ARTICLE

Tissue inhibitor of matrix metalloproteinase 1 (TIMP1)controls adipogenesis in obesity in mice and in humans

B. Meissburger & L. Stachorski & E. Röder &

G. Rudofsky & C. Wolfrum

Received: 21 December 2010 /Accepted: 20 January 2011 /Published online: 25 March 2011# Springer-Verlag 2011

AbstractAims/hypothesis Extracellular matrix reorganisation is acrucial step of adipocyte differentiation and is controlledby the matrix metalloproteinase–tissue inhibitor of matrixmetalloproteinase (TIMP) enzyme system. We thereforesought to define the role of TIMP1 in adipogenesis and toelucidate whether upregulation of TIMP1 in obesity hasdirect effects on adipocyte formation.Methods TIMP1 protein levels and mRNA were measuredin lean and obese mice with a focus on levels in adiposetissue. We also analysed the effect of recombinant murineTIMP1 on adipogenesis, adipocyte size and metaboliccontrol in vitro and in vivo.Results TIMP1 levels were increased in the serum andadipose tissue of obese mouse models. Recombinantmurine TIMP1 inhibited adipocyte differentiation in 3T3-L1 as well as in subcutaneous primary pre-adipocytes.Conversely, neutralising TIMP1 with a specific antibodyenhanced adipocyte differentiation. In vivo, injection ofrecombinant TIMP1 in mice challenged with a high-fat dietled to enlarged adipocytes. TIMP1-treated mice developedan impaired metabolic profile with increased circulatingNEFA levels, hepatic triacylglycerol accumulation andaccelerated insulin resistance. Altered glucose clearance inTIMP1-injected mice was due to changes in adipose tissue

glucose uptake, whereas muscle glucose clearanceremained unaffected.Conclusions/interpretation TIMP1 is a negative regulatorof adipogenesis. In vivo, TIMP1 leads to enlargedadipocytes in the state of overnutrition. This mightcontribute to the detrimental metabolic consequences seenin TIMP1-injected mice, such as systemic fatty acidoverload, hepatic lipid accumulation and insulin resistance.

Keywords Adipocyte hypertrophy . Adipogenesis .

Diet-induced obesity . Insulin resistance . TIMP1 .

Tissue inhibitor of metalloproteinase 1

AbbreviationsMMP Matrix metalloproteinaseSVF Stromal–vascular fraction of adipose tissueTIMP Tissue inhibitor of matrix metalloproteinase

Introduction

Obesity is the main risk factor for type 2 diabetes andhas become a growing health problem in the lastdecades [1–3]. Expansion of adipose tissue results froman imbalance between energy intake and energy expendi-ture. Secondary complications caused by obesity partlydepend on how well adipose tissue can adapt to anextended nutrient supply [4, 5]. One way of bufferingexcess nutrients in adipose tissue is the generation of fatcells through de novo differentiation. This hyperplasticresponse is preferable to a hypertrophic reaction, wherenutrients are stored in pre-existing adipocytes. Adipocytehypertrophy commonly leads to enlarged, insulin-resistantfat cells with a high lipolytic rate, thus promoting ectopic

B. Meissburger : L. Stachorski : E. Röder :C. Wolfrum (*)Swiss Federal Institute of Technology, ETH Zürich,Institute for ETH Zürich, Food Nutrition and Health,Schorenstrasse 16,8603 Schwerzenbach, Switzerlande-mail: wolfrum@imsb.biol.ethz.ch

G. RudofskyDepartment of Medicine I and Clinical Chemistry,University of Heidelberg,Heidelberg, Germany

Diabetologia (2011) 54:1468–1479DOI 10.1007/s00125-011-2093-9

lipid accumulation and systemic insulin resistance [6–9].New adipocytes can differentiate from precursor cells,which reside in the stromal–vascular fraction of adiposetissue (SVF) [10, 11]. For a fibroblast-like pre-adipocyteto become a lipid-laden adipocyte, restructuring of theextracellular matrix is indispensable [12–15]. Therefore,extracellular matrix remodelling has an important impacton adipocyte differentiation [16, 17]. The concomitantextracellular protein degradation is mediated by matrixmetalloproteinases (MMPs) and their regulatory counter-parts, the tissue inhibitors of matrix metalloproteinases(TIMPs) [17].

Altered levels of MMPs have been observed inadipose tissue of obese participants and the MMPsystem is tightly regulated during adipogenesis [18].MMPs influence the process of adipogenesis in arepressive or activating way depending on substratespecificity [17]. Mice deficient in MMP3, MMP11 orMMP19 exhibit increased adiposity during diet-inducedobesity [19–21]. In female mice after lactation MMPs alsoinfluence adipogenesis in the mammary gland. Loss ofMMP3, for example, leads to increased fat accumulation[22]. Moreover, lack of plasminogen, a precursor ofplasmin, a key enzyme activating MMPs, blocks adipocytedifferentiation [23]. Finally, mice treated with a globalMMP inhibitor display reduced adipose tissue weightconcomitant with a lower total number of adipocytes whenexposed to a high-fat diet [24]. This is consistent with thefact that the global MMP inhibitor BB-94 inhibits 3T3-L1adipocyte differentiation [18].

