Differential regulation of adipose tissue glucose transporters in genetic obesity (fatty rat) Selective increase in the adipose cell/muscle glucose transporter (GLUT 4) expression

Differential regulation of adipose tissue glucose

transporters in genetic obesity (fatty rat).

Selective increase in the adipose cell/muscle

glucose transporter (GLUT 4) expression.

I Hainault, … , C Guichard, M Lavau

J Clin Invest.

1991;

87(3)

:1127-1131.

https://doi.org/10.1172/JCI115077

.

Adipocytes from young obese Zucker rats exhibit a hyperresponsive insulin-mediated

glucose transport, together with a marked increase in cytochalasin B binding as compared

with lean rat adipocytes. Here, we examined in these cells the expression of two isoforms of

glucose transporter, the erythroid (GLUT 1) and the adipose cell/muscle (GLUT 4) types, in

rats aged 16 or 30 d, i.e., before and after the emergence of hyperinsulinemia. GLUT 1

protein and mRNA levels were identical in the two genotypes at both ages. In contrast, the

levels of GLUT 4 protein in obese rat adipocytes were 2.4- and 4.5-fold those of lean

littermates at 16 and 30 d of age, respectively, in perfect agreement with the genotype effect

on insulin-stimulated glucose transport activity. The levels of GLUT 4 mRNA per fat pad

were increased 2.3- and 6.2-fold in obese vs. lean rats 16- and 30-d-old, indicating a

pretranslational level of regulation. The obese phenotype was not associated with

overexpression of GLUT 4 mRNA in gastrocnemius muscle. This work indicates that the fa

gene exerts a differential control on the expression of GLUT 1 and GLUT 4 in adipose tissue

and provides evidence that independent of hyperinsulinemia, genotype is a major

regulatory factor of GLUT 4 expression in this tissue.

Research Article

Rapid Publication

Differential Regulation of Adipose Tissue

Glucose

Transporters

in

Genetic

Obesity (Fatty Rat)

Selective

Increasein the

Adipose

Cell/Muscle Glucose Transporter (GLUT 4)

Expression

1.Hainault,M.Guerre-Millo, C. Guichard, and M.Lavau

INSERM U 177, InstitutBiomedical desCordeliers, 75006 Paris, France

Abstract

Adipocytes from young obese Zuckerratsexhibit a hyperre-sponsive insulin-mediated glucose transport, together with a

marked increase incytochalasin B bindingascompared with lean ratadipocytes. Here, we examined in these cells the

ex-pression oftwoisoforms of glucose transporter, theerythroid (GLUT 1)and theadiposecell/muscle (GLUT4)types, inrats

aged 16or30d, i.e., before and after the emergence of hyperin-sulinemia. GLUT1proteinand mRNA levelswereidentical in thetwogenotypesatboth ages. In contrast, the levels of GLUT 4protein in obeseratadipocyteswere2.4- and4.5-fold those of lean littermatesat 16 and 30 d of age,respectively, inperfect agreement with the genotypeeffectoninsulin-stimulated

glu-cose transportactivity. The levelsofGLUT4 mRNA perfat padwereincreased 2.3- and 6.2-fold in obesevs.leanrats

16-and30-d-old, indicatinga pretranslationallevelofregulation. Theobese phenotypewasnotassociated with overexpression of GLUT4mRNA ingastrocnemiusmuscle. This workindicates that thefa geneexerts adifferentialcontrolontheexpression ofGLUT 1 and GLUT 4 inadipose tissue and provides evi-dence thatindependentofhyperinsulinemia,genotype isa

ma-jorregulatory factor of GLUT 4 expressionin this tissue. (J. Clin. Invest. 1991.

87:1127-1131.) Key

words:

glucose

trans-port *erythroid glucosetransporter

(GLUT 1)-

hyperinsuline-mia * muscle GLUT 4 *

suckling

Zuckerrat

Introduction

Adipocytes from younggenetically obese Zucker rats (fatty rats) exhibit, underphysiological concentrations ofinsulin, a severalfold increase in glucose transportactivityboth invitro and in

vivo,

ascomparedtofatcellsfrom theirleanlittermates (1, 2).Thisalteration could be responsiblefor thecompelling

Thiswork was presentedinpart atthe 1990 Annual Meeting of the

EuropeanAssociation fortheStudyof Diabetesin Copenhagen,

Den-mark, 10-13 September 1990andpublished inabstract form (1990.

