Glucagon-like peptides 1 and 2 in health and disease: A review

Peptides 44 (2013) 75–86

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Peptides journal homepage: www.elsevier.com/locate/peptides

Review

Glucagon-like peptides 1 and 2 in health and disease: A review Chinmay S. Marathe a,b,∗ , Christopher K. Rayner a,b , Karen L. Jones a,b , Michael Horowitz a,b a b

Discipline of Medicine, University of Adelaide, Royal Adelaide Hospital, Adelaide, Australia Centre of Research Excellence in Translating Nutritional Science to Good Health, University of Adelaide, Adelaide, Australia

a r t i c l e

i n f o

Article history: Received 21 January 2013 Received in revised form 30 January 2013 Accepted 30 January 2013 Available online 20 March 2013 Keywords: Glucagon-like peptides Postprandial glycemia Incretin secretion Gastric emptying

a b s t r a c t The gut derived peptides, glucagon-like peptides 1 and 2 (GLP-1 and GLP-2), are secreted following nutrient ingestion. GLP-1 and another gut peptide, glucose-dependent insulinotropic polypeptide (GIP) are collectively referred to as ‘incretin’ hormones, and play an important role in glucose homeostasis. Incretin secretion shares a complex interdependent relationship with both postprandial glycemia and the rate of gastric emptying. GLP-1 based therapies are now well established in the management of type 2 diabetes, while recent literature has suggested potential applications to treat obesity and protect against cardiovascular and neurological disease. The mechanism of action of GLP-2 is not well understood, but it shows promise as an intestinotropic agent. © 2013 Elsevier Inc. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discovery of glucagon-like peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Precursor gene and receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Secretion of GLP-1 and GLP-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. GLP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. The incretin effect in type 2 diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. GLP-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Metabolism of GLP-1 and GLP-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Actions of GLP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Effects on pancreatic beta and alpha cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Effects on gastrointestinal motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Appetite regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Therapeutic applications of GLP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Type 2 diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1. GLP-1 analogs: efficacy in type 2 diabetes and tachyphylaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.2. DPP-IV inhibitors: role in type 2 diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Cardiovascular disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.4. Neurological diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5. Bone metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Actions of GLP-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Therapeutic applications of GLP-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. Safety of GLP-1 and GLP-2 based therapies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Discipline of Medicine, Royal Adelaide Hospital, Adelaide 5000, Australia. Tel.: +61 431266075. E-mail address: [email protected] (C.S. Marathe). 0196-9781/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2013.01.014

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1. Introduction Glucagon-like peptides 1 and 2 (GLP-1 and GLP-2) are gutderived peptides secreted from specialized entero-endocrine ‘L’ cells located predominantly in the distal small intestine and colon, following exposure to ingested nutrients. They share with glucagon a common precursor molecule, proglucagon, but exhibit a diverse range of functions, both complementary and divergent. GLP-1 was discovered only 30 years ago, but its actions are now exploited in the management of type 2 diabetes, with a number of GLP-1 based drugs on the market. Apart from its insulinotropic and glucagonostatic properties, GLP-1 influences the function of multiple organ systems; chief among these are effects on gastrointestinal motility, and likely cardio-protective and neuro-protective effects. GLP-2, on the other hand, has intestinotropic properties and is likely to be useful in the management of chronic intestinal disorders such as short bowel syndrome and possibly inflammatory bowel disease. This review provides an overview of various aspects of these important gut peptides and summarizes their role in health and disease.

secretion at physiological doses, and it was renamed ‘glucosedependent insulinotropic polypeptide’ [37]. GIP can be therefore considered the first known ‘incretin’ hormone [5]. In 1983, Ebert et al. showed persistence of more than 50% of the incretin effect in rats after complete removal of GIP by radioadsorption, and therefore predicted the existence of additional insulinotropic factors derived from the gut [38]. Around this time, with the aid of gene sequencing techniques, Lund et al. [82] and Bell et al. [9] sequenced the proglucagon gene in anglerfish and humans respectively; this gene was shown to encode not only the 29 amino acid peptide, glucagon, but also other smaller peptides, including glicentin, oxyntomodulin, intervening peptides and, importantly, two additional sequences bearing 50% homology to glucagon. These were named glucagon-like peptides 1 and 2 (GLP-1 and GLP-2). GLP-1 was shown to have considerable insulinotropic activity at physiological levels [69,131], and thus became the second known incretin. GLP-2 does not have insulinotropic activity [131], but has generated substantial interest as an intestinal growth factor [35].

