Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line.

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.

m~ glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.
The GLUT2 glucose transporter isoform plays a major role in glucose-induced insulin secretion in the pancreatic p-cell by catalyzing the uptake of glucose into the cell (1). The regulated expression of this transporter has been studied in vitro by cell culture and in a number of animal models which have an imbalanced glucose homeostasis (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). In diabetes, the expres-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sion ofthis transporter is regulated in a tissue-specific manner. In particular, in every animal model of diabetes studied such as the diabetic Zucker faKa rat, the neonatal low-dose streptozocininduced diabetic rat, the GK rat, the db/db mouse or at the onset of diabetes in the BB/W rat, there is a strong reduction in GLUT2 gene expression which is restricted to the pancreatic p-cells while its expression in liver, intestine, or kidney is unaltered or slightly increased (8)(9)(10)(11)(12). From these data one could conclude that hyperglycemia of diabetic animals is responsible for the observed decrease in expression of GLUT2 in p-cells. However, exposure of pancreatic islets to high glucose in vitro leads to an increase in GLUT2 mRNA and protein levels. In diabetes it is therefore not clear what causes GLUT2 down-regulation. Experiments in which development of hyperglycemia was prevented by ascarbose treatment in Zucker diabetic rats showed that even in the presence of normoglycemia, GLUT2 expression steadily decreased over time (8). Taken together, the above observations suggest that in vitro glucose may directly stimulate GLUT2 gene expression while in the diabetic state the hyperglycemia causes a decreased GLUT2 expression.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankm/EMBL Data Bank with accession number(s1 X78722
To better understand the glucose-regulated expression of GLUT2, we studied its controlled expression by glucose in a highly differentiated insulinoma cell line and initiated the characterization of the promoter region of the transporter gene. We cloned and characterized the upstream regulatory region of the mouse GLUT2 gene and defined common initiation start sites in murine tissues which express GLUTS, i.e. pancreatic p-cells, liver, intestine, and kidney. The human and mouse promoters were functionally tested using a chloramphenicol acetyltransferase (CAT)' reporter system transiently or stably transfected into a highly differentiated p insulinoma cell line, INS-1 (13). By increasing the concentration of glucose in the culture medium, a concentration-dependent transcriptional activation of the human and mouse GLUT2 gene was shown.
Similarly, the endogenous GLUT2 gene expression was assessed in INS-1 cells by nuclear run-on and Northern blot analysis. As observed with CAT reporter constructs driven by the murine and human promoter, GLUT2 expression is transcriptionally regulated by glucose. Therefore, the 5"regulatory regions of the human and mouse GLUT2 genes contain carbohydrate-responsive element&) responsible for the glucoseinduced gene transcription of GLUT2 observed in INS-1 cells.

MATERIALS AND METHODS
Cell Culture-The transplantable x-ray-induced rat insulinoma INS-1 cell line was kindly provided by Asfari et al. (13). INS-1 cells were grown in RPMI 1640, 10% fetal calf serum, 10 m~ HEPES, 2 mM Lglutamine, 1 mM sodium pyruvate, 100 unitdm1 penicillin, 100 pglml The abbreviations used are: CAT, chloramphenicol acetyltransferase; MOPS, 4-morpholinepropanesulfonic acid; bp, base paids); kb, kilobase paids). Isolation of Nuclei and Cytoplasmic RNA-INS-1 cells from one 15-cm dish were used for each nuclear isolation. Nuclei were isolated as described by Marie et al. (14) with the exception that 5% (w/w) Nonidet P-40 was used. Before recovering the nuclear pellet, 3 ml of the cytoplasmic fraction were collected and RNA was isolated according to AUSubel et al. formamide, 0.1 M NaPO,, pH 6.5,0.5% SDS, 1 m M EDTA, 2 x Denhardt's (5 x for prehybridization), and 200 pg/ml yeast tRNA. Filters were washed in 2 x SSC, 0.1% SDS at room temperature followed by two washes for 20 min in 0.2 x SSC, 0.1% SDS at 55 "C and then exposed to Hyperfilm-MP (Amersham) at -70 "C for 7-10 