Proc. Natl. Acad. Sci. USA Vol. 89, pp. 5744-5748, July 1992 Cell Biology [Val12]HRAS downregulates GLUT2 in I6 cells of transgenic mice without affecting glucose homeostasis MICHAEL TAL*t, Y-JIAN Wut, MARGARITA LEISER§¶, MANJU SURANA§H, HARVEY LODISH*II NORMAN FLEISCHER§¶, GORDON WEIRS, AND SHIMON EFRAT$I,**,tt * for Biomedical Research, Cambridge, MA 02142; *Joslin Diabetes Center, Boston, MA 02215; Departments of § and **Molecular Pharmacology, and 1Diabetes Research and Training Center, Albert Einstein College of Medicine, Bronx, NY 10461; and I1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139-4307 Contributed by Harvey Lodish, February 26, 1992

ABSTRACT Glucose-induced insulin release from pancre- transgenic lineages, the disease develops predominantly in atic I cells depends on the (-cell metabolism of glucose, which male mice 5-8 months old and is characterized by the generates intracellular signals for secretion. The (3-cell glucose appearance of large cavities in the islets of Langerhans, transporter isotype GLUT2 and the glucose phosphorylating hypoinsulinemia, and hyperglycemia, without an autoim- enzyme glucokinase have both been implicated in coupling mune response. Most transgenic females do not manifest this insulin secretion to extracellular glucose levels. Here we present phenotype (11, 12). Ovariectomy and hormone replacement evidence that a pronounced decrease in P-cell GLUT2 has no reveal a protective effect of estrogen, whereas androgens immediate effect on glucose homeostasis. Analysis oftransgenic appear not to be involved in the increased male susceptibility mice overexpressing human [Val'2]HRAS oncoprotein under (12). Ultrastructural abnormalities, including engorged rough control of the insulin promoter reveals a great reduction in endoplasmic reticulum and accumulation of clear vesicles in plasma-membrane GLUT2 levels. These mice are nonetheless the cytoplasm (11), suggest that RAS overexpression impairs able to maintain normal fed and fasting plasma glucose and vesicular traffic in the (3-cell secretory pathway. insulin levels for a period of several months. Insulin secretion Analysis of glucose transporter expression in 2-month-old studied in isolated islets and the perfused pancreas is charac- RIP-Ras mice reveals a marked downregulation of P-cell terized by a normal incremental response to increasing glucose GLUT2 in transgenic males that precedes the appearance of concentrations. Glucose metabolism, as measured by glucose islet lesions by about 3 months. During this period the mice phosphorylation and oxidation in isolated islets, shows a nor- maintain normal glucose homeostasis, which suggests that mal dose dependence on extracellular glucose concentrations. GLUT2 is not essential for the normal response of ,B cells to These findings suggest that normal GLUT2 expression in P glucose. cells is not essential for glucose sensing. The transgenic mice provide an experimental system for studying the role ofglucose MATERIALS AND METHODS phosphorylation in regulation of insulin release in the absence Immunofluorescence. Pancreata were removed and fixed in of GLUT2. a paraformaldehyde/lysine/periodate fixative (13). Frozen sections (3 ,um) were stained with antisera to GLUT2 (14) and Pancreatic (3 cells secrete insulin in response to changes in GLUT1 (15) (both kindly provided by B. Thorens, Whitehead blood glucose concentrations. Glucose uptake and metabo- followed by visualization with a fluorescein- lism by the (3 cell are required for generating intracellular Institute), signals that trigger the exocytosis of insulin from prestored conjugated second antibody (14). The same sections were secretory vesicles (1). Glucose uptake into ,Bcells is mediated then double-stained with anti-insulin serum and visualized by the glucose transporter isotype GLUT2, a member of the with a rhodamine-conjugated second antibody (14). facilitated glucose transporter family (2, 3). A decreased Immunoblot Analysis. Islets were isolated by collagenase expression of (3-cell GLUT2 has been correlated with a infusion through the bile duct (16). The islets were disrupted reduced rate of insulin secretory response to glucose in in 5% (wt/vol) SDS/80 mM Tris-HCl, pH 6.8/5 mM EDTA/ several rodent models of diabetes (4-8), which supports the 5% (vol/vol) 2-mercaptoethanol/1 mM phenylmethylsulfo- hypothesis that GLUT2 is required for normal glucose sens- nyl fluoride. Cell lysates containing 30 ,ug of protein from ing. In contrast, there is evidence to suggest that glucose individual mice were resolved on two identical SDS/10o phosphorylation by glucokinase, rather than the transport of polyacrylamide gels and transferred to a nitrocellulose mem- glucose, is the rate-limiting step in glucose metabolism (1, 9, brane. The membrane was incubated with GLUT2 (14) and 10). Both GLUT2 and glucokinase have a relatively high Km glucokinase (17) antisera as described (18). The bound anti- for glucose, about 17 mM, which is in the range associated bodies were detected with 125I-labeled protein A. Autoradio- with insulin secretion, and have, therefore, been considered graphs were scanned with a Molecular Dynamics densitom- candidates for the (-cell glucose-sensing apparatus (1, 4). eter. Here we present evidence suggesting that normal expres- Insulin Secretion. Freshly isolated islets were preincubated sion of GLUT2 is not necessary for maintaining glucose in groups of 15 in 1 ml of Hepes-buffered Krebs-Ringer homeostasis. These findings originate from a study of trans- solution (HKRB) (19) containing 1.67 mM glucose for 1 hr at genic mice overexpressing human genes for the wild-type 37°C in a 5% C02/95% air humidified incubator. The medium [Gly12]HRAS or the oncogenic [Vall2]HRAS under control of was replaced with fresh medium containing the indicated the rat insulin II promoter (RIP-Ras). In these mice RAS glucose concentrations and the islets were incubated for 2 hr overexpression results in (-cell degeneration and diabetes at 37°C. Insulin in the incubation medium and cell extracts rather than abnormal cell proliferation (11). In multiple was measured by RIA in triplicate samples as described (19),

