department of nephrology, rheumatology, endocrinology and...

The anti-obesity action of ACAM by modulating the dynamics of cell adhesion

and actin polymerization in adipocytes

Kazutoshi Murakami1, 2, Jun Eguchi1, Kazuyuki Hida1, Atsuko Nakatsuka1, Akihiro

Katayama1, Miwa Sakurai3, Haruki Choshi1, Masumi Furutani4, Daisuke Ogawa3, Kohji

Takei5, Fumio Otsuka2, and Jun Wada1

Department of Nephrology, Rheumatology, Endocrinology and Metabolism1, Department of

General Medicine2, Department of Diabetic Nephropathy3, Central Research

Laboratory4, Department of Biochemistry5, Okayama University Graduate School of

Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan

Running title: Adipocyte adhesion molecule (ACAM) in obesity

Correspondence:

Jun Wada, MD, PhD Department of Nephrology, Rheumatology, Endocrinology and Metabolism Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences 2-5-1 Shikata-cho, Okayama 700-8558, Japan Tel: +81-86-235-7235 FAX: +81-86-222-5214 E-mail: [email protected]

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Summary

Coxsackie- and adenovirus receptor-like membrane protein (CLMP) was identified as the

tight junction-associated transmembrane protein of epithelial cells with homophilic binding

activities. CLMP is also recognized as adipocyte adhesion molecule (ACAM) and it

upregulated in mature adipocytes in rodents and human with obesity. Here, we present aP2

promoter-driven ACAM transgenic mice are protected from obesity and diabetes with the

prominent reduction of adipose tissue mass and smaller size of adipocytes. ACAM is

abundantly expressed on plasma membrane of mature adipocytes and associated with

formation of Phalloidin-positive polymerized form of cortical actin (F-actin). By electron

microscopy, the structure of zonula adherens with an intercellular space of ~10-20 nm was

observed with strict parallelism of the adjoining cell membranes over distances of 1 to 20 μm,

where ACAM and γ-actin are abundantly expressed. The formation of zonula adherens may

increase the mechanical strength, inhibit the adipocyte hypertrophy, and improve the insulin

sensitivities.

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INTRODUCTION

Quite a few adhesion molecules have been identified by molecular genetics as well as

gene expression profile studies in adipocytes derived from experimental models and human

studies. For instance, only cadherins were reported to be expressed in premature

adipocytes. In the cell lines, such as C3H10T1/2 and 3T3-L1 cells, N-cadherin and

cadherin-11 are expressed and they are prominently suppressed by the induction of

adipocyte differentiation and downregulated to very low levels after the full differentiation(1).

Transgenic expression of dominant-negative N-cadherin decreased bone formation,

delayed acquisition of peak bone mass and increased body fat(2). Although the information

of adhesion molecules in adipocyte biology is limited, we identified adipocyte adhesion

molecule (ACAM) from the visceral adipose tissues of OLETF (Otsuka Long-Evans

Tokushima fatty) rats by PCR-based cDNA suppressive subtraction methods(3). Mouse

ACAM was independently identified as adipocyte-specific protein 5 (ASP5) from 3T3-L1

cells by using signal sequence trap by a retrovirus-mediated expression screening

method(4). Human ACAM had been identified as coxsackie virus and adenovirus

receptor-like membrane protein (CLMP) by bioinformatics approaches and Raschperger et

al. demonstrated that CLMP is a component of the tight junction of epithelial cells and

colocalized with ZO-1 (zonula occludens-1)(5). ACAM/CLMP belongs to CTX (cortical

thymocyte marker in Xenopus) and they are characterized by extracellular variable (V-type)

and constant (C2-type) immunogloburin domains, which are involved in the homophilic

adhesion and aggregation of the cells. Although we reported the expression of ACAM

increased in mature adipocytes in genetically obese db/db and diet-induced obesity mice,

and also in adipose tissues in the subjects with obesity, the functional role of ACAM in

mature adipocytes and in obesity remained totally unknown. Here, we generated aP2

promoter-driven ACAM transgenic mice and they are protected from obesity and diabetes

with reduced accumulation of white and brown adipose tissues. In ACAM Tg mice under

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high-fat high-sucrose (HFHS) chow, ACAM is abundantly expressed on plasma membranes

of mature adipocytes, where the zonula adherens-like structure is formed. The adhesion

process and formation of zonula adherens are associated with the formation of actin

polymerization on the surface of the mature adipocytes. The formation of zonula adherens

may increase the mechanical strength, inhibit the adipocyte hypertrophy, alter the signaling

events, and improve the insulin sensitivities. Our finding provides the new therapeutic

modalities targeting the processes of cell adhesion and actin polymerization of adipocytes in

the treatment of obesity and diabetes.

RESEARCH DESIGN AND METHODS

Generation of ACAM transgenic mice: β-globin intron and human GH poly A signal were

ligated into BamHI-EcoRI and EcoRI-XhoI sites of pcDNA3.1Zeo vector (Invitrogen),

respectively (Figure S1a). Coding region of mouse ACAM cDNA was inserted into EcoRI

site by blunt end ligation after filling-in reaction. BamHI and XhoI fragment was subjected to

blunt end ligation into SmaI site of pBluescript SKII(+) (Stratagene), in which mouse aP2

promotor was inserted into EcoRV-PstI site by blunt end ligation. The insert was excised

with HindIII and NotI and transgene was generated. Microinjected C57BL/6JJcl one-cell

stage zygotes were oviduct-transferred and permitted to develop to term. Three transgenic

founders were obtained and Southern blot analysis was performed using EcoRI and SmaI

fragment of the transgene. Genotyping of Tg mice was performed by PCR using primers

5’-GACATTGAATGGCTGCTCACCG-3’ and 5’-GCTCTGCACATACTGTACAGTC-3’;

5’-GTTGGAACGCTGGGAACTCACACTGAGATC-3’ and

5’-GGTTCAGAACCTCTCACTTCCGGTCCTATG-3’.

Animals: Male C57BL/6JJcl mice were housed in cages and maintained on a 12-hour

light-dark cycle. For the animal experiments with mice, standard chow (NMF; Oriental Yeast)

and high fat-high sucrose (HFHS) diet (D12331; Research Diet) were used and the mice

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were sacrificed at 30 weeks of age. Oxygen consumption was measured using O2/CO2

metabolism measuring system (MK-5000, Muromachi). All animal experiments were

approved by the Animal Care and Use Committee of the Department of Animal Resources,

Advanced Science Research Center, Okayama University. Liver triglyceride was measured

by Folch’s method (Skylight Biotech, Tokyo).

Cell culture and adipocyte differentiation: Mouse 3T3-L1 fibroblasts (ATCC; American

Type Culture Collection) were cultured in vitro and differentiated into mature adipocytes.

3T3-L1 cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma)

supplemented with 10 % calf serum (Hyclone), 100 U/ml penicillin, and 0.1 mg/ml

streptomycin (Sigma). Confluent cells were induced to differentiate by the addition of 0.5

mM 1-methyl-3-isobutylxanthine (IBMX), 0.25 M dexamethasone (DEX), and 10 g/ml

insulin (INS) (Sigma). After 48 hours, induction medium was removed and cells were

cultured with DMEM supplemented with 10 % fetal bovine serum (Invitrogen) and 10 g/ml

insulin for 14 days. To determine the major inducer for ACAM expression, the confluent

preadipocytes were treated with DEX/IBMX/INS, DEX, IBMX, INS, DEX/IBMX, IBMX/INS,

DEX/INS, fetal bovine serum (FBS), pioglitazone (PIO), DEX/IBMX/INS/PIO,

8-bromoadenosine-cAMP (8-br-cAMP) (Calbiochem), or forskolin (Sigma) at the

concentrations indicated. In separate experiments, confluent preadipocytes were

preincubated with H89 (Sigma), SB203580 (Calbiochem), PD98059 (Calbiochem), and

LY294002 (Sigma) for 30min, respectively, in prior to induction of differentiation.

Poly (A+) RNA analysis of various tissues: For quantitative real time PCR analysis in

various tissues in mice, cDNA synthesized from 2 μg total RNA was analyzed in a Sequence

Detector (model 7900; Applied Biosystems) with specific primers and SYBR Green PCR

Master (Applied Biosystems). The relative abundance of mRNAs was standardized with

36B4 mRNA as the invariant control. The mRNA expression was determined by SYBR

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green. Gene specific primers are indicated in Table S1.

