Jung Eun Jang,1,2 Myoung Seok Ko,2 Ji-Young Yun,2 Mi-Ok Kim,2

Jin Hee Kim,2 Hye Sun Park,2 Ah-Ram Kim,2 Hyuk-Joong Kim,2

Bum Joong Kim,2 Young Eun Ahn,1,2 Jin Sun Oh,1,2 Woo Je Lee,1,2

Robert A. Harris,3 Eun Hee Koh,1,2 and Ki-Up Lee1,2

Nitric Oxide Produced byMacrophagesInhibits Adipocyte Differentiationand Promotes Profibrogenic Responsesin Preadipocytes to Induce AdiposeTissue FibrosisDiabetes 2016;65:2516–2528 | DOI: 10.2337/db15-1624

Fibrosis of adipose tissue induces ectopic fat accumula-tion and insulin resistance by inhibiting adipose tissueexpandability. Mechanisms responsible for the induction ofadipose tissue fibrosis may provide therapeutic targets butare poorly understood. In this study, high-fat diet (HFD)–fedwild-type (WT) and iNOS2/2 mice were used to examinethe relationship between nitric oxide (NO) produced bymacrophages and adipose tissue fibrosis. In contrast toWT mice, iNOS2/2 mice fed an HFD were protected frominfiltration of proinflammatory macrophages and adiposetissue fibrosis. Hypoxia-inducible factor 1a (HIF-1a) proteinlevel was increased in adipose tissue of HFD-fed WT mice,but not iNOS2/2 mice. In contrast, the expression of mito-chondrial biogenesis factors was decreased in HFD-fedWT mice, but not iNOS2/2 mice. In studies with culturedcells, macrophage-derived NO decreased the expression ofmitochondrial biogenesis factors, and increased HIF-1aprotein level, DNA damage, and phosphorylated p53 inpreadipocytes. By activating p53 signaling, NO suppressedperoxisome proliferator–activated receptor g coactivator1a expression, which induced mitochondrial dysfunctionand inhibited preadipocyte differentiation in adipocytes.The effects of NO were blocked by rosiglitazone. The find-ings suggest that NO produced by macrophages inducesmitochondrial dysfunction in preadipocytes by activatingp53 signaling, which in turn increases HIF-1a protein level

and promotes a profibrogenic response in preadipocytesthat results in adipose tissue fibrosis.

Low-grade inflammation in adipose tissue is a major factorin the development of obesity-associated insulin resistance(1). In addition to inflammation, adipose tissue fibrosis isexacerbated in obese human subjects and in high-fat diet(HFD)–fed rodents (2). Fibrosis limits the expandabilityof adipose tissue and contributes to ectopic fat accumulationand the development of insulin resistance (3). Inducible nitricoxide synthase (iNOS) is a member of the NOS family, whichproduces nitric oxide (NO). In addition to its role in thekilling of certain pathogens by macrophages, iNOS is induc-ible in many cell types and has been implicated in insulinresistance and various metabolic diseases (4,5). iNOS acti-vation is involved in the pathogenesis of inflammation andfibrosis (6). In particular, classically activated (M1) macro-phages have increased iNOS expression and contribute toadipose tissue inflammation (7,8). HFD increases iNOS mRNAtranscript levels in adipose tissue (5,9), but the role of iNOS inadipose tissue fibrosis is unknown.

Hypoxia is likely to be a major factor contributing toadipose tissue inflammation and fibrosis (2,10–12). Hypoxiastimulates the transcription of hypoxia-inducible factor (HIF)-

1Department of Internal Medicine, Asan Medical Center, University of UlsanCollege of Medicine, Seoul, Republic of Korea2Metabolism Research Unit, Asan Institute for Life Sciences, Seoul, Republic ofKorea3Richard L. Roudebush VA Medical Center and the Department of Biochemistryand Molecular Biology, Indiana University School of Medicine, Indianapolis, IN

Corresponding authors: Eun Hee Koh, ehk@amc.seoul.kr, and Ki-Up Lee, kulee@amc.seoul.kr.

Received 26 November 2015 and accepted 18 May 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1624/-/DC1.

J.E.J., M.S.K., and J.-Y.Y. contributed equally to this work.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered. More information is available at http://diabetesjournals.org/site/license.

2516 Diabetes Volume 65, September 2016

METABOLISM

1a and promotes stabilization of the HIF-1a protein byprolyl hydroxylase domain protein–dependent hydroxylation(13). Activation of HIF-1a inhibits preadipocyte differen-tiation and initiates adipose tissue fibrosis (2,3). HIF-1ais well known to upregulate the expression of iNOS (14).In addition, NO produced by iNOS can regulate HIF-1astability (13). A low NO concentration promotes HIF-1adegradation under hypoxic conditions (15), whereas ahigh level of NO increases HIF-1a stability through theinhibition of prolyl hydroxylase domain activity undernormoxic conditions (16). Furthermore, a high concentra-tion of NO inhibits the mitochondrial respiratory chain(17), whereas a low NO level promotes mitochondrial bio-genesis (18). Recent studies (19) have shown that mitochon-drial dysfunction can also induce pseudo-hypoxic HIF-1aactivation under normoxic conditions.

