title: Orexin A modulates INS-1E cell proliferation and insulin secretion via extracellular signal-regulated kinase and transient receptor potential channels.
abstract: Orexins A (OXA) and B (OXB) control energy homeostasis by regulating food intake, energy expenditure and sleep-wake cycle. Several studies showed that OXA stimulates insulin secretion and proliferation of beta cells. However, mechanisms of action are still not well understood. Here, we investigated whether ERK and transient receptor potential channels (TRPs) play a role in mediating the effect of OXA on cell growth, insulin production, and secretion using the established INS-1E cell line. Cell proliferation was measured using BrdU assay. Insulin mRNA expression was detected by real-time PCR. Insulin secretion was assessed using ELISA. Intracellular calcium levels were measured using fluorescence calcium imaging (fura-2/AM). Extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was detected by Western blot. TRP channel activity was blocked by lanthanum (III) chloride (La3+; 100 - 300 μM) or ruthenium red (RuR; 10 μM). OXA (100 nM) stimulated INS-1E cell proliferation, insulin secretion, intracellular Ca2+concentration and ERK1/2 phosphorylation, without changing insulin mRNA expression. Inhibition of ERK1/2 by 10 μM U0126 attenuated OXA-stimulated INS-1E cell proliferation. Blockade of TRP channel activity by La3+or RuR rendered OXA ineffective at modulating Ca2+regulation and insulin release. In contrast, the L-type channel blocker nifedipine (10 μM) failed to affect OXA-stimulated insulin release. Taken together, OXA increases INS-1E cell proliferation via ERK1/2-dependent mechanism. Furthermore, OXA stimulates insulin secretion from INS-1E cells. TRPs are relevant for OXA-stimulated insulin secretion and intracellular calcium regulation.
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznan University of Life Sciences, Poznan, Poland. mskrzyps@up.poznan.pl.
authors.forename: M
authors.initials: M
authors.lastname: Skrzypski
authors.affiliation: Institute for Experimental Pediatric Endocrinology, Charite, University-Medicine Berlin, Berlin, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Khajavi
authors.affiliation: Department of Ophthalmology, Charite University Medicine Berlin, Berlin, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Mergler
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Billert
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: D
authors.initials: D
authors.lastname: Szczepankiewicz
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: T
authors.initials: T
authors.lastname: Wojciechowicz
authors.affiliation: Department of Animal Physiology and Biochemistry, Poznan University of Life Sciences, Poznan, Poland.
authors.forename: K W
authors.initials: KW
authors.lastname: Nowak
authors.affiliation: Department of Hepatology and Gastroenterology and Interdisciplinary Centre of Metabolism-Endocrinology, Diabetes and Metabolism, Charite-University Medicine Berlin, Berlin, Germany.|Department of Internal Medicine-Gastroenterology, Park-Klinik Weissensee, Berlin, Germany.
authors.forename: M Z
authors.initials: MZ
authors.lastname: Strowski
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects; D064446:Orexin Receptors / Q000235:genetics; D000068797:Orexins / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D050051:Transient Receptor Potential Channels / Q000378:metabolism*
chemical_list: D007328:Insulin; D064446:Orexin Receptors; D000068797:Orexins; D012333:RNA, Messenger; D050051:Transient Receptor Potential Channels; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium