nucleoredoxin promotes adipogenic … nucleoredoxin promotes adipogenic differentiation through...

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Nucleoredoxin promotes adipogenic differentiation through regulation of Wnt/b-catenin

signaling

Young Jae Bahna,b, Kwang-Pyo Leeb, Seung-Min Leeb, Jeong Yi Choib, Yeon-Soo Seoa, and Ki-

Sun Kwonb,*

aDepartment of Biological Science, Korea Advanced Institute Science and Technology (KAIST),

Daejeon 305-701, Republic of Korea;

bAging Research Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB),

Daejeon 305-806, Republic of Korea

Running Title: Nucleoredoxin regulates adipogenesis.

*To whom the correspondence should be addressed: Ki-Sun Kwon, Aging Research Institute,

Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806,

Republic of Korea,Tel.: +82-42-860-4143, Fax: +82-42-879-8596, E-mail: [email protected].

by guest, on August 29, 2018

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ABSTRACT

Nucleoredoxin (NRX) is a member of the thioredoxin family of proteins that controls redox

homeostasis in cell. Redox homeostasis is a well-known regulator of cell differentiation into

various tissue types. We found that NRX expression levels were higher in white adipose tissue

of obese ob/ob mice and increased in the early adipogenic stage of 3T3-L1 preadipocyte

differentiation. Knockdown of NRX decreased differentiation of 3T3-L1 cells, whereas

overexpression increased differentiation. Adipose tissue-specific NRX (Adipo-NRX) transgenic

mice showed increases in adipocyte size as well as number compared to wild-type mice. We

further confirmed that the Wnt/β-catenin pathway was also involved in NRX-promoted

adipogenesis, consistent with a previous report showing NRX regulation of this pathway. Genes

involved in lipid metabolism were downregulated, whereas inflammatory genes, including those

encoding macrophage markers, were significantly upregulated, likely contributing to the obesity

in Adipo-NRX mice. Our results therefore suggest that NRX acts as a novel proadipogenic

factor and controls obesity in vivo.

Supplementary keywords: Nucleoredoxin, Wnt signaling, Adipogenesis, Obesity,

Inflammation


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INTRODUCTION

Adipose tissues play important roles in the regulation of whole-body energy homeostasis (1).

Excess energy intake increases the number and size of fat cells (2). These obese adipose tissues

facilitate chronic low-grade inflammation and insulin resistance, which result in severe obesity

and diabetes (3, 4). Thus, understanding adipocyte development and adipogenesis could lay the

groundwork for the development of efficient therapeutic strategies for preventing and treating

metabolic disorders associated with obesity.

Adipogenesis is controlled by a balance of internal and external factors that either stimulate

or repress adipogenic differentiation. In the early phase of adipogenic differentiation, C/EBPβ

(CCAAT/enhancer-binding protein β) and C/EBPδ induce expression of C/EBPα and PPARγ

(peroxisome proliferator-activated receptor gamma), which are the principal adipogenic

transcription factors that control the early differentiation of preadipocytes into lipid-

accumulating fat cells (5, 6). In addition, PPARγ appears to suppress canonical Wnt signaling by

accelerating proteasome-dependent degradation of β-catenin. Conversely, β-catenin, a

transcriptional coactivator in the Wnt signaling pathway, blocks adipogenesis by repressing

PPARγ and C/EBPα (7, 8). A number of reports have suggested a relationship between Wnt

signaling and diabetes and adipogenesis (9, 10). Adipose tissue-specific expression of Wnt10b

reduces adiposity and improves insulin sensitivity in the ob/ob obesity model (11). Although the

extensive down-regulation of β-catenin expression and its anti-adipogenic effects during

adipogenic differentiation have been characterized, less is known about the factors that

modulate β-catenin activity during adipogenesis.

Wnt signaling is initiated upon binding of Wnt ligands to transmembrane Frizzled receptors.

In the canonical Wnt signaling pathway, Frizzled receptors transduce signals through

Dishevelled (Dvl) to inhibit glycogen synthase kinase 3 (GSK3β), resulting in

hypophosphorylation and subsequent stabilization of β-catenin. Following nuclear translocation,

active β-catenin binds to and coactivates members of the T-cell factor/lymphoid-enhancer factor


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(TCF/LEF) family of transcription factors, leading to activation of target genes (12, 13).

Nucleoredoxin (NRX) is member of the thioredoxin (TRX) family of proteins. TRX family

proteins commonly possess a pair of redox-active, oxidation-sensitive cysteine residues in the

catalytic center that are directly involved in the reduction of disulfide bonds in target proteins

(14). Although NRX activity has been demonstrated in in vitro assays, whether the TRX-related

oxidoreductase activity of NRX plays a role in vivo is unknown (15). Previous studies have

shown that NRX is a multifunction protein that regulates target proteins through its direct

binding activity rather than its oxidoreductase activity (16, 17). Endogenous NRX protein is

predominantly localized in the cytosol of cells and its transcripts are widely expressed in all

adult tissues (18). Knockout of the NRX gene in mouse embryos is perinatally lethal; NRX-

knockout embryos (day 18.5) are smaller than their wild-type (WT) littermates and exhibit

craniofacial defects with short frontal regions (19). Interestingly, a genomic region around the

mouse NRX gene is involved in type 1 and type 2 diabetes (20). However, a role for NRX in

adipogenesis and obesity has not been reported.

Here, we investigated the role of NRX in preadipocyte differentiation and the obesity

phenotype using a 3T3-L1 preadipocyte differentiation system and adipose tissue-specific

transgenic mice. We show that NRX mediates adipogenesis by modulating β-catenin activity in

vitro and in vivo. Our findings suggest that NRX might act as a proadipogenic factor that is

involved in adipocyte differentiation and aspects of the obesity phenotype.

