Multiple symmetric lipomatosis may be the consequence of defective noradrenergic modulation of...

Journal of PathologyJ Pathol 2002; 198: 378–387.Published online 5 September 2002 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/path.1212

Original Paper

Multiple symmetric lipomatosis may be the consequenceof defective noradrenergic modulation of proliferationand differentiation of brown fat cellsEnzo Nisoli,1,2* Laura Regianini,1 Luca Briscini,1 Alessandra Bulbarelli,1 Luca Busetto,3 Alessandra Coin,3

Giuliano Enzi3 and Michele O Carruba1,2

1 Center for Study and Research on Obesity, Department of Preclinical Sciences, LITA Vialba, L. Sacco Hospital, University of Milan, Via G. B. Grassi 74,20157 Milan, Italy2 Istituto Auxologico Italiano, Via Spagnoletto 3, Milan 20149, Italy3 Division of Internal Medicine, University of Padua, Via Giustiniani 2, 35128 Padua, Italy

*Correspondence to:Enzo Nisoli, MD, PhD, Centro diStudio e Ricerca sull’Obesita,Dipartimento di ScienzePrecliniche, LITA Vialba,Ospedale L. Sacco, Via G. B.Grassi 74, 20157 Milano, Italy.E-mail: [email protected]

Received: 26 March 2002Accepted: 8 April 2002

AbstractMultiple symmetric lipomatosis (MSL) is an inherited disorder in which enlarging and unen-capsulated lipomas symmetrically develop in the subcutaneous tissue of the neck, shoulders,mammary, and truncal regions. In some cases, it is associated with mitochondrial DNAabnormalities. The pathogenesis of MSL is completely unknown, although the fat depositsmay be due to a neoplastic-like proliferation of functionally defective brown adipocytes. Ithas recently been demonstrated that the β3-adrenergic receptor is the functionally relevantadrenergic receptor subtype in brown adipocytes and that its stimulation by noradrenaline(NA) modulates the expression of genes, such as uncoupling protein (UCP)-1 and induciblenitric oxide synthase (iNOS), involved in fat cell proliferation and differentiation. Fur-thermore, Trp64Arg mutation of the β3-adrenoceptor has been implicated in lower NAactivity in adipose tissues. The aim of this study was to investigate the molecular and func-tional characteristics of MSL adipocytes and to analyse the effects of nitric oxide (NO) onthe proliferation/differentiation of MSL adipocytes in culture, and the relevance of puta-tive noradrenergic deficit in the development of lipomas in MSL patients. Cultured MSLadipocytes were able to synthesize UCP-1 (the selective marker of brown adipocytes), butunlike that of normally functioning brown fat cells, the expression of the UCP-1 gene wasnot significantly induced by NA. NA is also defective in inducing iNOS gene expression,thus leading to reduced NO production and a consequent reduction in the anti-proliferative,adipogenic (mitochondrial biogenesis) effects of NA on MSL cells. Furthermore, the tran-scriptional peroxisome proliferator-activated receptor γ co-activator-1 (PGC-1), which playsa key role in the sympathetic-stimulated mitochondrial biogenesis of brown adipocytes, isexpressed but not induced by NA in MSL cells, as it is in brown adipocytes. The study didnot find any association between β3-adrenoceptor gene polymorphism and noradrenergic sig-nalling defects in MSL subjects with or without mitochondrial DNA mutations. Copyright 2002 John Wiley & Sons, Ltd.

Keywords: multiple symmetric lipomatosis; lipodystrophies; brown adipose tissue; mito-chondrial biogenesis; uncoupling protein; nitric oxide; noradrenaline; leptin; PPARγ co-activator-1; mitochondrial DNA

Introduction

Multiple symmetric lipomatosis (MSL), or Launois–Bensaude’s disease, is an inherited disorder of mid-dle life that is not unusual in Mediterranean areas,where it has an incidence rate of 1 : 25 000 and prefer-entially occurs in males. It is clinically characterizedby enlarging, painless, extremely vascularized, sym-metric unencapsulated lipomas in the subcutaneoustissue of the posterior aspect of the neck and inter-scapular region that give rise to a grotesque physicalappearance [1–3]. Increasing evidence suggests thatvarious genetic abnormalities underlie MSL and that

some cases are associated with abnormal mitochon-drial DNA (mtDNA) and systemic mitochondrial dys-function [4,5].

An exciting hypothesis is that MSL fat depositsoriginate from functionally defective brown adiposetissue (BAT) accumulating an excess of lipids [6].BAT is a highly specialized tissue that exists inmost mammalian hibernators and humans, and pro-duces heat in response to cold exposure (non-shiveringthermogenesis) or after eating (diet-induced thermo-genesis) [7]. BAT accumulates in the interscapular,perirenal, axillary, and cervical regions of infants, inthe same areas as the MSL lipomas. Islands of brown

Copyright 2002 John Wiley & Sons, Ltd.

