©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Differences in Glucose Transporter Gene Expression between Rat Pancreatic - and -Cells Are Correlated to Differences in Glucose Transport but Not in Glucose Utilization (*)

Harry Heimberg (1)(§)(¶), Anick De Vos (1)(§), Daniel Pipeleers (2), Bernard Thorens (3), Frans Schuit (1)

From the (1) Department of Biochemistry and (2) Metabolism and Endocrinology, Diabetes Research Center, Vrije Universiteit Brussel, B-1090 Brussels, Belgium and the (3) Institut de Pharmacologie, Université de Lausanne, CH-1005 Lausanne, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Glucose exerts inverse effects upon the secretory function of islet - and -cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat - and -cells. -Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; -cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from - or -cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa -cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In -cells, the rates of glucose transport were 10-fold higher than in -cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5H]glucose utilization, was comparable in - and -cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between - and -cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.


INTRODUCTION

Mammalian glucose homeostasis is controlled by glucose sensor mechanisms in the endocrine pancreas which adjust release of insulin and glucagon to variations in extracellular glucose concentration (1) . Experiments on isolated rodent islets of Langerhans indicate that glucose metabolism is necessary for -cells to sense extracellular glucose (2, 3, 4) . The glucose transporter GLUT2 and glucokinase are candidate glucose sensor molecules in -cells (5, 6, 7, 8) . Both proteins are expressed in -cells and hepatocytes, and their low affinity for glucose results in catalytic rates that are linearly dependent on the blood glucose concentration. GLUT2 gene expression is down-regulated in islets of several diabetic animal models (6, 9) , seems required for glucose responsiveness in ATt-20 cells (7) , but appears not responsible for heterogeneity in glucose sensitivity of normal rat -cells (10) . Glucokinase gene expression has been correlated with the rates of glucose-induced insulin synthesis and release in intact islets (11, 12, 13) as well as with the intercellular differences in -cell function (10) . Virtually no information is available on the glucose sensor in -cells which represent 10% or less of the cell mass. The ability to purify -cells from rat pancreata creates the possibility of investigating this cell type (14) . Glucose inhibits glucagon release through a direct effect on -cells (15) . Glucose transport in -cells is slower than in -cells and therefore may qualify as a rate-limiting step for overall glucose metabolism (16) . This study compares purified rat - and -cell preparations for their kinetics of glucose transport and their metabolic flux through glycolysis. It investigates to which extent differences in glucose handling are caused by differential expression of glucose transporter genes.


EXPERIMENTAL PROCEDURES

Materials

D-[5-H]Glucose (21 Ci/mmol) was purchased from Amersham (Bucks, United Kingdom); L-[1-H]glucose (20 Ci/mmol), [C]urea (56 mCi/mmol), and 3-[H]OMG() (79 Ci/mmol) from DuPont NEN.

Purification and Culture of Cells from the Endocrine Pancreas

Preparations of dispersed islet cells (20% -cells and 65% -cells), purified -cells (>90% purity), isolated endocrine non--cells (>60% -cells and <20% -cells), and purified -cells (>80% -cells and <5% -cells) were isolated from adult male Wistar rats using previously published methods (14) . The purity of these preparations was routinely determined by immunocytochemistry and radioimmunoassays for insulin and glucagon. Cells were more than 90% viable as assessed by neutral red uptake (14) . Mean intracellular space was 622 ± 48 and 270 ± 34 pl/10cells for -cells ( n = 5) and non--cells ( n = 7), respectively. Dispersed islet cells, -cells, non--cells, and -cells were cultured for 16 h in Ham's F-10 medium, 1% bovine serum albumin, 2 mmol/liter glutamine, 50 µmol/liter isobutylmethylxanthine, and 6 mmol/liter glucose at 37 °C (17) .

Glucose Uptake Measurements

Zero- trans-3-OMG uptake was measured at 15 °C or 37 °C using 1 mmol/liter [C]urea (25 mCi/mmol) as an intracellular space marker and 2 mmol/liter L-[H]glucose (200 mCi/mmol) to correct for contamination with extracellular volume (16, 18) . Initial rates of 3-OMG uptake were measured at 15 s for islet cells and -cells (1 10cells per measurement) or at 1 min for non--cells (1.5 10cells per measurement). Uptake was stopped by the addition of 150 µl of ice-cold Earle's-Hepes buffer containing 50 mmol/liter glucose and 1 mmol/liter phloridzin and centrifuged through a 150-µl oil phase (4:1 dibutyl/dinonylphthalate). The bottom phase of the tubes was counted in liquid scintillation mixture. 3-OMG uptake was expressed per liter cellular volume after correction for the extracellular space. Apparent Vand Kof glucose uptake were determined by Eadie-Hofstee plots with the best-fit lines drawn using the method of least squares.

