GLUT2 Intracytoplasmic Loop Transmits Glucose Signaling 843

Journal of Cell Science 113, 841-847 (2000) 841 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0902

The large intracytoplasmic loop of the glucose transporter GLUT2 is involved in glucose signaling in hepatic cells

Ghislaine Guillemain1,2, Martine Loizeau1, Martine Pinçon-Raymond2, Jean Girard1 and Armelle Leturque1,2,* 1Endocrinologie Métabolisme et Développement, CNRS Ð UPR1524, 9 rue Jules Hetzel, 92190 Meudon, France 2Institut Biomédical des Cordeliers, INSERM-U505, 15 rue de l’Ecole de Médecine, 75006 Paris, France *Author for correspondence at address 2 (e-mail: [email protected])

Accepted 14 December 1999; published on WWW 14 February 2000

SUMMARY

The hypothesis that the glucose transporter GLUT2 can due to competitive binding of a protein that normally function as a protein mediating transcriptional glucose interacts with endogenous GLUT2. In addition, the GLUT2 signaling was addressed. To divert the putative interacting loop, tagged with green ﬂuorescent protein (GFP), was proteins from a glucose signaling pathway, two located within the nucleus, whereas the GFP and GFP- intracytoplasmic domains of GLUT2, the C terminus and GLUT2 C-terminal proteins remained in the cytoplasm. In the large loop located between transmembrane domains 6 living cells, a fraction (50%) of the expressed GFP-GLUT2 and 7, were transfected into mhAT3F hepatoma cells. loop translocated rapidly from the cytoplasm to the nucleus Glucose-induced accumulation of two hepatic gene mRNAs in response to high glucose concentration and conversely in (GLUT2 and L-pyruvate kinase) was speciﬁcally inhibited the absence of glucose. We conclude that, via protein in cells transfected with the GLUT2 loop and not with the interactions with its large loop, GLUT2 may transduce a GLUT2 C terminus. The dual effects of glucose were glucose signal from the plasma membrane to the nucleus. dissociated in cells expressing the GLUT2 loop; in fact a normal glucose metabolism into glycogen occurred concomitantly with the inhibition of the glucose-induced Key words: GLUT2, Glucose signaling, Living cell, Liver, Glucose transcription. This inhibition by the GLUT2 loop could be metabolism, Glucose-regulated transcription

INTRODUCTION interactions (Johnston, 1999). Extracellular glucose is detected by two nutrient sensors of low and high concentrations, Snf3p Glucose signaling in eukaryotic cells can be regarded as a and Rgt2p, cloned in Saccharomyces cerevisiae (Özcan et al., process involving the detection of extracellular glucose levels, 1996). These proteins are structurally similar to mammalian followed by the transduction to the nucleus of a glucose and yeast glucose transporters, except for a long cytoplasmic repression or induction signal. Hepatocytes and pancreatic β C terminus (Celenza et al., 1988; Özcan et al., 1996) that may cells both stimulate gene transcription in response to high serve as a signaling domain (Ko et al., 1993; Schmidt et al., glucose concentrations (Chen et al., 1990; Girard et al., 1997; 1999; Vagnoli et al., 1998). The nature of the glucose induction Towle and Kaytor, 1997; Vaulont and Kahn, 1994). In these signal is unknown, but it is not generated by glucose cells, glucose transport is mediated by the GLUT2 glucose metabolism, contrary to the glucose repression signal transporter (Thorens et al., 1988). A close correlation between (Johnston, 1999). GLUT2 levels and glucose-regulated gene expression is The aim of this study was to test the hypothesis that, in observed in hepatoma cells (Antoine et al., 1997), engineered addition to its transport activity, GLUT2 can function as a β cells (Newgard et al., 1997) and GLUT2-null mice (Guillam glucose sensor, triggering glucose-regulated gene expression. et al., 1997), suggesting that in liver and pancreatic β cells, the GLUT2 is a member of a family of proteins (GLUTs) whose presence of GLUT2 is required for the transcriptional effect of predicted structure consists of 12 transmembrane domains, and glucose. N- and C-terminal cytoplasmic domains. A large loop between The primary role of GLUT2 is to transport glucose inside transmembrane domains 6 and 7 is predicted to reside in the the cell, where it is metabolized. The prevailing idea is that the cytoplasm. The speciﬁcity of GLUT isoforms is determined by glucose signal in eukaryotic cells originates from its own the intracytoplasmic domains, which are not conserved in metabolism (Girard et al., 1997; Towle and Kaytor, 1997; terms of their amino acid sequences (Bell et al., 1990). Signal Vaulont and Kahn, 1994). A recent report, however, provides transduction from a transmembrane receptor protein requires evidence that the glucose-signaling pathway can be triggered the activation of its intracytoplasmic domain. If GLUT2 by other means. Originating from a plasma membrane sensor, transmits a glucose signal, one of its intracytoplasmic domains this pathway, described in yeast, is mediated by protein-protein must be part of the signaling pathway. In order to identify the 842 G. Guillemain and others