Molecular characterization of MSL 379

fat are normally seen in the visceral and periadrenalfat of adult humans. The thermogenic function ofbrown fat depends on the noradrenergic stimulationof mitochondrial uncoupling protein (UCP)-1, whichis exclusively expressed in brown adipocytes and can‘short circuit’ the oxidative phosphorylation pathway,thus leading to reduced ATP synthesis and the pro-duction of heat [8]. This action is primarily mediatedby β3-adrenoceptors, which are also highly expressedin brown adipocytes [9–11]. Selective activation ofβ3-adrenoceptors by synthetic agonists stimulates ther-mogenesis and normalizes energy balance in obeseanimals [10]. We have recently reported that the expo-sure of culture-differentiated rat brown adipocytes toβ3-adrenoceptor agonists stimulates the expression ofan inducible nitric oxide synthase (iNOS) isoformsimilar to the iNOS of macrophages and leads tonitric oxide (NO) synthesis and release [12]. Further-more, NO markedly inhibits proliferation, an eventthat seems to be essential for the differentiation ofproliferating brown adipocytes, and triggers the dif-ferentiation programme by modulating the expres-sion of peroxisome proliferator-activated receptor γ

(PPARγ ) [13], a gene known to play a priming rolein adipose tissues [14,15]. Puigserver et al. [16] havedemonstrated that transcriptional PPARγ co-activator-1 (PGC-1) is up-regulated in brown fat and skeletalmuscle following acute cold exposure, which is apotent stimulator of sympathetic nervous outflow tobrown fat [17]. In transient transfection assays, PGC-1 greatly increases the ability of PPARγ and thyroidhormone receptor to activate transcription from theUCP-1 promoter.16 Interestingly, PGC-1-expressingcells have increased levels of mitochondrial DNA,thus indicating that overall mitochondrial biogenesishas been stimulated.18

In order to investigate the molecular and func-tional characteristics of MSL adipocytes, we testedUCP-1, iNOS, and PGC-1 gene expression andtheir induction by NA. The results strongly sup-port the idea that MSL may derive from defec-tive noradrenergic regulation of iNOS and/or PGC-1 induction, leading to the dysregulated prolifera-tion and differentiation (i.e. mitochondrial biogene-sis) of a population of fat cells that has a num-ber of analogies with rodent and human brownadipocytes.

Materials and methods

Subjects

Ten unrelated patients with the typical appearance ofMSL, who were identified at the Department of Inter-nal Medicine (University of Padua), underwent surgi-cal lipoma excision. Specimens were taken from lipo-mas of the neck and shoulders and stored at −80 ◦Cor used to prepare cell cultures as described below.The study protocol was approved by the hospital

ethics committee and all the subjects gave informedconsent.

MSL adipocyte isolation and cell culture treatment

Samples of lipomatous tissue were digested with col-lagenase under sterile conditions and isolated as pre-viously described [19]. Three million cells seeded insix-well culture plates (Costar, Milan, Italy) were cul-tured in a water-saturated atmosphere of 5% CO2 inair at 37 ◦C in 2.0 ml of a culture medium consist-ing of Dulbecco’s modified Eagle’s medium (DMEM)supplemented with 4 mM glutamine, 10% w/v fetalcalf serum (Mascia Brunelli, Milan, Italy), 4 nM

insulin, 0.04 nM triiodothyronine, 10 mM HEPES, 50IU of penicillin, 50 µg/ml streptomycin, and 25 µg/mlsodium ascorbate (all from Flow Laboratories, Milan,Italy). The medium was completely replaced by freshpre-warmed medium on day 1 (when the cultures werefirst washed with pre-warmed DMEM), and on days 4and 8 (without washing).

In the experiments concerning the modulation ofgene expression, on culture day 8, the cells wereexposed once a day for a further 6 days (or 6 hfor iNOS expression) to noradrenaline (NA) freshlydiluted in buffers containing 0.1% ascorbic acidand then harvested. The medium was discarded; thewells were washed twice with ice-cold phosphate-buffered saline (PBS) (Sigma, Milan, Italy); and thecells were scraped out with a disposable cell scraper(Nunc, Milan, Italy) for mRNA extraction for RT-PCRanalysis. For cell counting, PBS containing 0.05%(w/v) trypsin and 0.02% (w/v) EDTA without cal-cium and magnesium was added to each well andthe cells were sedimented by 5 min of centrifuga-tion at 700 × g . The sedimented cells were dilutedin 0.5 ml of cultured medium and counted in a Burkerchamber.

