Endocrine Abstracts

Background and aim: Orexin-A (OXA) increases insulin secretion and inhibits glucagon secretion, suggesting a role in regulating glucose homeostasis. The effects of OXA on pancreatic A-cells on the cellular level have not yet been demonstrated. Aim of our study was therefore to characterise the underlying signal transduction pathways and to study the OXA effects on proglucagon gene transcription.

Methods: The effects of OXA on glucagon secretion were evaluated using an in situ perfused rat pancreas model and clonal pancreatic A-cells (InR1-G9). OXR-1 expression in InR1-G9 cells was detected by western blot and immunofluorescence. The effects of OXA on intracellular cyclic AMP, AKT, PDK-1, CREB and EGR-1 were measured by ELISA and western blots, intracellular calcium (Ca²⁺) by Fura-2. Proglucagon and Foxo1 mRNA levels were quantified by real-time PCR. Foxo1 was silenced using short interfering RNA (siRNA).

Results: Pancreatic A-cells express OX1R. OXA reduced glucagon secretion and proglucagon gene expression. OXA decreased intracellular cyclic AMP and Ca²⁺ concentrations, and increased the phosphorylation of AKT und PDK-1. PI-3 kinase inhibitor blocked the effects of OXA on proglucagon gene expression. OXA reduced the expression and phosphorylation of CREB, and EGR-1. Silencing of Foxo1 had no effects on basal proglucagon gene expression; however the inhibitory effect of OXA on glucagon gene expression was reversed.

Conclusions: We demonstrate for the first time the direct interaction of OXA with pancreatic A-cells and identify cAMP/AKT/PDK-1 and Ca²⁺ as intracellular target molecules for OXA action. We identify transcription factors Foxo1, CREB and EGR-1 as downstream targets for OXA signalling, suggesting a role in mediating the inhibitory effects of OXA on glucagon gene expression. We have now increasing evidence that OXA affects glucagon homeostasis.

Inhibition of glucagon secretion by OXA may have potential implication at lowering hyperglucagonemia frequently encountered in type 2 diabetes.