Journalof NeurochemistryLippi ncott—RavenPublishers,Philadelphia© 1997 InternationalSociety for Neurochemistry

Expression of the Na~—D-G1ucoseCotransporter SGLT1in Neurons

RobertPoppe,Ulrich Karbach,StepanGambaryan,*Heinrich Wiesinger,tMichael Lutzenburg,tMatthias Kraemer,~Otto W. Witte,

and HermannKoepsell

AnatomischesInstitut, BayerischeJulius-Maximilians-Universitat, Würzburg; ‘tPhysiologisch-CheniischesInstitut,

Eberhard-Karls-Universitat,TUbingen;and tNeurologischeKlinik, Heinrich-Heine-Universitat,Düsseldorf Germany

Abstract: In brains of the rabbit, pig, and human, expres-sion of the high-affinity Na~—D-glucosecotransporterSGLT1 and of the protein AS1, which alters the activityof SGLT1, was demonstrated. In situ hybridizationshowed that SGLT1 and RS1 are transcribed in pyramidalcells of brain cortex and hippocampus and in Purkinjecells of cerebellum. In neurons of pig brain SGLT1 proteinwas demonstrated by western blotting with synapto-somal membranes and by immunohistochemistry, whichshowed SGLT1 in pyramidal and Purkinje cells. To testwhether SGLT1 in neurons may be activated during in-creased D-glucose consumption, an epileptic seizure wasinduced in rat brain, and the uptake of specific nonmetab-olized substrates of SGLT1 {[14C]methyl-a-D-glucopyra-noside ([14C]AMG)} and of Nat-independent transport-ers {2-deoxy-D-[14C]glucose ([14C}2-DG)} was analyzedby autoradiography. During the seizure the uptake ofAMG and 2-DG was increased in the focus. Within twohours after the seizure 2-DG uptake in the focus returnedto normal. In contrast, the AMG uptake in the focus areawas still increased 1 day later. The data show that thehigh-affinity Na —D-glucose cotransporter SGLT1 isexpressed in neurons and can be up-regulated. KeyWords: Glucoseuptake—Sodium glucose cotransport—Neurons— Epileptic seizure—Regulation—lmmunohis-tochemistry.J. Neurochem. 69, 84—94 (1997).

GLUTI and GLUT3 are most relevant(Crone, 1965;Dick et al., 1984; Pardridgeet al., 1990; Nagamatsuet al., 1992, 1993; Maher et al., 1994). GLUT! ismainlyexpressedin the endothelialcells,whereastheexpressionof GLUT3 may be specific for neurons.

Transcriptionof some genesand gene fragmentswith homologyto thehigh-affinity Na~—D-glucoseco-transporterSGLTI (Hediger et al., 1987) has beenreportedin brain. Northernblot signalswereobtainedwith the functionally noncharacterizedeDNA rkSTl(Hitomi andTsukagoshi,1994) andwith two SGLTI-homologouscDNA fragments[RK-A andRK-I (Pajorand Wright, 1992)]. In addition, the gene of theSGLT1-homologousNa + —inyo-inositol cotransporterSMIT, which is engagedin osmoregulation,hasbeenrecently c!onedfrom the human (Kwon et al., 1992;Berry et a!., 1995), and the SGLT1-homologousgeneSNSTI, whichmediatessometransportof uridine,hasbeenisolated from rabbit kidney (Pajor and Wright,1992). Trying to elucidatewhether secondaryactiveSGLTI-typeNa~—glucosecotransportersmay partici-pate in cerebra!D-glucoseuptake,we studiedthe ex-pressionof SGLTI -type proteins.It wasdemonstratedthattheNa~—myo-inositolcotransporterSMITandthelow- and high-affinity Na~—D-glucose cotransportersSAAT1 (Kong et al., 1993; Mackenzieet al., 1994)

D-Glucoseis the major metabolic fuel in the CNS(Siesjö, 1978). For utilization in the brain D-glucosemust be transferredacrossthreeplasma membranes:the endothelialcell plasmamembranesof the blood—brain barrier(BBB) andtheplasmamembraneof glialor neurona!cells. Most D-glucosemay enter the glialcells, where lactate is producedandreleasedinto theinterstitium (Magistretti et al., 1995; TsacopoulosandMagistretti, 1996). Lactateand D-glucoseare trans-ported into neurons and metabolizedby oxidativephosphorylation.It has beendeterminedthat Na+-in-dependentglucosetransportersplay an importantrolein these translocationsteps and that the isoforms

ReceivedDecember12, 1996; revised manuscriptreceivedFebru-ary 27, 1997; acceptedFebruary27. 1997.

Addresscorrespondenceandreprint requeststo Dr. H. Koepsellat AnatomischesInstitut, Bayerische-Julius-Maximilians-Universi-tilt, Koellikerstrasse6, D-97070 Wurzburg,Germany.

The nucleotidesequencesreportedin this article have been sub-mitted to GenBankwith the accessionnumbersU41897(fragmenlof humanSAATI), U47672(fragmentof SMTT from the rat), andU41898(fragmentof rkSTl from the human).

Abbreviation,sused: AIB. a-aminoisobutyrate;AMG, methyl-a-D-glucopyranoside;BBB, blood—brainbarrier; BSA. bovine serumalbumin;2-DG, 2-deoxy-D-glucose;PBS,phosphate-bufferedsaline;PBS-T, phosphate-bufferedsaline containing 1% Tween20; SDS,sodium dodecyl sulfate; SSC. 0.15 M NaCI and 0.015 M sodiumcitrate,pH 7.0.

