Heterogeneous Expression of Glucokinase Among Pancreatic F8 Cells THOMAS L

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Proc. Natl. Acad. Sci. USA Vol. 89, pp. 2619-2623, April 1992 Physiology Heterogeneous expression of glucokinase among pancreatic f8 cells THOMAS L. JETTON AND MARK A. MAGNUSON* Departments of Molecular Physiology and Biophysics and of Medicine, 708 Light Hall, Vanderbilt University Medical School, Nashville, TN 37232 Communicated by Charles R. Park, December 23, 1991

ABSTRACT The cellular location of glucokinase (GK), a In addition to clarifying the islet-cell-specific expression of key component of the glucose-sensing mechanism of the pan- this enzyme, we sought to determine whether all P3 cells creatic islet, was determined using immunocytochemical tech- express similar levels of immunodetectable GK. d cells are a niques. In rat islets, GK imm noreactivity was detected only in neuroendocrine islet cell population that secrete insulin from (3 cells with no immunoreactivit detected in a, 8, or pancreatic ultrastructurally distinct secretory granules (10). Microscop- polypeptide-containing (PP) cells. However, within various (3 ical observations noting a relatively uniform pattern ofinsulin cells, GK immunoreactivity varied considerably. Most (3 cells immunoreactivity and evidence of electrochemical coupling displayed relatively low levels ofcytoplasmic immunoreactivity by intercellular gap junctions led to a prevailing hypothesis whereas other (3 cells stained intensely for this enzyme. Colo- that 3 cells were functionally homogeneous. However, evi- calization studies of GK and GLUT2, the hih K. glucose dence has been accumulating that /3 cells are not functionally transporter of( cells, confirmed that these proteins are located equivalent but may instead be segregated into distinct sub- in different subcellular domains of (3 cells. The lack of GK populations based on morphological and physiological crite- immunoreactivity in glucagon- and somatostatin-secreting cells ria including differential glucose sensitivities (11-13). For in islets suggests that these cells are not directly responsive to instance, a study by Schuit et al. (12) revealed that proinsulin glucose or utilize a fundamentally different mechanism for biosynthesis by flow-sorted /8 cells is stimulated by glucose sensing glucose fluctuations. Moreover, the differential expres- in a dose-dependent manner and individual (3 cells differ in sion of GK among pancreatic (3 cells suggests that glucose their response to glucose with some cells failing to synthesize phosphorylation is the probable enzymatic control point for the proinsulin even after culture in 20 mM glucose (12). The basis functional diversity of these cells. for the heterogeneous response of / cells to glucose has not been defined but could involve the differential expression of Glucokinase (GK) has been identified only in liver and the GK. pancreatic islets of Langerhans where distinct isoforms are generated due to tissue-specific alternate RNA splicing ofthe MATERIALS AND METHODS GK gene product (1, 2). In islet (3 cells, GK plays a key role Antibody Generation. Two antibodies directed against GK in the regulation ofinsulin secretion. This functionally unique were used for the immunodetection of this enzyme in nor- hexokinase catalyzes the high Km conversion of glucose to moglycemic rat pancreatic islets. An anti-glutathione S-trans- glucose 6-phosphate, the rate-limiting step in glucose utili- ferase (GST)-GK fusion protein antibody was produced in a zation by pancreatic (3 cells (3), thus translating fluctuations sheep as described (14). Briefly, a cDNA fragment encoding in intracellular glucose levels to changes in the rate of the rat GK B1 isoform was cloned into pGEX-2T and glycolysis. Increases in the rate of glucose usage by (3 cells, expressed in Escherichia coli, and the full-length fusion mediated by GK, are followed by changes in K' and Ca2" protein ("76 kDa) produced was purified by GST-agarose channel conductance, a resultant increase in intracellular affinity chromatography and used as an immunogen in sheep. [Ca2+], and then an increase in the