Glucose Utilization in Purified -Cells and Non--cells

Glucose utilization was measured as the conversion of D-[5-H]glucose into tritiated water using batches of 5 10cells and 2-h incubations at 37 °C (see Ref. 19). Cellular metabolism was stopped by the addition of 20 µl of 0.4 mol/liter citrate buffer, pH 4.9, containing 5 mmol/liter KCN, 10 µmol/liter antimycin A, and 10 µmol/liter rotenone. Tritiated water production was measured via liquid scintillation counting.

mRNA Analysis

Total RNA was isolated from 10cells by microadaptation of the guanidinium isothiocyanate/cesium chloride method (20) , separated according to its size on 1.5% agarose gels containing formaldehyde and transferred by capillary blot to nylon membranes (GeneScreen, Dupont NEN). Control RNAs were taken from tissues (brain, liver, and muscle) of the same rats as were used for -cell preparation. The blots were UV-cross-linked and hybridized with P-labeled cDNA probes (Megaprime, Amersham, Bucks, UK). Two stringent washings were done for 30 min in 0.2 SSC and 2.5% SDS at 65 °C. The intensity of the signals was scanned on an Ultroscan XL densitometer (Pharmacia, Uppsala, Sweden).

Analysis of Amplified cDNA

Total RNA extracted from the control tissues and from 2.5 10-cells or -cells was reverse-transcribed in 50 mmol/liter Tris-Cl, pH 8.3, 40 mmol/liter KCl, 6 mmol/liter MgCl, 1 mmol/liter dithiothreitol with 1 mmol/liter dNTP, 200 nmol/liter poly(dT), 50 units of RNase inhibitor, and 10 units of avian myeloblastosis virus reverse transcriptase for 1 h at 42 °C. The negative control contained no RNA. First strand cDNA was dialyzed on a 0.025-mm filter and amplified via polymerase chain reaction (21) in 20 mmol/liter Tris-Cl, pH 8.3, 25 mmol/liter KCl, 2 mmol/liter MgCl, 200 mmol/liter dNTP, 50 pmol of 5`- and 3`-primer mixes, and 2.5 units of Taq polymerase for 40 cycles in a Techne PHC1 thermocycler (Techne Ltd., Duxford, Cambridge, UK) with blanks in each assay. PCR products were controlled for their length on a 1.5% agarose gel. Gene-specific primers, recognizing sequences encoding the first extracellular and the third intracellular loop (22) , were: GLUT1-5` (codon 48-54), 5`AACCACCGCTATGGAGAG; GLUT1-3` (codon 222-228), 5`GCACACTCTTGGCCCGGT; GLUT2-5` (codon 78-84), 5`GCCTGGGAAGAAGAGACT; GLUT2-3` (codon 252-258), 5`AGCTTTTCTTTGCCCTGA; GLUT4-5` (codon 64-70), 5`GGTCCTGGGGGACCGGAC; GLUT4-3` (codon 238-244), 5`GACTCTTTCGGGCAGGCC, yielding amplified fragments of 540 base pairs. Mixed oligonucleotide primed amplification of cDNA (23) was performed using two degenerate primer sets, 5`-primer set: 5`GG(A/T)GCC(C/T)TGGG(A/C)AC(A/C/T)CT(C/G/T)(C/A)ACCA (codon 154-161 of GLUT1) and 3`-primer set: 3`GTCGTC(G/A)A(C/G)AG(C/A/T)CC(G/T)TAGTTAC (codon 282-289 of GLUT1), yielding an amplified fragment of 406 base pairs. Polymerase chain reaction products were diluted (1/3, 1/10, and 1/20) and dot-blotted to nylon membranes that were hybridized with 5`-radiolabeled oligonucleotides complementary to isoform-specific sequences encoding parts of the third intracellular loop: GLUT1-3`CTTGGCCCGGTTCTCACACG (codon 221-228), GLUT4-3`CCCCGGACGGGCTTTCTCAG (codon 237-244), and GLUT2-3`TCAGTCCCGTTTCTTTTCGA (codon 251-258). After three washing steps (twice in 3 SSC, 5% SDS at 50 °C for 30 min and once in 1 SSC, 1% SDS, at 50 °C for 30 min), the hybridized membranes were exposed for autoradiography. To facilitate subcloning, EcoRI and XbaI restriction sites were included in all the 5`- and 3`-primers, respectively. Polymerase chain reaction-derived fragments were eluted from agarose gel and cloned in XbaI and EcoRI cut pBluescript SK(Stratagene). Clones were sequenced via the dideoxy method using modified T7 DNA polymerase (Pharmacia, Uppsala, Sweden).