3. Precursor gene and receptors 2. Discovery of glucagon-like peptides The discovery of glucagon-like peptides 1 and 2 (GLP-1 and GLP-2) was the result of a deliberate and protracted search to characterize the gastrointestinal factors involved in blood glucose homeostasis, and capitalized on scientific advances, including radioimmunoassays and gene sequencing. The work of Bayliss and Starling [7], Moore and Edie [39], and others in the early 1900s had hinted that factors derived from the intestinal mucosa played a role in determining glycemic control in both health and diabetes. La Barre reported in 1932 that an extract from duodenal mucosa lowered the blood glucose concentration in rabbits and dogs when given orally [70], although such an outcome is difficult to explain on the basis of current knowledge, given that peptides are rendered ineffective when given by mouth. He named the substance, ‘incrétine’ or ‘incretin’. In the subsequent decades, the role of the gastrointestinal tract in regulating glucose homeostasis was seriously undermined by the work of influential American researcher Ivy and co-workers [26]. They reported that infusing duodenal extracts intravenously or stimulating the small intestine with hydrochloric acid did not lower blood glucose concentrations in fasting animals [76–78]. However, in discrediting the ‘incretin theory’, they failed to appreciate a number of characteristics of an ‘incretin’ which were subsequently defined by Creutzfeldt [25] as: (1) secretion following ingestion of nutrients, especially glucose; (2) glucose-dependency, i.e. the glucose-lowering activity of the incretins persists only when blood glucose levels are elevated; and (3) that glucose-lowering by stimulation of insulin takes place at physiological concentrations achieved following ingestion of nutrients. By the 1960s, it became possible to measure circulating insulin concentrations due to the development of specific and sensitive radioimmunoassay (RIA) techniques. In 1964, Mcintyre et al. [87] and Elrick et al. [42], working independently, renewed interest in the incretin concept by reporting that an oral glucose load considerably amplified the plasma insulin response, when compared to an intravenous glucose infusion that resulted in comparable blood glucose concentrations. This phenomenon is known as the ‘incretin effect’. In 1970, Brown and co-workers published a paper on the enterogastrone (i.e. inhibitory to gastric secretion and motility) properties of a gut-based peptide at supraphysiological levels, which they named ‘gastric inhibitory polypeptide’ (GIP) [15]. However, it was soon recognized that GIP had the capacity to stimulate insulin

A single gene, the preproglucagon gene (Gcg), is responsible for the production of glucagon and glucagon-like peptides in mammals. It belongs to the group of genes encoding the hormones of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily – a group of nine bioactive peptides that also includes GIP and secretin. Gcg is located on the long arm of chromosome 2 (2q36-q37) and consists of six exon regions, with exons 3, 4 and 5 encoding for glucagon, GLP-1 and GLP-2 respectively [132]. Gcg, is thought to have undergone repeated exon duplication during mammalian evolution – it has been suggested that the origin of glucagon dates from some one billion years ago, while diversification 700 million years ago resulted in GLP-1 and GLP-2 [62]. Expression of Gcg is evident in a number of organs including the pancreas (alpha cells of the islets of Langerhans), intestine (entero-endocrine L cells, especially in the distal small intestine and the colon), and the central nervous system (caudal brainstem and hypothalamus) [32,96]. Transcription of Gcg results in a common messenger RNA (mRNA) for the three peptides [5]. Translation of the common mRNA results in a 180 amino acid precursor, proglucagon [152]. It is, however, at the posttranslational stage that diversification takes place, resulting in organ-specific peptide profiles – while glucagon is the major peptide produced from proglucagon in the pancreas, glucagon-like peptides are produced in the intestine and brain. Prohormone convertase (PC) enzymes are responsible for this differential peptide profile [12,123]. In the pancreatic alpha cells, the enzyme PC 2 produces the end-products glucagon [124], intervening peptide1, glicentin-related polypeptide, and major proglucagon fragment. While glucagon plays a well-described role in glucose homeostasis, none of the other peptides has a known role in human physiology. On the other hand, PC 1 and 3 acting in the intestinal L cells and in the central nervous system respectively result in the products GLP-1, GLP-2, intervening peptide 2 (IP-2), glicentin and oxyntomodulin [123]. Oxyntomodulin suppresses appetite and leads to weight loss in humans, and is capable of increasing the intrinsic heart rate in rats [136]. While glicentin has intestinotropic effects in rodents [99], there is no known physiological role for IP-2 (see Fig. 1). A recent study found increased PC 2 positive cells in intestinal biopsies of type 2 diabetes patients compared with healthy controls [67]. The authors have suggested that the gut, particularly in patients with type 2 diabetes, could be an additional source of glucagon aside from the pancreas. This finding needs further

C.S. Marathe et al. / Peptides 44 (2013) 75–86

PROGLUCAGON GENE

77

Long Arm Chromosome 2

Transcription

PROGLUCAGON mRNA Translation

PROGLUCAGON

180 Amino Acid Precursor Protein

Tissue-specific post-translational processing

Pancreas

Intestine PC 1/3

PC 2

GRPP

Glucagon

IP-1

IP-2

Increases:

• Gluconeogenesis • Glycogenolysis • Lipolysis

GLP-1

GLP-2

• Insulinotropic • Glucagonostatic • Slows gastric emptying • Neuro-protective Cardio -protective •

• Intestinotropic • Glucagonotropic gastric • Slows emptying in

Glicentin

• Intestinotropic

rodents

Oxyntomodulin

• Suppresses appetite • Increases

intrinsic heart rate in rodents

Fig. 1. Simplified diagram illustrating the pleiotropy of the proglucagon gene: a number of peptides, GLP-1, GLP-2 and glucagon share a common precursor gene. Aside from the diversity of functions, these peptide products at times have counteracting actions. For example, glucagon and GLP-1 have opposing effects on blood glucose levels and while GLP-1 has glucagonostatic functions, GLP-2 may increase glucagon levels. IP-1 and IP-2 have no known physiological function. GRPP: glicentin-related polypeptide; IP-1 and IP-2: intervening peptide-1 and 2; PC 2: prohormone convertase enzyme 2; PC 1/3: prohormone convertase enzyme 1/3.

