Embryonic stem cells (ESCs) can be differentiated into insulin-producing cells by a five-stage procedure involving altering culture conditions and addition of nicotinamide. The amounts of insulin in these cells are lower than those found in pancreatic β cells. Glucagon-like peptide-1 (GLP-1) induces the differentiation of β cells from ductal progenitor cells. We examined the possibility of GLP-1, and its long-acting agonist exendin-4, enhancing the differentiation of insulin-producing cells from mouse ESCs (mESCs). A five-stage culturing strategy starting with embryoid bodies (EBs) was used in this study. mRNA for pancreatic duodenal homeobox gene 1 (PDX-1) and neurogenic differentiation (NeuroD) was detected from stage 1, hepatocyte nuclear factor 3 beta (HNF3β) and insulin 2 from stage 2, Ngn3 and glucose transporter 2 (GLUT2) from stage 3, and insulin 1 and other β-cell markers, at stages 4–5. Cells at stage 5 secreted C-peptide, being 0.68 ± 0.01 pmol/10⁶ cells per 2 days, and had an immunoreactive insulin content of 13.5 ± 0.7 pmol/10⁶ cells. Addition of GLP-1 (100 nM) and nicotinamide (10 mM) at stage 5 resulted in a 50% and 48% increase in insulin content and C-peptide secretion respectively compared with nicotinamide alone. Glucose-induced insulin secretion was enhanced 4-fold by addition of both growth factors. The GLP-1 receptor was present at all five stages of the culture. Addition of exendin-4 to cells at stage 2 resulted in a 4.9-fold increase in expression of the gene for insulin 1 and a 2-fold increase in insulin content compared with the effect of nicotinamide alone at stage 5. It is concluded that both GLP-1 and exendin-4 enhance the level of expression of insulin in glucose-responsive insulin-producing cells derived from the R1 mESC line.

ABSTRACT

β cells in the human fetal pancreas are immature in that they release little or no insulin in response to nutrients, such as glucose. The aim of this study was to examine further the immaturity of these cells, specifically regarding the storage and release of the precursor of insulin, proinsulin.

Explants of human fetal pancreas were cultured in vitro for 3 weeks. Levels of proinsulin remained relatively constant throughout at 0·04±0·002 (s.e.m.) pmol/mg per day with a molar ratio of proinsulin to insulin of 2·2 ± 0·11%. This low ratio was slightly greater than that observed in culture medium conditioned by adult human islets (0·3±0·1%), but similar to that found in acid–ethanol extracts of cultured explants (1·4 ± 0·3%). Passaging of human fetal pancreas for 3 months in diabetic nude mice, which should have caused some maturation of the fetal β cell, did not change the proportion of proinsulin present. Culture of explants in the presence of 12-O-tetra-decanoylphorbol-13-acetate resulted in some inhibition of proinsulin release, but much less than that for insulin, so that the molar ratio increased to 15·4±1·6% from the control 3·5 ± 0·3%. Static stimulation of cultured explants with 10 mmol Ca²⁺/l, 10 mmol theophylline/l, and these two agents together caused 15-, 4- and 10-fold enhancement respectively of proinsulin release; glucose, leucine, arginine and KCl had no effect. In contrast, all these agents caused significant insulin release, the last four to a much smaller extent (⩽ three fold) than the first three (10-, 19- and 65-fold respectively). Calcium channel blockers, verapamil and nifedipine, inhibited insulin but not proinsulin release.

The ability of different agents to modulate the release of proinsulin from the human fetal β cell, socalled regulated rather than constitutive secretion, as well as the low relative amount of proinsulin present show that this cell is more mature than was previously thought.

Journal of Endocrinology (1992) 132, 159–167

ABSTRACT

Insulin-like growth factor-II (IGF-II) is a polypeptide hormone thought to be involved in fetal development because of its wide distribution in fetal tissues and its presence in the fetal circulation. We have developed a highly sensitive radioreceptor assay for IGF-II using rat liver microsomal membranes and have used this assay to test for the presence of IGF-II in the human fetal pancreas and the release of IGF-II by the human fetal pancreas in organ culture. IGF-II was present in extracts of pancreatic tissue (0·056 ± 0·012 pmol/mg tissue, n= 5) and was released in culture at the rate of 0·027–0·134 pmol/mg tissue per day with release being maintained for at least 3 weeks in culture. The rate of release was not affected by the gestational age of the fetus over 14 to 20 weeks but was significantly related to the rate of insulin release (r = 0·712, P < 0·001, n = 34). Chronic exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA), which inhibits insulin release in the human fetal pancreas, caused an 85% drop in IGF-II production, which was reversed when TPA was removed. These studies demonstrate that IGF-II is produced by the human fetal pancreas in a pattern similar to that of insulin. We suggest that control of IGF-II release, like that of insulin, may involve protein-kinase C and that IGF-II may have a paracrine or autocrine role in the development of fetal pancreatic function.

Journal of Endocrinology (1989) 121, 367–373