Diabetologia. 33 Suppl.: A32).

Addresscorrespondenceto Dr. M.Lavau, INSERMU 177, Institut

BiomedicaldesCordeliers, 15ruedel'EcoledeMedecine,75006Paris,

France.

Receivedfor publication 30 July1990andinrevisedform 18 Oc-tober1990.

shuntingofglucoseintoadiposetissuetriglyceridesthat

charac-terizes the obese phenotype in the Zucker rat. Therefore, the

elucidation of theunderlyingmechanism of enhancedglucose transport activity in fatty rat adipocytes is of critical

impor-tance in the understanding of the biochemical lesion of this

genetic disorder.

Measurementsofglucose transporters bythecytochalasin

Bbinding method, have shown that the intracellularpool of

glucose transporterswasdramatically enlargedin those

adipo-cytes,allowingenhanced translocation in responsetoinsulin (1). Recent evidencesindicate that at least twoglucose

trans-porterspecies arepresentin adipocytes: the erythroid type (GLUT

1),'

anubiquitous protein which ispredominantly

ex-pressed inerythrocytes and brain(3-5), and the adipose

cell/

muscle type(GLUT 4), which is expressed exclusively in

tis-sues thatexhibitinsulin-dependent glucose transport: fat and

muscles (6-10). Differential regulation of these two glucose transportersin vivo has beendocumented recently in adipo-cytes;changes ininsulinemia levels, by streptozotocin and in-sulin treatment, induced parallel changes in GLUT4 protein andmRNA, whereas GLUT 1 expressionwasrelatively

unaf-fected(1 1-14). Likewise fasting and refeeding was shown to

specifically modulate the expression of the GLUT 4 gene (12, 13).

Wehaveaddressed here thequestion ofgenotype as a regula-tory factor in theexpressionofGLUT 1 and GLUT 4 at pro-tein and mRNA levels, in adipose tissue of Zucker rats. To

assessthe effectof thefattygenotypeindependently of the hy-perinsulinemia that characterizes postweaning obese Zucker rats, wehave compared the expression of these genes in 16-d-oldratsand30-d-old rats,i.e.,beforeandafter the emergence ofhyperinsulinemia(15). Our dataindicatethat thefatty geno-typeincreasesglucose transport through a selective pretransla-tional effect on GLUT 4 gene independent of hyperinsu-linemia.

Methods

Animals. Obese(fa/fa) Zucker rats andtheir lean (Fa/fa)littermates wereobtainedbybreeding heterozygous females and obese males. The

initial breeding pairswereobtained from Harriet G. Bird Memorial

Hospital, Stow, MA. Pupshad access to the dam's diet (normal rat

chow, UAR, Epinay/Orge, France)and wereseparated fromtheir mother at28 d ofage. For each experiment, two to six litters were

1.Abbreviations usedinthispaper:FA/fa,lean Zucker;rat,fa/fa,obese

Zuckerrat;GLUT1, erythroid-type glucosetransporter;GLUT 4,

adi-posecell/muscle-type glucosetransporter.

J.Clin.Invest.

© TheAmericanSocietyforClinicalInvestigation, Inc.

0021-9738/91/03/1127/05 $2.00

selected, with 10 pupsof both sexes in each. Male and female pups were usedundistinguished. Thegenotypes werediagnosed by plotting adi-posetissue weight against body weight in 16-d-old pups (16), andonthe basis of fat pad weight in 30-d-old rats.Ratswere killedbetween 09:00 and 1 1:00 h, bydecapitation.

Glucosetransport in isolatedadipocytes.For eachexperiment, sub-cutaneousinguinal adipose tissues,theonly fat depotstobedeveloped in young rats, were removedfrom several animals and pooled accord-ing to the rat genotype.Adipose cells were isolated by the collagenase methodof Rodbell(17)andglucose transportwasmeasured byusing6 14C glucose(50,uM),aspreviouslydescribed(18).Adiposecellsizewas

determined byamicrophotographicmethod(19).

