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Glycemic improvement in diabetic db/db mice

by overexpression of the human

insulin-regulatable glucose transporter (GLUT4).

E M Gibbs, … , A J Milici, J D McNeish

J Clin Invest.

1995;

95(4)

:1512-1518.

https://doi.org/10.1172/JCI117823

.

The effects of increased GLUT4 (insulin-regulatable muscle/fat glucose transporter)

expression on glucose homeostasis in a genetic model of non-insulin-dependent diabetes

mellitus were determined by expressing a human GLUT4 transgene (hGLUT4) in diabetic

C57BL/KsJ-db/db mice. A genomic hGLUT4 construct was microinjected directly into

pronuclear murine embryos of db/+ matings to maintain the inbred background. Four lines of

hGLUT4 transgenic mice were bred to homozygosity at the db locus and all showed a

marked reduction of both fasted and fed plasma glucose levels (to approximately 50 and

360 mg/dl, respectively) compared with age-matched nontransgenic db/db mice

(approximately 215 and 550 mg/dl, respectively), as well as an enhanced disposal of an

oral glucose challenge. In situ immunocytochemical localization of GLUT4 protein in muscle

from hGLUT4 db/db mice showed elevated plasma membrane-associated GLUT4 protein in

the basal state, which markedly increased after an insulin/glucose injection. In contrast,

nontransgenic db/db mice had low levels of plasma membrane-associated GLUT4 protein

in the basal state with a relatively small increase after an insulin/glucose challenge. Since

the intracellular GLUT4 levels in db/db mice were similar to nontransgenic db/+ mice, the

glucose transport defect in db/db mice is at the level of glucose transporter translocation.

Together, these data demonstrate that GLUT4 upregulation overcomes the glucose

transporter translocation defect and alleviates insulin resistance in genetically diabetic

mice, thus resulting in […]

Research Article

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(2)

Glycemic

Improvement in Diabetic dbldb Mice by Overexpression

of the

Human Insulin-regulatable

Glucose

Transporter

(GLUT4)

E. Michael Gibbs,*JeffreyL. Stock,* Scott C. McCoid,* Hans A.Stukenbrok, Jeffrey E. Pessin,IRalph W.Stevenson,* A. J.

Milici,*

and John D. McNeish*

Departments of *MetabolicDiseases, tMolecularGenetics, and§Cancer, Immunology andInfectiousDiseases, CentralResearch

Division, Pfizer, Inc., Groton, Connecticut 06340; andIDepartmentof Physiologyand Biophysics, TheUniversity ofIowa,

IowaCity, Iowa52242

Abstract

Introduction

The effects of increased GLUT4 (insulin-regulatable mus-cle/fat glucose transporter) expression on glucose homeo-stasis in a genetic model of non-insulin-dependent diabetes mellitus were determined by expressing a human GLUT4 transgene (hGLUT4) in diabetic C57BL/KsJ-db/db mice.

A genomic hGLUT4 construct was microinjected directly

into pronuclear murine embryos of dbl + matings to main-tain the inbred background. Four lines of hGLUT4 transgenic mice were bred to

homozygosity

at the db locus and all showed a marked reduction of both fasted and fed

plasma glucose

levels

(to

50 and 360

mg/dl,

respectively)

compared with age-matched nontransgenic db/db mice

(- 215 and 550mg/dl,respectively), as well as an enhanced

disposalofanoral glucose challenge. In situ immunocyto-chemical localization of GLUT4 protein in muscle from

hGLUT4 db/dbmice showed elevated plasma

membrane-associated

GLUT4

protein in the basalstate, which

mark-edlyincreasedafteraninsulin/glucose injection.Incontrast,

nontransgenic db/db mice had low levels ofplasma mem-brane-associated GLUT4 proteinin the basal statewitha

relatively small increaseafteraninsulin/glucosechallenge. Since the intracellular GLUT4 levels in db/db mice were similar to nontransgenicdb/+ mice, the glucose transport

