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The active metabolite of vitamin D], is a secosteroid hormone that not only regulates bone and calcium/phosphate metabolism but also exerts a number of other biological activities including immunomodulation (13) and insulinotropic effect in pancreatic β-cells (14) and vitamin D-deficient rats (15).1,25-(OH) cytotoxic cytokine challenge by decreasing major histocompatibility complex class I induction, IL-6 production, and nitrite release, a reflection of NO production (18).
They have been shown to induce apoptosis in purified primary human (4), rat (5), and mouse (6) β-cells, possibly by stimulating the synthesis of inducible nitric oxide synthase (7) and nitric oxide (NO) (8).Islet number was determined on samples of each preparation after dithizone staining and expressed as equivalent number of islets.Preparations used in this study exhibited a 75 ± 3% purity and an average yield of 4091 ± 1127 equivalent number of islets per gram pancreas.The rat insulin-producing β-cells RINm5F obtained from ATCC (Manassas, VA) were trypsinized every 5 d and subcultured (1 × 10 and maintained for 24 h in RPMI 1640 medium supplemented with 10% fetal calf serum and antibiotics.Cell processing consisted of treatments for 48 h in different media including cytokine-treated medium: basal medium plus IL-1β (50 IU/ml), IFN-γ (1000 IU/ml), and TNFα (1000 IU/ml; cytokines from Valbiotech, Ab Cys, Paris, France) and cytokine-treated medium containing 1,25-(OH)-arginine (NOi, 5 mm; Sigma-Aldrich Chemicals, Saint Quentin Fallavier, France), added for 1 h before stimulation with cytokines.After incubation, analyses were performed on free-floating cells pooled with the cells detached by mild trypsinization.
Human pancreases (mean age 38 ± 6 yr, n = 5) were harvested from adult brain-dead donors in accord with French regulations and the local Institutional Ethical Committee.
P.), Faculté de Médecine, 59045 Lille, France Cellular Therapy of Diabetes, Institut National de la Santé et de la Recherche Médicale, Equipe de Recherche et d’Innovation Méthodologique 0106 (R. In experimental models of islet transplantation, a loss of grafted β-cell mass has been observed and attributed, at least in part, to β-cell apoptosis (1).
We demonstrated for the first time, in both RINm5F cells and human islets, that 1,25-(OH) TRANSPLANTATION OF PANCREATIC islets of Langerhans is a promising therapy for type 1 diabetes, potentially restoring physiologic insulin secretion.
In the present study, we investigated the early mechanisms implied in β-cell death by first focusing on the rat insulin-producing RINm5F β-cell line, a model currently used for the study of pancreatic cell death (10, 19–24).
We particularly studied the disruption of the mitochondrial transmembrane potential (Δψm) and apoptotic features to ascertain the counteracting action of 1,25-(OH) on the expression of the antiapoptotic A20 gene and its induction by cytokines in RINm5F cells, and we subsequently extended these experiments to human pancreatic islet cells.
Insulin released in the culture media by cells was stored at −80 C before we performed the insulin assay using the RIA kit CT from CIS-Bio International (Gif-sur-Yvette, France).