Is There Appetite after GLP-1 and PACAP?

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Is There Appetite after GLP-1 and PACAP ?

JEAN CHRISTOPHEa

Department of General and Human Biochemistry, Institute ofPharmacy, Université Libre de Bruxelles, CP 205/3, Boulevard duTriomphe, B-1050 Brussels, Belgium

ABSTRACT: Anitobesity drugs must increase the sensitivity of the hypothalamic sati-ety center towards leptin and antagonize the synthesis and action of NPY. The arrayof pharmacologic tools available is vast and presently ineffective. Among peptideanalogs considered for evaluation [NPY-5 antagonists and CCK-A, bombesin, amylinand melanocyte-stimulating hormone-4 (or melanin-concentrating hormone?) ago-nists], is there a place for GLP-1 and PACAP? GLP-1 receptors present in ARC,PVN, VMN, and SON are the target for both central and blood-borne GLP-1 in thosehypothalamic neurons endowed with GLUT-2 and glucokinase. GLP-1, hyper-secreted by L-cells after a meal, is a potent insulinotropic agent and, together withglucose, reduces food intake and induces c-fos in the ARC. PACAP is present in theARC, PVN, and SCH, and its hypothalamic type I receptor elevates cAMP and inos-itol triphosphate in the PVN, where it may perhaps antagonize NPY-induced foodintake and hyperinsulinemia. However, irrelevant neuroendocrine, autonomic, andcircadian functions are also activated by this peptide, making it a less than ideal baseon which to build an obesity treatment.

CENTRAL ROLE OF HYPOTHALAMUS IN EATING PATTERNS

The “set point” hypothesis of food intake and energy expenditure (FIG. 1) considers thattwo vegetative centers in the hypothalamus are reciprocally regulated: the PVN-DMN(paraventricular-dorsomedial nucleus of the hypothalamus) in the mediodorsal area initi-ates and maintains feeding and has an anabolic role, while the satiety VMN (ventromedialhypothalamic nucleus) center in the laterobasal area stops feeding and plays a catabolicrole. Accordingly, goldthioglucose (GTG) or electrolytic lesions of the VMN lead tohyperphagia in mice and rats, respectively. The resulting dynamic obesity is accompaniedby cholinergic vagal hyperactivity with hyperinsulinemia,1 and decreased sympatheticactivity with diminished energy expenditure through body heat.

The subfornical organ, vascular organ of the lamina terminalis, and area postrema (AP)lack a blood-brain barrier so that the hypothalamus can respond to blood-borne peptidesand proteins. Systemic insulin crosses the saturable blood barrier by transcytosis throughfenestrated endothelia in the median eminence (ME) and choroid plexus. Together withglucose it acts as glucostat at the VMN level. Another afferent signal, the protein hormoneleptin secreted by white adipose tissue (WAT), acts as a lipostat at the arcuate nucleus(ARC) level, the important center producing neuropeptide tyrosine(1-36) (NPY). Thesesignals constantly indicate the quantity and composition of energy stores to the hypothal-amus. Efferent hypothalamic mechanisms control appetite through neuropeptides (such ascorticotropin-releasing factor) and other neurotransmitters, as well as energy expenditure(through the autonomic nervous system).

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Corresponding author: Tel.: 322-650.52.99; Fax: 322-650.53.24; E-mail: [email protected]

The hypothalamic disorders of obesity often include, beside hyperphagia, vagal hyper-insulinism, a low central orthosympathetic tone (with reduced thermogenesis), low sero-tonin efficacy, a hyperactive hypothalamo-hypophyso-adrenal axis, a hypoactiveGHRH-GH-IGF (growth hormone-releasing factor–growth hormone–insulin-like growthfactor) axis, and hypogonadism of central origin. Hyperlipogenesis, glucose intolerance,and excessive gluconeogenesis are secondary features.

