insulin regulates lipolysis and fat mass by upregulating growth/differentiation factor ... · 2019....
TRANSCRIPT
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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:
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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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
Brain Thymus Liver Spleen BAT epiWATingWAT BM Muscle
BMP3
**
0
2
4
6
8
10
12
14
Brain Thymus Liver Spleen BAT epiWATingWAT BM Muscle
Rel
ativ
eex
pres
sion
Inhibin-B
*
*
*
0
5
10
15
20
25
30
Brain Thymus Liver Spleen BAT epiWATingWAT BM Muscle
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
sion
+ + +
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
1.4
1.6
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0
1
2
3
4
5
6
B
BM
Insulin
WT WT KO KO
0
0.5
1
1.5
2
2.5
3
3.5
0
0.5
1
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
40
50
60
70
80
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
GDF5
0
50
100
150
200
250
300
350
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
GDF2 (BMP9)
0
500
1000
1500
2000
2500
3000
3500
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
GDF6 (BMP13)
0
200
400
600
800
1000
1200
1400
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
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
ssio
n
0
200
400
600
800
1000
1200
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
GDF7 (BMP12)
0
100
200
300
400
500
600
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
GDF8
0
1
2
3
4
5
6
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
GDF9
0
10
20
30
40
50
60
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
GDF9b (BMP15)
* *
**
* *
* **
*
*
-
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
GDF10 (BMP3b)
*
0
5
10
15
20
25
30
35
40
45
50
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
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
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
GDF15
0
2
4
6
8
10
12
14
16
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
BMP2
0
1
2
3
4
5
6
7
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
BMP4
0
20
40
60
80
100
120
140
160
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
BMP5
0
5
10
15
20
25
30
35
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
BMP6
0
50
100
150
200
250
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
BMP7
Rela
tive e
xpre
ssio
n
Supplemental Figure 1-2 (to be continued)
*
*
*
*
-
0
10
20
30
40
50
60
70
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
BMP8A
0
1000
2000
3000
4000
5000
6000
7000
8000
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
Inhibin-βE
0
500
1000
1500
2000
2500
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
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
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
BMP10
0
1000
2000
3000
4000
5000
6000
7000
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
Inhibin-α
0
10
20
30
40
50
60
70
80
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
Inhibin-βA
0
20000
40000
60000
80000
100000
120000
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
Inhibin-βC
0
2
4
6
8
10
12
14
16
18
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
TGF-β2
Supplemental Figure 1-3 (to be continued)
*
* * **
**
*
-
0
50
100
150
200
250
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
Nodal
0
0.5
1
1.5
2
2.5
3
3.5
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
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
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
AMH
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
Lefty1
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
Brain Thymus Liver Spleen BAT epiWAT ingWAT BM Muscle
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