Insulin regulates lipolysis and fat mass by upregulating growth/differentiation factor ... · 2019....

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Insulin regulates lipolysis and fat mass by upregulating growth/differentiation

factor 3 in adipose tissue macrophages

Yun Bu1, Katsuhide Okunishi1, Satomi Yogosawa1, Kouichi Mizuno1, Chester W.

Brown2, and Tetsuro Izumi1,3

1Laboratory of Molecular Endocrinology and Metabolism, Department of Molecular

Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi

371-8512, Japan; 2Division of Genetics, Department of Pediatrics, University of

Tennessee Health Science Center, Memphis, TN 38163; and 3Research Program for

Signal Transduction, Division of Endocrinology, Metabolism and Signal Research,

Gunma University Initiative for Advanced Research, Gunma University, Maebashi 371-

8512, Japan.

A short running title: Roles of the insulin-GDF3-ALK7 axis in obesity

Address correspondence to: Tetsuro Izumi, Phone: +81-27-220-8856; E-mail:

[email protected]. Or to Katsuhide Okunishi, Phone: +81-27-220-8877; E-mail:

[email protected].

The word count: 4077-Endnote92=3985>4000 words (Introduction to Discussion)

The number of figures: 6

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Abstract

Previous genetic studies in mice have shown that functional loss of activin receptor-like

kinase 7 (ALK7), a type I transforming growth factor (TGF)-β receptor, increases

lipolysis to resist fat accumulation in adipocytes. Although growth/differentiation factor

3 (GDF3) has been suggested to function as a ligand of ALK7 under nutrient-excess

conditions, it is unknown how GDF3 production is regulated. Here, we show that a

physiologically low level of insulin converts CD11c- adipose tissue macrophages

(ATMs) into GDF3-producing, CD11c+ macrophages ex vivo, and directs ALK7-

dependent accumulation of fat in vivo. Depletion of ATMs by clodronate upregulates

adipose lipases and reduces fat mass in ALK7-intact obese mice, but not in their ALK7-

deficient counterparts. Furthermore, depletion of ATMs or transplantation of GDF3-

deficient bone marrow negates the in vivo effects of insulin on both lipolysis and fat

accumulation in ALK7-intact mice. The GDF3-ALK7 axis between ATMs and

adipocytes represents a previously unrecognized mechanism by which insulin regulates

both fat metabolism and mass.

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Introduction

The worldwide prevalence of obesity increases morbidity and mortality and imposes a

growing public health burden. Most excess food intake is converted into fat, and

specifically into triglycerides (TGs), which is stored in adipocytes of white adipose

tissue (WAT). As adipocytes accumulate fat and increase in size, they start to secrete

proinflammatory adipocytokines, recruit or polarize macrophages and other

hematopoietic cells inside WAT, and cause chronic inflammation and obesity-related

disorders {Gregor, 2011 #37}. The TG content in adipocytes is determined by the

balance between the synthesis and breakdown of TG. While TG synthesis depends on

the uptake of nutrients, the rate of lipid removal through lipolysis is proportional to the

total fat mass as well as the activities of lipases, and is regulated by external factors,

such as catecholamine and insulin. It is important to understand the mechanisms of fat

accumulation to dissect the pathophysiology of obesity. Our previous genetic analyses

using F2 progeny between the Tsumura, Suzuki, obese diabetes (TSOD) and control

BALB/c mice revealed a naturally occurring mutation in Acvr1c encoding the type I

TGF-β receptor, ALK7, in BALB/c mice {Hirayama, 1999 #6}{Mizutani, 2006

#7}{Yogosawa, 2013a #3}{Yogosawa, 2013b #4}. The mutation gives rise to a stop

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codon in the kinase domain of ALK7. The congenic strain, T.B-Nidd5/3, is isogenic

with TSOD mice except for the BALB/c-derived ALK7 mutation and exhibits decreased

adiposity due to enhanced lipolysis. Activation of ALK7 downregulates the master

regulators of adipogenesis, C/EBPα and PPARγ, in differentiated adipocytes, which

leads to suppression of lipolysis and to increases in adipocyte size and TG content.

To understand the regulatory mechanisms associated with ALK7, it is essential

to determine its physiological ligand. TGF-β family members such as Nodal, inhibin-βB

(activin B or activin AB), GDF3, and GDF11 bind ALK7 and mediate its signals under

specific conditions {Reissmann, 2001 #19}{Tsuchida, 2004 #20}{Andersson, 2006

#21}{Andersson, 2008 #5}. Among them, GDF3 seems to function under nutrient-

excess conditions, because both GDF3- and ALK7-knockout mice attenuate fat

accumulation in the face of high-fat diet (HFD)-induced obesity {Andersson, 2008

#5}{Shen, 2009 #22}. However, it has not been shown that GDF3 directly activates

ALK7 in adipocytes. Besides, neither the producer nor the upstream regulator of GDF3

under nutrient-excess conditions is known. In the present study, we establish GDF3 as

the physiological ligand that activates ALK7 in adipocytes, and CD11c+ ATMs as the

main cell source of GDF3. We further demonstrate that insulin upregulates GDF3 in

ATMs ex vivo, and stimulates fat accumulation in vivo through the GDF3-ALK7

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signaling pathway. Our findings reveal a novel mechanism by which insulin regulates

adiposity through ATMs in addition to its classically defined direct effect on adipocytes.

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Research Design and Methods

Animal procedures. Animal experiments were performed in accordance with the rules

and regulations of the Animal Care and Experimentation Committee, Gunma University.

The TSOD mouse was originally established from an outbred ddY strain as an inbred

strain with obesity and urinary glucose {Suzuki, 1999 #32}. The congenic mouse strain,

T.B-Nidd5/3, was developed and characterized elsewhere {Mizutani, 2006

#7}{Yogosawa, 2013 #3}. The GDF3-knockout mouse with a genetic background of

C57BL/6J was previously described {Shen, 2009 #22}. C57BL/6N and BALB/cA mice

were purchased from CLEA Japan. Only male mice were phenotypically characterized

in the present study. Mice had ad libitum access to water and standard laboratory chow

(CE-2; CLEA Japan) in an air-conditioned room with 12-h light-dark cycles. A HFD

(55% fat, 28% carbohydrate, and 17% protein in calorie percentage; Oriental Yeast) was

given to mice from 4 weeks of age for the indicated duration. For macrophage

depletion, mice liposomes containing 110 mg/kg body weight of clodronate

(ClodronateLiposomes.org) was injected intraperitoneally twice per week. For the in

vivo insulin administration, saline or 0.75 U/kg body weight of insulin (Humulin R;

Lilly), the amount generally used for insulin tolerance tests, was injected

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intraperitoneally twice daily. For BM transplantation, recipient C57BL/6N mice at the

ages of 8-10 weeks were irradiated twice with an individual dose of 5.4 Gy with a 3-h

interval, and subsequently received an intravenous injection of 2 × 106 BM cells from

donor wild-type or GDF3-knockout mice. Mice were sacrificed after anesthetization by

isoflurane inhalation. Blood was collected from the inferior vena cava using 23-gauge

needles and syringes. Serum non-esterified fatty acid (NEFA) levels were measured as a

marker of lipolysis by NEFA C-test (Wako).