rate of insulin secretion An anti-peptide antibody targeted to the 10 C-terminal amino (4-6). The pivotal role of GK in regulating glucose metabo- acids of rat GK (ACKKACMLAQ) was produced in a rabbit lism ofthe P cell has been established by numerous biochem- as described (14). ical studies leading the enzyme to be termed the (-cell Antibody Purification. The IgG component ofboth antisera "glucose sensor" (3). were obtained by recombinant protein G-agarose chroma- Both glucagon and somatostatin secretion by the islet a and tography (Pierce) and purified further by sequential adsorp- 8 cells, respectively, are influenced by plasma glucose levels tion and elution from GST-GK fusion protein immobilized (7). Whether these cells are able to directly sense changes in onto either nitrocellulose or poly(vinylidene difluoride) mem- glucose concentration or, instead, may respond to paracrine branes (Immobilon-P; Millipore). Membrane strips corre- factors emanating from ( cells has not been resolved. Since sponding to the 76-kDa GST-GK band were excised and the only defined mechanism by which a cell can directly blocked with 3.0% (wt/vol) bovine serum albumin (BSA)/ respond to fluxes in glucose concentrations in the physiologic 0.05% Tween 20 in phosphate-buffered saline (PBS) for 24 hr range involves the expression of GK (3), we sought to at 4°C. After dialysis or desalting by cellulose chromatogra- determine whether a and 6 cells of the islet express this phy and equilibration with PBS, anti-GK antibodies were enzyme. A previous study measuring GK activity in strep- allowed to adsorb to the membrane-bound fusion protein for tozoticin-treated rats suggested that enzyme activity was not 24-72 hr at 4°C. After several washes in PBS supplemented restricted to the (3 cell but also was present in the glucagon- with 0.1% BSA and 0.05% Tween-20, GK-specific antibodies and/or somatostatin-secreting cells ofthe islet (8). However, were eluted with 100 mM glycine hydrochloride/0.2% BSA, the methods used in the study were indirect, and other pH 2.8, for 10-12 min with agitation at 20°C. The elution reports have shown that isolated a cells respond to glucose buffer was immediately neutralized with 3-5% of 1.0 M Tris differently than P cells (9), thus leaving this important issue (pH 9.5) and equilibrated with PBS by dialysis. for understanding islet function unresolved. Abbreviations: GST, glutathione S-transferase; GK, glucokinase; The publication costs of this article were defrayed in part by page charge BSA, bovine serum albumin; GLUT2, high Km glucose transporter; payment. This article must therefore be hereby marked "advertisement" PP cells, pancreatic polypeptide-containing cells. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2619 Downloaded by guest on September 26, 2021 2620 Physiology: Jetton and Magnuson Proc. Natl. Acad. Sci. USA 89 (1992) carried out with agitation for 1 hr at 20'C followed by incubation of the blots with the avidin-biotin complex for 1 hr. Prior to enzymatic development ofthe bound horseradish peroxidase, the blots were washed four times in PBS/0.05% Tween without NaN3. The peroxidase substrate contained 4..... 0.01% H202 and 0.003% 3-amino-9-ethylcarbazole in a 0.1 M sodium acetate (pH 5.2). 46 Immunocytochemistry. Tissues were removed from euglycemic male Sprague-Dawley rats that were killed by decap- itation. Liver and pancreas were rinsed in ice-cold PBS then FIG. 1. Protein blot detection ofGK. GK immersion-fixed in 4.0%6 (wt/vol) paraformaldehyde/0.1 M 3Yj --- was detected in total cytosolic protein ex- sodium phosphate buffer overnight at 40C. Tissues were tracts from rat insulinoma (7.5 &g; lane 1) or routinely processed for paraffin embedding with 4- to 5-,um liver (5 jg; lane 2) by SDS/PAGE protein blot analysis. The blotswere probed with the sections mounted on silanized slides. Immunocytochemical same affinity-purified antibody to the staining of GK and colabeling with appropriate pancreatic OST-GK fusion protein that was used forthe islet cell markers in normal rat tissues were accomplished immunolocalization studies. Positions of using immunoenzyme and immunofluorescent procedures, molecular