Is there a second GLP-1 receptor? This question continues to resonate largely due to the results of several studies demonstrating GLP-1 effects that are either "non-classical" in terms of binding, signal transduction, or the actions of GLP-1 may not be blocked by known GLP-1 receptor antagonists such as exendin(9-39). Alternatively, reports continue to accumulate of GLP-1 actions in tissues not yet clearly shown to express the known GLP-1 receptor.
Liver and portal system
Intraportal infusion of exendin(9-39) in rats did not modify the GLP-1-induced facilitation of neural afferents, leading these scientists to speculate that "the neural reception of tGLP-1 involves a receptor mechanism distinct from that in the well-known humoral insulinotropic action", as outlined in The hepatic vagal reception of intraportal GLP-1 is via receptor different from the pancreatic GLP-1 receptor. J Auton Nerv Syst. 2000 Apr 12;80(1-2):14-21.
Evidence for a structurally and functionally distinct GLP-1 receptor also derives from studies of GLP-1 action in the liver. Several studies have shown that GLP-1 binds to liver membranes, and is displaced by GLP-1(1-36)amide Glucagon-like peptide-1 binding to rat hepatic membranes. J Endocrinol. 1995 Jul;146(1):183-9. Experiments using human hepatoma cells have shown that GLP-1 modulates the cell content of radiolabelled glycosylphosphatidylinositols (GPIs), Inositolphosphoglycans are possible mediators of the glucagon-like peptide 1 (7-36)amide action in the liver. J Endocrinol Invest. 1996 Feb;19(2):114-8. Incubation of rat hepatocytes with GLP-1 resulted in inhibition of glucagon-stimulated glycogenolysis, an effect additive with insulin. The authors postulated that this effect is through a distinct receptor, as GLP-1 did not displace glucagon binding from liver membranes. See Glucagon-like peptide-1 inhibits glucagon-induced glycogenolysis in perivenous hepatocytes specifically. Regul Pept. 2003 Mar 28;111(1-3):207-10.
Similarly, GLP-1activated PI3K/PKB, p70s6k, p44 and p42 MAP-kinase in rat liver cells and the activation of PI3K/PKB, PKC and PP-1, but not PP-2A, appeared to mediate the GLP-1 stimulatory action on glycogen synthase a in rat hepatocytes, while MAPKs and p70s6k were postulated to participate in other GLP-1effects. Whether these cells expressed the known GLP-1 receptor remains unclear. See Cell signalling of the GLP-1 action in rat liver. Mol Cell Endocrinol. 2003 Jun 30;204(1-2):43-50.
Exendin-4 has also been shown to promote transdifferentiation of liver cells in the context of ectopic pdx-1 expression. Although expression of the known GLP-1R was not identified in liver cells, either prior to or following the transdifferentiation process, Ex-4 increased levels of cAMP in human liver cells, and increased levels of phosphoCREB, Akt, PKC, and ERK1/2. Following Pdx-1 transduction, Ex-4 increased insulin promoter activity and insulin gene expression in transduced liver cells. Similarly, Ex-4 induced expression of a broad number of transcription factors and proteins associated with specialized beta cell function. Ex-4 was also found to increase liver cell proliferation and likely increases the proportion of cells susceptible to transduction by Pdx-1 Exendin-4 promotes liver cell proliferation and enhances PDX-1-induced liver to pancreas transdifferentiation J Biol Chem. 2009 Sep 15. [Epub ahead of print]
Raab and colleagues assessed the actions of exendin-4 after treatment of neonatal rats with intrauterine growth retardation. Exendin-4 reduced basal hepatic glucose output and increased liver insulin sensitivity. Remarkably, the direct effects of exendin-4 on neonatal rat hepatocytes, namely reduce PEPCK expression and improved insulin signaling, were observed in the absence of detectable expression of the known GLP-1R-See Neonatal Exendin-4 Treatment Reduces Oxidative Stress and Prevents Hepatic Insulin Resistance in Intrauterine Growth Retarded Rats Am J Physiol Regul Integr Comp Physiol. 2009 Oct 21. [Epub ahead of print]
Gastrointestinal tract
A similar picture emerges from studies of GLP-1 action on gastrointestinal motility in dogs, where exendin (9-39) did not block the inhibitory actions of GLP-1 on phasic activity in response to electrical field stimulation, as shown in Local, exendin-(9-39)-insensitive, site of action of GLP-1 in canine ileum. Am J Physiol Gastrointest Liver Physiol. 2002 Sep;283(3):G595-602. Similarly, pretreatment of dogs with the antagonist exendin-(9-39) amide did not alter the magnitude of inhibition of gastric acid caused by exogenous GLP-1, as shown in Glucagon-like peptide-1-(7-36) amide and peptide YY mediate intraduodenal fat-induced inhibition of acid secretion in dogs. Endocrinology. 1998 Jan; 139(1): 189-94. These data, although intriguing, are somewhat difficult to interpret, as control experiments demonstrating the ability of exendin(9-39) to block known actions of GLP-1 on insulin or glucagon secretion, were not provided in these studies. Nevertheless, it seems reasonable to consider the possibility that GLP-1 may exert some neural effects through a second receptor that has not yet been isolated or well characterized.