defectin db/db mice is atthe level of glucose transporter

translocation. Together, these data demonstrate that

GLUT4 upregulation overcomes the glucose transporter

translocation defect andalleviates insulin resistance in

ge-netically diabeticmice, thusresultinginmarkedly improved glycemiccontrol.(J. Clin. Invest. 1995.95:1512-1518.) Key

words: non-insulin-dependent diabetes mellitus *

genetic

models * basalmetabolism * geneexpression *

upregulation

Non-insulin-dependentdiabetes mellitus

(NIDDM)'

is a meta-bolic disease that affects -5% of the population of the Western world. NIDDM is characterized by hyperglycemia in both the fed and fasted states and is predominantly associated with obe-sity. The disease is manifested by defects in both insulin secre-tion from the pancreas and insulin action in peripheral target tissues(i.e., liver, skeletal muscle, and adipose tissue). Skeletal muscle is the major site of post-prandial peripheral glucose disposal, and muscle from NIDDM patients displays markedly impaired glucose uptake in response to insulin, although the precise nature of the defect is unknown (1-3). The defect in glucose transport cannot be explained by a simple reduction in the level of gene expression of the major muscle glucose transporter isoform

(GLUT4),

aslittleor noreduction in skele-talmuscle GLUT4contenthas been found in NIDDMpatients

(3-6). Several lines of evidence suggest that elevated glucose levels directly affect insulin sensitivity in target tissues and contributetothedire long-term diabeticcomplicationssuchas

cardiovascular disease, retinopathy, neuropathy, and nephropa-thy (7, 8).

db/db mice are agenetic model ofNIDDM that display

many ofthecharacteristics of thehumandisease including hy-perglycemia, insulin resistance, and obesity (9-11 ). Importan-tly, asin humanNIDDM,db/dbmice haveamarked decrease in skeletal muscle glucose utilization due to amajor defect in glucose transport that isnot accompanied bysignificant alter-ations in GLUT4expression( 12, 13).Inthisstudy,thepossible beneficial effects of GLUT4 overexpression in this murine NIDDM modelweretestedby producing transgenic mice that expressa human GLUT4 transgene (14). These data indicate that GLUT4 upregulation in this genetic model of NIDDM reducedhyperglycemiaand resulted inimproved glycemic

con-trol.

Methods

Portions of this workwerepresentedasabstractsatthe1994Keystone SymposiaonMolecular Mechanisms CommontoTypesIandII Diabe-tes, Park City,UT on20January 1994 andatthe AnnualMeetingof

theAmericanDiabetesAssociation,NewOrleans,LAon14 June 1994. Address correspondence to Dr. E. Michael Gibbs, Department of MetabolicDiseases,Central ResearchDivision,PfizerInc.,Groton,CT 06340. Phone:203-441-4140;FAX:203-441-5719.

Receivedfor publication 18 August 1994 and in revisedform 9

December1994.

Transgenicmice. Pronucleartransgenic micewereproduced by estab-lishedprotocols (15, 16), however, embryos were obtained from the

inbred, mutant mousestrain,C57BL/KsJ-m+/+db (Jackson

Labora-tories,BarHarbor, ME),in which the recessivediabeticmutation,db,

is linked in repulsionto the recessive coatcolormutation, misty, m

(17). Thelinking of db andm mutations inrepulsionallows for the

identification of offspring genotypically wild-type (+/+) atthe db locusbythe presenceoftherecessivemisty (m/m)coatcolor(17).An

1. Abbreviations used in this paper: db, diabetes; GLUT4,

insulin-regulatable muscle/fatglucosetransporter; hGLUT4, humanGLUT4;

m,misty; NIDDM,non-insulin-dependentdiabetesmellitus;nt,

nucleo-tide. J. Clin.Invest.

©TheAmericanSocietyforClinicalInvestigation,Inc.