mass standards are indicated on respectively. However, since islet GK is present in low the left (kDa). concentrations, the sensitivity of an ABC method was required for detection. After testing several detection methods Wedern Blot Analysis. Male Sprague-Dawley rats (4150 g) with both paraffin and cryostat sections, the ABC peroxidase were fasted for 72 hr and then refed for 24 hr to boost the procedure was chosen detecting for GK immunoreactivity expression of hepatic GK. Livers were quickly removed, because it yielded consistent results with high sensitivity. rinsed in ice-cold PBS, and homogenized in 50 mM trietha- Affinity-purified GK antibodies were used at 3-25 pg/ml nolamine, pH 7.0/100 mM glucose/100 mM KCl/1 mM and diluted in 1% BSA/0.1% Triton X-100/PBS. The anti- dithiothreitol/5% (vol/vol) glycerol/5 mM EDTA/5 mM bodies were incubated for 2-24 hr at 40C. The GK and islet EGTA/50,LM phenylmethylsulfonyl fluoride/leupeptin (2.5 cell marker double-labeling experiments included the ABC mg/ml)/0.02% NaN3. The homogenate was centrifuged at horseradish peroxidase technique with either 3-3'-diami- 15,000 x g for 15 min and then stored at -70°C until used. An nobenzidine or 3-amino-9-ethylcarbazole and H202 as sub- extract from frozen rat insulinoma tissue, obtained from strates. Tissue sections were pretreated with 0.5% H202 in William Chick (University of Massachusetts), was prepared absolute methanol to quench endogeneous peroxidase activ- in the same manner. Tissue extracts were fractionated by ity and blocked in 5% (vol/vol) normal donkey serum before SDS/PAGE in 12% minigels under reducing conditions and each antibody step. Biotinylated secondary reagents, reared then electroblotted to Immobilon-P (Millipore) membranes. in donkey, were adsorbed against various animal proteins The blots were rinsed for two 15-min periods in PBS/0.05% (Jackson ImmunoResearch; ML grade). After immunostain- Tween 20 and then blocked in PBS/3.0% BSA/0.05% Tween ing for GK, sections of rat pancreas were incubated in for 6-24 hr at 4°C. The blots were incubated with the anti-GK antisera specific to each of the major islet cell types: a cells fusion protein antibodies (3 ,ug/ml) in PBS/3% BSA/0.05% were identified by rabbit anti-glucagon (ICN), (3 cells were Tween for 4-24 hr at 4°C followed by four 15-min washes in stained with a mouse monoclonal antibody to insulin (Zymed PBS/0.05% Tween. Binding of the primary antibodies was Laboratories), 6 cells were marked by rabbit anti- detected using a standard avidin-biotin complex (ABC) kit somatostatin (ICN), and pancreatic polypeptide-containing (Vector Laboratories). The secondary antibody was biotiny- (PP) cells were labeled with anti-pancreatic polypeptide (ICN lated rabbit anti-sheep antibody (Jackson ImmunoResearch) Biomedical), all diluted 1:100. An affinity-purified anti-GST- use;d at 2 ug/ml. Incubation of secondary antibodies was GLUT2 (rat) antibody (where GLUT2 is the high Km glucose

~,4,I*"ts FIG. 2. Localization of GK in rat pancreatic islets. Immunoperoxidase cy- *V: -% tochemistry for GK was used in conjunction with indirect immunofluorescence for islet cell markers. (A) Islet stained for GK A with anti-peptide antibody revealing heterogeneous immunoreactivity of islet core .. j. cells. (B) Same islet section as A immunostained for insulin showing GK colocaliza- -.3eq l. tion. Weaker immunofluorescence for insulin observed in cells with high levels of GK is attributable to the intensity of the W-V. h.;> immunoperoxidase reaction product that diminishes insulin immunoreactivity. (C) Higher magnification showing p cells stained for GK with anti-GST-GK fusion protein antibody. (D) ,B cells stained for GK with anti-peptide antibody. Control sections resulted in lack of islet-cell- specific staining. (Scale bars = 10 ,um.) Downloaded by guest on September 26, 2021 Physiology: Jetton and Magnuson Proc. Natl. Acad. Sci. USA 89 (1992) 2621