In the current study, we show that iNOS2/2 mice areprotected from HFD-induced adipose tissue fibrosis. In-terestingly, the expression of mitochondrial biogenesisfactors was significantly decreased in adipose tissue ofHFD-fed wild-type (WT) mice, and this alteration was re-versed in iNOS2/2 mice. The accumulation of DNA dam-age has been linked to aging and the onset of age-relateddiseases (20,21). p53 is a key player in the intrinsic cellularresponse to DNA damage, and p53 activation leads to cellcycle arrest, apoptosis, and senescence (22). In particular,the p53 signaling pathway represses the expression of per-oxisome proliferator–activated receptor g coactivator 1a(PGC-1a) (23). We thus examined the possibility that NOproduced by macrophages induces mitochondrial dysfunc-tion in preadipocytes by activating p53 signaling and thatthis is responsible for the accumulation of HIF-1a and thefibrogenic response in preadipocytes.

RESEARCH DESIGN AND METHODS

Animals and DietsEight-week-old male WT (C57BL/6J) and iNOS2/2

(C57BL/6-NOS2tm1Lau) mice (The Jackson Laboratory, BarHarbor, ME) were fed either a normal chow diet (ND;12 kcal% fat; Biopia, Gunpo, Korea) or an HFD (60 kcal%fat, 90% from lard and 10% from soybean oil; ResearchDiets, New Brunswick, NJ). Mice were housed at ambienttemperature (226 1°C) with a 12-h light-dark cycle and freeaccess to water and food. After 16 weeks of feeding, micewere fasted for 5 h in the morning before they were eutha-nized. Blood samples were collected for biochemical analysis,and epididymal white adipose tissue (eWAT) was rapidly re-moved, weighed, and stored at280°C. All animal experimen-tal protocols were approved by the Institutional Animal Careand Use Committee of the Asan Institute for Life Sciences.

Measurement of Metabolic ParametersPlasma glucose levels were determined using a glucose andlactate analyzer (YSI2300; Yellow Springs Instruments,Yellow Springs, OH). Plasma insulin and adiponectin levelswere measured using radioimmunoassay kits (Linco Research,St. Charles, MO).

Glucose and Insulin Tolerance TestingThe glucose tolerance test (GTT) and the insulin tolerancetest (ITT) were performed at 14 and 15 weeks of diet feeding,respectively. For the GTT, mice were fasted overnight andthen administered 1 g/kg glucose i.p. One week later, micewere fasted for 5 h in the morning and then injected with0.75 mU/kg i.p. regular human insulin for the ITT. Bloodwas collected before injection, and at 15, 30, 60, 90, and120 min after injection for blood glucose level measurements.

Determination of Body CompositionIn a separate set of experiments, body composition wasmeasured in WT mice and iNOS2/2 mice fed a ND or HFDfor 15 weeks (n = 5 each). Mice were anesthetized with keta-mine/xylazine (8 and 1.6 mg/kg i.p., respectively), and totalfat mass and lean body mass were measured using a PIXI-Mus Small Animal Densitometer (Lunar Corp., Madison, WI).

Histologic ExaminationTissues were fixed in 10% formalin, embedded in paraffin,and sectioned. Tissue sections were stained with hematoxylin-eosin (H-E). Immunohistochemistry for F4/80 protein wasperformed by incubating tissue sections with anti-F4/80antibody (ab6640; Abcam, Cambridge, MA). To detect con-nective tissue, additional sections were stained with Massontrichrome (MT) stain or 0.1% Sirius Red in saturated picric acid.

Isolation of Bone Marrow–Derived MacrophagesBone marrow–derived macrophages (BMDMs) were pre-pared as described previously (24). Nonadherent cells werecarefully removed, and fresh medium was added every 2days. On day 8, the cells were collected for experiments.

Isolation of Primary PreadipocyteseWAT from 4-week-old WT mice was digested withcollagenase (2 mg/mL; Sigma-Aldrich, St. Louis, MO) inHanks’ balanced salt solution, and adipose-derived stromal-vascular cells were isolated and cultured, as described pre-viously (25). Fresh medium was added every 2 days. Onday 8, the cells were collected for experiments.

Effect of NO on PreadipocytesRAW264.7 cells or BMDMs were treated with 10 ng/mLlipopolysaccharide (LPS) for 24 h, and the conditionedmedia were transferred to 3T3-L1 preadipocytes or primarypreadipocytes for 8 h. To examine the role of iNOS activa-tion, macrophages were cultured in media containing LPSwith 30 mmol/L S-methylisothiourea (SMT; Sigma-Aldrich).SMT is an iNOS-selective inhibitor that, at concentrationsup to 1 mmol/L, does not inhibit the activity of xanthineoxidase, diaphorase, lactate dehydrogenase, monoamineoxidase, catalase, cytochrome P450, or superoxide dismutase(26). In another set of experiments, 3T3-L1 preadipocytes weretreated with either of the NO donors sodium nitroprusside(SNP) or Deta-NONOate (Deta-NO; Sigma-Aldrich) for 8 h.

Effect of NO on Adipocyte Differentiation3T3-L1 preadipocytes were differentiated into matureadipocytes, as described previously (27), in the presenceor absence of SNP or macrophage-conditioned media with

diabetes.diabetesjournals.org Jang and Associates 2517

or without SMT. From day 0 of differentiation, 0.25 or0.5 mmol/L SNP was added, whereas RAW cell-conditionedmedia (one-fifth dilution) were supplemented from day 2of differentiation. Morphological changes in adipocyteswere observed by phase contrast microscopy.

