Sensitive and specific time-resolved fluorescence immunoassay of rat C-peptide for measuring hormone secretory and storage capacity of ß-cells in vivo and in vitro.

The limitations of current rat C-peptide assays led us to develop a time-resolved fluorescence immunoassay for measurements in plasma, incubation media, and tissue/cell extracts. The assay uses 2 monoclonal antibodies, binding to different parts of the C-peptide molecule, and allowing, respectively, capture of the peptide and its detection by europium-labeled streptavidin. It is performed on 25-µL samples for a dynamic range from 66pM up to 3900pM C-peptide and displays over 95% recovery of added peptide in the range of 111pM to 2786pM. Its inter- and intra-assay coefficients of variations are, respectively, lower than 7.6% and 4.8%. Cross-reactivities by rat insulin and by human and porcine C-peptide are negligible, and cross-reactivity by mouse C-peptide is 6% ± 2%. The assay has been validated for in vivo and in vitro measurements of C-peptide release and cellular content. Release patterns were similar to those for insulin and occurred in equimolar concentrations for both peptides. The molar C-peptide contents in purified ß-cells and isolated islets were similar to the corresponding insulin contents. This was also the case for pancreatic extracts containing protease inhibitors.

Protein markers for insulin-producing beta cells with higher glucose sensitivity.

BACKGROUND AND METHODOLOGY: Pancreatic beta cells show intercellular differences in their metabolic glucose sensitivity and associated activation of insulin production. To identify protein markers for these variations in functional glucose sensitivity, rat beta cell subpopulations were flow-sorted for their level of glucose-induced NAD(P)H and their proteomes were quantified by label-free data independent alternate scanning LC-MS. Beta cell-selective proteins were also identified through comparison with rat brain and liver tissue and with purified islet alpha cells, after geometrical normalization using 6 stably expressed reference proteins. PRINCIPAL FINDINGS: All tissues combined, 943 proteins were reliably quantified. In beta cells, 93 out of 467 quantifiable proteins were uniquely detected in this cell type; several other proteins presented a high molar abundance in beta cells. The proteome of the beta cell subpopulation with high metabolic and biosynthetic responsiveness to 7.5 mM glucose was characterized by (i) an on average 50% higher expression of protein biosynthesis regulators such as 40S and 60S ribosomal constituents, NADPH-dependent protein folding factors and translation elongation factors; (ii) 50% higher levels of enzymes involved in glycolysis and in the cytosolic arm of the malate/aspartate-NADH-shuttle. No differences were noticed in mitochondrial enzymes of the Krebs cycle, beta-oxidation or respiratory chain. CONCLUSIONS: Quantification of subtle variations in the proteome using alternate scanning LC-MS shows that beta cell metabolic glucose responsiveness is mostly associated with higher levels of glycolytic but not of mitochondrial enzymes.

Heterogeneity in distribution of amyloid-positive islets in type-2 diabetic patients.

Amyloid-containing (A+) islets are characteristic for type-2 diabetes (T2D), but their abundance seems variable among patients. It is unclear whether the distribution of A+ islets follows a certain pattern or occurs randomly throughout the pancreatic organ. We investigated the topography of A+ islets in eight pancreata of T2D patients and eight sex- and age-matched non-diabetic subjects. Transversal sections of head, body and tail segments were stained with synaptophysin combined with Congo red to map/quantify islet tissue and amyloid. In the eight T2D pancreata, the overall percentage of A+ islets varied from 4% to 85%. Further analysis in body and tail indicated that peripheral regions exhibited higher percentages of A+ islets than central regions (averages of, respectively, 30% and 17%, P<0.05). Non-diabetic control pancreata also exhibited A+ islets, albeit at a 25-fold lower frequency; a tendency towards higher percentage of A+ islets in peripheral versus central regions was also observed. The higher percentage A+ islets in peripheral regions was associated with a higher density and relative islet over exocrine surface area. These observations on heterogeneity in abundance and distribution of A+ islets need consideration when sampling tissue for studies on human islet amyloidosis. The present methodology allows us to further investigate the susceptibility to amyloidosis of islets in peripheral regions of the pancreas.