(3)

11.8-kb (NotIfragment) human genomic GLUT4transgeneincluding

5.3 kb of5'flanking sequenceandanSV40polyadenylationsequence (14) wasmicroinjectedat - 2.0

jLg/ml

intopronuclei of embryos

de-rived frommatings of C57BL/KsJ-m+/+db males and superovulated

females. 400injected embryosweretransferredtopseudopregnant CD-1 females. Of CD-135offspring, 13founder transgenic micewereidentified

by thepolymerasechainreaction(PCR) of total genomic DNA (datanot shown). Weobservedtwofounders withtherecessive, obesephenotype

(+db/+db)andtwofounderswith therecessivemistycoatcolor(m+ /

m+). The remaining nine founderswere m+/+db with blackcoatand lean phenotype. Independent lines were established by backcrossing

leanfoundermicewith C57BL/KsJ-m+/+dbmates.

RNaseprotection assay. Total RNAwasisolated from heart tissue

usingRNAzol'B(BIOTECX,Inc.Houston, TX) following the

manu-facturer's directions. The human-specific GLUT4 probe, PJW3 (14),

was linearized with EcoRl and its T3 promoterwasusedtogeneratea

[32P1UTP-labeled antisense probe.The RNA from each sample was

hybridized for 16 hat550C,treated with RNaseA,andelectrophoresed

on a6%polyacrylamide/7.5M ureagel.

Immunoblotting. Postnuclear membrane fractions were prepared

from frozen hearts and adipose tissue as described previously (18).

Samples weresolubilizedinLaemmliloading buffer,andproteins (50

,ugprotein)wereseparatedon10%reducing

SDS-polyacrylamide

gels

and then transferredtonitrocellulosemembranes. The membraneswere

incubated withaffinity-purifiedrabbit anti-GLUT4polyclonalantibodies (19) overnight at 4°C. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL; Amersham Corp., Arlington

Heights, IL)accordingtothemanufacturer'sinstructions. Bands were quantitated using a MasterScan interpretivedensitometer(Scanalytics,

aDivisionof CSPI, San Diego,CA).

Oralglucosetolerancetestand metabolite measurements. Nonanes-thetizedGLUT4transgenic (db/+,anddb/db)andage-matched

non-transgenic mice were fasted overnight and bled via the orbital sinus

(0.025 ml) immediatelybefore administration ofanoralglucose load

(1gglucose per kg of body wt) by gavage using a syringe equipped with a murine oral feeding needle(20gauge; Popper & Sons, Inc., New Hyde Park, NY).Mice weresubsequently bled after 30, 75, and 120 min. Plasmaglucoselevels were measuredusingthe VPSuperSystem Autoanalyzer(AbbottLaboratories, North Chicago, IL). Plasma insulin

wasdeterminedusingaradioimmunoassay (Binax,Portland, ME) with porcine insulin asstandard.

Immunofluorescence.Nontransgenic and GLUT4 transgenicdb/db

mice were either fastedovernightand left untreated or allowed to eat adlibitum and thengiven anintraperitonealinjection of glucose (1g/ kg) and insulin (8 U/kg) aspreviouslydescribed (20, 21). 30 min after theglucose/insulin challenge,micewereanesthetized and immediately

perfused throughtheleft ventricle with 25 ml of 4% formaldehyde plus 0.2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, at 23°C. After fixation in situ, cardiac and quadriceps muscle and pancreas wereremoved, fixed by immersion for 1 h, and processed for frozen

sectioningaspreviouslydescribed(22). Briefly,

0.5-jim

frozen sections wereincubated at 23°C with 4.8

jig/ml

rabbit anti-human GLUT4 IgG

(19)for 1 h followed by incubation with 5

jig/ml

donkey anti-rabbit

IgG conjugatedto Texas red(Jackson Laboratories). Optimal exposure timewas determined fortheinsulin/glucose-treated transgenic mice, and all other conditionswerephotographed with an identical exposure timeon aNikon FXA microscope.