GLUT2 domain that affects glucose sensing in the cell, the C terminus and large intracellular loop of GLUT2 were expressed in a hepatoma cell line. In large amounts, the intracytoplasmic domains would appear to compete with endogenous GLUT2 for binding to proteins involved in the glucose signaling pathway and thereby modify glucose-regulated gene expression. We assessed the impact of expression of the two domains on both a metabolic and a transcriptional effect of glucose.

MATERIALS AND METHODS

Cell culture The mhAT3F hepatoma cell line was derived from transgenic mice synthesizing the SV40 large T and small t antigens under the control of the antithrombin III promoter (Antoine et al., 1992). mhAT3F cells were grown in Dulbecco’s modified Eagle’s medium/Ham-F12, Glutamax (Life Technologies) supplemented with 100 ui/ml penicillin, streptomycin, 0.1 µM insulin, 1 µM dexamethasone, 1 µM triiodothyronine and 5% fetal calf serum. The mhAT3F cell line was chosen because GLUT2 is abundantly expressed and the stimulation of gene transcription by glucose is preserved (Antoine et al., 1997). The cells were cultured in 17 mM glucose unless otherwise stated. Cells were also cultured in glucose-free medium for 24 hours (it should be noted that some glucose was present in the fetal calf serum). Fig. 1. (A) Schematic representation of the secondary structure of When mhAT3F cells were cultured in the absence of glucose, the GLUT2 in the plasma membrane. (B) Transfection of mhAT3F with residual glucose concentration in the medium after 24 hours was intracytoplasmic domains of GLUT2 tagged at the N terminus with 0.03±0.01 mM. When mhAT3F cells were cultured with an initial GFP. (C) Western blot analysis of the fusion proteins GFP-GLUT2 glucose concentration of 17 mM, the residual glucose concentration loop and GFP-GLUT2 C terminus domain, revealed by an anti-GFP µ after 24 hours was 8.65±0.10 mM. antibody. Lanes 1,2: 10 g of homogenate of mhAT3F cells constitutively expressing GFP; lanes 3,4: 10 µg of homogenate of Plasmid constructs mhAT3F cells constitutively expressing GFP-GLUT2 C terminus; µ The coding regions of the intracellular loop between transmembrane lanes 5,6: 10 g of homogenate of mhAT3F cells constitutively domains 6 and 7 of the rat GLUT2 glucose transporter (amino acids expressing GFP-GLUT2 loop. In lanes 1, 3 and 5 the cells were 237-301) and the C terminus GLUT2 domain (amino acids 481-521) cultured in the absence of glucose and in lanes 2, 4 and 6 with 17 were amplified by means of PCR using a plasmid containing the full- mM glucose. The positions of molecular mass marker proteins are length GLUT2 cDNA (Thorens et al., 1988) as template. The shown. fragments were then inserted, in-frame, into the pEGFPc vector (Clontech), in fusion with green fluorescent protein (GFP). kinase and GLUT2 cDNAs and mRNAs were quantified by means of Transfection experiments computer-based scan analysis. An 18S rRNA probe was used to mhAT3F cells were plated and transfected by using the calcium visualize RNA loading (Postic et al., 1993). phosphate-DNA precipitation method (Chen and Okayama, 1988). The GLUT2 loop and GLUT2 C terminus in pEGFP vectors, and the Determination of radiolabeled glucose incorporation into vector alone, were transfected and harvested after 2 days for transient glycogen expression measurements. Transfected cells were selected with [14C]glucose (2 µCi, Amersham Pharmacia) incorporation into geneticin sulphate G-418 (0.4 mg/ml) (Gibco-BRL) for 3 weeks. glycogen was measured in confluent cells cultured in 30 mm dishes, Pooled colonies of stably transfected cell lines were established, and after 24 hours in the different experimental conditions, as previously individual clones were then obtained by clonal dilution. described (Kasus-Jacobi et al., 1997). Cells were washed twice with ice-cold 0.9% NaCl and scraped free in 30% KOH lysis buffer. Western blot analysis Glycogen was precipitated with ethanol and radioactivity was Proteins were separated by SDS-PAGE (10% acrylamide) and counted. Results are expressed as nmoles glucose incorporated into electrotransferred to a nitrocellulose membrane. After overnight glycogen/106 cells. incubation in 5% (w/v) non-fat milk in Tris-buffered saline (TBS) (20 mM Tris-HCl, pH 7.5, 150 mM NaCl), the blots were washed in TBS Fluorescence analysis containing 0.1% (v/v) Triton X-100 and incubated with a monoclonal Stable clones of mhAT3F cells were grown on Permanox four- anti-GFP (Clontech) or anti-GLUT2 C terminus antibody (East- chamber slides (Lab-Tek, Nunc) with 17 mM glucose. Cells were Acres). Immune complexes were detected by ECL (Amersham). washed three times with PBS (137 mM NaCl, 1.3 mM KCl, 16.1 mM Na2HPO4, 1.5 mM KH2PO4), fixed for 30 minutes in 4% Northern blot analysis paraformaldehyde in PBS, and quenched for 10 minutes in 50 mM Total RNA was purified from confluent mhAT3F cells after 24 hours NH4Cl. The nuclei were stained bright blue using Hoechst 33258 (0.5 of culture in the various experimental conditions, and northern µg/ml) for 5 minutes. After extensive washes in PBS the coverslips analysis was performed as previously described (Postic et al., 1993). were mounted in Mowiol (Hoechst). Fluorescence microscopy was The northern blots were hybridized with radiolabeled liver-pyruvate performed using an Olympus IMT2 inverted microscope, a GLUT2 intracytoplasmic loop transmits glucose signaling 843

Fig. 2. Effect of glucose on GLUT2 and liver-pyruvate kinase RNA accumulation in mhAT3F cells stably transfected with GFP, GFP- GLUT2 C terminus and GFP- GLUT2 loop. (A) This representative northern blot was hybridized with GLUT2 cDNA. After hybridization with L-PK cDNA, two bands were detected corresponding to liver-pyruvate kinase (L-PK) and muscle-pyruvate kinase (m-PK).The blots were also hybridized with an 18S rRNA probe as control. In mhAT3F cells, m-PK mRNA was constitutively expressed and served as a control. (B) Quantification was performed by scanning densitometry. *Significant increase in mRNA (P<0.05) in cells cultured with glucose (+) compared to cells (−) cultured in the absence of glucose; NS, no significant difference between the two groups (n=4).

fluorescence immersion lens (40×) and an FITC filter. Photographs in a circle (diameter of the region of interest: 5 µm) arbitrarily (Kodak Panther P1600 film) were taken with an Olympus MO-4Ti positioned inside the nucleus and in the cytoplasm of the cell. The camera adapted to the microscope. The location of the fusion proteins results, expressed as mean grey intensities, were recorded in each was quantified by using the Imagetool Software. captured image during the experimental period. We verified that the replacement of the circulating medium by a medium of the same Fluorescence imaging of GFP-GLUT2 loop composition did not affect fluorescence location (results not shown). A stable clone of GFP-GLUT2 loop was grown on glass coverslips Labtek (Nunc), with or without glucose added in the culture medium Statistical analysis for 24 hours. The experiments were performed at 25¡C in circulating Results are expressed as means ± s.e.m. Statistical analysis was PBS with or without glucose (17 mM). The microscopy was performed by using Student’s t-test for unpaired data. conducted using a confocal Zeiss LSM 510 microscope and a 63× objective lens. One 1 µm slice located in the nucleus of the cell was observed, and images were captured every 30 seconds during the RESULTS periods of the experiments. The PBS was circulating constantly; the total replacement of PBS in the culture chamber took 1 minute. Captured images were then analysed using the provider software. Expression of GLUT2 intracytoplasmic domains in Quantification of green fluorescence was established on a scale of grey mhAT3F cells intensity (256 grey levels). The mean grey intensities were measured The production of fusion proteins in clonal stable mhAT3F 844 G. Guillemain and others GLUT2 mRNA in mhAT3F Arbitrary Units

160 * wild type 120 *

80 GFP-GLUT2 loop

no no

xylose

glucose glucose

fructose fructose

Fig. 3. Effect of glucose, fructose and xylose on GLUT2 mRNA accumulation in wild-type mhAT3F cells and mhAT3F cells stably transfected with the GFP-GLUT2 loop. Northern blots were hybridized with GLUT2 cDNA and quantiﬁed by scanning densitometry. *Signiﬁcant increase (P<0.05) compared cells cultured for 24 hours in the absence (no) or presence of 17 mM sugar (n=4). GLUT2 mRNA amounts in mhAT3F cells expressing the GFP- GLUT2 loop were always statistically lower than GLUT2 mRNA amounts in wild-type cells (P<0.01; n=4).

