Insulin Regulates Lipolysis and Fat Mass by Upregulating Growth/Differentiation Factor 3 in Adipose Tissue Macrophages

Diabetes Volume 67, September 2018 1761

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 were sacrificed after anesthetization by isoflurane inhalation. other cells) were loaded onto polyacrylamide gels for electrophoresis. For imaging of whole-mount epiWAT, Blood was collected from the inferior vena cava using euthanized mice were perfused with 40 mL of fresh 1% 23-gauge needles and syringes. Serum nonesterified fatty formaldehyde in PBS via intracardiac injection over a few acid (NEFA) levels were measured as a marker of lipolysis minutes. EpiWAT was subdivided into small pieces by NEFA C-test (Wako). (;0.1 cm3) by scissors, and was then fixed in 1% form- Cell Fractionation of Epididymal WAT aldehyde in PBS and blocked in 5% BSA in PBS at room Epididymal WAT (epiWAT) was minced and digested with temperature for 30 min. For immunostaining of CD11c+ 1 mg/mL collagenase type I (Invitrogen) for 1 h at 37°C ATMs, cells attached on slide glasses by Cytospin (Thermo diabetes.diabetesjournals.org Bu and Associates 1763

Fisher Scientific) were fixed with 3.7% formaldehyde in metabolism from ALK7-intact C57BL/6 and ALK7- PBS for 30 min at room temperature. With permeabiliza- deficient BALB/c lean mouse strains fed either regular tion by 0.1% Triton X-100, the tissues or the cells were chow or an HFD. We also isolated these tissues from ALK7- incubated with 10 mg/mL anti-GDF3 antibody or control intact TSOD and ALK7-deficient T.B-Nidd5/3 obese mouse IgG overnight at 4°C followed by the Alexa Fluor 488– strains, both of which have the same genetic background conjugated secondary antibody (diluted at 1:500; Invitro- (3,4). Among the 33 members of the mammalian TGF-b gen) for 1 h at room temperature, and were observed under superfamily (13), GDF3, BMP3, inhibin-bB, and TGF-b1 a laser-scanning confocal microscope. The concentration of showed differential expression in WAT (Fig. 1A and Sup- GDF3 in a medium was measured by mouse GDF3 ELISA plementary Fig. 1). Their expression in WAT was strongly kit (Elabscience). upregulated in C57BL/6 mice fed an HFD compared with those fed regular chow. Some of them were also upregulated Vector Construction and Luciferase Reporter Assay in obese TSOD mice fed regular chow and even in ALK7- The binding site of Smad3 and Smad4 (CAGA) (12) was 14 deficient BALB/c mice fed an HFD. In contrast to the other inserted into the pGL4.10[luc2] vector (Promega). Human three ligands, GDF3 showed a remarkably high and specific embryonic kidney 293 T (HEK293T) cells cultured in expression in epiWAT of TSOD and HFD-fed C57BL/6 mice, DMEM containing 10% FBS and 1 mmol/L L-glutamine which is consistent with previous findings (4,10). We then were transfected with 20 ng of the reporter plasmid, 10 ng examined the ligand activity through ALK7 in HEK293T cells of the control plasmid pGLA474[hRluc/TK] (Promega), expressing a luciferase reporter containing a Smad3/4 re- 12.5 ng of plasmid containing ALK7 cDNA (4), and 6.25 ng sponsive element (12), which acts downstream of ALK7 in of that containing Cripto cDNA derived from mouse em- adipocytes (4). Consistent with a previous finding (9), GDF3 bryo, using Lipofectamine 2000 reagent (Invitrogen). After activated the reporter in a dose-dependent fashion only in 48 h, the recombinant proteins of human GDF3, bone the presence of exogenously expressed ALK7 and Cripto, morphogenetic protein 3 (BMP3), activin B, and TGF-b1 a coreceptor that enhances signaling via the type I and type II (R&D Systems) were added to the medium. After a further receptor kinase complex (14) (Fig. 1B). In contrast, BMP3 did 24 h, the luciferase activities were measured by the Dual- not show such enhancement. Activin B, a dimer of inhibin- Luciferase Reporter Assay System (Promega). The light bB, and TGF-b1 activated the reporter even in the absence units were normalized to Renilla luciferase activity. of ALK7 and Cripto, although both induced slight activa- Lipolysis Assay tion with the receptor expression. These findings make Isolated mouse adipocytes (600 mL) were incubated at GDF3 the most likely candidate ligand for ALK7. 37°C for 3 h in Krebs-Ringer HEPES buffer (20 mmol/L ALK7-deficient T.B-Nidd5/3 mice at 7 weeks of age HEPES, pH 7.4, 120 mmol/L NaCl, 5 mmol/L KCl, showed a significant reduction in epiWAT weight com- 2 mmol/L CaCl2, 1 mmol/L MgCl2, and 1 mmol/L pared with control TSOD mice (Supplementary Fig. 2A). KH2PO4) containing 2 mmol/L glucose and 1% fatty The mice exhibited increased levels of mRNA encoding acid–free BSA. Lipolysis was assessed by measuring the the transcription factors PPARg and C/EBPa,andtheir concentration of glycerol in the buffer using a Free Glyc- downstream genes encoding adipose TG lipase (ATGL) erol Determination Kit (Sigma-Aldrich). and hormone-sensitive lipase (HSL), as previously reported in older mice (4). Serum levels of NEFA reflecting Statistical Analysis enhanced lipolysis were also elevated relative to control 6 All quantitative data were expressed as the mean SD. TSOD mice. Therefore, the ALK7-deficient phenotypes Data analysis used GraphPad Prism software. The P values becomeovertat7weeksofage.GDF3inhibitedlipolysis were calculated using Student t test, one-way ANOVA with in adipocytes from TSOD mice at this age, whereas BMP3, Tukey multiple-comparison test, or repeated-measures activinB,orTGF-b1didnot(Fig.2A), which is consistent ANOVA with Bonferroni multiple-comparison test, as with the findings from the luciferase assays (Fig. 1B). fi appropriate, to determine signi cant differences between Importantly, GDF3 inhibited lipolysis and activated the group means. downstream Smad3 by phosphorylation only in ALK7- intact adipocytes from TSOD mice, but not in ALK7- RESULTS deficient adipocytes from T.B-Nidd5/3 mice (Fig. 2B and GDF3 Produced From CD11c+ ATMs Functions as C). These findings establish that GDF3 can signal through a Ligand of ALK7 in Adipocytes ALK7 in adipocytes. Because ALK7 knockout mice show reduced fat accumu- Because GDF3 is expressed in thymus, spleen, and BM lation when fed an HFD, but exhibit normal weight when as well as in WAT (Fig. 1A), as originally reported (15), it fed regular chow (9), the ALK7 signal could be activated might be expressed in hematopoietic cells rather than adi- under nutrient-excess conditions. We thus screened TGF-b pocytes in WAT. To identify the cell source of GDF3, we superfamily members that exhibit differential expressions first dissociated the epiWAT into the SVF and mature adi- depending on nutritional states and also between the pocytes, then further fractionated SVF cells by fluorescence- absence or presence of functional ALK7. For this purpose, activated cell sorting using fluorochrome-conjugated we isolated tissues potentially involved in nutritional antibodies targeting macrophage surface markers (16). 1764 Roles of the Insulin-GDF3-ALK7 Axis in Obesity Diabetes Volume 67, September 2018