MATERIAL AND METHODS

Generation of adipose tissue-specific NRX transgenic mice

The LSL-NRX transgenic mice were obtained by microinjection and germ-line transmission

of the transgenic construct (Fig. S1A). The LSL-NRX mouse strain was backcrossed with the

C57BL/6 strain for more than eight generations to create a uniform genetic background.

Adipose tissue-specific NRX transgenic mice (Adipo-NRX) were produced by crossing LSL-


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NRX mice with Adiponectin-Cre transgenic mice, generating mice with adipose tissue-specific

overexpression of hNRX. All animal experiments were performed according to protocols

approved by the Animal Care and Use Committee of the Korea Research Institute of Bioscience

and Biotechnology (KRIBB).

Potential founder mice were genotyped by polymerase chain reaction (PCR) analysis using

genomic DNA isolated from mouse tail clips. Primers used for the detection of the transgenic

product were 5′-TGC GAG ATT ACA CCA ACC TG-3’ and 5′-CCC ATA TGT CCT TCC GAG

TG-3’.

Isolation and culturing of primary adipocytes

Primary adipocytes were isolated from epididymal fat pads using the collagenase method, as

described previously (21). Briefly, freshly excised fat pads were minced and digested for 45

minutes to 1 h at 37°C in Krebs-Ringer bicarbonate (pH 7.4) containing 4% bovine serum

albumin and 1.5 mg/ml type I collagenase (Worthington, Lakewood, NJ). The digested tissue

was filtered through a 300-μm nylon mesh to remove undigested tissue and centrifuged at 500 ×

g for 5 minutes. The floating adipocyte fraction was removed, washed with buffer, and re-

centrifuged to isolate free adipocytes. The stromal-vascular pellet was resuspended in

erythrocyte lysis buffer (154 mM NH4Cl, 10 mM KHCO3, and 1 mM EDTA), filtered through a

45-μm nylon mesh to remove endothelial cells, and pelleted at 500 × g for 5 minutes. Enriched

cells were cultured in a growth medium composed of Dulbecco’s Modified Eagle Medium

(DMEM), 20% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin

(Gibco-Invitrogen, Carlsbad, CA) at 37°C in a humidified 5% CO2 atmosphere.

Cell culture and adipogenic differentiation

The preadipocytes cell line 3T3-L1, derived from mouse embryo fibroblasts, was grown at

37°C in DMEM containing 10% heat-inactivated bovine calf serum (Gibco-invitrogen), 100


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units/ml penicillin, and 100 mg/ml streptomycin in a humidified 5% CO2 atmosphere. 3T3-L1

cells were induced to differentiate into mature adipocytes according to the procedure of Student

et al.(22), with minor modifications. Briefly, 2 days after reaching confluence (day 0), cells

were placed in differentiation medium composed of DMEM, 10% fetal bovine serum and MDI,

a differentiation cocktail consisting of 0.5 mM IBMX, 1 μM dexamethasone and 10 μg/ml

insulin (Sigma, St. Louis, MO). The medium was replenished every other day.

Generation of stable 3T3-L1 cell lines

3T3-L1 cells stably expressing FLAG-tagged NRX were generated using a lentivirus-

mediated infection system. For expression of NRX, cDNA encoding FLAG-tagged NRX was

cloned into the multi-cloning site of the GFP-tagged pLentiM1.4 vector. Lentiviruses were

subsequently produced by transiently co-transfecting HEK293T cells with pLP1, pLP2, and

pVSV-G plasmid (Invitrogen, Carlsbad, CA) using Lipofectamine (Invitrogen). Forty-eight

hours after transfection, supernatants containing lentiviral particles were collected and used to

infect 3T3-L1 cells in the presence of 4 µg/ml polybrene. Infected cells were selected by

incubation with 2 µg/ml puromycin for 2–3 weeks, and used in experiments as indicated. NRX

cDNA was kindly provided by Dr. Tasuku Honjo (Kyoto University, Japan) (15). An NRX

mutant in which catalytic Cys205 and Cys208 residues were replaced with Ser (CS-NRX)

provided by Dr. Sung-Kyu Ju, was constructed using site-directed mutagenesis.

RNA interference

NRX knockdown in 3T3-L1 cells was accomplished using small hairpin RNA (shRNA)

against mouse NRX in pLKO.1-puro lentiviral vectors obtained from Sigma (clone ID

NM_022463.3-1358s1c1 and NM_022463.3-452s1c1), according to the manufacturer’s protocol.

Briefly, shRNA lentiviral particles were generated in HEK293T cells by transient transfection

with pLP1, pLP2, pVSV-G, and shRNA lentiviral vector or pLKO.1-scrambled (control) vector


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(SHC002V; Sigma) using Lipofectamine. Forty-eight hours after transfection, supernatants

containing lentiviral particles were collected and used to infect 3T3-L1 cells in the presence of 4

µg/ml polybrene. Infected cells were selected by incubation with 2 µg/ml puromycin for 2–3

weeks, and used in experiments as indicated. A shRNA-resistant NRX incapable of shNRX

binding and subsequent NRX degradation was constructed by site-directed mutagenesis of the

target region of shNRX. The construct was obtained by standard methods using the primers 5’-

CTT TTG TGA ATG ACT TCT TGG CTG AAA AAC TC-3’ and 5’-TCA GGC TTG AGT TTT

TCAGCCAAG AAG TCA TT-3’. Underlined regions indicate the sites that were mutated

without changing amino acid residues.

RNA extraction and real-time PCR analysis

Total RNA was extracted using the Easy-Blue reagent (iNtRON Biotechnology, Korea),

according to the manufacturer’s instructions, and treated with RNase-free DNase I (Takara,

shiga, Japan) to remove contaminating genomic DNA. cDNA was then synthesized from total

RNA by reverse transcription (RT) using a DiaStar RT kit (Solgent, Korea). Quantitative RT-

PCR analysis was performed using the Step One Plus real-time PCR system (Applied

Biosystems, Waltham, MA) with the corresponding primers.