Furthermore, both the "inactive" full length peptide GLP-1(1-36amide) as well as the bioactive GLP-1(7-36amide) peptides stimulated intestinal somatostatin secretion from rat intestinal cell cultures, implying the existence of a second GLP-1 receptor distinct from the currently well studied cloned pancreatic receptor, as described in Glucagon-like peptide-1-(7-36) amide and peptide YY mediate intraduodenal fat-induced inhibition of acid secretion in dogs. Endocrinology. 1998 Jan;139(1):189-94.
Central or peripheral infusion of exendin-4 suppresses levels of plasma ghrelin, a peptide produced largely in the stomach, in fasted rats. Intriguingly, the effects of exendin-4 on ghrelin secretion were not mimicked by native GLP-1, and they were not blocked by the classical GLP-1 receptor antagonist exendin(9-39). These findings implicate a role for a non-classical GLP-1R coupled to regulation of ghrelin secretion as outlined in Exendin-4 potently decreases ghrelin levels in fasting rats. Diabetes. 2007 Jan;56(1):143-51
Intriguingly, treatment of GK rats with exendin-4 was associated with a reduction in HbA1c, reduced weight gain, and an increase in small bowel mass. In contrast, treatment of GK rats with a DPP-4 inhibitor had no effect on body weight, food intake or small bowel mass. Plasma levels of GLP-2 were not increased by exendin-4 treatment. The mechanisms/receptor mediating the increase in small bowel mass in this experimental model remain unclear. See Exendin-4, but not Dipeptidyl Peptidase IV Inhibition, Increases Small Intestinal Mass in GK-rats. Am J Physiol Gastrointest Liver Physiol. 2007 Apr 12; [Epub ahead of print]
Fat and muscle
Several additional studies demonstrate GLP-1 effects that may be exerted through a distinct GLP-1 receptor in adipose tissue and muscle. Binding studies have demonstrated GLP-1
binding sites in membranes from both adipose tissue and muscle: Presence
and characterization of glucagon-like peptide-1(7-36) amide receptors in
solubilized membranes of rat adipose tissue. Endocrinology. 1993 Jan;132(1):75-9 and Glucagon-like
peptide-1 binding to rat skeletal muscle. Peptides.
1995;16(2):225-9.
GLP-1 exerts insulin-like effects via stimulation of glycogen synthesis, glycogen synthase a activity, and glucose oxidation and utilization, and inhibited glycogen phosphorylase a activity in strips of human skeletal muscle, all at physiological concentrations of the peptide. Paradoxically, both exendin-4 and exendin(9-39) both activated glycogen synthesis and synthase a activity, without significant effects on cAMP accumulation as shown in Glucagon-like peptide-1 (GLP-1) and glucose metabolism in human myocytes. J Endocrinol. 2002 Jun;173(3):465-73.
