One of the first actions identified for GLP-1 was the glucose-dependent stimulation of insulin secretion from islets in rodents, humans, or from islet cell lines. The classical original references for these findings include Insulinotropin: glucagon-like peptide I (7-37) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. J Clin Invest. 1987 Feb;79(2):616-9 and Glucagon-like peptide-1 7-36: a physiological incretin in man. Lancet. 1987 Dec 5;2(8571):1300-4 and Truncated glucagon-like peptide I, an insulin-releasing hormone from the distal gut. FEBS Lett. 1987 Jan 26;211(2):169-74 and Glucagon-like peptide I stimulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line. Proc Natl Acad Sci U S A. 1987 May;84(10):3434-8.
Following the detection of GLP-1 receptors on islet beta cells, a large body of evidence has accumulated illustrating that GLP-1 exerts multiple actions on various signaling pathways and gene products in the b-cell.
GLP-1 and beta-cell signal transduction
Although original studies demonstrated that GLP-1 activates cAMP in islet β cells, subsequent studies also demonstrated GLP-1-dependent changes in intracellular calcium. GLP-1R-/- β cells exhibit abnormalities in levels of glucose-stimulated cAMP and in glucose-stimulated calcium oscillations. See Altered cAMP and Ca2+ signaling in mouse pancreatic islets with glucagon-like peptide-1 receptor null phenotype. Diabetes. 1999 Oct;48(10):1979-86.
GLP-1 and to a lesser extent glucagon, stimulate coordinate oscillations in both intracellular calcium and cyclic AMP in b-cells, that are potentiated in the presence of elevated glucose concentrations. Cyclic AMP oscillations appear sufficient for stimulation of insulin exocytosis, whereas more sustained elevations in cyclic AMP are required for nuclear PKA translocation leading to CREB activation, and likely cell proliferation and survival. These findings illustrate a molecular mechanism differentiating transient vs sustained GLP-1R activation leading to differential downstream signal transduction events. See Oscillations of cyclic AMP in hormone-stimulated insulin-secreting -cells Nature 2006 439, 349-352
Furthermore, evidence for constitutive signaling of GLP-1 receptors in islet β cells is found in studies of islet cells incubated with the antagonist exendin (9-39) or in studies of immortalized GLP-1R-/- bTC cells. See Exendin-(9-39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and beta-cell glucose competence. Endocrinology. 1998;139(11):4448-54.
More recent experiments have indicated that although GLP-1 increases islet cAMP, many of the subsequent changes that occur in the β cell are PKA-independent. The growth effects of GLP-1 on islet cells may be mediated by the PI-3 kinase pathway, as shown in Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells. Diabetologia. 1999 Jul;42(7):856-64. The exact downstream signaling pathways utilized by GLP-1 in the islet β cell remains a subject of intense interest.
Several studies have implicated a role for cAMP-regulated guanine nucleotide exchange factors as downstream mediators of GLP-1 signaling in β cells. Intriguingly, experiments in INS-1 cells show that although PKA inhibitors such as H-89 may not abrogate many components of GLP-1R signaling, the cAMP antagonist 8-Br-Rp-cAMPS functions as a more complete inhibitor, likely as a result of its actions on cAMP-GEF II. Indeed, a dominant negative cAMP-GEF II cDNA blocked intracellular β cell calcium release mediated by forskolin and antisense oligonucleotides against cAMP-GEF II further reduced insulin secretion in the presence of H-89. See cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca(2+)-induced Ca(2+) release in INS-1 pancreatic beta-cells. J Physiol. 2001 Oct 15;536(Pt 2):375-85 and Critical role of cAMP-GEFII/Rim2 complex in incretin-potentiated insulin secretion. J Biol Chem. 2001 Oct 11. Hence the cAMP-GEF II signaling complex, interacting with Rim2, likely accounts for a substantial proportion of PKA-independent GLP-1R signaling in b-cells
GLP-1 exerts both direct and indirect incretin and non-incretin actions
To compare the relative incretin and non-incretin roles of GIP versus GLP-1, studies have been carried out in rats and mice using a receptor antagonist (antisera against the GIP receptor) or the GLP-1 antagonist exendin (9-39). The results agree with previous findings using GIPR-/- and GLP-1R-/- mice, and demonstrate that GLP-1, but not GIP, exerts both incretin and non-incretin actions in the regulation of blood glucose. See Glucose-dependent insulinotropic polypeptide confers early phase insulin release to oral glucose in rats: demonstration by a receptor antagonist. Endocrinology. 2000 Oct;141(10):3710-6. and Glucagon-like peptide-1, but not glucose-dependent insulinotropic peptide, regulates fasting glycemia and nonenteral glucose clearance in mice. Endocrinology. 2000 Oct;141(10):3703-9.