days. Films were quantified using a Molecular Dynamics scanner (Sunnyvale, CAI. Northern Blot Analysis-Cytoplasmic RNAs isolated from the cells for the nuclear run-on experiments were quantified and equal amounts (10 pg) were size-fractionated on 1 x MOPS, 1.2% agarose gels containing formaldehyde. Gels were transferred overnight by diffusion (10 x SSC) to Gene Screen membrane (Du Pont). Membranes were W crosslinked and baked 2 h at 80 "C. After prehybridization, the blots were hybridized with random primed (Boehringer, Mannheim, Germany) rat GLUT2 and actin probes in 5 x SSC, 100 m M NaPO,, pH 6.5, 5 x Denhardt's solution, 50% formamide, 10 m EDTA, 1% SDS, and 100 pg/ml yeast tRNA overnight at 42 "C. The blots were washed in 2 x SSC followed by 0.2 x SSC, 0.1% SDS at 60 "C. Directly after washing, the blots were quantified by electronic autoradiography with an Instant Imager 2024 (Packard Instrument Co.). Blots were then exposed to Hyperfilm-MP (Amersham). Isolation and Structural Analysis of the Mouse GLUT2 Promoter-The screening of a mouse genomic library with a mouse GLUT2 cDNA probe (17) identified 4 positive bacteriophage clones which together contained most of the GLUT2 gene. The 18-kb insert of clone 4 which encodes the promoter and the first three exons of GLUT2 was subcloned into a pBluescript K S ' (Stratagene, La Jolla, CA). Both strands of the promoter region, exons and exon-intron boundaries were sequenced using the Sequenase sequencing kit (U. S. Biochemical Carp.).
RNase Protection and Primer Extension-Mouse tissues were homogenized in 9 ml of 4 M guanidine isothiocynate buffer with a Kinametic polytron blender (Kriens, Switzerland) and layered onto a 4-ml 5.7 M CsCl cushion. RNAs were pelleted at 33,000 rpm for 17 h in a 50 Ti rotor. Mouse islet RNA was obtained from islets isolated by the method of Gotoh et al. (18). Antisense RNA probes were synthesized using subclones of the mouse promoter region (-1311 to +70 and -335 to +70 bp) as templates and T7 RNA polymerase (Promega). The RNase protection assay was carried out according to the protocol of the RPA I1 kit (Ambion, Austin, TX). Primer extension products were synthesized using an antisense primer 5'-CTTGTCTTCTGACA'I"l'GTGTG-3' which was end-labeled with [y-32PlATP and extended by reverse transcriptase (19). All probes and oligonucleotides were labeled with radionucleotides purchased from Amersham. The products of the RNase protection or primer extension assays were separated on a 6% polyacrylamiddurea sequencing gel. A sequencing reaction primed with the same oligonucleotide as used for the primer extension was run as a sizing marker.
lkanscriptional Reporter Constructs and Transfection Studies-Polymerase chain reaction-generated regions of the mouse promoter from -1311 to +49 or the human promoter from -1296 to +312 bp were subcloned into the SalYXbaI sites of the promoterless pCAT-basic vector (Promega).
The transcriptional reporter constructs were sequenced and then transiently transfected into INS-1 cells by liposome-mediated DNA transfection (DOTAP, Boehringer). Ten pg of either construct were usually co-transfected into 1-2 x lo6 cells, with a pSV, galactosidase reporter gene (pSV,GAL) as internal control. Twenty fours hours after transfection, glucose concentrations of the medium were changed (0,5, 10, and 20 mM) and the cells incubated for an additional 24 h. The cells were harvested and then ruptured by three freeze-thaw cycles, as described by Pothier et al. (20). After removal of cellular debris, the extracts were heated for 10 min at 65 "C to destroy endogenous deacetylating activity. Protein concentrations were determined using the BCA protein assay (Pierce). CAT assays were carried out using 100-150 pg of cell extracts and the acetylated chloramphenicol was separated on a thin layer chromatography plate. The results were normalized by the value of galactosidase activity measured from the co-transfection of pSV,GAL and/or adjusted to the protein concentration of the cell extracts. The CAT-enzyme-linked immunosorbent assay system (Boehringer) was also used in some studies to quantitate the CAT protein of the transfection studies. The correct initiation start site used in the fusion mouse promoter CAT constructs was determined by primer extension of transfected INS-1 cell RNA using a complementary oligonucleotide to the CAT gene (5'-TTACGATGCCATTGGG-3').