Cell fractionation of epiWAT. EpiWAT was minced and digested with 1 mg/ml

collagenase type I (Invitrogen) for 1 h at 37°C during shaking. The digested cells were

filtered through a 250-µm nylon mesh (Kyoshin Rikoh) and centrifuged at 50 × g for 10

min. The floating adipocytes were washed with PBS twice. After dispersing the pellet

containing the SVF, the medium was filtered through a 40-µm nylon mesh and

centrifuged at 300 × g for 10 min. The pellet was then incubated with erythrocyte-lysing

buffer consisting of 155 mM NH4Cl, 5.7 mM K2HPO4, and 0.1 mM EDTA at room

temperature for 1 min and washed with PBS twice.

The cells in the stromal-vascular fraction (SVF) were resuspended in PBS, 2

mM EDTA, and 2% FBS, and were incubated with excess Fc block (anti-CD16/CD32

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antibodies, BD Bioscience) to block Fc receptor-mediated, nonspecific antibody

binding. Cell surface markers were stained on ice in the dark for 20 min using CD11b-

phycoerythrin-Cy7, F4/80-allophycocyanin (TONBO Biosciences), and CD11c-

phycoerythrin (BD Biosciences) monoclonal antibodies. Some cells were stained as

negative controls with fluorochrome-matched isotype control antibodies. After

excluding dead cells by staining with 7-aminoactinomycin D, live cells were subjected

to characterization of cell populations or to sorting of specific cell populations by

FACSVerse or FACSAriaII flow cytometers (BD Biosciences).

RNA preparation and gene expression analyses. RNA was extracted using Sepasol-

RNA I Super (Nacalai Tesque). Total RNA (1 µg) was reverse-transcribed using oligo-

(dT)12-18 primer and Superscript III (Invitrogen). Quantitative PCR was performed with

SYBR premix Ex Taq (Takara Bio) using a LightCyler 480 (Roche). The results were

normalized against 36B4 mRNA expression. The primer sequences are listed in

Supplementary Table 1.

Antibodies, immunoblotting, and immunostaining. Rabbit polyclonal anti-ALK7

antibody was described previously {Yogosawa, 2013 #3}. Rabbit monoclonal

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antibodies toward Smad3, phospho-Smad3 (Ser 423/425), Akt, and phospho-Akt (Ser

473) were purchased from Cell Signaling Technology. Rat monoclonal anti-Cripto and

goat polyclonal anti-GDF3 antibodies were purchased from R&D Systems. Mouse

monoclonal antibodies toward β-actin and α-tubulin were purchased from Sigma-

Aldrich. For immunoblotting, isolated adipocytes and the SVF were lysed with buffer

(20 mM HEPES pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.2 mM EDTA, and 1 mM

dithiothreitol) containing protease and phosphatase inhibitors. The protein extracts (8-10

µg for macrophages and 20 µg for other cells) were loaded onto polyacrylamide gels for

electrophoresis. For imaging of whole-mount epididymal WAT (epiWAT), euthanized

mice were perfused with 40 ml of fresh 1% paraformaldehyde (PFA) in PBS via

intracardiac injection over a few minutes. EpiWAT was subdivided into small pieces

(~0.1 cm3) by scissors, and was then fixed in 1% PFA in PBS and blocked in 5% BSA

in PBS at room temperature for 30 min. For immunostaining of CD11c+ ATMs, cells

attached on slide glasses by Cytospin (Thermo Fisher Scientific) were fixed with 3.7%

PFA in PBS for 30 min at room temperature. With permeabilization by 0.1% Triton X-

100, the tissues or the cells were incubated with 10 µg/ml of anti-GDF3 antibody or

control IgG overnight at 4°C followed by the Alexa Fluor 488-conjugated secondary

antibody (diluted at 1:500; Invitrogen) for 1 h at room temperature, and were onserved

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under a laser scanning confocal microscope. The concentration of GDF3 in a medium

was measured by mouse GDF3 ELISA kit (Elabscience Biotechnology).

Vector construction and luciferase reporter assay. The binding site of Smad3 and

Smad4 (CAGA)14 {Dennler, 1998 #9} was inserted into the pGL4.10[luc2] vector

(Promega). HEK293T cells cultured in Dulbecco’s modified Eagle’s medium containing

10% FBS and 1 mM L-glutamine were transfected with 20 ng of the reporter plasmid,

10 ng of the control plasmid pGLA474[hRluc/TK], 12.5 ng of plasmid containing

ALK7 cDNA {Yogosawa, 2013 #3}, and 6.25 ng of that containing Cripto cDNA

derived from mouse embryo, using Lipofectamine 2000 reagent (Invitrogen). After 48

h, the recombinant proteins of human GDF3, bone morphogenetic protein (BMP) 3,

activin B, and TGF-β1 (R&D Systems) were added to the medium. After a further 24 h,

the luciferase activities were measured by the Dual-Luciferase Reporter Assay System

(Promega). The light units were normalized to Renilla luciferase activity.

Lipolysis assay. Isolated mouse adipocytes (600 µl) were incubated at 37°C for 3 h in

Krebs-Ringer Hepes buffer (20 mM HEPES pH 7.4, 120 mM NaCl, 5 mM KCl, 2 mM

CaCl2, 1 mM MgCl2 and 1 mM KH2PO4) containing 2 mM glucose and 1% fatty acid-

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free BSA. Lipolysis was assessed by measuring the concentration of glycerol in the

buffer using a Free Glycerol Determination Kit (Sigma-Aldrich).

Statistical analysis. All quantitative data were expressed as mean ± SD. Data analysis

employed GraphPad Prism software. The p values were calculated using Student’s t-test

or one-way ANOVA with Tukey’s multiple comparison test, as appropriate, to determine

significant differences between group means.

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Results

GDF3 produced from CD11c+ ATMs functions as a ligand of ALK7 in adipocytes.