Nitrite MeasurementNO production was estimated by measuring the medianitrite produced by RAW264.7 cells or BMDMs usingGriess reagent (Promega, Madison, WI).

Oil Red O StainingMature 3T3-L1 adipocytes treated with SNP were fixedwith 10% formaldehyde for 1 h. After a wash with PBS,the cells were stained with oil red O solution (Sigma-Aldrich) for 30 min. The slides were then washed severaltimes with water, and excess water was evaporated byheating the stained cultures to ;32°C.

ImmunofluorescenceTo measure DNA damage in the cells (28), cultured 3T3-L1 preadipocytes were fixed with 2% formaldehyde in PBSand stained with the primary histone H2AX (gH2AX)antibody (Millipore, Billerica, MA), followed by visualizationwith tetramethylrhodamine-conjugated anti-mouse IgGsecondary antibody (Invitrogen, Carlsbad, CA). For nucleistaining, cells were incubated with 0.5 mg/mL DAPI inPBS. Immunofluorescence staining for a-smooth muscleactin (aSMA), a marker of myofibroblasts, was performedusing an antibody from Abcam (ab5694). For the stainingof intracellular lipid droplets, cells were also stained withCellTrace BODIPY TR Methyl Ester (C34556; Invitrogen).

Measurement of the 3-Nitrotyrosine LevelNO can react with superoxide to form peroxynitrite (ONOO2),which can contribute to the fibrotic response (29). As amarker of oxidative damage mediated by peroxynitrite, wemeasured the 3-nitrotyrosine (3-NT) level using an ELISAkit (ab116691; Abcam).

Gene Expression AnalysisGene expression in tissues and cells was assessed using real-time PCR using gene-specific primers (Supplementary Table1) and the 7500 Fast Real-Time PCR System (Applied Biosys-tems, Foster City, CA) using an SYBR Green PCR Kit (AppliedBiosystems). Total RNA was isolated using TRIzol (Invitrogen),and 1 mg of each sample was reverse transcribed with randomprimers using a Reverse Aid M-MuLV Reverse TranscriptionKit (Fermentas, Hanover, MD). The relative expression levelsof each gene were normalized to those of 18S rRNA.

Western BlottingWestern blotting was performed as described previously (27).Antibodies against iNOS (catalog #610432; BD TransductionLaboratories, Lexington, KY), HIF-1a (catalog #NB100–134;Novus Biologicals, Littleton, CO), total p53 (catalog #2624;Cell Signaling Technology, Danvers, MA), and phospho-p53(catalog #9284; Cell Signaling Technology) were used. Forhousekeeping controls, we used a-tubulin (catalog #NB100–690; Novus Biologicals) for in vivo study and b-actin (catalog

#A5441; Sigma-Aldrich) for in vitro study, as we had difficultyusing b-actin as a housekeeping gene for in vivo samples.

Measurements of Cellular RespirationA XF24 extracellular flux analyzer (Seahorse Bioscience, NorthBillerica, MA) was used to measure the oxygen consumptionrate in 3T3-L1 preadipocytes, as described previously (30).

StatisticsData are presented as the mean 6 SEM. Statistical signif-icance was determined using an unpaired two-tailed t testor ANOVA. Data were analyzed using SPSS version 17(SPSS Inc., Chicago, IL). P , 0.05 was used as the thresh-old for statistical significance.

RESULTS

iNOS2/2 Mice Are Protected From HFD-InducedAdipose Tissue FibrosisIn agreement with previous studies (5,9), HFD feeding for16 weeks significantly increased iNOS mRNA expression ineWAT (Fig. 1A). This was associated with an increased levelof 3-NT, a marker of oxidative damage mediated by perox-ynitrite (29) (Fig. 1B). Compared with the ND, HFD feedingincreased body weight in both WT and iNOS2/2 mice. How-ever, the body weight and food intake of HFD-fed iNOS2/2

mice were significantly lower than those of WTmice (Fig. 1C).Fasting plasma glucose and insulin levels were reduced inHFD-fed iNOS2/2 mice compared with WT mice (Fig. 1D).Plasma adiponectin levels were significantly decreased byHFD feeding in WT mice, but not in iNOS2/2 mice (Fig. 1D).HFD-induced glucose intolerance and insulin resistance mea-sured by GTT and ITT, respectively, were attenuated in iNOS2/2

mice (Fig. 1E). H-E, F4/80 protein, MT, and Sirius Red stainingrevealed adipose tissue inflammation and fibrosis in HFD-fedWT mice, whereas iNOS2/2 mice showed significantly lessinflammation and fibrotic changes in the eWAT (Fig. 1F).

Interestingly, the weight of eWAT was significantly higherin HFD-fed iNOS2/2 mice than in HFD-fed WT mice (Fig.2A), in contrast to the body weight changes (Fig. 1A). Similarly,densitometer measurement of body composition revealed ahigher fat mass in iNOS2/2 mice than in WT mice (Fig.2B). Consistent with the notion that adipose tissue fibrosisleads to ectopic fat accumulation (3), hepatic steatosis in HFD-fed mice was significantly reduced in iNOS2/2 mice (Fig. 2C).

