Prokineticin Receptor 1 as a Novel Suppressor of Preadipocyte Proliferation and Differentiation to Control Obesity Ce´cilia Szatkowski1., Judith Vallet1., Mojdeh Dormishian1., Nadia Messaddeq2, Phillippe Valet3, Mounia Boulberdaa1, Daniel Metzger2, Pierre Chambon2, Canan G. Nebigil1* 1 Institute of Research and Biotechnology of Strasbourg, Centre national de la recherche scientifique, UMR7242, University of Strasbourg, Medalis/Labex, Drug Discovery Center, Illkirch, France, 2 Institute of Genetic and Molecular and Cellular Biology, Centre national de la recherche scientifique UMR7104, Institut National de la Sante´ et de la Recherche Me´dicale U964, University of Strasbourg, Illkirch, France, 3 Institutes of Cardiovascular and Metabolic Diseases, Institut National de la Sante´ et de la Recherche Me´dicale–University of Paul Sabatier UMR 1048, Toulouse, France Abstract Background: Adipocyte renewal from preadipocytes occurs throughout the lifetime and contributes to obesity. To date, little is known about the mechanisms that control preadipocyte proliferation and differentiation. Prokineticin-2 is an angiogenic and anorexigenic hormone that activate two G protein-coupled receptors (GPCRs): PKR1 and PKR2. Prokineticin- 2 regulates food intake and energy metabolism via central mechanisms (PKR2). The peripheral effect of prokineticin-2 on adipocytes/preadipocytes has not been studied yet. Methodology/Principal Findings: Since adipocytes and preadipocytes express mainly prokineticin receptor-1 (PKR1), here, we explored the role of PKR1 in adipose tissue expansion, generating PKR1-null (PKR12/2) and adipocyte-specific (PKR1ad2/ 2) mutant mice, and using murine and human preadipocyte cell lines. Both PKR12/2 and PKR1ad2/2 had excessive abdominal adipose tissue, but only PKR12/2 mice showed severe obesity and diabetes-like syndrome. PKR1ad2/2) mice had increased proliferating preadipocytes and newly formed adipocyte levels, leading to expansion of adipose tissue. Using PKR1-knockdown in 3T3-L1 preadipocytes, we show that PKR1 directly inhibits preadipocyte proliferation and differentiation. These PKR1 cell autonomous actions appear targeted at preadipocyte cell cycle regulatory pathways, through reducing cyclin D, E, cdk2, c-Myc levels. Conclusions/Significance: These results suggest PKR1 to be a crucial player in the preadipocyte proliferation and differentiation. Our data should facilitate studies of both the pathogenesis and therapy of obesity in humans. Citation: Szatkowski C, Vallet J, Dormishian M, Messaddeq N, Valet P, et al. (2013) Prokineticin Receptor 1 as a Novel Suppressor of Preadipocyte Proliferation and Differentiation to Control Obesity. PLoS ONE 8(12): e81175. doi:10.1371/journal.pone.0081175 Editor: Xiaoli Chen, University of Minnesota - Twin Cities, United States of America Received June 21, 2013; Accepted October 9, 2013; Published December 4, 2013 Copyright: ß 2013 Szatkowski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Centre National de la Recherche Scientifique, Fondation Recherche´ Me´dical, l9Association franc¸aise contre les myopathies, Medalis/labex and Re´gion Alsace. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction tissues (WATs). Preadipocytes are capable of proliferating and differentiating into an adipose deposit [7]. Stimulation of the Obesity causes many serious diseases such as type-2 diabetes proliferation of these cells may therefore result principally in an mellitus, cardiovascular diseases and certain types of cancer, and increase in adipocyte number. In mice, preadipocytes can has contributed to increases in mortality and morbidity rates [1]. proliferate and subsequently differentiate into mature adipocytes Identification of factors regulating white fat tissue growth provides [8]. Thus, understanding the mechanisms controlling preadipocyte an important strategy to combat these diseases. Obesity is proliferation and conversion to adipocyte provides insights into the characterized by an expansion of adipose tissue mass due to etiology and prevention of obesity and its associated pathologies. hypertrophy, an increase in adipocyte size [2], and hyperplasia, an Prokineticin-2 is a potent angiogenic [9] and anorexigenic increase in cell number [3]. The expended adipose tissue plays a hormone [10]. It binds two similar G protein-coupled receptors key role for the induction of insulin resistance commonly seen in (GPCRs): PKR1 and PKR2 [11]. This hormone, which is widely obesity [4]. The adipocytes increase in size is due to lipid distributed