Figure 1—Screening TGF-b superfamily members to identify ALK7 ligands. A: TSOD mice and their ALK7-deficient counterparts, T.B- Nidd5/3 mice, fed regular chow (RC) were sacrificed at 10 weeks of age. C57BL/6 (B6) and BALB/c (BALB) mice fed either RC or an HFD from 4 weeks of age were sacrificed at 14 weeks of age. Total RNA was isolated from the indicated tissues, including epiWAT, inguinal WAT (ingWAT), and brown adipose tissue (BAT), and mRNA levels of GDF3, BMP3, inhibin-bB, and TGF-b1 were quantified and normalized to the average values in epiWAT of C57BL/6 mice fed RC (n = 3). B: HEK239T cells were transfected with plasmids encoding ALK7 and/or Cripto. The protein levels of ALK7 and Cripto were examined at 48-h post-transfection by immunoblotting (left panel). HEK293T cells were transfected with plasmids encoding ALK7 and Cripto and simultaneously with a luciferase reporter fused with the Smad-binding promoter element. At 48-h post-transfection, different concentrations (0, 50, 150, and 400 ng/mL) of the indicated recombinant protein were added to the cells. The luciferase activities were measured after a further 24 h (middle panel: GDF3, n = 4; BMP3, n = 3; right panel, n = 3). *P , 0.05, **P , 0.01, ***P , 0.001; Student t test. +P , 0.05, ++P , 0.01, +++P , 0.001; repeated-measures ANOVA.