Changes in Macrophage Polarization in HFD-Fed MiceAre Reversed in iNOS2/2 MiceOf the various immune cells involved in inflammation,adipose tissue macrophages play a central role in the genesisof adipose tissue inflammation (31). At least two distinctpopulations of macrophages infiltrate the adipose tissue:proinflammatory (M1) and anti-inflammatory (M2). In agree-ment with previous studies (8), an HFD for 16 weeks signif-icantly increased the expression of M1 markers and some M2markers in the eWAT (Fig. 3A and B). This was associated withincreased expression of fibrosis markers (Fig. 3C). Changes inmost of the markers of macrophage polarization and fibro-sis were partially reversed in iNOS2/2 mice (Fig. 3A–C).

2518 iNOS in HFD-Induced Adipose Tissue Fibrosis Diabetes Volume 65, September 2016

HFD-Induced Changes in the Expression Levels ofHIF-1a Protein and Mitochondrial Biogenesis FactorsAre Reversed in the Adipose Tissue of iNOS2/2 Mice

Expression levels of HIF-1a mRNA and protein were sig-nificantly increased in WT mice receiving an HFD (Fig. 4A

and B) (12,32). Levels of HIF-1a mRNA transcripts werenot different in iNOS2/2 mice compared with WT mice,but HIF-1a protein levels were significantly reduced iniNOS2/2 mice receiving an HFD (Fig. 4B). On the otherhand, the expression levels of HIF-2a and HIF-3a significantly

Figure 1—iNOS2/2 mice are protected from HFD-induced insulin resistance and adipose tissue fibrosis. A: The expression levels of iNOS mRNAin the eWAT of WT and iNOS2/2 mice fed a ND or HFD, respectively. B: 3-NT levels in eWAT were measured by an ELISA kit. C: Body weight (left)and food intake (right) changes in WT and iNOS2/2 mice fed a ND or HFD. White circles, WT mice on a ND; white squares, WT mice on an HFD;black circles, iNOS2/2 mice on a ND; black squares, iNOS2/2 mice on an HFD. D: Fasting plasma glucose, insulin, and adiponectin levels. E: GTT(top) and ITT (bottom). F: Representative H-E, F4/80 immunohistochemical, MT, and Sirius Red staining of eWAT (top to bottom). The blue color inthe MT staining and the red color in the Sirius Red staining represent fibrotic changes. Scale bars, 50 mm. Data are presented as the mean6 SEMof 6–10 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. ND-fed WT mice; #P < 0.05, ##P < 0.01, and ###P < 0.001 vs. HFD-fed WT mice.

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decreased in WT mice fed the HFD but significantly in-creased in iNOS2/2 mice fed the HFD (Fig. 4A), support-ing the concept that HIF-2a or HIF-3a is compensatoryto HIF-1a (33).

Recent studies have suggested that mitochondrialdysfunction is an important pathogenic factor of fibrosis(34). Accordingly, the expression levels of genes encodingmitochondrial biogenesis factors, adiponectin, and mito-chondrial respiratory complex proteins were found to besignificantly decreased in the eWAT of HFD-fed WT mice,and these changes were partially reversed in iNOS2/2

mice (Fig. 4C and D).

Macrophage-Derived NO Decreases the Expressionof Mitochondrial Biogenesis Factors and Increasesthe HIF-1a Protein Level in PreadipocytesHFD feeding can cause changes in gut microbiota, resultingin increased plasma levels of LPS, which can exacerbateadipose tissue inflammation and obesity (35). Treatmentwith LPS (10 ng/mL) for 8 h significantly increased iNOSbut not HIF-1a protein levels in RAW264.7 macrophages(Fig. 5A) and BMDMs isolated from WT mice (Fig. 5B), and

accordingly increased the concentrations of nitrite in cul-ture supernatants (Fig. 5A and B). As expected, BMDMsisolated from iNOS2/2 mice did not show increased ex-pression of iNOS and nitrite in the culture supernatantafter LPS treatment (Fig. 5B).

Factors secreted by macrophages can promote aprofibrotic phenotype in preadipocytes and reduce adipo-cyte differentiation (36–39). Therefore, we tested the pos-sibility that NO produced by activated macrophages couldaugment the HIF-1a protein level and decrease the ex-pression of mitochondrial biogenesis factors in preadipo-cytes. Conditioned media from LPS-stimulated RAW264.7macrophages increased the expression of HIF-1a proteinand fibrogenic gene mRNA transcripts, and suppressedPGC-1a expression in 3T3-L1 preadipocytes (Fig. 5C).Treatment with LPS did not increase the HIF-1a proteinlevel in 3T3-L1 preadipocytes (Fig. 5D), indicating thatthe LPS remaining in the macrophage culture supernatantis not responsible for the changes. Cotreatment of mac-rophages with LPS and SMT did not completely reverseconditioned media-induced changes in preadipocytes, butsignificantly ameliorated them (Fig. 5C).

Interestingly, preadipocytes treated with conditionedmedia from LPS-treated BMDMs from iNOS2/2 miceshowed significantly less accumulation of HIF-1a protein(Fig. 5E) and higher levels of mitochondrial biogenesisfactors (Fig. 5F) than those treated with conditioned me-dia from BMDMs from WT mice.

In a further assessment of the role of NO, treatmentwith NO donors, SNP or Deta-NO, increased the proteinlevels of HIF-1a and the mRNA expression of profibro-genic genes, and suppressed PGC-1a expression in 3T3-L1preadipocytes (Fig. 5G).