Final concentrations of NO donor (GSNO) (100–1000 µM) or NOS inhibitor (L-NAME) (300 µM) wereadded to the cultures once a day from day 1 to day8. The cells were harvested 24 h after the addition ofthe agents.

RT-PCR assay

Total cytoplasmic RNA was isolated from 1 × 106 cul-tured cells using the RNazol method (TM Cinna Scien-tific, Friendswood, TX, USA). The RNAs were treatedfor 30 min at 37 ◦C with 5 U of RNase-free DNaseI in100 mM Tris–HCl, pH 7.5 and 50 mM MgCl2 in thepresence of 2 U/µl of placenta RNase inhibitor. Theconcentration of RNA was determined by absorbanceat 260 nm and its integrity was confirmed by meansof electrophoresis through 1% agarose gels contain-ing 0.1 mg/ml ethidium bromide. One microgram oftotal RNA was converted to cDNA using 200 U ofMoloney murine leukaemia virus reverse transcriptase(Promega, Madison, WI, USA) in 20 µl of Promega-supplied buffer containing 0.4 mM dNTPs, 2 U/µl

Copyright 2002 John Wiley & Sons, Ltd. J Pathol 2002; 198: 378–387.

380 E Nisoli et al.

RNase inhibitor, and 0.8 µg of oligo(dT)12–18 primer(Sigma, Milan, Italy); usually, 100 ng of cDNA wassynthesized from 1 µg of total RNA. A control exper-iment without reverse transcriptase was performed foreach sample in order to verify that the amplificationwas not due to any residual genomic DNA. An aliquot(5% vol., ∼20 ng) of the cDNA was amplified usingspecific primers and Taq DNA polymerase (Promega,Milan, Italy) in 25 µl of standard buffer [10 mM Tris-HCl (pH 9), 50 mM KCl, 0.1% Triton X-100, 2.5 mM

MgCl2, and 200 µM dNTPs]. The primers are shown inTable 1. For each gene, the PCR programme consistedof 1 min at 94 ◦C, a different number of amplification

cycles (each cycle consisting of 30 s at 94 ◦C, 30–35 sat a specific annealing temperature, and 30 s at 72 ◦C),and a final step of 7 min at 72 ◦C. The mRNA for con-stitutive β-actin was examined as the reference celltranscript using specific primers and conditions. After20 cycles, 5 µl of the PCR reaction mixtures obtainedfrom the different treatment groups was added to10 µl of the examined gene PCR products. Thesereaction mixtures were separated by electrophoresis(2.0% agarose gel in Tris–acetate–EDTA buffer con-taining 0.1 mg/ml ethidium bromide), revealed using aQuickImage-D (Camberra Packard, Milan, Italy), andanalysed densitometrically by means of Phoretix 1D

Table 1. PCR conditions used to amplify the gene products [(r), rat; (h), human]