Immunoblotting and immunoprecipitation

Cells were lysed in a lysis buffer containing 20 mM HEPES (pH 7.2), 150 mM NaCl, 0.5%

Triton X-100, 0.1 mM Na3VO4, 1 mM NaF, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride

hydrochloride (AEBSF), and 5 mg/ml aprotinin (Sigma). Soluble proteins in cell lysates were

separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and

analyzed by immunoblotting using anti-NRX (R&D Biosystems, Minneapolis, MN, AF5719),

anti-PPARγ (Cell Signaling Technology, Danvers, MA, #2430), anti-β-actin (Santa Cruz

Biotechnology, Santa Cruz, CA, SC-47778), anti-Dvl-1 (Santa Cruz Biotechnology, SC-8025),


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anti-lamin A/C (Santa Cruz Biotechnology, SC-6215), anti-cyclin D1 (Cell Signaling

Technology, 2922), anti-β-catenin (Cell Signaling Technology, 9562), anti-α-tubulin (Millipore,

05-829), anti-AKT (Santa Cruz Biotechnology, SC-1618), anti-phospho-AKT (Cell Signaling

Technology, 9271L), and anti-FABP4 (Cell Signaling Technology, 2120) antibodies. WAT from

ob/ob mice was kindly provided by Dr. Chul-Ho Lee (KRIBB). For immunoprecipitation,

lysates were incubated with anti-FLAG agarose (Sigma) or anti-NRX antibody at 4°C for

several hours, after which the beads were washed three times with cell lysis buffer. The beads

were resuspended in 1X SDS-PAGE sample buffer and the eluted proteins were resolved by

SDS-PAGE.

In vitro gene transfer

For Amaxa nucleofections, pellets containing 0.5–1.5 × 106 3T3-L1 cells were carefully

resuspended in 100 µl of Nucleofector solution (Lonza, Allendale, NJ), mixed with 1–2 µg of

plasmids, and subjected to nucleofection using T-30 Amaxa protocols. The cells were then

gently transferred into a 6-well plate and cultured until analysis.

Luciferase assays and reagents.

Luciferase activity was assessed using the Luciferase Assay system (Promega, Madison, WI),

according to the manufacturer’s instructions. 3T3-L1 cells were transduced with TOPflash and

FOPflash exogenous reporter constructs together with pCMV-β-galactosidase. Luciferase

activity was normalized to β-galactosidase activity to adjust for transfection efficiency.

Oil-red O staining

Cultured cells were washed twice with phosphate-buffered saline (PBS) and fixed by

incubating with 3.7% paraformaldehyde for 15 minutes at room temperature. The cells were

then washed with distilled water and stained for 30 minutes with 0.3% filtered Oil-red O


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solution in 60% isopropanol. The stained cells were washed twice with distilled water and

photomicrographed. Oil-red-O staining was then quantified as described previously (23).

Incorporated Oil-red O dye was extracted by adding absolute isopropanol to the stained cell-

culture dish and shaking the dish for 30 minutes. Triplicate samples were read at 510 nm using

an Ultrospec2000 Spectrophotometer (Pharmacia Biotech, Piscataway, NJ).

Hematoxylin and eosin staining

Adipose tissue, muscle, and liver samples were fixed for 12–16 h at room temperature in 10%

formalin (Sigma) and then embedded in paraffin. Five-micron sections cut at 50-μm intervals

were mounted on charged glass slides, deparaffinized in xylene, and stained with hematoxylin

and eosin (H&E). Adipocyte cross-sectional area was quantified for each adipocyte in each field

using NIH Image J software (http://rsb.info.nih.gov/ij/). To quantify cell number, adipocytes

were isolated from 100 mg of adipose tissue by using 2 mg/ml collagenase type II-S (Sigma)

digestion buffer and were counted using a cell counter (Logos Biosystems, Annandale, VA) (24).

Glucose and Insulin Tolerance Test

Glucose tolerance test (GTT) was performed with 8h fasted animals. After determination of

fasted blood glucose levels, each animal was injected intraperitoneally (i.p.) with 20% glucose

(1 g/kg). The insulin tolerance test (ITT) was performed, with an initial fasting for 4 h, and

subsequent i.p. injection of insulin (1U/kg). In all tests, tail blood glucose levels were measured

with glucometer (Roche Diagnostics, Mannheim, Germany) at the indicated times after injection.

Immunohistochemistry

Formalin-fixed, paraffin-embedded sections of 5 µm were mounted on charged glass slides,

deparaffinized in xylene, stained with hematoxylin, and processed for immunohistochemical

detection of F4/80 according to standard immunoperoxidase procedure using VECTASTAIN


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Elite ABC kit (Vector Labs, Burlingame, CA) and anti-F4/80 antibody (Abcam, Cambridge,

UK).

Statistical analysis

Quantitative data are presented as means ± SD unless indicated otherwise. Differences

between means were evaluated using Student’s unpaired t test. A P-value < 0.05 was considered

statistically significant.

RESULTS

NRX levels increase in the early stage of 3T3-L1 preadipocyte differentiation.

We analyzed the expression of NRX during 3T3-L1 cells differentiation induced by a

differentiation cocktail composed of IBMX (3-isobutyl-1-methylxanthine), dexamethasone, and

insulin. NRX transiently increased in the early stages of adipocyte differentiation at both mRNA

and protein levels. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

revealed that NRX mRNA reached a maximum 1 day after differentiation induction (Fig. 1A).