The publication costs of this article were defrayed in part by page charge tPresent address: Department ofCell Biology, Weizmann Institute of payment. This article must therefore be hereby marked "advertisement" Science, 76100 Rehovot, Israel. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ttTo whom reprint requests should be addressed.

5744 Downloaded by guest on October 3, 2021 Cell Biology: Tal et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5745 GLUT-2 insulin GLUT-1 insulin

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male RIP-cHras

FIG. 1. Immunofluorescence analysis ofglucose transporters in RIP-cHrasT1 mice. Pancreas sections from 8-week-old transgenic (I-L) and normal (A-D) males and from 12-week-old transgenic females (E-H) were stained with antisera to GLUT2 (A, E, and I) and GLUT1 (C, G, and K), followed by visualization with a fluorescein-conjugated second antibody. The same sections were then double-stained with anti-insulin serum (B, F, and J and D, H, and L, respectively) and visualized with a rhodamine-conjugated second antibody. (Bar = 30 Atm.) with rat insulin as standard, iodinated bovine insulin as Perfusates containing 16.7 mM glucose or 10 mM arginine tracer, and guinea pig anti-insulin serum. were introduced according to the time table shown in the For in situ pancreas perfusion, the mice were anesthetized, figure. After perfusion the mice were killed, and the pancreas cannulated through the aorta and portal vein, and perfused was stained with GLUT2 antiserum to ascertain the presence essentially as described (20) with oxygenated Krebs-Ringer of greatly reduced levels in the perfused transgenic mice. bicarbonate buffer containing 4% (wt/vol) dextran T70, 6.6 Glucose Phosphorylation. Isolated islets were homogenized mM glucose, 2 mM calcium, and 0.2% bovine serum albumin. and assayed for glucokinase and hexokinase activities by a After a 10-min equilibration period, samples were collected at fluorimetric method as described (9). Approximate Vmax and 1-min intervals into chilled glass tubes containing 4 mg of Km values were determined by Eadie-Scatchard plots (V EDTA and stored at -20TC until assayed for insulin by RIA. versus V/[S], where V is velocity and S is substrate) with the