te FIG. 3. Immunolocalization of GK in rat pancreatic islets in conjunction with islet markers. Arrows designate non-e cells that lack GK immunoreactivity. (A) Islet stained for GK with anti-GST-GK fusion protein antibody. (B) Same islet section as A stained for glucagon. (C) Islet stained for GK using anti-peptide antibody. (D) Same islet section as C stained for glucagon. (E) " .T, Islet periphery stained for GK with anti-peptide antibody. (F) Same islet section as in E stained for somatostatin. (G) Periphery of "pancreatic polypeptide-rich" islet stained for GK with anti-peptide antibody. (H) Same islet section as in G stained for pancreatic polypeptide. (Scale bars = 10 gim.) transporter) was obtained from R. Pipers and D. James a second antibody generated against the 10 C-terminal amino (Washington University) and used at =10 Ag/ml. acids of rat GK was also used. This sequence was chosen Secondary probes consisted of donkey anti-rabbit-Texas because it shares no homology with either hexokinase type I red and donkey anti-mouse-fluorescein isothiocyanate. In or II. We have demonstrated (14) the reactivity of this some experiments, the avidin-biotin technique was used anti-peptide antibody to GK expressed in bacteria. Although such that antibodies to islet cell markers were detected with much less reactive than the anti-GST-GK antibody on West- biotinylated secondary antibodies followed by the ExtrAvi- ern blots of tissue extracts, it also recognizes the same din fluorochrome (Sigma; 20 ,ug/ml). Control experiments to 50-kDa band (results not shown). Affinity purification ofboth determine specific GK immunoreactivity and method spec- antibodies by sequential binding and elution from purified ificity included staining adjacent tissue sections with preab- and immobilized GST-GK fusion protein was required to sorbed antibodies (anti-li-mer peptide antibody and li-mer obtain specific reactivity to the less abundant islet GK. The peptide), preimmune serum diluted 1:100 to 1:1000 (anti-11- specificity of both antibodies was established. Specific im- mer and anti-GST-GK antibodies), and omission of the munoreactivity of these antibodies to tissue sections was secondary antibody. Potential immunologic cross-reactivity observed only in pancreas and liver. No specific reaction of the antibodies to other hexokinases was examined by also products were observed in sections ofbrain, kidney, adipose, staining brain, adipose, and muscle. In these experiments, and muscle using the, ABC peroxidase method (results not specific immunoreactivity with these antibodies was detected shown). In addition, both antibodies detected a perivenous only in liver and islet core cells, as judged by the presence of distribution of immunoreactivity in the liver parenchyma peroxidase-generated reaction product. (data not shown), typical of GK (16, 17). Northern Blot Analysis. HIT M2.2.2 cells were obtained Pancreatic islets exhibited GK immunoreactivity within from R. Stein (Vanderbilt University) and a-TC-6 and a-TC-9 the cytoplasm of insulin-reactive cells, whereas no GK cells were obtained from E. Leiter (The Jackson Laborato- reactivity was seen in the pancreatic exocrine tissue sur- ries) and grown in Dulbecco's modified Eagle's medium rounding the islets (Fig. 2A and see Fig. 4A). Both antibodies supplemented with 10% (vol/vol) fetal calf serum. Total RNA to GK reacted only with a cytoplasmic component of the P3 was prepared using the AGCP method (15) and fractionated cell, although the fusion protein antibody appears more in agarose/formaldehyde gels. The blot was then transferred reactive, possibly reflecting a greater number ofepitopes that to a nitrocellulose membrane as described (14). were recognized with this antibody. No staining for GK was observed in a, 8, or PP cells as defined by double immuno- RESULTS staining with antisera specific for glucagon, somatostatin, or pancreatic polypeptide, respectively (Fig. 3). (3 cells stained A GST-GK fusion protein antibody produced in sheep re- with antibodies to insulin and GK showed distinct cellular acted predominantly with a protein band of -50 kDa in both immunolabeling patterns (Figs. 2 A and B and 4 A and B). liver and insulinoma tissue extracts (Fig. 1). Much weaker Whereas insulin immunoreactivity was generally homoge- reactivity of the GST-GK antibody to a 100-kDa band was neous throughout P cells, GK