MMPs are inhibited in 1:1 stoichiometry by the familyof TIMP proteins. TIMP1 inhibits most MMPs exceptMMP14, MMP16 and MMP19 [25]. Analysis in adiposetissue of lean and obese mice, and humans revealedsignificant upregulation of TIMP1 in obesity [18, 26].Conflicting data have been reported concerning the func-tional role of TIMP1 in adipose tissue. Whereas maleTIMP1-deficient mice kept on a high-fat diet develop lessadiposity, female TIMP1-deficient mice accumulate moreadipose tissue due to hyperphagia [27, 28]. On the otherhand, systemic adenoviral overproduction of the humanTIMP1 homologue in mice had no effect on adipose tissuemass [29]. In vitro, human TIMP1 did not influenceadipogenesis of murine 3T3-F442A cells, whereas inanother study, human TIMP1 accelerated 3T3-L1 differen-tiation [22, 29]. A certain drawback here is the use ofhuman TIMP1 in mouse model systems, which mightdisturb the interaction between MMPs and their inhibitorsdue cross species differences.

As TIMP1 levels are upregulated in obesity, the aim ofthis study was to define the role of TIMP1 in adipocyteformation and its potential influence on metabolism in thestate of obesity.

Methods

Real-time PCR cDNA was produced using a cDNA kit(Multi-MACS; Miltenyi, Bergisch Gladbach, Germany).mRNA expression was assessed using SybrGreen (Invitrogen,Basel, Switzerland) and normalised to Rplp0.

Primers (annealing temperature 60°C): Rplp0 forwardGCCGTGATGCCCAGGGAAGACA, Rplp0 reverseCATCTGCTT GGAGCCCACGTTG; Timp1 forwardGATATGCCCACAAGTCCCAGAACC, Timp1 reverseGCACACCCCACAGCCAGCACTAT; Pparγ (also knownas Pparg) forward AGGCGAGGGCGATCTTGACAG,Pparγ reverse AATTCGGATGGCCACCTCTTTG; A-Fabp(also known as Fabp4) forward ATGAAATCACCGCAGACGACAG, A-Fabp reverse GCCTTTCATAACACATTCCACCAC.

TIMP1 secretion from SVF SVF and adipocytes wereessentially isolated as described previously [30]. Adiposetissue was digested for 1 h with 2 mg/ml collagenasetype II. Adipocytes were cultured in 24-well 0.4 μmfilter inserts (Becton Dickinson, Franklin Lakes, NJ,USA). SVF was filtered through a 40 μm net anderythrocytes were lysed. Cells were seeded on collagen-coated plates at 80% density in high-glucose DMEMwith 10% (vol./vol.) fetal bovine serum and penicillin/streptomycin. The next day, the medium was changed toKrebs–Ringer buffer and collected after 24 h. TIMP1 inthe supernatant fraction was measured using an ELISAkit (Quantikine Mouse TIMP1; R&D Systems, Minne-apolis, MN, USA).

MMP activity assays Inhibition of MMP9 (Anaspec,Fremont, CA, USA) by TIMP1 was tested using a kit(SensoLyte 570 Generic MMP Assay Fluorimetric; Anas-pec, Fremont, CA, USA). Human or murine MMP9 wasused at 30 ng/ml corresponding to enzyme:substrate ratiosof 1:43 to 1:350. Human TIMP1 was purchased from R&DSystems (R&D Systems).

For global MMP activity from adipose tissue extracts,frozen adipose tissue was dounced in 300 μl hypotonic buffer(10 mmol/l KCl, 1.5 mmol/l MgCl2, 10 mmol/l HEPESpH 7.9), and 375 μl hypertonic buffer (20 mmol/l HEPES/KOH pH 7.9, 25% (vol./vol.) glycerol, 0.2 mmol/l EDTA,1.5 mmol/l MgCl2, 1.2 mol/l NaCl) was added, followed byincubation at 4°C for 30 min. Cleared lysates (50 μl) weretested for MMP activity using the SensoLyte kit (Anaspec,Fremont, CA, USA).

Recombinant murine Timp1 Murine Timp1 was clonedfrom adipose tissue cDNA and ligated into the pSecTagAvector (Invitrogen, Basel, Switzerland) using the followingprimers: murine Timp1 forward AAGCTTTTGTAG

Diabetologia (2011) 54:1468–1479 1469

CTGTGCCCCACCCCA, reverse CTCGAGTCGGGCCCCAAGGGATCTCC. For protein production HEK293Tcells were transfected with lipofectamine (Invitrogen,Basel, Switzerland). Opti-MEM supernatant fractionwas collected every 2 days. Murine TIMP1 was purifiedtwice via the his-tag using a Ni-NTA column (GEHealthcare, Glattbrugg, Switzerland) connected to anautomated fast protein liquid chromatography system.Murine TIMP1 was dialysed against PBS and analysedby western blot using anti-his tag antibody (CellSignaling, Danvers, MA, USA) or Coomassie-stained12% (wt/vol.) SDS-polyacrylamide gel.