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GLUT2 Glucokinase FIG. 2. Immunoblot analysis of GLUT2 and glucokinase in islets isolated from RIP-cHrasT1 mice. Cell lysates containing 30 jzg of protein from islets isolated from individual 8-week-old transgenic (lanes 4 and 5) and normal (lanes 6-8) males and from 12-week-old transgenic female mice (lanes 1-3) were resolved on two identical gels, transferred to a nitrocellulose membrane, and incubated with GLUT2 and glucokinase antisera, followed by detection with 125I-labeled protein A. Lanes 9 contain normal mouse liver protein. Molecular size standards are in kDa. Downloaded by guest on October 3, 2021 5746 Cell Biology: Tal et al. Proc. Natl. Acad. Sci. USA 89 (1992) 50 Table 1. Plasma glucose and insulin levels in RIP-cHrasT1 males cc- Fed Fasted a) 0o 40 Glucose, Insulin, Glucose, Insulin, c 0 4 Mice mg/dl ng/ml mg/dl ng/ml ) 30 Normal 192 ± 14 3.78 ± 1.78 122 ± 22 1.03 ± 0.54 0 Transgenic 200 ± 57 2.50 ± 1.53 101 ± 10 0.80 ± 0.68 0)c Normal and transgemnc male mice (8 week old) were bled before and after an overnight fast, and the plasma was analyzed for glucose cf 10 in a Beckman glucose analyzer and for insulin by RIA. Values represent mean ± SEM (N = 6 and 13 for fed and fasted mice, c' n , respectively). The differences between normal and transgenic mice u 10 20 are not significant (P > 0.1). Glucose, mM best-fitted lines drawn by the method of least squares. FIG. 3. Glucose-induced insulin secretion from RIP-cHrasT1 Glucose Oxidation. Isolated islets were preincubated in male islets. Islets isolated from 8-week-old normal (circles) and groups of 12 for 15 min in glucose-free HKRB in 1.5-ml transgenic (squares) male mice were incubated in the presence ofthe microcentrifuge tubes at 370C in a 5% C02/95% air humid- indicated glucose concentrations. Insulin was measured in the incu- ified incubator. The medium was then replaced with 150 tLd of bation medium and cell extracts with an RIA. Values are mean + = and HKRB containing 0.06-16.7 mM D-[1-14C]glucose for a 2-hr SEM (N 7 mice). The differences between normal transgenic mice at each glucose concentration are not significant by Student's incubation. Each tube was then placed in a sealed scintilla- t test (P > 0.1). tion vial and the 14CO2 formed was quantitated as described (21). contain about 50% of the amount in normal controls (Fig. 2). Analysis ofRNA from isolated islets reveals a commensurate RESULTS 8-fold reduction in GLUT2 mRNA levels in 2-month-old transgenic males, compared with normal controls (data not In the best characterized lineage ofRIP-Ras transgenic mice, that GLUT2 expression is affected by denoted RIP-cHrasT1, expression of initiates shown), suggesting [Val'I2HRAS RAS at the transcriptional or posttranscriptional levels. In during embryonic development. At 3 weeks of age the levels contrast with the observations with the trans- ofplasma membrane GLUT2 appear normal in cells ofboth [Val'2]HRAS, male and female transgenic mice, as judged by immunoflu- genic mice overexpressing wild-type [Gly12'HRAS in (3 cells orescence analysis of pancreas sections. However, the islet do not manifest decreased islet GLUT2 immunofluores- GLUT2 staining in 8-week-old males is decreased to unde- cence, even at older ages (data not shown). tectable levels (Fig. 1). In females, the reduction of GLUT2 Islet lesions and hyperglycemia do not develop in most occurs at a slower rate, with residual staining observed in a RIP-cHrasT1 males before 5 months of age. At 8 weeks of small number of cells through 12 weeks of age. The down- age, when the levels of GLUT2 are significantly reduced, the regulation of GLUT2 is not associated with an induction of mice maintain normal glucose homeostasis, as shown by the more ubiquitous GLUT1 isotype, as judged by immuno- normal fed and fasting plasma glucose and insulin levels fluorescence analysis. In both males and females, the islets (Table 1). They manifest a normal response in glucose express normal levels of insulin. Immunoblot analysis con- tolerance tests (data not shown). Insulin secretion from islets firms the immunofluorescence findings, showing a 10-fold isolated from these mice shows an incremental response to decrease in islet GLUT2 in the males, whereas female islets increasing glucose concentrations (Fig. 3). Similar results are