The actions of GLP-1 on the islet b-cell are likely partly indirect, via activation of sensory nerves or the portal glucose sensor, and partly direct, via activation of the islet β cell GLP-1 receptor. To review representative papers that address this physiology, see Glucose competence of the hepatoportal vein sensor requires the presence of an activated glucagon-like peptide-1 receptor. Diabetes. 2001 Aug;50(8):1720-8 and Sensory nerves contribute to insulin secretion by glucagon-like peptide-1 in mice. Am J Physiol Regul Integr Comp Physiol. 2004 Feb;286(2):R269-72. The extent to which these studies in mice are also relevant to understanding of GLP-1 action in humans remains uncertain.
GLP-1 and insulin gene expression
The GLP-1-stimulated increase in insulin mRNA is likely mediated in part via cAMP. It is well known that cAMP increases both insulin gene transcription and stabilizes insulin mRNA. Similarly, GLP-1 increases insulin mRNA in part via enhanced mRNA stability, and possibly through increased insulin gene transcription. See Glucagon-like peptide-1 affects gene transcription and messenger ribonucleic acid stability of components of the insulin secretory system in RIN 1046-38 cells. Endocrinology. 1995 Nov;136(11):4910-7 and Insulinotropic hormone glucagon-like peptide-I(7-37) stimulation of proinsulin gene expression and proinsulin biosynthesis in insulinoma beta TC-1 cells. Endocrinology. 1992 Jan;130(1):159-66.
The effect of GLP-1 on the insulin gene promoter appears to be mediated by two distinct cis-acting sequences, both in a PKA-dependent and PKA-independent manner, depending on the experimental model used; Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin I gene cAMP response element. Diabetes. 2000 Jul;49(7):1156-64. Inhibition of p38 mitogen-activated protein kinase (p38 MAPK) using a chemical inhibitor SB 203580 resulted in a marked increase in insulin promoter activity in response to GLP-1 stimulation, implying the existence of a functional interaction between GLP-1 and MAPK signaling pathways. Insulinotropic Hormone Glucagon-Like Peptide 1 (GLP-1) Activation of Insulin Gene Promoter Inhibited by p38 Mitogen-Activated Protein Kinase. Endocrinology. 2001 Mar 1;142(3):1179-1187.
Similarly the effects of exendin-4 on the induction of rat insulin I promoter activity in transfected INS-1 cells may be independent of the actions of 1) cAMP 2) PKA 3) the cAMP GEF Epac2, but is sensitive to inhibition by R0 31-8220, a serine/threonine PTK inhibitor. Mutational and deletional analyses demonstrated that the CRE is important for the effects of Ex-4 on RIP-luciferase activity. These results were not examined at the level of the endogenous insulin gene, and it remains unclear whether they are cell-line specific. Intriguingly, the conclusions reached about how Ex-4 exerts its effects on the rat insulin promoter are somewhat different from data obtained examining GLP-1R signal transduction, signaling inhibitors and other endpoints (insulin secretion). See Exendin-4 as a Stimulator of Rat Insulin I Gene Promoter Activity via bZIP/CRE Interactions Sensitive to Serine/Threonine Protein Kinase Inhibitor Ro 31-8220. Endocrinology. 2002 Jun;143(6):2303-13.