confirmation but challenges the traditional view that alpha cells are the only source of glucagon. Our understanding of proglucagon gene expression and regulation is limited to rodent models, and information about the human proglucagon gene has been obtained indirectly from transgenic mice and transfected rodent cell lines [5]. Rodent proglucagon gene expression in the pancreas is up-regulated during fasting and hypoglycemia, and down-regulated by insulin [5]. Conversely, intestinal proglucagon gene expression is reduced during fasting and increases with re-feeding [59]. The level of intracellular cAMP and activation of cAMP/PKA signaling are important determinants of both pancreatic and intestinal glucagon gene expression in rats [33], while transcription factor Pax-6 also appears to be essential [128,146]. Studies in transgenic mice indicate that 1.3 kilobase (kb) of rat proglucagon gene 5 flanking sequences are sufficient to direct pancreatic ␣ cell and brain rat glucagon gene expression [41], but ∼2.3 kb of proglucagon gene 5 flanking sequences are required for combined pancreatic ␣ cell, brain and intestinal rat glucagon gene expression [71], suggesting DNA sequences between −2.3 kb and −1.3 kb in the rat glucagon promotor are important for intestinal proglucagon gene expression in rats [5]. The factors responsible for human proglucagon gene expression are not clear. In a transgenic mouse model, 1.6 kb of human proglucagon gene 5 flanking sequences are sufficient to direct brain and intestinal glucagon gene expression [110] and sequences within 6 kb of the human proglucagon gene 5 flanking region were seen to be required for pancreatic expression when transfection techniques were employed [110]. More recently, it has become clear that a region in intron 1, named ‘evolutionary conserved noncoding region 3’ (ECR 3) is crucial for human pancreatic proglucagon expression [161]. Glucagon-like peptides bind to their respective receptors in a highly specific manner. The GLP-1 receptor (GLP-1 R) [141]

and GLP-2 receptor (GLP-2 R) [98] were both cloned from cDNA libraries using the technique of expression cloning. Both are classified as ‘family B’ G-protein coupled receptors (GCPR) and belong to the PACAP/glucagon receptor superfamily [31]. The GLP-1 R is expressed in multiple tissues, including the intestine, pancreas, central nervous system (CNS), lung, heart, kidneys, stomach and vagus nerve. The GLP-2 R, cloned from the intestinal and hypothalamic cDNA libraries, shows significant homology with GLP-1 R, but its expression is largely restricted to the intestine, with some exceptions, including the lungs and hypothalamus [126,159]. In the intestine, GLP-2 R expression is evident in a variety of cells including intestinal subepithelial myofibroblasts (ISEMF), enteroendocrine cells and enteric neurons [126]. Although the main function of GLP-2 is believed to be as an intestinal growth factor, GLP-2 receptor expression, perhaps surprisingly, is not seen on the proliferating epithelial crypt cells. It is believed, therefore, that GLP-2 may have indirect actions on intestinal growth through downstream mediators such as IGF-1 and ErbB ligand. 4. Secretion of GLP-1 and GLP-2 GLP-1 and GLP-2 are secreted by the intestinal L cells in response to nutrient ingestion. The L cell is a specialized enteroendocrine cell with maximum density in the ileum and colon, although L cells are present more proximally in the small intestine, including the jejunum and even duodenum [140]. The L cell is an open-type triangular epithelial cell; the basal side connects with the neuro-vasculature and the apical end incorporates cytoplasmic projections that are in direct contact with the intestinal lumen, providing the potential for luminal nutrients to influence GLP-1 and GLP-2 secretion [53]. A number of signaling mechanisms including ATP-sensitive potassium channel closure, sodium-glucose co-transporter-1, and activation of ‘sweet taste’

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receptors, have been postulated to account for glucose-sensing by L cells [156]. 4.1. GLP-1 Carbohydrates and fat are more potent stimuli for GLP-1 release than proteins, and in combination, they have an additive effect on insulin secretion, at least in animal studies [156]. The pattern of GLP-1 secretion also varies with the properties of carbohydrates, including their glycemic index; carbohydrates that are more slowly digested are likely to be exposed to more distal gut regions, with correspondingly later, but potentially more substantial, GLP-1 secretion [157]. GLP-1 secretion following a meal is bi-phasic with a shorter early phase beginning 10–15 min after ingestion, and a subsequent more sustained phase (from 30 to 60 min) [52]. The mechanism of ‘early’ GLP-1 release remains controversial, but it is possible that stimuli other than direct nutrient exposure to L cells may have a role, including neurotransmitters, gut peptides, and the vagus nerve [52,121]. There is substantial variation between species – for example, GIP has been shown to trigger GLP-1 secretion in rats [120], but not in humans [104]. Moreover, in a study looking at the effect of length of small intestine exposure on incretin secretion, GLP-1 was stimulated only when luminal glucose was allowed to access more than the proximal 60 cm of small intestine [75]. In contrast, there is evidence that the proximal small intestine may contain sufficient L cells to explain the early phase of GLP-1 release after direct contact with luminal nutrients [140]. The rate of gastric emptying also appears to influence GLP-1 secretion. Gastric emptying accounts for a third of the variance in the postprandial blood glucose response in both health and type 2 diabetes, since the stomach tightly regulates the entry of nutrients into the small intestine, where carbohydrates are absorbed. There is a relatively high inter-individual variation (between 1 and 4 kcal/min) [14], but low intra-individual variation [24], in the rate of gastric emptying in health. Patients with type 1 or type 2 diabetes are likely to display an even wider inter-individual range, with a high proportion having abnormally slow emptying, and a few emptying at rapid rates [58,65]. There an inverse relationship between postprandial GLP-1 levels and the rate of gastric emptying of an oral glucose load [154], since one of the actions of GLP-1 is to slow gastric emptying (discussed in more detail subsequently). In contrast, GIP levels have a direct relationship with the early rate of gastric emptying. The relationship between GLP-2 secretion and gastric emptying is not clear, but is likely to be similar to that of GLP-1. In studies employing intraduodenal infusions of glucose at varying rates within the physiological range, GLP-1 secretion was much greater during infusions at 4 kcal/min than at 1 or 2 kcal/min (which had comparable increases) in both health and type 2 diabetes [83,117] (see Fig. 2). This was in contrast with GIP levels, which increased steadily with increasing intraduodenal glucose loads, suggesting that the contribution of GLP-1 to the incretin effect increases at higher rates of small intestinal nutrient delivery. 4.1.1. The incretin effect in type 2 diabetes The incretin effect accounts for between 20 and 60% of total postprandial insulin secretion in health [92], with the magnitude of the incretin effect increasing with the size of the oral glucose load [105], which makes intuitive sense for limiting postprandial glycemic excursions [4]. However, the incretin effect is impaired in type 2 diabetes [101]. GLP-1 secretion after a mixed meal has been reported to be deficient in type 2 diabetes when compared to health [144], but this might be accounted for by differences in the rate of gastric emptying, and a number of subsequent studies have failed to show any reduction in GLP-1 levels [108,150]. Moreover, GIP concentrations are either normal or elevated in type 2