HYPOTHALAMIC GLUCOREGULATORY NEURONS, INSULIN, AND NPY

Glucose transporter 4 (GLUT 4) in the hypothalamus is sensitive to circulating insulin.2

In rats, intravenous insulin when given alone brings about glucoprivic feeding while thesame doses of insulin are anorexigenic if combined with doses of glucose that preventhypoglycemia (FIG. 1). In the rat there is a delay of 30 min between a single injection ofinsulin and the stimulation of food intake due to hypoglycemia. Glucopenia increases c-fos in the VMN, then elevates corticotropin-releasing factor(1-41) (CRF) and somato-statin (SS) levels in the PVN.3 This lack of glucose decreases the firing rate in the VMN,increases firing in lateral hypothalamus area (LH) and nucleus of the tractus solitarius(NTS) glucosensors, and increases sympathetic outflow through the splanchnic nerves,provoking increased hepatic glucose production. Intracerebroventricular 2-deoxy-D-glucose (2-DG) also produces central glucoprivation, but without hypoglycemia. Theresulting local hypothalamic glucopenia induces c-fos gene expression in 20% of NPYneurons in the ARC, and 3–6% of SS neurons in the ARC and PVN.4 A 3-h direct perfu-sion of the forebrain with a moderate glucose concentration via the carotid leads to a dou-bling in c-fos neurons in the VMN, parvocellular PVN (rich in CRF neurons), and DMN.5

An increase in portal vein glucose decreases the rate of vagal visceral signals to theNTS (FIG. 2). The NTS is a glucose-sensitive nucleus adjacent to the fourth ventricle in themedulla oblongata which is a relay center to the PVN, ARC and DMN. PVN axons project

324 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 1. The hypothalamic control of food intake and energy expenditure (Σ: sympathetic).

dorsocaudally to the locus ceruleus, central gray and lateral parabrachial nucleus (LPBN).The locus ceruleus and LPBN project to the NTS and the DMN. The central gray andLPBN areas project to sympathetic neurons in the intermediolateral cell column of thespinal cord. In addition, these complex autonomic connections interplay with several neu-ropeptides. For instance, the ARC contains NPY, GHRH, proopiomelanocortin (POMC),

CHRISTOPHE: APPETITE AFTER GLP-1 AND PACAP? 325

FIGURE 2. A scheme showing autonomic connections with gut and islets and resulting in vivoeffects (MR: muscarinic receptors; β and D cells secrete insulin and SS, respectively).

galanin (GAL), and norepinephrine (NE) cell groups. The dorsal parvocellular division ofthe PVN contains CRF and SS neurons. CRF neurons project to parasympathetic and sym-pathetic preganglionic cell groups in the brainstem and spinal cord.

NPY is the most potent and long-lasting orexigenic agent yet identified in rats. Thisabundant brain peptide is notably produced in the ARC, whose nerve fibers project into thePVN, DMN, VMN, PFH (perifornical hypothalamus), and MPOA (medial preoptic area).When administered centrally NPY releases insulin acutely via the efferent parasympa-thetic nervous system (from the LH to the vagus of the medulla then the pancreatic vagus),even in the absence of food.6,7 NPY-induced feeding is attenuated by peripheral glucoseinfusion.8 The injection of insulin into the third ventricle or hypothalamus prevents the risein NPY mRNA and NPY levels observed in fasting rats (FIG. 3). Thus, circulating insulinacts as a negative feedback loop to inhibit hypothalamic NPY mRNA levels and peptidesecretion as it readily enters the ARC, which contains many insulin receptors.9–17 On theother hand, dexamethasone treatment increases hypothalamic NPY mRNA and the NPYcontent in ARC and PVN through CORT receptors (while it suppresses CRF mRNA lev-els within the PVN).18,19 NPY synthesis also depends on cAMP20: CREB (cAMP-regulatedresponse element–binding protein), when phosphorylated by PKA (protein kinase A),dimerizes showing afterwards a tenfold higher affinity for CRE. Besides, activators ofCa2+- or PL (phospholipase)-dependent protein kinases, such as TPA and NGF (nervegrowth factor), also regulate NPY expression.21

NPY infusion into the PVN elicits feeding within 30 min, which is associated withhypothermia22 and c-fos expression, mostly in the parvocellular (SS-CRF–rich) region ofthe PVN but also in the magnocellular (arginine vasopressin–rich) PVN region, DMN,SON (supraoptic nucleus), and BNST (bed nucleus of the stria terminalis). At low con-


FIGURE 3. The control of NPY synthesis in the ARC and that of GLP-1 (and PACAP) on the PVNinfluence feeding (5-HT2BR and α2R: serotonin and α2 adrenergic-receptors).