To generate stably transfected INS-1 cells, the murine and human GLUT2 promoter CAT constructs were co-transfected with a pSV, neomycine plasmid (10 pg of CAT constructs and 2 pg of the neomycine plasmid) and the neomycine-resistant clones were then studied for integration of the CAT transgenes.
Statistics-All RNAor transfection studies were carried out in four or eight separate experiments, respectively. Data are expressed as mean +. S.E. and compared by the (nonparametric) Friedman test.

RESULTS
Identification of the Mouse Upstream Regulatory Region of the GLUT2 Gene-By screening a mouse genomic library with a GLUT2 cDNA probe, we isolated 4 overlapping bacteriophage clones which together contained most of the GLUT2 gene. The overall gene structure and exon-intron boundaries are similar to the recently published human GLUT2 gene (21). The 18-kb insert of clone 4 contains the first three exons of the gene and approximately 1.4 kb of the putative promoter region of GLUT2 (Fig. LA). The nucleic acid sequence of the mouse upstream regulatory region is shown in Fig. 1B. By computer analysis, several potential consensus sequences for various transcription factors (22-28) are located within the 1.3-kb promoter region.
The cis elements include a cyclic AMP responsive element (CRE = ACGTCA 6/61, an AP-1, AP-4, and a CCAAT-box-binding transcriptional factor (CTF-NF1 10/14 TGatgGTAAtCCAA). The role of these sequences in the control of GLUT2 gene expression needs to be established. A potential TATAA-like motif and CAAT box are located at -34 and -99 bp, respectively, of the major initiation start site and may be involved in mediating the basal transcriptional activity of the GLUT2 gene.
Nucleic acid comparison of the human and mouse 5"regulatory region of GLUT2 gene shows approximately 50% sequence identity between the human (-398 to + 222) and the murine (-579 to -25) promoters which suggests that these conserved regions are of functional importance (Fig. 1C).
Danscriptional Start Sites Are Common in GLUT2 Expressing Tissues-The initiation start sites were localized by RNase protection and primer extension assays. For the RNase protection assay, an antisense RNA probe corresponding to nucleotides -329 t o +70 of the mouse promoter sequence was hybridized in solution with RNAs extracted from different tissues and mouse promoter of GLUTS. A major initiation start site was localized at -55 bp from the first coding ATG which determined the +1 bp coding system (arrow and Tin bold character). Two minor start sites were also localized at -64 and -115 from the ATG encoding the first methionine. Potential TATAA and CAAT boxes are present at -34 and -99 bp, respectively, of the major transcriptional start. Several potential consensus sites for various transacting factors have been determined by computer analysis and shown with arrows (see results for description of the putative responsive elements). C, nucleic acid comparison of the human and mouse 5'-regulatory region of GLUT2 gene, Approximately 50% sequence identity was found between the human (-398 to +222) and the mouse (-579 to -25) promoters which may suggest that these conserved regions are of functional importance (-indicates identical sequences and A indicates gap in the sequence comparison).   +70 g t a c a g c O a c a t g e g g t c c t t t~u t t g g~g a g g~g t t~g a a t c t a t l l~~~~g a~a Fig. 2A shows a typical RNase protection assay, where the first coding ATG. The protected transcripts are present in the 399-base probe protects 3 major transcripts after RNase A and mouse islet, liver, intestine, and kidney RNAs but not in mouse T1 digestion, labeled 1,2, and 3 in the figure. These transcripts heart, whole pancreas (the endocrine pancreas representing a RNAprobe that contained the -329 to +70 bp region of the promoter. No protected transcripts were found in the negative controls which include tRNA, heart, and whole pancreas RNAs. A positive control was made from a sense RNA synthesizied in vitro. In all tissues expressing GLUT2, the probe protected 3 major transcripts defined as 1,2, and 3. Number 3 is the strongest signal and corresponds to the start site defined by primer extension, and is considered as the major initiation site for RNA polymerase 11. The intensity of the protected transcript corresponds to the expected amount of GLUT2 mRNA expression in these tissues: higher intensity is seen in islet and liver RNAs and lower intensity is found in the kidney and the