Rosiglitazone Treatment Increases MitochondrialRespiration and Decreases HIF-1a Protein Levelsand Fibrogenic Gene Transcription in PreadipocytesWe next examined the effect of rosiglitazone on NO-mediatedchanges in the HIF-1a protein level. Rosiglitazone is aperoxisome proliferator–activated receptor g (PPARg) ago-nist that increases mitochondrial biogenesis in differentiatedadipocytes (27). 3T3-L1 preadipocytes showed significantlylower expression of PPARg than differentiated adipocytes,and treatment with rosiglitazone did not significantly in-crease PPARg expression (data not shown). Nevertheless,rosiglitazone significantly increased mitochondrial respi-ration in SNP-treated 3T3-L1 preadipocytes (Fig. 6A).Treatment with rosiglitazone also reversed the SNP-mediatedincrease in the HIF-1a protein level (Fig. 6B), and changes inPGC-1a and fibrogenic gene transcription (Fig. 6C).

NO Suppresses Expression of PGC-1a by Activatingp53 SignalingA growing body of evidence suggests that DNA damage islinked to adipose tissue inflammation and systemicinsulin resistance (21), and that the p53 signaling path-way can repress PGC-1a expression (23). In particular,NO has been shown to increase p53 accumulation (40).

Figure 2—Activation of iNOS is responsible for limited adiposetissue expandability and ectopic fat accumulation. A: Weights ofeWAT from WT and iNOS2/2 mice after 16 weeks of ND or HFDfeeding. B: Changes in body composition. Lean body mass (left)and fat mass (right) are presented as the percentage of total bodymass. C: H-E staining of the liver of ND-fed or HFD-fed WT andiNOS2/2 mice. Scale bars, 50 mm. Data are presented as themean 6 SEM. **P < 0.01 and ***P < 0.001 vs. ND-fed WT mice;#P < 0.05 vs. HFD-fed WT mice.

2520 iNOS in HFD-Induced Adipose Tissue Fibrosis Diabetes Volume 65, September 2016

We thus tested the possibility that NO decreases mito-chondrial biogenesis via p53-dependent suppression ofPGC-1a. The highest activation state of p53 occurswhen it is phosphorylated on serine-15 (41). Conditionedmedia from LPS-treated RAW cells increased p53 phos-phorylation in 3T3-L1 preadipocytes, whereas condi-tioned media from SMT cotreated cells did not showthese effects (Fig. 7A). The mRNA expression levels ofp21, a downstream effector of p53 that regulates manycellular processes (42), were similarly altered (Fig. 7B).

gH2AX phosphorylation is a rapid and sensitive cellularresponse to the presence of DNA double-stranded breaks(28). Treatment with NO donors significantly increased

gH2AX staining in 3T3-L1 preadipocytes, indicating thatNO induces DNA damage in these cells (Fig. 7C). NO donorsalso significantly increased phosphorylated p53 protein levelsand p21 mRNA levels (Fig. 7D). Small interfering RNA(siRNA) directed against p53 reversed SNP-mediated changesin HIF-1a protein and phosphorylated p53 protein levels, andp21 and PGC-1a mRNA levels in 3T3-L1 preadipocytes (Fig.7E and F).

In agreement with these in vitro findings, significantlyhigher levels of phosphorylated p53 were noted in theeWAT of WT mice, but not iNOS2/2 mice, fed an HFD(Fig. 7G). In addition, the mRNA transcript levels of p21were significantly increased in the eWAT of WT mice

Figure 3—Effect of iNOS gene disruption on the polarization of adipose tissue macrophages and the expression levels of profibrogenicgenes. Relative mRNA expression levels of genes representing classically activated M1 macrophages, including tumor necrosis factor-a(TNF-a), IL-6, and monocyte chemoattractant protein 1 (MCP-1) (A), and alternatively activated M2 macrophages, including CD209,macrophage mannose receptor 1 (MRC-1), arginase 1 (ARG-1), macrophage galactose-type C-type lectin 2 (MGL-2), and IL-10 (B).C: Relative mRNA expression levels of genes responsible for fibrosis, including aSMA, transforming growth factor-b (TGF-b), collagen(Col) 1a, and Col3a. Data are the mean 6 SEM values from 8–10 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. ND-fed WT mice;#P < 0.05, ##P < 0.01, and ###P < 0.001 vs. HFD-fed WT mice.

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receiving an HFD, a change that was not observed inHFD-fed iNOS2/2 mice (Fig. 7H). This suggests thatNO can increase p53 signaling in the eWAT of HFD-fedmice as well as in preadipocytes cultured in vitro.

NO Inhibits the Differentiation of 3T3-L1 PreadipocytesInto AdipocytesWe next examined whether NO affects adipocyte differen-tiation in 3T3-L1 preadipocytes. SNP treatment significantlydecreased the expression of adipogenesis markers andincreased the expression of fibrosis markers (Fig. 8A andB). Phase-contrast imaging, oil red O and BODIPY staining,and immunofluorescent staining for aSMA in differentiated3T3-L1 adipocytes revealed an increased frequency of cells

with a profibrotic phenotype in SNP-treated cells, includinga flattened fibroblast-like morphology, decreased lipid drop-let content, and increased levels of aSMA expression (Fig.8C). Treatment with conditioned media from LPS-treatedRAW264.7 cells also decreased the expression of adipogen-esis markers and oil red O staining of lipid droplets in3T3-L1 adipocytes (Fig. 8D and E). Conditioned media fromcells cotreated with SMT did not completely reverse thesechanges, but significantly ameliorated them (Fig. 8D and E).

