METHODS: Caprine pancreatic tissues were collected from a local slaughterhouse and prior transported to the laboratory by maintaining the cold chain. Islets were obtained by a collagenase-based digestion and optimized isolation technique. Islet cell purity and viability were determined by dithizone and trypan blue staining, respectively. Islet clusters of different sizes were positively identified by staining methods and demonstrated 90% viability in the culture system. Following static incubation, an in vitro insulin secretion assay was carried out and analyzed by ELISA.
RESULTS: The islets remained satisfactorily viable for 5 days in the culture system following regular media changes. The current study has successfully optimized the isolation, purification and culture maintenance of caprine islets.
CONCLUSION: The successful yield, viability and functionality of islets isolated from the optimized protocol provide promising potential as an alternative source of islets for diabetes and transplantation researches.
METHODS: Caprine islets were isolated and purified. Islets were handpicked and the diameter of the islets was recorded using light microscopy. Viablility of the islets was analyzed by confocal microscopy. Insulin secretion assay was carried out and analyzed by ELISA.
RESULTS: When tested at 48 h after isolation, these small islets were 29.3% more viable compared to the large-sized islets. Large islets showed a high ratio (P insulin level under low glucose induction (3.3 mm) and simultaneously 2.92-fold (2.95 ± 0.33 ng/IE) more insulin under high glucose condition (16.7 mm) in comparison to large islets at the same islet equivalents (P
METHODOLOGY: After collection and purification of caprine islets with Euro-Ficoll density gradients, islets were considered for viability and functionality procedures with DTZ (dithizone) staining and GSIST (glucose-stimulated insulin secretion test) subsequently. Batches of islet were selected for immunostaining and study through confocal microscopy and flow cytometry.
RESULTS: Histological sections of caprine pancreatic islets showed that α-cells were segregated at the periphery of β-cells. In caprine islets, α- and δ-cells remarkably were intermingled with β-cells in the mantle. Such cytoarchitecture was observed in all examined caprine pancreatic islets and was also reported for the islets of other ruminants. In both small and large caprine islets (< 150 and > 150 μm in diameter, respectively), the majority of β-cells were positioned at the core and α-cells were arranged at the mantle, while some single α-cells were also observed in the islet center. We evaluated the content of β-, α-, and δ-cells by confocal microscopy (n = 35, mean ± SD; 38.01 ± 9.50%, 30.33 ± 10.11%, 2.25 ± 1.10%, respectively) and flow cytometry (n = 9, mean ± SD; 37.52 ± 9.74%, 31.72 ± 4.92%, 2.70 ± 2.81%, respectively). Our findings indicate that the caprine islets are heterogeneous in cell composition. The difference could be attributed to species-specific interaction between endocrine cells and blood.
CONCLUSIONS: Comparative studies of islet architecture may lead to better understanding of islet structure and cell type population arrangement. These results suggest the use of caprine islets as an addition to the supply of islets for diabetes research.
MATERIALS AND METHODS: Purified islets were treated with serum-free, serum, IBMX, tocopherol, or IBMX and tocopherol media. Quantitative polymerase chain reaction and Western blotting were carried out to compare the expression levels of PDX1 in treated purified islets cultured with different media.
RESULTS: Islets treated with IBMX/tocopherol exhibited the highest fold change in the relative expression of PDX1 on day 5 post-treatment (relative expression: 6.80±2.08), whereas serum-treated islets showed the lowest fold changes in PDX1 expression on day 5 post-treatment (0.67±0.36), as compared with the expression on day 1 post-treatment. Insulin production and viability tests of purified islets showed superiority of islet at supplemented serum-free media with IBMX/tocopherol compared to other cultures (53.875%±1.59%).
CONCLUSIONS: Our results indicated that supplemented serum-free medium with tocopherol and IBMX enhances viability and PDX1 gene expression compared to serum-added and serum-free media.
The aim: To establish possible associations of the insulin receptor substrate-1 (IRS-1) gene polymorphism with the severity of the metabolic syndrome components in patients with arterial hypertension (AH).
Material and methods: 187 patients with AH aged 45-55 years and 30 healthy individuals. Methods: anthropometry, reactive hyperemia, color Doppler mapping, biochemical blood analysis, HOMA-insulin resistance (IR), glucose tolerance test, enzyme immunoassay, molecular genetic method.
Results: Among hypertensive patients, 103 had abdominal obesity, 43 - type 2 diabetes, 131 - increased blood triglycerides, 19 - decreased high density lipoproteins, 59 - prediabetes (33 - fasting hyperglycemia and 26 - impaired glucose tolerance), 126 had IR. At the same time, hypertensive patients had the following distribution of IRS-1 genotypes: Gly/Gly - 47.9%, Gly/Arg - 42.2% and Arg/Arg - 10.7%, whereas in healthy individuals the distribution of genotypes was significantly different: Gly/Gly - 86.8% (p <0.01), Gly/ Arg - 9.9% (p <0.01) and Arg/Arg - 3.3% (p <0.05). Hypertensive patients with Arg/Arg and Gly/Arg genotypes had significantly higher HOMA-IR (p <0.01), glucose, insulin and triglycerides levels (p <0.05), than in Gly/Gly genotype. At the same time, body mass index, waist circumference, blood pressure, adiponectin, HDL, interleukin-6, C-reactive protein, degree of endothelium-dependent vasodilation, as well as the frequency of occurrence of impaired glucose tolerance did not significantly differ in IRS-1 genotypes.
Conclusions: In hypertensive patients, the genetic polymorphism of IRS-1 gene is associated with such components of the metabolic syndrome as hypertriglyceridemia and fasting hyperglycemia; it is not associated with proinflammatory state, endothelial dysfunction, dysglycemia, an increase in waist circumference and decrease in HDL.