84

EXPRESSIONOF SGLTJ IN NEURONS 85

Oligonucleotide

TABLE 1. Nucleotidesequencesof oligonucleotidesusedfor PCRexperiments

Sequence

5’-GC GCC AGC ACC CTC TTC AC-3’ (SGLTI rabbit, 1.222-1,240)5’-CAG CCC ACA AAA CAG GTC-3’ (SGLTI rabbit, 1,837-1,854)5’-TTC TGT GTC AGT GGC CTC C-3’ (SGLTI rabbit, 1,778-1,796)5’-GCC TCT CTS TTT GYY AGY AA-3’ (SAATI pig, 2 13-232)5’-TC ATC TGT GTG GCT TTG-3’ (SAATI pig, 451-467)5’-TT GTC CCC CTC GAC TAT G-3’ (SAATI pig, 725-742)5’-CT GTA CTG GAG ACA GCA G-3’ (SMIT dog, 8-25)5’-ATC CTG GTC ATG TGC AT-3’ (SMIT dog, 52-68)5’-C AAC GAT CAT AAG CAG AG-3’ (SMIT dog, 566-583)5’-GAG GCA GGC TCC AGA CTG-3’ (SNSTI rabbit, 22-39)5’-GCC CAC AGC CGG GTC CTC-3’ (SNSTI rabbit, 736-753)5’-TTT GTC ATC ATC GCGGG-3’ (SNSTI rabbit, 622-638)5’-G CAG CCC GCA GCT GCT G-3’ (rkSTl rabbit, 768-784)5’-GCC TCT CTSTTT GYY AGY AA-3’ (rkSTl rabbit, 241-260)5’-CCA GGC CAA CAT CAG CAC-3’ (rkSTl rabbit, 353-369)5’-G CAT GAG GCG GGC CCT C-3’ (RSl rabbit, authors’ unpublisheddata,534—550)5’-C GTA TGC TCT GTC TGT C-3’ (RSI rabbit, authors’ unpublisheddata, 933—949)5’-AAT CCS YTR ATG GAA GTR GA-3’ (RSI rabbit, authors’ unpublisheddata, 820—839)5’-CTG GAT CCA GAG GAG GAG GAC AT-3’ (SGLTI pig, 1,546-1,568)5’-GCC TGC AGG GCA CAG CCT GTG-3’ (SGLTI pig, 1,854-1,834)

Superscripts+ and — indicate whetherthesequencesbelongto theplus or minus DNA strand.The referencegeneswith the respectiveoligonucleotidepositions are indicated in parentheses(Hedigeret al.. 1987; Ohta et al.. 1990;Kwon et al., 1992; Pajor and Wright, 1992; Kong et al., 1993; Hitomi and Tsukagoshi,1994).

and SGLT1 andthe interactingprotein RS! (Koepselland Spangenberg,1994; Lambotte et al., 1996) aretranscribedin brain. The expressionof SGLT1 andRS1 waslocalizedto neuronalcells, anddataarepre-sented showing that Na~—D-glucose cotransport inbrain can be up-regulated.Part of the data hasbeenreportedin abstractform (Poppeet a!., 1994, 1996).

EXPERIMENTAL PROCEDURES

Library construction and screeningFor library constructionpBluescriptll SK(—) was di-

gestedwith PstI, o!igo(dT)-tailed, digestedwith BamHI,and separatedby agarosegel electrophoresis,and the largepBluescript BamHIIPstl-oligo(dT) fragment was recov-ered. From rabbit brain cortex mRNA was isolated byaffinity chromatographyon ol igo ( dT)-cellulose, and dou-ble-strandedeDNA wassynthesizedusingthe SuperScript-ReverseTranscriptaseKit of GibcoBRL (Eggenstein,Ger-many) with substitutionof oligo(dT)-tailed pBluescript!Iplasmidfor theoligo(dT) primer. The vector-tailedeDNAwassize-fractionatedby agarosegel electrophoresis,circu-larized,andtransformedin E,scherichiacoli strain DHIOB(GibcoBRL) by electroporation.The eDNA library wasscreenedunder high-stringencyconditions with a 32P-la-beled probe of the rabbit Na~—D-glucose cotransporterSGLTI [nucleotides 1,251—1,882(Hedigeret al., 1987)].

PCRsToinvestigatethemRNA distributionof SGLT-typetrans-

porterstotal RNA waspreparedfrom brain cortex of differ-ent speciesand oligo(dT)-primed; single-strandedeDNAwas synthesizedby reversetranscriptase.PCRs were per-formed using the primer pairs SI ~, S2; S4~,56; S7S9; Sl0~,S11; SIr, S14; and SI6~,S!7 (TableI). Controls were assayedwithout addition of reversetran-

scriptase.The PCRs were performed in 67 mM Tris-HCI(pH 8.4), 2 mM MgCI

2, 10 mM 2-mercaptoethanol,16.6mM(NH4)2S04,0.17 mg/ml bovineserumalbumin (BSA),0.2 mM each deoxynucleotidetriphosphate,0.4 1iM eachprimer, and20 U/mI Taq polymerase. After initial denatur-ation (5 mm, 90°C)35 PCRcycles(1 mm at 94°C;1 mmat 48—52°C;2 mm at 72°C)were performed.The sampleswere separatedby agarosegel electrophoresis, stained withethidium bromide, andhybridizedwith the internal primersS3 ~ S8~,SIS ~, andSI8~(Table I).

Subcloning and DNA sequencingFor sequencingof PCR-amplifiedcDNAs, the amplifica-

tion productswere gel-purified, blunt-endedwith Klenowpolymerase, phosphorylatedby T4 polynucleotidekinase,andsubclonedin pBluescriptl!plasmid.DNA sequencingofdouble-strandedDNA in pBluescript plasmids was per-formedby the chain terminationmethodusinga cycle se-quencingkit of Biozym (Hamein,Germany),~-

35S-dATP.andstandardprimersaswell assequence-relatedoligonucle-otideprimers.For sequencingof SGLT 1 clonesfrom arabbitbrain library exonucleaseII! deletioncloneswere preparedandsubcloned.

Synthesisof digoxigenin-labeledcRNAFor the generationof cRNA probesof SGLTI the ampli-

fication productsfrom therabbitandpig (primers51 ~, S2andSl9~,S20 ; TableI) weresubclonedinto pBluescriptllplasmid. To generate0.3-kb RSI probes,RSI in pBluc-script!! plasmid (Veyhl et al.. 1993) was restrictedwithEcoR!(sense)or Ncol (antisense),anddigoxigenin-labeledcRNAsweresynthesizedwith T3 (sense)andT7 (antisense)RNA polymerase.For the synthesisof antisenseandsensecRNAsof SGLTI from therabbitandpig theplasmidDNAswere linearizedwith EcoR! (rabbit antisense),BamH! (pigantisense),NotI (rabbit sense),and Pstl (pig sense),andthecRNAsweresynthesizedby T3andT7 RNApolymerase.

Sl~S2S3S4*S5+S657*S8~S9Sb’SI 1Sl2~S13SI 4~SISI 6S17518’Sl9~S20

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86 R. POPPE ET AL.

respectively.TheeRNAs werepurified by ethanolprecipita-tion andresuspendedin diethyl pyrocarbonatc-treatedwater.