diabetes [64,122]. GLP-1 retains its insulinotropic actions in type 2 patients, but the action of GIP is largely abolished, and the latter phenomenon is now generally believed to account for the impairment in the incretin effect [102]. 4.2. GLP-2 GLP-2 is also secreted from the L cells, by mechanisms that appear similar to those that stimulate GLP-1 [125]. The resulting biphasic secretory pattern of GLP-2 in humans is reported to peak initially at 30–60 min and again at 90–120 min [158]. GLP-1 and GLP-2 are released from the L cell in a 1:1 ratio [119], so the slightly delayed postprandial peaks in GLP-2 compared to GLP-1 remain to be explained. 5. Metabolism of GLP-1 and GLP-2 GLP-1 and GLP-2, like other members of the PACAP/glucagon superfamily, show significant homology at the N-Terminus in that they contain either alanine or proline residues. This makes them highly susceptible to degradation by a serine protease, dipeptidyl peptidase-IV, also known as CD 26 [95]. DPP-IV is distributed widely, with the greatest concentrations being found in the kidneys, liver, and at the brush border of enterocytes [95]. Importantly, however, DPP-IV is present on the endothelium of the entire vascular bed [79]. The presence of DPPIV on the endothelium of capillaries adjacent to the L cells [47] means that about a quarter of GLP-1 is inactivated before it leaves the gut [29], while another large fraction is degraded by the liver, so that only 10–15% of intact GLP-1 reaches the systemic circulation [52]. GLP-1 circulates mostly in its truncated form (GLP-1 (7,36)), although an equipotent form GLP-1 (7–37) also exists [114]. GLP-1 inactivation by DPP-IV in the systemic circulation is remarkably rapid, such that the half-life of intact GLP-1 is 1–2 min. The inactive metabolites, GLP-1 (9–36) or (9–37), are then rapidly cleared by the kidneys [92]. GLP-1 may also be susceptible to degradation by neutral endopeptidase 24.11 (NEP 24.11) [60], and in animal studies inhibition of both DPP-IV and NEP 24.11 improves the stability of intact GLP-1 [118]. The degradation of GLP-2 by DPP-IV is a little slower than for GLP-1, with a half-life of about 7 min, so that a higher proportion of intact GLP-2 reaches the systemic circulation. Like GLP-1, GLP-2 is cleared rapidly via the kidneys [49,53,115]. 6. Actions of GLP-1 Studies exploring the actions of GLP-1 have either examined the effects of exogenous GLP-1 infused at high (‘pharmacological’) doses, or have employed lower doses that simulate ‘physiological’ systemic concentrations. However, a more valid insight into the physiological, as opposed to pharmacological, actions of GLP-1 can be gained from studies utilizing the GLP-1 receptor antagonist, exendin (9–39) amide. 6.1. Effects on pancreatic beta and alpha cells GLP-1 stimulates insulin secretion in pancreatic beta cells in a blood glucose concentration-dependent manner. GLP-1 binds to GLP-1 receptors on beta cells, leading to a rise in cAMP levels, and resulting in insulin secretion by protein kinase A-dependent and -independent mechanisms. GLP-1 also enhances mRNA stability and insulin transcription, the latter through up-regulation of the transcription factor pancreas duodenal homeobox-1 (Pdx-1) and nuclear factor of activated T cells (NFAT) [151]. GLP-1 improves glucose sensitivity in glucose-resistant beta cells [56], possibly by

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Fig. 2. Blood glucose (A), plasma insulin (B), plasma glucagon-like peptide-1 (GLP-1) (C) and plasma glucose-dependent insulinotropic polypeptide (GIP) (D) concentrations in response to a 120-min intraduodenal glucose infusion at 1 kcal/min (G1), 2 kcal/min (G2), 4 kcal/min (G4) or (iv) saline control (S) in 10 healthy subjects and eight patients with type 2 diabetes. Data are mean ± SEM. *P < 0.05 vs. control, # P < 0.05 vs. G1, § P < 0.05 vs. G2. Reprinted with permission from Ma et al. [83].

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Table 1 Summary of motor effects of GLP-1 and incretin-based therapies on the gastrointestinal tract.