Protein Analysis

Protein blotting and analysis was performed as described before (5) with some minor adaptations. Cell and tissue samples were homogenized by sonication in 5% SDS, 5% -mercaptoethanol, 80 mmol/liter Tris-Cl (pH 6.8), 5 mmol/liter EDTA, and 10% glycerol in the presence of 1 mmol/liter phenylmethylsulfonyl fluoride. Homogenates were separated on a 10% SDS-polyacrylamide gel and electroblotted to nitrocellulose membranes. Membranes were incubated with anti-GLUT1 (1/1000) or anti-GLUT2 (1/4000) for 60 min at room temperature. The second antibody (anti-rabbit peroxidase) was incubated at room temperature for 50 min. Peroxidase activity was detected via chemiluminescence (ECL, Amersham, Bucks, UK). The intensity of the signals was quantified via laser densitometry.


RESULTS

3-O-Methylglucose Uptake Kinetics in Purified -Cells and Non--cells

Initial rates of zero- trans-3- O-methylglucose (3-OMG) uptake in islet cells, purified islet -cells, and non--cells were measured at 15 °C to slow down the process sufficiently for accurate calculations (Fig. 1). In islet cells, uptake approached saturation at 40 mmol/liter substrate (Fig. 1 a). Transformation of the data into Eadie-Hofstee plots (Fig. 1 b) and calculation of the least squares fit for either a one- or a two-component model revealed that the latter was more adequate ( p < 0.05). A predominant kinetic component ( K= 16 mmol/liter; V= 11 mmol/min/liter) was distinguished from a second component with apparent K= 3 mmol/liter and V= 3 mmol/min/liter. Uptake of 3-OMG in purified -cells was not saturated at 40 mmol/liter substrate (Fig. 1 c). In contrast to dispersed islet cells, Eadie-Hofstee transformation of data from pure -cells showed only one component of transport with V= 14 mmol/min/liter and K= 18 mmol/liter (Fig. 1 d). Transport in islet non--cells was at least one order of magnitude slower than that measured in -cells and dose dependently increased between 1 and 15 mmol/liter 3-OMG (Fig. 1 e). Kinetic analysis of 3-OMG transport in endocrine non--cells revealed one component with K= 8.5 mmol/liter and V= 0.8 mmol/min/liter (Fig. 1 f).


Figure 1: 3-OMG uptake in rat islet cells ( a and b), purified -cells ( c and d), and non--cells ( e and f). Initial uptake velocities were measured at 15 °C using zero- trans uptake conditions. Data represent mean values from five independent experiments for islet cells and purified -cells and from seven independent experiments for non--cells. One- or two-component regression analysis of the Eadie-Hofstee plots ( b, d, and f) of the data in a, c, and e was performed to calculate the K and Vvalues.



Comparison of 3-OMG Transport and Glucose Utilization

Initial velocities of 3-OMG transport were compared with overall rates of glycolysis in pure - and non--cells (). Data were expressed per liter of intracellular space to compensate for the differences in cell size between both cell populations. In contrast to -cells, 3-OMG uptake in non--cells was slow enough to be measured at 37 °C, so that direct comparison with glucose utilization was feasible. Rates of glucose utilization increased proportionally to the extracellular substrate concentration between 1 and 10 mmol/liter substrate and were virtually identical in - and non--cells (). On the contrary, 3-OMG uptake was approximately 10 times faster in -cells than in non--cells. Despite the fact that transport was measured at a lower temperature than utilization, rates of 3-OMG uptake in pure -cells exceeded the corresponding rates of glucose utilization at least by one order of magnitude (). In non--cells, the increase from 15 °C to 37 °C accelerated 3-OMG transport 3- to 6-fold, resulting in a mean calculated Qvalue of approximately 2. Glucose transport in non--cells was between 8 and 9 times more rapid than overall glucose utilization, both at 5 and 10 mmol/liter substrate ().

