAT in obesity by regulating UCP1 expression. Through these complementary
620
mechanisms adipocyte HIF2α counteracts BAT dysfunction, AT inflammation and
621
metabolic dysregulation and insulin resistance in obesity.
622 27
623 624
AUTHORS´ CONTRIBUTIONS
625
RGM designed and performed experiments, analysed data and wrote manuscript. KJC and
626
VIA designed experiments and analysed data. NQ, ME, MFRC, BG, KK, AZ, AC and JP
627
performed experiments. MB provided critical reagents. GE, GB, MEB, SRB, AEA and JH
628
participated in data interpretation and editing of the manuscript. TC conceived the project,
629
designed experiments and wrote the manuscript.
630 631
FUNDING SOURCES
632
This work was supported by the Deutsche Forschungsgemeinschaft (CH279/5-1 and
633
CH279/6-2 to TC), by the ERC (ENDHOMRET to TC), by the Else Kröner Fresenius-
634
Stiftung (to TC) and by a seed grant from the Center for Regenerative Therapies Dresden
635
(to TC, GB and MEB).
636 637
ACKNOWLEDGEMENTS
638
We would like to acknowledge S. Korten, S. Grossklaus, J. Gebler, M. Prucnal and C.
639
Mund for technical support.
640 641 642
DISCLOSURES
643
Authors declare no conflict of interest.
644 645
REFERENCES
28
646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691
1. 2. 3. 4. 5.
6.
7.
8.
9.
10. 11.
12.
13.
Majmundar, A. J., W. J. Wong, and M. C. Simon. 2010. Hypoxia-inducible factors and the response to hypoxic stress. Mol Cell 40:294-309. Rausch, M. E., S. Weisberg, P. Vardhana, and D. V. Tortoriello. 2008. Obesity in C57BL/6J mice is characterized by adipose tissue hypoxia and cytotoxic T-cell infiltration. Int J Obes (Lond) 32:451-463. Shimizu, I., T. Aprahamian, R. Kikuchi, A. Shimizu, K. N. Papanicolaou, S. MacLauchlan, S. Maruyama, and K. Walsh. 2014. Vascular rarefaction mediates whitening of brown fat in obesity. J Clin Invest 124:2099-2112. Ye, J., Z. Gao, J. Yin, and Q. He. 2007. Hypoxia is a potential risk factor for chronic inflammation and adiponectin reduction in adipose tissue of ob/ob and dietary obese mice. Am J Physiol Endocrinol Metab 293:E1118-1128. Pasarica, M., O. R. Sereda, L. M. Redman, D. C. Albarado, D. T. Hymel, L. E. Roan, J. C. Rood, D. H. Burk, and S. R. Smith. 2009. Reduced adipose tissue oxygenation in human obesity: evidence for rarefaction, macrophage chemotaxis, and inflammation without an angiogenic response. Diabetes 58:718-725. Lee, Y. S., J. W. Kim, O. Osborne, Y. Oh da, R. Sasik, S. Schenk, A. Chen, H. Chung, A. Murphy, S. M. Watkins, O. Quehenberger, R. S. Johnson, and J. M. Olefsky. 2014. Increased Adipocyte O2 Consumption Triggers HIF-1alpha, Causing Inflammation and Insulin Resistance in Obesity. Cell 157:1339-1352. Goossens, G. H., A. Bizzarri, N. Venteclef, Y. Essers, J. P. Cleutjens, E. Konings, J. W. Jocken, M. Cajlakovic, V. Ribitsch, K. Clement, and E. E. Blaak. 2014. Increased adipose tissue oxygen tension in obese compared with lean men is accompanied by insulin resistance, impaired adipose tissue capillarization, and inflammation. Circulation 124:67-76. Sung, H. K., K. O. Doh, J. E. Son, J. G. Park, Y. Bae, S. Choi, S. M. Nelson, R. Cowling, K. Nagy, I. P. Michael, G. Y. Koh, S. L. Adamson, T. Pawson, and A. Nagy. 2013. Adipose vascular endothelial growth factor regulates metabolic homeostasis through angiogenesis. Cell Metab 17:61-72. Gealekman, O., N. Guseva, C. Hartigan, S. Apotheker, M. Gorgoglione, K. Gurav, K. V. Tran, J. Straubhaar, S. Nicoloro, M. P. Czech, M. Thompson, R. A. Perugini, and S. Corvera. 2011. Depot-specific differences and insufficient subcutaneous adipose tissue angiogenesis in human obesity. Circulation 123:186194. Sun, K., I. Wernstedt Asterholm, C. M. Kusminski, A. C. Bueno, Z. V. Wang, J. W. Pollard, R. A. Brekken, and P. E. Scherer. 2012. Dichotomous effects of VEGF-A on adipose tissue dysfunction. Proc Natl Acad Sci U S A 109:5874-5879. Elias, I., S. Franckhauser, T. Ferre, L. Vila, S. Tafuro, S. Munoz, C. Roca, D. Ramos, A. Pujol, E. Riu, J. Ruberte, and F. Bosch. 2012. Adipose tissue overexpression of vascular endothelial growth factor protects against diet-induced obesity and insulin resistance. Diabetes 61:1801-1813. Halberg, N., T. Khan, M. E. Trujillo, I. Wernstedt-Asterholm, A. D. Attie, S. Sherwani, Z. V. Wang, S. Landskroner-Eiger, S. Dineen, U. J. Magalang, R. A. Brekken, and P. E. Scherer. 2009. Hypoxia-inducible factor 1alpha induces fibrosis and insulin resistance in white adipose tissue. Mol Cell Biol 29:4467-4483. Jiang, C., A. Qu, T. Matsubara, T. Chanturiya, W. Jou, O. Gavrilova, Y. M. Shah, and F. J. Gonzalez. 2011. Disruption of hypoxia-inducible factor 1 in
29
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14.