HybridizationsFor Southernhybridizationthe PCR amplification prod-

Licts were separatedby agarosegel electrophoresis,trans-ferred to Hyhond-N membranes (Amersham Buehler,BraLinschweig,Germany),andhybridizedwith oligonucleo-tides thatwere labeledby tailing with ddUTP-digoxigenin.Hybridizationwasperformed7°Cbelowthe meltingtemper-ature (Wilkinson, 1993) in the presenceof’ 5 >< SSC (I)< SSC is 0.15 M NaCI and 0.015 M sodium citrate, p1-I7.0),5 >< Denhardt’ssolution, 1%(wt/vol)blocking reagent(Boehringer,Mannheim, Germany),and 1% (wt/vol) so-dium dodecyl sulfate (SDS). For in situ hybridization 10-pm-thick cryostat-cutsectionswere fixed for 10 mm with4% (vol /vol) parafornialdehyde,rinsed with dicthyl pyro-carbonate-containingphosphate-bufferedsalinc (PBS), in-cubatedfor 10 mm with 0.1 M HCI, incubatedfor 30 mmwith 0.1 M triethanolamine-HCI(pH 8.0) containing0.25%(vol/vol) aceticanhydride,rinsedwith PBS, andair-dried.Prehybridizationwas performedfor 2 h (45°C)in thepres-enceof 50% (wt/vol) formamide,S >< Denhardt’ssolution,10 mM triethanolamine-HCI(pH 7.2), 25 mM EDTA, 50m/ll NaCI, 150 pg/mI denaturedsalmonspermDNA, 1,000pg/nil yeasttRNA, and 5 mM vanadylribonucleoside com-plex. For hybridizationthe sectionswere incubatedfor 16 h(45°C)with digoxigenin-labeledantisenseand senseeRNAin the presenceof 50% (wt/vol) formamide, 10% (wt/vol)dextran sulfate,2.5 >< Denhardt’ssolution, 10 mM trietha-nolamine-HCI (pH 7.2). 5 mM EDTA. 500 mM NaCI, ISOpg/nil denaturedsalmon sperm DNA, 1,000 pg/nil yeasttRNA. and 5 mM vanadylribonucleosidecomplex.Thereaf-ter the sectionswere washedfor 30 mm (45°C)in 5 >< SSCcontaining50% (wt/vol) formamide,60 mm (70°C)in IX SSC containing0.1% (wt/vol) Triton X-b00, and60 mm(70°C)in 0.1 >< SSC containing0.1% (wt/vol) Triton X-IOU and blocked (30 miii, 22°C)with 1% (wt/vol) BSA,1% (wt/vol) blocking reagent(Boehringer),150mMNaCI,and 100 mMTris-HCI. pH 7.5. Thesectionswerethen incu-bated(3 h, 22°C)with theFabfragmentof alkalinephospha-tase-conjugatedanti-digoxigeninantibody from a goat (di-lutecl 1:500), washedthreetimes (20 mm, 22°C)with 100mM Tris-HCI (pH 7.5) containing150 mM NaCI, andrinsedwith 100 mM NaCI, 50 mM MgCI2, and 100mM Tris-HCI,pH 9.5. The color wasdevelopedby incubationwith nitroblue tetrazolium chloride and 5—brorno—4—chboro—3—indolylphosphate.

Cell culture and preparation of synaptosomalmembranes

Neuron-richprimary culturesfrom fetal Wistar rat brainswere prepared(Loffler et al.. 1986). At the time of theexperimentthey contained< 10%astroglial cells. Astroglia-rich primaryculturesderived from neonatalWistarrat brainswere prepared as described (Hamprecht and Loftier, 1985).These cultures contain a majority of astroglial cells and oh-godendroglial, microghial. and ependyrnal cells as minorcomponents, hut definitely no neurons. Synaptosoinal mem-

branes from pig brain were isolated on a discontinuous Fi-coIl/sucrosegradient (Booth and Clark, 1978).

Western blots and antibody productionSynaptosomalmembranesfrom pig brain cortex (20 pg

of protein per lane) were applied to a discontinuous (5/7%)

polyacrylamideslab gel. Electrophoresiswas performedac-cording to the techniqueof Laemmli (1970), and the pro-teins were transferredto nitrocellulose.After incubatingthemembranesfor 1 h (22°C)with blocking buffer 1100 mMNaCI. 10 mM Tris-HCI (pH 8.0). 0.1% (wt/vol) Tween,1% (wt/vol) BSA. and0.5% (wt/vol) fish gelatin the blots

were developed with the polyclonal peptide antibodiesAbIand Ab2. AbI wasraisedin rabbits against peptide P1 (EAP-EETIEIEVPEEKKGC), which representsresidues525—542ofSGLTI from pig kidney (Ohtaet al., 1990). Theimmuni-zation was performed with Ph coupled to keyhole limpethemocyaninor to ovalbumin. The antiserumwas affinity-purified on peptidePb that was coupled to epoxy-activatedSepharose.Ab2 was provided by Dr. S. Shirazy-Beecheyand had been raised against residues402—420 of SGLTIfrom rabbit intestine(Lescale-Matysetal., 1993).The incu-bation with primary antisera(diluted 1:500) was performedfor 16 h (6°C)in the presenceof blockingbuffer. After threewasheswith 10 mM Tris-HCI (pH 8.0), 100mM NaCI, and0.1% (wt/vol) Tweenthe antibody binding was visualizedby incubationwith peroxidase—coupledanti—rabbit IgG anti-body (diluted 1:5,000)followed by chemiluminescencede-tectionwith theECL system(Aniersham).!n someexperi-mentsantibody binding was blockedby preincuhation(1 h.22°C)with 50 pg/mI peptide P1.

lmmunohistochemistryFor immunohistochemicaldetection of SGLTI in pig

brain, 3-pm-thick cryosectionsweretransferredto silanizedslides and fixed for 10 mm with absolutemethanol. Thesections were rinsed with PBS containing 1% (wt/vol)Tween 20 (PBS-T) and blocked for I h with casein. Forantibody reactionthe sectionswere incubatedfor I h (22°C)with antiserumAb I, whichwasdiluted 1:50 in PBS-T.Afterwashingwith PBS-T the sectionswere incubatedfor I h at22°Cwith tetramethylrhodamineisothiocyanate-labeledanti-rabbit lgG antibody (diluted 1:100; Sigma, Deisenhofen.Germany). After washing with PBS-T the sectionswereinspected.The specificity of the antibodyreactionwas con-trolled in parallelsectionsrising preimmuneserumandanti-serumAhI that wasblockedwith antigenicpeptide.

