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.
How important is the beta cell, relative to other cellular sites of GLP-1 action, for GLP-1R-dependent glucose control? Lamont and colleagues addressed this question in mice that expressed the human GLP-1 receptor under the control of the pdx-1 promoter in Glp1r-/- mice. Analysis of gene expression profiles revealed that islets from pdx1-hGLP-1:Glp1r-/- mice expressed normal levels of key GLP-1R-regulated islet genes important for control of GSIS. Furthermore restoration of functional GLP-1 activity in Glp1r-/- islets was illutrated by functional restoration of GLP-1-stimulated a) inulin secretion b) cAMP accumulation and c) Akt phosphorylation in islets from pdx1-hGLP-1:Glp1r-/- mice. Remarkably, despite the complete functional absence of CNS GLP-1R expression, restoration of islet GLP-1R expression was sufficient to normalize abnormal glucose tolerance in Glp1r-/- mice, and to confer robust GLP-1R dependent control of oral and intraperitoneal glucose tolerance and beta cell proliferation in pdx1-hGLP-1:Glp1r-/- mice. Nevertheless, a contribution for CNS GLP-1Rs in the control of glucose homeostasis was inferred by the failure of peripheral administration of the GLP-1R antagonist exendin(9-39) to impair glucose tolerance in pdx1-hGLP-1:Glp1r-/- mice Pancreatic GLP-1 receptor activation is sufficient for incretin control of glucose metabolism in mice J Clin Invest. doi:10.1172/JCI42497
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. Although GLP-1 increases islet cAMP, some 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.
Hodson and colleagues delineated the importance of cell to cell communication, pathways sensitive to lipotoxicity, for the GLP-1-mediated enahncement of calcium oscillations and augmentation of insulin secretion. The Gap Junction blocker, 18-α-glycyrrhetinic acid (AGA), disrupted synchronous calcium responses and impaired the beta cell response to GLP-1 in human islets. Palmitate reduced expression of islet connexin36, findings that correlated with impairment of insulin secretion and calcium oscillation in isolated islets in response to GLP-1 or GIP. Rodent islets were relatively resistant to the lipotoxi effects of high fat feeding in that they exhibited comparatively preserved incretin responses. A negative linear correlation was observed in human islet responses between donor BMI and the coordinated β cell responses to GLP-1 Lipotoxicity disrupts incretin-regulated human β cell connectivity J Clin Invest. doi:10.1172/JCI68459
Profiling of GLP-1/cAMP action with forskolin using INS-1 cells revealed a second wave of gene expression with delayed kinetics, increasing after 16h of exposure, with many of the genes corresponding to known targets of hypoxia inducible factor-a (HIF-1a). The second wave of forskolin-stimulated gene expression was sensitive to inhibitors of PKA and protein synthesis. Forskolin robustly increased protein levels of HIF-1a, and exendin-4 potentiated the high glucose-mediated induction of HIF-1a protein in INS-1 cells; the delayed actions of forskolin on HIF-1a-mediated gene expression were sensitive to rapamycin. Adenoviral expression of GLP-1 in mouse liver increased levels of phospho-S6 in murine b-cells, actions sensitive to rapamycin. The ability of Ad-GLP-1 expression to inhibit STZ-induced b-cell apoptosis was also blocked by rapamycin. mTOR links incretin signaling to HIF induction in pancreatic beta cells Proc Natl Acad Sci U S A. 2011 Sep 26.