15.
16.
17. 18.
19.
20. 21. 22. 23. 24.
25.
adipocytes improves insulin sensitivity and decreases adiposity in high-fat diet-fed mice. Diabetes 60:2484-2495. Zhang, X., K. S. Lam, H. Ye, S. K. Chung, M. Zhou, Y. Wang, and A. Xu. 2010. Adipose tissue-specific inhibition of hypoxia-inducible factor 1{alpha} induces obesity and glucose intolerance by impeding energy expenditure in mice. J Biol Chem 285:32869-2877. Matsuura, H., T. Ichiki, E. Inoue, M. Nomura, R. Miyazaki, T. Hashimoto, J. Ikeda, R. Takayanagi, G. H. Fong, and K. Sunagawa. 2013. Prolyl hydroxylase domain protein 2 plays a critical role in diet-induced obesity and glucose intolerance. Circulation 127:2078-2087. Rahtu-Korpela, L., S. Karsikas, S. Horkko, R. Blanco Sequeiros, E. Lammentausta, K. A. Makela, K. H. Herzig, G. Walkinshaw, K. I. Kivirikko, J. Myllyharju, R. Serpi, and P. Koivunen. 2014. HIF prolyl 4-hydroxylase-2 inhibition improves glucose and lipid metabolism and protects against obesity and metabolic dysfunction. Diabetes 63:3324-3333. Choe, S. S., K. C. Shin, S. Ka, Y. K. Lee, J. S. Chun, and J. B. Kim. 2014. Macrophage HIF-2alpha Ameliorates Adipose Tissue Inflammation and Insulin Resistance in Obesity. Diabetes 63:3359-3371. Taniguchi, C. M., E. C. Finger, A. J. Krieg, C. Wu, A. N. Diep, E. L. LaGory, K. Wei, L. M. McGinnis, J. Yuan, C. J. Kuo, and A. J. Giaccia. 2013. Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes. Nat Med 19:1325-1330. Wei, K., S. M. Piecewicz, L. M. McGinnis, C. M. Taniguchi, S. J. Wiegand, K. Anderson, C. W. Chan, K. X. Mulligan, D. Kuo, J. Yuan, M. Vallon, L. C. Morton, E. Lefai, M. C. Simon, J. J. Maher, G. Mithieux, F. Rajas, J. P. Annes, O. P. McGuinness, G. Thurston, A. J. Giaccia, and C. J. Kuo. 2013. A liver Hif2alpha-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition. Nat Med 19:1331-1337. Rankin, E. B., J. Rha, M. A. Selak, T. L. Unger, B. Keith, Q. Liu, and V. H. Haase. 2009. Hypoxia-inducible factor 2 regulates hepatic lipid metabolism. Mol Cell Biol 29:4527-4538. Lee, K. Y., S. Gesta, J. Boucher, X. L. Wang, and C. R. Kahn. 2011. The differential role of Hif1beta/Arnt and the hypoxic response in adipose function, fibrosis, and inflammation. Cell Metab 14:491-503. Cao, Y. 2010. Adipose tissue angiogenesis as a therapeutic target for obesity and metabolic diseases. Nat Rev Drug Discov 9:107-115. Harms, M., and P. Seale. 2013. Brown and beige fat: development, function and therapeutic potential. Nat Med 19:1252-1263. Fredriksson, J. M., J. M. Lindquist, G. E. Bronnikov, and J. Nedergaard. 2000. Norepinephrine induces vascular endothelial growth factor gene expression in brown adipocytes through a beta -adrenoreceptor/cAMP/protein kinase A pathway involving Src but independently of Erk1/2. J Biol Chem 275:13802-13811. Xue, Y., N. Petrovic, R. Cao, O. Larsson, S. Lim, S. Chen, H. M. Feldmann, Z. Liang, Z. Zhu, J. Nedergaard, B. Cannon, and Y. Cao. 2009. Hypoxiaindependent angiogenesis in adipose tissues during cold acclimation. Cell Metab 9:99-109.
30
738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783
26.
27. 28. 29.
30. 31.
32.
33. 34.
35. 36.
37.
Asano, A., M. Morimatsu, H. Nikami, T. Yoshida, and M. Saito. 1997. Adrenergic activation of vascular endothelial growth factor mRNA expression in rat brown adipose tissue: implication in cold-induced angiogenesis. Biochem J 328 ( Pt 1):179-183. Bagchi, M., L. A. Kim, J. Boucher, T. E. Walshe, C. R. Kahn, and P. A. D'Amore. 2013. Vascular endothelial growth factor is important for brown adipose tissue development and maintenance. Faseb J 27:3257-3271. Nikami, H., J. Nedergaard, and J. M. Fredriksson. 2005. Norepinephrine but not hypoxia stimulates HIF-1alpha gene expression in brown adipocytes. Biochem Biophys Res Commun 337:121-126. Matthias, A., K. B. Ohlson, J. M. Fredriksson, A. Jacobsson, J. Nedergaard, and B. Cannon. 2000. Thermogenic responses in brown fat cells are fully UCP1dependent. UCP2 or UCP3 do not substitute for UCP1 in adrenergically or fatty scid-induced thermogenesis. J Biol Chem 275:25073-25081. Cross, M., I. Mangelsdorf, A. Wedel, and R. Renkawitz. 1988. Mouse lysozyme M gene: isolation, characterization, and expression studies. Proc Natl Acad Sci U S A 85:6232-6236. Gothert, J. R., S. E. Gustin, J. A. van Eekelen, U. Schmidt, M. A. Hall, S. M. Jane, A. R. Green, B. Gottgens, D. J. Izon, and C. G. Begley. 2004. Genetically tagging endothelial cells in vivo: bone marrow-derived cells do not contribute to tumor endothelium. Blood 104:1769-1777. Langer, H. F., V. V. Orlova, C. Xie, S. Kaul, D. Schneider, A. S. Lonsdorf, M. Fahrleitner, E. Y. Choi, V. Dutoit, M. Pellegrini, S. Grossklaus, P. P. Nawroth, G. Baretton, S. Santoso, S. T. Hwang, B. Arnold, and T. Chavakis. 2011. A novel function of junctional adhesion molecule-C in mediating melanoma cell metastasis. Cancer Res 71:4096-4105. Judex, S., Y. K. Luu, E. Ozcivici, B. Adler, S. Lublinsky, and C. T. Rubin. 2010. Quantification of adiposity in small rodents using micro-CT. Methods 50:14-19. Phieler, J., K. J. Chung, A. Chatzigeorgiou, A. Klotzsche-von Ameln, R. Garcia-Martin, D. Sprott, M. Moisidou, T. Tzanavari, B. Ludwig, E. Baraban, M. Ehrhart-Bornstein, S. R. Bornstein, H. Mziaut, M. Solimena, K. P. Karalis, M. Economopoulou, J. D. Lambris, and T. Chavakis. 2013. The complement anaphylatoxin C5a receptor contributes to obese adipose tissue inflammation and insulin resistance. J Immunol 191:4367-4374. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402-408. Choi, E. Y., V. V. Orlova, S. C. Fagerholm, S. M. Nurmi, L. Zhang, C. M. Ballantyne, C. G. Gahmberg, and T. Chavakis. 2008. Regulation of LFA-1dependent inflammatory cell recruitment by Cbl-b and 14-3-3 proteins. Blood 111:3607-3614. Chatzigeorgiou, A., K. J. Chung, R. Garcia-Martin, V. I. Alexaki, A. Klotzsche-von Ameln, J. Phieler, D. Sprott, W. Kanczkowski, T. Tzanavari, M. Bdeir, S. Bergmann, M. Cartellieri, M. Bachmann, P. Nikolakopoulou, A. Androutsellis-Theotokis, G. Siegert, S. R. Bornstein, M. H. Muders, L. Boon, K. P. Karalis, E. Lutgens, and T. Chavakis. 2014. Dual role of B7 costimulation in
31
784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830
38.