GDF3 transcripts were enriched in the SVF, particularly in GDF3 antibody revealed that most of the CD11c+ ATMs CD11b+ F4/80+ macrophages (deﬁned as ATMs), with the express GDF3 (94.8 6 2.2%; n =3:;100 cells were greatest elevation seen in those expressing CD11c (Fig. 2D examined in total). GDF3+ cells were located around in- and Supplementary Fig. 2B). Immunostaining with anti- dividual adipocytes in WAT, consistent with localization diabetes.diabetesjournals.org Bu and Associates 1765

Figure 2—GDF3 acts on ALK7 within WAT. Primary adipocytes derived from epiWAT of 7-week-old TSOD or T.B-Nidd5/3 mice were incubated with the indicated recombinant protein (400 ng/mL) for 3 h (A and B) or 30 min (C). Glycerol release was measured and normalized to the average values of control TSOD adipocytes (A and B, n = 3). Phosphorylation of Smad3 in adipocytes was examined by immunoblotting with the indicated antibodies (C). The band with a black arrowhead in the p-Smad3 panel is a nonspeciﬁc protein. D: EpiWAT of 7-week-old TSOD mice were biochemically separated into adipocytes and the SVF. The SVF was then fractionated by FACS as shown in Supplementary Fig. 2B. GDF3 mRNA levels were quantiﬁed in each of the cell fractions (left panel: epiWAT, n = 4; adipocytes, n = 3; SVF, n = 4; CD11b2 cells in the SVF, n = 5; CD11b+ cells in the SVF, n = 4; CD11b+ F4/802 nonmacrophage cells, n = 3; CD11b+ F4/80+ macrophages, n = 3; CD11c2 macrophages, n = 4; and CD11c+ macrophages, n = 6). The extracts of CD11c+ and CD11c2 ATMs were immunoblotted with the indicated antibodies (middle top panel). CD11c+ ATMs (middle bottom panel: white bars, 10 mm) and whole-mount epiWAT (right panel: yellow bars, 50 mm) were immunostained with control IgG or anti-GDF3 antibody. ##P , 0.01, ###P , 0.001; one-way ANOVA. +P , 0.05, ++P , 0.01, +++P , 0.001; repeated-measures ANOVA.

in ATMs. In contrast, BMP3 and inhibin-bB were ex- monocyte chemotactic protein-1 (MCP-1), NLR family pyrin pressed mainly in mature adipocytes, whereas TGF-b1 domain containing 3 (NLRP3), and caspase-1 (Supple- was ubiquitously expressed in every cell fraction (Supple- mentary Fig. 1B and D). mentary Fig. 2C). Concomitant increases in the CD11c and GDF3 transcripts were also found in the SVF of HFD-fed Macrophage Depletion Reverses the Effects of ALK7 on C57BL/6 mice (Supplementary Fig. 1D). Although inflam- Adiposity masome activation has recently been shown to induce To evaluate the role of GDF3-producing ATMs in vivo, we GDF3 in ATMs from aged mice (17), the GDF3 induction intraperitoneally injected clodronate to deplete macro- in TSOD or HFD-treated C57BL/6 mice was not accom- phages (18). Clodronate treatment partially but signifi- panied by the upregulation of inflammasome activation– cantly decreased the percentage of ATMs, including that of related genes, such as tumor necrosis factor-a (TNF-a), CD11c+ ATMs, as well as the expression of F4/80, CD11c, 1766 Roles of the Insulin-GDF3-ALK7 Axis in Obesity Diabetes Volume 67, September 2018

Figure 3—Effects of macrophage depletion by clodronate. PBS or clodronate encapsulated in liposomes (CLO) was injected intraperitoneally into TSOD and T.B-Nidd5/3 mice twice a week for 3 weeks from 4 weeks of age. Three days after the ﬁnal injection at 7 weeks of age, the SVF was isolated from epiWAT. A: The mRNA level of GDF3 in SVF (TSOD, n = 8; T.B-Nidd5/3, n = 4), body weights at 4 weeks of age, and body and epiWAT weights and their ratio at 7 weeks of age (n = 5) in mice with or without CLO treatment. B: The mRNA levels of adipose transcription factors and lipases in epiWAT and serum NEFA concentrations normalized to the epiWAT weight (TSOD, n = 8; T.B-Nidd5/3, n = 4). #P , 0.05, ##P , 0.01, ###P , 0.001; one-way ANOVA.

