Differential Target Molecules for Toxicity Induced by Streptozotocin and Alloxan in Pancreatic Islets of Mice in Vitro

Abstract

Streptozotocin (STZ) and alloxan (ALX) are potent diabetogens in different species of laboratory animals. Here, we describe differential in vitro effects of STZ and ALX on β-cell molecules that are essential for glucose transport and metabolism, the glucose transporter 2 (GLUT2) and glucokinase (GK), respectively. Incubation of isolated pancreatic islets of C57 BL/6 mice with STZ or ALX for 30 min resulted in a concentration-dependent gradual loss of β-cell function as determined by basal and D-glucose (D-G)-stimulated insulin release. ALX concentration-dependently reduced the mRNA expression of GLUT2 and GK and the effect on GLUT2 was more marked. STZ, in contrast, did not affect the mRNA expression of GLUT2 and GK, but concentration-dependently reduced the GLUT2 protein expression. Both STZ and ALX failed to affect the mRNA expression of proinsulin and of β-actin. The deleterious effects of STZ and ALX were not due to β-cell loss, because the total RNA yields and protein contents as well as the proinsulin mRNA expression in isolated islets of the differentially treated islets did not differ significantly from controls. Furthermore, islets that had been exposed to STZ or ALX responded to the non-glucose secretagogue arginine in a pattern comparable to that of solvent-treated cultures. When preincubating islet cultures with either D-G or its chemically closely related analogue 5-thio-D-glucose (5-T-G), different effects were obtained after treatment with either ALX or STZ. Thus, preincubation with 5-T-G protected the cultures from STZ-induced GLUT2 protein reduction, whereas D-G failed to do so. Preincubation with D-G, however, protected the cultures from ALX-induced reduction of GLUT2 and GK mRNA expression, whereas 5-T-G, at best, exerted a modest protection against ALX at a concentration of 1 mmol/l. Apparently, in vitro, GLUT2 protein is a key target molecule for STZ, while GLUT2 mRNA and GK mRNA are target molecules for ALX.

Alloxan - streptozotocin - pancreatic islets - target molecules

References

• 1 Alonso S, Minty A, Bourlet X, Buckingham M. Comparison of three acting-coding sequences in the mouse; evolutionary relationships between the actin genes of warm-blooded vertebrates.  J Mol Evol. 1986;  23 11-22
• 2 Atkinson M A, MacLaren N K. The pathogenesis of insulin-dependent diabetes mellitus.  N Engl J Med. 1994;  331 1428-1436
• 3 Boquist L. A new hypothesis for alloxan diabetes.  Acta Path Microbiol Scand Sect A. 1980;  88 202-209
• 4 Chomczynski P, Sacchi N. Singlestep method for RNA isolation by acid guanidin thiocyanate-phenol-chloroform extraction.  Anal Biochem. 1987;  126 156-159
• 5 Crouch R K, Gandy S E, Kimsey G, Galbraith R A, Galbraith G M, Buse M G. The inhibition of islet superoxide dismutase by diabetogenic drugs.  Diabetes. 1981;  30 235-241
• 6 Elsner M, Guldbakke B, Tiedge M, Munday R, Lenzen S. Relative importance of transport and alkylation for pancreatic beta-cell toxicity of streptozotocin.  Diabetologia. 2000;  43 1528-1533
• 7 Elsner M, Tiedge M, Guldbakke B, Munday R, Lenzen S. Importance of the GLUT2 glucose transporter for pancreatic beta cell toxicity of alloxan.  Diabetologia. 2002;  45 1542-1549
• 8 Gille L, Schott-Ohly P, Friesen N, Schulte im Walde S, Udilova N, Nohl H, Gleichmann H. Generation of hydroxyl radicals mediated by streptozotocin in pancreatic islets of mice in vitro.  Pharmacol Toxicol. 2002;  90 317-326
• 9 Gunnarsson R, Berne C, Hellerstrom C. Cytotoxic effects of streptozotocin and N-nitrosomethylurea on the pancreatic B cells with special regard to the role of nicotinamide-adenine dinucleotide.  Biochem J. 1974;  140 487-494
• 10 Klinkhammer C, Popowa P, Gleichmann H. Specific immunity to streptozotocin: cellular requirements for induction of lymphoproliferation.  Diabetes. 1988;  37 74-80
• 11 Lenzen S, Panten U. Alloxan: history and mechanism of action.  Diabetologia. 1988;  31 337-342
• 12 Lenzen S, Brünig H, Münster W. Effects of alloxan and ninhydrin on mitochondrial Ca²⁺ transport.  Mol Cell Biochem. 1992;  118 141-151
• 13 Like A A, Rossini A A. Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus.  Science. 1976;  193 415-417
• 14 Malaisse-Lagae F, Sener A, Malaisse W J. Contrasting modes of action of D-glucose and 3-O-methyl-D-glucose as protectors of the rat pancreatic β-cell against alloxan.  Biochim Biophys Acta. 1983;  762 36-43
• 15 Murata M, Takahashi A, Saito I, Kawanishi S. Site-specific DNA methylation and apoptosis: induction by diabetogenic streptozotocin.  Biochem Pharmacol. 1999;  57 881-887
• 16 Oberley L W. Free radicals and diabetes.  Free Radic Biol Med. 1988;  5 113-124
• 17 Schnedl W J, Ferber S, Johnson J, Newgard C B. STZ transport and cytotoxicity: specific enhancement in GLUT2-expressing cells.  Diabetes. 1994;  43 1326-1333
• 18 Schulte im Walde S, Dohle C, Schott-Ohly P, Gleichmann H. Molecular target structures in alloxan-induced diabetes in mice.  Life Sci. 2002;  71 1681-1694
• 19 Sener A, Malaisse W J. Resistance to alloxan of tumoral insulin-producing cells.  FEBS. 1985;  193 150-152
• 20 Takasu N, Koniya I, Asawa T, Nagasawa Y, Yamada T. Streptozotocin- and alloxan-induced H₂O₂ generation and DNA fragmentation in pancreatic islets. H₂O₂ as mediator for DNA fragmentation.  Diabetes. 1991;  40 1141-1145
• 21 Thorens B, Weir G C, Leahy J L, Lodish H F, Bonner-Weir S. Reduced expression of the liver/beta-cell glucose transporter isoform in glucose-insensitive pancreatic beta cells of diabetic rats.  Proc Natl Acad Sci USA. 1990;  87 6492-6496
• 22 Wang Z, Dohle C, Friemann J, Green B S, Gleichmann H. Prevention of high- and low-dose STZ-induced diabetes with D-glucose and 5-thio-D-glucose.  Diabetes. 1993;  42 420-428
• 23 Wang Z, Gleichmann H. GLUT2 in pancreatic islets: crucial target molecule in diabetes induced with multiple low doses of streptozotocin in mice.  Diabetes. 1998;  47 50-56
• 24 Watson C J, Demmer J. Procedures for cDNA cloning. Glover DM, Hames BB DNA Cloning 1 - Core Techniques. New York; Oxford University Press 1995: 83-119
• 25 Zhang H, Brunk U. Alloxan cytotoxicity is highly potentiated by plasma membrane and lysosomal-associated iron - a study on a model system of cultured J-774 cells.  Diabetologia. 1993;  36 707-715
• 26 Zimny S, Gogolin F, Abel J, Gleichmann H. Metallothionein in isolated pancreatic islets of mice: induction by zinc and streptozotocin, a naturally occurring diabetogen.  Arch Toxicol. 1993;  67 61-65