Islet endocrine-cell behavior from birth onward in mice with the nonobese diabetic genetic background.

BACKGROUND: Glucagon-producing alpha cells play a crucial role during the perinatal period. Because of their peri-islet localization near the early dendritic and macrophage cell infiltration, we thought it pertinent to investigate alpha cells in greater depth in nonobese diabetic (NOD) mice, a well-recognized spontaneous model for human type I diabetes. MATERIALS AND METHODS: We determined alpha-cell distribution (glucagon immunohistochemistry and image analysis) and activity (real-time reverse transcriptase polymerase chain reaction [RT-PCR] and glucagon radioimmunoassay [RIA]), in relationship to glycemia in NOD and lymphocyte-deficient NODscid mice as compared to control mice (C57BL/6) from birth onward. RESULTS: NOD and NODscid mice, particularly at 1 day of age, had twice as many very small islets (<2,000 pixels) as C57BL/6 mice. During the postnatal period, the percentages of glucagon-positive areas in islets less than 2000 pixels were higher in NOD mice than C57BL/6; only a trend was found in NODscid. Pancreatic mRNA expression and glucagon content decreased in all strains at weaning. However, before weaning, pancreatic and blood glucagon levels were significantly lower in NOD and NODscid compared to C57BL/6 mice. Low basal nonfasting glycemia was observed in all strains before weaning with some strain differences: glycemia was significantly lower in NOD than C57BL/6, and higher in NODscid than NOD and C57BL/6. CONCLUSION: These data suggest that, before weaning, NOD and, to some extent NODscid pancreata contain more immature islets (as reflected by their small size and high percentages of glucagon-positive areas, concomitant with lower glucagon storage and basal secretion) than C57BL/6 pancreata.

Pancreatic hormone and glutamic acid decarboxylase expression in the mouse thymus: a real-time PCR study.

We devised a real-time RT-PCR method for the quantification of preproinsulin 1 and 2, proglucagon, prosomatostatin, and GAD 65 and 67 mRNAs in the thymus, using specific primers and internal probes. Corresponding standard cRNA synthesis and normalization to 18S ribosomal RNA allowed direct quantification. Then, during the first month of life, the expression of each substance of interest was measured in the thymus of NOD mice (a spontaneous model of type 1 diabetes), C57BL/6, BALB/c and lymphocyte-deficient mice (NODscid, NODrag, BALB/cscid and C57BL/6rag). In all mouse thymuses, preproinsulin 1 and GAD 65 were undetectable, preproinsulin 2 and proglucagon showed low expression, whereas that of GAD 67 and somatostatin were high. In 7-day-old mice, GAD 67 and prosomatostatin thymic expressions were lower in NOD than in C57BL/6, and at the same age, the scid mutation but not the rag mutation induced higher expression of all investigated genes compared to control mice. In conclusion, our data allowed the quantification of the expression of pancreatic factors in the mouse thymus. Investigations are underway to quantify, at the cellular level, i.e., in thymic dendritic/macrophage cells, the RNA expression of potential autoantigens, such as preproinsulin 2 and GAD 67.

Ontogeny of glutamic acid decarboxylase gene expression in the mouse pancreas.

Using two RT-PCR quantitative assays, we measured the pancreatic expression of the two isoforms of glutamic acid decarboxylase in the foetus, newborn and 14-, 21- and 35-day-old male and female NOD and C57BL/6 mice. In the C57BL/6 mouse, GAD 67 pancreatic expression is stable; in NOD mice, GAD 67 expression is similar to that found in control mice, except at 5 weeks of age, when pancreatic GAD 67 expression is about 2.5 times higher than in C57BL/6 mice. The pancreatic expression of GAD 65 is under the detection limit of the assay until 5 weeks of age. The overexpression of GAD 67 characterized in pancreas from 5-week-old NOD mice could be the result of beta cell hyperactivity, previously reported in this mouse strain.

