Máire E. Doyle

Glucagon-Like Peptide-1 Induces Cell Proliferation and Pancreatic-Duodenum Homeobox-1 Expression and Increases Endocrine Cell Mass in the Pancreas of Old, Glucose-Intolerant Rats

Glucose homeostasis in mammals is maintained by insulin secretion from the β-cells of the islets of Langerhans. Type 2 diabetes results either from primary β-cell failure alone and/or a failure to secrete enough insulin to overcome insulin resistance. Here, we show that continuous infusion of glucagon-like peptide-1 (7–36) (GLP-1; an insulinotropic agent), to young and old animals, had effects on the β-cell of the pancreas other than simply on the insulin secretory apparatus. Our previous studies on a rodent model of glucose intolerance, the aging Wistar rat, show that a plateau in islet size, insulin content, and β-cell mass is reached at 13 months, despite a continuing increase in body weight. Continuous sc infusion of GLP-1 (1.5 pm/kg·min), over 5 days, resulted in normal glucose tolerance. Our current results in both young and old rats demonstrate that treatment caused an up-regulation of pancreatic-duodenum homeobox-1 (PDX-1) expression in islets and total pancreas, induced pancreatic cell proliferat...

Pharmacological agents that directly modulate insulin secretion

Blood glucose levels are sensed and controlled by the release of hormones from the islets of Langerhans in the pancreas. The β-cell, the insulin-secreting cell in the islet, can detect subtle increases in circulating glucose levels and a cascade of molecular events spanning the initial depolarization of the β-cell membrane culminates in exocytosis and optimal insulin secretion. Here we review these processes in the context of pharmacological agents that have been shown to directly interact with any stage of insulin secretion. Drugs that modulate insulin secretion do so by opening the KATPchannels, by interacting with cell-surface receptors, by altering second-messenger responses, by disrupting the β-cell cytoskeletal framework, by influencing the molecular reactions at the stages of transcription and translation of insulin, and/or by perturbing exocytosis of the insulin secretory vesicles. Drugs acting primarily at the KATP channels are the sulfonylureas, the benzoic acid derivatives, the imidazolines, and the quinolines, which are channel openers, and finally diazoxide, which closes these channels. Methylxanthines also work at the cell membrane level by antagonizing the purinergic receptors and thus increase insulin secretion. Other drugs have effects at multiple levels, such as the calcineurin inhibitors and somatostatin. Some drugs used extensively in research, e.g., colchicine, which is used to study vesicular transport, have no effect at the pharmacological doses used in clinical practice. We also briefly discuss those drugs that have been shown to disrupt β-cell function in a clinical setting but for which there is scant information on their mechanism of action.

Glucagon-Like Peptide-1 Regulates the Beta Cell Transcription Factor, PDX-1, in Insulinoma Cells

Glucagon-like peptide-1 (GLP-1) enhances insulin biosynthesis and secretion as well as transcription of the insulin, GLUT2 and glucokinase genes. The latter are also regulated by the PDX-1 homeoprotein. We investigated the possibility that GLP-1 may be having its long-term pleiotropic effects through a hitherto unknown regulation of PDX-1. We found that PDX-1 mRNA level was significantly increased (p<0.01) after 2 hours and insulin mRNA level was subsequently increased (p<0.01) after 3 hours of treatment with GLP-1 (10 nM) in RIN 1046-38 insulinoma cells. Under these experimental conditions, there was also a 1.6-fold increase in the expression of PDX-1 protein in whole cell and nuclear extracts. Overexpression of PDX-1 in these cells confirmed the finding of the wild type cells such that GLP-1 induced a 2-fold increase in whole cell extracts and a 3-fold increase in nuclear extracts of PDX-1 protein levels. The results of electrophoretic mobility shift experiments showed that PDX-1 protein binding to the Al element of the rat insulin II promoter was also increased 2 h post treatment with GLP-1. In summary, we have uncovered a previously unknown aspect to the regulation of PDX-1 in beta cells. This has important implications in the physiology of adult beta cells and the treatment of type 2 diabetes mellitus with GLP-1 or its analogs.

Glucagon-Like Peptide-1 Causes Pancreatic Duodenal Homeobox-1 Protein Translocation from the Cytoplasm to the Nucleus of Pancreatic β-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 β-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 β-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 o...