39. 40. 41. 42.
43.
44.
45. 46. 47. 48. 49. 50.
obesity-related non-alcoholic steatohepatitis (NASH) and metabolic dysregulation. Hepatology 60:1196-1210. Kleiner, D. E., E. M. Brunt, M. Van Natta, C. Behling, M. J. Contos, O. W. Cummings, L. D. Ferrell, Y. C. Liu, M. S. Torbenson, A. Unalp-Arida, M. Yeh, A. J. McCullough, and A. J. Sanyal. 2005. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41:1313-1321. Mehlem, A., C. E. Hagberg, L. Muhl, U. Eriksson, and A. Falkevall. 2013. Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease. Nat Protoc 8:1149-1154. Xue, Y., S. Lim, E. Brakenhielm, and Y. Cao. 2010. Adipose angiogenesis: quantitative methods to study microvessel growth, regression and remodeling in vivo. Nat Protoc 5:912-920. Weir, J. B. 1949. New methods for calculating metabolic rate with special reference to protein metabolism. J Physiol 109:1-9. He, W., Y. Barak, A. Hevener, P. Olson, D. Liao, J. Le, M. Nelson, E. Ong, J. M. Olefsky, and R. M. Evans. 2003. Adipose-specific peroxisome proliferatoractivated receptor gamma knockout causes insulin resistance in fat and liver but not in muscle. Proc Natl Acad Sci U S A 100:15712-15717. Xu, J., D. J. Lloyd, C. Hale, S. Stanislaus, M. Chen, G. Sivits, S. Vonderfecht, R. Hecht, Y. S. Li, R. A. Lindberg, J. L. Chen, D. Y. Jung, Z. Zhang, H. J. Ko, J. K. Kim, and M. M. Veniant. 2009. Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in dietinduced obese mice. Diabetes 58:250-259. Chatzigeorgiou, A., T. Seijkens, B. Zarzycka, D. Engel, M. Poggi, S. van den Berg, O. Soehnlein, H. Winkels, L. Beckers, D. Lievens, A. Driessen, P. Kusters, E. Biessen, R. Garcia-Martin, A. Klotzsche-von Ameln, M. Gijbels, R. Noelle, L. Boon, T. Hackeng, K. M. Schulte, A. Xu, G. Vriend, S. Nabuurs, K. J. Chung, K. Willems van Dijk, P. C. Rensen, N. Gerdes, M. de Winther, N. L. Block, A. V. Schally, C. Weber, S. R. Bornstein, G. Nicolaes, T. Chavakis, and E. Lutgens. 2014. Blocking CD40-TRAF6 signaling is a therapeutic target in obesityassociated insulin resistance. Proc Natl Acad Sci U S A 111:2686-2691. Chatzigeorgiou, A., K. P. Karalis, S. R. Bornstein, and T. Chavakis. 2012. Lymphocytes in obesity-related adipose tissue inflammation. Diabetologia 55:25832592. Osborn, O., and J. M. Olefsky. 2012. The cellular and signaling networks linking the immune system and metabolism in disease. Nat Med 18:363-374. Altintas, M. M., A. Azad, B. Nayer, G. Contreras, J. Zaias, C. Faul, J. Reiser, and A. Nayer. 2011. Mast cells, macrophages, and crown-like structures distinguish subcutaneous from visceral fat in mice. J Lipid Res 52:480-488. Khan, T., E. S. Muise, P. Iyengar, Z. V. Wang, M. Chandalia, N. Abate, B. B. Zhang, P. Bonaldo, S. Chua, and P. E. Scherer. 2009. Metabolic dysregulation and adipose tissue fibrosis: role of collagen VI. Mol Cell Biol 29:1575-1591. Rankin, E. B., J. Rha, T. L. Unger, C. H. Wu, H. P. Shutt, R. S. Johnson, M. C. Simon, B. Keith, and V. H. Haase. 2008. Hypoxia-inducible factor-2 regulates vascular tumorigenesis in mice. Oncogene 27:5354-5358. Imtiyaz, H. Z., E. P. Williams, M. M. Hickey, S. A. Patel, A. C. Durham, L. J. Yuan, R. Hammond, P. A. Gimotty, B. Keith, and M. C. Simon. 2010. Hypoxia32
831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867
51.
52.
53.
