Associate editor: J. Wess
Mechanisms of action of glucagon-like peptide 1 in the pancreas

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Abstract

Glucagon-like peptide 1 (GLP-1) is a hormone that is encoded in the proglucagon gene. It is mainly produced in enteroendocrine L cells of the gut and is secreted into the blood stream when food containing fat, protein hydrolysate, and/or glucose enters the duodenum. Its particular effects on insulin and glucagon secretion have generated a flurry of research activity over the past 20 years culminating in a naturally occurring GLP-1 receptor (GLP-1R) agonist, exendin 4 (Ex-4), now being used to treat type 2 diabetes mellitus (T2DM). GLP-1 engages a specific guanine nucleotide-binding protein (G-protein) coupled receptor (GPCR) that is present in tissues other than the pancreas (brain, kidney, lung, heart, and major blood vessels). The most widely studied cell activated by GLP-1 is the insulin-secreting β cell where its defining action is augmentation of glucose-induced insulin secretion. Upon GLP-1R activation, adenylyl cyclase (AC) is activated and cAMP is generated, leading, in turn, to cAMP-dependent activation of second messenger pathways, such as the protein kinase A (PKA) and Epac pathways. As well as short-term effects of enhancing glucose-induced insulin secretion, continuous GLP-1R activation also increases insulin synthesis, β cell proliferation, and neogenesis. Although these latter effects cannot be currently monitored in humans, there are substantial improvements in glucose tolerance and increases in both first phase and plateau phase insulin secretory responses in T2DM patients treated with Ex-4. This review will focus on the effects resulting from GLP-1R activation in the pancreas.

Introduction

The incretin effect refers to the augmented insulin secretory response to a glucose load delivered to the gut relative to that achieved by intravenous glucose when the plasma levels of glucose, under both conditions, are comparable. This effect accounts for up to 60% of the insulin secretory response following an oral glucose load (Nauck et al., 1986) and is due to the insulinotropic effects of incretin hormones that are released from enteroendocrine cells of the gut. Glucose-dependent insulinotropic peptide (GIP, also referred to as gastric inhibitory polypeptide) and glucagon-like peptide 1 (GLP-1) are the main incretin hormones (Mojsov et al., 1987, Meier et al., 2002; see Table 1 for their amino acid sequences). GLP-1 results from a posttranslational cleavage of the product of the glucagon gene by the prohormone convertase PC1/3 (Dhanvantari et al., 2001). The majority of circulating biologically active GLP-1 in man is the GLP-1 (7-36) amide form, with lesser amounts of the bioactive GLP-1 (7-37) form also detectable (Orskov et al., 1994). The actions of GLP-1 have been extensively studied over the last 2 decades because its acute intravenous infusion or subcutaneous administration lowers blood glucose and increases insulin secretion. Most importantly, it does so in humans suffering from diabetes. Therefore therapeutic strategies based on activating the GLP-1 receptors (GLP-1R) on β cells and enhancing GLP-1 actions have been developed. One of the major drawbacks to the use of the native peptide in the clinic is its rapid degradation in serum due to the presence of a dipeptidyl peptidase IV (DPP-IV, also known as CD26) recognition site in the N-terminus (Hansen et al., 1999). This enzyme, which is present in the blood stream and on cell membranes, cleaves GLP-1 (7-36) peptide to yield the inactive GLP-1 (9-36) form. Therefore, many modifications have been made to GLP-1 to increase its biological half-life and consequently its efficacy in vivo. Exendin 4 (Ex-4, also called exenatide), a GLP-1R agonist is now available for treating type 2 diabetes mellitus (T2DM). This compound is synthesized in the salivary glands of the Heloderma suspectum or Gila monster lizard, native to Gila county in southern Arizona. Ex-4 does not possess the DPP-IV recognition site and is a potent insulinotropic agent. Another component of Gila monster saliva, exendin 9-39 (Ex-(9-39)) is an antagonist at the GLP-1R (Montrose-Rafizadeh, Yang et al., 1997) and, thus, has been useful in determining specificity of effects at this receptor in mechanistic studies.

Both acute and chronic treatment with GLP-1 and GLP-1R agonists are known to increase insulin secretion and decrease plasma glucose levels in T2DM. Their long-term effects on rodent β cells leading to increased β cell mass through increased β cell proliferation and differentiation in both nondiabetic and diabetic animals have also been extensively studied. However, given the current technical difficulties in assessing human islet mass, the latter properties of the compounds cannot be confirmed in humans.

Many aspects of GLP-1 biology remain unresolved. Here, we address a number of those issues including the evidence in the literature for GLP-1 expression in specific cell types of the pancreas, the downstream signaling of the GLP-1R in those cells, and the controversial link between intestinal dumping of food and hypersecretion of GLP-1 resulting in pathological overgrowth of islet β cells, as a postoperative complication in gastric bypass surgery. Another major issue surrounding the mechanism of action of GLP-1 on β cells is the importance of protein kinase A (PKA) versus other cAMP signaling pathways i.e., Epac (exchange protein associated with cAMP, also known as guanine nucleotide exchange factor [GEF]). Additionally, and most exciting to investigators in the field, as research on GLP-1 actions increases, many nondiabetologists are applying their sophisticated techniques to examine the molecular events consequent upon GLP-1R activation in β cells, and this has led to many interesting findings that we will cover in this review.

Here, we provide a comprehensive review of what is known to date of the molecular events consequent upon GLP-1R activation in the cells of the pancreas.