Glucose Transporter Gene Transcription in Islet Cells and in Purified -Cells and -Cells

GLUT1 cDNA hybridized with a 2.8-kb transcript in both - and -cells (Fig. 2). The ratio of GLUT1 mRNA over -actin mRNA was higher in -cells than in -cells. Hybridization of -cell RNA with a GLUT2 cDNA probe resulted in detection of two transcripts, one of 2.8 kb that was relatively abundant and also found in liver, and one of 3.9 kb that was found in -cells but not in liver. No GLUT2 expression was detected in -cells. GLUT4 was not transcribed in -cells or in -cells (data not shown).


Figure 2: RNA analysis of purified -cells and -cells. 5 µg of total RNA was Northern-blotted as described under ``Experimental Procedures'' and successively hybridized with cDNA probes specific for GLUT1, GLUT2, and -actin. Autoradiographic exposure times required to generate the signals that are shown were 12 days for GLUT1, 3 days for GLUT2, and 12 h for -actin. The blot shown here is representative of three independent experiments.



Isoform-specific oligonucleotide primers were used to amplify reverse-transcribed mRNA from - and -cells. Amplification of control cDNAs from brain, liver, and muscle resulted in DNA fragments of expected length (data not shown). While GLUT1 sequences were detected in all cDNA mixtures, GLUT2 and GLUT4 sequences were present only in liver and muscle cDNAs, respectively (data not shown). The partial sequences obtained from purified islet cells were identical with previously published sequences: -cells contained GLUT1 and GLUT2 sequences and -cells contained GLUT1 and no GLUT2 sequences (data not shown). To investigate whether new members of the GLUT family were present, we performed mixed oligonucleotide primed amplification of cDNA (23) with degenerate primer sets recognizing highly conserved sequences of the GLUT family (22) . Amplified cDNA was concentrated in a single ethidium bromide-stained band with the expected fragment length (data not shown). Dot blots of the polymerase chain reaction products were hybridized with oligonucleotide probes specific for GLUT1, GLUT2, and GLUT4 sequences (15% overall identity). This method was validated on amplified mRNA from brain (GLUT1-positive), liver (GLUT2-positive), and muscle (GLUT4-positive; Fig. 3 ). From brain mRNA, a fragment was amplified that reproducibly hybridized with the GLUT4-specific oligoprobe. In purified -cells, GLUT1-cDNA was detected reproducibly, but the predominant glucose transporter was GLUT2. Amplified cDNA from -cells was only positive for GLUT1 (Fig. 3). When the amplified DNA fragments were cloned and sequenced, both GLUT1 and GLUT2 sequences were found in -cells (1 GLUT1 clone for 100 GLUT2 clones), while the obtained -cell cDNA fragments contained GLUT1 sequences (10% of all clones), or sequences that were not homologous to any protein present in the PIR (Protein Identification Resource, Washington D. C.) or Swiss-prot data banks (remaining clones; data not shown).


Figure 3: Dot-blot analysis of cDNA from -cells and -cells after amplification via mixed oligonucleotide primed amplification of cDNA (23), starting from first strand cDNAs from 2.5 10 - or -cells and from 1 µg of control tissue RNA. The negative control contained no RNA. Per sample, 5 µl of amplified cDNA and a 3-, 10-, and 20-fold dilution was blotted. Compared to -cells and controls, 5 times more amplified cDNA from -cells was applied. Primers for amplification and probes for hybridization are described under ``Experimental Procedures.''



Glucose Transporter Protein Expression in Purified -Cells and -Cells

Translation products of the described mRNAs were studied via Western blotting (Fig. 4). Positive control tissues showed the expected 40-kDa band for brain GLUT1 and 62-kDa band for liver GLUT2. Isolated -cells were weakly positive for GLUT1 and strongly positive for GLUT2 immunoreactivity (Fig. 4). The GLUT1 protein appeared larger in -cells (approximately 43 kDa) than in brain. GLUT2 immunoreactivity was distributed over an intense band of approximately 58 kDa and a much weaker 62-kDa band (Fig. 4). In -cell preparations with 3-fold more cells, only weak GLUT1 and GLUT2 signals were detected. The ratio of GLUT1 over GLUT2 abundance in -cells was 60-fold higher than in -cells. Therefore, the GLUT2 signal in -cells probably originates from contaminating -cells, while the GLUT1 signal can be considered as -cell-specific. In -cells cultured for 16 h in serum-free medium complemented with 6 mmol/liter glucose, GLUT1 protein was 4-fold more abundant than in freshly isolated -cells (Fig. 4). A comparable induction of GLUT1 expression by culture in 6 mmol/liter glucose was observed in -cells.