and GDF3, in both TSOD and T.B-Nidd5/3 mice (Fig. 3A and ATMs isolated from epiWAT of TSOD mice showed ele- Supplementary Fig. 3A and B). However, clodronate de- vated levels of the GDF3 transcript during culture in FBS- creased total body weight, particularly epiWAT weight, only containing medium (Supplementary Fig. 4A), suggesting 2 in TSOD mice, indicating that the effects of the drug depend that some FBS component converts CD11c to CD11c+ on intact ALK7. Furthermore, clodronate increased the ATMs and concomitantly induces GDF3 expression. Be- PPARg, C/EBPa, ATGL, and HSL transcripts, and the serum cause obesity is frequently coincident with hyperinsuline- NEFA concentration normalized to the epiWAT weight, in mia, we suspected that insulin might upregulate GDF3. TSOD mice (Fig. 3B). Therefore, the effects of macrophage Plasma insulin concentrations are ;170 pmol/L in lean depletion from ALK7-intact TSOD mice are remarkably BALB/c mice and ;1.7 nmol/L in obese TSOD mice (2). similar to the phenotypic changes in ALK7-deﬁcient T.B- Ex vivo administration of 10 mU/mL insulin (61 pmol/L) Nidd5/3 mice when compared with control TSOD mice (3,4), increased expression of both CD11c and GDF3 after a 24-h 2 indicating that the GDF3-ALK7 axis represents a major link culture in CD11c macrophages derived from epiWAT of between macrophages and adipocytes in the regulation of TSOD mice, and wortmannin, an inhibitor of phosphati- whole-body lipid metabolism and fat accumulation. dylinositol 3-kinase, inhibited insulin-induced GDF3 upregulation (Fig. 4A). Insulin also increased the expression of Insulin Upregulates GDF3 in ATMs the typical M2 markers arginase and chitinase-like 3, but We next explored the external factors that increase GDF3 not that of the M1 markers TNF-a and MCP-1. Although 2 production under nutrient-excess conditions. CD11c 61 pmol/L insulin induced GDF3 in ATMs, it increased diabetes.diabetesjournals.org Bu and Associates 1767

Figure 4—Effects of insulin administered to CD11c2 ATMs or adipocytes. A: CD11c2 macrophages from epiWAT of 7-week-old TSOD mice (1.5 3 106 cells/24-well dish) were incubated with or without 61 pmol/L insulin in Krebs-Ringer HEPES buffer for 24 h (left panel; n = 7). Some were pretreated with the indicated concentration of wortmannin 10 min before the 24-h incubation (right panel; n = 3). The mRNA levels of the indicated genes were quantiﬁed and normalized to those without insulin incubation in each experiment (middle panel). Insulin-induced phosphorylation of Akt in CD11c2 macrophages pretreated with or without 100 nmol/L wortmannin was examined by immunoblotting with the indicated antibodies (right panel). B: Primary adipocytes isolated from epiWAT of TSOD or T.B-Nidd5/3 mice were incubated for 30 min with 0 mol/L, 61 pmol/L, or 25 nmol/L insulin or with 400 ng/mL GDF3. The cell extracts were immunoblotted with the indicated antibodies. The band with a black arrowhead in the p-Smad3 panel is a nonspeciﬁc protein. C: Adipocytes were incubated with or without 10 mmol/L isoproterenol plus 0 mol/L, 61 pmol/L, or 25 nmol/L insulin for 3 h. Glycerol levels in the medium were measured and normalized to the average value of TSOD adipocytes without insulin or isoproterenol incubation (n = 3). D and E: CD11c2 ATMs from TSOD mice were cultured with or without 61 pmol/L insulin for 24 h as in A. The conditioned medium of the macrophages was harvested after centrifugation of the culture plate at 300g for 10 min at 4°C, and the concentration of GDF3 was measured (D, left panel; n = 4). The conditioned medium warmed to 37°C was incubated with adipocytes of TSOD mice for 30 min to examine its effect on Smad3 phosphorylation (D, right panel), or with adipocytes of TSOD (n = 5) or T.B-Nidd5/3 (n = 3) mice for 3 h to examine its effect on glycerol release (E). *P , 0.05, **P , 0.01, ***P , 0.001; Student t test. #P , 0.05, ###P , 0.001; one-way ANOVA. +P , 0.05, ++P , 0.01, +++P , 0.001; repeated-measures ANOVA. Sups, supernatants.