RNA isolation and Northern blot analyses of 3T3-L1 cells: Total RNA was isolated from

3T3-L1 cells by using RNeasy Lipid Tissue Mini Kit (Qiagen). For Northern blot analysis, 10

µg total RNAs were subjected to 2.2 M formaldehyde 1% agarose gel electrophoresis and

capillary transferred to the Hybond XL nylon membranes (GE Healthcare Life Sciences).

The membranes were hybridized with [α-32P]dCTP-radiolabeled mouse ACAM, mouse

C/EBPβ, mouse C/EBPδ, mouse C/EBPα, mouse PPARγ, mouse LPL and mouse 18S

ribosomal RNA (ATCC) cDNAs at 68oC in ExpressHybTM Hybridization Solution (Clontech)

for 1 hour. Filters were washed at high stringency conditions, i.e. four times in 1 X SSC/0.1%

SDS at 20oC, followed by two times at 50oC in 0.1 X SSC/0.1% SDS.

Antibodies: Sheep polyclonal anti-ASAM (ACAM) (R&D systems), rat monoclonal

anti-F4/80 (Cl:A3-1) (AbD Serotec), rabbit polyclonal anti-nonmuscle myosin heavy chain

II-A (Covance), mouse monoclonal anti-γ-actin (Sigma) antibodies were used for

immunoblotting, immunofluorescence and immunogold studies. For secondary antibodies,

rabbit anti-sheep IgG, HRP conjugated (Millipore), Alexa Fluor 594 donkey anti-sheep IgG

(H+L), Alexa Fluor 488 goat anti-rat IgG (H+L), and Alexa Fluor 488 chicken anti-rabbit IgG

(H+L) (Invitrogen) were used.

LC-MS/MS (high performance liquid chromatography-tandem mass spectrometry)

and MALDI-TOF/MS (matrix assisted laser desorption/ionization): Recombinant

full-length of mouse ACAM tagged with calmodulin-binding peptide (CBP) and

streptavidin-binding peptide (SBP) (Adeno-pCTAP-mACAM) (InterPlay Mammalian TAP

system, Stratagene) were prepared by Adenovirus Expression Kit (Takara).

Adeno-pCTAP-mACAM was introduced to 3T3-L1 cells. Soluble proteins were purified by

CBP and SBP binding resin (InterPlay Mammalian TAP System, Stratagene) and subjected

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to SDS-PAGE and Coomassie blue staining. Visible bands were excised and in-gel-digested

with trypsin and analyzed with LC-MS/MS and MALDI-TOF/MS.

Cell fractionation of cultured 3T3-L1 cells: 3T3-L1 cells at 24 hours after the induction

were fractioned. Cells were washed in PBS and resuspended in suspension buffer (20mM

Tris-HCl; PH7.4, 1mM EDTA, 255mM sucrose) containing protease inhibitor cocktail

(Sigma) and homogenized with pre-cooled motor-driven Potter-Elvehjem grinder with 20

strokes at 1400 rpm. Nuclei were pelleted by centrifugation at 1,000g for 10min. The

post-nuclear supernatant (S1) was centrifuged at 16,000g for 20min. The pellet containing

mitochondria, peroxisomes and plasma membrane fractions was resuspended in 5 ml of

resuspension buffer and layered onto 5 ml of sucrose cushion (1.12 M sucrose, 1 mM EDTA,

20 mM Tris-HCl; PH7.4) and centrifuged at 101,000g for 25 min. The mitochondria and

peroxisomes were collected as a pellet and resuspended in suspension buffer. The plasma

membrane was collected at the interface, resuspended in 10 ml of suspension buffer,

centrifuged 16,000g for 15min and resuspended in suspension buffer. S1 was centrifuged at

48,000g for 20min and the pellet containing high density microsomes (HDM) was obtained.

The supernatant was centrifuged at 212,000g for 70 min, separated the pellet containing low

density microsomes (LDM) and the supernatant containing the cytosol. Pellets from

fractions containing HDM and LDM were resuspended in suspension buffer.

Immunoelectron microscopy: Adipose tissues and 3T3-L1 cells were fixed with 4%

paraformaldehyde and 0.05% glutaraldehyde in 0.01 M phosphate buffer, pH 7.4, for 30

minutes and then scraped from the dish, and a pellet was prepared with a microfuge. The

tissues and cell pellets were then dehydrated in a graded series of ethanol and they were

embedded and polymerized in LR-White (Polysciences, Inc.) at 50oC for 48 hours.

Approximately 60-nm thin sections were picked up 300 mesh nickel grid. The sections were

pretreated with 20 mM Tris-buffered saline, pH 8.2, containing 0.25% bovine serum albumin.

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They were incubated with anti-ACAM antibody and then with anti-sheep IgG conjugated with

10 nm colloidal gold particles for 4 hours at room temperature. They were washed with

Tris-buffered saline and stained with lead citrate and uranyl acetate and examined by an

electron microscope at accelerating voltage of 75 kv. Transmission and scanning electron

microscopy was performed as previously described(6).

Adenoviral vectors and infection: The adenoviral vector encoding constitutively active

form of C/EBPβ (LAP) were generously provided by Prof. Hiroshi Sakaue (Tokushima

University, Tokushima, Japan). For adenoviral infection, 3T3-L1 preadipocytes cultured to

90% confluency and were infected at a multiplicity of infection of 10 PFU/cell 48 hours

before induction of differentiation(7). The cells were collected at indicated time points and

were subjected to Northern blot analysis. As a control, the adenoviral vector encoding LacZ

(LacZ) was used.

Plasmid constructs and luciferase reporter assay: The ACAM 1869 bp (-1741 to +128)

luciferase plasmid was generated by ligating into the cloning site of the promorterless

luciferase reporter plasmid pGL3-Basic (Promega). Various 5’-deletion constructs -1341,

-584, -464, -235, -97, -79, -57 were made. The mutant constructs of promoter sequence

using -79 deletion construct: wild type (-72 to -59), 5’-AGCTAACCCCAAAC-3’; mutant 1

(MT1), 5’-AGCTAACAATGGCC-3’; and mutant 2 (MT2), 5’-AGCTAAACCCGAAC-3’, were

generated by PCR. 3T3-L1 preadipocytes were transfected using Neon electroporation

transfection system (Invitrogen). Transfections were performed using 10 g of pBIND

(Renilla luciferase)-SV40 (internal control) along with 10 g pGL3-Basic plasmids containing

the ACAM promoter or with pGL3-Basic plasmid. 48 hours after transfection, cells were

incubated with 0.5 mM IBMX for 6 hours, and luciferase reporter assays were performed

using the Dual Luciferase Reporter Assay System (Promega). Transfection efficiencies were

normalized to the Renilla luciferase activity. For the CMV-promoter-driven transient

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overexpression of Kruppel-like factor 4 (KLF4), GATA binding protein 1 and GATA binding

protein 6, EX-Mm19461-M02 (pReceiver-M02), EX-Mm02659-M02 (pEZ-M02) and

EX-Mm02664-M02 (pEZ-M02) (Genecopia, OmicsLink Expression Clone) were used,

respectively. For negative control, EX-NEG-M02 (pReceiver-M02CT) was used.

Statistical analysis: Data are expressed as the mean ± standard error (SE) and the

multiple comparisons were performed by a one-way ANOVA with Bonferroni and Tukey

corrections. A value of P < 0.05 was regarded as statistically significant. The data were

analyzed using the IBM SPSS Statistics software program (IBM).

RESULTS

ACAM Tg mice are protected from obesity: Three independent aP2-driven ACAM

transgenic lines of C57BL/6JJcl mice were established (Figure S1a). The copy numbers of

transgenes differed in L11 (high-), L22 (intermediate-), and L4 (low-expression) lines and the

protein expression of ACAM in both epididymal and subdermal fat tissues corresponded to

the copy numbers of transgene (Figure S1b). Under HFHS chow, mRNA expression of

ACAM was significantly augmented in epididymal, subdermal and brown adipose tissues,

and not in liver and skeletal muscle (Figure S1c). The body weight gains under HFHS chow

were prominently reduced in ACAM Tg mice in parallel with the expression of ACAM (Figure

S1d-f). Throughout the following experiments, intermediate-expression (L22) line was

employed, since the mice derived from high-expression (L11) line were completely protected

from obesity and the reduction of body weight may dominantly influence the phenotype such

as improvement of glucose metabolism. In ACAM Tg mice, the body and fat pad weight was

significantly reduced under HFHS chow compared with WT mice (Figure 1a and 1b) and

the adipocyte size was also significantly reduced (Figure 1c-e). In ACAM Tg mice, the

immunoreactivity of ACAM was enhanced on the cell surface of adipocytes in both

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immunofluorescence and immunoperoxidase studies (Figure 1f). The staining of ACAM

merged with F4/80 and the adipose tissue macrophages abundantly expressed ACAM

(Figure 1g). Both glucose tolerance and insulin sensitivity were significantly improved in

ACAM Tg mice under HFHS chow (Figure 2a-c) and serum small-sized LDL cholesterol

was significantly reduced compared with WT mice (Figure 2d). In contrast, the lipid droplets

in liver tissue, liver weight and triglyceride content were not altered in ACAM Tg mice under

HFHS chow (Figure 2e-g). The data suggested that ACAM Tg mice are protected from

obesity and insulin resistance.

