Engineering of Glucose-Stimulated Insulin Secretion and Biosynthesis in Non-Islet Cells (Glucose Transporters/Metabolc Regulafton/Transfecton) STEVEN D

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Proc. Nati. Acad. Sci. USA Vol. 89, pp. 688-692, January 1992 Biochemistry Engineering of glucose-stimulated insulin secretion and biosynthesis in non-islet cells (glucose transporters/metabolc regulafton/transfecton) STEVEN D. HUGHES*t, JOHN H. JOHNSON*I§, CHRISTIAN QUAADE*t, AND CHRISTOPHER B. NEWGARD*t¶ *Center for Diabetes Research, Gifford Laboratories and Departments of tBiochemistry, tInternal Medicine and §Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235 Communicated by Roger H. Unger, October 11, 1991

ABSTRACT The high-capacity glucose transporter known recently received strong experimental support from studies as GLUT-2 and the glucose phosphorylating enzyme glucoki- with spontaneous (17, 18) as well as experimentally induced nase are thought to be key components of the "glucose-sensing (19, 20) animal models of P-cell dysfunction. apparatus" that regulates insulin release from the 13 cells ofthe Insulin-dependent diabetes mellitus is caused by autoim- islets of Langerhans in response to changes in external glucose mune destruction of8 cells (see ref. 21 for review). Treatment concentration. AtT-20ins cells are derived from anterior pitu- of the disease requires insulin replacement, either by con- itary cells and are like 13 cells in that they express glucokinase ventional administration of the hormone or by transplanta- and have been engineered to secrete correctly processed insulin tion of insulin-secreting tissue. Since the latter strategy has in response to analogs of cAMP, but, unlike 13 cells, they fail to thus far relied largely on the use of scarce human pancreas as respond to glucose and lack GLUT-2 expression. Herein we the insulin source, it has not been feasible for general demonstrate that stable transfection of AtT-20ins cells with the application. Thus, we have recently begun to consider an GLUT-2 cDNA confers glucose-stimulated insulin secretion alternative approach, that of using molecular techniques to and glucose regulation of insulin biosynthesis and also results engineer an "artificial p cell"-i.e., a non-islet cell capable in glucose potentiation of the secretory response to non-glucose of performing glucose-stimulated insulin secretion. The an- secretagogues. This work represents a first step toward cre- terior pituitary cell line AtT-20ins is an intriguing candidate ation of a genetically engineered "artificial 13 cell." for such studies because of important similarities to p cells. (i) These cells have been modified for human insulin gene The pancreatic islets of Langerhans secrete glucoregulatory expression by stable transfection with a viral promoter/ hormones in response to changes in circulating levels of key proinsulin cDNA construct (22). (ii) AtT20ins cells are able metabolic fuels. In the case of glucose, transport into the 1 to process the proinsulin mRNA and preprotein and secrete cell and metabolism of this sugar are absolute requirements the correctly processed insulin polypeptide in response to for secretion, leading to the hypothesis that its specific analogs of cAMP (22). (iii) We have recently found that stimulatory effect is mediated by and proportional to its flux AtT-20ins cells contain significant amounts of the islet iso- rate through glycolysis and related pathways (1-6). In addi- form of glucokinase (23), making this the only cell type other tion to its acute effects on the release of prestored insulin, than liver or islets in which glucokinase gene expression has glucose stimulates de novo insulin biosynthesis by increasing been reported. insulin gene transcription, stabilizing insulin mRNA, enhanc- On the other hand, AtT-20ins cells differ from islets