Best Practice & Research Clinical Endocrinology & Metabolism
`Vol. 18, No. 4, pp. 531–554, 2004
`doi:10.1016/j.beem.2004.08.001
`available online at http://www.sciencedirect.com
`
`6 G
`
`lucagon-like peptide-1 and glucagon-like
`peptide-2
`
`Laurie L. Baggio PhD
`Research Associate
`
`Daniel J. Drucker* MD
`Director
`
`Department of Medicine, The Banting and Best Diabetes Centre, University of Toronto, Toronto General Hospital,
`200 Elizabeth Street, MBRW 4R-402, Toronto, Ont., Canada M5G 2C4
`
`The glucagon-like peptides (glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2))
`are released from enteroendocrine cells in response to nutrient ingestion. GLP-1 enhances
`glucose-stimulated insulin secretion and inhibits glucagon secretion, gastric emptying and feeding.
`GLP-1 also has proliferative, neogenic and antiapoptotic effects on pancreatic b-cells. More
`recent studies illustrate a potential protective role for GLP-1 in the cardiovascular and central
`nervous systems. GLP-2 is an intestinal trophic peptide that stimulates cell proliferation and
`inhibits apoptosis in the intestinal crypt compartment. GLP-2 also regulates intestinal glucose
`transport, food intake and gastric acid secretion and emptying, and improves intestinal barrier
`function. Thus, GLP-1 and GLP-2 exhibit a diverse array of metabolic, proliferative and
`cytoprotective actions with important clinical implications for the treatment of diabetes and
`gastrointestinal disease, respectively. This review will highlight our current understanding of the
`biology of GLP-1 and GLP-2, with an emphasis on both well-characterized and more novel
`therapeutic applications of these peptides.
`
`Key words: diabetes; obesity; food intake; intestinal disease; cell proliferation; apoptosis; insulin
`secretion.
`
`Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates blood glucose
`level through its combined actions on the stimulation of glucose-dependent insulin
`secretion and the inhibition of glucagon secretion, gastric emptying and food intake.1
`GLP-1 also increases b-cell mass via a stimulation of b-cell proliferation and neogenesis,
`and an inhibition of b-cell apoptosis.2 The observation that the pharmacological
`
`* Corresponding author. Tel.: C1 416 340 4125; Fax: C1 416 978 4108.
`E-mail address: d.drucker@utoronto.ca (D.J. Drucker).
`
`1521-690X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
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`532 L. L. Baggio and D. J. Drucker
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`administration of GLP-1 and related analogues can reduce elevated fasting and
`postprandial blood glucose levels in diabetic human subjects has generated intense
`interest in the development of GLP-1R agonist-based therapies for the treatment of
`diabetes mellitus.
`GLP-2 is a 33 amino acid peptide that regulates energy homeostasis via acute and
`chronic effects on gut motility, and nutrient ingestion and absorption. In rodents,
`exogenous GLP-2 administration promotes the growth and survival of epithelial cells
`within the small and large bowel mucosa via an inhibition of apoptotic cell death and a
`stimulation of cellular proliferation.1 GLP-2 also enhances barrier function and
`increases the resistance to and recovery from a variety of experimental models of gut
`injury.1 The reported actions of GLP-2 have fostered the initiation of clinical studies to
`assess the ability of GLP-2 to improve nutrient absorption and epithelial integrity and
`restore functional bowel mass in human patients with intestinal disease.
`The actions of GLP-1 and GLP-2 are mediated via unique G protein-coupled
`receptors. The GLP-2 receptor (GLP-2R) is expressed in a highly tissue-specific
`manner, predominantly in the gastrointestinal tract and brain.3 In contrast, the GLP-1
`receptor (GLP-1R) has a more widespread distribution and is expressed in a number of
`tissues,
`including the pancreas,
`intestine, stomach, central nervous system (CNS),
`heart, pituitary, lung and kidney.4–6
`Despite the potential therapeutic benefits of GLP-1 and GLP-2, the durations of
`their action are limited owing to a rapid inactivation of these peptides by the ubiquitous
`protease dipeptidyl peptidase-IV (DPP-IV). Consequently, the inhibition of DPP-IV
`activity or the development of DPP-IV-resistant glucagon-like peptide analogues offers
`additional therapeutic options for treating human disease.
