Diabetes Volume 67, September 2018 1761 Insulin Regulates Lipolysis and Fat Mass by Upregulating Growth/Differentiation Factor 3 in Adipose Tissue Macrophages Yun Bu,1 Katsuhide Okunishi,1 Satomi Yogosawa,1 Kouichi Mizuno,1 Maria Johnson Irudayam,2 Chester W. Brown,2 and Tetsuro Izumi1,3 Diabetes 2018;67:1761–1772 | https://doi.org/10.2337/db17-1201 Previous genetic studies in mice have shown that func- hematopoietic cells inside WAT, and cause chronic inflam- tional loss of activin receptor–like kinase 7 (ALK7), a type I mation and obesity-related disorders (1). The TG content in transforming growth factor-b receptor, increases lipol- adipocytes is determined by the balance between the syn- ysis to resist fat accumulation in adipocytes. Although thesis and breakdown of TG. Although TG synthesis depends growth/differentiation factor 3 (GDF3) has been sug- on the uptake of nutrients, the rate of lipid removal through gested to function as a ligand of ALK7 under nutrient- lipolysis is proportional to the total fat mass as well as the excess conditions, it is unknown how GDF3 production is activities of lipases, and is regulated by external factors, such METABOLISM regulated. Here, we show that a physiologically low level 2 as catecholamine and insulin. It is important to understand of insulin converts CD11c adipose tissue macrophages the mechanisms of fat accumulation to dissect the patho- (ATMs) into GDF3-producing CD11c+ macrophagesexvivo physiology of obesity. Our previous genetic analyses using and directs ALK7-dependent accumulation of fat in vivo. F2 progeny between the Tsumura, Suzuki, obese diabetes Depletion of ATMs by clodronate upregulates adipose (TSOD) and control BALB/c mice revealed a naturally oc- lipases and reduces fat mass in ALK7-intact obese mice, fi curring mutation in Acvr1c encoding the type I transforming but not in their ALK7-de cient counterparts. Furthermore, – depletion of ATMs or transplantation of GDF3-deficient growth factor-b (TGF-b) receptor activin receptor like ki- – bone marrow negates the in vivo effects of insulin on both nase 7 (ALK7) in BALB/c mice (2 5). The mutation gives rise lipolysis and fat accumulation in ALK7-intact mice. The to a stop codon in the kinase domain of ALK7. The congenic GDF3-ALK7 axis between ATMs and adipocytes repre- strain T.B-Nidd5/3 is isogenic with TSOD mice except for the sents a previously unrecognized mechanism by which BALB/c-derived ALK7 mutation and exhibits decreased ad- insulin regulates both fat metabolism and mass. iposity because of enhanced lipolysis. The activation of ALK7 downregulates the master regulators of adipogenesis, C/EBPa and peroxisome proliferator–activated receptor g (PPARg), in The worldwide prevalence of obesity increases morbidity differentiated adipocytes, which leads to the suppression of and mortality and imposes a growing public health burden. lipolysis and to increases in adipocyte size and TG content. Most excess food intake is converted into fat, and specif- To understand the regulatory mechanisms associated ically into triglycerides (TGs), which are stored in adipocytes with ALK7, it is essential to determine its physiological of white adipose tissue (WAT). As adipocytes accumulate fat ligand. TGF-b family members such as Nodal, inhibin-bB and increase in size, they start to secrete proinflammatory (activin B or activin AB), growth/differentiation factor adipocytokines, recruit or polarize macrophages and other (GDF) 3, and GDF11 bind ALK7 and mediate its signals 1Laboratory of Molecular Endocrinology and Metabolism, Department of Molecular Received 4 October 2017 and accepted 30 May 2018. Medicine, Institute for Molecular and Cellular Regulation, Gunma University, This article contains Supplementary Data online at http://diabetes Maebashi, Japan .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-1201/-/DC1. 2Division of Genetics, Department of Pediatrics, University of Tennessee Health © 2018 by the American Diabetes Association. Readers may use this article as Science Center, Memphis, TN long as the work is properly cited, the use is educational and not for profit, and the 3Research Program for Signal Transduction, Division of Endocrinology, Metabolism work is not altered. More information is available at http://www.diabetesjournals and Signal Research, Gunma University Initiative for Advanced Research, Gunma .org/content/license. University, Maebashi, Japan Corresponding author: Tetsuro Izumi, [email protected], or Katsuhide Okunishi, [email protected]. 1762 Roles of the Insulin-GDF3-ALK7 Axis in Obesity Diabetes Volume 67, September 2018 under specific conditions (6–9). Among these ligands, during shaking. The digested cells were filtered through GDF3 seems to function under nutrient-excess conditions, a 250-mm nylon mesh (Kyoshin Rikoh) and centrifuged at because both GDF3 and ALK7 knockout mice attenuate fat 50g for 10 min. The floating adipocytes were washed with accumulation in the face of high-fat diet (HFD)–induced PBS twice. After dispersing the pellet containing the obesity (9,10). However, it has not been shown that GDF3 stromal-vascular fraction (SVF), the medium was filtered directly activates ALK7 in adipocytes. Besides, neither through a 40-mm nylon mesh and centrifuged at 300g for the producer nor the upstream regulator of GDF3 under 10 min. The pellet was then incubated with erythrocyte- nutrient-excess conditions is known. In the current study, lysing buffer consisting of 155 mmol/L NH4Cl, 5.7 mmol/L we establish GDF3 as the physiological ligand that acti- K2HPO4, and 0.1 mmol/L EDTA at room temperature for vates ALK7 in adipocytes, and CD11c+ adipose tissue 1 min and washed twice with PBS. macrophages (ATMs) as the main cell source of GDF3. The cells in the SVF were resuspended in PBS, 2 mmol/L We further demonstrate that insulin upregulates GDF3 in EDTA, and 2% FBS, and were incubated with excess Fc ATMs ex vivo and stimulates fat accumulation in vivo block (anti-CD16/CD32 antibodies; BD Biosciences) to block through the GDF3-ALK7 signaling pathway. Our findings Fc receptor–mediated, nonspecific antibody binding. Cell reveal a novel mechanism by which insulin regulates surface markers were stained on ice in the dark for 20 min adiposity through ATMs in addition to its classically de- using CD11b-phycoerythrin-Cy7, F4/80-allophycocyanin fined direct effect on adipocytes. (Tonbo Biosciences), and CD11c-phycoerythrin (BD Bio- sciences) monoclonal antibodies. Some cells were stained RESEARCH DESIGN AND METHODS as negative controls with fluorochrome-matched isotype Animal Procedures control antibodies. After excluding dead cells by staining with 7-aminoactinomycin D, live cells were subjected to Animal experiments were performed in accordance with characterization of cell populations or to sorting of spe- the rules and regulations of the Animal Care and Exper- cific cell populations by a FACSVerse or a FACSAriaII Flow imentation Committee, Gunma University. The TSOD Cytometer (BD Biosciences). mouse was originally established from an outbred ddY strain as an inbred strain with obesity and urinary glucose RNA Preparation and Gene Expression Analyses (11). The congenic mouse strain T.B-Nidd5/3 was devel- RNA was extracted using Sepasol-RNA I Super (Nacalai oped and characterized previously (3,4). The GDF3 knock- Tesque). Total RNA (1 mg) was reverse transcribed using out mouse with a genetic background of C57BL/6J was oligo-(dT)12–18 primer and Superscript III (Invitrogen). described previously (10). C57BL/6N and BALB/cA mice Quantitative PCR was performed with SYBR Premix Ex were purchased from CLEA Japan. Only male mice were Taq (TaKaRa Bio) using a LightCyler 480 System (Roche). phenotypically characterized in the current study. Mice The results were normalized against 36B4 mRNA expres- had ad libitum access to water and standard laboratory sion. The primer sequences are listed in Supplementary chow (CE-2; CLEA Japan) in an air-conditioned room with Table 1. 12-h light/dark cycles. An HFD (55% fat, 28% carbohy- Antibodies, Immunoblotting, and Immunostaining drate, and 17% protein in calorie percentage; Oriental Rabbit polyclonal anti-ALK7 antibody was described pre- Yeast Co., Ltd.) was given to mice from 4 weeks of age viously (4). Rabbit monoclonal antibodies toward Smad3, for the indicated duration. For macrophage depletion, phospho-Smad3 (Ser 423/425), Akt, and phospho-Akt (Ser liposomes containing 110 mg/kg body weight clodronate 473) were purchased from Cell Signaling Technology. Rat (ClodronateLiposomes.org) were injected intraperitoneally monoclonal anti-Cripto and goat polyclonal anti-GDF3 twice per week. For the in vivo insulin administration, saline or 0.75 units/kg body weight insulin (Humulin R; antibodies were purchased from R&D Systems. Mouse monoclonal antibodies toward b-actin and a-tubulin Lilly), the amount generally used for insulin tolerance were purchased from Sigma-Aldrich. For immunoblotting, tests, was injected intraperitoneally twice daily. For bone isolated adipocytes and the SVF were lysed with buffer marrow (BM) transplantation, recipient C57BL/6N mice at (20 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 1% Triton 8–10 weeks of age were irradiated twice with an individual X-100, 0.2 mmol/L EDTA, and 1 mmol/L dithiothreitol) dose of 5.4 Gy with a 3-h interval, and subsequently 6 containing protease and phosphatase inhibitors. The pro- received an intravenous injection of 2 3 10 BM cells tein extracts (8–10 mgformacrophagesand20mgfor from donor wild-type or GDF3 knockout mice. Mice