Protein levels of NRX were proportional to mRNA levels (Fig. 1B). We next determined

whether hyperglycemic conditions affected NRX levels in ob/ob mice. Interestingly, NRX

protein was upregulated in white adipose tissue (WAT) of ob/ob mice compared to those in WT

mice (Fig. 1C). These results suggest that increased expression of NRX may be associated with

adipogenesis and, ultimately, obesity.

NRX overexpression leads to adipocyte hypertrophy combined with hyperplasia in mouse

adipose tissue.

To further investigate the function of NRX in adipogenesis in vivo, we generated loxP-stop-

loxP-NRX transgenic mice (LSL-NRX), then crossed these mice with adiponectin-Cre mice to

generate adipose tissue-specific NRX transgenic mice (Adipo-NRX).To validate the specific


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overexpression of NRX in adipose tissues, we analyzed NRX protein levels in several tissues

isolated from Adipo-NRX and WT mice. As expected, overexpression of NRX was observed

only in adipose tissues, including white and brown adipose tissue (Fig. S1B). To clarify activity

of the adiponectin promoter, we checked expression levels of adiponectin during adipogenesis.

We confirmed that the expression level of adiponectin was very low before induction of

differentiation, but were gradually increased after induction of differentiation (Fig. S1C), which

was consistent with previous report (25). We also observed that expression of adiponectin

promoter-driven NRX was induced at early differentiation stage in primary adipocytes isolated

from Adipo-NRX mice (Fig. S1D).

Although there was a slight increase in body weight in Adipo-NRX mice compared to WT

littermate controls, a comparison of organ weights revealed a notable increase in epididymal

WAT in Adipo-NRX mice (Fig. 2A). Epididymal and peri-renal fat masses were also

significantly larger in Adipo-NRX mice than in WT mice (Fig. 2B and C). There was no

difference in food intake between Adipo-NRX mice and WT littermate controls. An analysis of

adipocyte cross-sectional areas showed that epididymal fat of Adipo-NRX mice contained

hypertrophied adipocytes (Fig. 2D). A quantitative analysis revealed that adipose tissue

expansion of Adipo-NRX mice was caused by an increase of adipocyte numbers (Fig. 2E) as

well as an enlargement of adipocyte size, suggesting that hypertrophy was accompanied by

hyperplasia in adipose tissue of Adipo-NRX mice.

The plasma analysis revealed that blood glucose and insulin levels were higher in Adipo-

NRX mice than in WT mice (Table S1). We further assessed glucose homeostasis in WT and

Adipo-NRX mice via a glucose tolerance test and insulin tolerance test. Adipo-NRX mice

showed the tendency to glucose intolerance compared to WT mice (Fig. 2F), and exhibited

impaired insulin tolerance that was associated with reduced insulin sensitivity in WAT but not in

liver and skeletal muscle (Fig. 2G and H). Although the adipose tissue appeared lipid

accumulation, there was no significant change in lipid deposition in liver and skeletal muscle


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(Fig. S2). These data demonstrate that adipocyte specific NRX overexpression increases lipid

accumulation in WAT, resulting in insulin resistance.

NRX overexpression leads to increases adipogenic differentiation.

To evaluate whether overexpression of NRX facilitates adipogenic differentiation in cell

culure system, we infected 3T3-L1 preadipocytes with a lentivirus expressing GFP-tagged

mouse NRX or a control lentivirus. Stable overexpression of NRX was monitored by

immunoblotting (Fig. 3A) and fluorescence imaging. 3T3-L1 preadipocytes stably

overexpressing NRX showed increased accumulation of lipid droplets following induction of

adipocyte differentiation compared to control cells (Fig. 3B and C). We confirmed that the

adipogenic markers, PPARγ and FABP4 (fatty acid binding protein 4, adipocyte), were also

upregulated in NRX-overexpressing cells compared with control cells (Fig. 3D). To confirm that

NRX overexpression positively regulates adipocyte differentiation, we next isolated and

cultured primary adipocytes from epididymal fat pad of Adipo-NRX and WT mice.

Differentiation of primary adipocytes was increased in Adipo-NRX mice compared with that of

WT mice (Fig. 3E). Consistent with these morphological observations, expression of the

adipogenic marker, FABP4, was also increased in primary adipocytes from Adipo-NRX mice

(Fig. 3F). These data strongly suggest that NRX enhances adipogenesis.

Decreased lipid catabolism and increased inflammation and fibrosis in WAT of Adipo-

NRX mice

We found that expression of enzymes involved in lipid catabolism, including Atgl (adipose

triglyceride lipase), Mcad (acyl-Coenzyme A dehydrogenase, medium chain), and Cpt1a

(carnitine palmitoyltransferase 1a) were downregulated, indicating that adipocytes of Adipo-

NRX mice lacked the ability to burn excess fat. These results suggest that increased fat mass in

Adipo-NRX mice is due to decreased lipolysis and fatty acid oxidation. Since adipokine


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dysregulation is a hallmark of adipocyte impairment (26), we further measured adipokine

expression in WAT of Adipo-NRX mice. The mRNA levels of adiponectin were downregulated

and PAI-1 (plasminogen activator inhibitor type 1) was upregulated in WAT of Adipo-NRX

mice.

Obesity has been consistently associated with inflammation (4, 27). We examined mRNA

expression levels of several inflammatory genes in WAT of WT and Adipo-NRX mice. Among

the genes upregulated in Adipo-NRX mice were TNFa (tumor necrosis factor alpha) and

Cxcl10 (chemokine [C-X-C motif] ligand 10), which encode proinflammatory cytokines; Cybb

(cytochrome b-245, beta polypeptide), which is involved in phagocytosis; and Emr1 (EGF-like

module containing, mucin-like, hormone receptor-like 1) and Itgax (integrin alpha X), markers

of macrophage infiltration (Fig. 3G). Consistently, increased macrophage infiltration in WAT

was shown by immunostaining using a macrophage surface marker, F4/80 (Fig. 3H). Obesity is

associated with an overall increase in expression of several collagens that results in fibrotic state

(28, 29). Then, we tested mRNA levels of several fibrosis related genes, and found that Col1a1,

Col3a1, Col6a1, and Elastin were upregulated (Fig. 3G) in WAT of Adipo-NRX. These suggest

that adipocyte expansion is closely linked to inflammation and fibrosis in Adipo-NRX mice.