`
`SYNTHESIS, SECRETION AND METABOLISM OF GLP-1 AND GLP-2
`
`GLP-1 and GLP-2 are co-encoded within the proglucagon gene, which, in mammals,
`gives rise to a single mRNA transcript that is expressed in the a-cells of the endocrine
`pancreas, in the enteroendocrine L-cells of the intestine and in the hypothalamus and
`brainstem in the CNS.7,8 The proglucagon mRNA is translated into a single 160 amino
`acid precursor protein that undergoes tissue-specific post-translational processing to
`produce several biologically active proglucagon-derived peptides (PGDPs), including
`glucagon in the pancreatic a-cells and glicentin, oxyntomodulin, GLP-1 and GLP-2 in the
`intestine and brain (Figure 1). Glucagon is the major counter-regulatory hormone to
`insulin and is essential for maintaining blood glucose levels in the physiological range
`during the post-absorptive state. Oxyntomodulin has inhibitory effects on gastroin-
`testinal secretion and motility, and stimulatory effects on pancreatic enzyme secretion
`and intestinal glucose uptake.9 More recently,
`it has been demonstrated that
`oxyntomodulin can reduce food intake in both rodents and humans.10,11 In contrast,
`the physiological actions of glicentin are poorly defined. GLP-1 and GLP-2 exhibit
`trophic effects in the pancreas and intestine, respectively, and play important roles in
`the regulation of nutrient assimilation and energy homeostasis (see below).
`The PGDP sequences within the proglucagon precursor are flanked by pairs of basic
`amino acids, and the post-translational processing of proglucagon is carried out by
`prohormone convertases, which are endoproteolytic enzymes that cleave C-terminal
`to paired basic amino acid residues.12 Although the prohormone convertase enzymes
`responsible for the production of GLP-1 and GLP-2 in the CNS have not been
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`Glucagon-like peptide-1 and glucagon-like peptide-2 533
`
`GRPP
`
`Glucagon
`
`IP-1
`
`GLP-1
`
`IP-2
`
`GLP-2
`
`Glicentin
`
`MPGF
`
`Oxyntomodulin
`
`Pancreas
`Glucagon
`MPGF
`
`Intestine & Brain
`Glicentin
`Oxyntomodulin
`GLP-1
`GLP-2
`IP-1
`
`Figure 1. Mammalian proglucagon structure and tissue-specific post-translational processing of the
`proglucagon-derived peptides. GRPP, glicentin-related polypeptide; IP-1 and IP-2, intervening peptide-1 and -2;
`MPGF, major proglucagon fragment; GLP-1 and GLP-2, glucagon-like peptide-1 and -2.
`
`definitively established, prohormone convertase 1/3 has been localized to intestinal
`L-cells and is both necessary and sufficient for the post-translational processing of
`proglucagon to GLP-1 and GLP-2.13 In contrast, prohormone convertase 2 is expressed
`in the islet a-cell and is essential for the generation of 29 amino acid pancreatic
`glucagon.14
`GLP-1 and GLP-2 are secreted in a 1:1 ratio from intestinal L-cells15, the majority of
`which are located in the distal ileum and colon.16 The major stimulus for GLP-1 and
`GLP-2 secretion is the ingestion of nutrients, including glucose, fatty acids and dietary
`fibre.17 Fasting plasma levels of the biologically active forms of GLP-1 and GLP-2 in
`healthy humans are 5–10 and 15–20 pM, respectively, and increase 2–5-fold following
`food ingestion, the absolute peak level being dependent on the size and nutrient
`composition of the meal.18,19 When nutrients are ingested, the release of GLP-1
`and GLP-2 into the circulation occurs in a bi-phasic manner, consisting of a rapid (within
`10–15 minutes) early phase followed by a more prolonged (30–60 minutes) second
`phase.20 The distal location of most L-cells that produce GLP-1 and GLP-2 makes it
`unlikely that the rapid nutrient-stimulated increase in plasma levels of these peptides is
`due to a direct effect of nutrients on the L-cell. Indeed, studies in rodents and humans
`clearly indicate that the vagus nerve, the neurotransmitter gastrin-releasing peptide and
`the hormone glucose-dependent insulinotropic peptide all contribute to the rapid
`release of GLP-1 and GLP-2 from distal L-cells in response to nutritional stimuli.17 In
`contrast, the second phase of peptide secretion probably results from a direct
`stimulation of the L-cell by digested nutrients.21 Thus, nutrient-induced stimulatory
`signals are transmitted to intestinal L-cells indirectly, via neural and endocrine effectors,
`and also by direct interaction with these cells, to mediate the first and second phase,
`respectively, of GLP-1 and GLP-2 secretion.