Although the locomotor activity, food intake, and respiratory quotient (RQ) were not

altered in ACAM Tg mice under HFHS chow, the weight of brown adipose tissues was

significantly reduced in ACAM Tg mice under HFHS chow compared with WT mice (Figure

3a-3d). The light and electron microscopy demonstrated that lipid droplets were prominently

reduced in ACAM Tg mice under HFHS chow in brown adipose tissues (Figure 3d). The

oxygen consumption rate was significantly elevated in both dark and light periods in ACAM

Tg mice under HFHS chow (Figure 3e-g). Quantitative RT-PCR demonstrated that lipid

metabolism- (Fabp4, Acaca, Fasn, Lpl, Hsd11b1, Slc27a1), inflammation- (Adipoq, Cd36,

Retn), and glucose metabolism-related genes (Hk1, Slc2a4) significantly increased in ACAM

Tg mice compared with WT mice (Figure 4a). In brown adipose tissues, the gene

expression of Ucp1 and Cpt1a significantly increased in ACAM Tg mice compared with WT

(Figure 4c). In contrast, most of the genes expressed in subdermal adipose tissues, skeletal

muscle and liver were not altered in ACAM Tg mice under HFHS chow (Figure 4b, 4d-e).

Taken together, the overexpression of ACAM in adipocytes directly altered the biological

functions of adipose tissues in the status of obesity and it increased brown fat activity. We

also checked conversion of white to bright/beige adipocyte in subdermal WATs; however, we

did not observe such browning in the tissues (Figure S1g).

ACAM is differentially expressed in 3T3-L1 adipocytes: To explore the function of ACAM

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in adipocytes, we next investigated the mRNA expression during the 3T3-L1 adipocyte

differentiation. ACAM mRNA revealed the first peak at 6 hours after induction, i.e. the early

stage of differentiation, and down-regulated within 48 hours, which coincided with the

induction of Cebpb and Cebpd (Figure 5a and S2a). At 14 days, ACAM mRNA

demonstrated the second peak following the appearance of adipocyte maturation markers,

such as Cebpa, Pparg and Lpl (Figure 5b and S2b). By the fractionation of the 3T3-L1 cells,

ACAM localized mainly in the plasma membrane (PM) fraction, lesser extent in low-density

microsome (LDM) and high density microsome (HDM) fractions (Figure 5c and S2c).

Western blot analyses demonstrated the up-regulation of ACAM after the induction of

differentiation (Figure S2d). Adeno-pCTAP-mACAM was introduced to 3T3-L1 cells, soluble

proteins purified by CBP and SBP binding resin, and finally subjected to SDS-PAGE and

Coomassie blue staining (Figure 5d). Visible bands were excised and in-gel-digested with

trypsin and analyzed with LC-MS/MS and MALDI-TOF/MS, myosin II-A and γ-actin were

identified, respectively. We next confirmed the complex formation of ACAM and γ-actin by

the immunoprecipitation using 3T3-L1 cells (Figure 5e). Before the induction and at the

early stage of differentiation, ACAM expressed mainly on the cell processes of 3T3-L1 cells

revealed by immunofluorescence and immunogold studies (Figure 5f, 5g and 5h). Before

the induction, ACAM did not colocalized with myosin II-A; however, it merged with myosin

II-A concentrated on the cell surface as well as perinuclear cytosol when 3T3-L1 cells fully

differentiated to the mature adipocytes (Figure 5g).

We then searched the critical inducers for the up-regulation of ACAM mRNA expression.

The induction with IBMX or its combination with other inducers up-regulated ACAM mRNA

levels; the effects were almost comparable with DEX/IBMX/INS stimulation (Figure S3a).

IBMX induced ACAM mRNA expression in a dose- and time-dependent manner and it

peaked at 6 hours in the presence of 500 nM IBMX (Figure S3b and S3c). IBMX is known

to inhibit phosphodiesterases, stimulate adenyl cyclase activity, and increase the

intracellular cAMP accumulation(8). Both 8-br-cAMP and forskolin, a cAMP elevating agent,

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up-regulated ACAM mRNA levels in a dose dependent manner (Figure S3d and S3e). It is

also known that cAMP stimulates protein kinase A (PKA) activity(9) and ACAM mRNA

expression was reduced by specific PKA inhibitor (H89) in a dose-dependent manner

(Figure S3f). In contrast, SB203580 (p38 kinase inhibitor), PD98050 (MEK inhibitor), and

LY294002 (PI-3 kinase inhibitor) demonstrated no effects on IBMX-induced expression of

ACAM mRNA (data not shown).

The transcriptional regulation of ACAM mRNA seems to be dependent on IBMX-PKA

pathway, which is a known inducer of C/EBPβ(10). We found 13 consensus C/EBPβ-binding

sites by analyzing the -1741-bp ACAM promoter region (Figure S4a) and observed ~8-fold

induction of luciferase activities in -584, -464, -235, -97, -79 ACAM promoter constructs by

the treatments of IBMX or adenoviral vector encoding active form C/EBPβLAP,

liver-enriched activator protein) (Figure S4b and 4c). Possible C/EBPβ-binding consensus

site between -72 and -59, AGCTAACCCCAAAC, was mutated by using -79 ACAM promoter

construct to produce -79 (MT1) and -79 (MT2) ACAM promoter constructs (Figure S4a),

which resulted in a loss of transactivation induced by IBMX and LAP. KLF4 has been

reported to induce adipocyte differentiation by activating C/EBPβ. KLF4-expressing

plasmids in the combination with GATA1 and/or GATA6-expressing plasmids synergistically

enhanced luciferase activity using -79 ACAM promoter construct (Figure S4d).

ACAM promotes the polymerization of actin and forms zonula adherens in

adipocytes: We investigated the expression of ACAM and polymerized form of actin

(F-actin) by Phalloidin staining. In WT mice under HFHS chow, both F-actin and ACAM were

faintly stained surrounding the adipocytes in epididymal adipose tissues, while they

colocalized and accentuated in a patchy fashion on the adipocytes (Figure 6a-b). In WT

mice, the distance of plasma membranes between adjacent adipocytes was ~100-200 nm in

WT mice under HFHS chow (Figure 6C). In ACAM Tg mice under HFHS chow, we observed

the zone with an intercellular space of ~10-20 nm, the structure of zonula adherens (Figure

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6c, S5a, S7, black arrows). The zonula adherens was continuous and characterized by strict

parallelism of the adjoining cell membranes over distances of 1 to 20 μm (Figure S6 and

S7). The intercellular space was occupied by homogeneous and amorphous material of low

density, and conspicuous bands of dense material located in the subjacent cytoplasmic

matrix. In the zonula adherens, two layers of plasma membrane joined in a linear and

parallel pattern but they also demonstrated invagination or engulfment (Figure 6c, S5a and

S6, white arrows). The zonula adherens was associated with immunogold particles when

the sections were stained with γ-actin or ACAM-specific antibodies (Figure 6d and S5b,

arrows). In brown adipose tissues in ACAM Tg mice under HFHS chow, similar structures of

zonula adherens was observed and immunogold particles associated with ACAM were

demonstrated along the zonula adherens (Figure S8). The ACAM-mediated homophilic

adhesion of adipocytes and formation of zonula adherens promoted the actin polymerization.

The formation of zonula adherens inhibited the adipocyte hypertrophy by actin

polymerization, which resulted in the improvement of obesity and insulin resistance.

DISCUSSION

In 3T3-L1 adipocytes, IBMX is major inducer of ACAM mRNA expression and it is a

known inducer of C/EBPβ. We identified C/EBPβ binding sites in the promoter regions of

ACAM gene and luciferase-reporter gene assay demonstrated the promoter activities were

enhanced by C/EBPβ expressed by LAP. KLF4 functions as an immediate early regulator of

adipogenesis to induce C/EBPβ(11) and KLF4, GATA1 and GATA6 has been reported to

enhance the transcriptional activity of ACAM gene in Sertoli cells(12). In current study, we

demonstrated that the promoter activities in luciferase assay were further enhanced by the

expression of KLF4, GATA1 and GATA6 and they are the critical coactivator of the

transcription of the ACAM gene. Immunofluorescence and immunogold studies

demonstrated the presence of ACAM at the cell processes of the 3T3-L1 cells and we

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speculate that ACAM may serve the heterophilic binding by interacting with certain

extracellular matrix (ECM) glycoproteins and also with cytoskeletal components such as

γ-actin and myosin II-A complex.