in two ing translation of the insulin transcript into protein, and important ways: (i) they do not secrete insulin in response to stimulating the rate of conversion of proinsulin to insulin glucose and (ii) they express the low-Km GLUT-1 glucose (7-11). mRNA The lack of A substantial body of evidence has accumulated implicat- transporter mRNA and not GLUT-2 (23). ing a specific facilitated-diffusion-type glucose transporter glucose responsiveness in AtT-20ins cells could be explained known as GLUT-2 and the glucose phosphorylating enzyme either by deficient capacity or altered affinity of glucose glucokinase in the control of glucose metabolism in islet pB uptake relative to normal islets. To test this hypothesis, we cells. Both proteins are members of gene families; GLUT-2 have stably transfected AtT-20ins cells with the GLUT-2 is unique among the five-member family of glucose trans- cDNA, since its expression would be expected to increase porter proteins (GLUTs 1-5; see refs. 12 and 13 for review) both Km and Vmax of glucose transport, and analyzed the in that it has a distinctly higher Km and Vm. for glucose effects of this manuever on glucose regulation of insulin transport. Glucokinase (also known as hexokinase IV) is the synthesis and secretion. high Km, high Vm counterpart of GLUT-2 among the family of hexokinases (14). It has been proposed that these proteins METHODS work in concert as the "glucose-sensing apparatus" that modulates insulin secretion in response to changes in circu- AtT-20ins Cell Culture and Tissue Isolation. The AtT-20ins lating glucose concentrations (5, 15, 16). In normal f3 cells, cells used are similar to the line that was originally described glucose transport capacity is in excess relative to glycolytic (22) except that the Rous sarcoma virus long terminal repeat flux. Thus, the GLUT-2 transporter likely plays a largely was substituted for the simian virus 40 early gene promoter as permissive role in the control ofglucose metabolism, whereas the promoter/enhancer directing insulin gene expression. The glucokinase represents the true rate-limiting step (5, 15). cells were grown in Dulbecco's modified Eagle's medium Implicit in this formulation, however, is the prediction that (DMEM)/25 mM glucose supplemented with 10% fetal calf severe underexpression of GLUT-2 will result in loss of serum, 100 ,ug of streptomycin per ml, and 250 ,ug ofneomycin glucose-stimulated insulin secretion in islets, an idea that has per ml. Anterior pituitary and liver samples were excised from Wistar rats, and islets were isolated from groups of 10-20 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: GLUT-2, glucose transporter 2; ACTH, corticotropin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. 9To whom reprint requests should be addressed.

688 Downloaded by guest on September 24, 2021 Biochemistry: Hughes et al. Proc. Natl. Acad. Sci. USA 89 (1992) 689 animals as described (16, 24) and pooled for RNA extraction Glucose Phosphorylation Assays. Glucose phosphorylation or homogenization for glucose phosphorylation assays. and glucokinase activities were measured by conversion of Stable Transfection ofAtT-20ins Cells with GLUT-2. The rat [U-14C]glucose to [U-14C]glucose 6-phosphate, as described islet GLUT-2 cDNA (16) was cloned into the vector pCB-7 (a (method "B", ref. 27). Assays were performed in the pres- gift from Colleen Brewer and Michael Roth, University of ence and absence of 10 mM glucose 6-phosphate, which Texas Southwestern Medical Center), a derivative of vector potently inhibits hexokinase but has little effect on glucoki- pCMV4 (25), immediately downstream of its cytomegalovi- nase activity. rus promoter. AtT-20ins cells were transfected with this construct using electroporation. Stable transfectants were RESULTS selected with hygromycin, since the plasmid also contains a Expression of