Similar observations have been made using L6 myotubes, where GLP-1 enhanced insulin-stimulated glycogen synthesis and stimulating CO2 production and lactate formation, while actually decreasing the levels of cAMP. In contrast, stimulation of the cloned transfected cloned GLP-1 receptor in L6 myotubes did increase cAMP formation and inhibited glycogen synthesis. Both the biologically inactive GLP-1 (1-36amide) and exendin(9-39) displaced GLP-1 binding from non-transfected L6 cells. See GLP-1 action in L6 myotubes is via a receptor different from the pancreatic GLP-1 receptor. Am J Physiol. 1998 Sep;275(3 Pt 1):C675-83.
Similarly, GLP-1 increased basal and acute insulin-stimulated glucose uptake along with an increase in GLUT1 and GLUT4 protein levels in in fully differentiated 3T3-L1 adipocytes, however whether these cells express the known GLP-1 receptor has not been proven, and exendin(9-39) also acts as a GLP-1 agonist in these cells; see Regulation of glucose transporters and hexose uptake in 3T3-L1 adipocytes: glucagon-like peptide-1 and insulin interactions. J Mol Endocrinol. 1997 Dec;19(3):241-8 and Novel signal transduction and peptide specificity of glucagon-like peptide receptor in 3T3-L1 adipocytes. J Cell Physiol. 1997 Sep;172(3):275-83 and Glucagon-like peptide-1(7-36) amide (GLP-1) enhances insulin-stimulated glucose metabolism in 3T3-L1 adipocytes: one of several potential extrapancreatic sites of GLP-1 action. Endocrinology. 1994 Nov;135(5):2070-5.
The actions of GLP-1 and exendin-4 have been dissociated in studies of L6 myotubes and 3T3L1 preadipocytes. Both Ex-4 and GLP-1 enhanced insulin-stimulated 2-deoxyglucose uptake in L6 cells in a PI3 kinase-dependent manner. In contrast, Ex-4, but not GLP-1, enhanced insulin sensitivity in 3T3 adipocytes. See Exendin-4 increases insulin sensitivity via a PI-3-kinase-dependent mechanism: contrasting effects of GLP-1. Biochem Pharmacol. 2002 Mar 1;63(5):993-6
Effects of GLP-1 on cell signaling and glucose transport in normal human muscle cells have been described. Intriguingly, GLP-1, the agonist exendin-4, and the GLP-1R antagonist exendin(9-39) all had similar effects on glucose uptake and signal transduction in the muscle preparations. GLP-1, insulin, exendin-4, and exendin(9-39) activated PI3K/PKB and p42/44 MAPK enzymes, whereas p70s6k was activated only by GLP-1 and insulin. Differential blockade of peptide activities was seen with signal transduction inhibitors, implicating that the peptides do not exert identical effects in this muscle system. The identity of the receptor mediating these effects remains obscure. Effect of GLP-1 on glucose transport and its cell signalling in human myocytes. Regul Pept. 2005 Mar 30;126(3):203-211
In contrast, several studies have failed to demonstrate an effect of GLP-1 or exendin-4 on insulin sensitivity in man. For example, see Lack of effect of exendin-4 and glucagon-like peptide-1-(7,36)-amide on insulin action in non-diabetic humans. Diabetologia. 2002 Oct;45(10):1410-1415 and GLP-1 does not not acutely affect insulin sensitivity in healthy man. Diabetologia. 1996 Oct;39(10):1227-32 and Effect of glucagon-like peptide 1(7-36) amide on glucose effectiveness and insulin action in people with type 2 diabetes. Diabetes. 2000 Apr;49(4):611-7. Although a six week infusion of GLP-1 in patients with type 2 diabetes did improve insulin sensitivity as assessed by a hyperinsulinemic euglycemic clamp Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and beta-cell function in type 2 diabetes: a parallel-group study. Lancet. 2002 Mar 9;359(9309):824-30, it is possible that improvement in diabetic control and reduced glucose toxicity account for the findings in these subjects.