The mature adipocytes are surrounded by ECM glycoproteins such as collagens type I,

IV, V, VI, fibronectin and thrombospondin, and ECM environment regulates the adipogenesis

and adipocyte function(13). The remodeling of ECM surrounding the adipocytes takes place

and the basal lamina are thickened in omental adipose tissues of the rats fed with high-fat

diet(14). The administration of matrix metalloproteinase inhibitor, tolylsam, into the mice fed

with a high-fat diet resulted in lower body weight, lower subcutaneous and gonadal adipose

tissue mass(15). Thus, the peri-adipocyte ECM remodeling is tightly related to the

adipogenesis, tissue inflammation, fibrosis, insulin sensitivity and cardiovascular

diseases(13). Since peri-adipocyte ECM plays an important role in adipocyte biology and

the close cell-cell contact of the adipocytes has not been visualized in EM observation, the

role of ACAM in mature adipocytes remains elusive. ACAM/CLMP is expressed in tight

junction of epithelial cells and involved in the development and maintenance of epithelial

cells. Recently, we have developed ACAM knockout mice and ACAM-/- mice demonstrate

the elongation of small intestine, dilatation of bronchi, and formation of huge renal cysts

(submitted).

The presence of functional gap junction in cultured osteoblasts and adipocytes was

demonstrated; the inhibition of gap junctional communication by 18-α-glycyrrhetinic acid

(AGRA) blocks the maturation of pre-osteoblastic cells and converts to an adipogenic

phenotype(16). The later study further confirmed that the application of gap junction inhibitor

and small interfering RNA targeting connexin 43 inhibit the mitotic clonal expansion,

expression of Cebpb and adipocyte differentiation(17). Connexin 43 is a membrane

phosphoprotein forming gap junction and phosphorylation of connexin 43 is downregulated

after the induction of adipocyte differentiation. After the downregulation of phosphorylation,

connexin 43 is displaced from the plasma membrane and degraded by proteosomal

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pathway(18). Thus, gap-junctional intercellular communication is required in mitotic clonal

expansion and adipocyte differentiation. Historically, Farquhar and Palade observed EMs of

various epithelial cells and provided the identification of the zonula occludens (tight junction),

zonula adherens (intermediary junction), and the macula adherens (desmosome)(19). Gap

junction was not delineated from tight junction until the work of Revel and Karnovsky(20),

since extracellular or intermembrane gap is quite similar in tight and gap junctions(21). In

later study, the treatment of samples with uranyl acetate instead of lanthanum established

the presence of a 1.8 nm gap between the outer leaflets of the apposed membranes of gap

junction(21). Although the presence of functional gap junction in adipocytes has been

proved in previous studies, the morphological investigations by EM in adipose tissues has

not been published. We also extensively searched the adipose tissues of WT and ACAM Tg

mice under HFHS chow; however, we could not find the gap junction-like structures by EM.

Instead, we readily recognized zonula adherens with 10-20 nm intercellular space in ACAM

Tg mice under HFHS chow, in which transgenic overexpression of ACAM facilitated the

formation of zonula adherens. Originally, Farquhar and Palade defined zonula adherens by

the presence of an intercellular space (~20 nm) occupied by homogeneous, apparently

amorphous material of low density; by strict parallelism of the adjoining cell membranes over

distances of 0.2 to 0.5 μm; and by conspicuous bands of dense material located in the

subjacent cytoplasmic matrix(19), the observed structures ACAM Tg mice under HFHS

chow exactly corresponded to the definition of zonula adherens.

To further characterize the molecular organization of zonula adherens in adipocytes, we

performed TAP purification and immunoprecipitation studies, and demonstrated that ACAM

interacts with myosin II-A and γ-actin in 3T3-L1 cells. We further confirmed that ACAM and

γ-actin colocalized with zonula adherens observed in adipose tissues of ACAM Tg mice by

immunoelectron microscopy. Coxsackie and adenovirus receptor (CAR) is a homologue of

ACAM with 35% identity and it also interacts and binds to actin(22). CAR is strongly

expressed in developing nervous system, it uniformly expresses on all neural cells at an

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initial stage, and it downregulates and then restricts to axonal and dendritic surface at more

advanced stages(23). The membrane proximal Ig domain of CAR binds to a fibronectin

fragment and is involved in the heterophilic interactions, while two extracellular Ig domains

are involved in the homophilic interactions of CAR(23). Similarly, ACAM is localized on the

cell processes of preadipocytes, down-regulates after differentiation, and it re-distributes on

the plasma membrane of 3T3-L1 cells. In mature adipocytes in ACAM Tg mice, ACAM

colocalized with polymerized actin concentrated on the area of zonula adherens. Recently,

the actin cytoskeleton dynamics of polymerization and depolymerization cycles have been

reported to drive the adipocyte differentiation. Adipogenic stimuli downregulate RhoA-ROCK

signaling, induce the disruption of actin stress fibers, and result in the conversion to the

monomeric globular-actin (G-actin). The binding G-actin to MKL1 (megakaryoblastic

leukemia 1) inhibits the nuclear translocation of MKL1, the reduction of MKL1 in nuclei

activates the transcriptional activity of Pparg gene, and it results in adipocyte

differentiation(24). In fully-differentiated adipocytes, cortical F-actin is again formed and it

regulates the insulin-simulated translocation of GLUT4 from intracellular pool to the plasma

membrane. The overexpression of ACAM in transgenic mice prominently facilitates the

formation of cortical F-actin under the HFHS chow. One can speculate that the enhanced

formation of cortical F-actin may increase the mechanical strength per unit area and it

inhibits the adipocyte hypertrophy in the status of obesity induced by HFHS chow. In

addition, the formation of cortical F-actin enhances the translocation of GLUT4 and it may

improve the insulin sensitivity in ACAM Tg mice. We have also generated ACAM knockout

mice; however they were small for age and lethal around 14 weeks and we are unable to

analyze the size of adipocytes (submitted).

In 3T3-L1 preadipocytes, the expression of ACAM is induced by cAMP-PKA-C/EBPβ

pathway and it concentrates on the cellular processes. Once ACAM expression declines

and again appears on cell surface of the full-differentiated 3T3-L1 adipocytes. ACAM Tg

mice are protected from obesity and insulin resistance. The current investigation also

17

provides the evidence that the mature adipocytes develop the zonula adherens with the

molecular organization of ACAM and cortical F-actin in ACAM Tg mice under HFHS chow.

The promotion and maintenance of cortical F-actin by targeting ACAM is a candidate for the

new therapeutic modalities in the treatment of obesity and diabetes (Figure S9).

Funding.

This work was supported by JSPS Grant-in-Aid for Scientific Research, Grant numbers

(25126716, 26293218, 26461361, 26461362, and 40620753). KM is supported by The

Okayama Medical Foundation and recipient of Biological Study Award for Encouragement

(Ryobi Teien Memory Foundation).

Duality of Interests.

JW receives speaker honoraria from Astellas, Boehringer Ingelheim, Novartis, Novo Nordisk,

and Tanabe Mitsubishi, and receives grant support from Bayer, Daiichi Sankyo, Kyowa

Hakko Kirin, MSD, Novo Nordisk, Otsuka, Torii, Pfizer, Takeda, Taisho Toyama and

Tanabe Mitsubishi.

Author Contributions.

KM, JE, JW, and KH participated in the design of the whole study. AN, JW, JE, KM, and KH

participated in the generation of ACAM transgenic mice. Cell culture studies were performed

by KM, JE, AN, AK, MS, and HC. Western blot analyses were performed by DO, KT, and FO.

Electron microscopy was performed by MF and JW. KM, KT, FO, and JW and conceived of

the study, participated in coordination, performed the statistical analyses and helped to draft

the manuscript. All authors read and approved the final manuscript. JW takes full

responsibility for the work as a whole, including the study design, access to data, and the

decision to submit and publish the manuscript.