54.
55.
56. 57. 58. 59.
inducible factor 2alpha regulates macrophage function in mouse models of acute and tumor inflammation. J Clin Invest 120:2699-2714. Skuli, N., A. J. Majmundar, B. L. Krock, R. C. Mesquita, L. K. Mathew, Z. L. Quinn, A. Runge, L. Liu, M. N. Kim, J. Liang, S. Schenkel, A. G. Yodh, B. Keith, and M. C. Simon. 2012. Endothelial HIF-2alpha regulates murine pathological angiogenesis and revascularization processes. J Clin Invest 122:14271443. Tang, T., J. Zhang, J. Yin, J. Staszkiewicz, B. Gawronska-Kozak, D. Y. Jung, H. J. Ko, H. Ong, J. K. Kim, R. Mynatt, R. J. Martin, M. Keenan, Z. Gao, and J. Ye. 2010. Uncoupling of inflammation and insulin resistance by NF-kappaB in transgenic mice through elevated energy expenditure. J Biol Chem 285:4637-4644. Lee, K. Y., S. J. Russell, S. Ussar, J. Boucher, C. Vernochet, M. A. Mori, G. Smyth, M. Rourk, C. Cederquist, E. D. Rosen, B. B. Kahn, and C. R. Kahn. 2013. Lessons on conditional gene targeting in mouse adipose tissue. Diabetes 62:864-874. Haemmerle, G., A. Lass, R. Zimmermann, G. Gorkiewicz, C. Meyer, J. Rozman, G. Heldmaier, R. Maier, C. Theussl, S. Eder, D. Kratky, E. F. Wagner, M. Klingenspor, G. Hoefler, and R. Zechner. 2006. Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase. Science 312:734-737. Nguyen, K. D., Y. Qiu, X. Cui, Y. P. Goh, J. Mwangi, T. David, L. Mukundan, F. Brombacher, R. M. Locksley, and A. Chawla. 2011. Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis. Nature 480:104-108. Herrero, L., H. Shapiro, A. Nayer, J. Lee, and S. E. Shoelson. 2010. Inflammation and adipose tissue macrophages in lipodystrophic mice. Proc Natl Acad Sci U S A 107:240-245. Sun, K., C. M. Kusminski, K. Luby-Phelps, S. B. Spurgin, Y. A. An, Q. A. Wang, W. L. Holland, and P. E. Scherer. 2014. Brown adipose tissue derived VEGF-A modulates cold tolerance and energy expenditure. Mol Metab 3:474-483. Cannon, B., and J. Nedergaard. 2004. Brown adipose tissue: function and physiological significance. Physiol Rev 84:277-359. Feng, B., P. Jiao, Y. Nie, T. Kim, D. Jun, N. van Rooijen, Z. Yang, and H. Xu. 2011. Clodronate liposomes improve metabolic profile and reduce visceral adipose macrophage content in diet-induced obese mice. PLoS One 6:e24358.
33
868
FIGURE LEGENDS
869
Figure 1. HIF2α deletion in AdHIF2KO mice.
870
A, Epas1 (Hif2α) (left) and Hif1α (right) gene expression in subcutaneous (sc) and gonadal
871
(gon) white AT (WAT), brown AT (BAT), liver, quadriceps skeletal muscle and heart
872
(n=5-8/group). Gene expression of control mice was set as 1. B-C, Epas1 and Hif1α gene
873
expression in hypothalamus (B) and bone marrow-derived macrophages (BMDM) (C).
874
Gene expression of control mice was set as 1 (n=3-4/group).
875
Data are expressed as mean ± s.e.m. * P < 0.05.
876 877
Figure 2. Mice lacking HIF2α in adipocytes display worsened obesity-related
878
metabolic dysregulation.
879
A, Body weight of control and AdHIF2KO mice subjected to HFD. B, Mice were sacrificed
880
after 24 weeks of HFD, and subcutaneous (sc) and gonadal (gon) WAT, liver, spleen and
881
BAT were weighed. The tissue weights are presented as percentage of total body weight. C,
882
Lean and fat body mass was determined in obese (24 weeks on HFD) control and
883
AdHIF2KO mice by computed tomography (CT); data were normalized to control mice. D,
884
Glucose tolerance test of control and AdHIF2KO mice fed with HFD for 12 weeks. E,
885
Insulin tolerance test of control and AdHIF2KO mice fed with HFD for 14 weeks. F,
886
Glucose-stimulated insulin secretion of control and AdHIF2KO mice fed with HFD. G,
887
Control and AdHIF2KO mice were fed with a HFD for 15 weeks. Mice were fasted for 6
888
hours before an intraperitoneal injection of insulin (5 U/Kg of body weight), BAT was
889
harvested and analysed for phosphorylation of Akt at Ser473 and total Akt. Representative
890
blots (left) and densitometric quantification of the blots (right panel) are shown. Data
34
891
(densitometric analysis) are expressed as mean ± s.e.m. (n=6 mice per group). H-N, Plasma
892
insulin (H), blood glucose (I), plasma leptin (J), blood cholesterol (K), plasma adiponectin
893
(L), blood triglycerides (M) and plasma FGF-21 (N) of mice of the indicated genotypes fed
894
for 15 weeks with HFD and fasted overnight.
