Glucagon-Like Peptide-1 Causes Pancreatic Duodenal Homeobox-1 Protein Translocation from the Cytoplasm to the Nucleus of Pancreatic b-Cells by a Cyclic Adenosine Monophosphate/Protein Kinase A-Dependent Mechanism

Glucagon-like peptide-1 (GLP-1) enhances insulin secretion and synthesis. It also regulates the insulin, glucokinase, and GLUT2 genes. It mediates increases in glucose-stimulated insulin secretion by activating adenylyl cyclase and elevating free cytosolic calcium levels in the b-cell. In addition, GLP-1 has been shown, both in vitro and in vivo, to be involved in regulation of the transcription factor, pancreatic duodenal homeobox-1 protein (PDX-1), by increasing its total protein levels, its translocation to the nucleus and its binding and resultant increase in activity of the insulin gene promoter in b-cells of the pancreas. Here we have investigated the role of protein kinase A (PKA) in these processes in RIN 1046–38 cells. Three separate inhibitors of PKA, and a cAMP antagonist, inhibited the effects of GLP-1 on PDX-1. Furthermore, forskolin, (which stimulates adenylyl cyclase and insulin secretion), and 8-Bromo-cAMP, (an analog of cAMP which also stimulates insulin secretion), mimicked the effects of GLP-1 on PDX-1. These effects were also prevented by PKA inhibitors. Glucose-mediated increases in nuclear translocation of PDX-1 were not prevented by PKA inhibitors. Our results suggest that regulation of PDX-1 by GLP-1 occurs through activation of adenylyl cyclase and the resultant increase in intracellular cAMP, in turn, activates PKA, which ultimately leads to increases in PDX-1 protein levels and translocation of the protein to the nuclei of b-cells. (Endocrinology 142: 1820–1827, 2001) G PEPTIDE-1 (GLP-1) is an insulinotropic peptide secreted from the l-cells of gastrointestinal tract in response to ingestion of food (1). It binds to a specific G protein-coupled receptor on the b-cell resulting in activation of adenylyl cyclase. The subsequent rise in intracellular concentrations of cAMP enhances glucosemediated insulin secretion via protein kinase A (PKA)dependent and independent pathways (1–4). Activated PKA phosphorylates the Kir 6.2 and SUR1 subunits of the KATP channels resulting in increased channel activity (5). PKA influences the phosphylation state of the l-type voltagedependent Ca channel protein, resulting in a slower time course of Ca inactivation (6). Recent evidence indicates that some GLP-1-related events in b-cells are not necessarily mediated through PKA (7, 8). GLP-1 can elevate cytosolic calcium and stimulate insulin promoter activity independent of the PKA pathway. GLP-1 also leads to increases in tyrosine phosphorylation of SNAP25, a synaptic associated protein which is not PKA-mediated, because PKA phosphorylates serine/threonine residues only (9). Pancreatic duodenal homeobox-1 protein (PDX-1, also called IDX-1, IPF1, STF1, and IUF1) is a transcription factor with essential functions for pancreas development, islet formation and maintenance of the b-cell phenotype (10, 11). Absence of PDX-1 leads to pancreatic agenesis in rodents and humans (12, 13) and mutations in PDX-1 lead to MODY-type diabetes with reduced insulin secretion in humans (14, 15). In the adult pancreas, PDX-1 binds to A-box motifs of the insulin gene promoter and is involved in glucose-mediated up-regulation of the insulin gene (16). It is also involved in the transciptional regulation of the glucose sensing genes, GLUT2, and glucokinase (17). We and others (18, 19) have previously demonstrated that GLP-1 up-regulates PDX-1, both messenger RNA (mRNA) and protein levels, as well as its translocation to nuclei in an insulinoma cell line and this is concurrent with increased binding activity of PDX-1 protein to the rat insulin gene promoter (18). Glucose treatment results in PDX-1 translocation between 15–30 min with a return to basal levels by 2 h (16, 18). No increase in total PDX-1 protein levels has been seen with glucose treatment alone. GLP-1-mediated translocation reaches a maximum at 2 h (18, 19), total PDX-1 protein levels are significantly increased by 3 h and the amount of nuclear PDX-1 is still elevated 12 h after the addition of GLP-1 (18). Recent studies indicate that GLP-1, as well as increasing intracellular cAMP levels, activates phosphatidylinositol 3-kinase and mitogen activated protein (MAP) kinase in cultured INS-1 (19) and CHO cells (20). Here we concentrated on examining specifically whether GLP-1-mediated effects on PDX-1 are cAMP/PKA dependent. We have used inhibitors and stimulators of PKA activation and cAMP agonists to examine the involvement of PKA in the up-regulation of transcription, translation and translocation of the PDX-1 Received August 3, 2000. Address all correspondence and requests for reprints to: Josephine M. Egan, M.D., Diabetes Section, #23, NIA/NIH, 5600 Nathan Shock Drive, Baltimore, Maryland 21224. E-mail: [email protected]. 0013-7227/01/$03.00/0 Vol. 142, No. 5 Endocrinology Printed in U.S.A. Copyright © 2001 by The Endocrine Society