Because ALK7-knockout mice show reduced fat accumulation when fed a HFD, but

exhibit normal weight when fed regular chow {Andersson, 2008 #5}, the ALK7 signal

could be activated under nutrient-excess conditions. We thus screened TGF-β

superfamily members that exhibit differential expressions depending on nutritional

states and also between the absence or presence of functional ALK7. For this purpose,

we isolated tissues potentially involved in nutritional metabolism from ALK7-intact

C57BL/6 and ALK7-deficient BALB/c lean mouse strains fed either regular chow or a

HFD. We also isolated these tissues from ALK7-intact TSOD and ALK7-deficient T.B-

Nidd5/3 obese strains, both of which have the same genetic background {Mizutani,

2006 #7}{Yogosawa, 2013a #3}. Among the 33 members of the mammalian TGF-β

superfamily {Shi, 2011 #8}, GDF3 and BMP3, inhibin-βB, and TGF-β1 showed

differential expression in WAT (Fig. 1A and Supplementary Fig. 1). Their expression in

WAT was strongly upregulated in C57BL/6 mice fed a HFD compared with those fed

regular chow. Some of them were also upregulated in obese TSOD mice fed regular

chow and even in ALK7-deficient BALB/c mice fed a HFD. In contrast to the other

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three ligands, GDF3 showed a remarkably high and specific expression in epiWAT of

TSOD and HFD-fed C57BL/6 mice, consistent with previous findings {Shen, 2009

#22}{Yogosawa, 2013a #3}. We then examined the ligand activity through ALK7 in

HEK293T cells expressing a luciferase reporter containing a Smad3/4 responsive

element {Dennler, 1998 #9}, which acts downstream of ALK7 in adipocytes

{Yogosawa, 2013a #3}. Consistent with the previous finding {Andersson, 2008 #5},

GDF3 activated the reporter in a dose-dependent fashion only in the presence of

exogenously expressed ALK7 and Cripto, a co-receptor that enhances signaling via the

type I and type II receptor kinase complex {Chen, 2006 #42} (Fig. 1B). In contrast,

BMP3 did not show such enhancement. Activin B, a dimer of inhibin-βB, and TGF-β1

activated the reporter even in the absence of ALK7 and Cripto, although activing B

induced slight activation with the receptor expression. These findings make GDF3 the

most likely candidate ligand for ALK7.

ALK7-deficient T.B-Nidd5/3 mice at 7 weeks of age showed a significant

reduction in epiWAT weight (~ 0.6 g), which was almost equal to the reduction of body

weight, compared with control TSOD mice (Supplementary Fig. 2A). The mice

exhibited increased levels of mRNA encoding the transcription factors PPARγ and

C/EBPα, and their downstream genes encoding adipose triglyceride lipase (ATGL) and

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hormone-sensitive lipase (HSL), as previously reported in older mice {Yogosawa,

2013a #3}. Serum NEFA reflecting enhanced lipolysis were also elevated relative to

control TSOD mice. Therefore, the ALK7-deficient phenotypes become overt at 7

weeks of age. GDF3 inhibited lipolysis in adipocytes from TSOD mice at this age,

whereas neither BMP3, activin B, nor TGF-β1 did so (Fig. 2A), consistent with the

findings from the luciferase assays (Fig. 1B). Importantly, GDF3 inhibited lipolysis and

activated the downstream Smad3 by phosphorylation only in ALK7-intact adipocytes

from TSOD mice, but not in ALK7-deficient adipocytes from T.B-Nidd5/3 mice (Fig.

2B and C). These findings establish that GDF3 can signal through ALK7 in adipocytes.

Because GDF3 is expressed in thymus, spleen, and bone marrow (BM) as well

as in WAT (Fig. 1A), as originally reported {McPherron, 1993 #11}, it might be

expressed in hematopoietic cells rather than adipocytes in WAT. To identify the cell

source of GDF3, we first dissociated the epiWAT into the SVF and mature adipocytes,

then further fractionated SVF cells by fluorescence activated cell sorting using

fluorochrome-conjugated antibodies targeting macrophage surface markers {Gordon,

2005 #14}. GDF3 transcripts were enriched in the SVF, particularly in CD11b+ F4/80+

macrophages (defined as ATMs), with the greatest elevation seen in those expressing

CD11c (Fig. 2D and Supplementary Fig. 2B). Immunostaining with anti-GDF3 antibody

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revealed that most of the CD11c+ ATMs express GDF3 (94.8 ± 2.2%; n = 3:

approximately a hundred cells were examined in total). GDF-positive cells located

around individual adipocytes in WAT, consistent with its localization in ATMs. In

contrast, BMP3 and inhibin-βB were expressed mainly in mature adipocytes, whereas

TGF-β1 was ubiquitously expressed in every cell fraction (Supplementary Fig. 2C).

Concomitant increases in the CD11c and GDF3 transcripts were also found in the SVF

of HFD-fed C57BL/6 mice (Supplementary Fig. 1D). Although inflammasome

activation has recently been shown to induce GDF3 in ATMs from aged mice {Camell,

2017 #40}, the GDF3 induction in TSOD or HFD-treated C57BL/6 mice was not

accompanied by upregulation of inflammasome activation-related genes, such as tumor

necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP-1), NLRP3, and

Caspase 1 (Supplementary Fig. 1B and D).

Macrophage depletion reverses the effects of ALK7 on adiposity. To evaluate the

role of GDF3-producing ATMs in vivo, we intraperitoneally injected clodronate to

deplete macrophages {Van Rooijen, 1994 #33}. Clodronate treatment partially but

significantly decreased the percentage of ATMs, including that of CD11c+ ATMs, as

well as the expression of F4/80, CD11c, and GDF3, in both TSOD and T.B-Nidd5/3

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mice (Fig. 3A and Supplementary Fig. 3A and B). However, clodronate decreased total

body weight, particularly epiWAT weight, only in TSOD mice, indicating that the drug’s

effects depend on intact ALK7. Furthermore, it increased the PPARγ, C/EBPα, ATGL,

and HSL transcripts, and the serum NEFA concentration normalized to the epiWAT

weight, in TSOD mice (Fig. 3B). Therefore, the effects of macrophage depletion from

ALK7-intact TSOD mice are remarkably similar to the phenotypic changes in ALK7-

deficient T.B-Nidd5/3 mice when compared to control TSOD mice {Mizutani, 2006

#7}{Yogosawa, 2013a #3}, indicating that the GDF3-ALK7 axis represents a major link

between macrophages and adipocytes in the regulation of whole body lipid metabolism

and fat accumulation.

Insulin upregulates GDF3 in ATMs. We next explored the external factors that

increase GDF3 production under nutrient-excess conditions. CD11c- ATMs isolated

from epiWAT of TSOD mice showed elevated levels of the GDF3 transcript during

culture in FBS-containing medium (Supplementary Fig. 4A), suggesting that some FBS

component converts CD11c- to CD11c+ ATMs and concomitantly induces GDF3

expression. Because obesity is frequently coincident with hyperinsulinemia, we

suspected that insulin might upregulate GDF3. Plasma insulin concentrations are

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approximately ~170 pM in lean BALB/c mice and ~1.7 nM in obese TSOD mice

{Hirayama, 1999 #6}. Ex vivo administration of 10 µU/ml of insulin (61 pM) increased

both CD11c and GDF3 expressions after a 24-h culture in CD11c- macrophages derived

from epiWAT of TSOD mice, and wortmannin, an inhibitor of phosphatidylinositol-3-

kinase, inhibited insulin-induced GDF3 upregulation (Fig. 4A). Insulin also increased

the expression of the typical M2 markers, arginase (Arg1) and chitinase-like 3 (Ym1),

but not that of the M1 markers, TNFα and MCP-1. Although 61 pM insulin induced

GDF3 in ATMs, it increased GDF3 only weakly in macrophages derived from lung,

peritoneum, or BM of TSOD mice (Supplementary Fig. 4B). This was evident in the

low level of expression of the insulin receptor in these macrophages in contrast to that

in ATMs. These findings indicate the tissue selectivity of insulin sensitivity in

macrophages.

Although the above findings raise the possibility that insulin inhibits lipolysis

and accumulates fat in adipocytes through upregulation of GDF3 in ATMs, insulin is

generally believed to do so by directly acting on adipocytes. We next investigated the

effects of insulin on isolated adipocytes. We confirmed that insulin phosphorylates the

downstream Akt kinase, but does not activate Smad3 by a non-canonical pathway, in

adipocytes (Fig. 4B). However, the concentration of insulin (61 pM) we administered ex

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vivo to ATMs (Fig. 4A) did not inhibit basal or catecholamine-induced lipolysis in

adipocytes, although a higher concentration of insulin (25 nM) did so (Fig. 4C). These

findings indicate that a much higher dose of insulin is required to directly inhibit

lipolysis in adipocytes than is required to upregulate GDF3 in ATMs. Although ALK7

deficiency has been reported to enhance catecholamine-induced lipolysis in adipocytes

{Guo, 2014 #26}, we found that unstimulated lipolysis is already elevated, and that the

extent of stimulation by catecholamine is not changed, in ALK7-deficient adipocytes

(Fig. 4C). These findings confirm the previous finding that ALK7 deficiency elevates

basal lipolysis by affecting the expression levels of adipose lipases {Yogosawa, 2013a

#3}.

To reinforce the functional significance of insulin’s activity through GDF3

production from ATMs, we performed reconstitution assays by incubating adipocytes

with the supernatant of CD11c- ATMs that had been treated with or without 61 pM insulin.

Note that this concentration of insulin does not directly inhibit lipolysis in isolated

adipocytes (Fig. 4C). Insulin induced secretion of GDF3 into their supernatants (1-2

ng/ml), which dose-dependently increased the phosphorylation of Smad3 and inhibited

lipolysis in adipocytes of ALK7-intact TSOD mice, but not in those of ALK7-deficient

T.B-Nidd5/3 mice (Fig. 4D and E). We confirmed that 61 pM insulin did not change the

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expression levels of inhibin-βB and TGF-β1 in the ATMs (Supplementary Fig. 4C), both

of which can induce Smad3 phosphorylation in adipocytes. These findings indicate that

the insulin-stimulated release of GDF3 from ATMs successfully inhibits lipolysis in

adipocytes ex vivo.

Insulin inhibits lipolysis and accumulates fat in an ALK7-dependent manner in

vivo. To clarify whether insulin functions through the GDF3-ALK7 signaling pathway

in vivo, we intraperitoneally administered insulin twice a day for 2 weeks to TSOD and

T.B-Nidd5/3 mice. This in vivo insulin treatment elevated the WAT weight, and

decreased the levels of the ATGL and HSL transcripts and serum NEFA, in an ALK7-

dependent manner (Fig. 5A), suggesting that insulin inhibits lipolysis and accumulates

fat through the upregulation of GDF3 in ATMs.

In order to exclude the possibility that insulin’s effects via the GDF3-ALK7

axis are applicable only to the TSOD strain, for which the molecular pathogenesis of

obesity and diabetes is unknown {Hirayama, 1999 #6}, we administered insulin to a

commonly used C57BL/6 strain fed a HFD that indeed expressed ALK7 in WAT

(Supplementary Fig. 5A). Insulin increased adiposity in parallel with reductions in

expression of adipose lipases in epiWAT and serum NEFA concentration in C57BL/6

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mice (Fig. 5B). However, no such effects were found in ALK7-deficient BALB/c mice

fed a HFD. These findings indicate that insulin’s effects via ALK7 under nutrient-excess

conditions continue irrespective of the mouse strain.

GDF3 mediates the activity of insulin to promote adiposity in vivo. To further

substantiate the role of the GDF3-ALK7 axis in insulin activity in vivo, we injected

clodronate to deplete macrophages and then administered insulin to C57BL/6 mice fed a

HFD. We confirmed that neither clodronate nor insulin treatment alters the food intake

of mice (Supplementary Fig. 5B). Clodronate treatment markedly decreased ATMs,

including CD11c+ ATMs, and concomitantly reduced GDF3 levels in the SVF (Fig. 6A

and Supplementary Fig. 5B). Remarkably, it eliminated the in vivo effects of insulin to

increase CD11c and GDF3 in the SVF and adiposity in the whole body, and to decrease

adipose lipases and the serum concentration of NEFA. These findings demonstrate that

insulin can regulate fat metabolism and mass through its effects on macrophages in

vivo.

Finally, we performed BM transplantation experiments to directly prove the

involvement of GDF3 in the insulin activity. We transplanted the BM of GDF3-deficient

C57BL/6 mice {Shen, 2009 #22} to wild-type C57BL/6 mice to evade the cell

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elimination by the immune system due to major histocompatibility complex mismatch.

The recipient mice were then fed a HFD and treated with insulin. We confirmed that

GDF3 deficiency in BM cells does not affect the numbers of ATM (Supplementary Fig.

5C). In contrast to the mice harboring the wild-type BM, those harboring the GDF3-

deficient BM and thus losing GDF3 in the SVF failed to mediate the in vivo effects of

insulin to inhibit lipolysis in the WAT (Fig. 6B). These findings demonstrate that GDF3

production is necessary for insulin to regulate fat metabolism and mass under nutrient-

excess conditions.

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Discussion

We showed that GDF3 produced from CD11c+ ATMs acts as a ligand of ALK7 in

adipocytes to inhibit lipolysis and accumulate fat under nutrient-excess conditions. The

GDF3-ALK7 axis within WAT should represent the major interactive mechanism

between macrophages and adipocytes in the regulation of adiposity, because

nonselective macrophage depletion by clodronate highlights the ALK7-specific effects,

such as decreases in body and epiWAT weights, and increases in the expressions of

C/EBPα, PPARγ, ATGL, and HSL, as well as NEFA production in WAT, in ALK7-intact

TSOD mice, but not in their ALK7-deficient counterparts. While many studies have

focused on the effects of macrophages in the formation of chronic inflammation

associated with obesity, the present study demonstrates the role of ATMs in fat

accumulation per se. Although CD11c+ macrophages are conventionally understood to

be M1 macrophages that are recruited to and/or polarized in obese WAT to induce a

chronic inflammatory state {Lumeng, 2007 #15}, the GDF3-producing cells express a

substantial level of M2 markers. Similar to our findings, it has recently been shown that

a prototypical M2 marker, CD301b, as well as Arg1, is selectively expressed in CD11c+

mononuclear phagocytes including ATMs, and that depleting these cells leads to weight

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loss and increased insulin sensitivity in mice {Kumamoto, 2016 #28}.