Endogenous GLP-1 (physiological dose) Exogenous GLP-1 (pharmacological dose) GLP-1 receptor agonists (e.g., exenatide, liraglutide) DPP-4 inhibitors (e.g., sitagliptin, vildagliptin)

Gastric motility (delayed gastric emptying)

Small intestinal motility (delayed small intestinal transit)

Large intestinal motility (delayed colonic transit)

One positive study [30]

No studies available

No studies available

Strong evidence in human studies; healthy [103], obese [96], type 2 diabetic [84] Strong evidence with exenatide [11,33,70]. Some evidence with liraglutide [60] Negative human studies [132]

Positive evidence in animal studies [13,42]. Positive effect on fasting motility in humans [49] No studies available

Positive evidence in animal studies [43]. Only indirect evidence in humans [17] No studies available

No studies available

No studies available

Reproduced with permission from Marathe et al. [85].

up-regulating the expression of glucokinases and glucose transporters. In animal studies, GLP-1 has been shown to delay or reverse the progressive loss of beta cells which is characteristic of type 2 diabetes, by inhibiting apoptosis and stimulating their proliferation [92]. GLP-1 receptor knockout mice show impaired regeneration of beta cells and increased vulnerability to streptozotocin-induced apoptosis [73]. Low concentrations of GLP-1 suppress glucagon secretion when brought in contact with isolated perfused porcine pancreas [69]. Exogenous GLP-1 in both ‘high’ (pharmacological) and ‘low’ (physiological) doses has been shown to suppress glucagon in health [61], while exendin (9–39) elevates glucagon concentrations [40], suggesting that suppression of glucagon is a physiological action of endogenous GLP-1. It appears that the glucagonostatic properties of GLP-1 are preserved in type 2 diabetes [48] in both physiological and pharmacological concentrations. However, the mechanism by which GLP-1 suppresses glucagon remains controversial. GLP-1 receptor expression in the alpha cells of pancreas is considerably less than for beta cells – indeed, some investigators have failed to find any GLP-R expression [55] suggesting that the inhibitory action of GLP-1 might be indirect. In rodents, GLP-1 stimulates somatostatin secretion from pancreatic delta cells, which in turn can inhibit alpha cells [55]. The ‘intra-islet hypothesis’ proposes that the pancreatic vasculature is arranged so that the alpha cells in the mantle are supplied by blood already exposed to insulin from beta cells in the core. Postprandial hyperglucagonemia is common in type 2 diabetes [72] and this may, in part, be attributable to a defect in intra-islet postprandial glucagon suppression by insulin [91]. However, the ‘intra-islet’ theory has not been consistently supported in animal models [55]. Moreover, recent studies have shown that GLP1 can suppress glucagon even in patients with type 1 diabetes who have no residual beta cell activity [66]. In contrast to GLP-1, GIP has the capacity to stimulate glucagon in both health [89] and type 2 diabetes [81].

6.2. Effects on gastrointestinal motility Besides being an incretin, GLP-1 is also regarded as an enterogastrone because of its inhibitory effect on gastrointestinal motility and gastric acid secretion. It is well established that exogenous GLP-1 slows gastric emptying in both healthy subjects and patients with type 2 diabetes in a dose-dependent manner [88], and as a consequence, is associated with a decrease in, rather than stimulation of, postprandial insulin and C peptide concentrations, i.e. the slowing of gastric emptying induced by GLP-1 can outweigh its insulinotropic actions [107]. Reversal of the delay in emptying by the prokinetic agent, erythromycin, can ‘unmask’ the insulinotropic effects [90]. The effects of exogenous GLP-1 on gastric emptying are manifest even at relatively low doses (e.g. 0.3 pmol/kg/min), which yield

circulating concentrations comparable to physiological postprandial levels. Studies with exendin (9–39) confirm that GLP-1 has physiological actions to inhibit gastric antral motility and stimulate pyloric contractions. One study has confirmed that, accordingly, exendin (9–39) amide accelerates gastric emptying modestly in healthy humans [30], although three others failed to elicit an effect, possibly related to suboptimal techniques of measuring gastric emptying or insufficient caloric loads [111,127,155]. One of these negative studies did, however, show changes in the intragastric meal distribution with exendin 9–39 amide [127]. The physiological effects of GLP-1 on small intestinal and colonic motility have not been extensively studied although there is some evidence of impaired motility from animal studies [13,44] and indirect human evidence from a case study of a 60 year old woman with GLP-1 and GLP-2 producing tumor [17]. In rats, exendin (9–39) amide has been reported to reduce small intestinal motility induced by intraduodenal infusion of peptone, and to block the increase in fecal pellet output induced by intracerebroventricular infusion of GLP-1 [45]. Native GLP-1 has been shown to inhibit small intestinal transit in health [51], and a GLP-1 analog, ROSE-010 was more effective than placebo in controlling abdominal pain in patients with irritable bowel syndrome [50], presumably due to suppression of smooth muscle contractions. Further studies are indicated to define the effect of GLP-1 on small intestinal motility, particularly since this could represent an additional mechanism of glucose lowering (see Table 1).

6.3. Appetite regulation Exogenous GLP-1, administered intravenously, has been shown to suppress appetite in both normal weight [43] and obese individuals [100], as well as patients with type 2 diabetes [143]. More recently, exendin (9–39) was noted to induce hyperphagia in rats, suggesting a role for endogenous GLP-1 in the control of appetite [153]. It has not yet been clearly established whether endogenous GLP-1 can reduce food intake in humans, but a study employing a novel oral formulation of GLP-1, designed to mimic the endogenous GLP-1 profile by providing the greatest concentration of the peptide at the level of the gut [8], was recently reported to reduce food intake in healthy humans [137]. Following a meal, GLP-1 levels are elevated within 10–15 min, which would be consistent with a role as a ‘meal termination signal’ [92], acting through both peripheral and central mechanisms. In support of the latter, GLP-1 receptors are widely located in the central nervous system, including the hypothalamus (lateral, dorsomedial and ventromedial), which plays an important role in appetite regulation [129]. Intracerebroventricular infusion of GLP1 in rats induces satiation, which is reversed by administration of exendin (9–39) [149]. However, GLP-1 receptor knockout mice do not exhibit increased food intake or weight gain over wild type