DISCUSSION

In our current study, we found that iNOS2/2 mice wereprotected from HFD-induced adipose tissue fibrosis. The

Figure 4—HFD-induced changes in the expression of mitochondrial biogenesis factors and HIF-1a protein are reversed in the adiposetissue of iNOS2/2 mice. A: Expression levels of HIF-1a, HIF-2a, and HIF-3a mRNA in WT and iNOS2/2 mice fed a ND or HFD. B:Representative Western blots of HIF-1a protein and protein quantification in eWAT. C: Relative mRNA expression levels of mitochondrialbiogenesis factors, including endothelial NOS (eNOS), PGC-1a, and mitochondrial transcription factor A (mtTFA). D: Gene expression levelsof adiponectin and mitochondrial respiratory complexes, including cyclooxygenase (COX) I, COX IV, and COX V. Data are the mean6 SEMvalues of 8–10 mice. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. ND-fed WT mice; ##P < 0.01 and ###P < 0.001 vs. HFD-fed WT mice.

2522 iNOS in HFD-Induced Adipose Tissue Fibrosis Diabetes Volume 65, September 2016

HFD significantly increased both the mRNA and proteinexpression levels of HIF-1a in the eWAT of the mice. Thischange may have occurred because of adipose tissuehypoxia (2,10–12). It has been suggested that HIF-1a

induction by tissue hypoxia leads to an upregulation of“fibrotic response” genes, resulting in the local fibrosisand necrosis of adipocytes, which attracts classically acti-vated proinflammatory M1 macrophages and leads to

Figure 5—Macrophage-derived NO is responsible for PGC-1a suppression, HIF-1a accumulation, and increased profibrogenic gene transcription.A: Representative Western blots of iNOS and HIF-1a in RAW264.7 cells after treatment with 10 ng/mL LPS for 24 h and measurement of nitriteconcentration in the culture supernatant of macrophages after treatment with LPS, with or without 50 mmol/L SMT. B: Western blots of iNOS andHIF-1a and nitrite concentration of the culture supernatant in BMDMs from WT and iNOS2/2 mice after LPS treatment (10 ng/mL, 24 h). C: Effectsof conditioned media from LPS-treated macrophages. Conditioned media from RAW macrophages treated with LPS, with or without 50 mmol/LSMT, were transferred to 3T3-L1 preadipocytes. HIF-1a protein expression evaluated by Western blot and the relative mRNA expression levels ofgenes responsible for fibrosis and mitochondrial biogenesis factors in preadipocytes. D: Effect of LPS on iNOS and HIF-1a protein expression in3T3-L1 preadipocytes. Representative Western blots of HIF-1a (E) and mRNA expressions of mitochondrial biogenesis factors (F) in primarypreadipocytes treated with conditioned media from LPS-treated BMDMs isolated from WT and iNOS2/2 mice. G: Effects of 0.5 mmol/L SNP orDeta-NO on the HIF-1a protein level and the relative mRNA expression levels of genes responsible for mitochondrial biogenesis factors andfibrogenesis in preadipocytes. Data are the mean 6 SEM of three to five independent experiments. *P < 0.05. CON, control.

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metabolic dysfunction (2). Intriguingly, our study showedthat the HIF-1a protein levels, but not the transcriptlevels, were significantly reduced in iNOS2/2 versus WTmice receiving the HFD. Similarly, conditioned media fromactivated macrophages or treatment with NO donors sig-nificantly increased HIF-1a protein levels in preadipocytes.This finding suggested that the NO produced by activatedmacrophages amplifies and sustains the hypoxia-inducedfibrogenic responses in adipose tissue.

Notably, we found that the expression of mitochon-drial biogenesis factors was significantly decreased in theeWAT of WT mice, but not iNOS2/2 mice, on an HFD.NO produced by activated macrophages reduced mito-chondrial respiration in preadipocytes and their differenti-ation to mature adipocytes in a paracrine manner. Amongthe distinct cell types that reside in the adipose tissuestromal compartment, preadipocytes represent progeni-tor cells that are more committed to the adipocyte lineage(36). Accordingly, the administration of SNP or Deta-NOdecreased the expression of PGC-1a and increased profi-brotic responses in preadipocytes. In agreement with pre-vious studies (38,39), conditioned media from macrophagessignificantly increased profibrotic responses in preadipocytesand decreased the expression of mitochondrial biogenesisfactors. Importantly, HIF-1a protein was decreased, andmitochondrial biogenesis factors were increased in prea-dipocytes treated with conditioned media from LPS-treatedBMDMs from iNOS2/2 mice, suggesting an important roleof NO. However, SMT significantly but partially blockedthese responses in preadipocytes by conditioned mediafrom LPS-treated macrophages. This may be because LPS-stimulated macrophages produce various proinflammatorycytokines besides NO. A recent study (43) reported thatvarious proinflammatory cytokines produced by activatedmacrophages, such as tumor necrosis factor-a, interleukin(IL)-6, and IL-1b, decreased mitochondrial function in3T3-L1 adipocytes. Taken together, NO produced by acti-vated macrophages can promote a profibrotic phenotypein preadipocytes by decreasing mitochondrial function, inconcert with other cytokines.