`The half-life of circulating biologically active GLP-1 is less than 2 minutes22, whereas
`GLP-2 is more stable, with a half-life of approximately 5–7 minutes.23,24 The relatively
`short circulating half-lives of the bioactive forms of these peptides can be attributed to
`renal clearance and enzymatic inactivation. DPP-IV, a serine protease that cleaves
`dipeptides from the amino terminus of oligopeptides or proteins that have a proline or
`
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`534 L. L. Baggio and D. J. Drucker
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`alanine residue in the penultimate position25, is a critical determinant of GLP-1/GLP-2
`degradation. DPP-IV cleaves GLP-1 and GLP-2 at the alanine residue in position 2,
`yielding the inactive peptides GLP-1 (9–37/36NH2) and GLP-2 (3–33). DPP-IV
`expression is fairly widespread and is found on the surface of circulating white blood
`cells and in cells constituting the vascular endothelium of the small intestine, adjacent to
`the sites of GLP-1 and GLP-2 secretion.25,26 Thus, the majority of GLP-1 and GLP-2
`entering the portal circulation has already been inactivated by DPP-IV prior to entry
`into the systemic circulation. The kidney provides the major route of clearance for both
`GLP-1 and GLP-227, and patients with uraemia or chronic renal
`insufficiency have
`elevated levels of circulating GLP-1 relative to healthy control individuals.28,29
`
`GLP-1 AND GLUCOSE HOMEOSTASIS
`
`GLP-1 elicits multiple actions in the pancreas and in extra-pancreatic tissues that lead to
`the reduction of blood glucose (Figure 2). The first physiological action to be described
`for GLP-1 was the augmentation of glucose-stimulated insulin secretion.30–32 GLP-1
`binds to its specific receptor on the pancreatic b-cell and stimulates insulin secretion
`C
`through mechanisms that involve an inhibition of ATP-sensitive K
`channels (KATP) and
`subsequent b-cell depolarization, elevations in intracellular Ca2C
`level, an inhibition of
`channels and direct effects on the b-cell exocytotic
`voltage-dependent K
`machinery.33,34 The intracellular signalling events that modulate GLP-1-regulated
`insulin release include activation of the cAMP/protein kinase A (PKA), cAMP/guanine-
`nucleotide exchange factor and phosphatidylinositol-3 kinase (PI-3K)/protein kinase C
`(PKC)z pathways.34–36 Unlike other insulin secretagogues, GLP-1 also promotes insulin
`gene transcription, mRNA stability and biosynthesis, and thus has the capacity to
`replenish depleted b-cell insulin stores.37,38
`
`C
`
`GLP-1 Actions
`
`Pancreas
`
`CNS
`
`↑ Insulin synthesis & secretion
`↓ Glucagon secretion
`↑ Somatostatin secretion
`↑ Expression of genes that
`modify β-cell function
`↑ β-cell proliferation & neogenesis
`↑ β-cell survival
`
`↓ Food intake
`↑ Satiety
`↑ Neuronal cell proliferation &
`neogenesis
`↑ Neuronal cell survival
`↑ Learning & memory
`
`Stomach & Intestine
`
`Liver/Fat/Muscle
`
`Heart
`
`↓ Gastric emptying
`↓ Bowel motility
`
`↑ Glycogen synthesis
`↑ Lipogenesis
`
`Structure/ function
`Cardioprotection
`
`Figure 2. Pancreatic and extra-pancreatic glucagon-like peptide-1 receptor agonist-dependent actions. CNS,
`central nervous system.