intestine RNAs. B, the same RNA used to determine the start sites were hybridized with an actin antisense probe to show equivalent RNA loading in the various columns in the ribonuclease protection assay. small percentage of the organ), or tRNA which were used as negative controls. An actin antisense RNA probe was hybridized to the same RNAs to quantitate the amount of RNA (Fig.  2B ). The protected transcript mapped at -55 bases (number 3, Fig. 2 A ) is the major start site as confirmed by primer extension and several other RNase protection assays using a larger 1400-base antisense RNA probe (data not shown). Additional lower frequency start sites were also identified (transcripts 1 and 2 in Fig. 4A) in all tissues expressing GLUT2 which suggests the presence of multiple initiation start sites for the RNA polymerase 11. The intensity of the protected transcripts of Fig.   2A corresponds to the abundance of GLUT2 mRNA expressed in these tissues. The highest level being found in pancreatic islets and liver, whereas, in kidney and intestine, it is lower. By primer extension (using a complementary oligonucleotide to sequence +49 to +70 of the mouse promoter sequence) one major start site was identified (data not shown), which corresponds to the -55-bp transcript (transcript number 3, Fig. 2A),

-12 G C T C~C A G C T~~C A~~~C A G T A C A G G A C C T~G A~~~~~G~C
and was subsequently used to define the start site (bp +1 of the sequence shown in Fig. 1B).
Mouse and Human Promoters Are Functionally Active and Glucose-responsive When Dansiently or Stably Dansfected into a Differentiated p Insulinoma Cell Line-Regions of the mouse (-1311 to +49) and human (-1296 to +312) promoters were cloned into the eukaryotic expression vector pCAT Basic (Promega) and transiently transfected into a differentiated p insulinoma cell line, INS-1, which expresses a high level of GLUT2. Cells were then exposed for 24 h to a medium containing fetal calf serum and 0 or 20 mM glucose. As shown in Fig. 3, basal transcriptional activity, as measured by CAT assay, is higher for the mouse promoter in comparison to the promoterless vector pCAT Basic. Furthermore, the mouse promoter CAT fusion construct shows a glucose inducibility of 2-3-fold with 20 m M glucose in comparison to the basal transcriptional rate seen during incubation with 0 mM glucose. The pCAT basic vector did not show any change in activity when incubated with 0 or 20 mM glucose. Since all transfections studies were done in 5% fetal calf serum, the 0 mM glucose concentration corresponds to approximately 1-1.5 mM glucose as measured in the culture medium at the end of each experiment.
Similar transfection experiments were undertaken to determine if the human and the mouse regulatory regions have an identical glucose inducibility, and if so, whether it is concentration-dependent. A typical CAT assay is shown in Fig. 4A where CAT activities for both human and mouse promoter constructs were found to increase in a concentration-dependent manner during incubation with glucose concentrations of 0, 5, 10, and 20 mM. The transfections were repeated 6 times and CAT activity was normalized by protein content of the transfected cell extracts andor by the activity of P-galactosidase expressed from a co-transfected pSV, galactosidase reporter gene. Quan-  (pCAT-basic). The human and mouse promoters were glucose-responsive in a dose-dependent manner from 0 to 20 mM of glucose for an incubation time of 24 h. The arrows designated the mono-, bi-, and triacetylated chloramphenicol forms were separated by thin layer chromatography. B, quantitative assessment of transcriptional glucose responsiveness of the human and mouse GLUT2 promoters. Six different transfection experiments were carried out and CAT activity or protein measured by counting "C-acetylated chloramphenicol or by the CAT-enzyme-linked immunosorbent assay system. The CAT activity was normalized by protein content of cell extracts andor by the co-transfection of a pSV, galactosidase reporter gene. Glucose induced a dose-dependent transactivation of the mouse GLUT2 promoter: there was an increase in CAT activity of 1.4-, 2.5-, and 5.1-fold a t glucose concentrations of 5, 10, and 20 mM, respectively, compared to baseline (glucose = 0 mM). Similarly, glucose induced a 1.3-, 1.8-, and 2.8-fold transactivation of the human GLUT2 promoter when exposed to 5, 10, and 20 mM (* = p < 0.05, ** = p < 0.01, and *** = p < 0.001). Data are shown as the mean 2 S.E.