GeneForwardprimer

Reverseprimer

Fragmentlength (bp) PCR conditions Reference

β1-AR TCG TGT GCACCG TGT GGGCC

AGG AAA CGGCGC TCG CAGCTG TCG

265 30 cyclesannealing

58 ◦C, 30 s 38

β2-AR GCC TGC TGACCA AGA ATAAGG CC

CCC ATC CTGCTC CAC CT


58 ◦C, 30 s 38

β3-AR GCT CCG TGGCCT CAC GAGAA

CCC AAC GGCCAG TGG CCAGTC AGC


58 ◦C, 30 s 38

UCP-1 TAG GTA TAAAGG TGT CCTGG

CAC TTT TGTACT GTC CTGGTG G


58 ◦C, 35 s 39

UCP-2 TCG TGC AATGGT CTT GTAGG

CAA CAG CCACTG TGA AGTTCC


60 ◦C, 30 s 40

UCP-3 ACA GAT GTGGTG AAG GTCCG

TAC GAA CATCAC CAC GTTCC


60 ◦C, 30 s 41

Leptin (ob) CAC CAA AACCCT CAT CAAGAC

AGC CTG CTCAAA GCC ACCACC


60 ◦C, 35 s 42

TNF-α ATG AGC ACTGAA AGC ATGATC CGG GACGTG G

CAA TGA TCCCAA AGT AGACCT GCC CAGACT C


60 ◦C, 30 s 43

(r) iNOS ACG TGC GTTACT CCA CCAACA A

CAT AGC GGATGA GCT GAGCAT T


55 ◦C, 20 s 44

(h) iNOS TCC GAG GCAAAC AGC ACATTC A

GGG TTG GGGGTG TGG TGATGT


60 ◦C, 60 s 27

(r) PGC-1 AGT CGC AACATG CTC AAGC

TGT GGA AGAACA GAT GTGCC


60 ◦C, 30 s 16

(h) PGC-1 TCC TCT GACCCC AGA GTCAC

TAG AGT CTTGGA GCT CCTG


58 ◦C, 60 s 45

MELAS AGG ACA AGAGAA ATA AGGC

AAC GTT GGGGCC TTT GCGTAG


56 ◦C, 35 s 46

MERRF CCC CTC TAGAGC CCA CTGTAA AGC

GGG TGA TGAGGA ATA GTGTAA GG


56 ◦C, 35 s 47

4977-bp deletion CCC CTC TAGAGC CCA CTGTAA AGC

TAA GTT TGTTGG TTA GGTAG


56 ◦C, 35 s 48

β-actin TAA AGA CCTCTA TGC CAACAC AGT

CAC GAT GGAGGG GCC GGACTC ATC


60 ◦C, 30 s 49



version 3.0. The β-actin mRNA amplification productswere equivalent in all of the cell lysates. The identityof the PCR products was confirmed by hybridizationusing internal oligonucleotides obtained by means ofthe PCR amplification of cloned genes with the samespecific primers as those described.

MTT stainingFor colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), the cellswere seeded in 24-well plates (12.5 × 104 cells/cm2)and incubated in 0.4 ml of DMEM, supplementedas described above. They were incubated in mediumwith or without the compounds as indicated in thetext. At the indicated time points, the cells werewashed and 0.5 ml of filtered MTT stock solution(2.4 mM in RPMI 1640 without phenol red) was added,before the plates were incubated for 3 h at 37 ◦C. Atthe end of this incubation period, the untransformedMTT was carefully removed and the dye crystalswere solubilized in 1 ml of 2-propanol. As a negativecontrol, the absorbance of MTT was measured in cell-free plates treated in the same way. Absorbance wasread immediately in a UVKON 941 spectrophotometerusing a test wavelength of 570 nm and a referencewavelength of 690 nm.

Genomic DNA extraction and molecular geneticstudiesTotal DNA was prepared from frozen powdered MSLtissues by homogenization and from cultured MSLadipocytes. Both the tissues and the cells were lysedin buffer containing 10 mM Tris–HCl (pH 8), 20 mM

EDTA, and 0.5% sodium dodecyl sulphate, and occa-sionally shaken while on ice for 30 min. DNA wasextracted with a mixture of phenol and chloroform(Sigma, Milan, Italy) and precipitated by addingan equal volume of isopropanol and one-tenth vol-ume of 5 M sodium acetate. After storing at −20 ◦Covernight, DNA pellets were obtained by centrifuga-tion at 13 000 × g for 20 min at 4 ◦C and washedwith 75% ethanol. The pellets were dried, resuspendedin 50 µl of Tris–EDTA, and treated with RNase(100 µg/ml, Sigma) at 37 ◦C for 1 h. Genomic DNAwas first amplified by means of PCR (see Table 1 fordetails) using specific primers for mitochondrial DNAmutations (MELAS 3,243, MERRF 8,344, 4977-bpdeletion) and then digested with the restriction enzymeApa I, or automatically sequenced (M Medical, Flo-rence, Italy), or simply analysed by means of agarosegel electrophoresis.

Measurement of mitochondrial DNA contentThe cultured MSL cells were collected, counted,washed with saline buffer, and then pelleted. Mito-chondrial isolation was performed at 0–4 ◦C as pre-viously described [20]. Briefly, the pellet was resus-pended in 1.5 ml of ice-cold buffer solution contain-ing 0.25 M sucrose and 5 mM K-TES (pH 7.2) and

then homogenized. The homogenate was centrifugedat 8500 × g for 10 min; the supernatant was discarded;and the pellet was resuspended in the original volumeof the same buffer solution. The suspension was cen-trifuged at 700 × g for 10 min and the supernatant wasdecanted to a clean tube and centrifuged at 8500 × gfor 10 min. The isolated mitochondria were then lysedin 10 mM Tris–HCl (pH 8), 20 mM EDTA, and 0.5%Triton X-100 (pH 8), and occasionally shaken whileon ice for 30 min. The mitochondrial DNA (mtDNA)was extracted using a mixture of phenol and chloro-form, and then one-tenth volume of 5 mM NaCl wasadded to the solution and the mtDNA was precipitatedovernight by adding an equal volume of isopropanol.After overnight precipitation at −20 ◦C, the mtDNAwas pelleted by centrifugation, resuspended in sterilewater, and treated with 50 µg/ml RNase. In order toverify the yield, an aliquot of mtDNA was loaded onan ethidium bromide-stained 1.2% agarose gel. ThemtDNA concentration was determined by UV absorp-tion at 260 nm.