Preparationofmembranes and Western blot analysis. Plasma and low-density microsomal membranes were prepared from isolated adi-pocyte homogenates, aspreviously described (1), except that protease inhibitors(0.1 mMPMSF and 25,g/mlpepstatin) were added to ho-mogeneization medium. Total membraneswereprepared by

centrifu-gationof isolatedadipocytehomogenatesat200,000g for 60 min. The proteinswereassayed by the Bio-Rad Laboratories(Richmond, CA) protein determination method.Membraneproteinsweresubjected to SDS-PAGEusinga 12%polyacrylamide resolvinggel and transferred

ontoImmobilonfilters(MilliporeCorp.,Bedford,MA). The blotswere

immunoblotted, using either an antibody against the COOH-terminal peptide ofGLUT 1 obtained from Dr. S.W.Cushman(20),or a

mono-clonalantibody (1F8)against GLUT4obtained fromDr. P. F.Pilch (6). Immunolabeled bandswerevisualized and countedaspreviously described (2 1).

RNA isolation and Northern blotanalysis.Total RNA was ex-tracted by theguanidiniumisothiocyanate/LiClmethodof Cathalaet

al.(22), either from totalinguinal adiposetissueorfrom thetwo cellu-larfractions of this tissue, thecollagenase-isolated adipocytes and the stroma-vascular cellswhichwererecoveredbycentrifugationof

adipo-cyte infranatants(1,000gm^Xtfor 5 min).In somerats, the skeletal

mus-clegastrocnemiuswasalsodissectedoutforRNA preparation.RNA

was electrophoresed on 1.2%formaldehyde agarose gels, blotted, and fixedontonylon filters(Hybond N+, Amersham, LesUllis,France).

Hybridizationswereperformedeither witha ratbrain glucose

trans-porter cDNA obtained from Dr. M. J. Birnbaum (4)orwith a rat

muscleglucose transporter cRNA probe obtained from Dr. M. J. Charron (8), under stringentconditions. Blotswerealsohybridized

withj3-actincDNA. The relativeamountof eachmRNAwas

quanti-fied byscanningdensitometry(Cliniscan). Results

Bodyweight was only slightly increased in 16-d-old obese Zuckerrats ascomparedtolean littermates (22.4±0.27vs.

20.4±0.30 g; P<0.01)but theweight oftheinguinal adipose tissue (two pads) was already markedly higher than in lean pups(0.31±0.02 vs.0.13±0.01 g; P< 0.01). At 30 dofage, bodyweightswerehigher infa/faratsthan in leanlittermates (68.5±2.1 vs. 56.4±5.2g; P<

0.01)

and the

inguinal

adipose tissuewasmassivelyhypertrophied (2.0±0.12vs.0.5±0.02 g;P <0.01).Theincreaseininguinal adipose tissuein themutant rats wasfullyaccountedforbyanincreaseinfatcellsize(see TableI).Ithas been observed inour(23)and other(24) labora-toriesthatduringsuckling, insulinemia levelsweresimilarin obese and lean pups.Thishasbeensubsequently confirmedin manystudiesbydifferentgroups.Thus,insulin levelswere not

systematically investigated here,andwe onlymeasured afew samplesthatconfirmedthesimilarity ofboth genotypes, with insulinemiain the rangeof0. 13to0.16 nM,at16 dofage.At

30 dofage, obese ratshavedevelopedafrank hyperinsuline-mia (0.74vs.0.28nM). At both ages, obeseratswere normogly-cemic,with plasma glucoseconcentrationsin the rangeof7.9

to8.6mMforleanrats vs. 8.4 to9.1 mMforobese rats.

Table I. Cell Size and Glucose Transport Activity ofAdipose Cells from Young Zucker Rats

Cell size 614C Glucose uptake

nglipid/cell nmol/10 cells permin

16 d old Basal Insulin (0.72 nM)

Lean 22±3 (6) 34±4 (4) 254±34(4)

Obese 41±3(6) 65±11(4) 538±63 (4) 30 d old

Lean 44±8(7) 54±7 (6) 882±16 (6)

Obese 184±8(7) 155±23 (6) 3824±374 (6)

Data areexpressed asmean±SEM,with thenumberofexperiments in parentheses. Each value in obeseratis significantly different (P<0.05)from the corresponding value inlean control (Student's

Itest).

The rates of glucose uptake in adipocytes from lean and obese Zucker rats arepresented in Table I. At 16 d of age, both basal and insulin-stimulated glucose transport rates were al-ready two-fold higher in adipocytes from obese rats than in those from their lean littermates. The difference between the two genotypesenlargedafterweaningand, at 30 d of age, the maximalactivity of glucosetransport, asassessed in the pres-enceofphysiological concentrations of insulin, was more than fourfold higher in adipocytes from obese rats than in those from lean rats. It is noteworthy that adipocytes from suckling pups exhibit a substantialresponsivenesstoinsulin. However, both the absolute and the relative effects of the hormone on glucose transportactivityaremuch lower insucklingthan in weanedanimals, ingood agreementwithourprevious observa-tionson

adipocytes

fromratsfeda

high-fat

diet (25).