Exendin-4 differentiation of a human pancreatic duct cell line into endocrine cells: Involvement of PDX-1 and HNF3β transcription factors†‡

Exendin‐4 (EX‐4), a long acting agonist of GLP‐1, induces an endocrine phenotype in Capan‐1 cells. Under culture conditions which include serum, ∼10% of the cells contain insulin and glucagon. When exposed to EX‐4 (0.1 nM, up to 5 days), the number of cells containing insulin and glucagon increased to ∼40%. Western blot analysis detected a progressive increase in protein levels of glucokinase and GLUT2 over 3 days of EX‐4 treatment. We explored the sequence of activation of certain transcription factors known to be essential for the beta cell phenotype: PDX‐1, Beta2/NeuroD, and hepatocyte nuclear factor 3β (HNF3β). Double immunostaining showed that PDX‐1 coexisted with insulin and glucagon in EX‐4‐treated cells. Treatment caused an increase in PDX‐1 protein levels by 24 h and induced its nuclear translocation. Beta2/NeuroD protein levels also increased progressively over 24 h. HNF3β protein level increased twofold as early as 6 h after EX‐4 treatment. EMSA results indicated that EX‐4 caused a 12‐fold increase in HNF3β binding to PDX‐1 promoter area II. Beta2/NeuroD protein levels progressively increased after 24 h treatment. Differentiation to insulin‐producing cells was also seen when Capan‐1 cells were transfected with pdx‐1, with 80% of these cells expressing insulin 3 days after transfection. PDX‐1 antisense totally inhibited such conversion. During the differentiation of duct cells to endocrine cells, cAMP levels (EX‐4 is a ligand for the GLP‐1, G‐protein coupled receptor) and MAP kinase activity increased. Our results indicate that EX‐4 activates adenylyl cyclase and MAP kinase which, in turn, may lead to activation of transcription factors necessary for an endocrine phenotype. Published 2002 Wiley‐Liss, Inc.

Cannabinoids Inhibit Insulin Receptor Signaling in Pancreatic β-Cells

OBJECTIVE Optimal glucose homeostasis requires exquisitely precise adaptation of the number of insulin-secreting β-cells in the islets of Langerhans. Insulin itself positively regulates β-cell proliferation in an autocrine manner through the insulin receptor (IR) signaling pathway. It is now coming to light that cannabinoid 1 receptor (CB1R) agonism/antagonism influences insulin action in insulin-sensitive tissues. However, the cells on which the CB1Rs are expressed and their function in islets have not been firmly established. We undertook the current study to investigate if intraislet endogenous cannabinoids (ECs) regulate β-cell proliferation and if they influence insulin action. RESEARCH DESIGN AND METHODS We measured EC production in isolated human and mouse islets and β-cell line in response to glucose and KCl. We evaluated human and mouse islets, several β-cell lines, and CB1R-null (CB1R−/−) mice for the presence of a fully functioning EC system. We investigated if ECs influence β-cell physiology through regulating insulin action and demonstrated the therapeutic potential of manipulation of the EC system in diabetic (db/db) mice. RESULTS ECs are generated within β-cells, which also express CB1Rs that are fully functioning when activated by ligands. Genetic and pharmacologic blockade of CB1R results in enhanced IR signaling through the insulin receptor substrate 2-AKT pathway in β-cells and leads to increased β-cell proliferation and mass. CB1R antagonism in db/db mice results in reduced blood glucose and increased β-cell proliferation and mass, coupled with enhanced IR signaling in β-cells. Furthermore, CB1R activation impedes insulin-stimulated IR autophosphorylation on β-cells in a Gαi-dependent manner. CONCLUSIONS These findings provide direct evidence for a functional interaction between CB1R and IR signaling involved in the regulation of β-cell proliferation and will serve as a basis for developing new therapeutic interventions to enhance β-cell function and proliferation in diabetes.

Identification of novel GDNF isoforms and cis-antisense GDNFOS gene and their regulation in human middle temporal gyrus of Alzheimer disease

Background: Glial cell line-derived neurotrophic factor (GDNF) has been effective therapy in laboratory animal models of Parkinson disease. Results: A human GDNFOS (GDNF opposite strand) gene was discovered and potentially encodes a 105-amino acid peptide. Conclusion: GDNF sense and GDNFOS antisense genes are differentially regulated in human tissues. Significance: GDNF and GDNFOS isoforms may have roles in human neurodegenerative diseases. Primate-specific genes and isoforms could provide insight into human brain diseases. Our bioinformatic analysis revealed that there are possibly five isoforms of human GDNF gene with different pre- and pro-regions by inter- and intra-exon splicing. By using TaqMan primer probe sets, designed between exons, we verified the expression of all isoforms. Furthermore, a novel GDNFOS gene was found to be transcribed from the opposite strand of GDNF gene. GDNFOS gene has four exons that are spliced into different isoforms. GDNFOS1 and GDNFOS2 are long noncoding RNAs, and GDNFOS3 encodes a protein of 105 amino acids. To study human GDNF and GDNFOS regulation in neurodegenerative diseases, the protein and mRNA levels were measured by Western blot and RT-quantitative PCR, respectively, in postmortem middle temporal gyrus (MTG) of Alzheimer disease (AD) and Huntington disease (HD) patients in comparison with those of normal controls. In the MTG of AD patients, the mature GDNF peptide was down-regulated; however, the transcript of GDNF isoform from human exon 2 was up-regulated, whereas that of the conserved isoform from exon 1 remained unchanged in comparison with those of normal controls. In contrast, the mature GDNF peptide and the isoform mRNA levels were not changed in the MTG of HD. The findings of novel GDNF and GDNFOS isoforms and differences in tissue expression patterns dysregulated in AD brains may further reveal the role of endogenous GDNF in human brain diseases.