GLYCOGEN (nmol [U-14C ]glucose/106 cell / 24h) Fig. 5. Location of fluorescent proteins in cells stably transfected with GFP (A,B), GFP-GLUT2 C terminus (C,D) and GFP-GLUT2 GFP GFP- loop GFP- C term loop (E,F). (B,D,F) The nuclei were stained with Hoechst dye. 60 * (A,C,E) The green fluorescent fusion proteins are visualized by epifluorescence with a light microscope. 40 * 4 times higher than that of endogenous GLUT2 in 20 untransfected cells, as estimated by northern blotting (not shown). All mRNA signals were detected using the 0 radiolabeled full-length GLUT2 cDNA as probe. The probe 0517 0 517 0 5 17 mM glucose hybridized with about 10% of the mRNAs for GFP-GLUT2 C terminus and loop compared with 100% of GLUT2 mRNA, Fig. 4. Effect of the glucose concentration on glycogen synthesis in leading to a probable underestimation of the transgene mhAT3F cells stably transfected with GFP or GFP fusions of amounts. intracytoplasmic domains of GLUT2. Results are expressed in nmoles U14C-glucose incorporated into glycogen by 106 cells over Accumulation of GLUT2 mRNA in mhAT3F cells 24 hours. *Significant increase in cells transfected with GFP-GLUT2 The transcriptional effect of glucose on two glucose-dependent C terminus (P<0.05) compared to cells transfected with GFP-GLUT2 liver genes, encoding GLUT2 and L-pyruvate kinase, was loop (n=5-6 determinations). assayed in clonal stable mhAT3F transfectants (Fig. 2). The cells were cultured in the absence or presence of 17 mM glucose for 24 hours. In mhAT3F cells expressing GFP alone, transfectants was analyzed by using an antibody directed the level of GLUT2 and L-pyruvate kinase mRNAs increased against green fluorescent protein. The fusion proteins three- to fourfold in response to glucose, a level similar to that recognized by the anti-GFP antibody were of the expected observed in wild-type cells. Expression of the GFP-GLUT2 C sizes, i.e. 29 kDa for GFP alone, 32.5 kDa for GFP-GLUT2 C terminus did not affect the accumulation of GLUT2 and L- terminus and 34.8 kDa for GFP-GLUT2 loop (Fig. 1). The pyruvate kinase mRNA in response to glucose. In contrast, expression levels of the three fusion proteins were similar. As expression of the GFP-GLUT2 loop specifically abolished both necessary for this study, the expression of the fusion proteins GLUT2 and L-pyruvate kinase mRNA accumulation in was not affected by the presence of glucose in the culture response to glucose. After hybridization with L-PK cDNA, two medium. bands were detected: a 2.8-kb signal corresponding to liver Expression of the GFP-GLUT2 C terminus and loop was 3- pyruvate kinase (L-PK) and a 2.4-kb signal corresponding to GLUT2 intracytoplasmic loop transmits glucose signaling 845