Results and Discussion

Therelative expression ofhuman GLUT4 (hGLUT4) in eight

lines of

Fl

transgenic micewasdetermined by RNase protection

of total RNA from cardiac tissue (Fig. 1 A). Tissue-specific

expression of hGLUT4 RNA was examined by reverse

tran-scriptase-PCR of purified total RNA from four of the lines expressing highhGLUT4 levels. In additiontocardiac muscle,

appropriate expression (23, 24) of the

hGLUT4

transgene was detectedinhindquarter skeletal muscle, perirenal brown adipose

tissue, and white adipose tissue (epididymal and subcutaneous

for male and female mice, respectively; data not shown).We

also detected the hGLUT4 transgeneRNA intotalbrain homog-enates from the four lines analyzed, which is consistent with

the recent observation that GLUT4 is also expressed at low

levels in the brain (25,26).Asexpected,the hGLUT4 transgene RNAwas not detected in liver, kidney,spleen,orpancreas from hGLUT4 lines5,8,or12, however, thehighest

expressing

line (hGLUT4 line 6; Fig. 1 A) showedinappropriate expressionin thekidney and liver,presumablyduetochromosomal

position

effects of the transgene (27). With theexception of hGLUT4 transgeneexpressioninthebrain,the present resultsare

consis-tentwith earlier observations in hGLUT4

transgenic

mice pro-duced on a nondiabetic hybrid background (14) and confirm thatthehGLUT4 transgene isexpressedinparalleltothe endog-enous murine GLUT4 gene. Densitometric analysisof immu-noblots indicated that GLUT4 proteinwas overexpressed four-to sixfold in cardiac muscle and approximately threefold in white adipose tissue in hGLUT4db/db mice compared with nontransgenicdb/dbmice (Fig. 1,BandC).

The effect of GLUT4 overexpression on body weight of

db/

+ and

db/db

mice is shown in Fig. 2. Body weights of hGLUT4transgenic db/+ mice of bothsexesweresimilarto orslightly less than fornontransgenicdb/+ mice upto40 wk of age, which is consistent with our earlier observations in hGLUT4 transgenic mice produced on a hybrid background

(21). Body weights of both female (Fig. 2 A) and male (Fig. 2 B) hGLUT4 transgenic db/db mice were comparable with nontransgenicdb/dbmice up to - 10wk of age. However, the nontransgenic db/dbmice stopped gaining weightatthis age, and in fact slowly lost weight over the remaining course of theirlives, consistent with progression of the diabetic phenotype (10). In marked contrast, thehGLUT4db/db transgenic mice continuedtogain weight until - 15 wk of age and then

main-tained this weight until at least 35 wk of age. Since overex-pression ofGLUT4in muscle and fat is not a direct cause of obesityasevidenced by the results in hGLUT4db/+transgenic mice (Fig. 2) and hGLUT4 transgenic hybrid mice (14, 21), it islikely that GLUT4 overexpression in severely

insulin-resis-tantdb/dbmutantmice at least partially overcomes the insulin resistance and thus enhances the animals' ability to thrive.

Theimproved weight gain of the

hGLUT4db/db

transgenic mice was in direct contrast with a previous study in which GLUT4 was selectively overexpressed in adipose tissue using the aP2promoter in a different genetic mouse background (28). Although there was a marked increase in adiposity in these animals, this didnotoccurwhen

GLUT4

was overexpressed in both muscle and adipose tissue using the GLUT4 promoter (21). Thus, we hypothesize that either the overexpression of GLUT4 in both muscle and adipose tissue prevents fat cell hyperplasia or that these differences in adiposity arise due to the use of an early developmental aP2 promoter (29) versus thelate developmental GLUT4 promoter (30).