in mammalian tissues [12], has HIF-1 binding sites and accumulation [5]. Mature adipocytes are postmitotic [6]. Thus, is induced by low oxygen levels [13]. It is involved in diverse effects adipocyte hyperplasia in adults requires the generation of new in peripheral systems, including angiogenesis in the ovary, testis adipocytes from precursor cells (preadipocytes) and stem cells [14,15] and heart [16]. It also stimulates hematopoiesis [17] and resident in the stromal-vascular compartment of white adipose neurogenesis [18]. Prokineticins induce the differentiation of PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e81175 PKR1 Controls Preadipocyte Function murine and human bone marrow cells into the monocyte/ days to the L2/L2 and PKR1ad2/2 mice at the 3 weeks of age. macrophage lineage [19] and activate monocyte proliferation and The second tamoxifen injection was realized at the age of 13 differentiation [20] and macrophage migration [21]. Prokineticin- weeks. All the analyses were performed when the mice were 24 2/PKR1 restores the pluripotency of epicardial progenitor cells and 40 weeks old. Mice were placed in metabolic cages (1 mouse/ and triggers the differentiation of endothelial and vascular smooth cage) and acclimated for 2 days before the experiment. The body muscle cells [16]. By binding to PKR1, prokineticin-2 directly weight, water and food intake, and urine volume were performed promotes angiogenesis, by activating MAPK and Akt [16]. daily for an additional 3 days. The primers utilized in these studies Prokineticin-2 [22] is involved in appetite suppression [10] and for the genotyping analyses were shown in the Table 1. energy homeostasis, thermoregulation (15), via a direct hypotha- lamic mechanism [23]. The intracranial or peripheral injection of Glucose tolerance test (GTT) and insulin tolerance test prokineticin-2 has been shown to reduce food intake and body (ITT) weight in lean and obese mice at the levels of hypothalamus Prior to studies, mice were fasted for 5 h. For GTT, mice (central effect via PKR2) [10] and dorsal vagal complex received an intraperitoneal injection of glucose (1 mg/g body (peripheral effects via PKR1) [24]. However, the role of weight). In ITT studies, mice received an intraperitoneal injection prokineticin-2 and its receptors in adipocyte and preadipocyte of 0.75U of insulin per kg of body weight according to methods function is unknown. described elsewhere [27]. Blood samples were collected from the PKR1 is the principal receptor expressed by preadipocytes and tail vein (tail-snip technique) at various times after the glucose or adipocytes [12]. In this study, we explored the role of PKR1 in insulin load, as indicated. Blood glucose was immediately adipocyte function, in vivo using PKR1-null (PKR12/2) and ad2/2) determined on a Contour blood glucose monitoring system adipocyte-specific (PKR1 mutant mice, and in vitro, using (Bayer). murine (3T3-L1) and human preadipocyte cell lines (SGBS). Here we described multiple PKR1 functions regulates preadipocyte Insulin stimulation of Akt activity detected by Western proliferation and differentiation, controlling adipose tissue expan- sion. Blot assay Animals were anaesthetized with 1% pentobarbital and were H Materials and Methods followed by an IP injection of insulin (150 mU/g, Umaline , Rapide). 0 and 20 min after injection mice were scarified and the Ethics statement adipose tissues were harvested for protein extraction. Extracted The animal study was approved by the Animal Care and Use, proteins were transferred to nitrocellulose membranes and and ethics committees of the Perfecture du Bas-Rhin (Permit immunoblotted with the Phospho Akt (Ser-473 and Thr 308) Number: B67–274) with the recommendations in the Guide for Antibody kit (Cell Signaling Technology), following incubation the Care and Use of Laboratory Animals of the French Animal with horseradish peroxidase-conjugated goat anti-rabbit IgG. Care Committee, with European regulation-approved protocols. Phosphorylated protein was visualized by enzyme-linked chemi- The animal experimentation and housing were conducted at the luminescence (Amersham Biosciences) and quantified by scanning accredited Animal Experimentation and housing Facility of the laser densitometry, normalizing to total amounts of the Akt Institut de recherche de l9Ecole de biotechnologie de Strasbourg proteins. (Register number: C67-218-19). The human study was approved by the ethics committee of Histological and electron microscopy analyses Toulouse-Rangueil