The synergistic effect of GLP-1 and glucose, or the effect of forskolin, on activation of a transfected insulin promoter in INS-1 cells can be blocked by FK506, a selective calcineurin inhibitor. As calcineurin, the selective Ca2+/calmodulin-dependent phosphatase 2B binds NFAT, the synergy between glucose and GLP-1 for activation of insulin promoter activity may be mediated in part through NFAT, and mutation of potential NFAT binding sites in the insulin gene promoter produces considerable attenuation of the synergistic GLP-1 and glucose response. Hence, glucose and GLP-1, by increasing intracellular calcium, may potentiate insulin gene transcription in a calcineurin- and NFAT-dependent manner. See NFAT regulates insulin gene promoter activity in response to synergistic pathways induced by glucose and glucagon-like Peptide-1. Diabetes. 2002 Mar;51(3):691-8.
Studies using the MIN6 cell line have shown that the glucose-dependent GLP-1-mediated activation of Erk is dependent upon 1) protein kinase A and 2) cellular calcium entry via activation of L type voltage gated calcium channels, as the GLP-1 stimulated of Erk phosphorylation was inhibited by nifedipine, but not by dominant negative forms of Ras and Rap1. See cAMP dependent protein kinase and Ca++ influx through L-type voltage gated calcium channels mediate Raf independent activation of extracellular regulated kinase in response to glucagon like peptide-1 in pancreatic beta-cells. J Biol Chem. 2002 Oct 2
GLP-1 and endoplasmic reticulum stress
Type 2 diabetes is associated with gradual loss of insulin secretion and a progressive reduction in b-cell mass. Insulin resistance produces a sustained increase in demand for insulin, and over time, the b-cell is unable to sustain augmented levels of insulin biosynthesis and secretion. GLP-1 and GIP appear to maintain insulin biosynthesis via interaction with ER stress pathways in the b-cell. The GLP-1R agonist exendin-4 significantly reduced biochemical markers of islet ER stress in islets from db/db mice in vivo and both exendin-4 and GIP attenuated translational downregulation of insulin and improved cell survival in purified rat b-cells and in INS-1 cells following induction of ER stress in vitro. The actions of GLP-1 to enhance translation are mediated via induction of ATF-4, and accelerated recovery from ER stress-mediated translational repression in islet b-cells in a PKA-dependent manner. Exendin-4 also reduced ER stress-associated b-cell death in a PKA-dependent manner. See GLP-1 receptor activation improves beta cell function and survival following induction of endoplasmic reticulum stress. Cell Metab. 2006 Nov;4(5):391-406 and the accompanying Editorial EXtENDINg beta cell survival by UPRegulating ATF4 translation. Cell Metab. 2006 Nov;4(5):333-4.
GLP-1 and pdx-1 expression
Pdx-1 is a key regulator of pancreatic and islet growth and insulin gene transcription. Treatment of mice or rats with GLP-1 or exendin-4 increases pdx-1 expression in vivo. See 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. Endocrinology. 2000 Dec;141(12):4600-5 and Insulinotropic glucagon-like peptide 1 agonists stimulate expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes. 2000 May;49(5):741-8. A direct effect of GLP-1 on pdx-1 expression, both RNA and protein, has also been demonstrated using islet cell lines in vitro. See Glucagon-like peptide-1 promotes DNA synthesis, activates phosphatidylinositol 3-kinase and increases transcription factor pancreatic and duodenal homeobox gene 1 (PDX-1) DNA binding activity in beta (INS-1)-cells. Diabetologia. 1999 Jul;42:856-64
GLP-1 also stimulates the cytoplasmic to nuclear translocation of pdx-1 in a PKA-dependent manner in immortalized rat islet cells as shown in Glucagon-Like Peptide-1 Causes Pancreatic Duodenal Homeobox-1 Protein Translocation from the Cytoplasm to the Nucleus of Pancreatic beta-Cells by a Cyclic Adenosine Monophosphate/Protein Kinase A-Dependent Mechanism. Endocrinology. 2001 May 1;142(5):1820-1827