We found that a physiologically low concentration of 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 expands WAT in an

ALK7-dependent manner, which indicates that insulin regulates fat metabolism and

mass via the GDF3-ALK7 axis. Consistently, the in vivo effects of insulin on WAT are

absent after depletion of macrophages or transplantation of GDF3-deficient BM. It is

intriguing that ATMs appear to specifically express a high level of insulin receptor

compared with macrophages in other tissues. Although insulin is generally thought to

inhibit lipolysis directly in adipocytes by regulating the cAMP-mediated signaling

pathway {Burns, 1979 #29}{Choi, 2006 #30}{Lafontan, 2009 #24}{Degerman, 2011

#31}, and/or by suppressing transcription of adipose lipases {Kralisch, 2005

#16}{Kershaw, 2006 #17}{Kim, 2006 #18}, these actions in adipocytes have been

detected only at higher concentrations of insulin (1-100 nM) than those applied to ATMs

in the present study (61 pM). In fact, we observed that 25 nM insulin can directly inhibit

both basal and catecholamine-stimulated lipolysis in adipocytes, whereas 61 pM insulin

cannot. Therefore, insulin can differentially regulate fat metabolism and mass

depending on its local concentration in WAT.

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Given that GDF3 induction in ATMs requires a minimal concentration of

insulin, the GDF3-ALK7 pathway should be active at the beginning of

hyperinsulinemia under nutrient-excess conditions. This has a clinical implication for

the importance of “early intervention” in adiposity before the manifestation of insulin

resistance. Insulin resistance-related chronic hyperinsulinemia may accelerate fat

accumulation even under the same energy balance via activation of the GDF3-ALK7

axis, which makes it much harder for obese individuals to reduce adiposity. Future

research should focus on novel targeting strategies for this pathway, such as inhibitors

of GDF3 and ALK7, specific depletion of ATMs, and macrophage-specific inhibition of

insulin receptor expression.

In summary, we present a novel mechanism of obesity (Fig. 6C). Under

nutrient-excess conditions, insulin efficiently activates insulin receptor expressed on

CD11c- ATMs and converts them to CD11c+ ATMs to produce GDF3. GDF3 locally

stimulates ALK7 on adipocytes and activates Smads 2-4 to downregulate PPARγ,

C/EBPα, and also adipose lipases to store excess nutrient as fat {Yogosawa, 2013a #3}.

However, persistent activation of this physiological pathway enlarges adipocytes and

may change adipocytokine repertoires to cause chronic inflammation and insulin

resistance. In fact, ALK7-intact, aged obese mice exhibit elevated levels of

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proinflammatory MCP-1 and TNFα, a reduced level of insulin-sensitizing adiponectin,

and greater glucose intolerance, compared with their ALK7-deficient counterparts

{Yogosawa, 2013a #3}{Yogosawa, 2013b #4}. As such, the insulin-GDF3-ALK7 axis

plays a pivotal role in both physiological and pathological fat accumulation in WAT.

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Acknowledgments. We are grateful to T. Nara, E. Kobayashi and T. Ushigome for

colony maintenance of mice, S. Shigoka for assistance in preparing the manuscript, and

the staffs at Bioresource Center, Gunma University for their help in breeding of mice.

Funding. This work was supported by JSPS KAKENHI Grant Numbers JP24659442

and JP25126702 to T.I., and JP25860739 to S.Y. It was also supported by grants from

Japan Diabetes Foundation and from Novo Nordisk Insulin Study Award (to T.I.).

Duality of Interest. The authors have declared that no conflict of interest exists.

Author contributions. Y.B., K.O., S.Y., and K.M. performed experiments, C.W.B

provided experimental reagents, and K.O. and T.I. designed experiments and wrote the

paper.

The guarantor of the study. T.I. is the guarantor of this work and, as such, had full

access to all the data in the study and takes responsibility for the integrity of the data

and the accuracy of the data analysis.

27

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3. Mizutani S, Gomi H, Hirayama I, Izumi T. Chromosome 2 locus Nidd5 has a potent effect on adiposity in the TSOD mouse. Mamm Genome 2006;17:375-384

4. Yogosawa S, Mizutani S, Ogawa Y, Izumi T. Activin receptor-like kinase 7 suppresses lipolysis to accumulate fat in obesity through downregulation of peroxisome proliferator-activated receptor γ and C/EBPα. Diabetes 2013;62:115-123

5. Yogosawa S, Izumi T. Roles of activin receptor-like kinase 7 signaling and its target, peroxisome proliferator-activated receptor γ, in lean and obese adipocytes. Adipocyte 2013;2:246-250

6. Reissmann E, Jornvall H, Blokzijl A, Andersson O, Chang C, Minchiotti G, Persico MG, Ibanez CF, Brivanlou AH. The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development. Genes Dev 2001;15:2010-2022

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 Cell Endocrinol 2004;220:59-65

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T, Kawamura H. A new mouse model of spontaneous diabetes derived from ddY strain. Exp Anim 1999;48:181-189

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15. McPherron AC, Lee SJ. GDF-3 and GDF-9: two new members of the transforming growth factor-β superfamily containing a novel pattern of cysteines. J Biol Chem 1993;268:3444-3449

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17. Camell CD, Sander J, Spadaro O, Lee A, Nguyen KY, Wing A, Goldberg EL, Youm YH, Brown CW, Elsworth J, et al. Inflammasome-driven catecholamine catabolism in macrophages blunts lipolysis during ageing. Nature 2017;550:119-23

18. Van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods 1994;174:83-9319. Guo T, Marmol P, Moliner A, Björnholm M, Zhang C, Shokat KM, Ibanez CF. Adipocyte ALK7 links nutrient overload to catecholamine resistance in obesity. Elife 2014;3:e03245

20. Lumeng CN, Bodzin JL, Saltiel AR. Obesity induces a phenotypic switch in adipose tissue macrophage polarization. J Clin Invest 2007;117:175-184

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regulation of energy homeostasis in cyclic nucleotide phosphodiesterase 3B-null mice. J Clin Invest 2006;116:3240-3251

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26. Kralisch S, Klein J, Lossner U, Bluher M, Paschke R, Stumvoll M, Fasshauer M. Isoproterenol, TNFα, and insulin downregulate adipose triglyceride lipase in 3T3-L1 adipocytes. Mol Cell Endocrinol 2005;240:43-49

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28. Kim JY, Tillison K, Lee JH, Rearick DA, Smas CM. The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-α in 3T3-L1 adipocytes and is a target for transactivation by PPARγ. Am J Physiol Endocrinol Metab 2006;291:E115-127

30

Figure Legends

Figure 1. Screening TGF-β 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 a HFD from 4 weeks of age were sacrificed at 14 weeks of age.