CNS and pituitary
Discordant results have also been obtained in studies of the effects of exendin-4 and exendin(9-39) on suppression of TSH secretion in rats. A single subcutaneous injection of Exendin-4, but not GLP-1, glucagon, or GLP-2, suppressed levels of circulating TSH. Remarkably, exendin (9-39) also reduced levels of TSH, and did not prevent the exendin-4-dependent suppression of TSH. See Preproglucagon derived peptides and thyrotropin (TSH) secretion in the rat: Robust and sustained lowering of blood TSH levels in exendin-4 injected animals. Int J Mol Med. 2002 Sep;10(3):327-31
Administration of exendin-4, with and without exendin (9-39) to normal and diabetic rats led to the suggestion that experimental diabetes induces the expression of EX-4 receptors other than the classic GLP-1R, whose activation mediate the ACTH and aldosterone, but not corticosterone, secretagogue effects. These conclusions were drawn based on observations that Ex-4 stimulated ACTH and aldosterone in and exendin (9-39)-independent manner in STZ-diabetic but not normal rats. See Prolonged exendin-4 administration stimulates pituitary-adrenocortical axis of normal and streptozotocin-induced diabetic rats. Int J Mol Med. 2003 Oct;12(4):593-6
GLP-1(9-36) amide-A Bioactive peptide acting through a distinct receptor?
Several studies have raised the possibility that GLP-1(9-36) amide, which is proportionately more abundant than the full length bioactive peptide, may not simply represent an inert cleavage product, but may function either as an endogenous GLP-1R antagonist or a weak agonist (Glucagon-like peptide-1-(9-36) amide is a major metabolite of glucagon-like peptide-1-(7-36) amide after in vivo administration to dogs, and it acts as an antagonist on the pancreatic receptor. Eur J Pharmacol. 1996 Dec 30;318(2-3):429-35 and The inhibitory effect of glucagon-like peptide-1 (7-36)amide on antral motility is antagonized by its N-terminally truncated primary metabolite GLP-1 (9-36)amide. Peptides. 1998;19(5):877-82 and High potency antagonists of the pancreatic glucagon-like peptide-1 receptor. J Biol Chem. 1997 Aug 22;272(34):21201-6, or as a unique agonist with insulin-independent glucose-lowering properties, as hypothesized in GLP-1-(9-36) amide reduces blood glucose in anesthetized pigs by a mechanism that does not involve insulin secretion. Am J Physiol Endocrinol Metab. 2002 Apr;282(4):E873-9. Subsequent studies failed to demonstrate an insulin-like effect of GLP-1(9-360amide on insulin secretion or glucose tolerance/clearance in normal human subjects Effects of GLP-1-(7-36)NH2, GLP-1-(7-37), and GLP-1- (9-36)NH2 on intravenous glucose tolerance and glucose-induced insulin secretion in healthy humans J Clin Endocrinol Metab. 2003 Apr;88(4):1772-9. In contrast, infusion of GLP-1(9-36)amide exerted an insulin-like effect and possibly increased insulin sensitivity in obese but not lean human subjects, through incompletely understood mechanisms GLP-1 (9-36) Amide, Cleavage Product of GLP-1 (7-36) Amide, Is a Glucoregulatory Peptide Obesity (Silver Spring). 2008 Apr 17; [Epub ahead of print]
Remarkably, improvements in dog cardiovascular function have also been demonstrated using the truncated peptide GLP-1(9-36)amide, raising the possibility that a second functional receptor for GLP-1(9-36)amide may be critical for the cardiovascular actions of native GLP-1. See Active Metabolite of GLP-1 Mediates Myocardial Glucose Uptake and Improves Left Ventricular Performance in Conscious Dogs with Dilated Cardiomyopathy. Am J Physiol Heart Circ Physiol. 2005 Jul 15; [Epub ahead of print]
Moreover, emerging evidence suggests that the cardiovascular biology of GLP-1 receptor agonists and GLP-1-derived peptides is increasingly complex. Classical GLP-1R agonists exert cardioprotective actions dependent on the known GLP-1receptor. However, GLP-1(9-36)amide also appears to be cardioprotective in the ischemic mouse heart when infused post-ischemia, and these actions are independent of the known GLP-1R. Furthermore, even exendin-4 exerts some cardioprotective activity in a GLP-1R-independent manner. See Cardioprotective and Vasodilatory Actions of Glucagon-Like Peptide 1 Receptor Are Mediated Through Both Glucagon-Like Peptide 1 Receptor–Dependent and –Independent Pathways Circulation 2008: published online before print April 21, 2008, 10.1161/CIRCULATIONAHA.107.739938