Melanocortin-4 receptor activation stimulates hypothalamic brain-derived neurotrophic factor release to regulate food intake, body temperature and cardiovascular function.

In the present study, we aimed to investigate the neuromodulatory role played by hypothalamic brain-derived neurotrophic factor (BDNF) in the regulation of acute cardiovascular and feeding responses to melanocortin-4 receptor (MC4R) activation. In vitro, a selective MC4R agonist, MK1, stimulated BDNF release from isolated rat hypothalami and this effect was blocked by preincubation with the MC3/4R antagonist SHU-9119. In vivo, peripheral administration of MK1 decreased food intake in rats and this effect was blocked by pretreatment with an anti-BDNF antibody administered into the third ventricle. When anorexia was induced with the cannabinoid-1 receptor (CB1R) antagonist AM251, the anti-BDNF antibody did not prevent the reduction in food intake. Peripheral administration of MK1 also increased mean arterial pressure, heart rate and body temperature. These effects were prevented by pretreatment with the anti-BDNF antibody whereas the intracerebroventricular administration of BDNF caused changes similar to those of MK1. These findings demonstrate for the first time that activation of MC4R leads to an acute release of BDNF in the hypothalamus. This release is a prerequisite for MC4R-induced effects on appetite, body temperature and cardiovascular function. By contrast, CB1R antagonist-mediated anorexia is independent of the MC4R/BDNF pathway. Overall, these results show that BDNF is an important downstream mediator of the MC4R pathway.

Pharmacotherapy of obesity.

Obesity has become a significant health problem in industrialised and developing countries, and despite all nutritional and behavioural approaches, its prevalence is still increasing. In recent years, the identification and characterisation of central and peripheral mechanisms involved in the regulation of energy balance has made remarkable progress and provided numerous targets for novel anti-obesity agents. However, only few anti-obesity drugs are on the market and not many compounds have entered clinical development. In the present review, the clinically available agents are discussed and their pharmacological profiles are compared. Some of the drugs that are currently in clinical development are mentioned as examples of the possible future range of anti-obesity agents. Selected topics in drug discovery are presented to illustrate novel targets and concepts for the pharmacotherapy of obesity.

Molecular pathways to obesity.

Obesity results from a chronic imbalance between energy intake and energy expenditure. Environmental factors, such as the increased availability of high caloric food or the decreased need for physical activity, contribute to its development and their influence is amplified by genetic predisposition. In recent years remarkable progress has been made in the understanding of the pathophysiology of obesity. Although most of the insights into the regulation of energy balance have been obtained in rodent models, the rare clinical cases of monogenic obesity provided evidence for the importance of several of these mechanisms in humans. The identification of leptin as a factor originating from adipose tissue and informing the brain about the status of energy reserves firmly established the concept of long-term regulation of body fat stores. The disappointing therapeutic results with leptin in obese patients could be explained by the fact that during evolution this hormone developed rather as a starvation signal than as an adiposity signal. It is conceivable that the pharmacological interference with mechanisms downstream of leptin, for example with the melanocortin pathway, might be therapeutically more promising. The discovery of new molecular mechanisms involved in the regulation of the differentiation and proliferation of adipocytes and the elucidation of their paracrine and endocrine functions have changed the traditional view of adipose tissue as an inert depot for triglycerides. The identification of new uncoupling proteins could modify the current concepts of the regulation of thermogenesis in humans. The remarkable progress in the identification of novel targets involved in the regualtion of energy balance should have a positive impact on the search for new antiobesity agents.

Pharmacotherapy of obesity: targets and perspectives.

The search for anti-obesity agents has become one of the most exciting areas in drug discovery. Subsequent to an enormous increase in the number of possible molecular targets, the focus has shifted from target identification to target validation. Because important biological functions such as the regulation of energy intake and expenditure are controlled by complex systems, an improved understanding of pathophysiology is a prerequisite for the selection of successful development candidates for the treatment of obesity. Although most of the information on the regulation of energy balance has been obtained from rodents, various monogenic forms of human obesity provide clinical proof of concept for some of these mechanisms. However, it is still not known which are the most promising clinical approaches to lowering body weight and subsequently reducing morbidity and mortality.

Food intake in free-feeding and energy-deprived lean rats is mediated by the neuropeptide Y5 receptor.