Figure 4: Analysis of -cell- and -cell proteins for presence of GLUT1 and GLUT2. Western blots from 25 µg of protein from the control tissues or protein from 7 10-cells or 2.5 10-cells were performed as described under ``Experimental Procedures.'' Blotting efficiency was established by Ponceau staining of total protein. Exposure times were 30 min for GLUT1 and 5 min for GLUT2. The blot shown here is representative for four independent experiments. 16h G6 = 16 h of culture in serum-free medium supplemented with 6 mmol/liter glucose.



GLUT2 Protein Heterogeneity and Glucose Uptake

We then investigated whether the molecular heterogeneity of GLUT2 protein was correlated to the rate of glucose uptake. The ratio of the densitometric intensities of the 62-kDa versus the 58-kDa band was 10-fold lower in freshly isolated -cells (0.09 ± 0.05) than in 16-h cultured -cells (0.90 ± 0.10; p < 0.005; n = 3) (Fig. 5, lanes 1 and 2). This was not influenced by adding the trypsin inhibitor antipain (3 mg/ml) to the culture medium (Fig. 5, lane 3). Incubation of cultured -cells with trypsin (3 mg/ml, 10 min, 37 °C) resulted in a rapid disappearance of the 62-kDa band (Fig. 5, lane 4); this effect did not occur when antipain was present during trypsin exposure (Fig. 5, lane 5). Freshly isolated islet cells exhibited only the 58-kDa GLUT2 protein (Fig. 5, lane 6), but after 16 h of culture only 62-kDa GLUT2 immunoreactivity was found (Fig. 5, lane 7). As for isolated -cells, short trypsin treatment of cultured islet cells shifted the 62-kDa GLUT2 isoform to the 58-kDa isoform (Fig. 5, lane 8). The untreated and trypsin-treated cultured islet cell preparations, with, respectively, 62-kDa and 58-kDa GLUT2 protein, were compared for their glucose uptake kinetics, using the two-component model. Both preparations displayed low and high Kuptake with similar kinetic characteristics ().


Figure 5: The effect of -cell culture on GLUT2 expression. 10-cells were homogenized immediately after isolation ( lane 1), after 16 h of culture ( lane 2), after 16 h of culture in the presence of antipain ( lane 3), after 16 h of culture followed by 10-min treatment with trypsin (3 mg/ml, 37 °C) ( lane 4), and after 16 h of culture followed by addition of trypsin and antipain ( lane 5). Islet cells were used immediately after isolation ( lane 6) or after 16 h of culture ( lane 7), and after 16 h of culture followed by a 10-min incubation with trypsin ( lane 8). Ponceau staining of total protein was comparable in all lanes (not shown). Exposure time was 5 min. The blot shown here is representative for three independent experiments.




DISCUSSION

The present study confirms (18) that the kinetics of glucose uptake by unpurified islet cells is characterized by two components, one with a high Kand high V, the other with a low Kand low V. Analysis of glucose transport in purified - and non--cells indicates that the high Kcomponent is a feature of the -cells. The low Kcomponent is, however, not recovered in one of the two purified preparations, which suggests its association with islet cells, which are not selected during the purification procedure. Alternatively, low Kglucose uptake does occur in islet -cells, but the absence of non--cells alters the intrinsic activity of the expressed glucose transporters, for instance by altering their phosphorylation/dephosphorylation state. The preparation of endocrine non--cells displayed glucose transport with low capacity and intermediate affinity compared to the low V/low Ktransport observed in islet cells. Our data do not exclude that high affinity glucose transporters are expressed in non--cells and that their intrinsic activity and affinity for glucose are dependent on -cell factors.

Isolated -cells transcribe the GLUT1 gene and express the protein. The GLUT1 protein was more abundant after overnight culture at 6 mmol/liter glucose, which is compatible with previous data on isolated islets (24, 25) . Accumulation of GLUT1 through decreased protein degradation at 6 mmol/liter glucose has been proposed as part of the glucose sensing mechanism in fibroblasts (26) , but its biological relevance for -cells remains elusive. It is for instance conceivable that GLUT1 is a stress protein (27) which becomes more abundant during culture at low glucose levels. Since the -cell GLUT1 protein has not yet been detected in situ, it is possible that its expression was induced by the isolation procedure and maintained in cell culture. Whether this phenomenon influences glucose uptake has not been investigated. At both the mRNA and protein level, GLUT2 was the major glucose transporter in isolated -cells. The GLUT2(-like) mRNA occurred as a 2.8-kb and a 3.9-kb transcript. The cDNA corresponding to the 2.8-kb mRNA has been cloned (5) , but the sequence and significance of the larger transcript is not yet known. GLUT2 protein also displayed molecular heterogeneity, immunoblots reproducibly revealing proteins of 58 and 62 kDa. The 62-kDa protein can be rapidly converted to the 58-kDa form by proteolytic cleavage, indicating that the enzymatic procedures of islet isolation and cell preparation may have caused the appearance of the lower molecular weight form. Proteolytic cleavage occurs in the first extracellular loop of GLUT2 (28) . By combining functional and molecular measurements, we have now shown that shifts between the 62- and 58-kDa forms do not affect the glucose transport kinetics of -cells. Our data support the view that residues in the carboxyl-terminal region of GLUT2 determine its kinetic properties for glucose uptake (29) . They also indicate that the two components of glucose uptake in dispersed islet cells cannot be attributed to heterogeneity in GLUT2 protein. Recovery of the large GLUT2 isoform was more complete after culture of unpurified islet cell preparations than of pure -cells; it was also enhanced by culture at 20 mmol/liter glucose (data not shown). The presence of endocrine non--cells may add to the regulation of GLUT2 gene expression by the -cells. The glucose effect is probably caused by increased de novo synthesis, since glucose is known to increase GLUT2 gene expression (30) .

Endocrine islet non--cells did not exhibit specific GLUT2 expression, but they contained both GLUT1 mRNA and protein albeit at low abundance. Preparations with more than 80% -cells (14) also exhibited these GLUT1 signals, suggesting their association with -cells. Contamination with non--cells is still present, but we consider it unlikely that this explains the GLUT1 signals for the following reasons: ( a) preparations highly enriched in -cells were negative or very weakly positive for GLUT2, excluding a major contamination with -cells; ( b) cell culture which leads to selective loss of contaminating exocrine cells did not result in loss of GLUT1 expression in -cell preparations; and ( c) amplification of cDNA from -cells resulted in GLUT-nucleotide sequences that were identical with GLUT1. So far, GLUT1 protein has not been detected in -cells by immunocytochemical staining (24) but this may be related to the sensitivity limit of this technique. Since we sequenced only 15% of the GLUT1 sequence, the glucose transporter in -cells may be a GLUT1-related protein, with a specific amino- or carboxyl-terminal region, but cross-reacting on Western blots with an antibody directed against the carboxyl terminus of GLUT1. It has been proposed that insulin plays a permissive role for glucose-induced inhibition of glucagon secretion (31) . We therefore screened -cell RNA for the presence of sequences homologous to the insulin-responsive glucose transporter GLUT4. No GLUT4 sequences were found, which is compatible with the previous observation that insulin does not stimulate glucose transport rates in -cells (16) .

The absence of low K3-OMG transport in GLUT1-positive -cells may be explained by cryptic expression of the GLUT1 gene, but this seems unlikely, since GLUT1 protein has been localized in the plasma membrane of cultured islet -cells (24, 25) . Alternatively, rates of glucose uptake via GLUT1 in -cells may be too low to detect under conditions of simultaneously elevated rates of high K/high Vtransport via GLUT2. Rat pancreatic -cells display metabolic and functional heterogeneity in terms of glucose responsiveness (32) . The possibility that -cell subpopulations with different sensitivity for glucose differ in glucose transporter gene expression has been excluded (10) . Indeed, virtually all insulin-positive islet cells also contain membrane-bound GLUT2 immunoreactivity in islet sections (24) . GLUT1 and GLUT2 may be expressed on the same -cells at a different subcellular localization, for instance GLUT2 at the afferent pole (glucose sensor) and GLUT1 at the basal pole (33) . Increased GLUT2 immunoreactivity in the microvillous compartment of -cells supports this concept (34) . Subcellular immunodetection of GLUT1 and GLUT2 in -cells by electron microscopy will be required to further examine this possibility.