To get insight intothe molecular mechanism underlying the increasedglucosetransportactivity of adipose cells from the obese rats, therelativeamountofGLUT 1 andGLUT4

proteinswasdeterminedinfat cell membranes.The two types ofantibodyused in thisstudyrecognized proteinsinthe range of45 to 50 kD. As shown in Fig. 1, the apparent molecular weights oftheseproteinswere unchangedby thefa/fa geno-type. In both lean and obese rats, the proteinenrichment in

GLUT 1 washigherinplasma than inintracellular

mem-16

days

30days

Glut4 _

GIu

ti1"

* _mo

L L Ob Ob L Ob L Ob L Ob TOT TOT PM LDM

Figure1. Immunologicaldetection of GLUT IandGLUT4in

adipocyte membranes from 16-or30-d-old lean and obese Zucker

rats.Membraneswerepreparedasdescribed inMethods.Each lane

wasloaded with 100 ggofproteinsof total membranes(TOT), plasmamembranes(PM),orlow-densitymicrosomes(LDM).

Proteinswereimmunoblotted withapolyclonal antibodyraised

againstthecarboxy-terminalpeptideoftheerythroid glucose

transporter(GLUT

1)

orwithamonoclonalantibody

specific

forthe

adipocyte/muscle-type glucosetransporter(GLUT 4).Theseblotsare

representativeofatleast threeexperiments.L,lean rat;

Ob,

obeserat.

-qqqmwp--branes, whereastheprotein enrichmentinGLUT4 washigher inthe latter thaninthe former. Therelative amount of GLUT

1 per milligramoftotalmembrane proteins was similarin the twogenotypesat 16 dof age(Fig. 1and TableII), anddecreased

in obese as compared to leanratsin total membranesaswellas

in plasma and microsomal membranes at30d ofage (Fig. 1

and Table III).Incontrast,therelative amounts of GLUT 4per milligram of protein were increased in obese as compared to lean rat adipose cells in all the membrane fractions examined (Fig. 1 andTables II and III) at both ages. On a per-cell basis, the level of GLUT 1 protein was roughly unchanged by the

genotype, whereas the level ofGLUT4 wasincreased in obese rat fat cells, by 2.4-fold and 4.5-fold at 16 and 30 d of age, respectively (TablesIIand III).Therefore,asshown inFig.2, the genotypeeffectonadipocyteglucose transportactivitywas

totally accounted for by changesinGLUT4proteinmassper cell, in sucklingas wellasinweaning rats. It is of interestto

notethat theincreaseinGLUT4protein(2.4-fold)washigher than the increaseinfat cell size(1.9-fold)at16 of age and that thedifferencebetween thesetwoeffectsnarrowedat30 dofage (4.5-foldvs.4.2-fold). This lag of cell size enlargement behind GLUT 4 protein increase is suggestive ofa cause and effect relationshipbetween theincrease inGLUT4and theincrease incell size.

We next addressed thequestion ofthe molecular mecha-nism of this differential regulation ofthe two transporter sub-typesby thefattygenotypebyexaminingthe levelsof GLUT1

andGLUT4 mRNAinadipose tissue.Boththe GLUT1 and GLUT4probes used inthis study hybridizedeachto asingle mRNAtranscript, thesize ofwhich (- 2.7-2.8 Kb) was unal-teredbythefattygenotype(datanotshown).Asshown in Ta-bles II andIII,GLUT 1 mRNA levels per gram of totalRNA

weresimilar (16 dofage)ordecreased(30d ofage)in obese versus lean rats. In sharp contrast, the levels of GLUT 4 mRNA permicrogramof total RNAwere

markedly

increased in theobese rats, 2.1-and 3.6-foldat 16 and 30 d of age,

respec-TableIII. Effect oftheFatty Genotype onthe Levels of GLUT1 and GLUT 4 Proteins and mRNAs in Adipose Tissue

of30-d-oldZucker Rats

Lean Obese

GLUT 1 Protein*per

Ag

proteint

7.2±1.2(4) 3.1±0.9

(4)§

106fatcells 386±46 (4) 445±137 (4)

mRNAII

per

ggRNA 7.0±0.9(7) 4.3±0.8

(7)§

fatpad 1637±176(6) 1556±301(7)