Induction of an epileptic focus andautoradiography

In rat brain an epileptic focus was inducedby penicillinapplication, antI the uptake of 2-deoxy-u-[’C]glucose(I ‘

4C I 2-DG), [~4C]methyl-a-D-glucopyranoside(I ‘~CI -AMG), or a-I ‘4C 1 aminoisobutyrate([‘4C] AIB) into thefocusareawas determinedby autoradiography.The experi-ments were perfirmed as describedearlier (Witte et al..1994). In brief, adult male Wistar rats were anesthetizedwith 2% (vol/vol) isofiuraneduringthepreparationandwith1% (vol/vol) isofluraneduring the experiments.Two 1.8-mm-diameterholesweredrilled into the skull.theduramaterwas incised, and glass electrodescontaining artificial CSFwere inserted into the left and right frontal cortexes.!n theleft frontal cortex an epileptic focus was induced 1.0 mmanteriorto the bregmaand 1.0 mm lateral of the midline byreplacingthe electrodefor I h with an electrodecontaining50,000 IL/mI isotonic sodiumpenicillin solution. Thesec-ond electrodeat the right frontal cortex was usedas refer-ence.Throughout the experimentthe body temperaturewaskept at37°C.electrocortigramsandelectrocardiogramsweretaken,andpH, Pco:, andP0: in the blood were monitored.At 1 and 23 h after onsetof epileptic activity 80 pCi of

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EXPRESSIONOF SGLTI IN NEURONS 87

I 4C]AMG (n = 6 for each timepoint) wasgiven throughthe femoralvein as abotus.At 45 mm after theadditiontherats were decapitated,and the brain was immediately re-movedand frozen in chilled isopentane.The brainswere cutinto 20-p.m-thickcoronal slices, whichwere exposedfor 30days to ahigh-resolutionautoradiographyfilm (Hyperfilm-~max; RPN9; Amersham). The optical densities of the filmwere recordedwith a charge-coupled device cameracon-nected to anon-line videometry processorfor backgroundsubtractionand offset correction.This signalwasdigitizedand analyzedby an imaging program(NIH !mage). Forconfirmation of previously reporteddata on deoxyglueosemetabolism of penicillinfoci (Witte et al., 1994),22 h alterremoval of the penicillin electrode40 pCi of I ‘4C]2-DGwasadministeredin two animals. Quantitativeautoradiogra-phy wasperformedas describedheftre (Witte etal.. 1994).BBB integrity wastestedwith EvansBlue during epilepticactivity and 22 h after penicillin removal [n = 2 at bothtime points; 2%(wt/vol) Evans Blue, 0.66 ml iv.] andalso with [‘4C]A!B. The latter also is ableto detectBBBpermeabilityof small molecules(Blasberget al., 1983). Intheseexperiments40 pCi of [‘4C]AIB (n = 2 at both timepoints) was given intravenously.The ratswere decapitated20 mm after tracerinjection. Further processingwas thesame asdescribedfor I ‘4C I AMG autoradiography.

MaterialsI4C]AMG (10.3 GBq/mmol) was obtainedfrom Amer-

shamBuehler,and[‘4C]2-DG (2.1 GBq/mniol) and I’4C]-A!B (1.5 GBq/mmol) wereobtainedfrom Du Pont deNem-ours (Dreieich, Germany).Deoxynucleotidetriphosphates,nucleotidetriphosphates,ddUTP-digoxigenin,dUTP-digoxi-genin. goatanti-digoxigeninantibodycoupledwith alkalinephosphatase,5-bromo-4-chloro-3-indolyl phosphate,andni-tro blue tetrazoliumchloride wereprovidedby Boehringer.F. co/i DHIOB, terminal deoxynucleotidetransferase,Super-Script reversetranseriptase,Taqpolyrnerase,andDNA stan-dardswereobtainedfrom GibcoBRL. Theexonucleasedele-tion kit was purchased froni New England Biolabs(Schwalbaeh,Germany), and goat tetramethylrhodamineisothiocyanate-labeledanti-rabbit lgG antibody was fromSigma (Munchen,Germany).All other chemicalswere ob-tainedas describedearlier (Veyhlet al., 1993).

RESULTS

Detection of cDNA fragments that encodepresumed componentsof SGLT-type Na +

cotransport systemsin brainThe transcription oftheSGLT-type genesSGLTI,

SAATI, SMIT, rkSTl, and SNSTI (Hediger et al.,1987;Kwon et al., 1992;PajorandWright, 1992;Konget al., 1993; Hitomi andTsukagoshi,1994)andof theRS I gene,which encodesa protein that modifies theactivity of SGLT1 (KoepsellandSpangenberg,1994),wasinvestigated.ThemRNAs isolatedfrom brainsofthe rabbit, pig, dog, and human werereversetran-scribed,andspecificeDNA fragmentswere amplifiedby PCR. The amplificationproductswere identifiedbyhybridizationwith internal primersandgel-eluted,andthe nucleotide sequenceswere determined. Usingbrains from the speciesfrom which theoriginal geneshave been cloned, specific eDNA fragments for

FIG. 1. Amplification of specific cDNA fragments of presumedSGLT-type transporter components in brain of (a) the rabbit,pig, or dog and (b) the human. RNA was isolated from brain ofthe rabbit (a; lanes a, b, and g—m), pig (a; lanes c and d), dog(a; lanes e and f), and human (b), and eDNA was synthesizedafter oligo(dT) priming (a and b; lanes a, c, e, g, i, and I). Inlanes b, d, f, h, k, and m reverse transcriptase was omitted.Using specific primers amplification was attempted of eDNAfragments of SGLT1, SAAT1, SMIT, rkSTl, SNST1, and RS1,and the fragments were hybridized with specific internal primers.The primers used are shown in Table 1 (SGLT1, Si , S2-, S3SAAT1, S4 ‘, 56, S5~ SMIT, S7 , S9 , S8~SNST1, SlOSi1~, S12~rkSTl, 513, Si4~, S15 ; and RSi, 516’, S17,S18’). The amplification products were separated on agarosegels and analyzed by staining with ethidium bromide (upperparts of a and b) and by hybridization with internal primers(lower parts of a and b).