NRX knockdown attenuates adipogenic differentiation in 3T3-L1 preadipocytes.

To test whether endogenous NRX influences adipogenesis, we examined the effect of NRX

knockdown in 3T3-L1 preadipocytes. 3T3-L1 cells were infected with a lentivirus expressing a

small hairpin RNA (shRNA) targeting NRX (NRX shRNA) or non-targeting (scrambled)

shRNA. Depletion of endogenous NRX was confirmed by immunoblot analyses (Fig. 4A).

Knockdown of NRX attenuated differentiation of 3T3-L1 preadipocytes into mature adipocytes,

reducing accumulation of lipid droplets compared with control 3T3-L1 cells (Fig. 4B).Moreover,

expression levels of the adipocyte markers, PPARγ and FABP4, also were decreased in NRX-

depleted cells compared to control cells (Fig. 4C). Next, to exclude the possibility of off-target


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effects of shRNA targeting the NRX coding sequence, we transfected NRX-knockdown 3T3-L1

cells with an expression plasmid for an shRNA-resistant NRX (resNRX) with a codon usage

different from that of the original plasmid. resNRX expression restored PPARγ and FABP4

expression levels (Fig. 4E) as well as accumulation of lipid droplets (Fig. 4D). These results

clearly demonstrate that NRX is involved in adipogenic differentiation.

NRX regulates adipogenesis through modulation of β-catenin signaling in 3T3-L1

preadipocytes.

The conserved Wnt/β-catenin signaling pathway mediates a variety of cellular responses,

from cell proliferation to cell-fate determination (30, 31). Dvl transduces signals from the Wnt

receptor, Frizzled, to downstream components through inhibition of GSK3β, leading to the

stabilization and nuclear translocation of β-catenin (12). It has been reported that NRX regulates

Wnt/β-catenin signaling by directly binding and inhibiting Dvl, and, thereby controls cell

proliferation (16). Wnt/β-catenin signaling is also a central negative regulator of adipogenesis

(7). Here, we found that NRX is involved in adipogenic differentiation, prompting us to

examine whether NRX regulates adipogenesis through Wnt/β-catenin regulation. First, we

tested the interaction between NRX and Dvl in adipocyte. Endogenous Dvl interacted with

ectopically expressed WT NRX, but not with a cysteine-mutant NRX defective in thiol reducing

activity, in 3T3-L1 preadipocytes (Fig. 5A). Moreover, unlike WT NRX, ectopically expressed

cysteine-mutant NRX did not increase 3T3-L1 differentiation (Fig. 5B). These results suggest

that NRX controls adipogenesis by interacting with Dvl through a thiol-based mechanism. We

further observed that endogenous NRX-Dvl binding was dissociated by Wnt3a treatment (Fig.

5C), suggesting that NRX controls adipogenesis via Wnt/β-catenin signaling. To test this

hypothesis, we determined whether NRX controls the nuclear localization of β-catenin in 3T3-

L1 cells and primary adipocytes isolated from Adipo-NRX mice. Nuclear β-catenin levels and

transcriptional activity were increased in NRX-knockdown cells, but were decreased in these


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cells following restoration of NRX expression (Fig. 5D). Enhancing NRX expression by

increasing the amount of transfected plasmid induced a dose-dependent decrease in the levels of

nuclear β-catenin and its downstream effector cyclin D1 in Wnt3a-treated 3T3-L1 cells (Fig.

5E), resulting in recovery from Wnt3a-mediated inhibition of adipogenesis (Fig. 5F, third panel).

Consistent with the results obtained in 3T3-L1 preadipocytes (Fig. 5D and E), the expression of

β-catenin target genes was decreased in WAT of Adipo-NRX mice (Fig. 5G).

Next, we examined whether the interaction of NRX with Dvl plays a role in adipogenesis.

While single Dvl gene (Dvl-1, Dvl-2, or Dvl-3) knockdown showed no significant changes in

adipocyte differentiation compared with control group (Fig. S3), triple knockdown of all Dvl

genes enhanced differentiation of 3T3-L1 into mature adipocytes (Fig. 6A and B) accompanying

with an elevated expression of FABP4, an adipocyte differentiation marker, compared to control

(Fig. 6C). To test whether NRX role is dependent on Dvl, we checked the effect of NRX on

adipogenesis in the presence or absence of three Dvls. We found that the Dvls knockdown

suppressed reduced 3T3-L1 differentiation by NRX knockdown (Fig. 6D and E). NRX

knockdown reduced lipid accumulation by 31.2 %, while knockdown of Dvls together reduced

lipid accumulation only by 10.5 %. These results suggest that NRX regulates adipogenesis

through inhibition of Dvl-β-catenin axis in adipocyte differentiation.

DISCUSSION

Our study demonstrated that NRX mRNA and protein were increased in the early stages of

adipocyte differentiation and were also increased in WAT of ob/ob mice, a leptin-deficiency

model of obesity. Employing NRX-depleted and -overexpressing 3T3-L1 preadipocytes, we

showed that NRX is associated with adipogenic differentiation of 3T3-L1 cells. We found that

differentiation of primary adipocytes from Adipo-NRX mice was increased in vitro, and

epididymal and peri-renal fat mass were increased in Adipo-NRX mice in vivo. These

observations, taken together with our in vitro and in vivo adipogenesis data, thus uncover a


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novel role for NRX as a proadipogenic factor.