GLUT-2 mRNA in Transfected AtT-20ins resistance gene for this drug. Cells. Steady-state GLUT-2 mRNA levels were evaluated in RNA Blot Hybridization Analysis. RNA blot hybridization two GLUT-2-transfected AtT-20ins cell lines (CGT-5, CGT- analysis was carried out as described (19, 26). Blots were 6), untransfected AtT-20ins cells, and three primary tissues hybridized sequentially with 32P-labeled antisense GLUT-2 (Fig. 1). CGT-5 cells contained approximately half as much or 18S rRNA probes, with stripping of the blot between and CGT-6 cells contained an equal amount of GLUT-2 by boiling in 0.1% SDS for 30 min. mRNA compared to rat islets, and they contained 10 and 16 hybridizations times as much, respectively, as rat liver, measured by GLUT-2 Immunofluorescence in AtT-20ins Cells. Parental densitometric scanning and normalization to the signal ob- or GLUT-2-transfected AtT-20ins cells were harvested by tained with an 18S rRNA probe. Untransfected AtT-20ins incubation with a solution of 0.02% EDTA in phosphate- cells and primary anterior pituitary cells did not contain buffered saline (PBS) and transferred onto poly(L-lysine)- detectable amounts of GLUT-2 mRNA, consistent with our coated glass coverslips. The cells were then fixed with 3% previous work (23). paraformaldehyde in PBS; this was followed by incubation Immunocytochemistry of GLUT-2 in Transfected AtT-20ins with 0.1 M NH4Cl in PBS (pH 7.9). The cells were perme- Cells. We detected abundant GLUT-2 immunofluorescence abilized with 0.1% Triton X-100 for 5 min, and after preincubation with 2% bovine serum albumin, GLUT-2 antiserum (17) was applied at a dilution of 1:2500 in the presence or absence of an equal volume of the antigenic peptide (1 mg/ml). Cells were then incubated with fluorescein isothio- cyanate-conjugated goat anti-rabbit IgG and slides were visualized by fluorescent light microscopy. Glucose Transport Measurements. Cells were harvested by scraping with a rubber policeman and assayed for 3-0- methylglucose uptake under zero-trans conditions as described (24). Initial velocities of uptake were derived from duplicate measurements at 3, 6, and 15 sec for each concentration ofglucose with the transfected cell lines and 3, 15, and 30 sec for the parental cell line (due to slower transport rate in these cells). Insulin and Corticotropin (ACTH) Secretion. Parental or GLUT-2-transfected lines CGT-5 and CGT-6 were removed from growth plates by light trypsinization and replated in six-well dishes (Costar) at a density of 5 x 105 cells per well. The cells were then grown for 3 days in RPMI 1640 medium supplemented with 1 mM glucose. On the third day, cells were washed twice for 10 min each in Hepes balanced salt solution containing 1% bovine serum albumin (HBSS) but lacking glucose. Secretion experiments were initiated by addition of HBSS plus the secretagogues of interest. For assay of intracellular insulin, cells were collected in 1 ml of 5 M acetic acid, lysed by three cycles of freeze-thawing, and lyophilized. The dried lysate was then reconstituted in 5 ml of insulin assay buffer (50 mM NaH2PO4/0.1% bovine serum albumin/0.25% EDTA/1% aprotinin, pH 7.1) and aliquots were assayed for insulin by radioimmunoassay.

Cs CD } C3C: FIG. 1. GLUT-2 mRNA in C U tissues and AtT-20ins cell lines. O cc J-E Each lane contains 6 ,g of total N C,1)H RNA. Samples were prepared Probes > <- from liver, anterior pituitary, and islet tissues as well as irom untransfected (AtT-20ins) and GLUT-2 9 ~ * GLUT-2-transfected (AtT-20ins CGT-5 and CGT-6) AtT-20ins FIG. 2. GLUT-2 immunofluorescence in AtT-20ins cells. (A) cell lines. The blot was probed Cells from the GLUT-2-transfected cell line AtT-20ins CGT-6 treated with radiolabeled antisense with the anti-GLUT-2 antibody (17), showing immunofluorescence GLUT-2 cRNA and, as a control predominantly at the membrane surface of the