Comparison of overall flux via glucose utilization and 3-OMG uptake is possible since both processes were studied under the same experimental conditions and since the kinetic constants for glucose uptake and 3-OMG uptake are similar (35) . In pure rat -cells, glucose uptake is such a rapid process that its rate largely exceeds that of glycolysis even when measured at low temperatures. Abundant expression of GLUT2 renders the plasma membrane almost freely permeable to glucose, permitting the cytoplasmic glucose sensor to measure minute-to-minute variations in the extracellular glucose concentration. In -cells, absence of GLUT2 and low expression of GLUT1 result in glucose uptake rates which are 10-fold lower than in -cells, but they are still 10-fold higher than overall metabolic flux, strongly suggesting that transport in this cell type is not rate-limiting for overall glucose metabolism. It has been proposed that some glucose transporter isoforms may influence the process of glucose sensing via nonmetabolic properties, such as the structural organization of sensor proteins close to or in the plasma membrane (7) . Alternatively, enzymatic steps distal to glucose transport may be more directly responsible for glucose sensing in rat - and -cells.

  
Table: Glucose uptake and glucose utilization in - and non--cells

Data represent mean values ± S.E. of ( n) independent experiments. ND, not determined.


  
Table: Effect of trypsin on 3-O-methylglucose transport kinetics of islet cells

Apparent Vand Kwere determined by Eadie-Hofstee plot of the mean values from five independent experiments with the best-fit lines drawn using the method of least squares.



FOOTNOTES

*
This study was supported by Grant IUAP 15 from the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, FGWO Grant 3.0127.93 from the Belgian Fund for Medical Scientific Research, a grant from the Research Council of the Vrije Universiteit Brussel (OZR), and Concerted Action 92/97-1807 from the Flemish Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Both authors contributed equally to this study.

Present address: Dept. of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110.

The abbreviations used are: 3-OMG, 3- O-methylglucose; kb, kilobase pair(s).


ACKNOWLEDGEMENTS

We thank Erik Quartier for his excellent technical assistance, the personnel of the Dept. of Endocrinology and Metabolism for islet cell purification, and Dr. L. Kaufman for statistical advice. We gratefully acknowledge Dr. M. Birnbaum, Harvard Medical School, for providing the rat GLUT1 cDNA.


