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Glucagon-like Peptide-1 & Exendin
by: Suleiman Al-Sabah

Glucagon-like peptide-1 (GLP-1) is an endogenous peptide secreted into the blood stream by intestinal L cells. It is the most potent glucose incretin yet discovered. That is, it potentiates insulin secretion by pancreatic â-cells in a glucose-dependant manner (Kieffer & Habener, 1999).

What is an Incretin?

The incretin concept was introduced in 1930 to account for the observation that an oral dose of glucose resulted in a larger incremental increase in insulin secretion than an equal dose of glucose administered intravenously (Brubaker & Drucker, 2000). Unger & Creutzdeldt further refined the definition of an incretin as a gut hormone released in response to nutrient ingestion that stimulates glucose-dependent insulin secretion (Drucker, 2001). Glucose-dependent insulinotropic peptide (GIP) was the first incretin to be identified. In 1987 the second major incretin was discovered, and it was named GLP-1 (Ørskov & Nielson 1988). It is now known that together GIP and GLP-1 contribute equally to the incretin effect. GIP and GLP-1’s dependence on glucose to potentiate insulin secretion is an effective means for protection against hypoglycaemia. What makes GLP-1 a more attractive therapeutic agent however is that, unlike GIP, it can stimulate insulin secretion in subjects with type-2 diabetes.

A Bit More About GLP-1

GLP-1 secretion is regulated primarily by nutrients. Both carbohydrates and fats have been shown to stimulate GLP-1 secretion (Drucker, 2001). Recently free fatty acid activation of GPR120, a G protein?coupled receptor abundantly expressed in the intestines, has been shown to promote the secretion of GLP-1 both in vitro and in vivo (Hirasawa et al., 2005). GLP-1 release is biphasic in nature with a rapid response mediated by humoral and neural mechanisms and a delayed response stimulated by direct nutrient contact with intestinal L cells.

GLP-1 is a product of tissue specific post-translational proglucogon. Other peptide hormones derived from proglucagon include glucagon in the pancreas and oxyntomodulin, GLP-1 and GLP-2 in the intestines and brain. GLP-1 is inactive until cleaved further to yield GLP?1 (7-37) and GLP-1 (7-36) amide. Both these peptides are equipotent, however 90% of circulating active peptide is GLP?1 (7?36) amide (Drucker, 2001). For the purposes of this article the term GLP-1 will refer to the active peptide.

GLP-1 is degraded by N-terminal cleavage of the peptide’s first two amino acid residues (His1-Ala2) by dipeptidyl peptidase IV (DPP IV), and has a half-life of around 2 minutes (Kieffer et al., 1995). The cleaved product may have a relevant physiological function as an antagonist at the GLP-1 receptor (GLP-1R) (Wettergren et al., 1998).

GLP-1 is a member of the glucagons/PACAP family of homologous peptide hormones. This superfamily of peptide hormones has the honor of including secretin, which was the first substance to be identified as a hormone. The glucagons/PACAP family of peptides all function as hormones and neuropeptides and probably evolved from a single ancestral gene. A review of the glucagon/PACAP super-family is given in Sherwood et al. (2000).

Interestingly, in bony fish GLP-1 acts in a similar way to glucagon, stimulating glycogenolysis and gluconeogenesis in the liver (Mojsov, 2000). This suggests that GLP-1 only evolved into an incretin hormone after the divergence of bony fish and mammals.

Signalling in Pancreatic â-cells

Insulin secretion by pancreatic â-cells is biphasic in nature. The first sharp release is detectable within minutes following nutrient ingestion. An increase in glucose uptake by pancreatic â-cells via GLUT2 glucose transporters leads to an increase in glycolysis and subsequently an increase in the intracellular ATP:ADP ratio. This in turn causes the closure of ATP-sensitive K+ channels (KATP). Membrane depolarisation leads to an opening of L-type voltage?dependent calcium channels (VDCCs). An increase in intracellular calcium stimulates translocation and exocytosis of insulin-containing secretory granules (Ahérn, 1998). The events that mediate the second more sustained insulin release are less clearly understood, but intracellular calcium is involved, as is cAMP (Gromada et al., 1998).