Analysis of restriction-fragment-lengthpolymorphism of the β3-adrenoceptor

Blood samples of MSL patients were drawn forthe extraction of genomic DNA from leukocytes.Amplification of DNA by PCR was carried out understandard conditions. The amplified fragments weredigested with BstNI and analysed by agarose gelelectrophoresis, as described by Clement et al. [21].

Data analysis

The data are expressed as the mean values ± SD ofat least three independent experiments. Comparisonswere made using one-way analysis of variance fol-lowed by Student–Newman–Keuls post-hoc compar-isons. p values of less than 0.01 versus the controlvalue were considered significant.

Materials and drugs

The culture sera and media were purchased fromGIBCO, Basel, Switzerland. The type II collage-nase, fatty acid-free bovine serum albumin, andDNase I came from Boehringer Mannheim, Germany,and the S -nitroso-L-glutathione (GSNO) from Cal-biochem (San Diego, CA, USA). NA hydrochlorideand the remaining chemicals came from Sigma, Milan,Italy. Lipopolysaccharide (LPS) came from Sigma(Milan, Italy) and interferon γ (IFNγ ) from Strath-mann Biotech (Hannover, Germany). Except for NAhydrochloride (which was dissolved in a buffer con-taining 0.01% ascorbic acid to prevent oxidation), thedrugs were all dissolved in phosphate-buffered saline(PBS). Appropriate control solvents alone were usedin parallel in all of the experiments; in the exper-iments involving L-N G nitro-arginine methyl ester(L-NAME), control experiments using the less active


382 E Nisoli et al.

D-N G nitro-arginine methyl ester (D-NAME) enan-tiomer led to results that were similar to those of theuntreated controls.

Results

Morphological appearance and molecularcharacterization of MSL adipocytes in culture

Preadipocytes of the stromal-vascular fraction of lipo-matous and normal subcutaneous fat obtained from tendifferent MSL patients were cultured separately for8 days until confluence and then examined by meansof light and electron microscopy. The morphologicalappearance suggested that MSL adipocytes are moresimilar to brown than to white adipocytes (data notshown), as previously described in detail [6].

To confirm the morphological observations andcharacterize molecularly the MSL adipocytes, we usedRT-PCR to investigate the expression of some geneswhose products are critical for BAT function. Thisexperimental approach was chosen in order to increasethe detection limit. Gene expression was analysedduring the differentiation in culture of cells with orwithout the mtDNA mutations that often occur in MSLpatients. The results are the mean values ± SD, withthe controls being cultured white adipocytes from thenormal subcutaneous fat depots of the MSL patients.Although no real quantitative comparison betweenthe two cell types was possible using our approach,the data obtained raise some important considerationsconcerning the mRNA levels of the single genes in theMSL or control fat cells.

We first studied UCP-1 gene expression. Thisuncoupling protein is selectively expressed in themitochondrial inner membrane of mammalian brownfat [22], where it generates heat upon noradrenergicstimulation of β3-adrenoceptors by uncoupling oxida-tive phosphorylation [23]. We found that the UCP-1gene was expressed in the cultured MSL adipocytes,but not in the control adipocytes, of each of the tenpatients studied (Figure 1). This strongly suggests thatMSL cells express the principal biochemical marker ofbrown adipocytes.

The MSL cells expressed the β3-adrenergic receptorgene and its mRNA levels did not seem to be statisti-cally different from those of the cultured subcutaneouswhite adipocytes; similar results were obtained for β1-and β2-adrenoceptor subtypes (data not shown). More-over, both brown and white fat cells have been shownto express leptin, tumour necrosis factor (TNF)-α, andUCP-2 and UCP-3 [24,25]. This is also true of MSLcells and the mRNA levels were similar to those ofthe cultured control adipocytes (data not shown).

Effects of chronic NA treatment on UCP-1expression

It is widely known that UCP-1 gene expression in cul-tured human and rodent brown adipocytes is highly