GLUT 4 Proteinper

ugprotein 40±3.8(6) 56±4.3

(6)f

106 fat cells 2012±123(6) 9146±2142(6)'

mRNA per

ggRNA 23±1.0(9) 84±17(9)'

fat_{pad 4859±203 (9) 30206±6001 (9)'}

Data areexpressedasmean±SEM, with the number ofexperiments

in parentheses. *cpm.*_{Total membraneproteins.§P<0.05vs.lean}

(Student'sttest). 1"Arbitrarydensitometric units.'_{P<0.01 vs.lean}

(Student'sttest).

tively. Hybridization of the same blots with an actin cDNA

probe showed minor changes between thetwogenotypes (data

not shown), supporting the specificity ofthe variations in GLUT 1 andGLUT4mRNA. When normalizedtothe total RNA contentofthefatpad, we observedasimilarcontentof GLUT 1 mRNA in the two genotypes, whereas GLUT 4

mRNAwas2.3-fold and 6.2-foldhigherin obese rats than in lean controls at 16 and 30 d of age, respectively (Tables II and

550r 16 day-old

TableII. Effect of theFattyGenotypeonthe Levelsof GLUTI

and GLUT4 ProteinsandmRNAs inAdipose Tissue

of16-d-old ZuckerRats

Lean Obew

GLUT 1 Protein*per

Mgproteint 3.4±0.4(3) 3.7±0.1 (3) 106fat cells 184± 13(3) 268±40 (3)

mRNA§per

MgRNA 6.4(2) 6.9±0.8(3)

fatpad 286(2) 338±40

(3)

GLUT4

Proteinper

ugprotein 19±3.2 (6) 35±2.4(6)" 106fatcells 983±123(5) 2408±308(6)"

mRNA per

zgRNA 26±3.7 (6) 54±10(6)'

fatpad 1096±148(6) 2567±607

(6)'

Data are expressed asmean±SEM,with thenumber ofexperiments

inparentheses.*cpm. $ Total membrane proteins.§Arbitrary

den-sitometricunits. P1p<0.01and ' P<0.05vs.lean(Student's t test).

30 day-uld

450

[

c

-' 350

0

250

IU

co

150

c

5

-50

-50-ST ProteIn RNA ST Protein aNA

Figure 2. Genotype effectoninsulin-stimulated glucosetransport

(GT), GLUT 1 andGLUT4proteinsperfatcell (m) andGLUT 1

andGLUT4mRNA perfat pad(mn), in Zuckerrats 16 or30d old. Results areexpressed inpercentincreasesoverlean ratvalues.Data arecompiledoutoftheresultspresentedinTable I(GT),and2 and 3(GLUTI andGLUT4proteinsand mRNA, in 16-and30-d-old rats,respectively). Withthe sameexpressionofthe data, the genotype

III). As evidenced in Fig. 2, the changes in GLUT 4 mRNA per total tissue between the two genotypes paralleled closely the changes in GLUT 4 protein per fat cell, suggestingthat the increase in GLUT 4 mRNA mainly occurred within the adipo-cyte fraction of the tissue. The observation that GLUT 4 mRNA was virtually undetectable in the stroma-vascular cell fraction ofthe tissue whatever the rat genotype (data not shown) supportedthis conclusion.

To investigate whether the overexpression of GLUT 4 mRNA was a generalfeature of insulin-responsive tissues in the obese rat,weexamined GLUT 4 mRNA levels in a skeletal muscle,thegastrocnemius.Incontrast to adipose tissue, there was no consistent effect of the genotype on GLUT 4 mRNA expression in this muscle (Fig. 3). Further investigation of GLUT4mRNAlevels in gastrocnemius muscle demonstrated thattherewas nosignificant difference between the two groups ofrats(genotype effect, in percent over lean values: -4±20, for seven

pairs

of rats).

Discussion

The genetic obesity of the fa/fa rat, first described by Zucker and Zucker (26), isdue to a singlerecessive autosomal gene mutation, as yet unidentified. This rat develops a syndrome whichshares manyfeatureswith humanobesity:fat cell hyper-trophy,hyperphagia, hyperinsulinemia, hyperlipemia,and nor-moglycemia. This experimentalmodelof obesitymayprovide

auseful model for thehumandiseasewheregeneticfactorsplay

amajorrole(27).