`
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`Glucagon-like peptide-1 and glucagon-like peptide-2 535
`
`In addition to its ability to stimulate glucose-dependent insulin secretion by direct
`interaction with the pancreatic b-cell GLP-1R, GLP-1 may also stimulate insulin
`secretion indirectly via neural mechanisms. It has been estimated that more than half of
`the GLP-1 secreted from intestinal L-cells is inactivated by DPP-IV, while the majority of
`the remaining intact peptide is inactivated as it passes through the liver.26,39 It is thus
`likely that only small amounts of bioactive GLP-1 actually reach the pancreas intact.
`Studies using ganglionic blockers in rats or chemical denervation experiments in mice
`following capsaicin treatment suggest that endogenously released GLP-1 can stimulate
`insulin secretion in part by a sensory neural reflex that probably initiates in the
`hepatoportal system.40,41
`GLP-1 can also increase the steady-state levels of mRNA transcripts for key
`components of the molecular machinery involved in b-cell exocytosis, such as the
`sulphonylurea receptor and the inwardly rectifying potassium channel (Kir 6.2),
`molecular subunits of the b-cell KATP channel. Similarly, GLP-1 also regulates b-cell KATP
`channel function via the prevention of glucose-dependent inhibition of KATP channel
`activity.42
`Studies in both rodents and humans reveal that GLP-1 can improve the ability of the
`b-cell to sense and respond to glucose, thereby conferring glucose sensitivity to
`previously resistant b-cells.43,44 A potential mechanism whereby GLP-1 could restore
`b-cell glucose responsivity is suggested by studies demonstrating that GLP-1 can
`upregulate the expression of glucose transporters and glucokinases, components of the
`b-cell glucose sensor.45,46
`GLP-1 also lowers blood glucose levels by inhibiting the secretion of glucagon.47 The
`inhibitory effects of GLP-1 on glucagon secretion may occur through direct interaction
`with GLP-1 receptors on pancreatic a-cells48 or indirectly through GLP-1-mediated
`stimulation of insulin and/or somatostatin secretion.49
`Both animal and human studies demonstrate that GLP-1 delays gastric emptying
`and intestinal motility, thereby slowing the transit of nutrients from the stomach to
`the small
`intestine and attenuating meal-associated elevations in plasma glucose
`levels.50,51 The inhibitory effects of GLP-1 on the gut are likely to involve both CNS-
`and intestinal-derived GLP-1.52 The mechanism whereby peripheral GLP-1 inhibits
`gastrointestinal motility appears to involve either direct interaction with CNS
`centres that regulate visceral motility or an indirect mechanism via vagal afferent
`pathways.52
`GLP-1 may also regulate glucose disposal through peripheral actions on liver, skeletal
`muscle and adipose tissue. GLP-1 has been shown to increase glucose incorporation
`into glycogen in isolated rat hepatocytes and skeletal muscle53, and enhance insulin-
`stimulated glucose metabolism in 3T3 L1 adipocyte cultures and isolated rat
`adipocytes.54,55 However, subsequent studies have failed to support a direct extra-
`pancreatic role for GLP-1 in liver, muscle or adipose tissue.56,57 Whether GLP-1 has
`direct effects on glucose disposal independent of changes in the levels of islet hormones
`in humans remains unclear. A number of studies in healthy and diabetic humans suggest
`that GLP-1 can increase glucose disappearance,
`independent of
`insulin and
`glucagon58–60; in contrast, other studies indicate that GLP-1 has no direct influence
`on glucose disposition.61–65 Recent evidence suggests that the effects of GLP-1 may
`involve a suppression of endogenous glucose production rather than an increase in
`peripheral glucose disposal.66 The mechanism whereby GLP-1 could mediate these
`extra-pancreatic effects in humans, independent of changes in the insulin:glucagon ratio,
`remains uncertain. There is no consistent evidence from rodent or human studies that
`GLP-1 receptors are present in liver, fat or muscle tissues.67–71 Since GLP-1R binding,
`
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`536 L. L. Baggio and D. J. Drucker
`
`signal transduction pathways and the effects of GLP-1R agonists and antagonists in
`these non-pancreatic cell types differ from those observed with the known pancreatic
`GLP-1R, it seems likely that GLP-1 may mediate its effects in extra-pancreatic tissues via
