Abstract

BACKGROUND
Glutamic acid decarboxylase(GAD; EC 4.1.1.15), one of the major B-cell autoantigens in IDDM, is an enzyme which catalyzes the synthesis of major inhibitory neurotransmitter, r-aminobutyric acid (GARA), in the mammalian brain, pancreas and other organs. Two isoforms of GAD, GAD65 and GAD67, have been identified which differ in their intracellular localization. Autoantibodies to GAD have been detected several years before the clinical onset of IDDM, implicating GAD as a leading autoantigen which somehow correlated with the pathogenesis of IDDM. We have determined the characteristics of GAD isoform expression in the pancreas of NOD mouse, an animal model extensively employed in IDDM study, using RT-PCR and Southern blot methods. METHODS: Pancreas was obtained from female NOD mouse(neonate, 4, 8, 12, 16, 20 week-old) and age-matched female ICR mouse. Total cellular RNA was I.solated by acid guanidinium thiocyanate method and employed in the RT-PCR amplification using GAD65- and GAD67-specific primer designed in our laboratory. The PCR product was blotted onto the nylon membrane and subjected to Southern analysis using 32P-ATP labelled hybridization probe. RESULTS: In NOD pancreas, GAD67 was expressed six times higher than GAD65 at neonatal stage. Then, the expression was dramatically decreased from 4 weeks when the pancreatic insulitis begins to occur. After 12 weeks of age, both GAD67 and GAD65 expression was almost undetectable. However, in control ICR mouse, there were no significant differenees between GAD65 and GAD67 expression throughout the ages. And, the expression of both GAD65 and OAD67 was not decreased with ages in contrast to NOD mouse. CONCLUSION: In this experiment, we found that the expression of GAD isoforms in NOD mouse shows distinct pattern in comparison to that of control ICR mouse. The expression of GAD67 was significantly higher than GAD65 in neonatal NOD mouse while, in control ICR mouse, same level of GAD isoforrns expression was observed. This finding clearly suggested the possibility that the expression of GAD isoforms in diabetic NOD mouse is quite distinct and may somehow play a role in the pathogenesis of diabetes although the precise mechanism remains to be unveiled. In addition, our data also supported the hypothesis that expressional pattern, and, if possible, ' the etiophysiological function of GAD isoforms in NOD mouse pancreas may be quite different from that in human pancreas.

• Keywords: Insulin-dependent diabetes mellitus ( IDDM );non-obese diabetic ( NOD ) mouse;GAD65;GAD67;RNA;RT-PCR;Southern blot;ontogeny;Expression;