NO generated by iNOS has been shown to evoke p53accumulation (40). In agreement, we found that SNP orDeta-NO significantly increased DNA damage and the lev-els of phosphorylated p53 in preadipocytes, and thatsiRNA targeting p53 reversed the SNP-induced downregula-tion of PGC-1a. In support of the idea that p53 signalingcontributes to the reduced expression of mitochondrial bio-genesis factors that occurs in the adipose tissue of HFD-fedmice, the levels of phosphorylated p53 protein and p21transcript were significantly increased in the eWAT of WTmice, but not iNOS2/2 mice, on an HFD. Taken together,these data suggest that NO produced by activated macro-phages leads to p53 accumulation in preadipocytes, whichinduces mitochondrial dysfunction and profibrotic changes.

Although the inflammatory response that occurs duringtissue injury is necessary for proper tissue repair, it can leadto pathological tissue fibrosis if it becomes dysregulated(44). The fibrotic change is directly related to the degree ofinflammation, but also with the appearance of M2 macro-phages (45). In agreement, the expression of markers forM2 macrophages was significantly increased in long-termHFD-fed mice. iNOS is considered a marker of M1 macro-phages, but our present study showed that the increase inM2 markers in HFD-fed mice was significantly decreased inHFD-fed iNOS2/2 mice. This may contribute to the de-creased adipose tissue fibrosis in iNOS2/2 mice.

Figure 6—Rosiglitazone treatment increases mitochondrial respira-tion and decreases HIF-1a protein levels and fibrogenic gene tran-scription in preadipocytes. A: Effect of SNP (0.5 mmol/L) with orwithout rosiglitazone (20 mmol/L) for 8 h on mitochondrial respira-tion. Real-time oxygen consumption rates (OCRs) were measuredby an XF24 Extracellular Flux Analyzer. White circles, control (CON);white squares, SNP alone (SNP); black circles, SNP with rosiglita-zone (SNP + Rosi). During measurements, 1 mg/mL oligomycin(Oligo), 1 mmol/L carbonyl cyanide p-(trifluoromethoxy)-phenyl-hydrazone (FCCP), and 1 mmol/L rotenone (Rote) plus 2 mmol/Lantimycin A (AA) were sequentially added. Data are the mean 6SEM of three independent experiments. *P < 0.05 CON vs. SNP;#P < 0.05 SNP vs. SNP + Rosi. B and C: Effect of rosiglitazone onSNP-induced changes in preadipocytes. Effect of SNP with or with-out rosiglitazone on the HIF-1a protein level (B) and the mRNAexpression levels of PGC-1a and profibrogenic genes (C). Dataare the mean 6 SEM of five independent experiments. *P < 0.05.

2524 iNOS in HFD-Induced Adipose Tissue Fibrosis Diabetes Volume 65, September 2016

Figure 7—NO produced by activated macrophages decreases the expression of PGC-1a by activating p53 signaling and increases HIF-1aprotein expression in preadipocytes. A and B: Conditioned media from RAW264.7 cells treated with LPS, with or without SMT, weretransferred to 3T3-L1 preadipocytes. Changes in p53 phosphorylation (A) and the p21 mRNA expression level (B). C and D: Effect of NOdonors on the DNA damage response in 3T3-L1 preadipocytes. C: Immunofluorescence of gH2AX (red color, middle column) in 3T3-L1preadipocytes after treatment with NO donors for 8 h. Nuclear DNA was counterstained with DAPI (blue color, left column), and mergedimages are shown in the right column. D: Changes in the phosphorylated p53 protein level and p21 mRNA expression. ***P < 0.001 vs.control (CON). Effects of siRNA directed against p53 (si-p53) on SNP-induced changes in HIF-1a protein (E) and in the expression of p21and PGC-1a mRNA (F ). 3T3-L1 preadipocytes were transfected with si-p53 or control vector (si-CON) and treated with or without0.5 mmol/L SNP for 8 h. Data are the mean 6 SEM of five independent experiments. G and H: Changes in p53 signaling in the eWATof WT and iNOS2/2 mice fed a ND or HFD. Representative Western blots and quantification of total and phosphorylated p53 (G) and real-time PCR detecting the expression of p21 (H) are shown. The Western blot samples are identical to those in Fig. 4B. Data are the mean 6SEM of three to five independent experiments. *P < 0.05.

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Figure 8—NO inhibits the differentiation of 3T3-L1 preadipocytes to mature adipocytes. A: Effects of SNP on the mRNA transcript levels ofadiponectin and genes involved in adipogenesis, including SREBP-1c, PPARg, CCAAT/enhancer-binding protein (C/EBP)-a, and C/EBPbduring adipocyte differentiation. B: Effects of SNP on the relative mRNA transcript levels of genes involved in fibrosis, including aSMA,transforming growth factor-b (TGF-b), collagen (Col) 1a, and Col3a. 3T3-L1 preadipocytes were differentiated into adipocytes according toa standard protocol.C: Effects of SNP on morphological changes in differentiated 3T3-L1 adipocytes. Phase-contrast imaging (top row), oil redO staining (middle row), and double staining with BODIPY and immunofluorescence for aSMA (bottom row) were performed. The red colordenotes BODIPY-stained lipid droplets, and the green color denotes aSMA staining. D and E: Effects of conditioned media from LPS-treatedmacrophages on the differentiation of 3T3-L1 adipocytes. Diluted conditioned media from LPS-treated RAW cells with or without SMT wereadded from the second day of differentiation. D: The expression levels of genes responsible for adipogenesis. E: Oil red O staining after 5 daysof differentiation. Data are the mean6 SEM of three independent experiments. *P< 0.05 vs. control (CON); #P< 0.05 vs. LPS. F: A proposedmechanism for HFD-induced adipose tissue fibrosis. Adipose tissue hypoxia in HFD-fed mice upregulates the HIF-1a protein level by in-creasing transcription and protein stabilization. This contributes to the induction of fibrotic changes in preadipocytes. In addition, the over-production of NO by activated macrophages causes decreased mitochondrial biogenesis in preadipocytes via the activation of p53 signaling.In turn, this increases HIF-1a accumulation to increase fibrogenesis and also leads to defective adipocyte differentiation.