cells. (B) AtT-20ins 18S rRNA _ qW_jp la forgel loading, with an antisense CGT-6 cells treated with the anti-GLUT-2 antibody after its prein- oligonucleotide probe for 18S cubation with the hexadecapeptide antigen (17), showing specific rRNA (19). blocking of the signal. Downloaded by guest on September 24, 2021 690 Biochemistry: Hughes et al. Proc. Natl. Acad Sci. USA 89 (1992) in lines CGT-5 and CGT-6 at the cell membrane as well as Table 1. Insulin content in native (AtT-20ins) and GLUT-2- some intracellular signal that was mostly polarized to regions transfected (CGT-6) AtT-20ins cells grown at low or high glucose of cell-cell contact (Fig. 2A). The signal was blocked by Glucose in Insulin, milliunits per mg of total preincubation of the antibody with the hexadecapeptide growth protein antigen (Fig. 2B) and was not seen in untransfected cells or medium, in cells transfected with the vector lacking the GLUT-2 insert mM AtT-20ins CGT-6 (data not shown). Thus, AtT-20ins cells not only have the capacity to produce GLUT-2 mRNA and protein but also sort 1 1.77 ± 0.15 6.34 ± 0.35* the protein to the cell membrane, as occurs in islets and liver 25 2.10 ± 0.08t 11.24 ± 0.66*t (18, 28-30). Cells were grown for 3 days in RPMI 1640 medium supplemented Glucose Transport Measurements in Parental and GLUT- with the indicated concentrations of glucose and harvested for Cells. Fig. 3A shows a plot of the determination of intracellular insulin content. Each value represents 2-Expressing AtT-20ins the mean of 16 independent wells ± SEM. *, P < 0.001 (comparison concentration dependence ofglucose uptake into the various of CGT-6 cells to AtT-20ins cells); t, P = 0.06 (comparison of AtT-20ins cell lines. The CGT-5 and CGT-6 lines had appar- AtT-20ins cells at 25 mM glucose to AtT-20ins cells at 1 mM glucose); ent Km values for glucose of 16 and 17 mM and Vm. values *, P < 0.001 (comparison of CGT-6 cells at 25 mM glucose to CGT-6 of 25 and 17 mmol/min per liter of cell space, respectively cells at 1 mM glucose). (Fig. 3B). In contrast, the untransfected parental AtT-20ins line had an apparent Km for glucose of 2 mM and a Vmax of insulin release at 0 mM glucose was observed at the lowest 0.5 mmol/min per liter ofcell space (Fig. 3C), consistent with concentration ofglucose studied (5 ,uM); maximal stimulation its expression of the GLUT-1 mRNA (23). The transfected of -2.5-fold was observed at all higher concentrations over the AtT-20ins cells transport glucose with kinetics similar to range 10 ,uM-20 mM (P s 0.001). We found that glucose also isolated, dispersed islets of Langerhans, which have a Km of stimulated ACTH release from CGT-6 cells (but not parental 18 mM for glucose and a Vmx of24 mmol/min per liter ofcell AtT-20ins cells) with the same dose dependence as seen for space (16). Thus, the GLUT-2 cDNA is capable of transfer- insulin secretion (data not shown). It is highly unlikely that ring this activity into the AtT-20ins cell line. these results can be attributed to clonal selection of glucose- Insulin Content of Native and Engineered AtT-20ins Cells. responsive subpopulations of the parental AtT-20ins cells, Transfection of AtT-20ins cells with the GLUT-2 cDNA since cells transfected with vector lacking GLUT-2 failed to results in a substantial increase in intracellular insulin con- respond, whereas two independent GLUT-2-expressing lines tent. As shown in Table 1, the CGT-6 cells contained 3.6-fold (CGT-5 and CGT-6) gained glucose sensing. and 5.4-fold more insulin than the AtT-20ins cells after The secretion experiments involve static incubation of growth for 3 days at low (1 mM) and high (25 mM) glucose, with for 3 hr and thus provide little respectively (P < 0.001 for both comparisons). Furthermore, cells the secretagogue insulin content was approximately double in the CGT-6 cells information about the dynamics of insulin