GLUT2 Gene Expression
titative analyses are shown in Fig. 4B. Glucose concentrations of 5, 10, and 20 mM induced a significant 1.3-, 1.8-, and 2.8-fold, respectively, increase in CAT activity for the human promoter and a 1.4-, 2.5-, and 5.1-fold increase in CAT activity for the mouse promoter when compared to the baseline (defined as 0 mM glucose). The correct initiation start site used by the mouse promoter CAT construct was analyzed by primer extension of RNA from the INS-1 cell transfected with mAl311CAT. The major transcriptional start site corresponds to the +1 bp defined in Fig. 1B (data not shown).
To avoid variation of transfection efficiency in between experiments, we generated stably transfected INS-1 cells that integrated the murine promoter or human promoter CAT constructs together with a neomycine-resistant plasmid. Isolated clones were similarly studied for CAT activity when exposed to 0,5, 10, and 20 mM glucose for 24 and 48 h. The glucose inducibility of the CAT transgenes were quite similar to the transient transfection observed after 24 h. After 24 or 48 h of treatment, the maximum glucose inducibility was 3.6-fold higher than the 0 mM conditions. Therefore, these promoters were glucose-re-sponsive in a concentration-dependent manner (Fig. 5, A-C).
Glucose Effect on GLUT2 Gene Dunscription Rate and mRNA Accumulation-GLUT2 gene expression was studied in INS-1 cells exposed to 2,10, and 20 mM glucose over a 4-, 8-, and 24-h period. Glucose-induced GLUT2 gene transcription as assessed by nuclear run-on analysis (Fig. 6, A and B). A maximal 4-and 3.4-fold increase of GLUT2 was observed with 20 mM and 10 mM glucose, respectively, as compared to the 2 mM condition.
Cytoplasmic RNAs of the nuclear run-on experiments were collected and studied by Northern blot analysis. As expected from the dependence of GLUT2 transcription rate on the glucose concentration, GLUT2 mRNA accumulation increased in a concentration-dependent manner. A concentration of 20 mM glucose led to a 2.3-fold higher level of mRNA accumulation as compared to 2 mM glucose (Fig. 7, A and B). The experiments were repeated 3 times with similar results and all data were normalized to actin. DISCUSSION In this study, we have structurally characterized 1.4 kb of the mouse GLUT2 promoter and shown that this sequence shares u 48 hours regions of identity with the previously described human promoter (21). The presence of stretches of complete identity between the species interspersed with more divergent sequences suggests that such regions may be of functional importance in the control of gene expression. By RNase protection assay and primer extension we have defined common start sites in all mouse tissues expressing GLUTS. This finding is inconsistent with the use of alternative promoters or initiation start sites for cell-specific expression of GLUTB, as has been described for another gene involved in glucose-sensing, the glucokinase gene, where two different cell-specific promoters are found in the liver and p-cells (29). In experimental diabetes, GLUT2 mRNA expression increases in liver, whereas it decreases in P-pancreatic cells (1, [8][9][10][11][12] and it therefore seems unlikely that the use of alternative promoters or different start sites is responsible for this difference in cell-specific regulation of GLUT2. The structurally characterized mouse GLUT2 promoter region and the human GLUT2 B'-upstream region were then functionally tested using a CAT reporter system by transient or transcription was observed. The murine GLUTBCAT construct shows a maximum 2.5-fold increase and 5.1-fold increase for the 10 and 20 mM glucose concentration, respectively, when compared to the 0 mM glucose condition. The human promoter CAT transgene was similarly induced in a concentration-dependent manner. Although modest, the glucose inducibility was reproducible in transiently or stably transfected cells with both transgenes. These data suggest that the isolated murine and human GLUT2 promoters contain unidentified carbohydrateresponsive elements. Several genes, such as insulin, S14, and pyruvate kinase, have been shown to be transcriptionally regulated by glucose (14,(30)(31)(32)(33)(34)(35). In the rat 1 and human insulin genes, an AT-rich sequence has been proposed as the specific glucose-responsive element (34). However, in S14 and the pyruvate kinase genes, E boxes have been clearly shown to be responsible for the carbohydrate responsiveness with a common core element: CACGTG. The consensus is a putative binding site for MLTFNSF or other basidhelix-loop-helideucine zipper proteins. Diaz Guerra et al. (35) have recently shown that the L4 elements of the pyruvate kinase gene bind various nuclear proteins that include the ubiquitous MLTFNSF protein.