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mice, possibly reflecting the redundancy built into the control of appetite rather than the ineffectiveness of GLP-1 as an appetiteregulating signal [145]. GLP-1 receptors on vagal afferent nerve fibers in the gastrointestinal tract are also likely to be involved in mediating satiation, as ablation of vagal afferent pathways can neutralize the effects of GLP-1 on appetite [1,133]. 7. Therapeutic applications of GLP-1 The observation that the insulinotropic action of GLP-1, unlike GIP, remains relatively intact in type 2 diabetes has generated much interest in the development of GLP-1 based drugs. The extremely short half-life of GLP-1, however, makes the native peptide unsuitable for administration in the management of chronic conditions such as diabetes and obesity. Rather, the development of GLP-1 based therapy has involved two approaches – the development of incretin mimetics and incretin enhancers. Exenatide was the first incretin mimetic to become available, receiving US Food and Drug Administration (FDA) approval in 2005. Exenatide is a synthetic version of a naturally occurring peptide, exendin-4, found in the parotid gland of the Central American Gila monster lizard (Heloderma suspectum). Exenatide shares 53% homology with native GLP-1, and has a half-life of about 3 h. Liraglutide, the other FDA approved agent of this class, is much closer in structure to native GLP-1, and has a longer half-life of 11–15 h. GLP-1 agonists are available only in injectable form and common side effects include nausea and vomiting. Other drugs of this class undergoing clinical trials include lixisenatide, taspoglutide and albiglutide. An alternative approach to administering GLP-1 analogs is to prolong the action of endogenous GLP-1 and GIP by inhibiting DPPIV. DPP-IV inhibitors reduce serum DPP-IV activity by about 80% [2], have the advantage of oral administration, and are better tolerated than the GLP-1 agonists, albeit with apparently lower efficacy. DPP-IV inhibitors that are currently FDA approved are sitagliptin, vildagliptin, saxagliptin, and linagliptin. The application of GLP-1 based therapy to specific disorders is discussed in the following paragraphs. 7.1. Type 2 diabetes Intravenous or subcutaneous administration of native GLP-1 established the potential of incretin based therapy in type 2 diabetes [109]. A single subcutaneous dose of GLP-1 was shown acutely to normalize postprandial glycemia in type 2 patients [46]. Subsequently, 3 weeks administration of subcutaneous GLP-1 (three times daily) showed sustained results [142]. Continuous subcutaneous GLP-1 infusions improved HbA1c from 9.2% to 7.9% over a 6-week period in patients with poorly controlled type 2 diabetes [160]. Similarly, continuous subcutaneous GLP-1 monotherapy was shown to maintain a mean baseline HbA1c of 7.1% over a 3-month period in a group of well-controlled elderly type 2 diabetes patients who had been switched from their usual oral anti-diabetic therapy [94]. However, continuous infusions of GLP-1 are impractical and were soon superseded by GLP-1 receptor agonist drugs. 7.1.1. GLP-1 analogs: efficacy in type 2 diabetes and tachyphylaxis Analogs of GLP-1 have subsequently proven to be effective in longer-term clinical trials. All GLP-1 receptor agonists reduce HbA1c (ranging from 0.62% to 1.64% reduction according to one systematic review) when compared with placebo, with a higher percentage of patients achieving a HbA1c of less than 7% [134]. In the short-term, there does not seem to be much difference in efficacy amongst the various GLP-1 agonists available. GLP-1 agonists slow gastric emptying [11] and this seems to be the major mechanism of glucose-lowering when administered

Fig. 3. Change in T50 (ratio of exenatide T50/placebo T50) vs. placebo T50. 10 ␮g exenatide/placebo solid component. Regression line, r = −0.51, P = 0.0435. A longer t50 is indicative of slower gastric emptying. Reprinted with permission from Linnebjerg et al. [74].

acutely; the magnitude of glucose-lowering depends on the baseline rate of gastric emptying [74] (see Fig. 3). Exogenous GLP-1 has recently been reported to induce tachyphylaxis (a diminution in response with repeated dosing) with regards to its retarding action on gastric emptying [106]. ‘Longer acting’ GLP-1 agonists (exenatide LAR and liraglutide) likewise show a similar reduction in ability to slow gastric emptying with time [34,63]. Interestingly, the relatively ‘short-acting’ GLP-1 agonists (exenatide twice daily and lixisenatide) have not been found to exhibit tachyphylaxis [34,80]. Thus, both an individual’s baseline rate of gastric emptying and the choice of GLP-1 agonist (long or short acting) will have an impact on the glycemic control achieved in the long run. Deterioration in glycemic control in type 2 diabetes over time, in spite of conventional treatment (oral anti-diabetic agents and insulin) is attributed to the progressive loss of beta cell function [148]. GLP-1 agonists also appear to have a positive effect on beta cell health. Exenatide improved beta cell function (measured by hyperglycemic clamp techniques) in type 2 diabetes patients inadequately treated with metformin, while insulin glargine failed to do so [20]; this beneficial effect was sustained after a 4 week off-drug period following 3 years of exenatide treatment [19]. The LEAD-6 trial reported a comparable benefit on beta cell function with once daily liraglutide or twice daily exenatide [23]. 7.1.1.1. GLP-1 agonists and insulin: comparison and combination. Baseline HbA1c of trial patients appears to be important in determining superiority in head to head trials of GLP-1 agonists and insulin. Fasting blood glucose (which is mainly targeted by insulin) is the major contributor in poorly controlled patients, while the contribution of postprandial blood glucose (which is preferentially targeted by GLP-1 agonists) increases as HbA1c approaches 7% [97]. Accordingly, insulin was superior to a GLP-1 agonist in a trial involving poorly controlled patients (baseline HbA1c of 10.2%) [10], while exenatide fared better in patients with type 2 diabetes who had relatively better glycemic control [103]. As mentioned earlier, insulin acts primarily on fasting hyperglycemia, and postprandial blood glucose control may remain unsatisfactory in a number of type 2 patients despite insulin therapy. The combination of insulin and a GLP-1 receptor agonist, which would target both pre-prandial and post-prandial hyperglycemia, therefore holds substantial promise. A short acting GLP-1 agonist with sustained effects on gastric emptying would theoretically be the agent of choice in this situation. Recently, the FDA gave approval