Adipocyte differentiation 3T3-L1 pre-adipocytes and pri-mary cells from SVF were cultured on collagen-coatedblack 96-well plates in DMEM supplemented with 10%(vol./vol.) fetal bovine serum and penicillin/streptomy-cin. Adipogenesis was induced by treating 48 h post-confluent 3T3-L1 pre-adipocytes or 80% confluentstromal–vascular cells with induction medium (1 μg/mlinsulin, 1 μmol/l dexamethasone, 115 μg/ml isobutyl-methylxanthine) for 2 days, followed by 2 days ofinsulin medium (1 μg/ml) and 2 days of normalmedium. Murine TIMP1 at indicated concentrations,monoclonal rat anti-mouse TIMP1 antibody (R&DSystems) or rat IgG1 isotype control antibody (MBL,Woburn, MA, USA) at 0.5 μg/ml were added to themedium 24 h after cell seeding until the third day ofdifferentiation. Differentiated cells were fixed with 5%(wt/vol.) formaldehyde and stained with 4 μmol/l Hoechst(nuclei), 5 μmol/l Syto60 (cytosol) and 2 μmol/l Bodipy (lipiddroplets) or Oil Red O.We took nine pictures per well with anautomated microscope (CellWorx, Thermo Fisher Scientific,Pittsburgh, PA, USA). Pictures were analysed using CellProfiler Software [31].

Adipose tissue western blots Adipose tissue extracts wereprepared as described under MMP activity assays. Proteinconcentration was determined using the DC protein assay(BioRad, Reinach, Switzerland). Tissue lysates were sub-jected to 12% (wt/vol.) SDS-polyacrylamide gel electropho-resis and western blotting using the following antibodies: goatanti-mouse/rat MMP2 (1:1,000), goat anti-mouse MMP9(1:1,000; R&D Systems), mouse anti-mouse γ-tubulin(1:5,000; Sigma-Aldrich, Buchs, Switzerland).

TIMP1 levels in human obese participants TIMP1 serumlevels were measured in 20 obese men (BMI 32.9–54.9 kg/m2)using a kit (human TIMP1 Quantikine ELISA; R&DSystems). All patients enrolled in the present study gaveinformed consent to participate. The study was approved bythe ethical board of the University Hospital of Heidelberg,Germany.

Animal studies All animal studies were approved by theveterinary office of Zürich. Male C57BL/6J mice werekept on a 12/12 h light/dark cycle in a pathogen-freeanimal facility. C57Bl/6J-Lepob mice were obtained fromJackson Laboratory (Bar Harbor, ME, USA). For diet-induced obesity, mice were fed a high-fat diet (ProvimiKliba, Kaiseraugst, Switzerland) containing 60% energyfrom fat. High-fat diet was given for 14 weeks (tissueexpression and secretion studies) or 8 weeks (TIMP1serum concentrations). Control animals on a regular chowdiet were killed at the corresponding age. For murineTIMP1 injection studies, subcutaneous murine TIMP1(200 μg/kg) or PBS injections were administered dailyinto the scapular region of male C57BL/6J mice (age 6–8 weeks) for 3 weeks, during which mice were fed a high-fat diet. For analysis of short-term effects, mice wereinjected with murine TIMP1 and fed a high-fat diet forthree consecutive days, followed by metabolism cageanalysis of oxygen consumption (V

�O2), carbon dioxide

production (V�CO2), food intake and physical activity

using a metabolism cage system (Oxymax; ColumbusInstruments, Columbus, OH, USA).

Serum measurements Blood glucose was measured with aglycometer (Contour, Bayer HealthCare, Zürich, Switzer-land). For the insulin tolerance test, insulin (0.75 U/kg ofbody weight) was injected after 3 weeks of high-fat diet andmurine TIMP1 injections. Insulin was determined with a kit(Rat/Mouse Insulin ELISA; Crystal Chem, Downers Grove,IL, USA). Triacylglycerol and NEFAwere measured with thefollowing kits: Trig/GB (Roche/Hitachi, Basel, Switzerland);and NEFA-HR kit (Wako, Neuss, Germany). TIMP1 serumlevels were assessed with a kit (Quantikine Mouse TIMP1ELISA; R&D Systems).

Liver triacylglycerol Lipids from 150 mg liver wereextracted with 1 ml hexane:isopropanol (3:2) by homoge-nising the tissue in a TissueLyser. Supernatant fraction wastaken off and the extraction repeated with 0.5 ml hexane/isopropanol. We then added 0.5 ml 15% (wt/vol.) Na2SO4

and evaporated the upper organic phase overnight. Lipidswere dissolved in 2 ml Triton X-100/methanol/butanol(1:1:3) and measured with the Trig/GB Kit (Roche/Hitachi,Basel, Switzerland).