Adipose tissue expansion occurs through an enlargement in adipocyte size (hypertrophy)

and/or an increase in adipocyte number (hyperplasia). We showed that Adipo-NRX mice

exhibited hyperplasia in adipocytes, which is linked to enhanced adipogenic differentiation (32,

33), as well as hypertrophy (Fig. 2D and E). Adipo-NRX mice showed decreased expression of

enzymes involved in lipolysis and fatty acid oxidation in WAT, while unchanged in lipogenic

enzymes compared to WT mice. Decreased triacylglycerol catabolism is associated with the

occurrence of prevalent metabolic diseases, such as obesity and type 2 diabetes (34-36).

Hypertrophic adipocyte in our model is likely caused by the decrease of triacylglycerol

catabolism rather than an increase in triacylglycerol synthesis. However, the mechanism by

which NRX regulates fatty acid catabolism remains to be determined.

Lipid accumulation increased exclusively in WAT of Adipo-NRX mice, with no change in

liver and skeletal muscle compared to WT mice (Fig. S2). In plasma analysis, Adipo-NRX mice

exhibited increased fasting insulin level accompanying with increased fasting glucose level,

suggesting that pancreas function was normal (Table S1). Adipo-NRX mice showed the

tendency to glucose intolerance in GTT analysis (Fig. 2F), and also showed mild insulin

resistance as shown in impaired insulin tolerance and reduced insulin sensitivity in WAT but not

in liver and skeletal muscle (Fig. 2G and H). Therefore, insulin resistance in our model is likely

caused by excessive fat deposition in WAT.

Infiltration of adipose tissue by inflammatory cells has been described as a common feature

of obesity (27). We also found increased expression of inflammatory gene and macrophage

markers as well as dysregulation of adipokines in Adipo-NRX mice (Fig. 3G). Many rodent

models of obesity are associated with chronic inflammation, and it is known that obesity-

induced insulin resistance is strongly correlated with expression of inflammatory markers (37,

38). However, the molecular links between obesity and inflammation have not been completely

elucidated. Adipo-NRX mice might be a potential model system for detailed mechanistic studies


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of the relationship between obesity and inflammation.

Wnt signaling is an important regulator of fate decisions in mesenchymal cells (31). In

multipotent mesenchymal precursors in vitro, activation of Wnt/β-catenin signaling promotes

osteoblastogenesis, but inhibits adipogenesis (39). Given that NRX inhibits Wnt/β-catenin

signaling (16), it is possible that NRX reciprocally modulates adipogenic and osteogenic

differentiation. Activation of β-catenin in primary osteoblasts isolated from calvariae of NRX-/-

embryos results in enhanced osteoblastic differentiation (19). However, there have been no

reports of a role for NRX in adipogenesis. Here, we provide the first demonstration that NRX

positively regulates adipogenesis by inhibiting the Wnt/β-catenin pathway. Wnt10b is a likely

candidate for the endogenous Wnt that participates in adipogenesis. The expression level of

Wnt10b, which is highly expressed in preadipocytes, declined upon initiation of differentiation

(40), exhibiting a negative correlation with NRX expression (Fig. 1A).Wnt10b has been

reported to attenuate the development of obesity in ob/ob mice by stabilizing β-catenin and

subsequently inhibiting adipogenesis. Conversely, blocking Wnt signaling promotes

adipogenesis and obesity (11). Based on our data, it is likely that NRX is involved in obesity in

ob/ob mice through regulation of Wnt10b signaling.

Although current evidence suggests that β-catenin functions as a crucial regulator of

adipogenesis (41), how its expression and activity are regulated during adipogenic

differentiation, particularly the factors involved, remains largely unknown. During adipogenesis,

there is a considerable reduction in total β-catenin protein as well as a marked decrease in

nuclear β-catenin levels (8, 42).In addition to regulating β-catenin protein levels, we found that

NRX directly interacted with and inhibited the Wnt/β-catenin effector molecule Dvl during

adipogenesis. Both aspects of this negative regulation of Wnt/β-catenin signaling by NRX

enabled induction of the adipogenic transcription factor PPARγ, which, in turn, downregulated

β-catenin levels, ensuring complete terminal differentiation into mature adipocytes.

Our current findings show that the active site cysteine of NRX is required for proper


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inhibition of the Wnt/β-catenin pathway. However, we do not yet know how redox controls the

inhibitory function of NRX in Wnt/β-catenin signaling, although we presume that thiol-disulfide

exchange of the active site cysteine residues leads to a conformational change in the NRX

molecule that modulates its inhibitory function. Further studies will be needed to confirm this.

In conclusion, our study provides the first evidence that NRX acts as a proadipogenic factor

by regulating Wnt signaling and is associated with phenotypic manifestations of obesity.

Therefore, we propose that modulating Wnt/β-catenin signaling by blocking NRX may

ultimately prove to be an effective therapeutic strategy for managing obesity and metabolic

disorders such as diabetes.

ACKNOWLEDGMENTS

We thank Dr. Dae-Sik Lim (KAIST) for kindly providing Adiponectin-Cre transgenic mice. We

are grateful to Dr. Tasuku Honjo (Kyoto University, Japan) for NRX cDNA. This study was

supported by grants from the Bio & Medical Technology Development Program (20110030133

and 2013M3A9B6076413, to K.-S.K.) of the National Research Foundation funded by the

Korea government (MSIP), and the KRIBB Research Initiative Program.