`a ‘second’ or related GLP-1 receptor.72–76
`The importance of GLP-1 as a physiological regulator of glucose homeostasis is
`illustrated by studies in which GLP-1 action has been reduced or eliminated. Inhibition
`of GLP-1 activity with exendin (9–39), a specific GLP-1R antagonist, leads to impaired
`glucose tolerance and diminished levels of glucose-stimulated insulin in rodent, baboon
`and human studies.77–79 Similarly, mice with a targeted genetic disruption of the GLP-1
`K/K
`) are characterized by mild fasting hyperglycaemia, glucose
`receptor (GLP-1R
`intolerance in the face of an oral or intraperitoneal glucose challenge, and decreased
`circulating levels of insulin following glycaemic stimulation.80 Moreover, the relative
`importance of basal GLP-1 levels for glucoregulation is demonstrated in human studies
`in which the administration of exendin (9–39) produces significant elevations in the
`levels of fasting glucose and glucagon, suggesting that GLP-1 may have a tonic inhibitory
`effect on the a-cell.81
`More recent experiments have demonstrated that GLP-1 has proliferative and
`neogenic effects on pancreatic b-cells. Studies with the INS-1 b-cell line demonstrate
`that GLP-1 can activate the expression of immediate early genes, whose products are
`transcription factors that play a role in genetic programmes regulating islet cell
`proliferation and differentiation.82,83 The treatment of islet hormone-negative AR42J
`pancreatic exocrine cells with GLP-1 promotes their conversion into cells that produce
`and secrete insulin in a glucose-dependent manner.84 Moreover, the treatment of rat
`and human pancreatic ductal cell lines with GLP-1 causes them to differentiate into cells
`exhibiting endocrine properties.85,86 GLP-1 has also been shown to accelerate both the
`differentiation and maturation of human and porcine fetal islet cells.87,88 In both normal
`and diabetic rodents, short-term treatment with GLP-1R agonists leads to improved
`glucose tolerance, enhanced b-cell proliferation and neogenesis, leading to increased
`b-cell mass.89–91 In db/db mice and Goto-Kakizaki rats, rodent models of type 2
`diabetes, GLP-1R agonist administration is associated with increased b-cell mass and a
`delayed onset of diabetes.92,93 Moreover, GLP-1R agonist administration during the
`prediabetic neonatal period prevented the development of adult-onset diabetes in rats
`following experimentally induced intrauterine growth retardation.94 A key feature
`revealed by these animal studies is that the beneficial effects of GLP-1R agonists on
`glucose homeostasis may be sustained for prolonged periods following the suspension
`of GLP-1R agonist treatment.
`In addition to stimulating cell proliferation and differentiation, GLP-1 has also been
`shown to have cytoprotective effects. GLP-1 reduces apoptosis in rodent islets and islet
`lines exposed to cytotoxic agents95,96 and preserves morphology,
`cell
`improves
`glucose-stimulated insulin secretion and inhibits apoptosis in freshly isolated human
`islets.97 Similarly, GLP-1 reduces the lipotoxic effects of fatty acids in both human
`lines.98 GLP-1R agonist administration was associated with
`islets and rodent cell
`proliferative and anti-apoptotic effects in both the endocrine and exocrine
`compartments of the pancreas in Zucker diabetic rats99 and significantly reduced the
`number of apoptotic b-cells in db/db mice as well as following streptozotocin
`administration to wildtype mice.92,96
`A direct role for the GLP-1R in the proliferative, neogenic and anti-apoptotic actions
`of GLP-1 is supported by studies examining GLP-1-dependent effects in the presence of
`the GLP-1R antagonist exendin (9–39) in normal cells and rodents, or in separate
`K/K
`studies of GLP-1R
`mice. Exendin (9–39) blocks the GLP-1R agonist-mediated
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`Glucagon-like peptide-1 and glucagon-like peptide-2 537
`
`differentiation of human pancreatic ductal cells86 and inhibits the anti-apoptotic effects
`K/K
`of GLP-1 in the MIN6 b-cell line.95 GLP-1R
`mice have fewer large b-cell clusters,
`K/K
`islets exhibit an altered a-cell topography100, display an impaired ability
`and GLP-1R
`to regenerate their b-cell mass following partial pancreatectomy101 and manifest an
`increased susceptibility to streptozotocin-induced apoptosis.96
`The signal-transduction pathways downstream of GLP-1R activation coupled to