The importance of Pdx-1 for the pleiotropic actions of GLP-1 has been examined in studies of GLP-1 action in mice with b-cell-specific inactivation of the pdx-1 gene. These mice exhibit gradual excision of the pdx-1 gene in β cells and exhibit progressive loss of β cell function with increasing age. Although the GLP-1R agonist exendin-4 continues to lower glucose after oral glucose loading in these mice in keeping with preserved inhibition of gastric emptying, exendin-4 failed to increase levels of plasma insulin, pancreatic insulin content, and pancreatic insulin mRNA transcripts in b cellPdx1-/- mice. Furthermore, there was a complete loss of the proliferative and anti-apoptotic actions of Ex-4 in bcellPdx1-/- mice, and surprisingly, exendin-4 failed to inhibit glucagon secretion in these mice. Hence, appropriate expression of Pdx-1 in β cells is essential for multiple glucoregulatory actions of GLP-1R agonists in mice. See ß-Cell Pdx1 Expression Is Essential for the Glucoregulatory, Proliferative, and Cytoprotective Actions of Glucagon-Like Peptide-1. Diabetes 2005 54: 482-491
GLP-1 and glucose competence
One of the first clues to the pleiotropic beneficial effects of GLP-1 on islet β cells was the finding that GLP-1 enhanced the number of glucose-responsive islet cells in vitro. See Pancreatic beta-cells are rendered glucose-competent by the insulinotropic hormone glucagon-like peptide-1(7-37). Nature. 1993 Jan 28;361(6410):362-5. This effect of GLP-1 appears to extend to diabetic islets, as shown in Glucagon-like peptide-1(7-36)-amide confers glucose sensitivity to previously glucose-incompetent beta-cells in diabetic rats: in vivo and in vitro studies. J Endocrinol. 1997 Nov;155(2):369-76 and Glucagon-like-peptide-1 (7-36) amide improves glucose sensitivity in beta-cells of NOD mice. Acta Diabetol. 1996 Mar;33(1):19-24.
Nevertheless, GLP-1 receptor signaling is not an essential requirement for glucose-responsiveness in vitro or in vivo, as mice with genetic disruption of GLP-1R signaling exhibit intact insulin secretory responses to glucose in a perfused pancreas or perfused islet system. See Mouse pancreatic beta-cells exhibit preserved glucose competence after disruption of the glucagon-like peptide-1 receptor gene. Diabetes. 1998 Apr;47(4):646-52 and Enhanced glucose-dependent insulinotropic polypeptide secretion and insulinotropic action in glucagon-like peptide 1 receptor -/- mice. Diabetes. 1998 Jul;47(7):1046-52
GLP-1, and the "memory effect"
Intriguingly, several reports suggest that even a brief exposure to GLP-1 produces long lasting beneficial effects on β cell function which persist following discontinuation of The short half-life of glucagon-like peptide-1 in plasma does not reflect its long-lasting beneficial effects. Eur J Endocrinol. 2002 Jun;146(6):863-9
It is not clear whether the same type of memory effect will be observed in human diabetic subjects treated with Absence of a Memory Effect for the Insulinotropic Action of Glucagon-like Peptide 1 (GLP-1) in Healthy Volunteers. Horm Metab Res. 2003 Sep;35(9):551-6
GLP-1 and proinsulin processing
Does GLP-1 enhance the processing of proinsulin to insulin in normal subjects or patients with IGT or diabetes? This question is being studied in multiple clinical trials. Human subjects with IGT infused with GLP-1 exhibit a decreased circulating ratio of proinsulin to insulin. Nevertheless, the rapid kinetics of these changes does not permit any definite conclusions about GLP-1-regulated proinsulin processing, versus regulation of secretion or clearance etc. See Evidence against a Rate-Limiting Role of Proinsulin Processing for Maximal Insulin Secretion in Subjects with Impaired Glucose Tolerance and beta-Cell Dysfunction. J Clin Endocrinol Metab. 2001 Mar 1;86(3):1235-1239.
GLP-1, sulfonylureas, the SUR, and insulin secretion
What are the targets for GLP-1 action in the islet b cells, and how does it restore sensitivity to sulfonylureas in patients with type 2 diabetes? Equally importantly, how does GLP-1 receptor activation modify the activity of the SUR or its components?