Adipocyte and fat pad size Adipose tissue was fixedovernight in 5% (wt/vol.) paraformaldehyde before paraffinembedding and sectioning to 10 μm slices. Haematoxylin andeosin staining of sections was performed according to standardprocedures [32]. Microscopic pictures were taken and cell sizeanalysed using Cell Profiler Software [31]. Fat pads weremeasured with an animal computed tomography scanner(LaTheta; Echo Medical Systems, Houston, TX, USA).

1470 Diabetologia (2011) 54:1468–1479

Ex vivo glucose uptake Ex vivo glucose uptake wasperformed essentially as described previously [33]. Aftera 6 h fast during the dark period, extensor digitorumlongus muscles and adipose tissue were dissected andincubated for 20 min at 37°C in KRB buffer containing0.1% (wt/vol.) BSA, 5 mmol/l HEPES and 10mmol/l glucosewith or without 10 nmol/l insulin. Tissues were rinsedand glucose transport assessed during 75 min at 37°C using2-deoxy[14C]glucose (0.07 MBq; Perkin Elmer, Schwerzen-bach, Switzerland) in KRB with or without insulin. Tissueswere incubated for another 10 min at 37°C in KRB toremove extracellular radiolabelled glucose. Tissues wereincubated overnight at −20°C in 1 mol/l NaOH. Sampleswere dounced and centrifuged at 13,000 g for 10 min.Radioactivity was determined by liquid scintillation counting(Beckman Coulter, Brea, CA, USA) using a liquid scintil-lation cocktail (Ultima Gold; Perkin Elmer).

Statistical analysis Results are given as mean ± standarddeviation. Statistical analyses were performed using a two-tailed Student’s t test.

Results

It is known that obesity leads to a decreased capacity ofcompensatory de novo adipogenesis [34, 35], which isinfluenced by paracrine or endocrine factors. Wetherefore analysed whether Timp1 mRNA expressionwas deregulated in mouse models of obesity. In micewith diet-induced obesity mRNA expression of Timp1 wasupregulated in several tissues, including liver, kidney,heart, colon, thymus and brain. The most prominentincrease in Timp1 mRNA expression was seen in adiposetissue (Fig. 1a). These data were corroborated againstTIMP1 protein levels in adipose tissue and heart muscle.TIMP1 protein levels were increased two- to fourfold insubcutaneous and visceral adipose tissue of obese com-pared with lean mice, whereas TIMP1 protein in heartmuscle was only mildly affected. TIMP1 protein wasgenerally more abundant in visceral than in subcutaneousadipose tissue compartments (Fig. 1b). As TIMP1 is acirculating factor, we also measured TIMP1 in the serumof lean and obese mice. In ob/ob mice and mice with diet-induced obesity, TIMP1 serum concentrations were upre-gulated three- to fourfold compared with lean mice(Fig. 1c).

To get further insight into TIMP1 secretion from adiposetissue, protein secretion was measured in different fatdepots and cell fractions. TIMP1 secretion was higher invisceral than in subcutaneous adipose tissue (Fig. 1d).TIMP1 secretion was also higher from SVF than fromadipocytes, though direct comparisons between adherent

SVF and adipocytes based on cellular protein content aredifficult. TIMP1 secretion of SVF from mice fed a high-fatdiet for 14 weeks did not differ compared with SVF fromlean mice. However, adipocytes from animals on high-fatdiet showed higher levels of TIMP1 secretion than thosefrom mice fed a standard chow diet (Fig. 1d).

As it has been reported that TIMP1 serum levels arehigher in obese than lean patients [26], we analysedwhether TIMP1 serum levels in participants with severeobesity also correlated with type 2 diabetes. In an obesecohort of 20 men (BMI 32.9–54.9 kg/m2), there was asignificant positive correlation between TIMP1 serumlevels and HOMA index, whereas BMI did not correlatewith circulating TIMP1 levels in these obese patients(Fig. 1e, f). In female patients there was no significantcorrelation (data not shown).

Whereas TIMP1 serum levels also increased in mice inthe context of obesity (Fig. 1c), TIMP1 serum levels in amouse model of type 1 diabetes (streptozotocin-induced)did not differ compared with control mice. In summary,these results indicate that the increase in TIMP1 serumlevels is predominantly associated with obesity.

To shed light on a possible role of TIMP1 in adipo-genesis, we analysed Timp1 mRNA expression in thecourse of 3T3-L1 adipocyte differentiation. Expression ofTimp1 mRNA dropped at the time of induction and wasvery low in post-confluent differentiating 3T3-L1 pre-adipocytes (Fig. 1g).