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to a combination regimen of basal insulin and exenatide, and the combination of twice daily exenatide with insulin glargine has been shown to have additive benefits for glycemic control, without increasing the risk of hypoglycemia [21]. Longer duration trials are now warranted. 7.1.2. DPP-IV inhibitors: role in type 2 diabetes DPP-IV inhibitors also reduce HbA1c in patients with type 2 diabetes when compared with placebo, but the reduction tends to be a little less than with GLP-1 agonists. For example, sitagliptin reduces HbA1c by 0.3–1% and vildagliptin by 0.5–1%, when given alone or in combination with other anti-diabetic agents [134]. The slightly lower efficacy than for GLP-1 agonists might relate to the fact that these drugs produce a relatively modest increase in postprandial levels of intact GLP-1, and have minimal, if any, effect on the rate of gastric emptying [138]. A novel approach to GLP-1 based therapy in type 2 diabetes is to use dietary strategies, such as macronutrient ‘preloads’ given 30–60 min before the main meal, to enhance the secretion of endogenous GLP-1 and insulin and slow gastric emptying of the meal. For example, a whey protein preload can achieve a substantial acute reduction in postprandial glycemia in patients with type 2 diabetes by these mechanisms [84]. The use of alternative preloads that entail minimal additional energy intake is the subject of ongoing investigation [156]. 7.2. Obesity A key advantage of incretin-based therapies over insulin and sulfonylureas is their impact on body weight. GLP-1 agonists including exenatide and liraglutide have consistently been associated with modest weight loss, and their combination with insulin appears not to be associated with the weight gain that is seen with insulin alone. DPP-IV inhibitors, on the other hand, are weight neutral [28]. The effects of GLP-1 based therapy on weight are discussed below. Clinical trials with twice daily exenatide given for 30 weeks yielded modest weight loss in type 2 diabetes (∼2 kg) compared with placebo, and the effect was sustained for 52 weeks with the once weekly formulation [22]. Similar effects are apparent with liraglutide, and are associated with loss of visceral fat. The nausea that frequently accompanies the initiation of therapy with a GLP-1 agonist is usually transient and not thought to account for weight loss [145]. Whether this effect of GLP-1 agonists could be exploited for the treatment of obesity has now been evaluated in trials involving non-diabetic obese subjects, where liraglutide achieved greater weight loss than placebo or orlistat over 20 weeks (7 kg vs. 2.8 kg and 4.1 kg respectively) [3]. 7.3. Cardiovascular disease GLP-1 receptors are widely expressed in the cardiovascular system, being found in the cardiomyocyte, endothelium and smooth muscle cells of the myocardial vasculature [2]. Rodent studies have indicated the capacity for exogenous GLP-1 to induce ‘pre-ischemic conditioning’ and limit the size of a subsequent infarct [27]. There have been reports of improvements in left ventricular ejection fraction and other cardiac indices in heart failure patients treated with GLP-1, but outcomes have been inconsistent between trials. GLP-1 agonists are also observed to reduce blood pressure, and improve endothelial function and lipid profiles. Some of these cardiovascular effects of GLP-1 may be mediated indirectly, possibly through GLP-1 metabolites, rather than through the GLP-1 receptor, so that the effects of DPP-IV-resistant agonists may differ from those of native GLP-1 [27]. Lowering of blood glucose and modest weight loss associated with GLP-1 based therapy are themselves likely to be beneficial for cardiovascular function [68], and moreover,