895
In A-F and H-N, data are expressed as mean ± s.e.m. (n=6-15 mice/group). * P < 0.05.
896 897
Figure 3. Lean AdHIF2KO do not show metabolic dysregulation.
898
A, Body weight of control and AdHIF2KO mice fed with normal diet (ND) (10% Kcal
899
from fat). B, Weights of subcutaneous (sc) and gonadal (gon) WAT, liver, spleen and BAT
900
from control and AdHIF2KO fed with ND for 24 weeks. C, Insulin tolerance test of control
901
and AdHIF2KO fed with ND for 13 weeks.
902
Data are expressed as mean ± s.e.m. (n=6-9 mice/group).
903 904
Figure 4. Obese AdHIF2KO mice show altered lipid metabolism.
905
A, Expression of metabolic genes in gonadal adipocyte fraction from control and
906
AdHIF2KO mice fed a HFD for 24 weeks is shown. Gene expression of control mice was
907
set as 1 (n=6-7/group). B, Subcutaneous (sc) WAT and gonadal (gon) WAT explants from
908
obese control and AdHIF2KO mice were cultured for 2h at 37ºC in the presence or absence
909
of 10 μM isoprenaline and release of FFA to the medium was quantified (n=4-6/group). C-
910
F, Control and AdHIF2KO were fed with a HFD for 6 weeks and placed in metabolic cages.
911
After 16 hours of adaptation, respiratory exchange ratio (RER) (C), food intake (D), oxygen
912
consumption (E) and energy expenditure (EE) (F) were monitored (n=6/group). G, Oral
913
lipid tolerance test of control and AdHIF2KO mice fed with a HFD for 15 weeks (n=12-
914
13/group). 35
915
Data are expressed as mean ± s.e.m. * P < 0.05.
916 917
Figure 5. Obese AdHIF2KO mice show enhanced WAT inflammation and reduced
918
WAT vascularity.
919
A-C, Mice were sacrificed after 24 weeks on a HFD. A, Representative pictures in gonadal
920
(gon) WAT (left) and quantification from subcutaneous (sc) and gon WAT (right panel) of
921
immunohistochemistry for F4/80 (n=5/group). Macrophage accumulation of control mice
922
was set as 1. Scale bar is 200 μm. B, Representative pictures of TUNEL
923
immunohistochemistry from the gonWAT of obese control and AdHIF2KO mice (24 weeks
924
on HFD), and the respective quantification (right panel) are shown (n=5/group). WAT
925
apoptosis of control mice was set as 1. Scale bar is 200 µm. C, Representative pictures of
926
Masson Trichrome staining for fibrosis detection in gonWAT from control and AdHIF2KO
927
mice. Scale bar is 200 μm. D and E, Representative images of staining for CCl-103F
928
(Hypoxiprobe-F6) in gonWAT (D) as well as quantification of hypoxia staining (E) from sc
929
and gonWAT from obese control and AdHIF2KO mice (16 weeks on HFD) are shown
930
(n=5-6/group). Black arrows indicate hypoxic areas. Scale bar is 200 µm. F-J, Mice were
931
sacrificed after 24 weeks on a HFD. F, ScWAT and gonWAT were digested with
932
collagenase, and flow cytometry analysis for CD31+CD45- cells was performed to analyse
933
endothelial cell numbers (n=10-17). The absolute endothelial cell number per g of tissue
934
was quantified. Data are shown relative to control; data of control mice were set as 1. G and
935
H, Representative images of scWAT (G) of isolectin B4 staining in whole mounts and
936
quantification (H) of isolectin B4 staining in whole mounts from scWAT and gonWAT
937
(n=5-7). Vascularization of control mice was set as 1. Scale bar is 200 μm. I, Vegf-a gene
36
938
expression in subcutaneous and gonadal adipocyte fractions from control and AdHIF2KO
939
mice (n=6-7). Gene expression of control mice was set as 1. J, VEGF-A protein levels
940
measured in scWAT and gonWAT lysates from control and AdHIF2KO mice, normalized
941
over total protein content (n=4-7).