REFERENCES
  1. Gerich, J. E., Charles, M. A., and Grodsky, G. M. (1974) J. Clin. Invest. 54, 833-841 [Medline] [Order article via Infotrieve]
  2. Meglasson, M. D., and Matschinsky, F. M. (1986) Diabetes Metab. Rev. 2, 163-214 [Medline] [Order article via Infotrieve]
  3. MacDonald, M. J. (1990) Diabetes 39, 1461-1466 [Abstract]
  4. Randle, P. J. (1993) Diabetologia 36, 269-275 [Medline] [Order article via Infotrieve]
  5. Thorens, B., Sarkar, H. K., Kaback, H. R., and Lodish, H. F. (1988) Cell 55, 281-290 [Medline] [Order article via Infotrieve]
  6. Unger, R. H. (1991) Science 251, 1200-1205 [Medline] [Order article via Infotrieve]
  7. Hughes, S. D., Quaade, C., Johnson, J. H., Ferber, S., and Newgard, C. B. (1993) J. Biol. Chem. 268, 15205-15212 [Abstract/Free Full Text]
  8. Matschinsky, F., Liang, Y., Kesavan, P., Wang, L., Froguel, P., Velho, G., Cohen, D., Permutt, M. A., Tanizawa, Y., Jetton, T. L., Niswender, K., and Magnuson, M. A. (1993) J. Clin. Invest. 92, 2092-2098 [Medline] [Order article via Infotrieve]
  9. Thorens, B. (1992) Int. Rev. Cytol. 137A, 209-238 [Medline] [Order article via Infotrieve]
  10. Heimberg, H., De Vos, A., Vandercammen, A., Van Schaftingen, E., Pipeleers, D., and Schuit, F. (1993) EMBO J. 12, 2873-2879 [Abstract]
  11. Epstein, P. N., Boschero, A. C., Atwater, I., Cai, X., and Overbeek, P. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12038-12042 [Abstract]
  12. German, M. S. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1781-1785 [Abstract]
  13. Efrat, S., Leiser, M., Wu, Y.-J., Fusco-DeMana, D., Emran, O. A., Surana, M., Jetton, T. L., Magnuson, M. A., Weir, G., and Fleischer, N. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2051-2055 [Abstract]
  14. Pipeleers, D. G., in't Veld, P. A., Van De Winkel, M., Maes, E., Schuit, F. C., and Gepts, W. (1985) Endocrinology 117, 806-816 [Abstract]
  15. Pipeleers, D. G., Schuit, F. C., Van Schravendijk, C. F. H., and Van De Winkel, M. (1985) Endocrinology 117, 817-823 [Abstract]
  16. Gorus, F., Malaisse, W. J., and Pipeleers, D. (1984) J. Biol. Chem. 259, 1196-1200 [Abstract/Free Full Text]
  17. Ling, Z., Hannaert, J. C., and Pipeleers, D. (1994) Diabetologia 37, 15-21 [CrossRef][Medline] [Order article via Infotrieve]
  18. Johnson, J. H., Newgard, C. B., Milburn, J. L., Lodish, H. F., and Thorens, B. (1990) J. Biol. Chem. 265, 6548-6551 [Abstract/Free Full Text]
  19. De Vos, A., Schuit, F. C., and Malaisse, W. J. (1991) Biochem. Int. 24, 117-121 [Medline] [Order article via Infotrieve]
  20. Rappolee, D. A., Brenner, C. A., Schultz, R., Mark, D., and Werb, Z. (1988) Science 241, 1823-1825 [Medline] [Order article via Infotrieve]
  21. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K., Horn, G., Erlich, H. A., and Arnheim, N. (1985) Science 230, 1350-1353 [Medline] [Order article via Infotrieve]
  22. Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941-945 [Medline] [Order article via Infotrieve]
  23. Lee, C. C., Wu, X., Gibbs, R. A., Cook, R. C., Muzny, D. M., and Caskey, T. (1988) Science 239, 1288-1290 [Medline] [Order article via Infotrieve]
  24. Tal, M., Liang, Y., Najafi, H., Lodish, H. F., and Matschinsky, F. M. (1992) J. Biol. Chem. 267, 17241-17247 [Abstract/Free Full Text]
  25. Tal, M., Thorens, B., Surana, M., Fleischer, N., Lodish, H. F., Hanahan, D., and Efrat, S. (1992) Mol. Cell. Biol. 12, 422-432 [Abstract]
  26. Haspel, H. C., Wilk, E. W., Birnbaum, M. J., Cushman, S. W., and Rosen, O. M. (1986) J. Biol. Chem. 261, 6778-6789 [Abstract/Free Full Text]
  27. Wertheimer, E., Sasson, S., Cerasi, E., and Ben-Neriah, Y. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2525-2529 [Abstract]
  28. Thorens, B., Gérard, N., and Dériaz, N. (1993) J. Cell Biol. 123, 1687-1694 [Abstract]
  29. Katagiri, H., Asano, T., Ishihara, H., Tsukuda, K., Lin, J. L., Inukai, K., Kikuchi, M., Yazaki, Y., and Oka, Y. (1992) J. Biol. Chem. 267, 22550-22555 [Abstract/Free Full Text]
  30. Chen, L., Alam, T., Johnson, J. H., Hughes, S., Newgard, C. B., and Unger, R. H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4088-4092 [Abstract]
  31. Greenbaum, C. J., Havel, P. J., Taborsky, G. J., and Klaff, L. J. (1991) J. Clin. Invest 88, 767-773 [Medline] [Order article via Infotrieve]
  32. Pipeleers, D. G. (1992) Diabetes 41, 777-781 [Abstract]
  33. Mueckler, M. (1990) Diabetes 39, 6-11 [Abstract]
  34. Orci, L., Thorens, B., Ravazzola, M., and Lodish, H. F. (1989) Science 245, 295-297 [Medline] [Order article via Infotrieve]
  35. Gould, G. W., Thomas, H. M., Jess, T. J., and Bell, G. I. (1991) Biochemistry 30, 5139-5145 [Medline] [Order article via Infotrieve]

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