18

References

1. Shin CS, Lecanda F, Sheikh S, Weitzmann L, Cheng SL, Civitelli R: Relative abundance of different cadherins defines differentiation of mesenchymal precursors into osteogenic, myogenic, or adipogenic pathways. Journal of cellular biochemistry 2000;78:566-577 2. Castro CH, Shin CS, Stains JP, Cheng SL, Sheikh S, Mbalaviele G, Szejnfeld VL, Civitelli R: Targeted expression of a dominant-negative N-cadherin in vivo delays peak bone mass and increases adipogenesis. Journal of cell science 2004;117:2853-2864 3. Eguchi J, Wada J, Hida K, Zhang H, Matsuoka T, Baba M, Hashimoto I, Shikata K, Ogawa N, Makino H: Identification of adipocyte adhesion molecule (ACAM), a novel CTX gene family, implicated in adipocyte maturation and development of obesity. The Biochemical journal 2005;387:343-353 4. Tsuruga H, Kumagai H, Kojima T, Kitamura T: Identification of novel membrane and secreted proteins upregulated during adipocyte differentiation. Biochemical and biophysical research communications 2000;272:293-297 5. Raschperger E, Engstrom U, Pettersson RF, Fuxe J: CLMP, a novel member of the CTX family and a new component of epithelial tight junctions. The Journal of biological chemistry 2004;279:796-804 6. Makino H, Yamasaki Y, Haramoto T, Shikata K, Hironaka K, Ota Z, Kanwar YS: Ultrastructural changes of extracellular matrices in diabetic nephropathy revealed by high resolution scanning and immunoelectron microscopy. Laboratory investigation; a journal of technical methods and pathology 1993;68:45-55 7. Sakaue H, Ogawa W, Matsumoto M, Kuroda S, Takata M, Sugimoto T, Spiegelman BM, Kasuga M: Posttranscriptional control of adipocyte differentiation through activation of phosphoinositide 3-kinase. J Biol Chem 1998;273:28945-28952 8. Parsons WJ, Ramkumar V, Stiles GL: Isobutylmethylxanthine stimulates adenylate cyclase by blocking the inhibitory regulatory protein, Gi. Mol Pharmacol 1988;34:37-41 9. Chernogubova E, Cannon B, Bengtsson T: Norepinephrine increases glucose transport in brown adipocytes via beta3-adrenoceptors through a cAMP, PKA, and PI3-kinase-dependent pathway stimulating conventional and novel PKCs. Endocrinology 2004;145:269-280 10. Cao Z, Umek RM, McKnight SL: Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev 1991;5:1538-1552 11. Birsoy K, Chen Z, Friedman J: Transcriptional regulation of adipogenesis by KLF4. Cell metabolism 2008;7:339-347 12. Sze KL, Lee WM, Lui WY: Expression of CLMP, a novel tight junction protein, is mediated via the interaction of GATA with the Kruppel family proteins, KLF4 and Sp1, in mouse TM4 Sertoli cells. Journal of cellular physiology 2008;214:334-344 13. Chun TH: Peri-adipocyte ECM remodeling in obesity and adipose tissue fibrosis. Adipocyte 2012;1:89-95 14. Aslan H, Altunkaynak BZ, Altunkaynak ME, Vuraler O, Kaplan S, Unal B: Effect of a high fat diet on quantitative features of adipocytes in the omentum: an experimental, stereological and ultrastructural study. Obesity surgery 2006;16:1526-1534 15. Van Hul M, Lupu F, Dresselaers T, Buyse J, Lijnen HR: Matrix metalloproteinase inhibition affects adipose tissue mass in obese mice. Clinical and experimental pharmacology & physiology 2012;39:544-550 16. Schiller PC, D'Ippolito G, Brambilla R, Roos BA, Howard GA: Inhibition of gap-junctional communication induces the trans-differentiation of osteoblasts to an adipocytic phenotype in vitro. The Journal of biological chemistry 2001;276:14133-14138 17. Yanagiya T, Tanabe A, Hotta K: Gap-junctional communication is required for mitotic clonal expansion during adipogenesis. Obesity 2007;15:572-582 18. Yeganeh A, Stelmack GL, Fandrich RR, Halayko AJ, Kardami E, Zahradka P: Connexin 43 phosphorylation and degradation are required for adipogenesis. Biochimica et biophysica acta 2012;1823:1731-1744

19

19. Farquhar MG, Palade GE: Junctional complexes in various epithelia. The Journal of cell biology 1963;17:375-412 20. Revel JP, Karnovsky MJ: Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. The Journal of cell biology 1967;33:C7-C12 21. Grosely R, Sorgen PL: A history of gap junction structure: hexagonal arrays to atomic resolution. Cell communication & adhesion 2013;20:11-20 22. Huang KC, Yasruel Z, Guerin C, Holland PC, Nalbantoglu J: Interaction of the Coxsackie and adenovirus receptor (CAR) with the cytoskeleton: binding to actin. FEBS letters 2007;581:2702-2708 23. Patzke C, Max KE, Behlke J, Schreiber J, Schmidt H, Dorner AA, Kroger S, Henning M, Otto A, Heinemann U, Rathjen FG: The coxsackievirus-adenovirus receptor reveals complex homophilic and heterophilic interactions on neural cells. The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30:2897-2910 24. Nobusue H, Onishi N, Shimizu T, Sugihara E, Oki Y, Sumikawa Y, Chiyoda T, Akashi K, Saya H, Kano K: Regulation of MKL1 via actin cytoskeleton dynamics drives adipocyte differentiation. Nature communications 2014;5:3368

20

FIGURE LEGENDS

Figure 1 ACAM transgenic (Tg) mice are resistant to obesity. a. Wild type (WT) and

ACAM Tg male C57BL/6JJcl mice were fed with high fat-high sucrose (HFHS) and standard

(STD) chow. b. Fat pad weight of WT and ACAM Tg mice at 30 weeks of age. c. The

average size of adipocytes in epididymal adipose tissues of WT and ACAM Tg mice under

HFHS diet. The size distribution of adipocytes (d), Periodic acid-Schiff (PAS) staining (e. left

panels) and scanning electron micrographs (e. right panels), immunofluorescence (f. left

panels) and immunoperoxidase (f. right panels) staining of ACAM, and double

immunofluorescence staining of F4/80 and ACAM (g) in epididymal adipose tissues of WT

and ACAM Tg mice under HFHS diet. All data are presented as mean ± standard error (SE).

n=8. *p<0.05 vs. WT mice.

Figure 2 Glucose tolerance and insulin sensitivity are improved in ACAM Tg mice. a.

Glucose tolerance test by intraperitoneal injection of glucose in WT and ACAM Tg mice

under STD and HFHS diet. Glucose tolerance was improved in ACAM Tg mice under STD

and HFHS chow. Insulin tolerance test. Insulin sensitivity was improved in ACAM Tg mice

under HFHS (b) but not under STD (c) chow. d. Serum cholesterol levels of lipoprotein

fractions separated by High-performance liquid chromatography (HPLC) at 25 weeks of age.

e. PAS and Oil Red O staining of liver tissues in WT and ACAM Tg mice under HFHS chow.

There were no significant differences in both liver weight (f) and triglyceride contents (g). All

data are presented as mean ± SE. n=8. *p<0.05 vs. WT mice.

Figure 3 Oxygen consumption rate increases in ACAM Tg under HFHS chow.

Locomotor activities (a), food intake (b), and respiratory quotient (RQ) (c) were not altered in

WT and ACAM Tg mice fed with HFHS and STD chow. d. The weight of brown adipose

tissues was significantly reduced in ACAM Tg mice compared with WT mice under HFHS

21

chow. The size of lipid droplets was prominently reduced in ACAM Tg mice demonstrated by

light and electron microscopy. e-g. Oxygen consumption rate (V・

O2) was significantly

increased in ACAM Tg mice during both dark and light periods compared with WT mice

under HFHS chow. All data are presented as mean ± SE. n=8. *p<0.05 vs. WT mice.

Figure 4 Quantitative RT-PCR for genes related to glucose and lipid metabolism in

various tissues in WT and ACAM Tg mice under HFHS chow. a. Epididymal adipose

tissues. Many genes, such as Fabp4, Acaca, Fasn, Lpl, Hsd11b1, Slc27a1, Adipoq, Cd36,

Retn, Hk1 and Slc2a4, were upregulated in ACAM Tg mice compared with WT mice under

HFHS chow. b. Subdermal adipose tissues. c. Brown adipose tissues. Ucp1 and Cpt1a

were up-regulated in ACAM Tg mice compared with WT mice under HFHS chow. d. Skeletal

muscle. e. Liver. All data are presented as mean ± SE. n=4. *p<0.05 vs. WT mice under

HFHS.