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FOOTNOTES

Abbreviations: Fabp4, fatty acid binding protein 4; C/EBPα, CCAAT/enhancer-binding protein

α; PPARγ, peroxisome proliferator-activated receptor gamma; TNFa, tumor necrosis factor

alpha; Cxcl10, chemokine [C-X-C motif] ligand 10); Cybb, cytochrome b-245, beta polypeptide;

Emr1, EGF-like module containing, mucin-like, hormone receptor-like 1; Itgax, integrin alpha


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X; Atgl, adipose triglyceride lipase; Mcad, acyl-Coenzyme A dehydrogenase, medium chain;

Cpt1a, carnitine palmitoyltransferase 1a; PAI-1, plasminogen activator inhibitor type 1; FAS,

fatty acid synthase; HSL, hormone sensitive lipase; Ccl5, Chemokine (C-C motif) ligand 5;

MMP-7, matrix metalloproteinase-7; NRX, nucleoredoxin; Dvl, Dishevelled; Adipo-NRX mice,

adipose tissue-specific NRX transgenic mice; LSL-NRX mice, loxP-stop-loxP-NRX transgenic

mice.

FIGURE LEGENDS

Fig. 1. NRX expression during adipogenic differentiation of 3T3-L1 preadipocytes and in

WAT of obese mice. (A-B) 3T3-L1 cells were induced to differentiate into adipocytes by

treatment with MDI medium. A) Expression of NRX was analyzed using real-time quantitative

RT-PCR. Total RNA was extracted at the indicated days of differentiation. β-Actin was used as

an internal control. B) NRX expression was analyzed by Western blotting at the indicated days

of differentiation. PPARγ and FABP4 were used as adipocyte differentiation markers. β-Actin

was used as a loading control. C) Western blot analysis of NRX expression in visceral WAT of

WT and ob/ob mice. Data are presented as mean fold-changes ± SD (*P<0.05, **P<0.01).

Fig. 2. Adipo-NRX transgenic mice have larger amounts of adipose tissue compared to WT

mice. A) Body weights and organ weights of 6-month-old Adipo-NRX and WT mice were

determined (n=6). B, C) Representative pictures of epididymal and peri-renal fat dissected from

fat pads of Adipo-NRX and WT mice. D) H&E staining of WAT from epididymal fat pads of

Adipose-NRX and WT mice (left). Quantitative analysis of cell numbers/mm2 in sections of

WAT (right) E) Total cell number of adipocytes per fat fad isolated from WT and Adipo-NRX

mice (n=3). F) Glucose tolerance test and G) Insulin tolerance test in WT and Adipo-NRX mice.

Total area under curve (AUC) of each graph was measured (insets). Fasted mice were injected


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with glucose (1g/kg i.p.) or insulin (1U/kg i.p.). Blood glucose levels (mg/dL) were determined

at the indicated time points. Values are means ±SD of 6-8 mice. H) Western blotting analysis of

phospho-AKT (AKT-S473 phosphorylation) in indicated tissues isolated from WT and Adipo-

NRX mice (n=3) after i.p. injection of insulin (10U/kg). The symbol “ ̶ ” or “+” means without

or with insulin stimulation, respectively. Data are presented as mean percentages ± SD (*P<0.05,

**P <0.01).

Fig. 3. Overexpression of NRX promotes adipogenic differentiation of 3T3-L1

preadipocytes and primary adipocytes. 3T3-L1 cells were infected with lentiviruses

expressing NRX (pLenti-NRX-GFP-flag) or control (pLenti-GFP-flag), selected with

puromycin, and induced to differentiate using MDI medium. A) Ectopic expression of NRX was

monitored by Western blot analysis with anti-NRX antibody. B) After 8 days of differentiation,

the degree of lipid accumulation was visualized by staining cells with Oil red-O. C) Staining in

cells was quantified using a dye-extraction solution (n=4). D) Whole-cell lysates were extracted

on day 0 or day 8 for Western blot analysis of NRX, PPARγ, FABP4, and β-actin. E) Primary

white adipocytes isolated from Adipo-NRX and WT mice were cultured in MDI medium

containing rosiglitazone to induce differentiation into adipocytes. After 10 days of

differentiation, cells were stained with Oil red-O dye. F) Whole-cell lysates were extracted from

differentiated white adipocytes on day 10 for Western blot analysis of NRX, FABP4, and β-actin

(n=4). G) Relative mRNA abundance was examined in WAT from WT and Adipo-NRX mice by

real-time quantitative RT-PCR (n=3). Data are presented as mean fold-changes ± SD (*P<0.05,

**P<0.01, ***P<0.001). H) Frozen sections of WAT isolated from WT and Adipo-NRX were

immunostained with antibody againstF4/80, a macrophage surface marker. Images were taken at

magnification X200 (upper panels) and X400 (lower panels).

Fig. 4. NRX shRNA attenuates adipogenic differentiation in 3T3-L1 preadipocytes. A)


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NRX protein levels in NRX-knockdown and control, non-target (NT) 3T3-L1 cells were

monitored by Western blot analysis with anti-NRX antibody. B) NRX-knockdown and control

3T3-L1 cells were differentiated for 8 days in MDI medium and then stained with Oil red-O dye.

C) After induction of differentiation, whole-cell lysates were prepared from non-target and

NRX-knockdown cells on the indicate days for Western blot analysis of NRX, PPARγ, FABP4,

and β-actin. β-Actin was used as a loading control. D) Rescue experiments were performed by

transfecting NRX-knockdown 3T3-L1 cells with plasmids encoding shRNA-resistant NRX

(resNRX). Eight days after differentiation, cells were stained with Oil red-O dye. E) Analysis of

protein levels during adipogenic differentiation of NRX-knockdown and NRX-rescued cells.

Whole-cell lysates were extracted on day 8 for Western blot analysis of NRX, PPARγ, FABP4,

and β-actin.