immunoreactivity varied con- also observed in insulinoma cells that was the right size to siderably among a cells. Most cells displayed only low-to- represent another hexokinase. Therefore, to ensure that GK moderate levels of GK immunostaining, although some was being detected in these immunolocalization experiments, cells exhibited relatively high levels of cytoplasmic immu- Downloaded by guest on September 26, 2021 2622 Physiology: Jetton and Magnuson Proc. Natl. Acad. Sci. USA 89 (1992) noreactivity (Fig. 2 C and D). This heterogeneous pattern of Additional support for the (3-cell-specific distribution of immunoreactivity was observed using both antisera to GK GK in the islet was obtained from an RNA blot experiment but was more striking using the anti-C-terminal peptide using a GK cDNA probe. GK mRNA was not detected in two antibody. clonal sublines of a-TC-1 cells, a cell line derived from Within the cytoplasm of individual P cells, GK immuno- transgenic mice bearing a glucagon-simian virus 40 large reactivity was often asymmetrically displaced toward one tumor antigen fusion gene (Fig. 5, lanes 3 and 4). However, side of the cell, possibly reflecting the polarized position of in the same RNA blot, GK mRNA was detected in RNA the nucleus and the resultant displacement of most of the isolated from liver- and (-cell-derived HIT M2.2.2 cells (Fig. cytoplasm (18). Since the N-terminal ends of the hepatic and 5, lanes 1 and 2). P-cell GK isoforms are different (1, 19), it has been suggested that these different sequences could confer partitioning to DISCUSSION different subcellular domains. To determine whether this might be the case, we performed a triple-immunolabeling We have developed two polyclonal antibodies that show in specific reactivity to GK. In this study we have used these experiment where GK, insulin, and GLUT2 were localized antibodies to immunolocalize GK in the pancreatic islets of the same islet (Fig. 4). Although insulin and GK showed euglycemic rats. In spite of the low amount of GK immuno- patterns of immunoreactivity consistent with a cytoplasmic reactivity in the islet and the need for amplification tech- distribution, GLUT2 immunolabeling revealed surface stain- niques for reliable detection of this enzyme, the techniques ing consistent with the localization of the transporter in the used are able to provide semiquantitative data on the abun- (3-cell membrane (20, 21). dance of GK in the various cells of the islet. We found GK immunoreactivity restricted to (3 cells of pancreatic islets with no detectable immunoreactivity found in a, 8, or PP cells. In addition, we also found that the amount ofGK varies Al. A greatly among different ( cells. Given the widely perceived role ofGK determining the rate ofglucose metabolism by the I 1:II^ ..1,, islet, which is linked subsequently to the rate of insulin U. secretion, these two observations, the (-cell-specific expression of the enzyme and the heterogeneous nature of its ; t expression, have distinct implications for islet function. The restriction ofGK immunoreactivity to ( cells suggests that only the insulin-secreting cells of the islet contain the necessary biochemical components to sense physiologic glucose levels [e.g., GK, a high Km glucose phosphorylating enzyme, and GLUT2, a high Km glucose transporter (21)]. A However, the subcellular distribution ofthe GK and GLUT2 in the ( cell is different. The lack of any apparent circum- ferential localization pattern for GK seems to exclude any absolute requirement for the spatial coupling of GK to GLUT2, as has been suggested (19, 22). Moreover, the absence of both a high Km glucose transporter and a high Km glucose phosphorylating enzyme in a and 8 cells of the islet suggests that these cell types are either incapable of directly responding to physiologic glucose concentrations or use a fundamentally different mechanism to do so. Glucagon- secreting cells might, therefore, respond to glucose by an indirect mechanism, possibly the release ofa paracrine factor by ( cells. A candidate is the inhibitory neurotransmitter -aminobutyric acid, which is synthesized and secreted by the (3 cell (23, 24). yAminobutyric acid, by increasing CI- A