GDF3 only weakly in macrophages derived from lung, in ATMs. These ﬁndings indicate the tissue selectivity of peritoneum, or BM of TSOD mice (Supplementary Fig. insulin sensitivity in macrophages. 4B). This was evident in the low level of expression of the Although the above ﬁndings raise the possibility that insulin receptor in these macrophages in contrast to that insulin inhibits lipolysis and accumulates fat in adipocytes 1768 Roles of the Insulin-GDF3-ALK7 Axis in Obesity Diabetes Volume 67, September 2018

Figure 5—Effects of insulin administered to a whole body. A: Saline or insulin (0.75 units/kg body weight) was injected intraperitoneally into TSOD (top panels; n = 10) and T.B-Nidd5/3 (bottom panels; n = 8) mice twice daily for 2 weeks from 5 weeks of age. Shown are the weight ratio of epiWAT to total body, mRNA levels of ATGL and HSL in epiWAT, and serum NEFA concentration normalized to the epiWAT weight. B: Saline or insulin was injected to C57BL/6 (top panels; n = 8) and BALB/c mice (bottom panels; n = 8) that had been fed an HFD for 3 weeks from 4 weeks of age, and the effects of insulin were examined as in A.*P , 0.05, **P , 0.01; Student t test.

through the upregulation of GDF3 in ATMs, insulin is adipocytes. We conﬁrmed that insulin phosphorylates the generally believed to do so by directly acting on adipocytes. downstream Akt kinase, but does not activate Smad3 by We next investigated the effects of insulin on isolated a noncanonical pathway, in adipocytes (Fig. 4B). However, diabetes.diabetesjournals.org Bu and Associates 1769

Figure 6—Insulin regulates fat metabolism and mass through the upregulation of GDF3 in ATMs. A: C57BL/6 mice fed an HFD (n = 7 per group) were treated with PBS or clodronate (CLO) from 4 weeks of age for 3 weeks, as described in Fig. 3, and were also treated with saline or insulin from 5 weeks of age for 2 weeks, as described in Fig. 5B. Shown are GDF3 mRNA levels in the SVF, the weight ratio of epiWAT to total body, serum NEFA concentration normalized to the epiWAT weight, and ATGL and HSL mRNA levels in epiWAT. B: The BM of WT or GDF3 knockout (KO) C57BL/6 mice were transplanted into WT C57BL/6 mice at 8–10 weeks of age. The recipient mice were fed an HFD for 3 weeks and treated with insulin for 2 weeks from 6 and 7 weeks after the BM transfer, respectively (n =6–9/group). #P , 0.05, ##P , 0.01, ###P , 0.001; one-way ANOVA. C: Scheme of the insulin-GDF3-ALK7 axis. See text in DISCUSSION. IR, insulin receptor. 1770 Roles of the Insulin-GDF3-ALK7 Axis in Obesity Diabetes Volume 67, September 2018 the concentration of insulin (61 pmol/L) we adminis- GDF3 Mediates the Activity of Insulin to Promote tered ex vivo to ATMs (Fig. 4A) did not inhibit basal or Adiposity In Vivo catecholamine-induced lipolysis in adipocytes, although a To further substantiate the role of the GDF3-ALK7 axis in higher concentration of insulin (25 nmol/L) did so (Fig. 4C). insulin activity in vivo, we injected clodronate to deplete These findings indicate that a much higher dose of insulin macrophages and then administered insulin to C57BL/6 is required to directly inhibit lipolysis in adipocytes than mice fed an HFD. We confirmed that neither clodronate is required to upregulate GDF3 in ATMs. Although ALK7 nor insulin treatment alters the food intake of mice deficiency has been reported to enhance catecholamine- (Supplementary Fig. 5B). Clodronate treatment markedly + induced lipolysis in adipocytes (19), we found that unsti- decreased ATMs, including CD11c ATMs, and concomi- mulated lipolysis is already elevated and that the extent of tantly reduced GDF3 levels in the SVF (Fig. 6A and stimulation by catecholamine is not changed in ALK7-de- Supplementary Fig. 5B). Remarkably, it eliminated the ficient adipocytes (Fig. 4C). These findings confirm the in vivo effects of insulin to increase CD11c and GDF3 previous finding that ALK7 deficiency elevates basal lipol- in the SVF and adiposity in the whole body, and to decrease ysis by affecting the expression levels of adipose lipases (4). adipose lipases and the serum concentration of NEFA. To reinforce the functional significance of the activity These findings demonstrate that insulin can regulate fat of insulin through GDF3 production from ATMs, we per- metabolism and mass through its effects on macrophages formed reconstitution assays by incubating adipocytes in vivo. 2 with the supernatant of CD11c ATMs that had been Finally, we performed BM transplantation experiments treated with or without 61 pmol/L insulin. Note that this to directly prove the involvement of GDF3 in the insulin concentration of insulin does not directly inhibit lipolysis activity. We transplanted the BM of GDF3-deficient in isolated adipocytes (Fig. 4C). Insulin induced the secre- C57BL/6 mice (10) to wild-type C57BL/6 mice to evade tion of GDF3 into their supernatants (1–2 ng/mL), which the cell elimination by the immune system due to MHC dose-dependently increased the phosphorylation of Smad3 mismatch. The recipient mice were then fed an HFD and and inhibited lipolysis in adipocytes of ALK7-intact TSOD treated with insulin. We confirmed that GDF3 deficiency in mice, but not in those of ALK7-deficient T.B-Nidd5/3 mice BM cells does not affect the number of ATMs (Supple- (Fig. 4D and E). We confirmed that 61 pmol/L insulin did mentary Fig. 5C). In contrast to the mice harboring the not change the expression levels of inhibin-bB and TGF-b1 wild-type BM, those harboring the GDF3-deficient BM, in the ATMs (Supplementary Fig. 4C), both of which can and thus losing GDF3 in the SVF failed to mediate the induce Smad3 phosphorylation in adipocytes. These find- in vivo effects of insulin to inhibit lipolysis in the WAT ings indicate that the insulin-stimulated release of GDF3 (Fig. 6B). These findings demonstrate that GDF3 produc- from ATMs successfully inhibits lipolysis in adipocytes tion is necessary for insulin to regulate fat metabolism and ex vivo. mass under nutrient-excess conditions.