Figure 1. UCP-1 mRNA expression in cultured MSL cells (MSL)and white adipocytes from normal subcutaneous fat (C) of thesame MSL patients. mRNA was extracted from the cells on day8 of culture. Representative agarose gel showing the RT-PCRanalysis of UCP-1 and β-actin mRNA content (top). A controlwithout reverse transcriptase (lanes — ) was used for eachsample to verify that the amplification was not due to residualgenomic DNA. β-actin primers were added to each reaction.Using scanning densitometry, the abundance of UCP-1 mRNAwas normalized to arbitrary units by assigning the value of 1 tothe UCP-1 mRNA abundance in MSL cells (bottom). The barsrepresent the mean values ± SD of ten separate experiments,one for each MSL patient

stimulated by NA exposure [26]. RT-PCR analysis ofcultured MSL adipocytes treated with 10 µM NA for6 days revealed no significant stimulation of UCP-1 gene expression, even if this NA-induced UCP-1stimulation was present in some patients, unlike theresults obtained in rat brown adipocytes (Figure 2).These findings suggest that NA is less effectivein inducing UCP-1 expression in MSL cells thanin mammalian BAT cells, a defective efficacy thatmay be due to alterations in the adrenergic sig-nalling system. Mutations of the gene encoding theβ3-adrenoceptor are unlikely, since no MSL patienthad the Trp64Arg mutation [21] that has been impli-cated in lower NA activity in adipose tissues, andso, as previously suggested, events downstream ofthe ligand–receptor interaction seem to be morelikely.

Defective induction of iNOS and PGC-1 geneexpression in MSL adipocytes

When we investigated the ability of NA to mod-ulate iNOS mRNA levels in MSL adipocytes, wefound that 10 µM NA (for 6 h or 6 days) was muchless effective in MSL adipocytes than in rat brownadipocytes (Figure 3) and did not increase NO pro-duction measured in terms of nitrite/nitrate accu-mulation (data not shown). However, 6 h treatment



Figure 2. Effects of noradrenaline (NA) exposure of culturedMSL and rat brown fat (BAT) cells on UCP-1 gene expression.On culture day 8, 10 µM NA was added to the standardmedium for a further 6 days. The abundance of mRNA wasnormalized to arbitrary units by assigning the value of 1 to theUCP-1 mRNA abundance in NA-treated rat brown adipocytes.The bars represent the mean values ± SD of three separateexperiments using cells obtained from three MSL patients(MSL) or six rats (BAT). *p < 0.001 vs. saline (S)-treated brownadipocytes

with 10 ng/ml LPS plus 100 U/ml IFNγ did induceiNOS gene expression in MSL cells, as has beendescribed in human macrophages by Reiling et al. [27](Figure 3).

Interestingly, the expression of PGC-1, a sympa-thetically inducible protein involved in the mitochon-drial biogenesis of brown adipocytes and other celltypes [18], was unchanged or slightly decreased inMSL adipocytes after acute (6 h) or chronic (6 days)NA treatment (Figure 4), thus further confirming thelack of NA stimulation in such cells.

Effects of the GSNO NO donor and the L-NAMENOS inhibitor on MSL adipocyte proliferation anddifferentiation

In order to investigate the effect of NO on prolifera-tion, cultured MSL adipocytes were treated daily fromday 1 to day 8 with or without different concentrationsof GSNO (ranging from 100 to 1000 µM), which liber-ates NO in solution [28], and the cells were counted ina Burker chamber. GSNO did not affect the viability ofthe MSL cells, as tested by trypan blue exclusion, andit was maximally effective in inhibiting cell prolifera-tion at the low concentration of 200 µM. As shown inFigure 5A, there were approximately ten times morecells on day 8 than after GSNO treatment (p < 0.001for all treatment days). The fact that cell proliferationwas restored once the NO sources had been washedaway confirms that the decrease in cell number wasdue to reduced cell proliferation (cytostasis) and notcytotoxicity.

To verify whether the inhibition was mediated bythe generation of NO or was due to other actionsof GSNO, cell proliferation experiments were car-ried out in the presence of oxyhaemoglobin (Hb),which binds and inactivates NO [29]. As shown inFigure 5A, GSNO completely lost its inhibitory effecton cell proliferation when chronically administered

Figure 3. Effects of noradrenaline (NA) and lipopolysaccharide (LPS) plus interferon γ (IFNγ ) exposure of cultured MSL and ratbrown fat (BAT) cells on human (h) and rat (r) iNOS gene expression. On culture day 8, either 10 µM NA or 10 ng/ml LPS plus100 U/ml IFNγ were added to the standard medium for 6 h. The mRNA abundance was normalized to arbitrary units by assigningthe value of 1 to the (r)iNOS mRNA abundance in NA-treated rat brown adipocytes. The bars represent the mean values ± SDof three separate experiments using cells obtained from three MSL patients or six rats. *p < 0.001 vs. saline (S)-treated brownadipocytes


384 E Nisoli et al.

Figure 4. Effects of the noradrenaline (NA) exposure ofcultured MSL and rat brown fat (BAT) cells on human (h)and rat (r) PGC-1 gene expression. On culture day 8, 10 µMNA was added to the standard medium for a further 6 days.The mRNA abundance was normalized to arbitrary units byassigning the value of 1 to the (r)PGC-1 mRNA abundancein NA-treated rat brown adipocytes. The bars represent themean values ± SD of three separate experiments using cellsobtained from three MSL patients or six rats. *p < 0.001 vs.saline (S)-treated brown adipocytes

in a medium supplemented with Hb (50 µM; seeref. 30).

