Determination of Glutamic Acid Decarboxylase (GAD65) in Pancreatic Islets and Its In Vitro and In Vivo Degradation Kinetics in Serum Using a Highly Sensitive Enzyme Immunoassay

Glutamic acid decarboxylase GAD65 autoantibodies (GADA) are an established marker for autoimmune diabetes. Recently, the autoantigen GAD65 itself was proposed as biomarker of beta-cell loss for prediction of autoimmune diabetes and graft rejection after islet transplantation. Therefore, the GAD65 content in pancreatic islets of different species and its serum degradation kinetics were examined in this study using a sensitive immunoassay. GAD65 was found in quantities of 78 (human), 43.7 (LEW.1A rat) and 37.4 (BB/OK rat) ng per 1,000 islets, respectively, but not in mouse islets. The in vitro half-life of porcine GAD65 and human recombinant GAD65 ranged from 1.27 to 2.35 hours at 37°C in human serum, plasma and blood, and was unaffected by presence of GAD65 autoantibodies. After injecting 2,000 ng recombinant human GAD65 into LEW.1A rats, the in vivo half-life was 2.77 hours. GAD65 was undetectable after 24 hours in these animals, and for up to 48 hours following diabetes induction by streptozotocin in LEW.1A rats. Estimated from these data, at least 13 islets in rat and 1,875 in human must be simultaneously destroyed to detect GAD65 in circulation. These results should be taken into consideration in further studies aimed at examining the diagnostic relevance of GAD65

Phenotypic and gene expression differences between DA, BN and WOKW rats.

BACKGROUND: Because inbred rat strains are widely used as laboratory models, knowledge of phenotypic and genetic variations between strains will be useful to obtain insight into the relationship between different strains. METHODS AND RESULTS: We studied phenotypic traits: of each strain--BN/K, DA/K and WOKW--10 male rats were studied for body weight and serum constituents at an age of 10 and 30 weeks. In addition, a total of 95 rats were studied for life expectancy. At an age of 30 weeks, these male rats were killed by an overdose of anesthetic (Sevofluran, Abbott), and the subcutaneous and visceral adipose tissue as well as bone tissue were removed to study the expression of 20 genes. There were significant differences in body weight, serum lipids and leptin at an age of 30 weeks between strains. Regarding life expectancy, BN rats lived longest (1072±228d). The highest gene expression was found in bone of BN rats. In adipose tissues, Nfkb1 is only expressed in subcutaneous adipocytes, and 5 genes, Col2a1, Mmp9, Tnfa, Ins1 and Cyp24a1, are not expressed in adipocytes. The ranking BN = DA>WOKW was observed in only one gene in subcutaneous (Fto) and visceral adipocytes (Col6a1). There were no significant differences in gene expression of one gene in subcutaneous adipocytes and of 3 genes in visceral adipocytes. Comparing the gene expression in visceral and subcutaneous adipocytes, only one gene showed a comparable behavior (Bmp1). CONCLUSION: From these results, it can be concluded that obvious phenotypic differences are caused by genetic differences between three rat strains, BN, DA and WOKW, as supported by gene expression studies in bone and adipose tissues. Especially BN rats can be used to study the genetic basis of long life

Increase in Bone Mass Before Onset of Type 1 Diabetes Mellitus in Rats

Background/Aim: The typical insulin deficiency in type 1 diabetes mellitus has general effects on metabolism and also affects bone quality. Materials and Methods: Two diabetic rat lines (BB/OK; BB.6KWR) and two non-diabetic rat strains (KWR and BB.14+18KWR), as control group, were included in the study. Bone mineral density, bone mineral content and body structure measurements were performed. The measurements took place before the onset of diabetes mellitus Results: A comparison of the groups showed increased bone density values of the diabetic rats in relation to the control groups. A new finding of increased bone density in the diabetic rats occurs. Conclusion: Diabetic rats showed no osteoporotic bone metabolism before the onset of clinically relevant type 1 diabetes mellitus, but rather increased bone metabolic activity

Relative gene expression in bone (A), subcutaneous (B) and visceral (C) adipocytes of 30 weeks old males (mean ± SEM).

<p>* BN, DA and WOKW are significantly different at 1 ** or 0.1*** per cent level. + BN and DA are significantly different to WOKW at 1++ or 0.1+++per cent level. § BN is significantly different to DA and WOKW at 1++ or 0.1+++per cent level. # BN is significantly different to WOKW at 1 ## or 0.1 ### per cent level. $BN is significantly different to DA at 1$$or 0.1$$$ per cent level. & DA is significantly different to WOKW at 1 && or 0.1 &&& per cent level.</p

Significant correlations between fat ratio and gene expression in subcutaneous (Sub) and visceral (Vis) adipocytes of BN, DA and WOKW rats.

<p>Significant correlations between fat ratio and gene expression in subcutaneous (Sub) and visceral (Vis) adipocytes of BN, DA and WOKW rats.</p