SGLTI, SAAT 1, SMIT, rkSTI, andRS1 weredetected(Fig. Ia). In rabbit brain no eDNA for SNST1 wasfound. The nueleotide sequences ofthe brain eDNAfragmentsrelatedto SGLTI. SAATI, andSMIT wereidenticalto thosethathavebeen determined fromsmallintestineandkidney of the rabbit [SGLTI nueleotides1,270—1,864(Hediger et al., 1987)], LLC-PK

1 cellsof thepig [SAAT1 nucleotides240—733(Kong et al.,1993)], andMDCK cells of thedog [SMIT nucleo-tides 503—1,042(Kwon et al., 1992)]. ComparingtherkSTI-related eDNAfragment fromrabbit brain withrkSTl from rabbit kidney [nucleotides 261—767(Hi-tomi and Tsukagoshi,1994)1, one nueleotide ex-changewasobserved atposition 586 (A/G, Ile/Val).In theRSI fragmentfrom rabbitbrainthreenucleotideexchangesat positions 687 (T/C), 704 (G/A, Arg/His), and 798 (CIT) were observedin comparisonwith RSI from rabbit intestine (authors’ unpublisheddata).

To test whetherSGLTI, SAAT I, SMIT, rkST1, andRSl werealso expressedin human brain thePCR cx-perimentsdescribedabovewere repeatedwith eDNAprepared fromthefrontal cortex ofhumanbrain. Figurelb shows that specific eDNA fragments related toSGLTI, SAAT I, SM!T, rkSTI, andRS I couldbe am-

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R. POPPE ET AL.

plified. SNSTI was not detected in human brain. Theamplified human eDNA fragmentswere sequenced.The eDNA fragmentsof SGLTI andRSI from humanbrain were 100%identical to the respectivefragmentsof SGLTI and RSI from human intestine [SGLTInueleotides1,230—1,830(Hediger et al., 1989); RS1nueleotides2,574—2,989 (Lambotte et al., 1996)].Comparing the amplified eDNA fragment of SMITwith the recently clonedhuman gene(Berry et al.,1995), six nueleotide exchanges were observed at thefollowing positionsin theopenreadingframe: 18 (GIC, Glu/Asp), 92 (C/G, Ser/Cys), 148 (GIA, Ala!Thr), 157 (GIA), 573 (TIC), and 580 (G/A, Ala!lie). The fragment of human SAATI shows 86% nu-eleotideidentity with theSAATI from thepig (Konget al., 1993) and 83% identity with the encoded aminoacid sequence.The human rkSTl fragmenthas85%nueleotideand89% amino acididentity with the rabbitgene(Hitomi and Tsukagoshi, 1994).The datashowthat SGLTI, SAATI, SMIT, rkSTl, andRSI aretran-scribedin mammalianbrain.

Isolation and sequencingof the complete SGLT1cDNA from rabbit brain

To verify whetherSGLT1 andnot a partial identicalgeneis transcribedin brain,thecompleteopenreadingframe of SGLTI was isolated from a rabbit braineDNA library. A positiveclonewith an insert of 2.2 kbwas isolated that containedthecompleteopenreadingframe of SGLTI. Nueleotidesequencing showed thatthis eDNA wasidentical with rabbit SGLT1 (Hedigeret al., 1987)with theexceptionof onenueleotidemis-matchat position 2,167, whereT was replacedby A.This indicatesthat in the rabbitSGLT1 is transcribedin brain.

in situ hybridizations in brainTo localize the transcriptionof SGLTI in brain in

situ hybridization was performed in the rabbit (Fig. 2)and pig (Fig. 3). The nucleotide sequences ofthemRNA hybridization probesused [rabbit SGLTI nu-eleotides 1,222—1,854 (Hediger et al., 1987); pigSGLTI nucleotides1,546—1,854(Ohta et al., 1990)]were between87 and 89% identical to SGLTI se-quences ofdifferent species.The nueleotideidentitiesto pig SAAT1 and to the other members of the SGLT1genefamily were 72% and<63%, respectively.Be-causeunderthehybridizationstringencyusedtheanti-sense probes did not cross-react with SGLTI of otherspecies (data not shown), they were specific forSGLTI within the respectivespecies.Figures 2 and3show that SGLTI is transcribedin neuronsof hippo-campus,brain cortex, and cerebellum.Hybridizationwasobtainedwith antisenseprobesin pyramidalcellsof the eornu ammonis and in granularcells of thedentategyrus, whereasno reaction was found withsense eRNAs(Figs.2aandband3a andb). In cerebralcortex of the rabbit and pig specific hybridizationswere observedin pyramidal cells (seeFigs. 2c and3c). In cerebellumstrong specific reactionscould be

FIG. 2. Cellular localization of SGLT1 transcription in rabbitbrain. Sections (10 pm thick) through hippocampus (a and b),cortexof frontal brain (c), and cerebellum (d and e) were hybrid-ized with specific antisense (a and c—e) or sense (b) cRNAprobes of SGLT1 (see Experimental Procedures). The digoxi-genin-labeled probes were visualized by the phosphatase reac-tion of alkaline phosphatase-coupled antibodies against digoxi-genin. Hybridization is observed in pyramidal cells of the cornuammonis (arrows in a), in granular cells of the dentate gyrus(arrowhead in a), in neurons of parietal cortex (a, upper rightcorner), in pyramidal cells of the frontal cortex (c), and in Pur-kinje cells of cerebellum (arrows in d and e). No hybridizationwas observed with the sense cRNA probe (b). Bars = 500 pmin (a) and (b), 100 pm in (c) and (d), and 50 pm in (e).

localizedto Purkinje cells (Figs. 2d andeand3d ande). Some reaction was also observedin the stratumgranulareof cerebellum,which may be localized togranularcells (Fig. 3d and e). We then investigated

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FIG. 3. Cellular localization of SGLT1 transcription in pig brain.Sections through hippocampus (a and b), brain cortex (c), andcerebellum (d and e) were hybridized with specific antisense (aand c—e) and sense (b) probes of porcine SGLT1. The experi-ments were performed as described in Fig. 2. In hippocampushybridization was observed in pyramidal cells of the cornu am-monis (arrow in a) and in granular cells of the dentate gyrus(arrowhead in a). In different layers of brain cortex pyramidalcells were labeled (c). Staining was observed in Purkinje cellsof the cerebellum (arrowheads in d and e) and in granular cells(arrows in e). Bars = 500 p.m in (a—c), 100 pm in (d), and 50pm in (e).

cerebral cortex, and in Purkinje cells of cerebellum(Fig. 4).

