Characterization of cochlear nucleus principal cells of Meriones unguiculatus and Monodelphis domestica by use of calcium-binding protein immunolabeling.

Antibodies directed against calcium-binding proteins (CaBPs) parvalbumin, calbindin-D28k and calretinin were used as neuronal markers to identify and characterize different principal cell types in the mammalian cochlear nucleus. For this purpose, double immunofluorescence labeling and the combination of CaBP-labeling with pan-neuronal markers were applied to analyze the CaBPs distribution in neurons of the cochlear nucleus (CN) of the Mongolian gerbil (Meriones unguiculatus) and the gray short-tailed opossum (Monodelphis domestica). Despite of the fact, that these two mammalian species are not closely related, principal cell types in the CN of the two species showed many corresponding morphological features and similarities in immunolabeling of the CaBPs. Parvalbumin seems not to be suited as a differential neuronal marker in the CN since it is expressed by almost all neurons. In contrast, calbindin and calretinin were more restricted to specific cell types and showed a mostly complementary labeling pattern. As one of the most interesting findings, calbindin and calretinin were predominantly found in subpopulations of globular bushy cells and octopus cells in the ventral CN. Such a neuron-specific CaBP-expression in subpopulations of morphologically defined cell types argues for a more refined classification of CN cell types in Meriones and Monodelphis. Additionally, other cell types (cartwheel cells, unipolar brush cells, fusiform cells) were marked with calbindin or calretinin as well. Calretinin staining was predominantly observed in auditory nerve fibers and their endings including endbulbs of Held in Meriones. Spherical bushy cells showed a different calretinin-immunolabeling in Meriones and Monodelphis. This species-specific difference may be related to adaptive differences in auditory function.

Circadian and age-dependent expression patterns of GLUT2 and glucokinase in the pancreatic beta-cell of diabetic and nondiabetic rats.

Alterations in glucose sensing are well-known in both humans and animal models of non-insulin-dependent diabetes mellitus. However, the circadian- and age-dependent expression of glucose-sensing genes has not previously been investigated in vivo. In the present paper, we show a progressive loss of beta-cell GLUT2-mRNA and, by immunocytochemistry, a gain of soluble, cytoplasmic GLUT2-protein in Goto-Kakizaki rat islets. We report that GLUT2-mRNA shows significant diurnal variation, which is stronger in metabolically healthy rats. We also demonstrate the significant diurnal variation of glucokinase-mRNA, with higher levels in the pancreas of 6-week-old Goto-Kakizaki rats than in Wistar rats. This leads to a maximum glucose phosphorylation capacity in-phase with food intake, enhanced glucose-stimulated insulin secretion, and prevents postprandial hyperglycemia. Perfusion experiments showed a reduction in glucose-stimulated insulin secretion in Goto-Kakizaki rat islets with an impaired first phase. Hyperglycemia and hypoinsulinemia in newborn and up to 3-week-old Goto-Kakizaki rats are thus probably due to reduced pancreatic beta-cell content, reduced beta-cell insulin content and impaired glucose sensing. The de-compensation of the metabolic situation in 42-week-old Goto-Kakizaki rats is likely to be caused by beta-cell destruction accompanied by negligible accumulation of GLUT2 in the cell membrane and further reduction of glucokinase expression.

Circadian changes of ether-a-go-go-related-gene (Erg) potassium channel transcripts in the rat pancreas and beta-cell.

Evidence has previously been presented that circadian rhythms play a role in islet hormone secretion. Here, RT-PCR was used to monitor the circadian expression of ether-a-go-go-related gene (Erg) potassium channel isoforms and Erg1 splice variants. Immunohistochemistry was used to identify the pancreatic distribution patterns of ERG1a and ERG1b, as well as ERG2 and ERG3. The influence of ERG on insulin secretion was monitored by perfusion of rat INS-1 beta-cells with the blockers E-4031 and rBeKm-1. We identified Erg1a, Erg1b, Erg2 and Erg3 transcripts in islets and INS-1 cells. Immunohistochemistry showed differential expression of ERG isoforms in the islet. Ca(2+) imaging and electrophysiological recordings of INS-1 cells during ERG blocking by E-4031 indicated functional ERG channels. Serum shock treatment of INS-1 cells elicited a time-dependent expression response for Erg transcripts. These results add to the current understanding of the function of ERG channels in beta-cells and the circadian secretion processes of insulin.

Did the gradual loss of GLUT2 cause a shift to diabetic disorders in the New Zealand obese mouse (NZO/Hl)?

The New Zealand obese mouse (NZO/Hl) is characterised by hereditary obesity and type-2 diabetes, including insulin resistance, hyperinsulinaemia, and glucose intolerance. In other diabetic models, it has been revealed that the proper functioning of the glucose transporter isoform 2 (GLUT2) is essential for adequate secretion of insulin. The aim of this study was to compare the distribution of islet cells and GLUT2, as well as the expression of GLUT2-mRNA, in the pancreas of NZO mice and metabolically unimpaired NMRI (Naval Medical Research Institute) mice. Pancreas tissue was obtained from different stages of development. For molecular determination of the expression level of GLUT2-mRNA, total-RNA was extracted from the pancreas and analysed by quantitative real-time RT-PCR. All investigated NZO mice displayed increased weight, elevated hyperinsulinaemia, and slightly enhanced blood glucose levels compared with the NMRI control mice. By means of immunofluorescence microscopy drastically reduced insulin levels were detected, which might be compensated by the observed islet cell hyperplasia and hypertrophy. Furthermore, the normally peripheral localisation of the alpha-cells within islets was disturbed. By contrast, there were no changes in somatostatin cell distribution. However, considerable differences appeared with regard to GLUT2: whereas the beta-cells of NMRI mice showed dense immunostaining of the GLUT2 transporter on the cell surface, in all age groups of NZO mice, GLUT2 on the plasma membranes was reduced and dispersed in the cytoplasm. These findings agree with the molecular biological results, which displayed decreased mRNA-expression of GLUT2. In summary, the observed alteration of islet morphology and of GLUT2 expression in diabetic mice complements our previous results from a superfusion protocol and further clarifies the mechanisms of diabetogenesis in NZO mice.