Glutamic acid decarboxylase (GAD 67) gene expression in the pancreas and brain of the nonobese diabetic mouse.

A quantitative PCR was developed to measure GAD 67 mRNA levels in pancreas from NOD mice. Two nonautoimmune mouse strains were used as controls. An internal standard, rat GAD 67 mRNA, was prepared to quantify the reaction. In NOD mice GAD 67 expression in the pancreas was in the order of 2 x 10(6) molecules/microgram of total RNA; this expression was five times less in the two control mouse strains, whatever the age. The overexpression of GAD 67 characterized in the NOD mouse could be restricted to the pancreas since similar analysis performed in mouse brain did not reveal a large difference between the three mouse strains studied.

Gene expression of pancreatic glutamic acid decarboxylase in the nonobese diabetic mouse.

The 65 kDa isoform of glutamic acid decarboxylase (GAD 65) is an autoantigen implicated in type I diabetes. We have developed a quantitative PCR method to measure GAD 65 mRNA in the pancreas of nonobese diabetic (NOD) mice. Two other nonautoimmune mouse strains were used as controls. In all mice, pancreatic GAD 65 expression declined with age. In 5-week-old NOD mice, a clear difference was detected between males and females. This sexual dimorphism may explain the absence of tolerance to GAD 65 in the NOD female which could contribute to autoimmune destruction of beta cells in the pancreas and subsequent development of diabetes.

Prevention of autoimmune diabetes in nonobese diabetic female mice by treatment with recombinant glutamic acid decarboxylase (GAD 65).

The nonobese diabetic (NOD) mouse spontaneously develops insulin-dependent diabetes (IDDM or type I diabetes), resulting from T-lymphocyte-mediated destruction of pancreatic beta cells. This autoimmune phenomenon includes mononuclear cell infiltration of the islets of Langerhans (insulitis) and the presence of circulating autoantibodies. The specificity of the autoantibodies and of the autoreactive T cells was investigated and several autoantigens were proposed, in particular glutamic acid decarboxylase (GAD). This enzyme exists in two forms (GAD 65 and GAD 67) encoded by two independent genes. To explain the role of GAD in type I diabetes, we prepared recombinant rat GAD 65 as fusion protein, produced in an Escherichia coli expression system, and we treated NOD female mice from 4 to 7 weeks of age by repeated intraperitoneal injections of 5 micrograms fusion protein (3 injections per week); control groups received the fusion partner, maltose binding protein (MBP) or dissolving agent (NaCl 0.9%). We investigated two parameters, the degree of insulitis 5 weeks after the last injection and the overall incidence of the disease. Histological examination of the pancreata from GAD-treated mice revealed a significant reduction in the severity of insulitis compared with the two control groups. Furthermore, we observed that the time of onset and the frequency of diabetes in NOD females injected with GAD fusion protein differed significantly from the control groups receiving MBP or NaCl (P < 0.0001). These results show that a 3-week treatment of NOD female mice starting at 4 weeks of age protects them from diabetes, again emphasizing the crucial role of GAD as autoantigen in type I diabetes.

Quantitative analysis of glutamate decarboxylase (GAD 65) gene expression in NOD mouse pancreas.

Glutamate decarboxylase (GAD), especially the GAD 65 isoform, is a major autoantigen in autoimmune diabetes. To determine the role of GAD 65 in the pathogenesis of the disease, we developed a quantitative PCR method allowing to establish the absolute number of GAD 65 mRNA molecules expressed in pancreas of male and female non-obese diabetic (NOD) mice at 5 weeks of age, in comparison of the 2 non autoimmune mouse strains. It appeared that pancreatic expression of GAD was similar in the 3 strains (around 30,000 molecules/micrograms total RNA) in males. However, in the NOD mouse, sexual dimorphism was observed with low GAD 65 expression in the female known to show higher incidence of the disease than the male. This finding could contribute to the absence of GAD 65 tolerance in the female and suggests an hormonal control of GAD 65 gene expression.