80 6.6 G 16.7 G G ARG 6.6 G

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FIG. 4. Glucose-induced insulin secretion from in situ-perfused pancreas of RIP-cHrasTl males. The pancreas of 8-week-old transgenic and normal male mice was perfused with buffer containing 6.6 mM glucose. After a 10-min equilibration period, perfusates containing 16.7 mM glucose or 10 mM arginine were introduced as shown. Values are mean ± SEM. The secretion at both 6.6 and 16.7 mM glucose was significantly different between normal and transgenic mice (P < 0.001). The mean output during the arginine challenge did not differ significantly between normal and transgenic mice. Downloaded by guest on October 3, 2021 Cell Biology: Tal et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5747 Table 2. Glucose phosphorylation activity in islets from impaired in the transgenic males to limit glucose metabolism, RIP-cHrasTl males as demonstrated by a normal rate ofglucose phosphorylation Glucokinase Hexokinase and oxidation in isolated islets. Calculations have suggested that glucose transport must be reduced by >95% to interfere Vmax, units/g Km, Vmax, units/g Km, with glucose metabolism (22). The significance of the in- Mice of protein mM of protein mM creased insulin secretion in the transgenic, compared to Normal 1.88 7.8 0.65 0.058 normal mice, that was observed in the pancreas perfusion Transgenic 1.83 8.1 0.65 0.038 analyses remains to be clarified, given that the transgenic Islets isolated from 8-week-old normal and transgenic male mice mice do not manifest elevated plasma insulin levels. Aug- were homogenized and assayed for glucokinase and hexokinase mented secretion at low glucose concentrations has been activities. Approximate Vmax and Km values were determined by found in other rodent models with reduced GLUT2 (4-8, 14). Eadie-Scatchard plots (V versus V/[S]) with the best-fitted lines mRNA levels in the transgenic male drawn by the method of least squares. In both normal and transgenic The reduced GLUT2 mice, the glucokinase and hexokinase activities account for 74 and islets suggest that RAS overexpression downregulates 26% of the total glucose phosphorylation, respectively. (1 unit = 1 GLUT2 transcriptionally or posttranscriptionally. The Ras ,umol of product per min at 220C.) oncoprotein has been previously shown to have an opposite stimulatory effect on the mRNA levels ofthe low-Km glucose observed with the in situ-perfused pancreas (Fig. 4). The transporter isotype GLUT1 in cultured rodent fibroblasts transgenic mice respond vigorously to the change of perfus- (23). The extent to which the decreased levels of GLUT2 ate glucose from 6.6 to 16.7 mM. The incremental change in contribute to (-cell degeneration in these mice remains to be insulin release between baseline and stimulating glucose established. Notably, while (3-cell degeneration occurs pre- levels or arginine is not significantly different between normal dominantly in male transgenic mice, both males and females and transgenic mice. However, more insulin is secreted by show a decrease in GLUT2 expression, although the effect in the transgenic mice at both 6.6 and 16.7 mM glucose, the females is delayed and less-pronounced compared to the compared with the controls. males. In addition, [Val12]HRAS and wild-type [Gly12]HRAS In contrast to the decreased levels of GLUT2, no major are equally effective in inducing (-cell degeneration, whereas changes are detected in the levels of glucokinase protein in only [Val12]HRAS causes downregulation of GLUT2 expres- the RIP-Ras mice, compared to normal controls (Fig. 