Transcription of SGLT1, SAAT1, SMIT, andrkSTl in cultured neuronal and glial cells

Using PCR with reversetranscribedmRNAs fromrat primary cultures ofneuronal and glial cells, weinvestigatedthe transcription of SGLTI in culturedneuralcells. In theseexperimentsthe amplification ofspecificeDNA fragmentsof SAAT1, SMIT, SNST1,

the transcriptionof the previously described proteinRSI, which altersNa~—D-glueosecotransportactivityexpressedby SGLT1 (Koepsell and Spangenberg,1994; Lambotteet al., 1996). Performingin situ hy-bridizations with 0.3-kb antisenseand senseeRNAfragmentsof porcineRS1, transcriptionwas found inthe sameneuronsas SGLT1, i.e., in pyramidal andgranularcells of hippocampus,in pyramidal cells of

FIG. 4. Cellular localization of RS1 transcription in pig brain. Insitu hybridization with 0.3-kb antisense (a and c—e) and sense(b) cRNA probes of RS1 from the pig was performed as de-scribed in Experimental Procedures. With the antisense probehybridization was observed in pyramidal cells of the cornu am-monis (arrow in a), in granular cells of the dentate gyrus (arrow-head in a), in pyramidal cells of brain cortex (c), in Purkinje cellsof cerebellum (arrowheads in d and e), and in granular cells ofcerebellum (arrow in e). Bars = 500 pm in (a) and (b), 250 pmin (c), 100 pm in (d), and 50 pm in (e).

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FIG. 5. Amplification of specific eDNA fragments of SGLT-typeproteins from cultured neuronal and glial cells of the rat. RNAwas isolated from neuron-rich (lanes a, c, e, g, and i) andastroglia-rich primary cultures of rat brain (lanes b, d, f, h, andk). Using oligo(dT) priming and reverse transcriptase, eDNA wassynthesized, and PCRs and internal hybridizations were per-formed with the primers listed in Table 1 (SGLT1, S1~,S2,53; SAAT1, S4~,S6,S5’; SMIT, S7~,S9, S8; SNST1,510,511 ,S12~andrkST1,S13,S14,S15 ).Theampli -fication products were separated by agarose gel electrophoresisand stained with ethidium bromide (a) or hybridized with theinternal primers (b).

andrkSTl was alsoattempted(Fig. 5). The specificityof the amplificationproductswas verified byhybridiza-tion with specific internal primersand by DNA se-quencing. As in total brain no amplification of anSNST1 eDNA fragmentcould be achieved. Inneuronalcells, but not in glial cells, eDNAfragmentsof SGLT!and SAAT! were amplified. In contrast, eDNAfrag-mentsof SMIT and rkSTl could be amplifiedfrombothneuronalandglial cells.

Expression of SGLTI protein in brainWestern blotsof brain synaptosomalmembranes

weredevelopedwith theSGLTI-specific peptideanti-body Ab I andwith thepeptideantibody Ab2. Ab1 wasraised againstthe 18-amino-acidpeptide P1, whichrepresentsresidues5 18—535 of SGLTI from the pig(Ohta et al., 1990). P1 is specific for SGLTI becausethe respective regions of SAATI, rkSTl, and SMITcontain only seven, six, and twoidenticalamino acidsthat aredistributed withinP1. Abi is specificfor por-cine SGLTI (data not shown). Ab2 is less specificand cross-reactswith SGLTI of different species.Itwas raised against amino acids 402—420 of rabbitSGLTI and may cross-react with SAATI. When syn -aptosomal membranes fromporcinebrain cortexwereseparatedby SDS-polyacrylamidegel electrophoresisand blotted ontonitrocellulose membranesand themembranes weredevelopedwith antiserumAb2, themajorreaction productwas observed at ~=75kDa (Fig.6, lane a) as has beendescribed for Ab2 in smallintestine (Lescale-Matyset al., 1993).With antiserumAbi a strongreactionwasobserved atthe sameposi-tion; however, severaladditional polypeptides werestained(Fig. 6, lane b). These nonspecific reactions

were absentwhenAbl wasaffinity-purified with pep-tide P1 (Fig. 6, lane e). Also, Abi binding to the75-kDaband was specificallysuppressedif AbI waspreabsorbedwith the antigenic peptide (data notshown). To determine the cellular localization of’SGLT1 protein in pig brain, lightmicroscopicimmu-nohistoehemistrywas performedwith AbI (Fig. 7).On sections throughhippocampus,cerebral cortex,andcerebellumSGLTI protein was detected in thesametypesof neuronal cellswhereSGLTI mRNA hadbeenlocalized: in pyramidalcells of brain cortex (Fig.7a).in pyramidal and granular cells of hippocampus (Fig.7b ande), andin Purkinjecells of cerebellum(Fig. 7eand f). The immunostainingwas completelyabolishedwhen Ab! was preabsorbed with the antigenic peptideP1 (see, e.g., Fig. 7d). The subeellular localizationofSGLT1 protein in different neurons is the topic offurtherstudies. The lightmicroscopicdatasuggestthatin the neurons of braincortex andcerebellumSGLTIprotein is mainly localized intracellularly (Fig. 7a, e.andf), whereas in the neurons ofhippocampusSGLTIproteinis mainly localized in plasma membranes (Fig.7c). In cerebellum,localization of SGLT1 at nervefibers was observed (Fig.7e).

Up-regulation of Na~—D-glucosecotransportactivity after experimental induction of anepileptic focus

Attempts to demonstrate transport activity ofSGLTI in neuronal plasmamembranevesicles pre-paredfrom cerebral cortex ofhealthy pigs andrabbitsshowedno or low Nat-dependentandphloridzin-in-hibitable uptakeof [14C]AMG (datanot shown).Be-causethis suggestedthat SGLTI proteinis functionallyinactive, we tried to elucidate whetherthe Na~—D-glucose cotransporter can be up-regulatedwhen themetabolism is changed.As a model for metabolicchangeswe induced an epileptic focus in rat brain inwhich theD-glucoseconsumption ofneuronsincreasesduring andshortly after theseizure(Witte et al., 1994).The epileptic focus was induced in the frontal cortexby applying penicillin for I h through a glass electrode.After applicationof penicillin to the corticalsurface,epileptic activity started within minutes (see Fig. 8;top panel). It always disappeared within 60 mm afterpenicillin removal. As has been described previously.