`stimulation of cell proliferation have been examined in islets and islet cell lines and
`appear to include trans-activation of the epidermal growth factor receptor, which leads
`to increased PI-3K activity and a subsequent activation of PKCz.102 The precise
`mechanisms involved in GLP-1-dependent b-cell differentiation/neogenesis are poorly
`defined but may involve the activation of PKC and mitogen-activated protein kinase.84
`Notably, a common observation following the GLP-1R agonist treatment of b-cells is an
`increase in levels of PDX-1, a transcription factor essential for pancreatic development
`function.84–87,103,104 The molecular mechanisms implicated in GLP-1-
`and b-cell
`dependent anti-apoptotic pathways appear to be diverse and involve multiple signalling
`pathways. Studies in rodents and experiments with isolated islets or b-cell
`lines
`demonstrate that the anti-apoptotic effects of GLP-1 are associated with reduced levels
`of the pro-apoptotic markers active caspase-3 and poly-ADP-ribose polymerase
`cleavage, and an upregulation of anti-apoptotic factors including Bcl-2, Bcl-xL and
`inhibitor of apoptosis protein-2.92,95,98,99,105 The cumulative experimental evidence
`indicates that the intracellular signalling pathways that mediate GLP-1-dependent
`cytoprotective effects include:
`
`1. cAMP/PKA;
`2. PI-3K-dependent activation of the prosurvival kinase Akt/protein kinase B;
`3. activation of nuclear factor-kappa B activity;
`4. cAMP/PKA-mediated phosphorylation and activation of cAMP response element
`binding protein (CREB), subsequent CREB-dependent activation of insulin receptor
`substrate-2 and induction of Akt/protein kinase B.92,98,106,107
`
`As b-cell mass is reduced and b-cell apoptosis is increased in autopsy studies of
`pancreata from patients with type 2 diabetes108, the ability of GLP-1 to promote b-cell
`proliferation and neogenesis, and inhibit b-cell apoptosis, raises the possibility that
`GLP-1 therapy has the potential to preserve or even restore functional b-cell mass in
`diabetic individuals.
`
`GLP-1 AND FEEDING BEHAVIOR
`
`Numerous studies in rodents have demonstrated that the central (intracerebroven-
`tricular) or peripheral administration of GLP-1R agonists leads to the inhibition of
`food intake and reductions in body weight.109–111 Moreover, GLP-1 also inhibits food
`intake and promotes satiety in normal, obese and diabetic humans112–114, suggesting
`that GLP-1 could play an important role in controlling appetite and body weight.
`K/K
`However, GLP-1R
`mice exhibit normal
`feeding behaviour and body weight,
`indicating that GLP-1R signalling may not be essential for the regulation of satiety and
`maintenance of body weight.80
`There is considerable interest in how GLP-1 mediates its effects on ingestive
`behaviour, and experimental evidence indicates that GLP-1 probably modifies food
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`538 L. L. Baggio and D. J. Drucker
`
`It has been proposed that the
`intake via a number of different mechanisms.
`inhibitory effects of GLP-1 on food intake can be mediated indirectly, by its ability to
`slow gastric emptying, thereby promoting gastric distension and a sensation of
`satiety. In addition, GLP-1 receptors are expressed in the hypothalamus and nucleus
`of the solitary tract (NTS), CNS regions that are thought to be important for
`regulating appetite and satiety.5,115 Thus, GLP-1 could also modify feeding by direct
`interaction with these CNS centres. Alternatively,
`it has been suggested that the
`inhibitory effect on food intake could reflect a secondary physiological response to
`the GLP-1-dependent activation of aversive signalling pathways that produce visceral
`illness.116–118
`Binding sites or receptors for GLP-1 have been detected in numerous CNS
`regions, including the hypothalamus, NTS, subfornical organ (SFO) and area postrema
`(AP).5,115,119,120 The SFO and AP represent blood–brain barrier-free CNS locations and
`have also been shown to play a role in the regulation of feeding.121 Although GLP-1 is
`produced in CNS neurons located in the NTS, studies in rats have demonstrated that
`peripherally administered GLP-1 can also gain access to the CNS through interaction
`with GLP-1 receptors in the SFO and AP.122 However, the relative importance of
`peripheral versus central GLP-1 for appetite regulation remains to be elucidated.