If NO acts as an anti-proliferative factor in grow-ing MSL cells, the inhibition of NOS should increasecell proliferation. Treatment with L-NAME at a con-centration of 300 µM, which is likely to inhibit allthree enzyme isoforms [31], increased MSL cell pro-liferation in comparison with untreated MSL cells

(Figure 5A). Under the same experimental condi-tions, the less active D-NAME enantiomer left cellproliferation unchanged in comparison with controls(Figure 5A). Furthermore, the stimulatory effect ofL-NAME was completely antagonized by 1 mM L-arginine (Figure 5A).

The mitochondrial conversion of 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)to formazan was taken as an index of mitochon-drial biogenesis and consequently MSL cell differen-tiation [13]. Figure 5B shows that chronic treatmentwith GSNO (200 µM) reduced the MTT signal by only45%, despite the very marked inhibition of cell pro-liferation (Figure 5A). This suggests that each MSLcell was more differentiated than the control cells.The presence of 50 µM Hb blocked the inhibition bythe NO donor (Figure 5B). Interestingly, the activeproliferative effect of L-NAME was not accompaniedby a marked increase in MTT staining (Figure 5B),thus indicating that the differentiation mechanism wasnot enhanced in actively proliferating MSL cells. D-NAME (300 µM) also did not affect MTT staining(data not shown). These results were confirmed byanalysis of mtDNA levels after chronic GSNO treat-ment of MSL cells (150 ± 10% over untreated cells).

Mitochondrial DNA mutations in MSL patients

None of the patients studied have the point mutationsof the transfer ribonucleic acid (tRNA) for leucineat position 3,243 (MELAS mutation) or lysine atposition 8,344 [myoclonic epilepsy with ragged-redfibres (MERRF) mutation] [4], but three had thelarge-scale DNA deletion (i.e. the 4977-bp mtDNAdeletion) [4].

Figure 5. Effects of prolonged NO donor and L-NAME exposure on cell number (A) and the mitochondrial conversion of MTTto formazan (B) in cultured MSL cells. The cells were grown in culture medium without any further addition (C, controls), orwith the daily addition of 300 µM GSNO, or of 300 micro µ L-NAME or D-NAME alone or in combination with 50 µM Hb or 1 mML-arginine. Hb and L-arginine did not have any effect per se. The experiments with Hb (n = 4) were not performed in parallel.Each point represents the mean values ± SEM of three experiments performed in triplicate using cells obtained from three MSLpatients. *p < 0.01 vs. untreated cells



Discussion

In this study, we used cultured precursor adipocytesobtained from the lipomatous and non-lipomatous fatof ten MSL patients to study the molecular and func-tional features of MSL cells, and the effects of NA andNO on their gene expression, proliferation, and differ-entiation. The main findings demonstrate that MSLcells express specific molecular markers of brownadipocytes (including the mitochondrial inner mem-brane protein UCP-1) and that NA has no stimulatingeffect on their expression. Furthermore, the failure ofNA to induce NO production seems to be involved inthe dysregulated proliferation and differentiation (i.e.mitochondrial biogenesis) of MSL cells.

The presence of UCP-1 gene expression and its non-induction by NA seem to be most consistent with thehypothesis that MSL adipocytes are brown fat cellswith defective sympathetic signalling. Ultrastructuralanalysis of intact tissue and isolated preadipocytesshowed that unlike typical white adipocytes, MSLadipocytes are not monovacuolar but resemble thelargest adipocytes that can be found in rat and humanbrown fat [6]. Furthermore, it has been found that theyhave a defective lipolytic response to catecholamines,as measured by glycerol release in the medium [19].Interestingly, there was no significant difference inβ-adrenoceptor subtype mRNA levels between thecultured MSL cells and the control subcutaneousadipocytes, and no functional mutation in the β3-adrenoceptor [21]. It has also been reported that MSLcells have a normal lipolytic response to cAMP [19].The altered response of MSL adipocytes to NAcould therefore be due to an abnormal amount ordefective function of G-proteins, which couple surfaceβ-adrenoceptors to adenylyl cyclase, or the defectmay be in the catalytic subunit of adenylyl cyclaseitself. These hypotheses are supported by our findingthat NA does not induce UCP-1 gene expression inMSL cells. However, it is important to emphasizethe fact that unlike the control white subcutaneousadipocytes, the cells did express the UCP-1 marker ofbrown adipocytes. Kazumi et al. [32] failed to detectany UCP-1 mRNA in MSL lipomas, but this mayhave been because they used northern blots; our RT-PCR analysis is a more sensitive detector of poorlyexpressed UCP-1 mRNA, as has also been shown byVila et al. [33] in one MSL patient.