centration (0.15–15 nM) NPY and [Pro34]-NPY increase discharge rates in single PVNneurons.23 Among extrahypothalamic sites, NPY-induced feeding augments c-fos in thelateral subdivisions of the central amygdaloid nucleus and solitary tract.24,25

NPY may also inhibit the satiety center in VMN after food deprivation. The electro-physiological unit activity in VMN neurons is intensively suppressed by NPY applied invitro.26 Besides, the orexigenic effect of intracerebroventricularly administered NPY islacking in fasted rats with bilateral electrolytic lesions in the VMN (and too much NPYalready produced in the ARC),27,28 while administration of NPY antibodies abolisheshyperphagia in these VMN-lesioned rats.29 GTG-sensitive VMN neurons play little role inthe induction of NPY mRNA by fasting in the ARC, as fasting increases NPY levelsmRNA in the ARC of GTG-treated mice.30

NPY-induced feeding is mediated by Y5 receptors. The rat hypothalamic Y5 receptormRNA encodes a 456–467 amino acid protein with less than 33% overall identity to the otherNPY receptors and is found primarily in the PVN and lateral hypothalamus. The Y5 receptoroccupancy stimulates food intake, being therefore the main “feeding” receptor subtypeinvolved in obesity.31,32 The mouse NPY Y5 receptor shares 60% amino acid identity to the Y1receptor and its pharmacology resembles that of the anxiolytic Y1 receptor. It is abundantlyexpressed in the PVN, but also within the VMN, SCH (suprachiasmatic nucleus), BNST, andanterior hypothalamus.33 Hypothalamic NPY receptors are coupled to cAMP-dependent PKAand Ca2+/calmodulin-dependent protein kinase II that activate CREB, allowing increased CREbinding. Indeed, NPY injected in the rat PFH provokes an intense phosphoCREB signal in thePVN and VMN of rats, similar to that observed after food deprivation.34

GLP-1 IN THE ENTERO-HYPOTHALAMIC AXIS

Most frequently in obese rodents and obese humans the ARC reacts poorly to the lep-tin hypersecreted by adipose tissue, so that the local synthesis of NPY is unchecked (FIG. 3). Feeding is induced neither in ob/ob mice by intracerebroventricular injection of2-DG nor in fa/fa rats by intracerebroventricular injection of insulin. This may reflect asecondary reduced insulin receptor concentration and/or impaired glucose utilization in thehypothalamus of these genetically obese rodents.15,35 Anti-obesity drugs must thereforeincrease the sensitivity of the ARC towards leptin and insulin, thereby antagonizing thesynthesis of NPY. They could as well inhibit the action of NPY on the PVN. Is GLP-1worth evaluation in this respect?

In rat, pro-GLP-1–glucagon mRNA and GLP-1 are localized in both open-type L cellsof the distal intestine and in NTS neurons, whose efferent fibers go to the hypothala-mus36 –38 (FIG. 2). Rat brain GLP-1 receptors may therefore act as the target for blood borneas well as for central GLP-1 in those hypothalamic nuclei involved in appetite.39– 41 ThemRNAs for GLP-1 receptors, GLUT-2, and GK are indeed colocalized in the PVN, ARC,VMN, and SON42– 44 and there is only one GLP-1 receptor isoform known.45,46

Intracerebroventricular administration of GLP-1 powerfully inhibits feeding in fastedrats as well as angiotensin II-induced drinking behavior and induces c-fos accumulation inthe PVN and central amygdaloid nucleus. Intracerebroventricular administration ofexendin-4(9-39)amide, the N-shortened derivative of exendin-4 (a peptide from helodermasuspectum venom), acts as a specific GLP-1 antagonist. It blocks the inhibitory effect ofGLP-1 on food intake, doubles food intake in satiated rats, and augments the feedingresponse to NPY.43,47– 49 GLP-1 produces an immediate secretion of aspartic acid and glut-amine (from astrocytes?) in the VMN.50

Are peripheral effects of GLP-1 contributing to satiety: perhaps but indirectly (FIG. 2).There is a pulsatile secretion of GLP-1 from intestinal L cells in fasted man with a frequency


of 5–7/h. GLP-1 is hypersecreted after a carbohydrate or fat-rich meal. More specifically, theamplitude of plasma GLP-1 pulses increases after a 100 g oral glucose load but atropine infu-sion delays and reduces this increased amplitude,51 suggesting a direct muscarinic (M3?)52

stimulatory control of L cells. Hence, an oral glucose load may provoke a higher GLP-1response when the vagal celiac branch allows a cholinergic-sensitizing effect.