release. We have grown at high glucose compared with the same cells grown at recently succeeded in growing AtT-20ins cell lines in liquid low glucose (P < 0.001). In the untransfected AtT-20ins cells, culture, thus allowing their secretory properties to be studied in contrast, high glucose caused only a 20% increase in insulin by perifusion. In this configuration, insulin was released from content. CGT-6 cells within minutes of the start of glucose perifusion Glucose-Stimulated Insulin and ACTH Secretion from AtT- (5 mM), and the secretion response exhibited a first and a 20ins Cells. Fig. 4A compares glucose-stimulated insulin re- second phase as is characteristic of normal (3 cells (unpub- lease from AtT-20ins cells and CGT-6 cells. Consistent with lished data). our previous results (23), glucose had no significant effect on In normal islets, glucose potentiates the insulin secretory insulin release from parental AtT-20ins cells. AtT-20ins cells response to various ,B-cell secretagogues, including agents transfected with the pCB7 vector lacking a GLUT-2 insert that increase intracellular cAMP levels (31, 32). Glucose has were also unresponsive to glucose (data not shown). GLUT- a modest stimulatory effect on forskolin-stimulated insulin 2-transfected cells, in contrast, are clearly glucose responsive release from parental AtT-20ins cells, shown by expressing (data are shown for line CGT-6 only; results for line CGT-5 the data either as insulin release per mg of cellular protein were qualitatively identical). A submaximal but statistically (Fig. 4A) or as insulin release per zg of cellular DNA (Fig. significant (P = 0.002) increase in insulin release relative to 4B). In contrast, glucose had a powerful potentiating effect on B C A A CGT-5 Km-l6nM Vmax-25 mmo9/mniniter O Parental Cell Line Kmin 2mM Vmax-o.5 rnmol/min/lwer O CGT-6 Km,1 7mM Vmax-17 mmoA/miniter 180r 160 D 81.4 140 1.2 _ 120 .t 1.0 100 _ /0 . 2, 0.8 a 2 80 -iE -*E 0.6 E 60 1 0.4 E 40 -0.2 20 I ..d I -0.4 -0.2 0 0.2 0.4 0.6 0.8 1.0 -8 -4 0 4 8 12 16 20 34-OCH3-Glucose (mM) 143-0CH3-Gluce] 143-0-CH3-GkooseJ

FIG. 3. Glucose transport into AtT-20ins cells. (A) Measurements of 3-O-methylglucose (3-O-CH3-Glucose) uptake as a function of glucose concentration into untransfected AtT-20ins cells (parental) and GLUT-2-transfected lines CGT-5 and CGT-6. (B) Reciprocal plot of glucose uptake versus 3-O-CH3-Glucose concentration for GLUT-2-transfected lines CGT-5 and CGT-6. The calculated Km and Vm. values for glucose transport are indicated. (C) Reciprocal plot ofglucose uptake versus 3-O-CH3-Glucose concentration for untransfected AtT-20ins cells (parental cell line). The calculated Km and Vmax values for glucose transport are indicated. Note the difference in scales between B and C. Downloaded by guest on September 24, 2021 Biochemistry: Hughes et al. Proc. Natl. Acad. Sci. USA 89 (1992) 691

800- A Table 2. Glucose phosphorylating activities in tissues and AtT-20ins cell lines * CGT-6 Total glucose Glucokinaset units Z 600- phospho- per g of protein rylation,* Gluco- tm * * * * ** * units per g of 50 mM 15 mM kinase, % E 400 - Cell type protein glucose glucose of total m AtT-20ins 03 200- Parental 9.19 ± 0.27 0.63 ± 0.06 0.43 ± 0.08 6.8 C Line CGT-6 8.09 ± 0.20 0.86 ± 0.18 0.40 ± 0.05 10.6 Islet 9.61 ± 2.10 2.31 ± 0.35 24.0

0 Liver 8.42 ± 1.09 7.19 ± 1.31 85.4 0 .005 .01 .05 .1 .5 2.5 11 20 F F+G Values represent the means ± SEM for three independent determinations for liver and islets and four independent determinations for [Glucosel untransfected (parental) and GLUT-2-transfected (line CGT-6) AtT- 20ins cells. B *Measured in 14,000 x g supernatant of crude homogenates, at 50 4 * mM as described z -Glc, -Fors glucose (method "B", ref. 27). 0 El +Glc, -Fors tDetermined with the same assay as used for total glucose phos- co El -Gic, +Fors phorylation at 50 or 15 mM glucose, except in the presence of 10 mM E] +Gic, +Fors to m glucose inhibit hexokinase. =L 6-phosphate