Figure 5 ACAM expression is spatio-temporally regulated in the differentiation of

3T3-L1 adipocytes. a and b. ACAM mRNA revealed its peak at 6 hours after the induction

of differentiation and down-regulates within 48 hours. Again, ACAM mRNA expression

up-regulated ~14 days after induction at the late stage of differentiation. c. 3T3-L1 cells at

18 hours after hormonal induction were homogenized and fractionated by sucrose gradient

centrifugation. A protein (40 μg) from each fraction was separated by SDS/PAGE and

immunoblotted using anti-ACAM. Fractions are nucleus (Nuc), mitochondria/peroxisomes

(Mito/Per), plasma membrane (PM), low density microsome (LDM), high density microsome

(HDM), and cytosol (Cyto). d. Adenovirus vector expressing ACAM C-terminal tagged with

calmodulin and streptavidin-binding peptides (Adeno-pCTAP-mACAM) was applied to

3T3-L1 cells and purified products were analyzed by SDS-PAGE. Excised bands were

subjected to in-gel digestion with trypsin, analyzed by LC-MS/MS and MALDI-TOF/MS, and

myosin II-A and γ-actin were identified. e. Immunoprecipitation study. Cell lysates of 3T3-L1

22

cells were immunoprecipitated with anti-γ-actin and blotted with anti-ACAM antibodies. f.

Before the induction of adipocyte differentiation (Day 0), ACAM localized on the cell

processes and did not merge with myosin II-A. g. In mature 3T3-L1 adipocytes at 14 days

after the induction (Day 14), ACAM merged with myosin-IIA, and they coloalized on cell

surface. h. The immunogold studies using ACAM antibody demonstrated that they were

preferentially expressed on the cell process of 3T3-L1 cells at 2 days after the induction

(Day 2).

Figure 6 Formation of zonula adherence in ACAM Tg mice associated with ACAM and

γ-actin. a and b. Double immunofluorescence staining of phalloidin and ACAM in WT and

ACAM Tg mice under HFHS chow. F-actin demonstrated by phalloidin staining was faintly

visualized in WT mice. In contrast, F-actin was prominently increased and it merged with

ACAM. c. In WT mice, the distance of plasma membranes between adjacent adipocytes

was ~100-200 nm in WT mice under HFHS chow. In ACAM Tg mice under HFHS chow, the

structure of zonula adherens with an intercellular space of ~10-20 nm was observed (black

arrows). Two layers of plasma membrane joined in a linear and parallel pattern but they also

demonstrated invagination or engulfment (white arrows). d. The zonula adherens in ACAM

Tg mice under HFHS chow was associated with immunogold particles of γ-actin or

ACAM-specific antibodies (black arrows).

Figure 1

WT HF 278

Tg HF 402 kirei

WT WAT 390 20x10

Tg WAT 402 20x10-2

a b

*

*

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

WT (HFHS) ACAM Tg (HFHS)

0

2,000

4,000

6,000

8,000

WT(HFHS)

ACAMTg

(HFHS)

Adi

pocy

te s

ize

of e

pidi

dym

alfa

t (%

)

c

d e

*

Mea

n si

ze o

f epi

didy

mal

adip

ocyt

es (m

2)

WT (HFHS)

f gWT (HFHS)WT (HFHS)

ACAM Tg (HFHS)ACAM Tg (HFHS)

0.0

0.5

1.0

1.5

2.0

2.5

Fat

pad

wei

ght (

g)


WT (STD) ACAM Tg (STD)

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

Bod

y w

eigh

t (g)



* **

100m

ACAM Tg (HFHS)

100m

100m

100m

ACAM Tg (HFHS)

WT (HFHS)

100m100m100m

100m100m100m

100m100m

100m 100mACAM AbACAM Ab

WT (HFHS)

ACAM Tg (HFHS)

F4/80 ACAM Ab Merge

Figure 2

WT8 x400

Tg liver 289 10x20

WT liver 279 20x10

**

* * *

Glucose tolerance test Insulin tolerance test (0.75 IU/kg)

Insulin tolerance test (0.50 IU/kg)

a b c

*

d

0

10

20

30

WT(HFHS)

ACAM Tg(HFHS)

WT (STD) ACAM Tg(STD)

LDL

chol

este

rol (

mg/

dl)

0

100

200

300

400

0 30 60 90 120min

Blood glucose (mg/dl)



0

50

100

150

200

250

300

0 15 30 60 90 120min


WT (HFHS)

ACAM Tg (HFHS)

0

50

100

150

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250

0 15 30 60 90 120min


WT (STD)

ACAM Tg (STD)

0

5

10

15

20

25

30

Cho

lest

erol

(m

g/dl

)

WT (HFHS) ACAM Tg (HFHS) WT (STD) ACAM Tg (STD)

WT (HFHS)

ACAM Tg (HFHS)

e

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

WT(HFHS)

ACAMTg

(HFHS)

WT(STD)

ACAMTg

(STD)

f g

Live

r w

eigh

t (g

)

Live

r tr

igly

cerid

e co

nten

ts (

mg/

g)

0

20

40

60

80

100

120

140

WT(HFHS)

ACAMTg

(HFHS)

Oil Red O

100m

100m

100m

100m

WT (HFHS)

ACAM Tg (HFHS)

PAS

WT BAT 310 20x10

Tg BAT 281 10x20-2

0

2,000

4,000

6,000

8,000

10,000

WT(HFHS)

ACAMTg

(HFHS)

WT(STD)

ACAMTg

(STD)

0.0

2.0

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WT(HFHS)

ACAMTg

(HFHS)

WT(STD)

ACAMTg

(STD)

0.00

0.05

0.10

0.15

0.20

0.25

WT(HFHS)

ACAMTg

(HFHS)

WT(STD)

ACAMTg

(STD)

0.03

0.04

0.05

0.06

0.07

Bro

wn

adip

ose

tissu

e (g

)*

VO

2 (m

l/g/d

ay)

a b c

*

e f

d WT (HFHS)

ACAM Tg (HFHS)

Figure 3

*

Dark Light


* ** *****

** **** **

0.00

0.02

0.04

0.06

0.08

0.10

VO

2 (m

l/g/d

ay)

RQ

(w

hole

day

)

0

10

20

30

WT(HFHS)

ACAMTg

(HFHS)

WT(STD)

ACAMTg

(STD)

(ml/g

/day

)

VO2 (whole day)

0

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ACAMTg

(HFHS)

WT(STD)

ACAMTg

(STD)

(ml/g

/12h

ours

)

VO2 (dark time)

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ACAMTg

(HFHS)

WT(STD)

ACAMTg

(STD)

(ml/g

/12h

ours

)

VO2 (light time)

Foo

d in

take

(g/

3da

ys)

Loco

mot

orac

tivity

(co

unts

)

0.0

0.2

0.4

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WT(HFHS)

ACAMTg

(HFHS)

WT(STD)

ACAMTg

(STD)


Dark Light

100m

100m

10m

10m

WT (HFHS)

ACAM Tg (HFHS)

* *

g

*

*

*

** ****

** ****

**

**

**

*

Epididymal adipose tissuesa

b Subdermal adipose tissues

Brown adipose tissues

Liver

c d

e

Figure 4

0

0.5

1

1.5

2

2.5

3


Rel

ativ

e m

RN

A e

xpre

ssio

n

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0.5

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1.5

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ativ

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RN

A e

xpre

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Skeletal muscle

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Hours after induction

0 0.25 0.5 1 2 4 6 8 10 12 24 48 96

a

Cebpd

ACAM

Cebpb

Cebpa

Pparg

18S

18S

Pparg

Cebpa

ACAM

Lpl

-2 0 2 4 6 8 10 12 14 16

Days after inductionbFigure 5

f

g

Day

0D

ay 1

4

Day

2

50m50m50m

50m50m50m 200nm

200nm

h

Day

2

ACAM Ab

ACAM Ab

Myosin II-A Ab

Myosin II-A Ab

Merge

Merge

ACAM Ab

3T3-L1

3T3-L1

3T3-L1

3T3-L1

ACAM Ab

ACAM

c

250‐150‐100‐75‐

50‐

37‐

25‐

20‐

15‐

(kDa)Myosin II-A

(LC-MS/MS)

γ-actin(MALDI-TOF/MS)

d

e

250‐150‐100‐75‐50‐37‐

25‐20‐15‐

(kDa)

IB: anti-ACAM

IP: γ-actin IgG γ-actin IgG Input

+ + - - +Cell lysates

A-0027

WT-actin-0024

TG2-3-0016ASAN-Tg2-0026

ASAM-WT1-0005

Figure 6

ACAM Tg (HFHS) ACAM Tg (HFHS)

WT (HFHS)WT (HFHS)


WT (HFHS) WT (HFHS)

c d

γ-actin Ab

γ-actin Ab

ACAM Ab

ACAM Ab

Phalloidin ACAM Ab Merge 100μm

WT (HFHS)

ACAM Tg (HFHS)

100μm

100μm

100μm

100μm

100μm

a

10μm

10μm

0.2μm

0.2μm

Phalloidin ACAM Ab Merge

b

0.2μm 0.2μm

0.2μm 0.2μm

Supplementary Figures and Tables

Supplementary Figure S1, related to Figure 1

Production of adipocyte adhesion molecule transgenic (ACAM Tg) mice. a. Schematic

diagram showing structure of transgene consisting of aP2 promotor, β-globin intron, coding

region of ACAM cDNA and hGH polyA tail. EcoRI and SmaI-digested fragment of cDNA was

used for genomic Southern blot analysis. b. Genomic Southern blot analyses of 3 transgenic

lines and Western blot analyses of ACAM in epididymal and subdermal fat tissues. c.