`
`GLP-1 AND THE CARDIOVASCULAR SYSTEM
`
`Studies using anaesthetized rats or conscious calves and central or intravenous GLP-
`1 administration are associated with increases in heart rate and systolic, diastolic and
`mean arterial blood pressure.123,124 Consistent with these effects, GLP-1 receptors
`are expressed in the heart and in the NTS and AP, CNS regions known to regulate
`cardiovascular function.125–127 The stimulatory effects of GLP-1 on the rat
`cardiovascular system are independent of catecholamine action, are blocked by
`administration of the GLP-1R antagonist exendin (9–39) and appear to be mediated
`by both direct and indirect GLP-1R-dependent effects on the nervous system and
`heart.124 More recent studies using telemetric monitoring to measure heart rate and
`blood pressure in freely moving rats demonstrated that GLP-1R-dependent increases
`in heart rate and blood pressure are associated with the activation of (i) neuronal
`activity in several autonomic control regions of the rat CNS, (ii) GLP-1-sensitive
`hypothalamic and medullary catecholamine neurons that
`innervate sympathetic
`preganglionic neurons, and (iii) neuronal activity in the adrenal medulla, suggesting
`that central GLP-1 regulates cardiovascular function by activating the sympathetic
`nervous system.128 An essential role for GLP-1R signalling in the maintenance of
`K/K
`normal cardiac structure and function is demonstrated in studies using GLP-1R
`K/K
`mice. GLP-1R
`hearts are characterized by increased septal and posterolateral
`K/K
`myocardial wall
`thickness, and GLP-1R
`mice display abnormal cardiac
`haemodynamic responses to external stress.129
`In contrast to its stimulatory effects on the cardiovascular system in animals, limited
`studies in humans indicate that GLP-1R agonist administration has no significant effects
`on heart rate or blood pressure.130–132 Moreover, GLP-1 may also have cardiopro-
`tective effects under certain conditions. A 14 day infusion of GLP-1 prevents the
`development of hypertension, improves endothelial function and reduces renal and
`cardiac damage in Dahl salt-sensitive rats maintained on a high-salt diet.133
`The antihypertensive effect of GLP-1 in these hypertension-prone rats is attributed
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`Glucagon-like peptide-1 and glucagon-like peptide-2 539
`
`to a GLP-1-dependent increase in salt and water excretion133, actions recently
`demonstrated in human subjects.134 Additionally, a 72 hour infusion of GLP-1 in
`patients with acute myocardial infarction and severe left ventricular systolic dysfunction
`following successful reperfusion with primary angioplasty led to improved regional and
`global left ventricular function and was associated with reduced in-hospital mortality
`rate and length of hospitalization.135
`
`GLP-1 AND NEUROPROTECTION
`
`Comparable to actions observed in studies of pancreatic b-cells, GLP-1 also exerts
`proliferative, neogenic and anti-apoptotic effects on neuronal cells. GLP-1R agonist
`treatment of PC12 cells stimulates neurite outgrowth, enhances nerve growth factor-
`induced differentiation and improves cell survival after nerve growth factor with-
`drawal.136 GLP-1R agonists prevent glutamate-induced apoptosis in cultured rat
`hippocampal neurons and restore cholinergic marker activity in the basal forebrain of
`ibotenic acid-treated rats, a rodent model of neurodegeneration.137 Furthermore,
`GLP-1R-activated pathways seem to be important for learning and memory. GLP-1R
`agonist administration is associated with enhanced learning in rats, an effect that can be
`K/K
`blocked by the co-administration of exendin (9–39).138 In contrast, GLP-1R
`mice
`exhibit deficits in learning that can be overcome by hippocampal Glp1r gene transfer.138
`K/K
`GLP-1R
`mice are also more susceptible to kainate-induced seizures and
`hippocampal neuronal degeneration, whereas GLP-1R agonist treatment prevents