Fig. 5. NRX regulates adipogenesis via modulation of β-catenin in 3T3-L1 cells. (A-B) WT

NRX interacts with Dvl and increases 3T3-L1 differentiation. A) 3T3-L1 cells were transfected

with vectors expressing FLAG-tagged WT or mutant (Mut) NRX, lysed and

immunoprecipitated with anti-FLAG antibody, and analyzed by immunoblotting with anti-Dvl

antibody. B) After 8 days of incubation in differentiation media, cells were stained with Oil red-

O dye. C) 3T3-L1 cell lysates were immunoprecipitated using anti-NRX antibodies after

treatment with 200 ng/ml Wnt3a followed by immunoblotting, as indicated. (D-F) NRX inhibits

β-catenin activity and promotes recovery from Wnt3a-mediated inhibition of adipogenesis. D)

Reporter gene (TOPflash) expression assays in NRX-knockdown 3T3-L1 cells after

cotransfection of increasing amounts of resNRX. Nuclear (N) and cytosolic (C) fractions were

analyzed by immunoblotting. Lamin A/C was used as a control for the nuclear fraction. E)

3T3-L1 cells transfected with vectors expressing FLAG-tagged NRX were treated with Wnt3a.

Cytosolic and nuclear proteins were isolated for Western blot analysis of FLAG-NRX, β-catenin,

lamin A/C, and cyclin D1. F) 3T3-L1 cells overexpressing NRX were induced to differentiate


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into adipocytes with Wnt3a for the first 2 days. After 8 days of differentiation, cells were stained

with Oil red-O dye. Whole-cell lysates were prepared on day 8 for Western blot analysis of

NRX, FABP4, and β-actin. G) β-Catenin target genes were examined in WAT from WT and

Adipo-NRX mice by real-time quantitative RT-PCR (n=3). Data are presented as mean fold-

changes ± SD (*P<0.05, **P<0.01)

Fig. 6. Dvls Knockdown suppresses reduced adipogenesis in NRX-depleted 3T3-L1 cells. A)

After transfection with three siRNAs targeting Dvl-1, 2 and 3 together siRNAs (Dvls siRNA) in

3T3-L1, relative mRNA levels of Dvls were analyzed using real-time quantitative RT-PCR

compared to control siRNA transfected cells. B) Representative images of lipid accumulation of

control or Dvls siRNA transfected cells were taken by Oil red O staining at 8 days post

adipogenic differentiation. C) Relative mRNA levels of FABP4 were analyzed using real-time

quantitative RT-PCR at 4 (left) or 8 (right) day post adipogenic differentiation. D)

Representative Oil red-O stained images of control or NRX-depleted 3T3-L1 cells transfected

with indicated siRNAs. At 8day post adipogenic differentiation, cells were stained with Oil red-

O dye. E) Oil red-O Stained cells were quantified using a dye-extraction solution (n=3).


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Figure 1

Bob/obWT

β-Actin

NRX

C

DM(days)

PPARγ

NRX

β-Actin

0 1 2 4 6 8

FABP4

A

*

WT ob/ob

*

*

**


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G

WT

Adip

o-N

RX

Figure 2

AW

T

Adip

o-N

RX

WT

Adip

o-N

RX

WT

Adip

o-N

RX

WT

Adip

o-N

RX

WT

Adip

o-N

RX

WT Adipo-NRX

F

WT Adipo-NRX

Epididymal fat

Adipo-NRXWT

Peri-renal fat

D E

H

B C

Sk. Muscle

+ + +

WT Adipo-NRX

WAT

Insulin

Liverp-AKT

AKT

p-AKT

AKT

+ + +

p-AKT

AKT

** * *

*

Adip

o-N

RX

WT

***


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NRX

anti-FLAG

FABP4

β-Actin

GFP

PPARγ

NRXGFP

GM (Day 0) DM (day 8)

GFP NRX

IB: NRX

Endo.

Exo.

GFP NRX

A

E

C

WT Adipo-NRX

D

F

BFigure 3

FABP4

NRX

β-Actin

WT Adipo-NRX

G

* **

*

*

*

*

***

****

H WT Adipo-NRX

F4/80

(X400)

(X200)

***

**


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Figure 4

β-actin

NRX

NT shNRXNT shNRX

NT

FABP4

shNRX

PPARγ

NRX

β-Actin

DM(days)0 1 2 4 6 80 1 2 4 6 8

A B

C

NT shNRX shNRX +resNRX

PPARγ

β-Actin

FABP4

NT shNRX

GM (Day 0) DM (day 8)

NRX

shNRX+ resNRX NT shNRX shNRX+

resNRX

D

E by guest, on A

ugust 29, 2018w

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Figure 5

A

IP:anti-FLAG

FLAG-WT NRX

FLAG-Mut NRX+

+

WCL

NRX

Dvl-1

anti-FLAG

Dvl-1

--

--

B

0 15 30 (min)

WCL

IP: NRX

NRX

Dvl-1

Wnt3a

NRX

Dvl-1

C

NT shNRX

Flag-NRX Nuc.β-catenin

NRX

Lamin A/C

CyclinD1

+ ++--

N

C

D

F

GFP NRX

+ Wnt3a(mock) + Wnt3a

Nuc.β-catenin

anti-FLAG

FLAG-NRX

Lamin A/C

+ Wnt3a

CyclinD1N

C

+ ++- +++

E

G

NRX

β-Actin

FABP4

Wnt3a+ +

GFP

-NRX

Control WT NRX Mut NRX

1 2 3 4 1 2 3 4

*

*

**

*

*

β-catenin target genesTopFlash


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Figure 6

A B

C

shNRX +Dvls siRNA

D

contr. siRNA

DvlssiRNA

NT +Dvls siRNA

E

shNRX +Contr. siRNA

NT +Contr. siRNA

contr. siRNA Dvls siRNA


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nucleoredoxin promotes adipogenic … nucleoredoxin promotes adipogenic differentiation through...

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