Hedgehog signals inhibit postnatal beta cell neogenesis from adult rat exocrine pancreas in vitro.

AIMS/HYPOTHESIS: Transdifferentiation of pancreatic exocrine cells into insulin-producing beta cells may represent an important alternative to islets required for diabetes cell therapy. Rat pancreatic acinar cells are known to transdifferentiate into functional beta cells, with recapitulation of several pancreas developmental features. Considering the inhibitory functions of hedgehog signalling in early and mid-stage pancreatic development, we questioned whether it also operates in transdifferentiating acinar cells and whether its modulation would influence postnatal beta cell neogenesis in vitro. METHODS: Rat exocrine cells were precultured in suspension for 4 days and then incubated with EGF and leukaemia inhibitory factor (LIF) for 72 h. The hedgehog signalling pathway was modulated during this, and its effects analysed by RT-PCR, immunocytochemistry and western blot. RESULTS: Our data indicate induction of Dhh and Ihh, but not Shh, expression during acinar cell culture, resulting in activation of hedgehog targets (Ptc1, Gli1). Exposure of the metaplastic cells to EGF and LIF induced beta cell differentiation without affecting endogenous hedgehog activity. Whereas blocking endogenous hedgehog only slightly increased beta cell neogenesis, exposure to embryoid body-conditioned medium activated hedgehog signalling as well as other pathways such as Notch, resulting in severe blockade of beta cell neogenesis. Interestingly, this effect was partially rescued by treatment with the hedgehog inhibitor, 3-keto-N-(aminoethyl-aminocaproyl-dihydrocinnamoyl)-cyclopamine (KAAD-cyclopamine), alone. CONCLUSIONS/INTERPRETATION: We report here Dhh/Ihh-dependent activation of hedgehog targets during pancreatic exocrine cell metaplasia in vitro and a persistent inhibitory function of hedgehog signalling in a model of postnatal beta cell differentiation.

Hedgehog signals in pancreatic differentiation from embryonic stem cells: revisiting the neglected.

Recent demonstrations of insulin expression by progenies of mouse and human embryonic stem (ES) cells have attracted interest in setting up these cells as alternative sources of beta-cells needed in diabetes cell therapy. It is widely acknowledged that information gathered in the field of developmental biology as applied to the pancreas is of relevance for designing in vitro differentiation strategies. However, looking back at the protocols used so far, it appears that the natural route toward the pancreas, which goes via the definitive endoderm, was usually bypassed. As a consequence Hedgehog signaling, the earliest inhibitor of pancreas initiation from the endoderm, was generally not considered. A recall of the status of this pathway during ES cell differentiation appears necessary, especially in the light of findings that Activin A treatment of mouse and human ES cells coax them into definitive endoderm, a lineage showing wide Hedgehog ligands expression with the potential to hinder pancreatic programming.

In vitro generation of insulin-producing beta cells from adult exocrine pancreatic cells.

AIMS/HYPOTHESIS: Transplantation of insulin-producing beta cells from donors can cure diabetes, but they are available in insufficient quantities. In this study, we investigated the possibility of generating insulin-producing cells from adult rat exocrine cells cultured in the presence of growth factors. METHODS: Rat exocrine pancreatic cells were isolated and treated in vitro with epidermal growth factor (EGF) and leukaemia inhibitory factor (LIF). Analysis was performed by immunocytochemistry, DNA measurement and radioimmunoassay. Cells were transplanted to alloxan-treated (70 mg/kg) nude mice and glycaemia was monitored for 21 days. Nephrectomy was performed on day 15. RESULTS: In a 3-day culture period, addition of LIF plus EGF to the medium resulted in an 11-fold increase of the beta cell mass. This could not be attributed to the very low mitotic activity of contaminating beta cells. Furthermore, when contaminating beta cells were initially destroyed with alloxan, this effect was even more pronounced. The newly formed cells secreted insulin in response to glucose and were immunoreactive for C-peptide-I, Pdx-1 and GLUT-2, which are characteristics of mature beta cells. Electron microscopy showed that they also contained insulin-immunoreactive secretory granules. Some insulin-positive cells were immunoreactive for amylase and cytokeratin-20, or were binucleated, which are characteristics of exocrine cells. The cells were able to restore normoglycaemia when transplanted to alloxan-diabetic mice, and hyperglycaemia recurred upon removal of the graft. CONCLUSIONS/INTERPRETATION: Our study shows that functional beta cells can be generated from exocrine tissue by transdifferentiation and thereby may offer a new perspective for beta cell therapy.

Metabolic and hormonal effects of five common African diets eaten as mixed meals: the Cameroon Study.

OBJECTIVE: To evaluate glycaemic and insulinaemic index and in vitro digestibility of the five most common Cameroonian mixed meals consisting of rice+tomato soup (diet A), bean stew+plantains (B), foofoo corn+ndolé (C), yams+groundnut soup (D), and koki beans+cassava (E). SUBJECTS: Ten healthy non-obese volunteers, aged 19-31 y, with no family history of diabetes or hypertension. INTERVENTIONS: A 75 g oral glucose tolerance test followed by the eating of the test diets with carbohydrate content standardized to 75 g every 4 days with blood samples taken at 0, 15, 30, 60, 120 and 180 min. In vitro digestion of each diet according to Brand's protocol. MAIN OUTCOME MEASURES: Plasma glucose, cholesterol, triglyceride, insulin and C-peptide, with calculation of glycaemic and insulinaemic index defined as the area under the glucose and insulin response curve after consumption of a test food divided by the area under the curve after consumption of a control food containing the same amount of carbohydrate, and digestibility index. RESULTS: Glycaemic index (GI) varied from 34.1 (diet C) to 52.0% (diet E) with no statistical difference between the diets, and insulinaemic index varied significantly from 40.2% (C) to 70.9% (A) (P=0.03). The digestibility index varied from 18.9 (C) to 60.8% (A) (P<0.0001), and did not correlate with glycaemic or insulinaemic indices. However, carbohydrate content correlated with GI (r=0.83; P=0.04), digestibility index (r=-0.70; P<0.01), and insulinaemic index (r=0.91; P<0.01). Plasma C-peptide and plasma lipids showed little difference over 180 min following the ingestion of each meal. CONCLUSIONS: Glycaemic index of these African mixed meals are relatively low and might not be predicted by in vitro digestibility index.