In any case, we next determined the functional expression of the hGLUT4 transgene on glucose

homeostasis

in vivo by assessingthephysiological responses of both nondiabetic

(db/

+) and diabetic

(db/db)

mice to an oral glucose challenge

(4)

overex-B

GLUT4

---,

A

NTG LINE8

I

-I

--

I

1 2 3 4 5 6 7 8 9 10 11

ADIPOSE

TISSUE

i

-4-250 nt

GLUT4

HEART

4000

2000

-'A__, __ HEART MALE

Figure 1.Expression of hGLUT4 in transgenicdb/dbmice.(A) RNase protection assay of hGLUT4 lines. 15 ug of heart total RNA was probed with5 x 105cpm of the humanGLUT4 specific probe, pJW3, to determine

hGLUT4mRNAexpressionasdescribed in Methods. Lanes1,2, 3, 4, 5, 7,

8, and 9correspondtoindividual mice (7-15 wk of age) from hGLUT4 lines4, 3, 7, 5, 6,12, 8, and11,respectively. Lane 6 is a nontransgenic negative control mouse,whilelane 10is apositivecontrolmouse from the

previously characterized hGLUT4Cline (14,21,31). Thetransgenic GLUT4 protectedfragmentis 241 nucleotides (nt) and migrates just ahead of a 250-ntsize marker(lane11).Thedriedgel was exposed to x-ray film at-700C

with anintensifying screenfor3 d.(B)Immunoblotanalysis of hGLUT4

transgenicline 8.50

/sg

of adiposetissue (top) orcardiacmuscle(bottom)

postnuclearmembraneproteinwasanalyzed for GLUT4 proteincontentusing

anti-GLUT4antibodies as described in Methods.NTG indicatesthree samples from femaledb/dbnontransgenic mice, andLINE8indicates threesamples

from femalehGLUT4 line 8db/dbtransgenic mice. (C)Relativeexpression of GLUT4protein.GLUT4bands fromtheimmunoblots of female mice

shown in B and also from immunoblots ofcardiacmuscle of male

non-transgenic(n=3)andhGLUT4db/db transgenic(n=5)mice were

quanti-tated

by

densitometric

analysis. Lightly

shaded barsrepresent

nontransgenic

mice, and hatched barsrepresent

transgenic

mice. All mice

analyzed

were

between9and 12 wkofage. Significant differences from sex-matched non-HEARTFEMALE ADIPOSE FEMALE transgenic miceareindicated, *P<0.05, * P< 0.01,and P= 0.07.

pressinglinesdisplayedreducedfasting plasma glucose, which

remained below 100mg/dlafter theglucoseload.One hGLUT4

transgenic line did not have reduced fasting plasma glucose comparedwith thenontransgenic mice, but showedessentially

noincrease inplasma glucoseafter theglucoseload(hGLUT4

line8; Fig.3A).The male mice used in theexperimentshown

inFig. 3 Arangedfrom 7to 16 wk ofage;similarresultswere

obtained in female mice and also inoneline of hGLUT4 dbl

+ mice (line 8) that was tested at6 and 90 wk ofage (data

notshown). Further, as was observed in ourprevious studies

(21, 31), functional expression of the GLUT4 transgene in

skeletal andcardiac musclewasconfirmedbyatwo- to fourfold

increase in glycogen content in hGLUT4 transgenic mice

(Gibbs, E. M., S. C. McCoid, W. J. Zavadoski, J. L. Stock,

J. D. McNeish, and R. W. Stevenson, unpublished

observa-tions).Thus, highlevelexpressionof hGLUT4proteinin

nondi-abetic, db/+ heterozygotes produced a phenotype similar to

GLUT4 transgenic mice on anondiabetic hybridbackground (21, 31),and the effect of thetransgene was maintainedover

thecourseof the animals' lives.

Nontransgenic db/db mice (8-9 wk of age) had fasting hyperglycemia (- 250mg/dl) that markedlyincreased up to

- 600-700mg/dl, after theglucosechallenge (Fig.3B). Re-markably, age-matched db/db mice expressing the hGLUT4

-

AS w.u

_ .

_~~~Io

C

z

Z_A 0

*-uc

O

:>n

(5)

1 0 20 30 40

AGE (weeks)

B

60

50

a

0

m

40

30

-

20-0 10 20 30

AGE (weeks)

transgene (line 12)hadfasting plasma glucoselevels of - 50 mg/dl, which typically remained below 100mg/dl even after

the oral glucose challenge (Fig. 3B; similar results were

ob-tained for three other hGLUT4lines,notshown).

