Orexin-A Inhibits Glucagon Secretion and Gene Expression through a Foxo1-Dependent Pathway

Abstract

Orexin-A (OXA) regulates food intake and energy homeostasis. It increases insulin secretion in vivo and in vitro, although controversial effects of OXA on plasma glucagon are reported. We characterized the effects of OXA on glucagon secretion and identify intracellular target molecules in glucagon-producing cells. Glucagon secretion from in situ perfused rat pancreas, isolated rat pancreatic islets, and clonal pancreatic A-cells (InR1-G9) were measured by RIA. The expression of orexin receptor 1 (OXR1) was detected by Western blot and immunofluorescence. The effects of OXA on cAMP, adenylate-cyclase-kinase (AKT), phosphoinositide-dependent kinase (PDK)-1, forkhead box O-1 (Foxo1), and cAMP response element-binding protein were measured by ELISA and Western blot. Intracellular calcium (Ca(2+)(i)) concentration was detected by fura-2and glucagon expression by real-time PCR. Foxo1 was silenced in InR1-G9 cells by transfecting cells with short interfering RNA. OXR1 was expressed on pancreatic A and InR1-G9 cells. OXA reduced glucagon secretion from perfused rat pancreas, isolated rat pancreatic islets, and InR1-G9 cells. OXA inhibited proglucagon gene expression via the phosphatidylinositol 3-kinase-dependent pathway. OXA decreased cAMP and Ca(2+)(i) concentration and increased AKT, PDK-1, and Foxo1 phosphorylation. Silencing of Foxo1 caused a reversal of the inhibitory effect of OXA on proglucagon gene expression. Our study provides the first in vitro evidence for the interaction of OXA with pancreatic A cells. OXA inhibits glucagon secretion and reduces intracellular cAMP and Ca(2+)(i) concentration. OXA increases AKT/PDK-1 phosphorylation and inhibits proglucagon expression via phosphatidylinositol 3-kinase- and Foxo-1-dependent pathways. As a physiological inhibitor of glucagon secretion, OXA may have a therapeutic potential to reduce hyperglucagonemia in type 2 diabetes.

Orexin A regulates plasma insulin and leptin levels in a time-dependent manner following a glucose load in mice

“…7. Previous studies have demonstrated that OXR1 is clearly expressed in pancreatic islets and beta cells of pigs, rats and mice [9][10][11]. Here, we showed that OXA influenced insulin secretion in pancreatic islets through OXR1, but not OXR2.…”

“…In the fasting state, OXA potently inhibits glucagon secretion in perfused rat pancreas [11] and rats in vivo [14]. Consistently, OXA decreases blood glucose levels in fasting mice [15].…”

“…Moreover, leptin potently inhibits insulin secretion [21][22][23] whereas OXA exerts the opposite effect [11]. These findings suggest that leptin and orexins are strongly correlated with each other and that they have counteracting roles in the regulation of appetite and plasma insulin levels.…”

“…OXRs have been demonstrated not only in central neurons but also in some peripheral endocrine cells. Recent studies have demonstrated that OXR1 is expressed in the pancreatic alpha and beta cells of pigs, rats and mice [9][10][11] and in human and rat adipocytes [12,13]. Therefore, it is likely that orexins contribute to important endocrine functions.…”

“…However, conflicting findings have been reported on the effect of OXA on insulin secretion in the fasting state in vivo [14][15][16][17]. A recent study demonstrated that OXA potentiates glucose-stimulated insulin secretion (GSIS) in perfused rat pancreas [11]. However, little is known about the mechanism of OXA's action in GSIS and the role of OXA in the regulation of blood glucose levels after a glucose load.…”