Fig. 6. Intracellular movements of GFP-GLUT2 loop in one living mhAT3F cell. These results were obtained by confocal microscopy. Bar, 10 µm. In the same cell, images of the GFP-GLUT2 loop were recorded in the absence (A) and presence (B) of 17 mM glucose. The fluorescence was quantified (see Materials and Methods), in the nucleus (black squares) and the cytoplasm (open squares) as mean grey intensity in an area of 5 µm diameter (right part). (A,B) Two captured images from the same cell at the times indicated on the right. This experiment was representative of three different experiments. muscle pyruvate kinase (M-PK). m-PK is constitutively MEAN GREY INTENSITY IN 6 NUCLEI expressed in mhAT3F cells and served as a control (Fig. 2). Inhibition of the glucose effect was also observed in a pool 250 with without glucose of stable mhAT3F cells constitutively expressing GFP-GLUT2 loop (not shown), indicating that the inhibition was due solely 200 to the fusion protein, and not to a peculiar insertion of the GFP- GLUT2 loop construct in the mhAT3F cell genome. The intracellular loop of GLUT2 was thus identified as the 150 protein domain that interferes with the transcriptional signaling triggered by glucose. 100

Effect of glucose, fructose and xylose on GLUT2 50 mRNA accumulation To determine the nature of the triggers that stimulate the 0 glucose signaling pathway, several sugars were tested. In wild- 0 2 4 6 8 10 12 14 min type mhAT3F cells, 17 mM fructose, a hexose transported by GLUT2 (although less efficiently than glucose; Gould et al., Fig. 7. Intracellular movements of GFP-GLUT2 loop in six nuclei 1991), mimicked the effect of glucose on GLUT2 mRNA from living transfected mhAT3F cells. These results were obtained accumulation. With 17 mM xylose, a pentose poorly by confocal microscopy. Images were captured in the GFP-GLUT2 transported by GLUT2 (Gould et al., 1991), no significant loop cells incubated for 7 minutes in the presence of 17 mM glucose and then for 7 minutes in the absence of glucose. GFP-GLUT2 loop stimulation of GLUT2 mRNA accumulation was detected in fluorescence in the nuclei was quantified as mean grey intensity (see wild-type mhAT3F cells (Fig. 3). A correlation between the Materials and Methods), the dark line represents the mean values of amount of transported sugar and GLUT2 mRNA accumulation six different cells (pale lines). This experiment was reproduced three was thus observed. No stimulation in GLUT2 mRNA times. accumulation was observed in mhAT3F cells expressing GPF- GLUT2 loop with any type of sugar (Fig. 3). GLUT2 C terminus or GFP-GLUT2 loop, were cultured for 24 Incorporation of radioactive glucose into glycogen hours with increasing glucose concentrations (0, 5 and 17 in mhAT3F cells mM), incorporation of radioactive glucose into glycogen was To assess the impact of GLUT2 domains upon a metabolic dependent upon the concentration of glucose (Fig. 4). When pathway, glycogen synthesis was studied using glucose as a compared to GFP-transfected cells, the incorporation of substrate. When mhAT3F cells, transfected with GFP, GFP- radioactive glucose into glycogen did not vary in the presence 846 G. Guillemain and others of GFP-GLUT2 loop or GFP-GLUT2 C terminus in transfected targeting of GLUTs has been attributed to the N- and C- cells, thus demonstrating that the metabolic pathway was not terminal domains, whereas the substrate binding site and its altered (Fig. 4). In contrast, a slight increase in glucose specificity are mainly due to transmembrane domains incorporation into glycogen was observed in cells transfected (Saravolac and Holman, 1997). Expression of intracellular with GFP-GLUT2 C terminus when compared to GFP-GLUT2 domains of GLUT4, the insulin-sensitive glucose transporter, loop (Fig. 4). The reason for this difference remained demonstrated that the C terminus of GLUT4 transfected into unexplained but could be due to the intrinsic property of adipocytes