Insulin Inhibits Lipolysis and Accumulates Fat in an DISCUSSION ALK7-Dependent Manner In Vivo We showed that GDF3 produced from CD11c+ ATMs acts To clarify whether insulin functions through the GDF3- as a ligand of ALK7 in adipocytes to inhibit lipolysis and ALK7 signaling pathway in vivo, we intraperitoneally accumulate fat under nutrient-excess conditions. The administered insulin twice a day for 2 weeks to TSOD GDF3-ALK7 axis within WAT should represent the major and T.B-Nidd5/3 mice. This in vivo insulin treatment interactive mechanism between macrophages and adipo- elevated the WAT weight and decreased the levels of cytes in the regulation of adiposity, because nonselective the ATGL and HSL transcripts and serum NEFA in an macrophage depletion by clodronate highlights the ALK7- ALK7-dependent manner (Fig. 5A), suggesting that insulin specific effects, such as decreases in body and epiWAT inhibits lipolysis and accumulates fat through the upre- weights, and increases in the expressions of C/EBPa, gulation of GDF3 in ATMs. PPARg, ATGL, and HSL, as well as NEFA production in In order to exclude the possibility that the effects of WAT, in ALK7-intact TSOD mice, but not in their ALK7- insulin via the GDF3-ALK7 axis are applicable only to the deficient counterparts. Although many studies have fo- TSOD strain, for which the molecular pathogenesis of cused on the effects of macrophages in the formation of obesity and diabetes is unknown (2), we administered chronic inflammation associated with obesity, the current insulin to a commonly used C57BL/6 strain fed an HFD study demonstrates the role of ATMs in fat accumulation that indeed expressed ALK7 in WAT (Supplementary Fig. per se. Although CD11c+ macrophages are conventionally 5A). Insulin increased adiposity in parallel with reductions understood to be M1 macrophages that are recruited to in the expression of adipose lipases in epiWAT and serum and/or polarized in obese WAT to induce a chronic in- NEFA concentrations in C57BL/6 mice (Fig. 5B). However, flammatory state (20), the GDF3-producing cells express no such effects were found in ALK7-deficient BALB/c mice a substantial level of M2 markers. Similar to our findings, fed an HFD. These findings indicate that the effects of it has recently been shown that a prototypical M2 marker, insulin via ALK7 under nutrient-excess conditions con- CD301b, as well as arginase, are selectively expressed in tinue irrespective of the mouse strain. CD11c+ mononuclear phagocytes including ATMs, and diabetes.diabetesjournals.org Bu and Associates 1771 that depleting these cells leads to weight loss and increased (4,5). As such, the insulin-GDF3-ALK7 axis plays a pivotal insulin sensitivity in mice (21). role in both physiological and pathological fat accumulation We found that a physiologically low concentration of in WAT. 2 insulin alters the properties of CD11c ATMs ex vivo by increasing the expressions of CD11c and GDF3. Moreover, in vivo insulin administration inhibits lipolysis and Acknowledgments. The authors thank the members of the Laboratory of expands WAT in an ALK7-dependent manner, which indi- Molecular Endocrinology and Metabolism, Gunma University, particularly T. Nara, cates that insulin regulates fat metabolism and mass via E. Kobayashi, and T. Ushigome for colony maintenance of mice and S. Shigoka the GDF3-ALK7 axis. Consistently, the in vivo effects of for assistance in preparing the manuscript. The authors also thank the staffs at Bioresource Center, Gunma University for help in the breeding of the mice. insulin on WAT are absent after the depletion of macro- Funding. This work was supported by the Japan Society for the Promotion of phages or the transplantation of GDF3-deficient BM. It is fi Science KAKENHI grant JP25860739 to S.Y. and grants JP24659442 and intriguing that ATMs appear to speci cally express a high JP25126702 to T.I. and grants from the Japan Diabetes Foundation and the level of insulin receptor compared with macrophages in Novo Nordisk Insulin Study Award (to T.I.). other tissues. Although insulin is generally thought to Duality of Interest. No potential conflicts of interest relevant to this article inhibit lipolysis directly in adipocytes by regulating the were reported. cAMP-mediated signaling pathway (22–25) and/or by sup- Author Contributions. Y.B., S.Y., and K.M. performed experiments. K.O. pressing the transcription of adipose lipases (26–28), these performed experiments, designed experiments, and wrote the paper. M.J.I. and actions in adipocytes have been detected only at higher C.W.B. provided experimental reagents. T.I. designed experiments and wrote concentrations of insulin (1–100 nmol/L) compared with the paper. T.I. is the guarantor of this work and, as such, had full access to all the those applied to ATMs in the current study (61 pmol/L). data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Infact,weobservedthat25nmol/Linsulincandirectly inhibit both basal and catecholamine-stimulated lipolysis References in adipocytes, whereas 61 pmol/L insulin cannot. There- 1. Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. Annu Rev fore, insulin can differentially regulate fat metabolism Immunol 2011;29:415–445 and mass depending on its local concentration in WAT. 2. Hirayama I, Yi Z, Izumi S, et al. Genetic analysis of obese diabetes in the The novel pathway through ATMs might explain the TSOD mouse. Diabetes 1999;48:1183–1191 exquisite sensitivity of lipolysis to suppression by insulin 3. Mizutani S, Gomi H, Hirayama I, Izumi T. Chromosome 2 locus Nidd5 has infused into human subjects (half-maximal suppression at a potent effect on adiposity in the TSOD mouse. Mamm Genome 2006;17: 375–384 101 pmol/L) (29). 4. Yogosawa S, Mizutani S, Ogawa Y, Izumi T. Activin receptor-like kinase Given that GDF3 induction in ATMs requires a minimal 7 suppresses lipolysis to accumulate fat in obesity through downregulation of concentration of insulin, the GDF3-ALK7 pathway should peroxisome proliferator-activated receptor g and C/EBPa. Diabetes 2013;62: be active at the beginning of hyperinsulinemia under 115–123 nutrient-excess conditions. This has a clinical implica- 5. Yogosawa S, Izumi T. Roles of activin receptor-like kinase 7 signaling and its tion for the importance of “early intervention” in adi- target, peroxisome proliferator-activated receptor g, in lean and obese adipocytes. posity before the manifestation of insulin resistance. Adipocyte 2013;2:246–250 Insulin resistance–related chronic hyperinsulinemia may 6. Reissmann E, Jörnvall H, Blokzijl A, et al. The orphan receptor ALK7 and the accelerate fat accumulation even under the same energy Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate balance via activation of the GDF3-ALK7 axis, which makes development. Genes Dev 2001;15:2010–2022 it much harder for obese individuals to reduce adiposity. 7. Tsuchida K, Nakatani M, Yamakawa N, Hashimoto O, Hasegawa Y, Sugino H. Activin isoforms signal through type I receptor serine/threonine kinase ALK7. Mol Future research should focus on novel targeting strategies Cell Endocrinol 2004;220:59–65 for this pathway, such as inhibitors of GDF3 and ALK7, fi fi 8. Andersson O, Reissmann E, Ibáñez CF. Growth differentiation factor speci c depletion of ATMs, and macrophage-speci c in- 11 signals through the transforming growth factor-b receptor ALK5 to regionalize hibition of insulin receptor expression. the anterior-posterior axis. EMBO Rep 2006;7:831–837 In summary, we present a novel mechanism of obesity 9. Andersson O, Korach-Andre M, Reissmann E, Ibáñez CF, Bertolino P. Growth/ (Fig. 6C). Under nutrient-excess conditions, insulin effi- differentiation factor 3 signals through ALK7 and regulates accumulation of 2 ciently activates insulin receptors expressed on CD11c adipose tissue and diet-induced obesity. Proc Natl Acad Sci U S A 2008;105: ATMs and converts them to CD11c+ ATMs to produce 7252–7256 GDF3. GDF3 locally stimulates ALK7 on adipocytes and 10. Shen JJ, Huang L, Li L, Jorgez C, Matzuk MM, Brown CW. Deficiency of activates Smads 2–4 to downregulate PPARg, C/EBPa, and growth differentiation factor 3 protects against diet-induced obesity by selectively – also adipose lipases to store excess nutrient as fat (4). acting on white adipose. Mol Endocrinol 2009;23:113 123 11. Suzuki W, Iizuka S, Tabuchi M, et al. A new mouse model of spontaneous However, persistent activation of this physiological pathway diabetes derived from ddY strain. Exp Anim 1999;48:181–189 enlarges adipocytes and may change adipocytokine reper- fl 12.DennlerS,ItohS,VivienD,tenDijkeP,HuetS,GauthierJM.Direct toires to cause chronic in ammation and insulin resistance. binding of Smad3 and Smad4 to critical TGF b-inducible elements in the In fact, ALK7-intact, aged obese mice exhibit elevated levels promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 1998; of proinflammatory MCP-1 and TNF-a, a reduced level of 17:3091–3100 insulin-sensitizing adiponectin, and greater glucose intoler- 13. Shi M, Zhu J, Wang R, et al. Latent TGF-b structure and activation. Nature ance, compared with their ALK7-deficient counterparts 2011;474:343–349 1772 Roles of the Insulin-GDF3-ALK7 Axis in Obesity Diabetes Volume 67, September 2018