0 engineered to exhibit three distinct types of regulatory re-

0 **8 r- sponses to glucose that are characteristic of islet /8 cells- 0 1 namely, acute stimulation U, of insulin secretion, enhancement c of insulin biosynthesis, and a potentiated response to the

(a combination of glucose plus non-glucose secretagogues. These functions are central to the control of glycemia and prevention of diabetes in humans as well as other mammals. AtT-2Cins CGT-6 The AtT-20ins cell line represents a particularly useful model system because ofa number ofbiochemical similarities FIG. 4. Insulin release from AtT-20ins cells in response to between these cells and /3 cells of the islets of Langerhans. glucose and glucose potentiation of forskolin-induced secretion. (A) Foremost among these is the capacity of AtT-20ins cells to Insulin release was measured from untransfected (AtT-20ins) and target the proinsulin precursor to the regulated pathway of GLUT-2-transfected (CGT-6) AtT-20ins lines incubated with various secretion, where it is packaged into secretory granules and glucose concentrations over the range of 0-20 mM or with 0.5 ILM forskolin (F) or 0.5 JLM forskolin plus 2.5 mM glucose (F+G) for a correctly processed to mature insulin (22, 33, 34). In the period of 3 hr. Data are normalized to the total cellular protein current study, GLUT-2-transfected AtT-20ins cells were present in each secretion well and represent the mean SEM for found to gain glucose-stimulated insulin and ACTH secre- three to nine independent secretion wells per condition. *, P < 0.001 tion. The magnitude and glucose concentration dependence compared to secretion at 0 mM glucose; #, P = 0.002 compared to of the responses were highly similar for the two hormones. secretion at 0 mM glucose. (B) Insulin release was measured from These findings, coupled with the clear that untransfected (AtT-20ins) and GLUT-2-transfected (CGT-6) AtT- demonstration 20ins lines incubated with 0.5 t.M forskolin (Fors) and 2.5 mM insulin and ACTH are colocalized in the same secretory glucose (Glc) in combinations indicated by the legend. Data are granules in AtT-20ins cells (33), strongly suggest that glucose normalized to total cellular DNA in each secretion well and are stimulates insulin release from GLUT-2-expressing AtT- expressed as the mean ± SEM for three to nine independent 20ins cells via a mechanism involving a glucose-regulated measurements. Statistically significant increases in secretion relative signal for secretory granule membrane fusion and exocytosis, to the -Glc, -Fors control are indicated by the symbol * (P < 0.001). as in the islet /-cell. Transfection of AtT-20ins cells with GLUT-2 also dramat- forskolin-stimulated insulin release from transfected CGT-6 ically increases their intracellular insulin content relative to cells. The response was unchanged by glucose concentration untransfected cells and confers glucose regulation of insulin over the range of 1-5 mM, and similar potentiating effects of biosynthesis. This is so despite the fact that insulin gene glucose on dibutryl cAMP-(5 mM) and isobutylmethylxan- expression in AtT-20ins cells is directed by the Rous sarcoma thine- (0.1 mM) induced secretion were also observed (data virus long terminal repeat sequence. Glucose has no effect on not shown). the activity ofthis promoter/enhancer element in transfected Glucose Phosphorylation in AtT-20ins Cells. As shown in primary fetal islet cells (35) or HIT cells (36) in culture, even Table 2, total glucose phosphorylating capacity and glucoki- though HIT cells naturally express GLUT-2 (37). The most nase activity are not significantly different in transfected (line likely explanation for the enhanced insulin synthesis in CGT-6) versus untransfected (parental) AtT-20ins