Research from the Habener laboratory demonstrates a direct effect of GLP-1 in Type 2 diabetes in vivo, and its actions at the molecular level on the islet b cell. See Regulated Expression of Adenosine Triphosphate- Sensitive Potassium Channel Subunits in Pancreatic b-Cells. Endocrinology 2001 Jan 1;142(1):129-138 and Glucagon-Like Peptide-1 Inhibits Pancreatic ATP-Sensitive Potassium Channels via a Protein Kinase A- and ADP-Dependent Mechanism. Mol Endocrinol. 2002 Sep;16(9):2135-44
Complementary studies using SUR1-/- mice have illustrated the functional importance of intact SUR biological activity for at least some aspects of β cell. SUR1 null mice exhibit an impaired insulin secretory response to glucose and β cell, as outlined in Sulfonylurea receptor type 1 knock-out mice have intact feeding-stimulated insulin secretion despite marked impairment in their response to glucose. J Biol Chem. 2002 Oct 4;277(40):37176-83. Similarly, β cells exhibit a PKA-independent defect in completely sensing and responding to the increased levels of cAMP following incretin stimulation. See cAMP-Activated Protein Kinase-Independent Potentiation of Insulin Secretion by cAMP Is Impaired in SUR1 Null Islets. Diabetes. 2002 Dec;51 (12): 3440-9
Although the actions of GLP-1 to stimulate insulin secretion are normally highly glucose-dependent, administration of GLP-1 in the presence of a sulfonylurea agent leads to enhanced insulin secretion even at normal or low glucose concentrations. Studies in the perfused rat pancreas demonstrate that GLP-1 augments the tolbutamide-mediated stimulation of insulin secretion at 3 mM glucose. GLP-1 also increased somatostatin and decreased glucagon secretion at both 3 and 11 mM glucose in the same experiments and the secretion of SMS and glucagon were inversely correlated. These findings confirm that substantial inhibition of the KATP channel by an agent such as tolbutamide uncouples the glucose-dependent action of GLP-1 on the b-cell. See Sulfonylurea compounds uncouple the glucose dependence of the insulinotropic effect of glucagon-like Peptide 1. Diabetes. 2007 Feb;56(2):438-43
GLP-1 b-cells in a cyclic AMP- and PKA-dependent manner. The effect of Glucagon-like Peptide-1 Stimulates GABA Formation by Pancreatic Beta Cells at Level of Glutamate Decarboxylase. Am J Physiol Endocrinol Metab. 2006 Dec 26; [Epub ahead of print]
What are the effects of GLP-1 on free fatty acid generation in the islet b cell? In a series of original experiments, Yaney and colleagues demonstrated that GLP-1 releases FFAs from intracellular stores and stimulates FFA oxidation in HIT cells. The contribution of this effect to GLP-1-stimulated insulin secretion clearly merits further investigation. See Glucagon-like peptide 1 stimulates lipolysis in clonal pancreatic beta-cells (HIT). Diabetes. 2001 Jan;50(1):56-62. Nevertheless, despite observations that GLP-1 activates lipase activity in the β cell, studies using HSL-/- mice demonstrate that GLP-1 is fully capable of increasing glucose-stimulated insulin secretion despite the absence of β cell lipase, as described in Hormone-sensitive lipase has a role in lipid signaling for insulin secretion but is nonessential for the incretin action of glucagon-like Peptide 1. Diabetes. 2004 Jul;53(7):1733-42.
Summary of β Cell genes and proteins activated by GLP-1
| Experimental Model | Gene or Protein |
| INS-1 cells | Akt and IRS proteins |
| INS-1 cells | Glucokinase |
| RIN1046-38 cells | GLUT-1 RNA |
| RIN1046-38 cells | Hexokinase I RNA |
| INS-1 cells | Immediate early genes |
| Multiple cell models | Insulin RNA |
| INS-1 cells | Kir 6.2 RNA |
| Multiple islet cell lines | Pdx-1 RNA and protein |
| RIN1046-38 cells | SNAP-25 phosphorylation |
| INS-1 cells | Calcineurin and NFAT |
| Diabetic rodents | Foxo1 |
| INS-1 cells | EGF receptor |
| Multiple cell models | IRS-2 |
| Rodent islets | PTB1 |
| Rat islets | GAD and GABA |