To test whether murine TIMP1 also plays a functionalrole in adipogenesis, we purified recombinant mouseTIMP1 (murine TIMP1) from the cell culture supernatantfraction (Fig. 2a). As seen in Fig. 2b, c, increasing amountsof murine TIMP1 added to the medium in the range ofendogenously secreted TIMP1 from 3T3-L1 pre-adipocytes(1.27±0.14 μg/ml) led to a progressive decrease in 3T3-L1adipocyte formation. We next tested whether this inhibitoryeffect could also be seen in primary cells from murineadipose SVF. Whereas addition of murine TIMP1 did notchange the differentiation capacity of visceral SVF, it didlower the degree of differentiation in subcutaneous SVF(Fig. 3a–c). The opposite effect was achieved using aneutralising antibody against TIMP1, which increasedadipocyte differentiation in subcutaneous SVF only(Fig. 3d,e).

Previous reports using the human analogue of TIMP1(human TIMP1) in cell culture models of adipogenesis didnot observe any influence on adipocyte differentiation. Ashuman and mouse TIMP1 only share 72% homology, wehypothesised that the human and murine isoforms ofTIMP1 inhibit MMPs to differing degrees. We thereforecompared the ability of human TIMP1 to inhibit murineMMP9 or human MMP9 in an MMP activity assay. Asshown in Fig. 3f, human TIMP1 inhibited proteolytic

Diabetologia (2011) 54:1468–1479 1471

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Fig. 1 TIMP1 changes in obe-sity. a Tissue mRNA expressionof Timp1 in male C57Bl/6J miceon chow diet (black bars) orafter 14 weeks of high-fat diet(white bars). Expression wasnormalised to Rplp0; n=5.b TIMP1 protein levels in tissueextracts of subcutaneous fat,visceral fat and heart musclefrom male C57Bl/6J mice onchow diet (black bars) or high-fat diet (grey bars) or crossedinto the ob/ob background(white bars). TIMP1 proteinconcentration was measured byELISA and normalised to totalprotein concentration; n=4.c TIMP1 protein concentrationsin serum of lean and obese micemeasured by ELISA; n=5.d TIMP1 protein secretion frommurine SVF or adipocytes ofC57Bl/6J mice on chow diet(black bars) or high-fat diet(grey bars). Supernatant frac-tions were collected after 24 hand TIMP1 concentrations mea-sured by ELISA; n=5. e HumanTIMP1 serum concentrations inobese men plotted againstHOMA index or (f) BMI; n=20.e R2=0.34, p=0.009;(f) R2=0.00, p=0.96.g mRNA expression of Timp1(inverted triangles), Pparγ(triangles) and A-Fabp (squares)normalised to Rplp0 in the courseof 3T3-L1 differentiation; n=5.Values represent mean ± SD;*p<0.05, **p<0.01,***p<0.001

1472 Diabetologia (2011) 54:1468–1479

activity of human MMP9 more efficiently than that ofmurine MMP9. In addition, we analysed the efficacy ofhuman and murine TIMP1 to inhibit murine MMP9activity. As seen in Fig. 3g, murine TIMP1 showed ahigher inhibition of murine MMP9 than of human TIMP1.

To analyse whether murine TIMP1 also affected adipo-cyte plasticity in vivo we treated male C57BL/6J mice fed ahigh-fat diet with murine TIMP1. Treatment involved dailyinjection of 200 μg/kg body weight of murine TIMP1 orPBS into subcutaneous, scalpular adipose tissue for a timeperiod of 3 weeks. Apart from local accumulation in thesubcutaneous fat, this injection regime also led to increasedcirculating blood levels of TIMP1 approximately threefoldover baseline levels (Fig. 4a). To assess the effect of murineTIMP1 injection on adipose tissue development, wemeasured whole body weight as well as fat pad weight bycomputed tomography scanning (Fig. 4b, c). Body and fatpad weight gain did not differ between PBS-injected andmurine TIMP1-injected mice. However, we did observe anincrease in mean adipocyte size and a shift in the sizedistribution towards larger adipocytes in mice injected withmurine TIMP1 (Fig. 4d–f). This is in line with theobservation that murine TIMP1 inhibits adipocyte forma-tion, thus causing adipocyte hypertrophy.

Moreover, mice injected with murine TIMP1 showedhigher blood glucose and insulin levels, as well asdecreased insulin sensitivity in an insulin tolerance testcompared with control animals, indicating increased insulinresistance (Fig. 5a–c). In line with this, NEFA serum

concentrations and liver triacylglycerol content were in-creased in mice treated with murine TIMP1 (Fig. 5d,e),whereas serum triacylglycerol levels were unchanged(Fig. 5f). To analyse whether murine TIMP1 injectionsaltered glucose uptake specifically in adipose tissue, weperfomed ex vivo glucose uptake studies in fat and musclefrom mice that had been injected with murine TIMP1 orPBS for 3 weeks in combination with a high-fat diet. Asseen in Fig. 5g, murine TIMP1 injections led to decreasedinsulin-stimulated glucose uptake predominantly into sub-cutaneous and, to a minor extent, into visceral adiposetissue, whereas muscle glucose uptake was unaffected.