cells. Both CGT-6 cells is that GLUT-2 transfection unmasks posttran- lines have a total glucose phosphorylating capacity that is scriptional control mechanisms of insulin biosynthesis in similar to that in liver and islets but contain only 32% of the AtT-20ins cells similar to those operative in the islet 83 cell glucokinase activity in islets and 10% of that in liver. More- (9-11). over, glucokinase represents only 9%o of the total glucose Maximal insulin release from GLUT-2-transfected AtT- phosphorylating activity of AtT-20ins cells, as compared to 20ins cells occurs at a much lower glucose concentration 24% in normal islets and 86% in normal liver. ('10 ,LM glucose) than required for normal islets, which do not respond to glucose at concentrations of <4-5 mM. The DISCUSSION fact that CGT-5 and CGT-6 cells respond to subphysiological levels of glucose, despite expression of glucokinase and The data presented in this study demonstrate that the non- GLUT-2, suggests that these cells contain metabolic deter- islet cell line AtT-20ins can, by a single manipulation, be minants that can override the regulatory function of the Downloaded by guest on September 24, 2021 692 Biochemistry: Hughes et al. Proc. Natl. Acad. Sci. USA 89 (1992) high-Km components. The increased sensitivity of GLUT-2- 8. Nielsen, D. A., Welsh, M., Casadaban, M. J. & Steiner, D. F. expressing AtT-20ins cells to glucose relative to islet f3 cells (1985) J. Biol. Chem. 260, 13585-13589. can be explained by the fact that expression of the GLUT-2 9. Welch, M., Nielsen, D. A., MacKrell, A. J. & Steiner, D. F. (1985) J. Biol. Chem. 260, 13590-13594. transporter not only increases the Km for transport but also 10. Welch, M., Scherberg, N., Gilmore, R. & Steiner, D. F. (1986) the transport capacity at all glucose concentrations studied Biochem. J. 235, 459-467. (see Fig. 3A); In the face of increased rates of glucose 11. Gold, G. (1989) in Insulin Secretion, eds. Draznin, B., Melmed, transport, even at low glucose, glucose phosphorylation S. & LeRoith, D. (Liss, New York), pp. 25-35. likely becomes the rate-limiting step for generation of glu- 12. Bell, G. I., Kayano, T., Buse, J. B., Burant, C. F., Takeda, J., Lin, D., Fukumoto, H. & Seino, S. (1990) Diabetes Care 13, cose-related signals. Although AtT-20ins cells express the 198-208. mature islet glucokinase transcript (23), which when pre- 13. Thorens, B., Charron, M. J. & Lodish, H. F. (1990) Diabetes pared as cDNA and expressed in bacteria clearly encodes Care 13, 209-218. glucokinase activity (38), their predominant glucose phos- 14. Weinhouse, S. (1976) Curr. Top. Cell. Regul. 11, 1-50. phorylating enzyme is hexokinase [Km for glucose of ='10 ILM 15. Newgard, C. B., Quaade, C., Hughes, S. D. & Milburn, J. L. (39)]. Support for the notion that hexokinase can exert a (1990) Biochem. Soc. Trans. 18, 851-853. 16. Johnson, J. H., Newgard, C. B., Milburn, J. L., Lodish, H. F. dominant effect is provided by a recent study showing that & Thorens, B. (1990) J. Biol. Chem. 265, 6548-6551. overexpression of hexokinase I in normal islets results in a 17. Johnson, J. H., Ogawa, A., Chen, L., Orci, L., Newgard, shift in the glucose dose-response curve such that the trans- C. B., Alam, T. & Unger, R. H. (1990) Science 250, 546-549. fected cells are maximally responsive to glucose at subphys- 18. Orci, L., Ravazzola, M., Baetens, D., Inman, L., Amherdt, M., iological levels (40). Peterson, R. G., Newgard, C. B., Johnson, J. H. & Unger, Islet transplantation has been extensively investigated as a R. H. (1990) Proc. Natl. Acad. Sci. USA 87, 9953-9957. strategy for curing insulin-dependent diabetes mellitus (41) 19. Chen, L., Alam, T., Johnson, J. H., Hughes, S., Newgard, C. B. & Unger, R. H. (1990) Proc. Natl. Acad. Sci. USA 87, but suffers from the difficulties associated with procuring 4088-4092. sufficient quantities of tissue. This problem could potentially 20. Thorens, B., Weir, G. C., Leahy, J. L., Lodish, H. F. & be circumvented if a cell type that is available in unlimited Bonner-Weir, S. (1990) Proc. Natl. Acad. Sci. USA 87, 6492- quantity could be engineered to secrete insulin in response to 6496. metabolic signals. In the current study, AtT-20ins cells, 21. Rossini, A. A., Mordes, J. P. & Like, A. A. (1985) Annu. Rev. which can secrete insulin, but are blind to glucose, are shown Immunol. 