Activation of GLP-1R activates Gs which stimulates adenylate cyclase and results in an increase in intracellular cAMP (Bode et al., 1999). This rise in cAMP levels activates protein kinase A (PKA). It has been shown that inhibition of PKA impairs the ability of GLP-1 to potentiate insulin secretion. This indicates that phosphorylation events mediated by PKA are an essential step in the stimulus/secretion coupling (Holz et al., 1999). GLP-1 also potentiates glucose-induced inhibition of KATP channels, which leads to the depolarisation of the cell membrane, activation of VDCC and in increase in intracellular calcium (Holz et al., 1993). There are clearly other signalling pathways involved that have yet to be determined, and some recent work has implicated nitric oxide.

GLP-1 and the Treatment of Type-2 Diabetes

As I’m sure most readers are aware diabetes mellitus is rapidly becoming one of this century’s largest threats to human health. Global figures for people suffering the disease are set to rise from an estimated value of 150 million in 2001 to 220 million in 2010 and 300 million in 2025 (Zimmet et al., 2001).

The ability of GLP-1 to lower blood glucose and promote satiety makes it ideal to alleviate the type 2 diabetic phenotype. Unlike sulphonylurea treatment, GLP-1 treatment does not result in the side effects of hypoglycaemia or weight gain. The therapeutic potential of GLP-1 has been demonstrated in diabetic patients (Moller, 2001) but it is not without its drawbacks. Being a peptide, GLP-1 has to be injected (which is inconvenient) and it is rapidly degraded by DPP IV. The use of DPP IV inhibitors has been shown to result in an increase in circulating GLP-1 levels in clinical trials (Holst & Deacon, 1998). However other peptides such as GLP-2, PACAP, GIP, GNRH and NPY are also metabolised by DPP IV, so the use of DPP IV inhibitors is likely to result in a host of other physiological consequences.

Enter the Gila Monster

The venom of the Gila monster (Heloderma suspectum) contains a family of peptides known as the exendins. They include helospectin-1, helospectin-2, helodermin, exendin-3 and exendin-4. Helodermin shares 53% sequence identity with PACAP and 42% with VIP, and is an agonist at the VIP and secretin receptors. exendin-3 and exendin–4 are GLP-1 receptor agonists (Goke et al., 1997). The gene that codes exendin-4 in the Gila monster is distinct from that which codes GLP-1 and there are no mammalian homologues of these reptilian peptides (Raufman, 1996). Exendin was named by Eng et al. because it is an exocrine product with endocrine actions (Eng et al., 1990).

GLP-1 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGREx-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

Figure 1.
A sequence alignment of GLP-1 and exendin-4. Residues shown in bold are conserved in relation to GLP-1.

The N-terminal region of GLP-1 and exendin-4 (Ex-4) are highly conserved (Figure 1) and it is this similarity that makes Ex-4 a GLP-1 receptor agonist. The single amino acid substitution of Ala2 to Gly makes Ex-4 several orders of magnitude more resistant to DPP IV digestion compared to GLP-1 (Thum et al., 2002). This increased resistance to DPP IV makes Ex-4 more potent than GLP-1 in vivo (Drucker, 2001). As a result, Ex-4 is a good candidate for the treatment of type 2 diabetes.

It is not known why the Gila monster injects an agent that glucose-dependently potentiates insulin secretion and reduces appetite into its victims. One theory is that Ex-4 actually acts in the Gila monster itself. Another theory is that any potential predator that decides to have a Gila monster for lunch will find that it is not hungry once it has been bitten by the Gila monster.

Conclusion

Liraglutide (Novo Nordisk) is a DPP IV resistant analogue of GLP-1 intended for once-daily administration and is now entering Phase 3 clinical trials. Exanatide (Amylin Pharmaceuticals Inc.) is synthetic Ex-4. Phase 3 clinical trials were completed in November 2003. Both these compounds have potential in the treatment of type-2 diabetes, however both compounds require subcutaneous injection. The race is on for a non-peptide GLP-1 receptor agonist that is orally active.

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