Unlike in typical brown adipocytes [12], NA didnot stimulate the expression of iNOS in MSL cells.The consequent defective NO production could beresponsible for the altered proliferation and differen-tiation of MSL adipocytes. This was strongly sug-gested by the fact that proliferation of immature MSLcells was markedly inhibited by chronic treatment withNO donor, while chronic treatment with the selectiveinhibitor of the NOS enzyme stimulated cell prolifera-tion. Our results also suggest that chronic GSNO treat-ment of cultured MSL adipocytes only weakly inhibitsthe mitochondrial conversion of MTT, although it

markedly slows cell proliferation. This may indicatethat mitochondrial biogenesis has already occurredand that the single cells are markedly differentiated.This was further confirmed by studying mtDNA lev-els in MSL cells after NO-donor treatment. Arrestedgrowth is a necessary step for cell differentiation [12]and in actively proliferating MSL cells (such as thosechronically treated with L-NAME), mitochondrial bio-genesis is blunted. It is important to note that NAdid not increase PGC-1 mRNA levels in MSL cells,as it does in brown adipocytes. Various lines of evi-dence identified PGC-1 as a key component of theregulatory pathway involved in controlling uncoupledmitochondrial respiration and thermogenesis. In rela-tion to the adipose lineage, PGC-1 expression makeswhite fat cells more like brown fat cells [16]. A cru-cial question that must be answered is whether theexpression of PGC-1 itself determines or helps todetermine whether the cells are white fat or brownfat cells. Given that albeit low levels of PGC-1 areexpressed in the MSL cells, they may be more sim-ilar to brown adipocytes, and ultrastructural exam-ination showed that a few of the MSL adipocytescontained typical brown adipocyte mitochondria. Onthe other hand, the lack of any stimulatory effectof NA on PGC-1 expression suggests a mechanis-tic molecular process for defective mitochondrial bio-genesis and therefore the defective differentiation ofMSL adipocytes.

The reasons for selective adipose tissue prolifera-tion remain largely unknown. Some authors have sug-gested that mtDNA mutation may induce anomalousnuclear gene expression leading to fat cell prolifer-ation [34]. Our results suggest that the presence ofmtDNA mutations causing mtDNA abnormalities inMSL adipocytes and systemic mitochondrial dysfunc-tion in MSL patients does not correlate significantlywith the investigated features of the disease. But itseems to be more likely that adipose growth dependson changes of intermediary metabolism. Brown fat hasa high oxidative mitochondrial metabolism coupledwith lipolysis, and lipid storage may be secondary tooxidative dysfunction [5] due to impaired adenosinetriphosphatase generation in MSL fat, which couldlead to reduced lipolysis and thus explain adiposegrowth [35].

The other genes expressed in MSL cells, such asleptin, UCP-2, UCP-3, and TNF-α, require furtheranalyses to correlate them with the disease, althoughthe lack of any marked changes in their mRNAlevels raises doubts as to their particular involvementin the pathogenesis of the disease. However, thedemonstration that TNF-α mRNA levels in lipomatoustissue are comparable to those seen in non-lipomatousfat is in line with the observation that MSL patientshave normal insulin sensitivity (Enzi, unpublishedresults), as adipose-derived TNF-α has been widelyinvolved in the pathophysiology of insulin-resistancein obese subjects [36]. Furthermore, circulating leptinlevels in MSL patients are similar to those observed


386 E Nisoli et al.

in normal weight subjects (Enzi, unpublished results)and our measurements of leptin mRNA levels in MSLlipomatous tissue seem to confirm this. Finally, noconclusive results have yet been obtained concerningthe physiological role(s) of UCP-2 and UCP-3 [37],and so the relevance of their gene expression in MSLcells remains to be elucidated.

New methodological approaches, such as DNAand/or protein arrays, will improve our understandingof the molecular basis of this lipodystrophic diseaseand any possible genetic predisposition to develop-ing it. As things stand now, MSL can be consid-ered an intriguing biological model for investigatingthe molecular mechanisms leading to other lipodys-trophic disorders.

Acknowledgements

We wish to thank Maura Digito for helpful technical assistance,Alessandra Valerio and Cristina Tonello for general discussion,and Paolo Mantegazza for encouragement and support. Thiswork was supported by a grant (E.0721) from TelethonFoundation (Italy).

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