3, 289-320. to gain glucose responsiveness upon introduction of the gene 22. Moore, H.-P., Walker, M. D., Lee, F. & Kelly, R. B. (1983) for the GLUT-2. further Cell 35, 531-538. high-Km glucose transporter Clearly, 23. Hughes, S. D., Quaade, C., Milburn, J. L., Cassidy, L. C. & molecular manipulations, including alteration of the glucoki- Newgard, C. B. (1991) J. Biol. Chem. 266, 4521-4530. nase/hexokinase ratio to correct the enhanced glucose sen- 24. Johnson, J. H., Crider, B. P., McCorkle, K., Alford, M. & sitivity of the cells, and elimination of cosecretion of ACTH Unger, R. H. (1990) N. Engl. J. Med. 322, 653-659. will be required in order for engineered AtT-20ins cells to be 25. Andersson, S., Davis, D. L., Dahlback, H., Jornvall, H. & considered as a therapeutic reagent in the treatment of Russell, D. W. (1989) J. Biol. Chem. 264, 8222-8229. insulin-dependent diabetes mellitus. Nevertheless, this study 26. Newgard, C. B., Nakano, K., Hwang, P. K. & Fletterick, R. J. shows that critical elements of the islet glucose-sensing (1986) Proc. Natl. Acad. Sci. USA 83, 8132-8136. 27. Kuwajima, M., Newgard, C. B., Foster, D. W. & McGarry, apparatus can be isolated and transferred to candidate cells J. D. (1986) J. Biol. Chem. 261, 8849-8853. and cell lines and thus represents a first step toward engi- 28. Thorens, B., Sarkar, H. K., Kaback, H. R. & Lodish, H. F. neering of an artificial 13 cell. (1988) Cell 55, 281-290. 29. Orci, L., Thorens, B., Ravazzola, M. & Lodish, H. F. (1989) We thank Drs. Roger Unger, Joseph Goldstein, and Kenneth Science 245, 295-297. Luskey for critical reading of the manuscript and Janet Turner, Lee 30. Tal, M., Schneider, D. L., Thorens, B. & Lodish, H. F. (1990) Bryant, Joan McGrath, and Dr. Chris McAllister for expert technical J. Clin. Invest. 86, 986-992. assistance. We are also grateful to Drs. Regis Kelly, Linda Matsuu- 31. Ullrich, S. & Wollheim, C. B. (1984) J. Biol. Chem. 259, chi, and Lois Clift-O'Grady for provision of the AtT-20ins cells and 4111-4115. helpful advice about their use, Sara K. McCorkle and Dr. Thomas 32. Malaisse, W. J., Garcia-Morales, P., Dufrane, S. P., Sener, A. Pieber for assistance with insulin radioimmunoassays, and Dr. John & Valverde, I. (1984) Endocrinology 115, 2015-2020. Porter and Ms. Brenda Hoyt for assistance with ACTH radioimmu- 33. Orci, L., Ravazzola, M., Amherdt, M., Perrelet, A., Powell, noassays. The studies were supported by a Research and Develop- S. K., Quinn, D. L. & Moore, H.-P. (1987) Cell 51, 1039-1051. ment Award from the American Diabetes Association (to C.B.N.) 34. Gross, D. J., Halban, P. A., Kahn, R. C., Weir, G. C. & and National Institutes of Health Grant PO1-DK42582 (to C.B.N. Villa-Komaroff, L. (1989) Proc. Natl. Acad. Sci. USA 86, and J.H.J.). C.Q. is supported by Training Grant 11-7458 from the 4107-4111. Danish Natural Science Research Council. 35. German, M. S., Moss, L. G. & Rutter, W. J. (1990) J. Biol. Chem. 265, 22063-22066. 1. Prentki, M. & Matschinsky, F. M. (1987) Physiol. Rev. 67, 36. Moss, L. G., Walker, M. D. & Rutter, W. J. (1987) Endocri- 1185-1248. nology 120, 31 (abstr.). 2. Turk, J., Wolf, B. A. & McDaniel, M. L. (1987) Prog. Lipid 37. Seino, Y., Inagaki, N., Yasuda, K., Inoue, G., Kuzuya, H. & Res. 26, 125-181. Imura, H. (1991) Diabetes 40, 173 (abstr.). 3. Ashcroft, S. J. 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