942
Data are expressed as mean ± s.e.m. * P < 0.05.
943 944
Figure 6. No role of myeloid or endothelial HIF2α in obesity-related metabolic
945
dysregulation.
946
A, Effective deletion of Epas1 (Hif2α) but not of Hif1α in bone marrow-derived
947
macrophages from control and MyeHIF2KO mice. Gene expression of control mice was set
948
as 1. B, Body weight of HFD-fed control and MyeHIF2KO mice. C, Tissue weights of
949
obese control and MyeHIF2KO mice. D, Insulin tolerance test of obese MyeHIF2KO and
950
control mice. E, FACS staining for endothelial cells (CD31+CD45-) in the SVF of
951
subcutaneous (sc) and gonadal (gon) WAT of obese MyeHIF2KO and littermate control
952
mice. The absolute endothelial cell number per g of tissue was quantified. Data are shown
953
relative to control; data of control mice were set as 1. A-E: Data are expressed as mean ±
954
s.e.m. (n=4-5 per group). * P < 0.05.
955
F, Effective deletion of Epas1 (Hif2α) but not of Hif1α was assessed in sorted lung
956
endothelial cells from control and EndHIF2KO mice. Gene expression of control mice was
957
set as 1. G, Body weight of HFD-fed control and EndHIF2KO mice. H, Tissue weights of
958
obese EndHIF2KO and littermate control mice. I, Insulin tolerance test of obese
959
EndHIF2KO and littermate control mice. J, FACS staining for endothelial cells
960
(CD31+CD45-) from the SVF of scWAT and gonWAT of obese EndHIF2KO and
37
961
littermate control mice. The absolute endothelial cell number per g of tissue was quantified.
962
Data are shown relative to control; data of control mice were set as 1. F-J: Data are
963
expressed as mean ± s.e.m. (n=5 per group). * P < 0.05.
964 965
Figure 7. Obese AdHIF2KO mice develop enhanced hepatosteatosis.
966
Mice were sacrificed after 24 weeks on a HFD. A, Representative pictures of Oil-Red-O
967
(ORO) staining of livers from control and AdHIF2KO mice and quantification (n=5/group).
968
Steatosis (ORO staining) of control mice was set as 1. Scale bar is 200 μm. B,
969
Representative images (H&E staining) of obese control and AdHIF2KO livers. Scale bar is
970
200 µm. C, Triglyceride content was determined in livers from control and AdHIF2KO
971
mice fed for 24 weeks with HFD (n=6-7). D, Histological scoring for steatosis,
972
hepatocellular ballooning, lobular inflammation and NAFLD Activity Score (NAS) of
973
livers from obese control and AdHIF2KO mice (n=5). E-G, Gene expression analysis for
974
lipolytic (E) and lipogenic markers (F), as well as transporters for FFA (Cd36) and glucose
975
(Glut2) (G) in livers from obese control and AdHIF2KO mice (n=6-7). Gene expression of
976
control mice was set as 1. H, Serum free fatty acids (FFA) from control and AdHIF2KO
977
mice fed a HFD (n=8-12).
978
Data are expressed as mean ± s.e.m. * P < 0.05.
979 980
Figure 8. Dysfunction of BAT in obese AdHIF2KO mice.
981
A, Representative images of BAT of obese control and AdHIF2KO mice. B, Representative
982
H&E staining of BAT from control and AdHIF2KO mice upon HFD for 24 weeks. Scale
983
bar is 100 μm. C, Quantification of brown adipocyte cell diameter and fitting curve of
38
984
control (grey line) and AdHIF2KO mice (black line). D, Gene expression analysis for
985
lipogenic markers in BAT from obese control and AdHIF2KO mice fed a HFD for 24
986
weeks. Gene expression of control mice was set as 1. E, Quantification of F4/80
987
immunohistochemistry in BAT from control and AdHIF2KO mice upon HFD for 24 weeks.
988
Macrophage accumulation of control mice was set as 1. F, Gene expression of pro-
989
inflammatory markers in BAT from control and AdHIF2KO mice on HFD for 24 weeks.
990
Gene expression of control mice was set as 1. G, Representative images of isolectin B4
991
staining in whole mounts from BAT of control and AdHIF2KO mice on HFD for 24 weeks.