could cause insulin-like stimulation of glucose GLUT2 C terminus, which is responsible for the high Km for transport (Lee and Jung, 1997). glucose of GLUT2 protein (Katagiri et al., 1992). The potential signaling functions of intracytoplasmic domains of GLUT2 were studied in an mhAT3F cell line. The Location of the overexpressed proteins in mhAT3F effect of glucose on the accumulation of mRNA encoding two cells glucose-sensitive hepatic genes, GLUT2 and L-pyruvate The subcellular location of the fusion proteins was studied kinase, was abolished in cells expressing the large loop but not using the green fluorescence of GFP (Fig. 5). GFP and GFP- the C terminus of GLUT2. The GLUT2 loop was thus GLUT2 C terminus were distributed uniformly in mhAT3F identified as a GLUT2 domain mediating the transcriptional cells. In contrast, the GFP-GLUT2 loop was concentrated in glucose signal. Overexpression of the GLUT2 loop relative to the nuclei, as visualized by superimposition of the green (GFP) endogenous GLUT2 was sufficient to divert the glucose signal and bright blue (nuclear) fluorescence. This specific location from its usual pathway, suggesting that the GFP-GLUT2 loop was observed regardeless of the position of the tag, i.e. GFP- competitively binds a protein that normally binds to GLUT2 loop (Fig. 5) or GLUT2 loop -GFP (result not shown). endogenous GLUT2. We therefore created a dominant- The fluorescence of the fusion proteins was carefully negative mutant mammalian cell, allowing the investigation of quantified. The ratio of mean grey intensity of identical areas in the effects of glucose on gene transcription. the nucleus and cytoplasm showed that the GFP alone and GFP- The effect of glucose on GLUT2 mRNA accumulation was C terminus proteins had the same cellular location. Their mean mimicked by sugars that are efficiently transported by GLUT2 grey ratios were, respectively, 1.51±0.05, n=10 and 1.51±0.10, in wild-type cells. Therefore, sugar transport by GLUT2 n=10. In contrast, the mean grey ratio of GFP-GLUT2 loop in appears to be necessary to initiate the pathway and control its AT3F cell was 2.15±0.11, n=12, showing a nuclear accumulation amplitude. In contrast, in cells expressing the GFP-GLUT2 of the fusion protein. This specific location was observed with loop, the entry of glucose and fructose mediated by the light microscope (Fig. 5) and further confirmed by confocal endogenous GLUT2 was not sufficient to trigger the (Fig. 6) microscopic observations. transcription signal. In bacteria, yeast and plants, complex signal transduction Intracellular movements of GFP- GLUT2 loop in networks are activated in response to glucose, and the living cells in response to glucose transcriptional effects of glucose appear to be dissociated from Using confocal microscopy in living cells, intracellular the metabolic effects. In mammalian cells, the transcriptional movements of the GFP-GLUT2 loop were detected despite the and metabolic processes have always been closely related in high level of transgene expression. The translocations of GFP- glucose signaling. By expressing the intracytoplasmic domain GLUT2 loop in mhAT3F were followed in response to of GLUT2, we discriminated between the dual effects of switches of glucose concentrations in the culture medium. In glucose. Indeed, increasing extracellular glucose cells equilibrated at 25¡C in PBS, the GFP-GLUT2 loop was concentrations modulated glucose incorporation into glycogen located in the nucleus and to a lesser extent in the cytoplasm in both wild-type and transfected cells. This showed that (Fig. 6). Upon a switch from PBS to PBS containing 17 mM glucose metabolism via glucose-6-phosphate was preserved glucose, the fluorescence from GFP-GLUT2 loop decreased in when the loop domain was transfected. Taken together, these the cytoplasm and increased in the nucleus during the 14 results show that the transfected intracytoplasmic loop of minutes