In the case of the murine 5"regulatory region of GLUTS, we found no perfect consensus for the core CACGTG defined as E boxes. At -879 to -873 or -472 to -466 of the murine GLUT2 sequence two CACaGGG sequences share partial analogy to the L4 proximal element defined in the pyruvate kinase gene (CACGGG). At -592 to -587, a GTGCCA sequence has a weak analogy to the distal L4 element of the pyruvate kinase gene (GTGCCC). Several AT-rich stretches are present in the murine and human promoters but these structurally defined sequences need to be functionally tested to see if they are able to confer glucose responsiveness on heterologous promoters.
We have demonstrated by nuclear run-on analysis that INS-1 cells exposed to medium with 10 and 20 mM glucose have a maximum 3.4-and 4-fold increase, respectively, in endogenous GLUT2 gene transcription. Furthermore, cytoplasmic RNA of the run-on analysis have shown a parallel induction of GLUT2 gene expression to a maximum 1.7-and 2.3-fold for the 10 and 20 mM glucose concentrations, respectively. Therefore the endogenous GLUT2 gene is transcriptionally regulated by glucose as is the case for the murine and human GLUT2 promoters. The modest differences between transcription rate and mRNA expression could possibly be explained by either a delay in the transfer of the transcripts from the nuclei to the cytoplasm andor an alteration in GLUT2 mRNA stability induced by glucose. We have measured GLUT2 mRNA half-life in INS-1 cells in 11 mM glucose and determined that it was approximately 8 h. Further work will be necessary to demonstrate a possible role of glucose in altering GLUT2 mRNA stability.
Several reports have previously shown that GLUT2 gene expression is modulated by glucose in vivo and in vitro. In primary cultures of rat hepatocytes, Asano et al. mRNA expression in p-cells of rats maintained hyperglycemic for 5 days. More recently, a maximal 10-fold increase in GLUT2 expression was described by Ferrer et al. (7) when rat islets were incubated in 11 mM glucose in comparison to 2 mM. In this study, a time course experiment of the effect of glucose on GLUT2 has shown a 2.5-fold induction of GLUT2 mRNA only after 8 h of culture in 16.7 mM glucose. Taken together, these observations clearly demonstrate that high glucose concentrations positively modulate GLUT2 gene expression in p-cells and hepatocytes.
In diabetic animal models, a drastic P-cell-specific reduction of GLUT2 expression has been reported for the NIDDM Zucker fdfa, the neonatal low-dose STZ-induced diabetic rat, the Wistar Kyoto, GK rats, db mice, and the autoimmune diabetic BB rats (8)(9)(10)(11)(12). By cross-transplanting islets from db/db mice into control mice or vice versa, the decreased GLUT2 expression was shown to be reversible and induced by the diabetic environment of the animals (36). The pathogenic significance of the decrease in p-cell GLUT2 is controversial: it has been claimed that this loss may be the primary cause of the specific glucose-induced secretory abnormality encountered in the diabetic state (1, 8,11) although others consider this possibility unlikely (37). Whatever the pathogenic significance of the loss of GLUT2 expression, it is one of the earliest biochemical markers of the diabetic state. Since glucose regulates positively GLUT2 gene transcription in the normal state and since the carbohydrate-responsiveness of GLUT2 is selectively lost in the p-cell of diabetic rodents, further work to identify the cis elements and trans-acting factors involved in the control of GLUT2 gene transcription is needed and this may lead to a better understanding of the pathogenic events involved in the early diabetic state.