The importance of the nine-amino acid C-terminal sequence of exendin-4 for binding to the GLP-1 receptor and for biological activity

Exendin-4, a 39-amino acid (AA) peptide, is a long-acting agonist at the glucagon-like peptide-1 (GLP-1) receptor. Consequently, it may be preferable to GLP-1 as a long-term treatment for type 2 diabetes mellitus. Exendin-4 (Ex-4), unlike GLP-1, is not degraded by dipeptidyl peptidase IV (DPP IV), is less susceptible to degradation by neutral endopeptidase, and possesses a nine-AA C-terminal sequence absent from GLP-1. Here we examine the importance of these nine AAs for biological activity of Ex-4, a sequence of truncated Ex-4 analogs, and native GLP-1 and GLP-1 analogs to which all or parts of the C-terminal sequence have been added. We found that removing these AAs from Ex-4 to produce Ex (1-30) reduced the affinity for the GLP-1 receptor (GLP-1R) relative to Ex-4 (IC50: Ex-4, 3.22+/-0.9 nM; Ex (1-30), 32+/-5.8 nM) but made it comparable to that of GLP-1 (IC50: 44.9+/-3.2 nM). The addition of this nine-AA sequence to GLP-1 improved the affinity of both GLP-1 and the DPP IV resistant analog GLP-1 8-glycine for the GLP-1 receptor (IC50: GLP-1 Gly8 [GG], 220+/-23 nM; GLP-1 Gly8 Ex (31-39), 74+/-11 nM). Observations of the cAMP response in an insulinoma cell line show a similar trend for biological activity.

Insertion of an N-Terminal 6-Aminohexanoic Acid after the 7 Amino Acid Position of Glucagon-Like Peptide-1 Produces a Long-Acting Hypoglycemic Agent

The use of glucagon-like peptide-1 (GLP-1) as a routine treatment for type 2 diabetes mellitus is undermined by its short biological half-life. A cause of degradation is its cleavage at the N-terminal HAE sequence by the enzyme dipeptidyl peptidase IV (DPP IV). To protect from DPP IV, we have studied the biological activity of a GLP-1 analog in which 6-aminohexanoic acid (Aha) is inserted between histidine and alanine at positions 7 and 8. We have compared the biological activity of this new compound, GLP-1 Aha8, with the previously described GLP-1 8-glycine (GLP-1 Gly8) analog. GLP-1 Aha8 (10 nm) was equipotent with GLP-1 (10 nm) in stimulating insulin secretion in RIN 1046-38 cells. As with GLP-1 Gly8, the binding affinity of GLP-1 Aha8 for the GLP-1 receptor in intact Chinese hamster ovary (CHO) cells expressing the human GLP-1 receptor (CHO/GLP-1R cells) was reduced (IC50: GLP-1, 3.7± 0.2 nm; GLP-1 Gly8, 41 ± 9 nm; GLP-1 Aha8, 22 ± 7 nm). GLP-1 Aha8 was also shown to stimulate intracellular cAMP produ...

Human CB1 Receptor Isoforms, present in Hepatocytes and β-cells, are Involved in Regulating Metabolism

Therapeutics aimed at blocking the cannabinoid 1 (CB1) receptor for treatment of obesity resulted in significant improvements in liver function, glucose uptake and pancreatic β-cell function independent of weight loss or CB1 receptor blockade in the brain, suggesting that peripherally-acting only CB1 receptor blockers may be useful therapeutic agents. Neuropsychiatric side effects and lack of tissue specificity precluded clinical use of first-generation, centrally acting CB1 receptor blockers. In this study we specifically analyzed the potential relevance to diabetes of human CB1 receptor isoforms in extraneural tissues involved in glucose metabolism. We identified an isoform of the human CB1 receptor (CB1b) that is highly expressed in β-cells and hepatocytes but not in the brain. Importantly, CB1b shows stronger affinity for the inverse agonist JD-5037 than for rimonabant compared to CB1 full length. Most relevant to the field, CB1b is a potent regulator of adenylyl cyclase activity in peripheral metabolic tissues. CB1b blockade by JD-5037 results in stronger adenylyl cyclase activation compared to rimonabant and it is a better enhancer of insulin secretion in β-cells. We propose this isoform as a principal pharmacological target for the treatment of metabolic disorders involving glucose metabolism.