14. Chen C, Ware SM, Sato A, et al. The Vg1-related protein Gdf3 acts in a Nodal 22. Burns TW, Terry BE, Langley PE, Robison GA. Insulin inhibition of lipolysis of signaling pathway in the pre-gastrulation mouse embryo. Development 2006;133: human adipocytes: the role of cyclic adenosine monophosphate. Diabetes 1979; 319–329 28:957–961 15. McPherron AC, Lee SJ. GDF-3 and GDF-9: two new members of the 23. Choi YH, Park S, Hockman S, et al. Alterations in regulation of energy transforming growth factor-b superfamily containing a novel pattern of cysteines. homeostasis in cyclic nucleotide phosphodiesterase 3B-null mice. J Clin Invest J Biol Chem 1993;268:3444–3449 2006;116:3240–3251 16. Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat Rev 24. Lafontan M, Langin D. Lipolysis and lipid mobilization in human adipose Immunol 2005;5:953–964 tissue. Prog Lipid Res 2009;48:275–297 17. Camell CD, Sander J, Spadaro O, et al. Inﬂammasome-driven catecholamine 25. Degerman E, Ahmad F, Chung YW, et al. From PDE3B to the regulation of catabolism in macrophages blunts lipolysis during ageing. Nature 2017;550:119– energy homeostasis. Curr Opin Pharmacol 2011;11:676–682 123 26. Kralisch S, Klein J, Lossner U, et al. Isoproterenol, TNFalpha, and insulin 18. Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: downregulate adipose triglyceride lipase in 3T3-L1 adipocytes. Mol Cell Endocrinol mechanism of action, preparation of liposomes and applications. J Immunol 2005;240:43–49 Methods 1994;174:83–93 27. Kershaw EE, Hamm JK, Verhagen LA, Peroni O, Katic M, Flier JS. Adipose 19. Guo T, Marmol P, Moliner A, et al. Adipocyte ALK7 links nutrient overload to triglyceride lipase: function, regulation by insulin, and comparison with adipo- catecholamine resistance in obesity. eLife 2014;3:e03245 nutrin. Diabetes 2006;55:148–157 20. Lumeng CN, Bodzin JL, Saltiel AR. Obesity induces a phenotypic switch 28. Kim JY, Tillison K, Lee JH, Rearick DA, Smas CM. The adipose tissue tri- in adipose tissue macrophage polarization. J Clin Invest 2007;117:175– glyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-a in 3T3-L1 184 adipocytes and is a target for transactivation by PPARgamma. Am J Physiol 21. Kumamoto Y, Camporez JPG, Jurczak MJ, et al. CD301b(+) mononuclear Endocrinol Metab 2006;291:E115–E127 phagocytes maintain positive energy balance through secretion of resistin-like 29. Campbell PJ, Carlson MG, Nurjhan N. Fat metabolism in human obesity. Am J molecule alpha. Immunity 2016;45:583–596 Physiol 1994;266:E600–E605