As expected, fedplasmainsulin levels werereduced in

fe-male transgenic dbl+ mice compared with female

non-Figure 2. Body weightversusagein

non-transgenic andtransgenic hGLUT4db/+ and

db/db mice.Body weightsof female(A)and male(B) dbl+ (squares) and db/db (cir-cles) nontransgenic (open symbols) and hGLUT4 transgenic (closed symbols)mice

arepresentedasmeans±SEM(n = 3-15,

withamediann=5). Most data points

rep-resentmeansobtained fromsingle

measure-mentsof individual mice.However,because of thelimiting numberof hGLUT4db/db

transgenicmice thatwasavailable,a sub-groupofthisphenotypewassetaside for this

40 study (n= 22and 17, for female and male

mice, respectively)andwasreweighedat 2-wkintervalscovering the 15-35 wkage range.

transgenic dbl+ mice (28.3±2.5 vs 39.5±3.4 1U/ml; P

< 0.025; micewere6-11 wk ofage) and in maletransgenic

micedbl+comparedwithnontransgenic dbl+mice(37.1 +3.1

vs65.5±9.3 MzU/ml;P < 0.005;micewere6-10 wk ofage).

Theinsulin-lowering effect in thetransgenic micepresumably

occursin responsetothehypoglycemia produced byincreased

A

60

-50

-

40-Im

I--z

a

0 0

30

-

(6)

A

-I

_E

0

0 -I

8.

C

IL

600

-

400-

200-0 30 60 90 120

TIME (min)

0 30 60 90 120

TIME (min)

Figure 3. Effect ofanoralglucose challengeonplasmaglucose levels in hGLUT4 transgenic mice. Micewerefastedovernightand bled via the

orbital sinus(0.025 ml) immediately before administrationofanoral glucose load (1 gglucose/kg body wt) andat30, 75, and 120minthereafter.

(A) Oral glucose tolerance in male nontransgenic and hGLUT4transgenicdbl+ mice. Valuesareexpressedas meansobtained from the analysis

ofn= Sdbl+ (open circles),n= 4hGLUT4db/+ line 5 (closedcircles),n =4hGLUT4db/+ line 6 (closed squares),n = 3hGLUT4db/

+ line 8 (closed triangles), andn= 3hGLUIT4db/+ line 12 (open triangles). Error barsareomitted for clarity. Plasma glucosevaluesfor

transgenic mice significantly different from nontransgenic miceare asfollows: all 4 lineswereloweratboth the 75- and 120-mintime points(P

< 0.005), lines 8 and 12wereloweratthe 30-min time point(P< 0.05),andlines 5,6,and 12 had lower fasting plasma glucose (P<0.025).

(B) Oral glucose tolerance in nontransgenic and hGLUT4 transgenicdb/dbmice.Valuesareexpressedasmeans+SEMobtained from the analysis

ofn= Sdbl+ (squares),n= Sdb/db (triangles), andn=3 hGLUT4db/dbline 12(circles)male(closedsymbols) and female (open symbols)

mice, respectively. All glucose valuesweresignificantly lower in the transgenic mice compared with both db/db mice(P< 0.005)exceptfor the

120-min timepoint for the female mice (P< 0.025), and with dbl+ mice (P< 0.05).

glucose disposal. Surprisingly, fed plasma insulin levels in hGLUT4db/db transgenicmaleand female mice were signifi-cantly higherthan nontransgenic db/dbmice,whereas fasting

plasma insulin levels werelower-in transgenic db/db mice of bothsexes (Table I). The increased fedplasmainsulin levels in thetransgenicdb/db micelikely result froma(partial)

pro-Table I. ComparisonofPlasma Glucose and Insulin Levels betweenNontransgenic db/db and hGLUT4 Transgenic

db/dbMice

Male Female

hGLUT4 hGLUT4 dbldb dbldb dbldb dbldb

Fedglucose (mg/dl) 537±18 363±44* 566±30 352±40*

(n) (22) (12) (16) (14)