2526 iNOS in HFD-Induced Adipose Tissue Fibrosis Diabetes Volume 65, September 2016

iNOS2/2 mice in our colony were resistant to theHFD-induced body weight gain. Even though the originalarticle on iNOS and insulin resistance did not report dif-ferences in body weight (5), a later study (46) showed thatthe deletion of iNOS in ob/ob mice increased energy expen-diture. iNOS2/2 mice in our colony consumed significantlyless food than WT mice by a presently unknown mechanism.We did not perform pair-fed experiment to examine howmuch difference in the body weight results from decreases infood intake or increases in energy expenditure in HFD-fediNOS2/2 mice. However, during the early period of HFDfeeding, the body weight of HFD-fed iNOS2/2 mice wasnot significantly different from that of WT mice, eventhough iNOS2/2 mice consumed significantly less foodthan WT mice. This suggests that, during this early periodof HFD feeding, decreased energy expenditure compensatesfor decreased food intake. Thus, increases in energy expen-diture may not be the major cause of the body weight de-crease in iNOS2/2 mice. The cause of this difference betweenstudies is currently unknown. However, body weight reduc-tion in iNOS2/2 mice may explain reduced insulin resistancein these mice.

Alternatively, reduced adipose fibrosis may also explainreduced insulin resistance in iNOS2/2 mice. Interestingly,the weight of the eWAT depot in HFD-fed iNOS2/2 micewas significantly higher than that of HFD-fed WT mice,even though the iNOS2/2 mice were resistant to theHFD-induced body weight gain. The measurement ofbody composition also showed that fat mass was higherin HFD-fed iNOS2/2 mice than in WT mice. These findingsare unexpected but are consistent with the notion thatadipose fibrosis limits adipose tissue expandability. Im-paired adipose tissue expandability may lead to the devel-opment of ectopic fat accumulation and insulin resistance(3). Consistently, we found that HFD-induced steatosis inthe liver, a representative site of ectopic fat accumulation,was ameliorated in iNOS2/2 mice.

Our present study contradicts a previous report (47)showing that iNOS deficiency in myeloid cells does notprevent diet-induced insulin resistance. In that study, iNOSdeficiency in macrophages did not attenuate adipose tissueinflammation or the LPS-induced inflammatory responsein macrophages. The cause of this discrepancy is presentlyunclear. However, the degree of tissue injury, inflammation,and fibrosis may not correlate with each other (6), and itwould be interesting to see whether these mice with myeloidiNOS deficiency are protected from adipose tissue fibrosis.

It should also be noted that the role of iNOS in fibrosisis highly controversial and depends on the tissue and ex-perimental protocol. For example, accelerated liver fibro-sis was found in HFD-fed iNOS2/2 mice (48,49), whereasiNOS2/2 mice were protected from cholesterol-inducedliver fibrosis (50). Interestingly, iNOS2/2 mice have shownincreased hepatic injury but decreased fibrosis after long-term carbon tetrachloride administration (6).

Taken together, we suggest that NO produced by activatedmacrophages induces p53-dependent PGC-1a suppression

and mitochondrial dysfunction in preadipocytes; andthat this is responsible for the increased HIF-1a accumu-lation, defective adipocyte differentiation, and increasedfibrogenesis (Fig. 8F). Interactions between macrophageiNOS and preadipocytes may therefore potentially repre-sent a novel therapeutic target for restoring healthy adi-pose tissue function.

Acknowledgments. The authors thank Dr. Kevin Clayton (Boston BioEdit)for English proofreading.Funding. This study was supported by the National Research Foundationof Korea (NRF), which is funded by the Ministry of Education, Science, andTechnology (grants NRF-2006-2005412 and 2009-0091988 to K.-U.L.; grant2012R1A1A3012626 to E.H.K.).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. J.E.J. designed and conducted the study,performed experiments, analyzed and interpreted the data, and wrote themanuscript. M.S.K. and J.-Y.Y. performed in vivo and in vitro experiments andinterpreted the results. M.-O.K. performed some parts of the in vitro experiments.J.H.K., H.S.P., A.-R.K., H.-J.K., and B.J.K. contributed to the data analysis orperformed some parts of experiments. Y.E.A., J.S.O., W.J.L., and R.A.H. criticallyreviewed the manuscript, provided suggestions, and contributed to thediscussion. E.H.K. and K.-U.L. conceptualized and designed the study andanalyzed the data. E.H.K. and K.-U.L. are the guarantors of this work and, assuch, had full access to all the data in the study and take responsibility for theintegrity of the data and the accuracy of the data analysis.

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