[Val12]HRAS Downregulates GLUT2 in I6 Cells of Transgenic Mice

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 *Whitehead Institute for Biomedical Research, Cambridge, MA 02142; *Joslin Diabetes Center, Boston, MA 02215; Departments of §Medicine 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 &. 4 .S * normal 4;, . .; .X ** ,I female RIP-cHras 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 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 - 200 - - 97- 0 ems -.z-68- S- - 43 - Vp - 25 - 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.

Load more