Fasting glucose (mg/dl) 246±19 60±10* 185±17 36±5*

Wn (1) (6) (I11) (6)

Fedinsulin

(ILU/ml)

118±8 263±38* 80±6 174±40*

(n) (29) (19) (25) (12)

Fastinginsulin

(tU/ml)

101±7 69±9t 109±7 81±5t

(n) (10) (9) (15) (14)

Datafromtransgenic mice between7 and11 wkofage fromhGLUT4

lines5, 6, 8, and 12werepooled fortheseanalyses. Thevalue in

parenthesesindicates the number of animals used for eachanalysis.

Significantdifferences fromage-and sex-matchednontransgenicmice areindicated,*P< 0.005, *P< 0.0005.

tection from the glucose-dependent pancreatic atrophy that oc-curs in nontransgenic db/db mice as they age (9-11, 32).

Alongthisline,wehaveobserved that the isletsurfaceareain

paraffin sectionsofpancreatictissue isapproximately threefold

greater in hGLUT4db/db mice compared with age-matched (- 30 wk) nontransgenic db/db mice (Milici, A. J., D. N.

Scampoli, S. C. McCoid, J. L. Stock, J. D. McNeish, R. W. Stevenson, and E. M. Gibbs,unpublished observations). Since

inappropriate GLUT4expressionin,B cellsmightbeexpected

to alter insulinsecretion, itwas importantto demonstrate that thisdidnotoccur.hGLUT4 RNAwasnotdetected inpancreatic

RNAusing the sensitive reversetranscriptase-PCR

methodol-ogy. In addition, no difference in GLUT4expression was ob-served in frozen sections ofpancreatic tissue from transgenic

micecomparedwithnontransgenicmiceasassessedby

immu-nofluorescence (data not shown). Taken together, these data suggestthatexpression ofthehGLUT4 transgenein an

appro-priate tissue-specific manner in db/db mice results in more

functionalpancreatic cells thatcanefficientlyrespondtothe

partiallyelevated fedplasma glucoselevels thannontransgenic

db/db mice. Thesedata suggest thatlowering plasma glucose byincreased GLUT4expressionreducesstressonthepancreas

and maintains its functional integrity, thus preventing the

mice from progressing into the late hypoinsulinemic stage of

NIDDM.

To assess GLUT4 subcellular localization in vivo under

fasting and maximally insulin-stimulated states (20, 21), we

examined the cardiacmyocytedistribution oftheGLUT4

pro-teinbyimmunofluorescence usingaGLUT4specific antibody

(19) coupledwithaTexasred-conjugated secondary antibody

140-10

0 E

UA

co

0 0.

120

100

80

60

-40

-20

(7)

Figure 4. Immunofluorescentlocalization of cardiac myocyte GLUT4 protein expression in nontransgenic and transgenic hGLUT4db/dbmice. Female nontransgenic (A and C) andhGLUT4 line 8 transgenic (B and D)db/dbmice (9 wk of age)were either fasted overnight and left untreated (A and B) or allowed to feed adlibitum and given an intraperitoneal injection of insulin (8 U/kg) and glucose(1 g/kg) (C and D). Arrowheads mark several locations of punctatecytoplasmic staining (A and B) in myocytes from the fasted animals, and arrows mark isolated myocytes in which the entire membrane was labeled in a fasted transgenic animal (B). Arrows mark locations of membrane-associated GLUT4 (C and D) in myocytes after insulin/glucosetreatment. Insulin induced a very patchy translocation of GLUT4 in the nontransgenic mice (C), whereas in the transgenic mice (D) there was anintense uniform labeling of the entire myocyte plasmalemma. Essentially identical results were also obtained fromdb/dbhGLUT4 line 12transgenic mice (8 wk of age; data not shown). No significant fluorescent signal could be detected in any tissue sections from eithernontransgenic ortransgenic mice labeled with equal concentrations of nonimmune rabbit IgG (data not shown). x900.