2). In sion. addition, the activities ofglucokinase and hexokinase appear The demonstration that a normal glucose phosphorylation to be normal in 2-month-old transgenic males (Table 2). activity is sufficient for maintaining the correct (-cell respon- Accordingly, the rate of glucose utilization as a function of siveness to glucose, even in the presence of greatly reduced the extracellular concentration of glucose in 2-month-old GLUT2 levels, emphasizes the importance of glucokinase in transgenic males, as measured by oxidation of [1-14C]glucose glucose sensing in (3 cells. The RIP-Ras mice provide an to 14CO2 in isolated islets, closely resembles that of control experimental system for addressing questions related to mice (Fig. 5). Thus, a normal pattern of glucose phosphor- glucokinase regulation in response to various physiological ylation is sufficient for maintaining a correct 83-cell respon- in the of normal GLUT2 function. siveness to glucose, even in the presence of greatly reduced conditions absence levels of GLUT2. We thank 0. A. Emran and D. Fusco-DeMane for technical assistance. This work was supported by a grant from the Juvenile DISCUSSION Diabetes Foundation to S.E., National Institutes of Health Grant DK-20541 to the Albert Einstein Diabetes Research and Training These results demonstrate that normal expression ofGLUT2 Center, and National Institutes of Health Grant DK-35449 to G.W. in (3 cells is not necessary for maintaining their ability to M.T. was supported by a fellowship from the European Molecular accurately sense extracellular glucose concentrations and to Biology Organization. adjust the rate of insulin secretion in response to changing glucose levels. Glucose transport is probably not sufficiently 1. Meglasson, M. D. & Matschinsky, M. F. (1986) Diabetes/ Metab. Rev. 2, 163-214. 10 2. Bell, G. I., Kayano, T., Buse, J. B., Burant, C. F., Takeda, J., Lin, D., Fukumoto, H. & Seino, S. (1990) Diabetes Care 13, 198-208. 3. Thorens, B., Charron, M. J. & Lodish, H. F. (1990) Diabetes a) U) Care 13, 209-218. 0 4. Unger, R. H. (1991) Science 251, 1200-1205. 5. Chen, L., Alam, T., Johnson, J. H., Hughes, S. & Newgard, -0 C. B. (1990) Proc. Natl. Acad. Sci. USA 87, 4088-4092. N 6. Johnson, H. J., Ogawa, A., Chen, L., Orci, L., Newgard, C. B., Alam, T. & Unger, R. H. (1990) Science 250, 546-549. 0 7. Orci, L., Unger, R. H., Ravazzola, M., Ogawa, A., Komiya, I., Bactens, D., Lodish, H. F. & Thorens, B. (1990) J. Clin. Invest. 86, 1615-1622. 8. Thorens, B., Weir, G. C., Leahy, J. L., Lodish, H. F. & Bonner-Weir, S. (1990) Proc. Natl. Acad. Sci. USA 87, 6492- 0 5 10 15 20 6496. Glucose, mM 9. Liang, Y., Najafi, H. & Matschinsky, F. M. (1990) J. Biol. Chem. 265, 16863-16866. FIG. 5. Glucose oxidation in islets of RIP-cHrasT1 males. Islets 10. Meglasson, M. D., Manning, C. D., Najafi, H. & Matschinsky, isolated from 8-week-old normal (circles) and transgenic (squares) F. M. (1986) Diabetes 35, 1340-1344. male mice were incubated in the presence of the indicated concen- 11. Efrat, S., Fleischer, N. & Hanahan, D. (1990) Mol. Cell. Biol. trations of D-[1-14C]glucose for 2 hr and the amounts of 14CO2 formed 10, 1779-1783. were quantitated. Values for oxidized glucose are expressed as nmol 12. Efrat, S. (1991) Endocrinology 128, 897-901. of glucose oxidized per hr by 100 ,ug of islet protein and represent 13. McLean, I. W. & Nakane, F. P. (1974) J. Histochem. 61, mean + SEM (N = 3). 1077-1083. Downloaded by guest on October 3, 2021 5748 Cell Biology: Tal et al. Proc. Natl. Acad. Sci. USA 89 (1992)

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