PDX-1 and cell-cell contact act in synergy to promote delta-cell development in a human pancreatic endocrine precursor cell line.

Cell lines from the fetal and adult pancreas that were developed by retroviral transfer of the SV40T and ras(val12) oncogenes lose insulin expression but retain extremely low levels of somatostatin and glucagon mRNA. In contrast to expanded populations of primary human islet cells, none of them express the homeodomain transcription factor PDX-1. When that factor was expressed in the cell lines by retroviral-mediated gene transfer, one of the cell lines, TRM-6, derived from human fetal islets, exhibited a 10- to 100-fold increase in somatostatin gene expression. This is the first report of induction of the endogenous somatostatin gene by PDX-1. Promotion of cell-cell contact by aggregation of TRM-6/PDX-1 into islet-like clusters produced a further 10- to 100-fold increase in somatostatin mRNA, to a level similar to that of freshly isolated islets, which resulted in production of somatostatin protein. Thus, we demonstrate here that signals induced by cell-cell contact act in synergy with PDX-1 to up-regulate the endogenous somatostatin promoter in an immortalized cell line from human fetal islets. This system provides a powerful model for studying human islet cell development and, particularly, the role of cell-cell contact in the differentiation process.

Erythrocyte Na/K ATPase activity and diabetes: relationship with C-peptide level.

Erythrocyte Na/K ATPase activity is decreased in Type I diabetic patients; for Type II diabetic patients, literature data are controversial. Therefore, we have compared this enzymatic activity in 81 patients with Type I diabetes mellitus, 87 with Type II diabetes mellitus and 75 control subjects. Mean erythrocyte Na/K ATPase activity was lower in the Type I diabetic patients (285 +/- 8 nmol Pi x mg protein(-1) x h(-1)) than in the control subjects (395 +/- 9 nmol Pi x mg protein(-1) x h(-1)) whereas that of the Type II diabetic patients did not differ from that of control subjects. Sex, age, body mass index, and HbA1c levels did not influence erythrocyte Na/K ATPase activity. The 25 Type II diabetic patients treated with insulin, however, had lower Na/K ATPase activity than the 62 on oral treatment (264 +/- 18 vs 364 +/- 16 nmol Pi x mg protein(-1) x h(-1), p < 0.001) but similar to that of Type I diabetic patients. Among the Type II diabetic patients, stepwise regression analysis showed that fasting C-peptide level was the only factor independently correlated with Na/K ATPase activity; it explained 23% of its variance. In fact, in the insulin-treated patients, those with almost total endogenous insulin deficiency (C-peptide < 0.2 nmol x l(-1)) had the lower Na/K ATPase activity (181 +/- 21 vs 334 +/- 17 nmol Pi x mg protein(-1) x h(-1), p < 0.0001). The biological effects of treatment with C-peptide have recently led to the suggestion that this peptide could have a physiological role through the same signalling pathway as insulin, involving G-protein and calcium phosphatase and thus restoring Na/K ATPase activity. The relationship we describe between endogenous C-peptide and this activity is a strong argument for this physiological role.

Effects of proinsulin C-peptide on nitric oxide, microvascular blood flow and erythrocyte Na+,K+-ATPase activity in diabetes mellitus type I.

This study was conducted to evaluate the influence of proinsulin C-peptide on erythrocyte Na(+),K(+)-ATPase and endothelial nitric oxide synthase activities in patients with type I diabetes. In a randomized double-blind study design, ten patients with type I diabetes received intravenous infusions of either human C-peptide or physiological saline on two different occasions. C-peptide was infused at a rate of 3 pmol.min(-1).kg(-1) for 60 min, and thereafter at 10 pmol.min(-1).kg(-1) for 60 min. At baseline and after 60 and 120 min, laser Doppler flow (LDF) was measured following acetylcholine iontophoresis or mild thermal stimulation (44 degrees C), and venous blood samples were collected to determine plasma cGMP levels and erythrocyte membrane Na(+),K(+)-ATPase activity. The LDF response to acetylcholine increased during C-peptide infusion and decreased during saline infusion [18.6+/-19.2 and -13.2+/-9.4 arbitrary units respectively; mean+/-S.E.M.; P<0.05). No significant change in LDF was observed after thermal stimulation. The baseline plasma concentration of cGMP was 5.5+/-0.6 nmol.l(-1); this rose to 6.8+/-0.9 nmol.l(-1) during C-peptide infusion (P<0.05). Erythrocyte Na(+),K(+)-ATPase activity increased from 140+/-29 nmol of P(i).h(-1).mg(-1) in the basal state to 287+/-5 nmol of P(i). h(-1).mg(-1) during C-peptide infusion (P<0.01). There was a significant linear relationship between plasma C-peptide levels and erythrocyte Na(+),K(+)-ATPase activity during the C-peptide infusion (r=0.46, P<0.01). No significant changes in plasma cGMP levels or Na(+),K(+)-ATPase activity were observed during saline infusion. This study demonstrates an effect of human proinsulin C-peptide on microvascular function, which might be mediated by an increase in NO production and an activation of the erythrocyte Na(+),K(+)-ATPase. These mechanisms are compatible with the previous observed microvascular effects of C-peptide in patients with type I diabetes.