992
Scale bar is 100 μm. H, VEGF-A protein levels, normalized over total protein content, were
993
measured in BAT lysates from control and AdHIF2KO mice on HFD for 24 weeks.
994
Data in C-F and H are expressed as mean ± s.e.m. (n=5-7 mice per group). * P < 0.05.
995 996
Figure 9. Reduced UCP1 expression in the BAT of obese adipocyte-specific
997
HIF2α deficient mice.
998
A, Representative images of UCP1 immunohistochemistry in BAT from 24 week-HFD-fed
999
control and AdHIF2KO mice. Scale bar is 100 μm. B, Representative western blot for
1000
UCP1 and tubulin (left) and quantification (right) from BAT protein lysates from control
1001
and AdHIF2KO mice upon HFD for 24 weeks. The UCP1/tubulin ratio of control mice was
1002
set as 1 (n=4-5/group). C, Gene expression of Pgc1α, Prdm16, Dio2, Cidea and Adrb3 in
1003
BAT from control and AdHIF2KO mice fed a HFD for 24 weeks is shown. Gene
1004
expression of control mice was set as 1 (n=7-14/group). D and E, Obese control and
1005
AdHIF2KO mice were exposed to either room temperature (RT) (D) or 4ºC (E) and
1006
temperature was measured (n=3-4 for RT, n=9-12 for 4ºC). F, Gene expression analysis of
39
1007
the BAT from the mice displayed in D and E. Gene expression of control RT mice was set
1008
as 1.
1009
Data in B-F are expressed as mean ± s.e.m. * P < 0.05.
1010 1011
Figure 10. Metabolic dysregulation of WAT and BAT in AdHIF2KO mice in early
1012
stages of diet-induced obesity.
1013
Control and AdHIF2KO mice were fed with HFD for 4 weeks. A, Subcutaneous (sc) and
1014
gonadal (gon) WAT and BAT were weighed. The tissue weights are presented as
1015
percentage of total body weight. B, Representative images from H&E staining (upper
1016
panels) and UCP1 immunohistochemistry (lower panels) in BAT from control and
1017
AdHIF2KO mice. Scale bar is 100 μm. C, Quantification of immunohistochemistry for
1018
F4/80 in BAT from both genotypes. Macrophage accumulation of control mice was set as 1.
1019
D, Quantification of immunohistochemistry for F4/80 in sc and gonWAT from control and
1020
AdHIF2KO mice. Macrophage accumulation of control mice was set as 1. E, Vessels were
1021
stained for isolectin B4 in whole mounts of scWAT and gonWAT from control and
1022
AdHIF2KO mice and quantified. Vascularization of control mice was set as 1. F,
1023
Representative images from H&E staining in livers from control and AdHIF2KO. Scale bar
1024
is 200 μm. G-H, Gene expression analysis for lipolytic (G) and lipogenic markers (H), as
1025
well as transporters for FFA (Cd36) and glucose (Glut2) (I) in livers from obese control and
1026
AdHIF2KO mice. Gene expression of control mice was set as 1.
1027
Data in A, C-E and G-I are expressed as mean ± s.e.m. (n=6 mice per group). * P < 0.05.
1028
40
1029
Figure 11. VEGF administration reverses metabolic dysregulation of the BAT
1030
associated with adipocyte HIF2α deficiency.
1031
Control and AdHIF2KO were fed with HFD for a total of 8 weeks; for the last 3 weeks of
1032
the experiment, mini-osmotic pumps were implanted subcutaneously for administration of
1033
murine VEGF or PBS as a control. A, BAT weight after PBS or VEGF treatment is shown.
1034
The tissue weight is presented as percentage of total body weight. B, Representative images
1035
for UCP1 staining in BAT from control and AdHIF2KO mice. Scale bar is 100 μm. C,
1036
Quantification of immunohistochemistry for F4/80 in BAT from control and AdHIF2KO
1037
mice treated with either PBS or VEGF. Macrophage accumulation of control mice was set
1038
as 1. D, Quantification of immunohistochemistry for F4/80 in subcutaneous (sc) and
1039
gonadal (gon) WAT from control and AdHIF2KO mice treated with either PBS or VEGF.
1040
Macrophage accumulation of control mice was set as 1 in each case. E, Flow cytometry
1041
analysis for CD31+CD45- cells was performed to analyse absolute endothelial cell numbers.
1042
The absolute endothelial cell number per g of tissue was quantified. Data are shown relative
1043
to control mice; data of control mice were set as 1 in each case.
1044
Data in A, and C-E are expressed as mean ± s.e.m. (n=3-6 mice per group). * P < 0.05.
41