,. I..>( _ !,. -I , r * _ FIG. 5. Northern blot analysis forGK mRNA. Total RNA (20ug) was loaded in each lane. The source of the RNA is as follows. FIG. 4. Immunolocalization ofGK with insulin and GLUT2 in rat Lanes: 1, liver; 2, HIT M2.2.2; 3, pancreatic islets. (A) Islet stained for GK using anti-peptide anti- aTC-6; 4, aTC-9. (A) Autoradio- body. Arrows denote islet core cells that stain intensely for GK. (B) gam of blot that was probed with Same islet section immunostained for insulin showing a uniform 32P-labeled GK cDNA. (B) Ethid- pattern of immunostaining. A diminution in insulin immunofluores- B ium bromide-stained RNA gel cence among some * cells (arrows) is due to intensity of immuno- prior to blotting to demonstrate Same islet section that amounts of RNA were peroxidase reaction product. (C) immunostained T equal for GLUT2. Note the uniform pattern of immunofluorescence on the present in each lane. kb, surface of , cells. (Scale bars = 10 Am.) S Kilobases. Downloaded by guest on September 26, 2021 Physiology: Jetton and Magnuson Proc. Natl. Acad. Sci. USA 89 (1992) 2623 channel conductivity, has been shown to hyperpolarize a the role that (3-cell heterogeneity plays in islet function, both cells, thereby inhibiting glucagon secretion (25). in euglycemic and hyperglycemic states. The lack of any detectable GK mRNA in two clonal cell lines that secrete glucagon supports our immunocytochemi- We thank A. Khoor for performing the Northern blot experiment. cal observations that localize GK to (3 cells and not a cells. We also thank M. Tamkun and A. Powers for helpful comments, W. Chick for rat insulinoma tissue, E. Leiter and R. Stein for cells, and Both of these observations contradict the previous study of D. James and R. Pipers for the GLUT2 antibody. These studies were Bedoya et al. (8) that measured GK activity in the islets ofrats supported by grants from the Public Health Service (DK42612 and made diabetic with streptozotocin. However, it should be DK42502) and the Juvenile Diabetes Foundation. noted that the possibility remains that GK levels may be below the threshold of detection in the non-e cells of eugly- 1. Magnuson, M. A. & Shelton, K. D. (1989) J. Biol. Chem. 264, cemic islet but be induced to a detectable level in the diabetic 15936-15942. animal. Since this study used only euglycemic animals, the 2. Iynedjian, P. B., Pilot, P. R., Nouspikel, T., Milburn, J. L., latter is not as Quaade, C., Hughes, S., Ucla, C. & Newgard, C. B. (1989) possibility excluded, although viewed unlikely. Proc. Natl. Acad. Sci. USA 86, 7838-7842. In addition, because ofthe relative scarcity of8 cells, it is also 3. Meglasson, M. D. & Matschinsky, F. M. (1986) Diabetes possible that a small population of cells expressing GK might Metab. Rev. 2, 163-214. not have been detected. 4. Ashcroft, F. M., Harrison, D. E. & Ashcroft, S. J. H. (1984) The finding of a marked heterogeneous distribution pattern Nature (London) 312, 445-448. of GK among (3 cells strengthens the concept of functional 5. Cook, D. L. & Hales, C. N. (1984) Nature (London) 311, heterogeneity of islet 18 cells. Furthermore, this finding 271-273. that the differential of GK in the ( 6. Boyd, A. E. I., Hill, R. S., Oberwetter, J. M. & Berg, M. suggests expression cell, (1986) J. Clin. Invest. 77, 774-781. as determined by GK immunostaining in intact islets, may 7. Schauder, P., McIntosh, C., Arends, J., Arnold, R., Frerichs, serve as the functional basis for conferring graded glucose M. & Creutzfeld, W. (1976) FEBS Lett. 68, 225-227. responses to different (3 cells. Other studies noting (-cell 8. Bedoya, F. J., Oberholtzer, J. C. & Matschinsky, F. M. (1987) diversity include reports of striking size differences (26), J. Histochem. Cytochem. 35, 1089-1093. differences in immunoreactivity to certain neuronal markers 9. Wesslen, N., Pipeleers, D., Van De Winkel, M., Rorsman, P. (27, 28), the ability to elaborate neurites in culture (29), & Hellman, B. (1987) Acta Physiol. Scand. 131, 230-234. variable proinsulin synthesis and immunoreactivity (12, 30), 10. Orci, L. (1982) Diabetes 31, 538-565. 