To elucidate whether murine TIMP1 injections influ-enced MMP levels in adipose tissue, we analysed produc-tion of MMP2 and MMP9, which are known to inhibitadipogenesis [36]. Although MMP levels varied betweenmice, we saw a general trend towards upregulation ofMMP, with higher production of the inactive pro-form ofMMP9 and MMP2 in mice injected with murine TIMP1(Fig. 6a–f). These data were corroborated against MMPactivity in adipose tissue extracts. As seen in Fig. 6g,murine TIMP1 injections led to a significant decrease inglobal MMP activity in subcutaneous as well as visceraladipose tissue. Interestingly, global MMP activity as suchwas considerably lower in visceral than in subcutaneousadipose tissue.

Since it has been reported that some of the observedeffects of TIMP1 might be due to a central regulation ofenergy balance, we tested respiration as well as activity of

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Fig. 2 TIMP1 inhibits 3T3-L1adipocyte differentiation.a Coomassie staining (CS) andwestern blot (WB) of purifiedrecombinant murine TIMP1.Murine TIMP1 was detectedusing an anti his-tag antibody.b 3T3-L1 pre-adipocytes weredifferentiated into adipocytes inthe presence of differentamounts of murine TIMP1 aslabelled. Lipid droplet formationwas assessed by fluorescentstaining with automated imageacquisition and analysis; n=4.Values represent mean ± SD;*p<0.05, **p<0.01,***p<0.001. c Representativeimages of differentiated 3T3-L1adipocytes, with treatments asindicated. Green, lipid droplets;blue, nuclei; red, cytoplasm.Scale bar 100 μm

Diabetologia (2011) 54:1468–1479 1473

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bFig. 3 TIMP1 inhibits primaryadipocyte differentiation.a Subcutaneous and visceralSVF from male C57BL/6J micewere differentiated in the pres-ence of different amounts ofmurine TIMP1 (mTIMP1); n=4.b Representative images offluorescent staining of differen-tiated subcutaneous and visceralmurine SVF. c Oil Red O stain-ing of differentiated subcutane-ous SVF in the presence ofdifferent amounts of murineTIMP1, as indicated. d Subcu-taneous and visceral SVF frommale C57BL/6J mice weredifferentiated in the presence ofanti-TIMP1 (grey bars) orcontrol antibody (black bars);n=4; with (e) representativeimages of fluorescent staining.f Inhibition of murine MMP9(black bars) or human MMP9(grey bars) by human TIMP1(hTIMP1). MMP9 activity wasmeasured as fluorescence permin using a fluorescence reso-nance energy transfer (FRET)substrate peptide; n=3.g Inhibition of murine MMP9(mMMP9) by different amountsof human TIMP1 (grey bars) ormurine TIMP1 (black bars) wasmeasured using a FRET sub-strate peptide; n=3. Scalebars (b, c, e) 100 μm. Values(a, d, f, g) represent mean ± SD;*p<0.05, **p<0.01,***p<0.001

1474 Diabetologia (2011) 54:1468–1479

the mice in a metabolism cage system after short-terminjections of murine TIMP1 (3 days) and after 3 weeks ofmurine TIMP1 treatment. Neither short- (data not shown)nor long-term (Fig. 7) murine TIMP1 injections led to

alterations in oxygen consumption, feeding or physicalactivity, implying that murine TIMP1 injections do notaffect regulatory mechanisms of energy homeostasis in thecentral nervous system.

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Fig. 4 Murine TIMP1 leads toadipocyte hypertrophy in vivo.a Serum concentration ofTIMP1 measured by ELISAduring daily injections of murineTIMP1 (triangles) or PBS(squares) into subcutaneous adi-pose tissue; n=5. b Body weightbefore (black bars) and after(grey bars) injection of murineTIMP1 or PBS into maleC57BL/6J mice in combinationwith a high-fat diet for 3 weeks;n=5. c Fat pad weight measuredby computed tomography ofmice injected daily with murineTIMP1 (grey bars) or PBS(black bars), and fed a high-fatdiet for 3 weeks; n=5. d Meanadipocyte size of subcutaneousfat at the site of injection, and inventral subcutaneous and omen-tal fat in male C57BL/6J micethat were injected daily for3 weeks with murine TIMP1(grey bars) or PBS (black bars)during high-fat diet; n=5.e Adipocyte size distribution ofsubcutaneous fat at the site ofinjection after 3 weeks of mu-rine TIMP (grey bars) or PBS(black bars) injections; n=5.f Adipocyte size distribution ofventral subcutaneous fat (murineTIMP, grey bars; PBS, blackbars); n=5. Values representmean ± SD; *p<0.05,**p<0.01, ***p<0.001

Diabetologia (2011) 54:1468–1479 1475

In summary, we show here that TIMP1 abundance isupregulated in obesity. TIMP1 inhibits adipocyte differen-tiation and leads to enlarged adipocytes in vivo. Miceinjected with murine TIMP1 mice show impaired NEFAand glucose profiles, increased hepatic triacylglycerolaccumulation and faster development of insulin resistance.