GLP-1 is the most potent insulinotropic hormone known and is also able to induceproinsulin biosynthesis. Accordingly, exendin-4(9-39) reduces insulin secretion in vivo inrats after a standard meal.53 In vitro, in the presence of glucose, the immediate effects ofGLP-1 include an elevation of cAMP and a potentiation of membrane depolarization. Thisβ cell depolarization is due to (1) the closure of ATP-sensitive K+ channels by ATP derivedfrom glycolysis that provokes Ca2+ influx through the opening of voltage-dependent Ca2+

channels (VDCCs) and (2) the PKA phosphorylation of VDCCs increasing opening prob-ability and the activation of non selective cation channels (NSCCs) promoting an inwardNa+ (Ca2+)-dependent current.54,55 This glucose-dependent insulin secretion is enhanced bycarbachol releasing Ca2+ from intracellular pools through phospholipase C.56 In fa/fa pan-creas the glucose threshold for the insulinotropic action of GLP-1 is reduced and, in thepresence of 4.4 mM glucose, GLP-1 increases insulin secretion 25-fold, whereas there isonly a fivefold stimulation in Fa/? pancreas, showing increased GLP-1 sensitivity in the βcells from obese animals.57 GLP-1 inhibits glucagon secretion in vivo in rat and pig,another way to lower blood glucose. This inhibitory action of GLP-I on intact islets ismediated by a paracrine release of SS from D cells.58,59

GLP-1 also inhibits gastric acid secretion, delays gastric emptying and secondaryexocrine pancreatic secretion, and inhibits upper gastrointestinal secretion and motility inhumans (the “ileal-brake” effect).60– 62 The inhibitory effects of GLP-1 on the stomach areobserved not only after pentagastrin stimulation but also under vagal induction throughsham feeding in man. A centrally mediated mechanism is implied, considering the absenceof effects in vagotomized subjects.63,64 In isolated rat parietal cells GLP-1 stimulates H+

production (aminopyrine accumulation) via cAMP, a finding not duplicated in vivo in rat,65

suggesting that the direct stimulatory effect on parietal cells is overcome by SS release thatmediates Gi-adenylyl cyclase inhibitory transduction.66,67

PACAP AS A POSSIBLE LINK IN OBESITY

The neuropeptide pituitary adenylyl cyclase activating peptide (PACAP), first isolatedfrom ovine hypothalamus,68 is widely distributed in the brain. It is notably present in therat hypothalamus. PACAP perikarya are found in the PVN, ARC, SCH, VMN, SON, pre-optic periventricular zone, retrochiasmatic area, tuber cinereum, and mammillary nuclei.PACAP axons are present in an ascending pathway from the SCH (the brain’s biologicalclock) to the PVN and in the BNST (a relay between limbic and autonomic structures).

PACAP type 1 receptors (PACAP-R) are distributed even more homogeneously inbrain. Such receptors are present in the PVN, SON, SCH, retrochiasmatic area, ME, andposterior periventricular nucleus as well as the BNST, dorsal vagal nucleus, and amyg-daloid complex. The PACAP type I receptor gene is expressed in both the magnocellu-lar (arginine vasopressin) and parvocellular (SS/CRF) parts of the PVN and in the SONin rats.69-72 Alternative splicing of two exons for the region encoding the third intracellu-lar loop of rat PACAP type 1 receptors generates five isoforms with differential signaltransduction properties. A sixth splice variant produces a 21 amino acid deletion in theN-terminal extracellular domain showing altered receptor selectivity with respect toPACAP-27 and -38 binding.73 PACAP present in nerve terminals around and within ratislets activates L-type Ca2+ channels through a seventh short PACAP receptor isoformwith discrete sequences in TM-II and TM-IV.74