Quantitative RT-PCR for ACAM mRNA in ACAM Tg and wild type (WT) mice at 30 weeks of

age. All data are presented as mean ± standard error (SE). n=8. *p < 0.05 WT vs. ACAM Tg

mice. d. e. f. Body weight of WT and ACAM Tg lines under high fat high sucrose (HFHS)

chow. All data are presented as mean ± s.e.m. n = 8. **p<0.01 and *p<0.05 vs. WT mice.

Line 4 (n=20), Line 11 (n=8) and Line 22 (n=20). g. PAS staining of subdermal adipose

tissues (WATs) in ACAM Tg and WT mice fed HFHS chow. Browning of adipocytes are not

seen in ACAM Tg mice.


a. b. c. Uncropped images for the blots shown Figure 5a, 5b and 5c. d. Western blot

analyses of ACAM before (Day 0) and after differentiation (Day 6 and 14) of 3T3-L1 cells.


The effect of various inducers on ACAM mRNA expression in 3T3L1 adipocytes a.

Confluent 3T3-L1 cells were cultured for 6 hours and stimulated with various combination of

inducers. The addition of IBMX alone or the combination with other inducers up-regulates

ACAM mRNA levels. (IBMX, 0.5 mM 1-methyl-3-isobutylxanthine; DEX, 0.25 M

dexamethasone; INS, 10 μg/ml insulin; FBS, 10% fetal bovine serum; PIO, pioglitazone) b

and c. IBMX up-regulated ACAM mRNA levels in a dose dependent manner and the peak

was observed at 6 hours and declined within 48 hrs. d and e. cAMP (8-bromoadenosine-

cAMP) and Forskolin increased ACAM mRNA levels. f. Effect of H89 (PKA inhibitor) on

DEX/IBMX/INS induced expression of ACAM mRNA. 3T3-L1 preadipocytes were pretreated

with H89 for 30 minutes and stimulated with DEX/IBMX/INS for 6 hours. H89 inhibited the

up-regulation of ACAM mRNA in a dose dependent manner.

Supplemental Figure S4, related to Figure 5

Luciferase reporter assay of ACAM promoter. a. Several possible CCAAT/enhancer-

binding protein β (C/EBPβ)-binding sites are indicated in ACAM promoter region (underlined).

We prepared deletion constructs of mouse ACAM promoter and ligated into pGL3 vector.

Mutations were also introduced into the -79 construct between -72 to -59; -79 (Mutant 1; MT

1) and -79 (Mutant 2; MT 2). b. ~8-fold induction of luciferase activity was observed in -584

to -79-bp ACAM promoter constructs by IBMX (1-methyl-3-isobutylxanthine) stimulation and

the luciferase activity was reduced to the basal level in the -57-bp ACAM promoter construct.

-79 (MT 1) and -79 (MT 2) constructs showed loss of transactivation. c. Constitutively active

form of C/EBPβ (LAP) also significantly increased luciferase activity in -584 to -79 constructs

and it was lost in -57, -79 (MT 1) and -79 (MT 2) constructs. d. The transfection of expression

vectors for Kruppel-like factor 4 (KLF4), somatic globin transcription factor 1 and 6 (GATA1

and GATA6) enhanced the luciferase activities. *p<0.01 vs. control basic vectors.


Transmission and immunogold electron microscopy of epididymal fat tissues in

ACAM Tg mice under HFHS chow. a. The zomula adherens was continuous and

characterized by strict parallelism of the adjoining cell membranes over distances of 1 to 20

μm. The intercellular space was occupied by homogeneous and amorphous material of low

density, and conspicuous bands of dense material located in the subjacent cytoplasmic

matrix. In the zonula adherens, two layers of plasma membrane joined in a linear and

parallel pattern (black arrows) but they also demonstrated invagination or engulfment (white

arrows). b. Immunogold particles associated with γ-actin and ACAM antibodies were aligned

along the zonula adherens.


Transmission electron microscopy of epididymal fat tissues in ACAM Tg mice under

HFHS chow. a-f. The zomula adherens was often observed at the corner of polygonal-

shaped adipocytes. The length of zonula adherens expanded from 1 to 20 μm and it was

frequently associated with invagination of the two layers of plasma membranes as indicated

by white arrows (b, c, d, e, and f).


Transmission electron microscopy of epididymal fat tissues in ACAM Tg mice under

HFHS chow. a-c. The zomula adherens was observed at the corner of polygonal-shaped

adipocytes and it traveled longer distance about 20 μm indicated black arrows.


Transmission electron microscopy of brown adipose tissues in ACAM Tg mice under

HFHS chow. a-g. As observed in epididymal adipose tissues in ACAM Tg mice under HFHS

chow, similar structures of zonula adherens were observed in brown adipose tissues. Dense

material in the subjacent cytoplasmic matrix along the two layers of plasma membranes was

observed. Immunogold particles associated with ACAM were observed along the zonula

adherens in brown adipose tissues.

a

c

d e

Supplementary Figure S1

15

20

25

30

35

40

45

50

Bod

y w

eigh

t (g)

15

20

25

30

35

40

45

50

Bod

y w

eigh

t (g)

f

L4 L11

**

ACAM Tg(HFHS)

ACAM Tg(HFHS)

WT (HFHS) WT (HFHS)

aP2 promoter -globin hGH polyAACAM cDNA

5.4 kb 0.65 kb 0.52 kb

Eco

RI

Pst

I

Bam

HI

Sm

aI

Xho

I

Hin

dIII

Eco

RI

Sm

aI

Not

I

Southern Probe (641 bp)

1.1 kb

Eco

RV

15

20

25

30

35

40

45

50

Body weight (g)

L22

*

ACAM Tg(HFHS)

WT (HFHS)

Southern blot analyses (Epididymal fat)

Epididymal fat (HFHS) Subdermal fat (HFHS)

Western blot analyses

L11 L22 L4 WTL11 L22 L4 WT

40kDa

ACAM mRNA

0

200

400

600

800

1000

Epididymaladiposetissues

Subdermaladiposetissues

Brownadiposetissues

Liver Skeletalmuscle

WT (HFHS) ACAM Tg (HFHS)WT (STD) ACAM Tg (STD)

Rel

ativ

e m

RN

A e

xpre

ssio

n*

* * * *

*

b

-641 bp

WT L4 L4 WT L11 L11 WT L22 L22

g

WT Sub WAT 297

Tg Sub WAT 282

WT (HFHS)

100m

ACAM Tg (HFHS)

100m

a b

Cebpd

ACAM

Cebpb

Cebpa

Pparg

18S

18S

Pparg

Cebpa

ACAM

Lpl

c

Figure 5a Original figures Figure 5b Original figures

Figure 5c Original figure


250‐150‐100‐75‐50‐37‐

25‐20‐15‐

(kDa)

Day 6 Day 14

Western blot analyses (3T3-L1 cells)

Day 0

d

-40kDa

a

d f

c

e

bD

EX

/IBM

X/IN

SD

EX

IB

MX

INS

DE

X/IB

MX

DE

X/IN

SIB

MX

/INS

PIO

DE

X/IB

MX

/INS

/PIO

FB

S

ACAM

Cebpb

18S

0 n

M1

nM

5 n

M50

nM

250

nM

500

nM

ACAM

Cebpb

18S

0 h

r3

hr

6 h

r12

hr

24 h

r48

hr

ACAM

Cebpb

18S

IBMX (6 hrs) IBMX (500 nM)

0 μ

M10

μM

100

μM

250

μM

500

μM

1 m

M

cAMP (6 hrs)

18S

ACAM

Cebpb

Forskolin (6 hrs)

5 μ

M

0 n

M5

nM

50 n

M50

0 n

M

50μ

M

H89 (6 hrs) 0

μM

1 μ

M2.