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. Similarly, the GLP-1R-dependent activation of glucokinase activity in INS-1 cells and rat islets is mimicked by non-selective activators of cAMP and Epac, and knockdown of either Epac2, Rim2, or Rab3A with siRNA in INS-1 cells completely eliminated the effects of GLP-1 on glucose uptake, cellular ATP and GK activity. Glucagon-Like Peptide-1 Enhances Glucokinase Activity in Pancreatic β-Cells through the Association of Epac2 with Rim2 and Rab3A Endocrinology. 2011 Dec 6. [Epub ahead of print]
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
Song and colleagues examined the separate importance of PKA signaling and snapin phsophorylation for GLP-1 action starting with generation of a mouse with activated islet PKA activity (increased CREB phosphorylation) due to conditional b-cell ablation of the Prkar1a ( the abundant protein kinase A regulatory subunit in islets). Islet and b-cell mas was normal in Prkar1a KO mice, however glucose tolerance and GSIS were improved as assessed in mice in vivo and in insulin secretion experiments using islets ex vivo. A greater number of insulin granules (somewhat larger in size) were detected in b-cells from the KO mice. The heterozygous mice exhibited enhanced sensitivity to the insulinotropic effects of Ex-4 in islets. Remarkably, glucose tolerance tests carried out in 7 subjects with inactivating Prkar1a mutations exhibited a similar phenotype with improved glucose tolerance and increased levels of insulin. Ex-4 increased snapin phosphorylation at serine 50 in a cAMP and PKA-dependent manner. Snapin is an exocytosis modulating protein initially identified in neuronal synapses that is regulated by PKA. Phosphosnapin exhibited increased association with SNAP-25, collectrin, and Epac2, and these interactions were GLP-1R- and glucose-dependent, enhanced by Ex-4. Islet transduction of a virus encoding the snapin phsphorylation mimic S50D enhanced GSIS at 10 mM glucose whereas lentiviral knockdown of snapin in murine islets impaired insulin secretion at all glucose levels and abrogated the stimulatory response to Ex-4. Levels of phsophorylated snapin were reduced in islets from DIO mice, and the S50 phosphorylation site was found to be subject to O-GlcNAcylation, and Ex-4 modulated the ratio of snapin subjected to phsophorylation (increased by Ex-4) vs. O-GlcNAcylation. Hence the available evidence suggests that the incretin-like favorable effects on GSIS are mimicked by snapin S50 phosphorylation Snapin mediates incretin action and augments glucose-dependent insulin secretion Cell Metab. 2011 Mar 2;13(3):308-19
Gheni and colleagues examined the basis for defective incretin action in murine b-cells. They identified that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation is transported into insulin granules by cAMP/PKA signaling, which the leads to amplification of insulin granule exocytosis. Metabolomic analysis of incretin-responsive and non-responsive cells identified higher activity of the mealeate aspartate shuttle in b-cells that responded to incretin hormones. Aminooxyacetate(AOA), a shuttle inhibitor, abolished insulin secretory response to GLP-1 and GIP, whereas GLP-1 increased the insulin granule content of glutamate via PKA-sensitive mechanisms. Furthermore, dimethylglutamate mimicked the effects of incretin/cAMP signaling on insulin secretion and the presence or absence of glucose-stimulated islet glutamate production correlated with the degree of incretin responsivity. These findings highlight the role of cytosolic glutamate as a key signal linking glucose metabolism to incretin/cAMP action and amplification of insulin secretion. Glutamate Acts as a Key Signal Linking Glucose Metabolism to Incretin/cAMP Action to Amplify Insulin Secretion Cell Reports 2014 http://dx.doi.org/10.1016/j.celrep.2014.09.030
GLP-1R expression in beta cells may be regulated by ubiquitination and SUMO proteins. High glucose upregulated the expression of SUMO mRNA transcripts and the SUMO conjugating enzyme Ubc-9 in islet cells; enhanced expression of transfected SUMO-1 diminished the exendin-4-dependent stimulation of cAMP in transfected MIN6 cells. Functional FRET and co-immunoprecipitation studies implied a direct covalent interaction between SUMO-1 and the GLP-1R. and enhanced SUMO-1 expressed reduced cell surface expression of the GLP-1R in MIN6 cells. Partial knockdown of Ubc-9 improved exendin-4 stimulated insulin secretion SUMO down-regulates GLP-1 stimulated cAMP generation and insulin secretion Am J Physiol Endocrinol Metab. 2012 Jan 10.
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.