The new neuropeptide Y (NPY) Y5 receptor antagonist CGP 71683A displayed high affinity for the cloned rat NPY Y5 subtype, but > 1, 000-fold lower affinity for the cloned rat NPY Y1, Y2, and Y4 subtypes. In LMTK cells transfected with the human NPY Y5 receptor, CGP 71683A was without intrinsic activity and antagonized NPY-induced Ca2+ transients. CGP 71683A was given intraperitoneally (dose range 1-100 mg/kg) to a series of animal models of high hypothalamic NPY levels. In lean satiated rats CGP 71683A significantly antagonized the increase in food intake induced by intracerebroventricular injection of NPY. In 24-h fasted and streptozotocin diabetic rats CGP 71683A dose-dependently inhibited food intake. During the dark phase, CGP 71683A dose-dependently inhibited food intake in free-feeding lean rats without affecting the normal pattern of food intake or inducing taste aversion. In free-feeding lean rats, intraperitoneal administration of CGP 71683A for 28 d inhibited food intake dose-dependently with a maximum reduction observed on days 3 and 4. Despite the return of food intake to control levels, body weight and the peripheral fat mass remained significantly reduced. The data demonstrate that the NPY Y5 receptor subtype plays a role in NPY-induced food intake, but also suggest that, with chronic blockade, counterregulatory mechanisms are induced to restore appetite.

Neuropeptide Y Y1 receptor antisense oligodeoxynucleotides enhance food intake in energy-deprived rats.

In the literature, conflicting data on the effect of NPY Y1 antisense oligodeoxynucleotides (ODNs) on food intake have been reported, describing either an increase or a decrease in feeding in antisense-treated animals. In the present studies antisense oligodeoxynucleotides targeted to the Y1 receptor (Y1 antisense ODNs) were used to re-investigate the functional importance of this receptor subtype in vivo in the regulation of feeding in rats. We used phosphothioate-terminal protected derivatives of two ODN sequences used in previous reports. In addition, as one of these sequences was not tested in vitro, we demonstrated its efficacy in LMTK-cells transfected with the Y1 receptor subtype. In vivo, repeated intracerebroventricular (i.c.v.) injections of Y1 antisense ODNs did not affect basal food intake or the increase in food intake after i.c.v. injection of neuropeptide Y (NPY, 300 pmol). Y1 antisense ODNs given intracerebroventricularly enhanced food intake in energy-deprived rats (+175% and +60% vs. control scrambled and sense sequences, respectively after 2 h of refeeding). Analysis of the structure of feeding behaviour revealed that Y1 antisense ODNs enhanced fasting-induced food intake during the first hour of refeeding by inducing increases in meal size (+143% and +155% vs. sense and scrambled ODNs) but not meal duration. These data suggest that the NPY Y1 receptor is not directly implicated in feeding in the rat when calorie intake is normal but might be specifically activated during energy deprivation.

Inhibition of food intake by neuropeptide Y Y5 receptor antisense oligodeoxynucleotides.

The recently discovered rat neuropeptide Y (NPY) receptor, the Y5 subtype, has been proposed to mediate the NPY-induced feeding response and therefore plays a central role in the regulation of food intake. These conclusions were based on studies with peptidic agonists. We now report studies in which phosphothioate end-protected antisense oligodeoxynucleotides (ODNs) targeted to prepro NPY (prepro NPY antisense ODNs) or to the Y5 receptor (Y5 antisense ODNs) were used to assess the functional importance of this novel receptor subtype in vivo. NPY antisense ODNs given intracerebroventricularly to rats prevented the increase in hypothalamic NPY levels during food deprivation and inhibited fasting-induced food intake. Likewise, repeated intracerebroventricular injections of Y5 antisense ODNs prevented fasting-induced food intake in rats. Moreover, two Y5 antisense ODNs, targeted to different sequences of the receptor, significantly decreased basal food intake and inhibited the increase in food intake after intracerebroventricular injection of NPY. These effects proved to be selective, since the feeding response to galanin was not affected. Analysis of the structure of feeding behavior revealed that prepro NPY and Y5 receptor antisense ODNs reduced food intake by inducing decreases in meal size and meal duration analogous to the orexigenic effects of NPY that are mediated by increases in these parameters. Although changes in Y5 receptor density could not be measured, the results with Y5 antisense ODNs strongly suggest that this receptor subtype mediates the feeding response to exogenous and endogenous NPY. Selective Y5 antagonists may therefore be of therapeutic value for the treatment of obesity and eating disorders.

A receptor subtype involved in neuropeptide-Y-induced food intake.

Neuropeptide Y (NPY) is a powerful stimulant of food intake and is proposed to activate a hypothalamic 'feeding' receptor distinct from previously cloned Y-type receptors. This receptor was first suggested to explain a feeding response to NPY and related peptides, including NPY2-36, that differed from their activities at the Y1 receptor. Here we report the expression cloning of a novel Y-type receptor from rat hypothalamus, which we name Y5. The complementary DNA encodes a 456-amino-acid protein with less than 35% overall identity to known Y-type receptors. The messenger RNA is found primarily in the central nervous system, including the paraventricular nucleus of the hypothalamus. The extent to which selected peptides can inhibit adenylate cyclase through the Y5 receptor and stimulate food intake in rats correspond well. Our data support the idea that the Y5 receptor is the postulated 'feeding' receptor, and may provide a new method for the study and treatment of obesity and eating disorders.