Previouswork hasshown thatadipocytes fromyoung obese Zuckerratsexhibitahyperresponsive insulin-mediatedglucose transporttogether withamarked increase in glucose

transport-ers asassessedby

using cytochalasin

Bbinding (1).The purpose of this workwastoelucidatethemolecularbasis of this alter-ation, more

specifically

toaddress thequestion of which

glu-cosetransporterisoformwasinvolved.The present data show that thegenotype-linked increaseininsulin-stimulatedglucose transport activity is paralleled byacorresponding increase in the cell amountofGLUT 4protein,whereas the abundance of GLUT 1 proteinisunchanged. Furthermore, GLUT4mRNA

WAT

Muscle

* *

*

L

Ob

L Ob

Figure 3. Northern-blot analysisofGLUT 4 mRNAfromadipose

tissueandmuscle from Zuckerrats30dold. RNAwasextracted frominguinal fat pad (WA T)orfrom thegastrocnemiusmuscle

(Muscle)of thesame rats. Northernanalysiswasperformedas

described in Methods. 20,gof totalRNAwereloaded in eachlane

and blotswerehybridizedwithacRNAprobespecificfor theadipose cell/muscle-type glucosetransporter(GLUT 4).These blotsarefrom twodifferent experiments. L, lean rat; Ob, obese rat.

levels per fat pad are increased proportionately to changes in GLUT 4 protein, whereas GLUT 1 mRNA is not affected by the fatty genotype. All together our data clearly show that the two adipocyte glucose transporter species are subject to differ-ential regulation in the fatty rat model.

A similar pattern of regulation, in which an increase in GLUT 4 expression is associated with unchanged levels of GLUT 1 expression, has been previously reported in adipo-cytes of rats whose insulinemia levels were increased (insulin treatment of streptozotocin diabetes or fasting-refeeding, 11-14), suggesting that insulin could be a major regulatory factor in determining GLUT4gene expression. Our observation that GLUT 4 is overexpressed in 1 6-d-old normoinsulinemic suck-ling obese pups conclusively establishes that the fa gene regu-lates GLUT 4 expression independent of hyperinsulinemia. This is the first demonstration that GLUT 4 expression in adi-pose tissue is under genotype control. The finding that the lev-els of GLUT 4 mRNA are unchanged in obese rat gastrocen-mius muscle discloses the tissue specificity of this genotypic regulation.

We questioned whether the amplification of the genotype effect observed after weaning could be ascribed to insulin. How-ever, recently, several sets of observations have made the role of hyperinsulinemia in chronic regulation of adipocyte GLUT 4 expression unclear. It has been shown that, in chronic insulin treatment of streptozotocin-diabetic rats, GLUT 4 mRNA lev-els which are initially elevated twofold above control rats in adipose tissue, decline to normal steady-state level within 7 d of insulin therapy, and then remain constant (28). Hyperinsuline-mic 5-wk-old db/db Hyperinsuline-mice do not exhibit any alteration in GLUT 4 gene expression in adipose tissue (29). Finally chronic insulin treatment of either 3T3 Ll (30) or 3T3 F442A (21) cultured adipose cells has no significant effect on GLUT 4 ex-pression.

The overexpression of GLUT 4 found here in adipocytes of Zucker mutant pups could play a determinant role in the in-duction of fat cell hypertrophy, an early phenotypic trait that develops before the onset of hyperphagia and hyperinsulinemia (31). The abundance of GLUT 4 protein would entail a hy-perresponsive glucose transport activity to insulin, providing large supplies of lipid synthesis precursors. In addition, through an increased level of glycolytic intermediates, the overexpres-sion of GLUT 4 might be responsible for the induction of the array of enzymes, such as lipoprotein lipase, fatty acid synthe-tase, and malic enzyme, that has been observed in adipocytes of very young obese pups (23, 31) because those enzymes have been reported to be under the regulation of specific interme-diary metabolites (32-34). Thus, GLUT 4 overexpression would act as a "metabolic pull" channeling substrates into adi-pose tissue triglycerides whatever the food intake levels, a prom-inent metabolic feature of the obese Zucker rat (35-37).

Wewishtothank Drs.S. W.Cushman,P. F.Pilch,M.J.Charron, and

M.J.Birnbaumfor theirkind gift of probes.

Thiswork wassupportedbya grantfrom theFondationpourla RechercheMedicale.

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