Although GLP-1 increases islet cAMP, some 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
Adenylyl cyclase 8 appears to be an important molecular mediator of the deleterious effects of glucose on b-cell responsivity to GLP-1 in rat and human islets. INS-1 cells, rat or human islets were exposed to varying glucose levels for 3 days; hyperglycemia (11-20 mM glucose)reduced the GLP-1 responses (calcium, cyclic AMP and insulin secretion) in association with significant reduction (4-fold) in the expression of adenylyl cyclase 8. Adenoviral re-expression of adenylate cyclase 8, but not the GLP-1 receptor, restored the calcium response to GLP-1; conversely knockdown of Adcy8 (shADCY8) markedly reduced the calcium and cyclic AMP responses to exogenous GLP-1 Adenylyl cyclase 8 is central to glucagon-like peptide 1 signalling and effects of chronically elevated glucose in rat and human pancreatic beta cells Diabetologia. 2011 Feb;54(2):390-402
GLP-1 may also exert its effects on insulin exocytosis in part through rapid potentiation of the posttranslational activation of glucokinase. GLP-1 rapidly potentiated glucose metabolism, increased glucokinase activity and S-nitrosylation of GCK, a reflection of GCK activation. GLP-1-induced GCK activation in a L-NAME-sensitive manner, findings suggestive of a role for NOS. The ability of GLP-1 to promote granule exocytosis in b-cells was diminished in the presence of a GCKV367M mutant protein
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 islet inflammation
Pugazhenthi and colleagues examined the effects of exendin-4 treatment of human islets from non-diabetic donors. Exendin-4 modestly suppressed the expression of chemokines (CXCL9, CXCL10, and CXCL11) following induction of an inflammatory response by treatment of islets with IFN-g. Small reductions in STAT-1 were also observed and these anti-inflammatory effects were mimicked by forskolin and the effects of exendin-4 were potentiated by phosphodiesterase inhibitors. Similar findings were obtained in Min-6 insulinoma cells in a PKA-independent manner independent of H-89. Anti-inflammatory action of exendin-4 in human islets is enhanced by phosphodiesterase inhibitors: potential therapeutic benefits in diabetic patients Diabetologia. 2010 Jul 16. [Epub ahead of print]
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
Takahashi and colleagues studied the importance of Epac2 for the interaction between GLP-1 and SUs in the control of insulin secretion. Although GLP-1 alone did no augment insulin secretion at normoglycemia, GLP-1 enhanced the insulin-stimulating actions of SUs at normal glucose levels in mouse islets. Thse stimulatory effects were markedly diminished in Epac2-/- islets. The insulin-stimulatory effects of gliclizide were preserved but those of glimeipiride were attenuated in Epac2-/- islets and selective agonists for 1) PKA and 2) Epac2 revealed that Epac2, rather than PKA couples GLP-1 action to enhancement of SU action in islets. Not all SUs act equally in this pathway, as Rap1 activation was markedly enhanced by combination of an Epac-selective cAMP analog with glibenclamide or glimepiride but not gliclazide, consistent with findings made in Epac2-/- mice using liraglutide plus glimepiride Role of Epac2A/Rap1 signaling in interplay between incretin and sulfonylurea in insulin secretion Diabetes. 2014 Oct 14. pii: DB_140576.
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.
Deletion of the transcription factor COUP-TFII in mice impaired glucose homeostasis, in association with reduced GLP-1 action in islet cells. The ability of GLP-1 to induce b-catenin signaling and increase expression of cyclin D1 required COUP-TFII expression in islet cells. Furthermore Glp1r mRNA transcripts were reduced in knockout islets. COUP-TFII Controls Mouse Pancreatic β-Cell Mass through GLP-1-β-Catenin Signaling Pathways PLoS One. 2012;7(1):e30847.
Summary of β Cell genes and proteins activated by GLP-1
|Experimental Model||Gene or Protein|
|INS-1 cells||Akt and IRS proteins|
|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|
|INS-1 cells||EGF receptor|
|Multiple cell models||IRS-2|
GAD and GABA
|Rodent islet cells||COUP-TFII|