(Fig. 4). Cardiac muscle tissue

from

fasted nontransgenicdb/

db mice exhibitedapunctate cytoplasmicGLUT4 immunoflu-orescence(Fig. 4 A). Both thelevel of GLUT4

immunofluo-rescence and localizationin the basalstate weresimilarto our

previous observations in normal mice (21). With insulin and glucosetreatment,nontransgenicdb/db mice showedaminimal redistribution of GLUT4 immunofluorescence from the

con-densed cytoplasmic vesicles to the plasmalemma (Fig. 4 C). This contrasts with our previous data indicating substantial GLUT4 translocationin nondiabetic mice (21) andis adirect demonstration that one manifestation of the insulin resistance

in the db/db mice is aGLUT4 translocation defect. Overex-pression of GLUT4 protein in vivo resultedin aspecific immu-nofluorescent signal thatwasreadily detected inboththe plasma membrane and intracellular vesicles from cardiac myocytes of

the fastednon-insulin-treated hGLUT4db/db line8transgenic

mice (Fig. 4 B). Furthermore, GLUT4 db/db transgenic mice

treatedwith insulinandglucosedemonstrated amarked increase in cell surface GLUT4 proteincontent with anaccompanying decrease in the punctate intracellular GLUT4

immunofluores-cence(Fig.4 D).Qualitatively andquantitatively similar immu-nofluorescent data were obtained in quadriceps muscles ob-tained from thesamemice usedintheseexperiments (datanot

shown), which indicates that the cardiac myocyte analysis is representative ofGLUT4translocationin skeletal muscle.

Quantitative data obtainedby imageanalysis of cardiac my-ocytes from nontransgenic db/db mice demonstrated only a

1.2-fold increaseinplasmalemma GLUT4 immunofluorescence after insulin stimulation. In contrast, basal levels of plas-malemma GLUT4 in the transgenic db/db mice were much greater than those observed in insulin-treated nontransgenic

(8)

chal-lenge. These data indicate that in the hGLUT4db/db transgenic mice relatively high levels of GLUT4 protein reside at the plasma membrane in the basal state and that insulin directly induces an increase in plasma membrane-associated human GLUT4 protein in a manner consistent withtranslocationfrom anintracellular vesicular pool. This GLUT4 redistribution oc-curs in spite of the insulin resistance (9-13) and translocation defect (see above) in the nontransgenic db/db mice.

Thus, if the defect in insulin action in db/db mice occurs in the insulin signaling pathway, it does not appear to be rate limiting in preventing GLUT4 translocation from the

intracellu-larpool to the plasma membrane. Since the defect in the insulin signaling pathway in db/db mice appears to be overcome by increased GLUT4 expression, it is possible that increased glu-cosedisposal reduces hyperglycemia and the "glucose toxic-ity" proposed by DeFronzo (7) as a contributing factor to insulin resistance.

Insummary, these data demonstrate that high level expres-sionof the human insulin responsive GLUT4/muscle-fat spe-cific facilitative glucose transporter transgene in ageneticmodel ofNIDDMresults in a high level of cell surface GLUT4 protein localization. Additionally, the increased GLUT4expression re-sulted in arestoration of insulin-stimulated GLUT4 transloca-tion from the intracellular storage site to the plasma membrane.

As aconsequence, after a large oral glucose load, circulating glucose levels were markedly reduced in the hGLUT4 transgenic db/db mice compared with the nontransgenic db/

dbmice, indicating a substantially greater degree of glycemic control. Mostimportantly, these results suggestthatincreasing GLUT4 levelsattheplasma membrane by either genetic manip-ulation orby pharmacologic intervention may be an effective therapy for humanNIDDM as improved glycemic control

oc-curs evenin thepresence of severe insulin resistance and pan-creatic defects.

Acknowledgments

Wethank Dr. Art Franke for continued support and BenCapers and Ramon Bermudez for their excellent assistance with the animal

hus-bandry.

This workwassupportedin partbygrants from the National

Insti-tutesof HealthtoJ. E. Pessin.

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