`kainate-induced apoptosis in wildtype animals.138 These observations have led to the
`suggestion that GLP-1R agonists may potentially be useful
`for the treatment of
`neurological disorders,
`including the neuropathy that results as a secondary
`complication of diabetes.139 In contrast, endogenous GLP-1 has also been implicated
`in the pathogenesis of b-amyloid protein-induced neurotoxicity as a continuous co-
`infusion of a GLP-1R antagonist with b-amyloid protein prevented memory impairment
`and hippocampal apoptosis in rats.140
`
`GLP-1 AND THE TREATMENT OF TYPE 2 DIABETES
`
`Numerous studies have demonstrated that GLP-1 can enhance glucose-stimulated
`insulin secretion and lower fasting and postprandial blood glucose levels in individuals
`with type 2 diabetes. The administration of GLP-1 by continuous subcutaneous infusion
`for 6 weeks increased insulin secretion, reduced fasting and postprandial glucose levels,
`lowered haemoglobin A1c (HbA1c) values, and decreased food intake and body weight
`in patients with type 2 diabetes141, indicating that GLP-1 retains its effectiveness, even
`with continuous long-term treatment. As obesity can be a contributing factor to the
`pathogenesis of diabetes, the ability of GLP-1 to reduce food intake and promote satiety
`and weight loss provides an additional means for improving glycaemia in these
`individuals. GLP-1 also lowers fasting and postprandial glucose and reduces the meal-
`associated insulin requirement in human patients with type 1 diabetes, probably via its
`inhibitory effects on glucagon secretion and/or gastric emptying.142–144 Moreover, the
`insulinotropic and glucagonostatic effects of GLP-1 are glucose dependent145–147; thus
`under conditions of normoglycaemia, GLP-1 has no effect on the levels of plasma insulin
`or glucagon.
`
`MPI EXHIBIT 1069 PAGE 9
`
`MPI EXHIBIT 1069 PAGE 9
`
`

`

`540 L. L. Baggio and D. J. Drucker
`
`The ability of GLP-1 to lower blood glucose levels and promote weight loss,
`combined with its potential to preserve or restore functional b-cell mass, has generated
`considerable interest in the use of GLP-1 as a therapeutic agent for the treatment of
`diabetes. Moreover, the observation that GLP-1 secretion is deficient in type 2 diabetics
`suggests that the restoration of normal GLP-1 concentrations in these patients may be
`beneficial.19,148 However, because the plasma half-life of native GLP-1 is very short,
`owing to its rapid inactivation by DPP-IV, continuous infusion or multiple injections of
`GLP-1 are required to attain adequate glycaemic control. Consequently, alternative
`therapeutic strategies have been devised focused on the generation of GLP-1 analogues
`that are resistant to DPP-IV and the development of compounds that inhibit DPP-IV
`activity.
`
`DPP-IV-RESISTANT GLP-1R AGONISTS AND DPP-IV INHIBITION
`AS ALTERNATIVE THERAPEUTIC STRATEGIES
`
`A number of structurally unique GLP-1R agonists have been developed with
`prolonged activity such that once- or twice-daily injections of these molecules are
`potentially as efficacious as continuous GLP-1 infusion for the treatment of
`experimental or clinical diabetes. Exendin-4 is a naturally occurring, DPP-IV-
`resistant GLP-1R agonist originally isolated from the venom of the Heloderma
`suspectum lizard149, and exenatide is a synthetic version of exendin-4. A single
`subcutaneous injection of exenatide in patients with poorly controlled type 2
`diabetes significantly reduced fasting and postprandial glucose concentrations,
`in
`association with increased levels of plasma insulin and decreased plasma
`glucagon.150 Four weeks of twice-daily exendin-4/exenatide treatment significantly
`reduced postprandial glucose and HbA1c levels151, and, when used in combination
`with metformin and/or sulphonylurea treatment, also lowered postprandial plasma
`glucose and HbA1c in diabetic patients with poor glycaemic control.132 The results
`of recently completed phase 3 clinical trials demonstrated that the exenatide
`treatment