experimental period (Fig. 6). When the cells GLUT2 inhibits the transcriptional effect of glucose on equilibrated in PBS containing 17 mM glucose were switched glucose-sensitive genes independently of glucose storage into to PBS, the fluorescence from GFP-GLUT2 loop decreased by glycogen and suggests the existence of a parallel pathway in twofold in the nuclei of six recorded cells, during the mammalian cells, as in other cells. experimental period (Fig. 7). A fraction (50%) of the GFP- The intracellular location of the fusion protein might help to GLUT2 loop translocated within a few minutes from the clarify its inhibitory role on glucose-induced transcription. cytoplasm to the nucleus in response to high glucose, and Indeed, we showed that the GFP-GLUT2 loop specifically conversely during glucose deprivation. accumulated in the nucleus of mhAT3F cells, whereas the C terminus remained in the cytoplasm. It is known that a globular protein under 50 kDa (about 9 nm) can freely cross the nuclear DISCUSSION pore. The nuclear location of the GFP-GLUT2 loop was probably not size-dependent, as the GFP and GFP-GLUT2 C The presence of the glucose transporter isoform GLUT2 is terminus are smaller than the GFP-GLUT2 loop. Using the required for appropriate responses to glucose in both pancreatic PSORT II program, the GLUT2 loop (not the C terminus) was β cells and hepatocytes. We studied the role of two GLUT2 shown to contain 20% basic amino acids, which are a target protein domains in the transduction of the glucose signal in for nuclear import machinery. The location of the fusion hepatic cells. Specific functions have been attributed to protein might be affected by extracellular glucose domains of glucose transporters. For example, subcellular concentrations. In response to a high glucose concentration and GLUT2 intracytoplasmic loop transmits glucose signaling 847 within a few minutes, a fraction of the expressed GFP-GLUT2 (1992). Gene expression in hepatocyte-like cell lines established by targeted loop translocated from the cytoplasm to the nucleus in living carcinogenesis in transgenic mice. Exp. Cell Res. 200, 175-185. mhAT3F cells. These regulated movements favor the Bell, G. I., Kanyano, T., Buse, J. B., Burant, C. F., Takeda, J., Lin, D., Fukumoto, H. and Seino, S. (1990). Molecular biology of mammalian hypothesis that GFP-GLUT2 loop translocates via a complex glucose transporters. Diabetes Care 13, 198-208. mechanism involving other proteins. In these cells, the ratio of Celenza, J. L., Marshall-Carlson, L. and Carlson, M. (1988). The yeast GFP-GLUT2 loop versus endogenous GLUT2 favored the SNF3 gene encodes a glucose transporter homologous to the mamalian former in the competition for protein binding. The proteins that protein. Proc. Natl. Acad. Sci. USA 85, 2130-2134. Chen, C. and Okayama, H. (1988). Calcium phosphate-mediated gene would normally interact with the wild-type GLUT2 protein in transfer: a highly efficient system for stably transforming cells with plasmid the plasma membrane would now bind cytoplasmic GFP- DNA. Biotechniques 6, 632-638. GLUT2 loop. The higher nuclear location of the GFP-GLUT2 Chen, L., Alam, T., Johnson, J. H., Hughes, S. and Newgard, C. B. (1990). loop in response to glucose accompanied inactivation of Regulation of §-cell glucose transporter gene expression. Proc. Natl. Acad. transcription of glucose-sensitive genes in transfected cells. Sci. USA 87, 4088-4092. Girard, J., Ferré, P. and Foufelle, F. (1997). Mechanisms by which This suggests that, in wild-type cells, the protein that binds to carbohydrate regulate expression of genes for glycolytic and lipogenic endogenous GLUT2 has to be released from its anchoring site enzymes. Annu. Rev. Nutr. 17, 325-352. on the plasma membrane to activate the transcription of Gould, G. W. and Seatter, M. J. (1997). Introduction to the Facilitative glucose-sensitive genes inside the nucleus. Glucose Transporter Family. Heidelberg: R. G. Landes Company. Gould, G. W., Thomas, H. M., Jess, T. J. and Bell, G. I. (1991). Expression There are several mechanisms by which the large intracellular of human glucose transporters in Xenopus oocytes: kinetic characterization loop of GLUT2 may transduce a signal from the plasma and substrate specificities of the erythrocyte, liver, and brain isoforms. membrane to the nucleus. A recent discovery in the Notch-1 Biochemistry 30, 5139-5145. pathway shows that growth factors stimulate intracytoplasmic Guillam, M.