Discussion

Tight regulation of extracellular matrix remodelling byMMPs and their inhibitors such as TIMP1 is crucial for theadipogenic process. The fact that TIMP1 is upregulated inobesity partly due to increased abundance in adipose tissueled to the assumption that TIMP1 influences adipose tissueplasticity in the state of overnutrition.

We confirm here that TIMP1 is highly abundant in theSVF of adipose tissue and that levels are higher in visceral

than in subcutaneous adipose tissue [18, 26, 37–39]. Inobese animals, TIMP1 secretion from mature adipocyteswas increased in all fat depots, whereas TIMP1 secretionfrom SVF was not altered. These data conflict to someextent with a report from Maury et al. [39], which showsthat TIMP1 secretion is higher from SVF and adipocytes inhuman omental adipose tissue of obese patients comparedwith lean participants. These differences might be due tospecies differences or due to different culture and prepara-tion conditions. Based on our observations, it can beassumed that the increase of circulating TIMP1 levels inobesity observed here and by others [26] is at least partlydue to increased secretion from mature adipocytes, asTIMP1 levels in other TIMP1-producing tissues areincreased to a much lower extent.

As reported previously, TIMP1 production wasblunted at induction of adipocyte differentiation, hintingat an inhibitory role of TIMP1 in adipogenesis [18]. We

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95 72 68 50

kDa

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nsity

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nsity

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Fig. 6 a Western blot for MMP2, MMP9 and γ-tubulin of proteinextracts from subcutaneous injected adipose tissue, with quantification(b); murine TIMP, grey bars; PBS, black bars. c, d As above (a, b) forsubcutaneous adipose tissue and (e, f) visceral adipose tissue. g GlobalMMP activity in adipose tissue extracts from mice injected with PBS(black bars) or TIMP1 (grey bars) after 3 weeks of high-fat diet. MMPactivity was measured using a fluorescence resonance energy transfersubstrate peptide; n=4. Results are representative of three independentinjection experiments with independent protein preparations; n=5.Values (b, d, f, g) are mean ± SD; *p<0.05, **p<0.01, ***p<0.001,†p=0.07

PBS TIMP1 PBS TIMP1 PBS TIMP10

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FA (

mm

ol/l)

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PBS TIMP10123456789

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od g

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se (

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ol/l)

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cose

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of i

nitia

l)

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)

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Injected Subcut. Visceral Muscle

adipose tissue

Insu

lin-s

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ated

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ke (

%)

******

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TIMP1 TIMP1 TIMP1PBS PBS PBS

PBS TIMP10

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Insu

lin (

pmol

/l)

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0.00

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TAG

(m

mol

/l)

a b c

d

g

e f

Fig. 5 Mice injected with murine TIMP1 showed an impairedmetabolic profile. a Fasting blood glucose and (b) insulin serumlevels in mice injected with murine TIMP1 or PBS, and on a high-fatdiet. c Insulin tolerance test of mice injected with murine TIMP1(triangles) or PBS (squares) and fed a high-fat diet for 3 weeks.Insulin dose: 0.75 U/kg body weight. a–c n=5. d Serum NEFA, (e)liver triacylglycerol (TAG) content and (f) serum triacylglycerol ofmice injected with PBS or TIMP1 after 3 weeks of high-fat diet; n=5.g Insulin-stimulated glucose (glc) uptake into adipose tissue andmuscle from mice injected with PBS or TIMP1 after 3 weeks of high-fat diet. Basal, black bars; 10 nmol/l insulin, grey bars. n=4. *p<0.05,**p<0.01, ***p<0.001

1476 Diabetologia (2011) 54:1468–1479

showed here that murine recombinant TIMP1 repressedadipogenesis in 3T3-L1 pre-adipocytes and in subcutane-ous SVF, whereas neutralisation of TIMP1 led to enhanceddifferentiation. As TIMP1 inhibits a broad spectrum ofMMPs, these results are consistent with the fact that globalMMP inhibitors repress adipocyte differentiation in vitro[18, 36]. In a previous study, Demeulemeester et al. [29]reported that overabundant human TIMP1 had no effecton adipocyte differentiation in 3T3-F332A pre-adipocytes, whereas Alexander et al. [22] reportedenhanced adipogenesis using the MMP inhibitorGM6001 or human TIMP1. The differences betweenthe various reports suggest that the balance betweeninhibition of different classes of MMPs plays a crucialrole. In fact, different classes of MMPs are known tohave opposite effects on adipogenesis. Whereas MMP9and MMP2 are reported to promote adipocyte formation[36, 40, 41], MMP3 or MMP11 inhibit the latter [22, 42].Additionally, TIMP1 can have MMP-independent effectson cell growth [43]. One future challenge will therefore beto study the ‘mechanics’ of how TIMP1 influencesadipogenesis.