FIG. 6. Immunological demonstration ofSGLT1 protein in plasma membranes ofpig brain by western blot. The polyclonalpeptide antibodies Abi and Ab2 wereused to identify SGLT1 protein in synapto-somal membrane vesicles isolated frompig brain. Lane a was developed with anti-serum Ab2, lane b with antiserum Abi,and lane c with Abi that was affinity-puri-fied with the antigenic peptide P1. Thelines indicate the positions of the molecu-lar mass marker proteins at 116 (8-ga-lactosidase), 97 (phosphorylase a), 66(BSA), and 45 kDa (ovalbumin).

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EXPRESS/ON OFSGLTJ IN NEURONS 9’

beenshownto be specificallytransportedby facilitateddiffusion systems of theGLUT family but not bySGLTI and SAATI. In contrast, theNa~—D-glueoseeotransporters SGLTIand SAATI specifically trans-port AMG (Ekedaet al., 1989;Mackenzieet al., 1994).Unlike [‘4C]2-DG, [‘4C]AMG uptakewas not onlyincreasedin the focal area during epilepticactivity,but also 21 h after the end ofepileptic spiking (Fig.8, panel AMG). Experiments with Evans Blue didnot reveal any increase of BBB permeability duringepilepticactivity or 21 h later,whereas[‘4C]AIB auto-radiography showed a weak BBB leakage of the activefocus (datanot shown).Also, with [‘4C]AIB therewasno indication for an alteration of the BBB perme -ability 21 h after the end ofepilepticactivity (Fig. 8,panelAIB).

DISCUSSION

FIG. 7. Light microscopic immunological localization of SGLT1protein in pig brain. Brain cortex (a), hippocampus (b—d), andcerebellum (e and f) were stained with Abi. Immunostainingwas performed as described in Experimental Procedures. Thespecificity of the observed immune reactions was verified bynegative controls, which were obtained with preimmune serumor when Abi was preabsorbed with the antigenic peptide P1. In(d) the preabsorbed control of (c) is shown as an example. WithAbi specific immunostaining was observed in pyramidal cells ofbrain cortex (a), in pyramidal cells of the cornu ammonis (arrowin b), in granular cells of the dentate gyrus (arrowhead in b),and in Purkinje cells of cerebellum (e and arrowhead in f). In thegranular layer of cerebellum nerve fibers are stained (arrows ine). Bars = 250 pm in (a), 500 pm in (b), and 50 p.m in (c—f).

during epileptic activity the deoxyglucoseconsump-tion increasedin the focal areaby 279 ± 67% (Witteet al., 1994). When[14C]2-DG autoradiography wasperformed 22 h after removalof the penicillin elec-trode, no focal increaseof 2-DG consumptioncouldbe detectedany more (Fig. 8, panel2-DG). 2-DG has

In this study we demonstrate the transcription ofseveral membrane proteins in brain that belong to theSGLT1 family. The Na~—myo-inositol cotransporterSMIT(Kwon et al., 1992) was detected in neurons andglial cells,whereasthe high- andlow-affinity Na~—D-

glucose cotransportersSGLT1 and SAATI (Hedigeret a!., 1987; Kong et a!., 1993; Mackenzie et al., 1994)were only found in neurons.In theease ofSGLTI thecomplete eDNAwas isolated from rabbit brain. By insitu hybridizationin the rabbit and pig SGLTI mRNAwasdetectedin pyramidal cells of neoeortex andhip-pocampus, ingranularcells of thedentategyrus, andin Purkinjecellsof cerebellum.Recentlysimilar resultswere alsoobtained in the rat (authors’unpublisheddata).The neuronallocalizationof SGLT1 protein wasdemonstratedin the pig by using an SGLTI -specificantibodyfor western blotting andimmunohistochemis-try. With this antibody SGLT1 protein was localizedto thesametype ofneuronsin which SGLTI transcrip-tion was found. The lightmierographsshowed that inpyramidalcells of neocortexand in Purkinje cells ofcerebellumSGLT1 protein is mainly localizedintracel-lularly andsuggestedthat in pyramidalcells ofhippo-campus SGLTI protein is associated with the plasmamembrane.The intracellular SGLTI protein is sup-posedto be associated withintracellularvesieles.Byfutureelectronmicroscopic investigationsandby im-munological studieson fractionated cellular mem-branes thesubeellularlocalization of SGLTI proteinhas to be defined. Itremainsto be elucidated whetherSGLT1 from intracellularvesicles is incorporated intothe plasmamembraneduring metabolicstressas hasbeendescribedduring up-regulationof Na~—D-glu-cosecotransportin HT24 cells (Delézayet al., 1995)andwhetherthetranslationandtranscriptionof SGLT 1are up-regulated.

Various facilitated diffusion systems forD-glucOsebelongingto theGLUT family have been described inbrain (Maheret al., 1994).GLUT1 hasbeen detectedin luminal and basal membranes of the endothelialcells

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FIG. 8. Increased [14C]AMG uptake in rat

brain after induction of anepileptic focus. Toppanel: Electrocorticogram of an experimen-tally induced epileptic focus during penicillinapplication. Second panel, AMG: Autoradio-gram shows [‘4C]AMG uptake patterns 22 hafter removal of the penicillin electrode. Astrong increase of [‘4C]AMG uptake was ob-served on the left frontal cortex where the epi-leptic focus had been induced. In the righthemisphere the [14C]AMG uptake was not in-creased. Third panel, 2-DG: [14C]2-DG up-take pattern 22 h after removal of penicillin.The uptake in the focus area was not signifi-cantly different from the uptake in the righthemisphere. Bottom panel, AIB: [14C]AIBup-take pattern 22 h after removal of penicillin.The experiment shows the intactness of theBBB in the focus area. The scales indicate theradioactivity of the tissue (panels AMG andAIB) or the regional cerebral metabolic rate ofglucose as calculated by the quantitative auto-radiographic 2-DG method (panel 2-DG).