Total RNA was isolated from the indicated tissues, including epididymal WAT

(epiWAT), inguinal WAT (ingWAT), and brown adipose tissue (BAT), and mRNA levels

of GDF3, BMP3, inhibin-βB, and TGF-β1 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 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’s t-test. #p < 0.05, ##p < 0.01, ###p <

31

0.001; one-way ANOVA.

Figure 2. GDF3 acts on ALK7 within WAT

(A-C) 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, B)

or 30 min (C). Glycerol release was measured and normalized to that of control TSOD

adipocytes (A, 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. 1B. GDF3 mRNA levels were

quantified in each of the cell fractions (left panel: epiWAT, n = 4; adipocytes, n = 3;

SVF, n = 4; CD11b- cells in the SVF, n = 5; CD11b+ cells in the SVF, n = 4; CD11b+

F4/80- non-macrophage cells, n = 3; CD11b+ F4/80+ macrophages, n = 3; CD11c-

macrophages, n = 4; and CD11c+ macrophages, n = 6). #p < 0.05, ##p < 0.01, ###p <

0.001; one-way ANOVA.

Figure 3. Effects of macrophage depletion by clodronate

32

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 concentration 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.

Figure 4. Effects of insulin administered to CD11c- ATMs, adipocytes.

(A) CD11c- macrophages from epiWAT of 7-week-old TSOD mice (1.5 × 106 cells/24-

well dish) were incubated with or without 61 pM 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 CD11c-

macrophages pretreated with or without 100 nM wortmannin was examined by

33

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

M, 61 pM, or 25 nM of insulin or with 400 ng/ml of 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

µM isoproterenol plus 0 M, 61 pM, or 25 nM insulin for 3 h. Glycerol levels in the

medium were measured and normalized to those of TSOD adipocytes without insulin or

isoproterenol incubation in each experiment (n = 3). (D, E) CD11c- ATMs from TSOD

mice were cultured with or without 61 pM insulin for 24 h as in (A). The conditioned

medium of the macrophages was harvested after centrifugation of the culture plate at

300 × g for 10 min at 4°C, and the concentration of GDF3 was measured (D, upper

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, lower

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’s t-

test. #p < 0.05, ##p < 0.01, ###p < 0.001; one-way ANOVA.

Figure 5. Effects of insulin administered to a whole body.

34

(A) Saline or insulin (0.75 U/kg body weight) was injected intraperitoneally into TSOD

(upper panels; n = 10) and T.B-Nidd5/3 (lower 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 (upper panels; n = 8) and

BALB/c mice (lower panels; n = 8) that had been fed a 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’s t-test.

Figure 6. Insulin regulates fat metabolism and mass through upregulation of GDF3

in ATMs

(A) C57BL/6 mice fed a HFD (n = 7 per group) were treated with PBS or clodronate

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 wild-type (WT) or GDF3-knockout (KO) C57BL/6 mice at 8-

10 weeks of age were transplanted into wild-type C57BL/6 mice. The recipient mice

35

were fed a HFD for 3 weeks and treated with insulin for 2 weeks from 6 and 7 weeks

after the BM transfer, respectively (n = 6-9 per 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.

0

5

10

15

20

25

30

35

40

Brain Thymus Liver Spleen BAT epiWATingWAT BM Muscle

Rel

ativ

eex

pres

sion

GDF3

*

**

B6-HFDB6-RC

T.B-Nidd5/3-RC

BALB-HFDBALB-RC

TSOD-RCALK7(+)

ALK7(-)

***

Figure 1

B

ALK7

Cripto

-actin

ALK7

Cripto

++

++

A

***0

1

2

3

4

5

6

7

8


BMP3

**

0

2

4

6

8

10

12

14


Rel

ativ

eex

pres

sion

Inhibin-B

*

*

*

0

5

10

15

20

25

30


TGF-β1

** ** * *

0

20

40

60

80

100

120

140

ALK7+

Cripto

GDF3 BMP3

##

Rel

ativ

e lu

cife

rase

act

ivity 0 50 150 400

(ng/mL)

+ +

#

0

5

10

15

20

25

30

350 50 150 400

(ng/mL)

###

##

#####

GDF3 Activin B TGF-1

Rel

ativ

e lu

cife

rase

act

ivity

### ######

###

+ + +

#

###

0

5

10

15

20

25

epiW

AT

adip

ocyte

SV

F

CD

11b

-

CD

11b

+

F4

/80

-

F4

/80

+

CD

11c-

CD

11

c+

epiWAT SVF CD11b+ CD11b+F4/80+

GDF3 mRNA expression

A C

Rela

tive e

xpre

ssio

n

TSOD CD11c+ ATM

ATM

control IgG anti-GDF3

###

###

###

##

a-tubulin

TSOD ATM

GDF3

50kD

37kD

CD11c

+ -

Figure 2

TSOD epiWAT

control IgG

anti-GDF3

0

0.5

1

1.5

2

- + - +

TSOD T.B-Nidd5/3R

ela

tive g

lycero

l re

lease

#

Primary adipocyte

GDF3

######

###

B

p-Smad3

total-Smad3

b-actin

GDF3 ++

TSOD T.B-Nidd5/3

Primary adipocyte

: non-specific bandRela

tive g

lycero

l re

lease

0

0.2

0.4

0.6

0.8

1

1.2

TSOD adipocyte

D

Control GDF3 BMP3 Activin B TGF-b1

## ##

###

0

0.2

0.4

0.6

0.8

1

1.2

1.4

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

0

1

2

3

4

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

0

0.5

1

1.5

2

2.5

3

3.5

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

05

1015202530354045

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

BW (g) (after 3-wk Tx)

Figure 3

05

1015202530354045

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

BW (g)(at the start)

00.2

0.40.60.8

1

1.21.41.6

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

epiWAT (g)

ALK7 +

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

Ratio epiWAT/BW

####

####

#######

ALK7 + ALK7 + ALK7 +

0

0.2

0.4

0.6

0.8

1

1.2

1.4

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

0

1

2

3

4

5

6

7

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

0

1

2

3

4

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

mRNA expression in epiWATB

Rel

ativ

e ex

pres

sion

#

#

#####

CEBP

###

###

###

###

######

ATGL HSL

######

NEFA/epiWAT(mM/g)