In contrast to subcutaneous SVF, no effect of murineTIMP1 or TIMP1 neutralisation could be seen in visceralSVF. It is known that subcutaneous and visceral adiposetissue show distinct gene expression patterns and thatvisceral fat is a higher risk factor for insulin resistance[11, 44]. As the differentiation capacity of visceral SVF assuch was seen here and elsewhere [45] to be much lower, itis likely that other inhibitory factors apart from TIMP1additionally control adipogenesis in visceral fat.

In vivo, global knockout of Timp1 leads to sex-specificeffects in mice with diet-induced obesity, giving rise to lessfat and smaller adipocytes in male animals, whereas femalemice with a null mutation develop bigger fat pads andlarger adipocytes [27, 28]. One issue is the impact of Timp1ablation on the central nervous system, leading to hyper-phagia in female mice, which might mask direct effects onadipose tissue development. Male Timp1 knockout mice donot show altered energy balance, but do display lower bodyweights as early as at weaning, implying a role for TIMP1in development. We did not observe any changes in energybalance in murine TIMP1-injected mice, nor did murineTIMP1 injections influence fat pad growth during high-fatdiet feeding. This suggests that the observed increase inadipocyte size goes in hand with a decrease in adipocytenumber, which is possibly a result of decreased compensa-tory adipogenesis in mice injected with murine TIMP1.

Demeulemeester et al. reported that chronic overabun-dance of human TIMP1 had no effect on adipose tissuedevelopment [29], whereas Alexander et al. observed more,hypertrophied adipocytes in the mammary gland [22]. Here,we were able to show that injection of murine TIMP1 intosubcutaneous fat during 3 weeks of high-fat diet led toenlarged adipocytes in subcutaneous fat pads. Discrepan-cies can be partially attributed to species-specific differ-ences, as human TIMP1 is less efficient in inhibitingmurine MMP9 than human MMP9. Furthermore, humanTIMP1 is also less efficient than murine TIMP1 inregulating murine MMP9 activity. In addition, overabun-dance in the liver [29] might also change activity of humanTMP1 by alternate mechanisms.

Murine TIMP1 injections under a high-fat diet also ledto an impaired metabolic profile. It is known that enlargedadipocytes become insulin-resistant and have a highlipolytic rate [8, 9, 46, 47]. As murine TIMP1 injectionsdid not alter muscle glucose uptake, it can be excluded thataltered muscle function contributed to the observed changesin glucose metabolism. Although it cannot be ruled out thatTIMP1 acts on other metabolic tissues, the elevated serumlevels of NEFA seen here hint at a contribution of disturbedadipose tissue function to this phenotype. Elevated NEFAlevels in turn cause increased hepatic triacylglycerolaccumulation, promoting insulin resistance [4]. Interesting-ly, a positive correlation was also found between circulatingTIMP1 levels and insulin resistance in our group of obesemen. This correlation supports the link between TIMP1levels and insulin resistance.

Murine TIMP1 injections led to decreased MMP activityin adipose tissue, suggesting that the observed metaboliceffects are at least partly due to altered adipose tissue MMPactivity. Interestingly, MMP activity in visceral vs subcu-taneous adipose tissue was much lower in itself, a findingthat is consistent with the increased endogenous levels of

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Fig. 7 Murine TIMP1 does not affect central energy regulation.a Food intake, (b) movement and (c) respiration in male C57BL/6Jmice injected with murine TIMP1 (grey bars) or PBS (black bars)and fed a high-fat diet for 3 weeks; n=5

Diabetologia (2011) 54:1468–1479 1477

TIMP1 in visceral fat. Given the fact that murine TIMP1inhibited adipogenesis and increased adipocyte size, espe-cially in subcutaneous adipose tissue where endogenousTIMP1 levels are low, it can be speculated that TIMP1injections increase subcutaneous adipose tissue insulinresistance under high-fat diet feeding, a process that isnormally seen predominantly in visceral adipose tissue.This is supported by the fact that insulin-stimulated glucoseuptake was lower in visceral than in subcutaneous adiposetissue in control animals and much less affected by TIMP1treatment.

It is well known that proinflammatory cytokines andadipokines are upregulated in adipose tissue of obeseparticipants and contribute to insulin resistance [48, 49].In 3T3-L1 adipocytes, TIMP1 can be induced by cytokinessuch as TNFα, IL-6 and IL1-β [50, 51]. It is thereforeintriguing to speculate that increased TIMP1 production inobesity is driven by inflammatory signals, thus inhibitingdifferentiation of new adipocytes. In fact, it has been shownthat the differentiation capacity of SVF from obese mice isimpaired, which could in turn lead to adipocyte hypertro-phy and increased insulin resistance [52].

Based on these findings, TIMP1 might serve as a targetfor modulation of adipose tissue plasticity in response toadiposity. Inhibition of TIMP1 would result in an improvedhyperplastic response of adipose tissue, which is known toreduce the risk of insulin resistance.

Acknowledgements These studies were supported by ERC-Adipodif,FP7-LipidomicNet, SNF and the Roche Research Foundation.

Duality of interest The authors declare that there is no duality ofinterest associated with this manuscript.

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