GLP-1 can suppress the expression of receptors for advanced glycation end products; the latter contribute to the pathogenesis of diabetic vascular complications [27]. 7.4. Neurological diseases Over the last decade, animal studies have pointed toward GLP-1 playing a protective role in neuro-degenerative disorders including Alzheimer’s disease, Parkinson’s disease and stroke syndromes. GLP-1 reduces endogenous amyloid-beta, accumulation of which is involved in neuronal apoptosis leading to Alzheimer’s disease [116]. Exendin-4 can arrest deterioration, or even reverse established nigrostriatal lesions, in two distinct rodent models of Parkinson’s disease, and has protective effects on dopaminergic neurons, with preservation of dopamine levels and improved motor function in a third rodent model [54]. Exendin-4 also reduces cortical damage and improves functional outcomes in a transient middle cerebral artery stroke model in rats [139]. 7.5. Bone metabolism GLP-1 agonists appear capable of reversing the deficient bone formation and structure associated with glucose intolerance. Exendin-4 acts via the Wnt signaling pathway to promote bone formation in rodent models of type 2 diabetes and insulin resistance [113]. GLP-1 also increases markers of bone turnover including osteocalcin (OC), osteoprotegerin (OPG) and RANKL, which are deficient in diabetic rats [112]. Human studies are not currently available and it remains to be seen if GLP-1 agonists have a role in the treatment of osteoporosis. 8. Actions of GLP-2 GLP-2 can increase intestinal crypt cell proliferation and reduce apoptosis in animal models, resulting in increased mucosal thickness and surface area, and greater crypt cell number and depth [147], in both the small and large intestine. In accordance with a repair function, GLP-2 reduces the concentrations of inflammatory markers, including IFN-gamma, TNF-gamma and IL-1 beta, in models of ileitis/colitis induced by chemical injury [57]. In contrast to the glucagonostatic actions of GLP-1, GLP-2 increases glucagon levels in both the fasted and postprandial states [93], although to what extent GLP-2 contributes to the hyperglucagonemia observed in patients with type 2 diabetes remains uncertain. Peripheral administration of GLP-2 in rats has recently been reported to reduce gastric emptying and food intake in the short term [6], but any effects of GLP-2 on gastric emptying in humans appear minimal [130]. 9. Therapeutic applications of GLP-2 A number of recent studies have explored the role of GLP-2 based therapy in gastrointestinal disorders. Short bowel syndrome, which follows extensive intestinal resection or loss (e.g. from pediatric necrotizing enterocolitis), is characterized by up-regulation in absorptive capacity of the remnant intestine – a process where GLP-2 is believed to be central [135]. Teduglutide, a DPP-IV resistant analog of GLP-2, can improve absorptive function in patients with short bowel syndrome when administered subcutaneously, and may reduce reliance on parenteral nutrition. A multicentre trial showed beneficial effects of teduglutide in short bowel syndrome patients on total parenteral nutrition although there was no dosedependent benefit [86]. Larger international multicentre trials are currently underway.

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GLP-2 based therapy also shows potential in the treatment of inflammatory bowel disease, and teduglutide showed dose dependent effects to reduce disease activity and induce remission in patients with moderate to severe Crohn’s disease [18]. Finally, the cytoprotective effects of GLP-2 could potentially be beneficial in chemotherapy-induced gastrointestinal mucositis. 10. Safety of GLP-1 and GLP-2 based therapies The diverse expression of GLP-1 receptors in multiple organ systems raises the possibility of unintended consequences of GLP-1 based therapy. Prolonged administration of GLP-1 agonists in rats has been associated with C-cell proliferation and an increased incidence of medullary thyroid carcinoma. Primates, however, have much lower expression of GLP-1 receptors in the thyroid than rats, so that prolonged administration of liraglutide was devoid of this effect in monkeys, and two years’ therapy with liraglutide had no impact on serum calcitonin, a marker of C-cell proliferation, in patients with type 2 diabetes. Animal studies also suggested an increased risk of acute pancreatitis with GLP-1 based therapy, but no definite association has been established in humans [36]. The cytoproliferative and anti-apoptotic effects of GLP-2 have raised concerns regarding carcinogenesis, with some evidence for an increased risk of colon cancer in animal studies, although whether GLP-2 receptors are expressed in human colon cancer is controversial [16]. 11. Summary GLP-1 and GIP (collectively known as incretin hormones) play a major role in prandial insulin secretion in health and GLP-1 retains its insulinotropic activity in type 2 diabetes. GLP-1 based therapy is widely used today in the management of type 2 diabetes following FDA approval of exenatide in 2005 and several GLP-1 agonist drugs are currently in development. The efficacy of GLP1 therapy would be considerably improved by understanding the properties of these different agonists (including duration of action and tendency for tachyphylaxis) and the complex inter-dependent relationships GLP-1 shares with postprandial glycemia and gastric emptying. In addition to its role in glycemic control, recent research has suggested further beneficial attributes, such as cardioand neuro-protective functions of GLP-1, and further research is needed to clarify this. The mechanism by which GLP-2 exerts its intestinotropic effects are not fully understood but initial results in the treatment of chronic bowel disorders such as short bowel syndrome and Crohn’s disease are promising. References [1] Abbott CR, Monteiro M, Small CJ, Sajedi A, Smith KL, Parkinson JR, et al. The inhibitory effects of peripheral administration of peptide YY(3–36) and glucagon-like peptide-1 on food intake are attenuated by ablation of the vagal–brainstem–hypothalamic pathway. Brain Res 2005;1044:127–31. [2] Ahren B. The future of incretin-based therapy: novel avenues – novel targets. Diabetes Obes Metab 2011;13(Suppl. 1):158–66. [3] Astrup A, Rossner S, Van Gaal L, Rissanen A, Niskanen L, Al Hakim M, et al. Effects of liraglutide in the treatment of obesity: a randomised, double-blind, placebo-controlled study. Lancet 2009;374:1606–16. [4] Bagger JI, Knop FK, Lund A, Vestergaard H, Holst JJ, Vilsboll T. Impaired regulation of the incretin effect in patients with type 2 diabetes. J Clin Endocrinol Metab 2011;96:737–45. [5] Baggio LL, Drucker DJ. Biology of incretins: GLP-1 and GIP. Gastroenterology 2007;132:2131–57. [6] Baldassano S, Bellanca AL, Serio R, Mule F. Food intake in lean and obese mice after peripheral administration of glucagon-like peptide 2. J Endocrinol 2012;213:277–84. [7] Bayliss WM, Starling EH. The mechanism of pancreatic secretion. J Physiol 1902;28:325–53. [8] Beglinger C, Poller B, Arbit E, Ganzoni C, Gass S, Gomez-Orellana I, et al. Pharmacokinetics and pharmacodynamic effects of oral GLP-1 and PYY336: a proof-of-concept study in healthy subjects. Clin Pharmacol Ther 2008;84:468–74.

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