#

+ALK7 +

A

##

###

PPAR

+ + +ALK7 ALK7 ALK7 ALK7

Rel

ativ

e ex

pres

sion

###

#####

GDF3 mRNA in SVF

#

ALK7 +

0

5

10GDF3 mRNA in TSOD-

CD11c- ATM

00.5

11.5

22.5

33.5

4

- 61pM

25nM

- 61pM

25nM

- 61pM

25nM

- 61pM

25nM

TSOD T.B-Nidd5/3

TSOD T.B-Nidd5/3

Basal Isoproterenol 10 μM

# #

0

1

2

3

4

Gene induction by ex vivo Insulin

***

0

0.2

0.4

0.6

0.8

1

InsulinWortmannin

(nM) 100 1000

A

Rel

ativ

e ex

pres

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+ + +

CD11c GDF3 TNF MCP1 Arg1 Ym1

Rel

ativ

e ex

pres

sion

Rel

ativ

e gl

ycer

ol re

leas

eCB

p-AKT

β-actin

Insulin

TSOD T.B-Nidd5/3

Primary adipocyte

AKT

61pM

p-Smad3total-Smad3

25nM

ALK7 +

TSOD

+

GDF3

400ng/ml

61pM

25nM

* **

**

## #####

Insulin

# #

#

###

D

p-Smad3

-actin

total-Smad3

CD11c-ATM

Insulin

++

Sups

E

Sups

Rel

ativ

e gl

ycer

ol re

leas

e

CD11c-ATM

Insulin

+

+

+

TSOD T.B-Nidd5/3###

## #

0

0.2

0.4

0.6

0.8

1

1.2

GDF3 in the sups

0

500

1000

1500

2000

pg/m

L

TSOD adipocyte

#

###

###

Figure 4

###

+

Insulin + +

TSOD CD11c- ATM

Wortmannin (nM)

100

p-AKT

AKT

β-actin

CD11c-ATM

Insulin

++

Sups

+

++

+

00.20.40.60.8

11.21.41.61.8

0

0.5

1

1.5

0

0.01

0.02

0.03

0

0.5

1

1.5

00.20.40.60.8

11.21.41.6

00.20.40.60.8

11.2

ARatio

epiWAT/BWATGL mRNA

in epiWATHSL mRNA in epiWAT

NEFA/epiWAT(mM/g)

B

00.0050.01

0.0150.02

0.0250.03

0.035

0

0.5

1

1.5

0

0.5

1

1.5

Rel

ativ

e ex

pres

sion

0

1

2

3

C57BL/6: ALK7 (+)

BALB/c: ALK7 (-)

0

0.01

0.02

0.03

0

0.5

1

1.5

0

0.5

1

1.5

2

0

1

2

3

4

Rel

ativ

e ex

pres

sion

*

** *

TSOD: ALK7 (+)

T.B-Nidd5/3: ALK7 (-)

0

0.01

0.02

0.03

0.04

Insulin

***

Rel

ativ

e ex

pres

sion

*

0

0.5

1

1.5

*

Insulin

Rel

ativ

e ex

pres

sion

Figure 5

++++

Ratio epiWAT/BW

ATGL mRNA in epiWAT

HSL mRNA in epiWAT

NEFA/epiWAT(mM/g)

++++

Ratio epiWAT/BW

ATGL mRNA in epiWAT

HSL mRNA in epiWAT

NEFA/epiWAT(mM/g)

Insulin ++++

Ratio epiWAT/BW

ATGL mRNA in epiWAT

HSL mRNA in epiWAT

NEFA/epiWAT(mM/g)

Insulin ++++

0

0.5

1

1.5

2

0

0.2

0.4

0.6

0.8

1

1.2

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1.6

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0.01

0.015

0.02

0.025

0.03

0.035

0

1

2

3

4

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6

B

BM

Insulin

WT WT KO KO

0

0.5

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2.5

3

3.5

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1.5

2

2.5

A

CLO

Insulin ++

+

+

Rela

tive e

xpre

ssio

nGDF3 mRNA

in SVF

epiWAT/BW

ratio

NEFA/epiWAT

(mM/g)

ATGL mRNA

in epiWAT

HSL mRNA

in epiWAT

#

#

###

## ###

0

0.5

1

1.5

2

2.5

##

##

######

###

#

###

###

Rela

tive e

xpre

ssio

n

###

Figure 6

Insulin

Lipolysis

Obesity

Insulin

Resistance

CD11c+ATMIR

CD11c-ATM

GDF3

Inflammation

ALK7Adipocyte

Fat Accumulation

C

+

+

+

+

++

+

+

+

+

+

++

+

+

+

GDF3 mRNA

in SVF

epiWAT/BW

ratio

NEFA/epiWAT

(mM/g)

ATGL mRNA

in epiWAT

HSL mRNA

in epiWAT

### ###

###

Rela

tive e

xpre

ssio

n

+ +WT WT KO KO

+ +WT WT KO KO

+ +

##

Rela

tive e

xpre

ssio

nWT WT KO KO

+ +

#

##

###

WT WT KO KO

+ +

##

###

###

0

0.5

1

1.5

2

2.5

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0

0.5

1

1.5

2

2.5

3

0

10

20

30

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60

70

80

Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle

GDF5

0

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GDF2 (BMP9)

0

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GDF6 (BMP13)

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GDF1

*

B6-HFD

B6-NC

T.B-Nidd5/3-NC

BALB-HFD

BALB-NC

TSOD-NC

ALK7(+)

ALK7(-)

Supplemental Figure 1-1 (to be continued)

Rela

tive e

xpre

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GDF7 (BMP12)

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GDF9b (BMP15)

* *

**

* *

* **

*

*

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8


GDF10 (BMP3b)

*

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15

20

25

30

35

40

45

50


GDF11 (BMP11)

B6-HFD

B6-NC

T.B-Nidd5/3-NC

BALB-HFD

BALB-NC

TSOD-NC

ALK7(+)

ALK7(-)

0

20

40

60

80

100

120

140

160


GDF15

0

2

4

6

8

10

12

14

16


BMP2

0

1

2

3

4

5

6

7


BMP4

0

20

40

60

80

100

120

140

160


BMP5

0

5

10

15

20

25

30

35


BMP6

0

50

100

150

200

250


BMP7

Rela

tive e

xpre

ssio

n


*

*

*

*

0

10

20

30

40

50

60

70


BMP8A

0

1000

2000

3000

4000

5000

6000

7000

8000


Inhibin-βE

0

500

1000

1500

2000

2500


BMP8BR

ela

tive e

xpre

ssio

n

B6-HFD

B6-NC

T.B-Nidd5/3-NC

BALB-HFD

BALB-NC

TSOD-NC

ALK7(+)

ALK7(-)

0

100

200

300

400

500

600


BMP10

0

1000

2000

3000

4000

5000

6000

7000


Inhibin-α

0

10

20

30

40

50

60

70

80


Inhibin-βA

0

20000

40000

60000

80000

100000

120000


Inhibin-βC

0

2

4

6

8

10

12

14

16

18


TGF-β2


*

* * **

**

*

0

50

100

150

200

250


Nodal

0

0.5

1

1.5

2

2.5

3

3.5


TGF-β3R

ela

tive e

xpre

ssio

n

B6-HFD

B6-NC

T.B-Nidd5/3-NC

BALB-HFD

BALB-NC

TSOD-NC

ALK7(+)

ALK7(-)