Section snippets

GLP-1R in the pancreas

GLP-1R is a specific 7-transmembrane receptor guanine nucleotide-binding protein (G-protein) coupled receptor (GPCR). It was first cloned from rat pancreatic islets (Thorens, 1992) and later from a human pancreatic insulinoma (Dillon et al., 1993, Thorens et al., 1993) and a gut tumor cell line (Graziano et al., 1993). The rat and human GLP-1R exhibit a 95% amino acid homology and are 90% identical (Thorens, 1992, Thorens et al., 1993), differing at 42 amino acid positions (Tibaduiza et al.,

Stimulation of cAMP production

The GLP-1R is coupled to the Gsα subunit and therefore agonist engagement with the receptor results in activation of AC with consequent production of cAMP (Drucker et al., 1987). At least 9 different mammalian membrane-bound isoforms of AC (AC I–AC IX) are known to exist (Hanoune & Defer, 2001). Leech and coworkers performed RT-PCR on extracts from whole rat and human islets showing that AC III, AC IV, AC V, AC VI and AC VII were present in rat islets and AC V and AC VI and were found in human

Glucose-induced insulin secretion

When blood glucose increases postprandially, it equilibrates across the membrane of the β cell through GLUT2 and GLUT1 transporters. It is rapidly phosphorylated to glucose 6-phosphate by glucokinase, which thereafter determines the rate of glycolysis that acts as the glucose sensor and pyruvate generation for entry into the tricarboxylic acid (TCA) cycle in mitochondria. Subsequent oxidative metabolism provides the link between the products of glucose metabolism and insulin secretion. The

Chronic effects of GLP-1 on insulin synthesis and secretion

Drucker et al. (1987) initially demonstrated the effect of GLP-1 on increasing insulin mRNA levels in 1987. In 1992 Fehmann and Habener showed that GLP-1 (10 nM) treatment induced the proinsulin gene using a chloramphenicol-acetyltransferase (CAT) reporter gene assay, and it increased insulin mRNA levels and insulin content in the βTC-1 cell line following 24 hr of treatment (Fehmann & Habener, 1992). In 1995 it was shown that prolonged treatment of rat insulinoma cells with GLP-1 (1 or 10 nM

Regulation of β cell mass

β cell mass is regulated by a balance between β cell proliferation and death. Islet neogenesis is a controversial subject as there is no direct evidence for the existence of a specific pancreatic endocrine stem cell and we reserve discussion of this topic for Section 6.2.1. Studies in rodents and humans have and continue to illustrate that the incretin hormones play a central role in the homeostasis of pancreatic β cell mass as well as function and that these 2 parameters are closely

Effects on glucagon secretion: are they direct or indirect?

As already explained above (Section 2) there is controversy about the presence of GLP-1Rs on α cells and if present, they are on but a few cells. The functional assays examining effects of GLP-1 on α cells vary. Heller and Aponte (1995) performed dose–response analysis of GLP-1 treatment of whole islets and did not see any increase in glucagon secretion. Moens et al. (1996) also failed to elicit cAMP production in rat α cells in response to 1 nM GLP-1, but it should be remembered that it can be

Exocrine pancreatic secretion

Fehmann and colleagues were the first to study the effect of GLP-1 on pancreatic acinar secretions (Fehmann et al., 1990). They examined the synergistic action of GLP-1 (10 pM) and cholecystokinin-8 (CCK-8, 1 nM to 1 pM) on isolated rat pancreatic acini and found that GLP-1 had no impact on CCK-induced amylase secretion. Eng and colleagues performed a dose response curve and found that both Ex-4 and GLP-1 could increase cAMP levels in dispersed guinea pig acini but did not actually increase

GLP-1R−/− mice

GLP-1R−/− mice display abnormally high blood glucose levels after an intraperitoneal glucose challenge demonstrating that GLP-1 is important for clearance of the glucose load, irrespective of the site of glucose entry into the circulation (Scrocchi et al., 1996). As anticipated from the known actions of GLP-1, they also exhibit mild fasting hyperglycemia and glucose intolerance after oral glucose that is associated with reduced glucose-stimulated insulin secretion. Despite evidence that

GLP-1 as an insulinotropic agent

In 1986 and 1987 GLP-1 was shown to have insulinotropic properties in rodents (Holst et al., 1986, Mojsov et al., 1987) and there was little if any doubt about its potency from these early experiments. The first human experiments were performed by Bloom and co-workers in 1987 (Kreymann et al., 1987). They showed that infusing GLP-1 intravenously so as to reach plasma concentrations in the apparently physiological range lead to increased glucose-dependent insulin secretion. This

Summary

GLP-1R activation has many beneficial effects on acute insulin secretion and the maintenance of correct β cell glucose sensing, transcriptional synthesis, proliferation and survival. This is most likely due to the activation and integration of multiple pathways consequent upon engagement of GLP-1 agonists with the receptor. Therefore for clinicians the use of GLP-1R agonists would seem to be the perfect treatment for chronic β cell failure in T2DM. However there is as yet only 1 GLP-1R agonist,

Acknowledgments

MED would like to thank Ammon B. Peck. JME would like to thank Byung-Joon Kim and Olga D. Carlson for Fig. 5 data and Tina Roberson for editorial assistance. JME was supported by the Intramural Research program of the NIH, National Institute on Aging. The author have no conflict of interest in the writing of this review.

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