11. Solomon, D. & Meda, P. (1986) Exp. Cell Res. 162, 507-520. alterations in membrane potential (13), and the differential 12. Schuit, F. C., IntVeld, P. A. & Pipeleers, D. G. (1988) Proc. insulin secretory activity of individual (3 cells (11). Such Nati. Acad. Sci. USA 85, 3865-3869. heterogeneity may be an important aspect of overall islet 13. Dean, P. M. & Matthews, E. K. (1970) J. Physiol. (London) function. Given the role of GK in determining glucose usage 210, 255-264. in (3 cells, the variable expression of this enzyme could 14. Liang, Y., Jetton, T. L., Zimmerman, E., NAjafi, H., Matschin- explain several aspects of their apparent functional hetero- sky, F. M. & Magnuson, M. A. (1991) J. Biol. Chem. 266, geneity. In contrast, heterogeneity in GLUT2 expression was 6999-7007. (3 15. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, not observed among cells, as determined in this and other 156-159. studies (20, 21). Therefore, the expression pattern ofGLUT2 16. Lawrence, G. M., Jepson, M. A., Trayer, I. P. & Walker, in P cells is unlikely to account for the functional differences D. G. (1986) Histochem. J. 18, 45-53. among various (3 cells. 17. Trus, M., Zawalich, K., Gaynor, D. & Matschinsky, F. (1980) The mechanisms underlying both the (-cell-specific ex- J. Histochem. Cytochem. 28, 579-581. pression and the heterogeneous expression of GK are not 18. Bonner-Weir, S. (1988) Diabetes 37, 616-621. known. Glucose is able to regulate the expression of GK 19. Newgard, C. B., Quaade, C., Hughes, S. D. & Milburn, J. L. in cultured rat islets in a (1990) Biochem. Soc. Trans. 18, 851-853. activity concentration-dependent 20. Thorens, B., Sarkar, H. K., Kaback, H. R. & Lodish, H. F. manner that is thought to involve either a translational or (1988) Cell 55, 281-290. posttranslational mechanism (31). Whether the same mech- 21. Orci, L., Thorens, B., Ravazzola, M. & Lodish, H. F. (1989) anism that mediates the response of GK to glucose is also Science 245, 295-297. involved in determining its heterogeneous expression is not 22. Unger, R. H. (1991) Science 251, 1200-1205. known. 23. Baekkeskov, S., Aanstoot, H.-J., Christgau, S., Reetz, A., The typical mammalian islet consists of an ordered ar- Solimena, M., Cascalho, M., Folli, F., Richter-Olesen, H. & rangement of a, 8, and PP cells forming a peripheral mantle DeCamilh, P. (1990) Nature (London) 347, 151-156. around a core of(3 cells that receive the afferent blood supply 24. Garry, D. J., Appel, N. M., Garry, M. G. & Sorenson, R. L. This architecture create (1988) J. Histochem. Cytochem. 36, 573-580. (32). may various microenviron- 25. Rorsman, P., Berggren, P.-O., Bokvist, K., Ericson, H., ments that could allow specific patterns of cell-to-cell sig- Mohler, H., Ostenson, C.-G. & Smith, P. A. (1989) Nature naling to occur. For instance, the glucose response of a and (London) 341, 233-236. 8 cells might be determined by their relative proximity to 26. Brelje, T. C., Scharp, D. W. & Sorenson, R. L. (1989) Diabe- glucose-sensing and nonsensing ( cells. In addition, the tes 38, 808-814. number of glucose-sensitive (3 cells, determined by the 27. Alpert, S., Hanahan, D. & Teitelman, G. (1988) Cell 53, amount of GK they contain, may affect the threshold for 295-308. insulin secretion for a given islet. The overall insulin secre- 28. Teitelman, G., Alpert, S. & Hanahan, D. (1988) Cell 52, 97-105. tory response may depend upon the number of ( cells that 29. Teitelman, G. (1990) Dev. Biol. 142, 368-379. exceed a threshold of GK and their 30. Orci, L. (1986) Diabetes Metab. Rev. 2, 71-106. specific activity relative 31. Liang, Y., Najafi, H. & Matschinsky, F. M. (1990) J. Biol. locations within the islet core, as suggested by their regional Chem. 265, 16863-16866. differences in insulin immunoreactivity and degranulation 32. Weir, G. C. & Bonner-Weir, S. (1990) J. Clin. Invest. 85, after in vivo stimulation with glucose and glibenclamide (33). 983-987. Thus, it should now be useful to use GK immunoreactivity as 33. Stefan, Y., Meda, P., Neufeld, M. & Orci, L. (1987) J. Clin. an in situ marker for (-cell metabolic diversity so as to study Invest. 80, 175-183. Downloaded by guest on September 26, 2021