0

200

400

600

800

1000

1200


AMH

0

500

1000

1500

2000

2500

3000

3500

4000

4500


Lefty1

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000


Lefty2

Supplemental Figure 1-4

*

* *

*

Figure S2

B

A

Rela

tive e

xpre

ssio

n

0

0.5

1

1.5

2

2.5

3

3.5

4

TSOD T.B-Nidd5/3

HSL

**

Rela

tive e

xpre

ssio

n

Rela

tive e

xpre

ssio

n

Rela

tive e

xpre

ssio

n

0

10

20

30

40

50

TSOD T.B-Nidd5/3

BW(g)

0

0.5

1

1.5

2

TSOD T.B-Nidd5/3

epiWAT weight (g)

***

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

TSOD T.B-Nidd5/3

Ratio epiWAT/BW

***

0

0.5

1

1.5

2

TSOD T.B-Nidd5/3

NEFA (mM)

**

0

0.5

1

1.5

2

2.5

TSOD T.B-Nidd5/3

NEFA/epiWAT(mM/g)

**

0

1

2

3

4

5

6

7

8

TSOD T.B-Nidd5/3

PPARg

*

0

1

2

3

4

5

6

7

8

TSOD T.B-Nidd5/3

CEBPa

*

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

TSOD T.B-Nidd5/3

ATGL

**

mRNA in epiWAT

0

1

2

3

4

5

6

7

8

TSOD T.B-Nidd5/3

Food Intake(g/day)

CD11c

TSOD ATM

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

CD11c- CD11c+

*

D

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

epiWAT

weight (g)

**

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

Ratio

epiWAT/BW

***

mRNA in SVF

0

0.2

0.4

0.6

0.8

1

1.2

1.4

C57BL/6 (3 wks NC vs HFD)

TNFa

*

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6MCP1

0

1

2

3

4

5

Arg1

0

1

2

3

4

5

6

*

Ym1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

CD11c- CD11c+

TNFa

Rela

tive e

xpre

ssio

n

0

0.2

0.4

0.6

0.8

1

1.2

1.4

CD11c- CD11c+

MCP1

mRNA

0

0.5

1

1.5

2

2.5

CD11c- CD11c+

Arg1

0

0.5

1

1.5

2

2.5

CD11c- CD11c+

Ym1

0

0.4

0.8

1.2

1.6

NLRP3

0

0.5

1

1.5

2

CAS1

RC HFD RC HFD RC HFD RC HFD RC HFD RC HFD RC HFD RC HFD

0

5

10

15

20

25

0

1

2

3

4

5

6

GDF3 CD11c

Rela

tive e

xpre

ssio

n

***

RC HFDRC HFD

CD11b-PE-Cy7

F4

/80

-AP

C

CD

11

c-P

E

CD11b-PE-Cy7

CD11b+

F4/80+ ATM

73.7%

CD11c+

ATM

17.8%

C

0

0.5

1

1.5

2

2.5

ep

iWA

T

ad

ipocyte

SV

F

CD

11

b-

CD

11

b+

F4/8

0-

F4/8

0+

CD

11

c-

CD

11

c+

SVF CD11b+ CD11b+F4/80+

BMP3 mRNA

Rela

tive e

xpre

ssio

n

ATM

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

epiWAT

adipocyte

SVF

CD11b-

CD11b+

F4/80-

F4/80+

CD11c-

CD11c+

SVF CD11b+ CD11b+

F4/80+

Inhibin-bB mRNA

ATM

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

epiWAT

adipocyte

SVF

CD11b-

CD11b+

F4/80-

F4/80+

CD11c-

CD11c+

SVF CD11b+ CD11b+

F4/80+

TGF-b1 mRNA

ATM

##

###

###

###

###

###

###

###

0

10

20

30

40

50

60

70

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

% of ATM in SVF

###

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

0

0.5

1

1.5

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

Figure S3

0

1

2

3

4

5

6

7

8

9

10

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

% of CD11c+ ATM in SVF

0

2

4

6

8

10

12

14

16

PBS CLO PBS CLO

TSOD T.B-Nidd5/3

% of CD11c+ in ATM

##

##

##

A

B

ALK7 +

ALK7 + +

Rela

tive

exp

ressio

n

Rela

tive

exp

ressio

n

ATM

56.3%

CD11c+

11.49%

F4

/80

-AP

C

CD11b-

PE-Cy7

PBS

CD

11

c-P

E

CD11b-

PE-Cy7

CLO

ATM

35.26%

CD11c+

4.35%

T.B-Nidd5/3

ATM

58.49%

ATM

40.84%

CD11c+

6.57%

CD11c+

2.8%

###

++

TSOD

PBS CLO

####

##

###

F4/80 mRNA

in epiWAT

# #

#

#

CD11c mRNA

in epiWAT

#

##

0

1

2

3

4

0

0.5

1

1.5

2

Lung.MΦ P.MΦ BMDM

Figure S4

A

Rela

tive e

xpre

ssio

n

B

TSODR

ela

tive e

xpre

ssio

n

CD11c- ATM

C

Rela

tive e

xpre

ssio

n

Gene induction

by 61 pM insulin

0

0.5

1

1.5

Inhibin-bB TGF-b1

TSOD

CD11c- ATM

TSOD

GDF3 mRNA induced

by 61 pM insulin

TSODTSOD

0

0.2

0.4

0.6

0.8

1

1.2

1.4

CD11c-ATM

Lung.MΦ P.MΦ BMDM

IR mRNA

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

CD11c-ATM

Lung.MΦ P.MΦ BMDM

GDF3 mRNA (after 24 h culture without insulin)

no

culture

24 h culture

FBS(-) FBS(+)

GDF3 mRNA

###

##

#

####

###

###

0

1

2

3

Figure S5

C

0

10

20

30

40

50

60

70

% of ATM in SVF

BM

Insulin + +WT WT KO KO

+ +WT WT KO KO

0

1

2

3

4

numbers of SVF cells

(x106 cells/g)

B

++

+

++

++

+0

10

20

30

40

50

60

70

% of ATM in SVF

##

###

0

1

2

3

4

5

% of CD11c+ ATM in SVF

##

###

Insulin

CLO

######

++

+

+0

1

2

3

4

5

Food intake (g/day)

ATSOD T.B-Nidd5/3 C57BL/6 BALB/c

ALK7

b-actin

Rela

tive e

xpre

ssio

n

++

+

+

CD11c mRNA

in SVF

##

### ###

Diabetes Figure 1 New (2 column) 20180406Diabetes Figure 2 New (2 column) 20180406Diabetes Figure 3 New (2 column) 20180406Diabetes Figure 4 New (2 column) 20180406Diabetes Figure 5 New (2 column) 20180406Diabetes Figure 6 New (2 column) 20180406-newDiabetes Supplemental Figures N 20180406-new

Insulin regulates lipolysis and fat mass by upregulating growth/differentiation factor ... · 2019....

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Transcript of Insulin regulates lipolysis and fat mass by upregulating growth/differentiation factor ... · 2019....