of the BBB and in plasmamembranesof glial cells,whereas GLUT3was found in some populationsofneurons.D-Glucose leaving the capillaries is mainlytransported into the surrounding astrocytes, where it ismetabolizedby glycolysis,which is up-regulateddur-ing neuronalactivity (Magistretti et al., 1995;Tsaco-poulos and Magistretti, 1996). Lactate generated dur -ing glycolysis is releasedfrom glial cells and may betransportedinto neuronsand metabolizedby oxidativephosphorylation (Hampreeht and Dringen, 1995).Also, D-glucosefrom the interstitium may bedirectly

transportedinto the neurons.Underresting conditionswith normal interstitial concentrationsof D-glueoseand oxygen supply, sugartransportto neuronsis proba-bly mediatedby GLUT3 (Nagamatsuet aI., 1992,1993),and thefacilitative diffusion of D-glueosemaybe sufficient for energy supply. Thismay not be theeasewhenthe energy supplyis reducedor the energyconsumptionincreasedduring ischemia, hypoxemia.hypoglycemia, or an epileptic seizure. Under theseconditions the glycogen stores in the glial cellsmaybe depleted,and the lactateconcentrationin the inter-

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EXPRESS/ONOF SGLTI IN NEURONS 93

stitium may be reduced.The ri-glucoseconcentrationin the microenvironmentof firing neuronsmay dropfar below the millimolar K,,, value of GLUT3. It hasbeenshownthat theextraeellularD-glucoseconcentra-tion in the microenvironment of firing neuronsis muchlower than themeann-glucoseconcentrationin braintissue. Using microdialysis and mieroeleetrodetech-niques interstitial D-glucose concentrationsbetween0.47 and2.4 mM weredetermined,and it was shownthattheD-glucoseconcentrationin theinterstitium wasinversely related to neuronal activity (Fellows et al.,1992;SilverandEreciuiska,1994). During hypoglyce-mia and isehemiainterstitial n-glucoseconcentrationsof 0.16 and 0.03 mM, respectively, were observed(Silver and Ereciñska,1994).

Thedetectionof SGLTI in neuronsraisedthe ques-tion of its physiologicalrole. Consideringtheapparentintracellular localization of SGLTI in most neuronstogetherwith our difficulties in detectingNa’ —n-glu-cosecotransportactivity in neuronalplasmamembranevesieles, we proposed the hypothesis that SGLTI niaybeactivatedduringmetabolicstresswhenthe intersti-tial n-glucoseconcentrationaroundthe neurons maydropfarbelow theK,,, valueof GLUT3. As an examplebr cerebralmetabolicchangeswe inducedan epilepticfocus in rat brain. Due to the increasedneuronalactiv-ity andglucoseconsumptionin an epileptic focus, asignificant reductionof the interstitial D-glucosecon-centration is expected,which may be underestimatedby measurementsof D-glueoseconcentrationsin brainhomogenates(Nakanishiet al., 1996). Becausenon-metabolizedsugarsare availablethat are specificallytransportedby Nat-independentGLUT transporters([‘4C] 2-DGor 3-0- [ “tC] methylglucose)or by Na~—

n-glucoseeotransporters([‘4C ] AMG), transportac-tivities of both transportertypes canbe analyzedsepa-rately. In the well-established2-DG model for mea-surementof cerebral glucosemetabolism,the phos-phorylation of 2-DG within the cells may bethe rate-limiting stepundernormoglycemicconditions.Duringincreasedglucoseconsumptiontransportof 2-DG maybecomerate-limiting.3-0-I ‘4ClMethylglucoseis alsotransportedby thefacilitateddiffusion systemsfor glu-cosebut not phosphorylated.It is thereforeusedasamarker of brain glucosecontent. [‘4C]AMG is spe-cifically transportedby Na~—n-glucosecotransportersand not phosphorylated(Warfield and Segal, 1976;Ikeda et al., 1989; Mackenzieet al., 1994). The in-creaseduptakeof[’4C]AMG in theareaof an epilepticfocusas shown in the presentstudyrequiresthepres-enceof an alternativetransportsystemacrossthe BBBbecauseAMG is not transportedby facilitated diffu-sion through the membersof the GLUT family andbecauseit cannotbe attributedto a leak in the BBB,which wasexcludedexperimentally.Probably I ‘4C]-AMG passestheBBB via SGLTI. In blood vesselsofbovine brain cortex some sodium-dependentglucosetransporthas been reported(Nishizaki et al., 1995),and we were recently ableto demonstratealso fran-

seription of SGLTI in brain capillariesof thepig (au-thors’ unpublisheddata). Our observationthat I ‘4C I -

AMG uptake in the focus areawas increasedI dayafter an epileptic focus hadbeeninducedmostproba-bly reflects up-regulation of SGLT1 in neurons. Inbrain SGLT1 is mainly expressedin neurons,andpre-liminary datashowedthat in the epileptic focus theamount of SGLT1 protein in pyramidal cells is in-creased(authors’ unpublisheddata).Our datasuggestthat the secondary active Na~—n-glucose cotransportsystem(s) concentraten-glucosein neuronsandpro-tect them from degenerationat reducedenergysupplyand/or increasedglucoseconsumption.This may bemore important thanthe relatively small up-regulationof GLUT3 expressionin neurons that hasbeen ob-servedduringhypoglycemiaandseizures(Christensenet al., 1981; Cornford et aI., 1994; Nakanishi et al.,1996). Experimentsareplannedto elucidatethe func-tional activity of SGLTI duringdifferentphysiologicaland pathological conditions. Specifically, a possibleup-regulation of SGLT I by a decreaseof n-glucosecontent as observed in LLC-PK

1 cells (Moran et al.,1983;Ohtaet al., 1990)will be determinedas well asa possible involvement of protein kinase C, mRNA-stabilizing proteins,and/or trafficking of intracellularvesicles (Shioda et al., 1994; Delézay et al., 1995;PengandLever, 1995). Also, the protein RSI, whichhas been localized to the same type of neurons asSGLTI, may be involved. RSI is able to alter theactivity of SGLTI within the plasma membrane(Koepsell and Spangenberg,1994; Lambotte et al.,1996) and has recently beenshown to participate inthesugar-dependentregulationof Na~—D-glucoseco-transportactivity (authors’ unpublisheddata).

Acknowledgment: We thank I. Schatzfor experttechni-cal assistanceand M. Christoffor preparingthe figures. Pro-vision of the peptideantibody Ab2 againstSGLTI by Prof.S. Shirazy-Beecheyis gratefully acknowledged.This workwassupportedby SFB 174, grant A 17, and SFB 194, grantB2, from the DeutscheForschungsgemeinschaft.

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