5 μ

M5

μM

10 μ

M20

μM

ACAMACAM

Cebpb Cebpb

18S 18S


a

d

*

Luciferase activity (Firefly/Renilla)

*

*

** *

b c

6 hrs after IBMX stimulation


* **

* *


* **

* *

0.0

0.1

0.2

0.3

0

0.1

0.2

0.3

Lac Z LAP

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

-584 ggtacccctagcgatttcataccgacctaaccagacctggaaggagaagagaaataagag -525-524 gctgggggaaacaagaagagagaggaagaagggaggagacaagagcaagaaagggaacaa -465-464 gatctggtgtagtgatgcgctcagcactaggtatatctatgtgagttcgagaccagccta -405-404 gtctacgtagacttccagaccaacaaagagctacagagtgcgaccctgtctaccaccacc -345-344 ccagtcatcctcgggagggggaggagagagagagagagagagagagagagagagagacat -285-284 aaagcattgtgagagacaaaaatatacaaaagagcagagagattagagagatcaaggaga -225-224 aaaaagacagaattgaaaaggccgcatccaccggttcttggcccaagtgctgcggattct -165-164 ctcctgcaccgcatcagttggcaccgagcgcccccctcgatcaccccgggccgcggccct -105-104 ccctcccgggtgggaggtaaagcagccccagcagctaaccccaaacgcagagagggaggg - 45- 44 cgggagcgaagagaagggagggcgaggggagggatggagggaggGGCTGCGGTAGGAGGC 1617 AGCCGTGTGGTTCCAGCTCATTTTTTTTCCCTCTTTCTCCAGTCGGTTTTCTTTCCAAAC 7677 AGGGAAAAGTGTTCCACGAAGCGGTAGCTCCTTGCCGCCTCGCCTTCTCCTCCCTAACCC 136

137 TGGGCCCGGCCCCCGTCCCGGCGCGAGCTGGTGGAGCCAGGGCTAGAAGCCCTCGGTGCC 196197 CCCGGAGCGCAGCGCGCAGGGGACCCGGGCGCGGGGCCAGCGCCCGCACATGGCTGCAGC 256257 CCCCCGCGCGCACCCCGAGGCGCCGCGCCCTGCTCACAGAAGGTCCGTCGGCTGGGCTCG 316317 GTCGCCCTGCAGCCAGGCTGCGCTGAGCCGGGAAGTGCCCGTGTCCGGAGATCGGGATGT 376

M S377 CCCTCTTCTTCCTCTGGCTA 396

L F F L W L

luciferase

-584 -79-235 -97-464 -57

Consensus NKNTTGCNYAAYNNWild type AGCTAACCCCAAACMutant 1 AGCTAACAATGGCCMutant 2 AGCTAAACCCGAAC


1 μm 0.2 μm



0.5 μm0.5 μmγ-actin Ab ACAM Ab

a

b



Tg3-D-0012 Tg3-D-0013

2.0 μm2.0 μm

b

Tg3-D-0007 Tg3-D-0006

5.0 μm 1.0 μm

a

Tg3-D-0041 Tg3-D-0042

2.0 μm 1.0 μm

f

Tg3-D-0038 Tg3-D-0039

2.0 μm 2.0 μm

e

Tg3-D-0009 Tg3-D-0010

5.0 μm 1.0 μm

d

Tg3-D-0004Tg3-D-0002

1.0 μm 0.2 μm

c


10 μm

2.0 μm

2.0 μm

a

b

c

2.0 μm

5 m


1.0 μm

2.0 μm

1.0 μm2.0 μm

0.5 mACAM AbACAM Ab

a b

c

d e

f g


Preadipocytes

F-actin

ACAM/CLMP

ACAM/CLMP

Adherens junction

Cortical actin

Mature adipocytes with hypertrophy in obesity

Mechanical strength, Inhibition of hypertrophy, Signal transduction

1

Supplementary Table 1

Gene Symbols

Forward primers Reverse primers

Acaca 5’-GGATGACAGGCTTGCAGCTATG-3’ 5’-GGAACGTAAGTCGCCGGATG-3’

Acc1 5’-GGATGACAGGCTTGCAGCTATG-3’ 5’-GGAACGTAAGTCGCCGGATG-3’

Aco 5’-TGACCTGCCGAGCCAGCGTAT-3’ 5’-GACAGAAGTCAAGTTCCACGCCACT-3’

Adipoq 5’-CTACGACCAGTATCAGGA-3’ 5’-GAAAGCCAGTAAATGTAGAG-3’

Cd36 5’-AAGCTATTGCGACATGATT-3’ 5’-GATCCGAACACAGCGTAGAT-3’

Clmp (ACAM)

5’-AGCCGTCATGTCTACAATAACTTGA-3’ 5’-GGGCTTGGATGGTCTCACTAGCACT-3’

Cpt1a 5’-GCTGCTTCCCCTCACAAGTTCC-3’ 5’-GCTTTGGCTGCCTGTGTCAGTATGC-3’

Fabp4 5’-CCGCAGACGACAGGA-3’ 5’-CTCATGCCCTTTCATAAACT-3’

Fasn 5’-GCTGGCATTCGTGATGGAGTCGT-3’ 5’-AGGCCACCAGTGATGATGTAACTCT-3’

G6pc 5’-AGCCTCCGGAAGTATTGTCTCA-3’ 5’-TCCACCCCTAGCCCTTTTAGTAG-3’

G6pdh 5’-CCGGTGTTTGAACGTCATCT-3’ 5’-CCGGAGGCTGGCATTGTAG-3’

Gck 5’-TACGACCGGATGGTGGATGA-3’ 5’-ACCAGCTCGCCCATGTACTTTC-3’

Hk1 5’-TGCTACATGGAGGAACTGCGACACAT-3’ 5’-ATGCCGCTCACCATCTTCTCGAACAG-3’

Hsd11b1 5’-GCTCACTACATTGCTGGCACTATGG-3’ 5’-TCTTCGCACAGAGTGGATGTCGTCA-3’

Irs2 5’-GGAGAACCCAGACCCTAAGCTACT-3’ 5’-GATGCCTTTGAGGCCTTCAC-3’

Leptin 5’-CACCAGGCTCCCAAGAATCATGTA-3’ 5’-GGGATGGCTCTTATCTCTACTTGCT-3’

Lpl 5’-AGGACCCCTGAAGACAC-3’ 5’-GGCACCCAACTCTCATA-3’

Pck1 5’- CCACAGCTGCTGCAGAACA-3’ 5’- GAAGGGTCGCATGGCAAA-3’

Pgc1a 5’-ATACCGCAAAGAGCACGAGAAG-3’ 5’-CTCAAGAGCAGCGAAAGCGTCACAG-3’

Pparg 5’-GTTTTATGCTGTTATGGGTG-3’ 5’-GTAATTTCTTGTGAAGTGCTCATAG-3’

Retn 5’-AACTCCCTGTTTCCAAATGCAATAA-3’ 5’-GGGCTGCTGTCCAGTCTATCCTT-3’

Rplp0 (36B4)

5’-GACAATGGCAGCATCTACAG-3’ 5’-CAACAGTCGGGTAGC-3’

Slc2a4 5’-CACAGAAGGTGATTGAACAGAGC-3’ 5’-TCCGGTCCCCCAGGA-3’

Slc27a1 5’-GCCGATGTGCTCTATGACT-3’ 5’-CGGCAGATTTCACCTATGTA-3’

Srebf1 5’-GAGCCATGGATTGCACATTT-3’ 5’-CACGGACGGGTACATCT-3’

Tnfa 5’-CCCACACCGTCAGCCGATTT-3’ 5’-GTCTAAGTACTTGGGCAGATTGACC-3’

Ucp1 5’-TACCAAGCTGTGCGATGTCCA-3’ 5’-CACACAAACATGATGACGTTCC-3’

Ucp2 5’-TGCCTTCCTTTCTCCGCTTGG-3’ 5’-GAAAGGTGCCTCCCGAGATTGGTAG-3’

Ucp3 5’-CAGGATTCTGGCAGGCTGCACGACA-3’ 5’-TTCCTCCCTGGCGATGGTTC-3’

department of nephrology, rheumatology, endocrinology and...

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