The type-1 PACAP-R and heavier PACAP-R-hop splice variant mRNAs operating inthe rat PVN75 stimulate cAMP and inositol triphosphate formation. PACAP as well as feed-ing inhibits strongly the binding of rat hypothalamic nuclear extracts to CRE while NPYadministration or food deprivation exerts the opposite effect, suggesting that CREBdephosphorylation is the satiety signal that negatively modulates genes involved in feed-ing.34 In food-deprived mice, intracerebroventricularly administered PACAP reduces foodintake and increases motor activity (rearing and grooming through CRF release?). It alsoantagonizes NPY-induced food intake: an intrahypothalamic injection of PACAP 10 minprior to the injection of NPY reduces feeding during the 4-h measurement period.76 –78

PACAP is the most potent glucose-dependent insulinotropic neuropeptide known.79 Itstimulates insulin and glucagon secretion in vivo and in vitro in rat and mouse.80,81 Themode of action of PACAP on β cells is similar to that exerted by GLP-1 acting via its ownreceptor (it augments insulin secretion glucose-dependently by increasing cAMP and Ca2+;see above).

PACAP present in the gut “little brain” (in submucous more frequently than in myen-teric ganglia)82 inhibits pentagastrin- and histamine-stimulated gastric acid secretion byinteracting with histamine effects on parietal cells.83 In the rat jejunal mucosa PACAP pro-motes anion secretion via submucous neurons.84,85 PACAP also relaxes smooth muscle inall portions of the rat gastrointestinal tract by activating apamin-sensitive K+ channelsthrough PACAP type I receptors and inhibits the contraction mediated by VIP (acting ontype II receptors in cholinergic neurons).85,86 The involvement of PACAP in several moreautonomic and neuroendocrine functions, as well as regulation of the circadian pacemaker,is demonstrated by several observations (this volume and Christophe87).

GLP-1 AGONISTS AS THERAPEUTIC ANOREXIGENIC AGENTS?

The United States population (with 33% obese adult subjects) spends 33 billion dol-lars each year on weight loss products and services while drug companies are spendinghundreds of millions of dollars on developing obesity treatments. Alongside other pep-tides considered for evaluation (FIG. 4) as anorexigenic agents [NPY-5 antagonists andcholecystokinin A (CCK-A), bombesin, amylin, and melanocyte-simulating hormone-4(or melanin-concentrating hormone?) agonists], is there any clinical usefulness con-ceivable for GLP-I or PACAP? The half-life of GLP-I in human blood is only 5 min, dueto its rapid degradation by enzymes derived from plasma cell membranes, such as


FIGURE 4. The positions of GLP-1 as a candidate among other anorexigenic agents (Σ: sympathetic tone).

dipeptidyl-peptidase IV, producing the desHis-Ala derivative.88 Besides endopeptidase24.11 could cleave GLP-1 at Phe6-Thr7, Tyr13-Leu14, and Glu21-Phe22. Therefore,drug design for longer acting, peptidase-resistant GLP-I/exendin-4 analogs with pro-tracted efficacy after subcutaneous, sublingual, or oral administration may be requiredbefore considering efficient clinical trials.

The multiple irrelevant neuroendocrine, autonomic of PACAP, and circadian functions,including those exerted through PACAP-VIP type 2 receptors of PACAP, prevent thedevelopment of obesity treatment based on this anorexigenic neuropeptide.

SUMMARY

Antiobesity drugs must inter alia increase the sensitivity of the hypothalamic satietycenter towards leptin and antagonize the synthesis and action of NPY. Unfortunately, thearray of pharmacologic tools available is as vast as, presently, ineffective. Among peptideanalogs considered for evaluation [NPY-5 antagonists and CCK-A, bombesin, amylin andmelanocyte-simulating hormone-4 (or melanin-concentrating hormone?) agonists], isthere a place for GLP-1 and PACAP? GLP-1 receptors present in ARC, PVN, VMN, andSON are the target for both central and blood-borne GLP-1 in those hypothalamic neuronsendowed with GLUT-2 and glucokinase. GLP-1, hypersecreted by L-cells after a meal, isa potent insulinotropic agent and, together with glucose, reduces food intake and inducesc-fos in the ARC. Concerning PACAP present in the ARC, PVN, and SCH, its hypothala-mic type I receptor elevates cAMP and inositol triphosphate in the PVN where it may per-haps antagonize NPY-induced food intake and hyperinsulinemia. However, irrelevantneuroendocrine, autonomic and circadian functions are also activated by this peptide.

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