-T., Hümmler, E., Schaerer, E., Wu, J.-Y., Birnbaum, M. J., cleavage of a transmembrane receptor, and the fragment peptide Beermann, F., Schmidt, A., Dériaz, N. and Thorens, B. (1997). Early diabetes and abnormal postnatal pancreatic islet development in mice released mediates the Notch-1 signaling pathway (Schroeter et lacking Glut-2. Nature Genet. 17, 327-330. al., 1998). Cleavage of the intracellular loop of the endogenous Johnston, M. (1999). Feasting, fasting and fermenting. Glucose sensing in glucose transporter by the action of an external signal is unlikely, yeast and other cells. Trends Genet. 15, 29-33. since the glucose transporter GLUT2 is an integral plasma Kasus-Jacobi, A., Perdereau, D., Tartare-Deckert, S., Van Obbergen, E., membrane protein with 12 transmembrane domains, and has Girard, J. and Burnol, A.-F. (1997). Evidence for a direct interaction between Insulin Receptor Substrate-1 and Shc. J. Biol. Chem. 272, 17166- never been found in the cytoplasm or nucleus of hepatocytes or 17170. pancreatic β cells. Alternatively, endogenous GLUT2 could Katagiri, H., Asano, T., Ishihara, H., Tsukuda, K., Lin, J.-L., Inukai, K., sequester a cytoplasmic protein, on receipt of a sugar signal; the Kikuchi, M., Yasaki, Y. and Oka, Y. (1992). Replacement of intracellular protein would then be released from the plasma membrane, C-terminal domain of GLUT1 glucose transporter with that of GLUT2 increases Vmax and Km of transport activity. J. Biol. Chem. 267, 22550-22555. unmasking a site for mediation of the transcriptional effect of Ko, C. H., Liang, H. and Gaber, R. F. (1993). Roles of multiple glucose glucose. The external signal that provokes glucose signaling from transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 638-648. glucose transporter GLUT2 might be the sugar itself. Indeed, Lee, W. and Jung, C. (1997). A synthetic peptide corresponding to the glucose crosses the cell membrane by means of structural GLUT4 C-terminal cytoplasmic domain causes insulin-like glucose changes (inward- or outward-facing substrate binding sites) of its transport stimulation and GLUT4 recruitment in rat adipocytes. J. Biol. Chem. 272, 21427-21431. transporters (Gould and Seatter, 1997). In this study, however, we Newgard, C. B., Clark, S., BeltrandelRio, H., Hohmeier, H. E., Quaade, cannot rule out a role for one of the early glucose metabolites. C. and Normington, K. (1997). Engineered cell lines for insulin In conclusion, we provide evidence that GLUT2, through its replacement in diabetes: current status and future prospects. Diabetologia large intracytoplasmic loop, mediates a glucose signaling 40 Suppl. 2, S42-47. Özcan, S., Dover, J., Rosenwald, A. G., Wölf, S. and Johnston, M. (1996). pathway from the plasma membrane to the nucleus of Two glucose transporters in Saccharomyces cerevisiae are glucose sensors hepatoma cells. that generate a signal for induction of gene expression. Proc. Natl. Acad. Sci. USA 93, 12428-12432. We thank Edith Brot-Laroche and Anne-Françoise Burnol for Postic, C., Burcelin, R., Rencurel, F., Pégorier, J.P., Loizeau, M., Girard, helpful discussions. We also thank Dominique Perdereau for DNA J. and Leturque, A. (1993). Evidence for a transient inhibitory effect of sequencing, and Françoise Vialat (CNRS UPR 9068, Nogent) and insulin on GLUT2 expression in the liver: studies in vivo and in vitro. 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