1008 Diabetes Volume 66, April 2017 Yasuhiro Onogi,1 Tsutomu Wada,1 Chie Kamiya,1 Kento Inata,1 Takatoshi Matsuzawa,1 Yuka Inaba,2,3 Kumi Kimura,2 Hiroshi Inoue,2,3 Seiji Yamamoto,4 Yoko Ishii,4 Daisuke Koya,5 Hiroshi Tsuneki,1 Masakiyo Sasahara,4 and Toshiyasu Sasaoka1 PDGFRb Regulates Adipose Tissue Expansion and Glucose Metabolism via Vascular Remodeling in Diet-Induced Obesity Diabetes 2017;66:1008–1021 | DOI: 10.2337/db16-0881 Platelet-derived growth factor (PDGF) is a key factor in The physiological roles of the vasculature in adipose tissue angiogenesis; however, its role in adult obesity remains have been attracting interest from the viewpoint of adipose unclear. In order to clarify its pathophysiological role, tissue expansion and chronic inflammation (1,2). White we investigated the significance of PDGF receptor b adipose tissue (WAT) such as visceral fat possesses the (PDGFRb) in adipose tissue expansion and glucose unique characteristic of plasticity; its volume may change metabolism. Mature vessels in the epididymal white several fold even after growth depending on nutritional adipose tissue (eWAT) were tightly wrapped with peri- conditions. Enlarged adipose tissue is chronically exposed cytes in normal mice. Pericyte desorption from vessels to hypoxia (3,4), which stimulates the production of angio- and the subsequent proliferation of endothelial cells genic factors for the supplementation of nutrients and were markedly increased in the eWAT of diet-induced oxygen to the newly enlarged tissue area (5). Selective ab- obese mice. Analyses with flow cytometry and adipose lation of the vasculature in WAT by apoptosis-inducible tissue cultures indicated that PDGF-B caused the de- peptides or the systemic administration of angiogenic in- PATHOPHYSIOLOGY tachment of pericytes from vessels in a concentration- hibitors has been shown to reduce WAT volumes and result dependent manner. M1-macrophages were a major type in better glucose metabolism profiles in obese mice (1,2,6). of cells expressing PDGF-B in obese adipose tissue. In contrast, pericyte detachment was attenuated and vas- Regarding angiogenic factors, vascular endothelial growth cularity within eWAT was reduced in tamoxifen-inducible factor (VEGF) has been characterized in the most detail conditional Pdgfrb-knockout mice with decreases in ad- and is known to play crucial roles in the neovascular de- – ipocytesizeandchronicinflammation. Furthermore, velopment of adipose tissue with obesity (7 13). Pdgfrb-knockout mice showed enhanced energy expen- PDGF-B is another growth factor and its homodimer diture. Consequently, diet-induced obesity and the asso- activates an intracellular signaling cascade by binding to ciated deterioration of glucose metabolism in wild-type its receptor, PDGF receptor b (PDGFRb) (14,15). It plays mice were absent in Pdgfrb-knockout mice. Therefore, an essential role in fetal vascular development and also PDGF-B–PDGFRb signaling plays a significant role in the contributes to wound healing and tumor growth via development of adipose tissue neovascularization and angiogenic actions in adults (16). PDGFRb is almost re- appears to be a fundamental target for the prevention of strictively expressed in perivascular mesenchymal cells, obesity and type 2 diabetes. particularly in vascular smooth muscle cells and pericytes 1Department of Clinical Pharmacology, University of Toyama, Toyama, Japan Corresponding authors: Toshiyasu Sasaoka, [email protected], and 2Department of Physiology and Metabolism, Brain/Liver Interface Medicine Tsutomu Wada, [email protected]. Research Center, Institute for Frontier Science Initiative, Kanazawa University, Received 19 July 2016 and accepted 17 January 2017. Ishikawa, Japan This article contains Supplementary Data online at http://diabetes 3Metabolism and Nutrition Research Unit, Innovative Integrated Bio-Research .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0881/-/DC1. Core, Institute for Frontier Science Initiative, Kanazawa University, Ishikawa, Japan © 2017 by the American Diabetes Association. Readers may use this article as 4Department of Pathology, University of Toyama, Toyama, Japan long as the work is properly cited, the use is educational and not for profit, and the 5Department of Internal Medicine, Kanazawa Medical University, Ishikawa, work is not altered. More information is available at http://www.diabetesjournals Japan .org/content/license. diabetes.diabetesjournals.org Onogi and Associates 1009 (14,15). Endothelial cell (EC)–derived PDGF-B promotes variable amounts of the 40% glucose solution to maintain the migration of pericytes to neovessels for maturation blood glucose levels at 110 6 20 mg/dL. by covering blood vessels, and these pericytes regulate vascular permeability and sprouting (17). The systemic Cell and Adipose Tissue Cultures deletion of the Pdgfb or Pdgfrb gene results in a lack of Bone marrow–derived macrophages (BMDMs) were pre- pericytes in the vasculature and embryonic lethality with pared, as described previously (4). Bone marrow cells vascular dysfunction (15,18–20). However, it currently re- were treated with 100 nmol/L 4-hydroxytamoxifen to de- mains unclear whether the angiogenic effects of PDGF are lete the Pdgfrb allele for 48 h and differentiated into involved in the mechanisms responsible for the develop- FL-BMDM and Pdgfrb-KO-BMDM, respectively. In adipose ment of adult obesity. tissue cultures, epididymal WAT (eWAT) isolated from lean Therefore, we herein used tamoxifen-inducible condi- 8- to 12-week-old male mice was cut into small pieces tional Pdgfrb-knockout (KO) mice in which PDGFRb was (;3 3 3 mm) and cultured in serum-free DMEM. To ob- deleted during adulthood without affecting vascular de- tain Pdgfrb-KO tissue, adipose tissue pieces from Cre-ER/ fl fl fl fl velopment in the fetal and growing periods for the selec- Pdgfrb ox/ ox or Pdgfrb ox/ ox control mice were incubated tive ablation of Pdgfrb. These mice were fed a normal with 100 nmol/L 4-hydroxytamoxifen for 48 h. These ad- chow diet or a high-fat diet (HFD), and the effects of ipose tissues were treated with or without PDGF-BB (Life the Pdgfrb deletion on adipose tissue vasculature and glu- Technologies) for 24 h and were used for the analysis of cose metabolism were investigated. whole-mount immunofluorescence. RESEARCH DESIGN AND METHODS Immunofluorescence in Sections and Whole-Mount Materials Adipose Tissue fl Human recombinant insulin was provided by Novo Nordisk Immuno uorescence analyses were performed in accor- fi Pharma Ltd. Antibodies and ELISA kits used in this study dance with previous methods with minor modi cations (28). m were listed in Supplementary Table 1. All other reagents of Sections (30- or 40- m slices) were obtained from the mid- analytical grade not elsewhere specified were obtained from dle part of the eWAT using a cryostat (CM 3050S-IV; Leica Sigma-Aldrich or Wako Pure Chemical Industries. Microsystems) and were used in experiments of Fig. 3D and I, and Supplementary Fig. 4D. To conduct whole-mount Mice and Metabolic Analyses immunofluorescence, tissues obtained from the tip region All experimental procedures used in this study were of eWAT, an active area for neoangiogenesis (29), were approved by the Committee of Animal Experiments at stained with antibodies and subsequently incubated with fl fl the University of Toyama. Pdgfrb ox/ ox mice on a C57BL/6 fructose solution as an optical clearing agent to reduce the background were crossbred with Cre-estrogen receptor amount of light scattering in tissues (30) and were used in transgenic mice (The Jackson Laboratory). Their off- experiments shown in Fig. 3A and F.Theimmunofluores- fl fl spring, Cre-ER/Pdgfrb ox/ ox, were orally administered cent images were randomly obtained by a confocal micro- tamoxifen at 9 weeks old (2.25 mg/10 g body wt for 5 scope (TCS-SP5; Leica Microsystems). Blood vessel areas, consecutive days; Cayman) to produce conditional systemic pericyte association levels, and PDGFRb-like immunoreac- D Pdgfrb SYS-KOmice,asshowninFig.1E (21,22). To conduct tivities in each mouse were measured with the ImageJ soft- fl fl experiments appropriately, Pdgfrb ox/ ox littermates of the ware (National Institutes of Health), and the data were same age and sex were treated identically and used as con- averaged. trols (FL). Mice were housed in a temperature-controlled colony room (23 6 3°C) with free access to food and water Real-Time PCR and Western Blotting under a 12-h light-dark cycle (lights on at 7:00 A.M.). Mice The method used for real-time PCR and Western blotting were fed control PicoLab Rodent Diet 20 (chow 3.43 kcal/g; was described previously (23). Primer sequences are listed PMI Nutrition International) or 60 kcal% HFD (5.24 kcal/g; in Supplementary Table 2. D12492; Research Diets Inc.) for 12 weeks, and physiological experiments were performed thereafter. Male mice were Flow Cytometry used in all experiments. The glucose tolerance test (GTT) The fractionation of the stromal vascular fraction (SVF) of andinsulintolerancetest(ITT)wereconductedafter6h eWAT was analyzed by FACSCanto II and FACSAria II for of fasting, as described previously (23,24). Body composition cell sorting (BD Biosciences), as previously described was analyzed with MRI (MRmini SA; DS Pharma Biomedical, (26,31). Data were analyzed by FACS Diva 6.1.2 (BD Bio- Osaka, Japan), as described previously (25,26). sciences) or FCS Express (De Novo Software). Hyperinsulinemic-Euglycemic Clamp Measurement of Energy Consumption and Locomotor Hyperinsulinemic-euglycemic clamp studies were performed Activity after 7–8 days of recovery from cannulation, as described VO2,VCO2, locomotor activity, and food consumption were previously (27). During clamp studies, human insulin at a measured with metabolic chambers (MK-5000RQ; Muroma- concentration of 2.5 mU/kg/min was infused into mice with chi Kikai, Tokyo, Japan), as described previously (25,26). 1010 Role of PDGF-PDGFRb in Adipose Tissue Expansion Diabetes Volume 66, April 2017 Figure 1—Systemic Pdgfrb KO mice exhibited resistance to diet-induced obesity. Mice were fed a chow diet or an HFD from 8 weeks of age.