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.