Mechanism of interaction between neuropeptide Y and angiotensin II in the rabbit femoral artery.

Neuropeptide Y has direct vasoconstrictor actions and potentiates the effects of other vasoconstrictor agents. To find out whether both effects of neuropeptide Y are mediated via the same receptor and intracellular mechanism, the interaction between neuropeptide Y and angiotensin II was studied in rabbit femoral arteries. In this preparation, neuropeptide Y, but not its 13-36 fragment, induced constriction. Only neuropeptide Y potentiated the vasoconstrictor response to angiotensin II and the associated rise in inositol-1-phosphate. These potentiating effects of neuropeptide Y were totally prevented by removal of extracellular Ca2+, partially prevented by a Ca(2+)-channel blocker and mimicked by a Ca(2+)-channel activator. Pharmacological modulation of adenylate cyclase had no effect. These results suggest that the direct and indirect vascular effects of neuropeptide Y are mediated via Y1 receptors and depend on the influx of extracellular Ca2+. The rise in inositol-1-phosphate seems to be secondary to an increase in intracellular Ca2+, while modulation of adenylate cyclase is apparently not involved.

Effects of a V1-vasopressin antagonist on ACTH release following vasopressin infusion or insulin-induced hypoglycemia in normal men.

Experimental evidence indicates that arginine vasopressin contributes to the release of adrenocorticotropic hormone under certain conditions. We studied for the first time the AVP antagonist [d(CH2)5 Tyr(Me)AVP] in 6 normal men in order to evaluate the possible role of AVP as an ACTH-releasing hormone during insulin-induced hypoglycemia. To test the agent's capacity to inhibit an ACTH release by exogenous AVP, we compared the ACTH response to an infusion of 300 ng AVP/min a. 30 min after injection of 5 micrograms/kg of the antagonist, b. after injection of placebo (0.9% NaCl). Plasma ACTH levels during AVP infusion rose from 17.2 +/- 1.6 ng/l (3.8 +/- 0.35 pmol/l) to 31.7 +/- 4.2 ng/l (7.0 +/- 0.92 pmol/l) at 40 min after injection of the antagonist, the difference to the control-group (increment from 16.5 +/- 1.2 ng/l (3.6 +/- 0.26 pmol/l) to 41.8 +/- 3.5 ng/l) (9.2 +/- 0.77 pmol/l) being significant (p less than 0.05). Peak plasma cortisol levels were 323 +/- 42 and 529 +/- 52 nmol/l, respectively (p less than 0.05). We then tested the compound in the same subjects during an insulin-induced hypoglycemia; 30 min after administration of 10 micrograms/kg of the AVP antagonist or placebo, all subjects received 0.12 IU/kg of normal insulin, thus inducing a fall of blood glucose levels below 2 mmol/l. The AVP antagonist caused a moderate but insignificant reduction of the rise in plasma ACTH and a slightly greater, significant reduction of the increment in plasma cortisol (350 +/- 19 nmol/l with antagonist and 469 +/- 90 nmol/l with placebo, p less than 0.05) during insulin-induced hypoglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Haemodynamic effects of acute and chronic renin inhibition in marmosets.

We have developed marmoset models for the in vivo evaluation of primate-specific inhibitors of human renin. After acute intravenous administration to normotensive sodium-depleted marmosets, renin inhibitors of different structural types induced a maximum hypotensive response of a magnitude similar to that induced after angiotensin converting enzyme (ACE) inhibition. The response was prevented by pretreatment with an ACE inhibitor. A close relationship between the inhibition of plasma renin activity (PRA) and the fall in blood pressure was observed with most of the inhibitors. CGP 29,287, a synthetic renin inhibitor, and R-3-36-16, a monoclonal antibody, both induced a selective increase in renal blood flow similar to that induced by an ACE inhibitor. A sustained reduction in blood pressure was observed during continuous administration of CGP 29,287 or R-3-36-16 over 14 days, despite an increase in immunoreactive renin and an apparent recovery of PRA. A similar blood pressure fall and an increase in plasma renin was observed during continuous administration of an ACE inhibitor. The renin inhibitor CGP 29,287 also lowered blood pressure after acute administration to hypertensive marmosets with normal PRA. Our studies demonstrate that renin inhibitors have similar haemodynamic effects to ACE inhibitors, and indicate that they may have a similar antihypertensive efficacy.

Renin inhibitors as possible antihypertensive agents.

The blockade of the renin-angiotensin system by inhibition of angiotensin-converting enzyme has become an established principle in the treatment of hypertension. This has stimulated interest in the inhibition of renin, the enzyme which catalyzes the first and rate-limiting step in the formation of angiotensin II. During recent years, considerable progress has been made in the development of renin inhibitors. Several potent and selective compounds have been synthesized and studied in vitro and in vivo. In experimental animals with a stimulated renin-angiotensin system the haemodynamic effects of renin inhibitors have been shown to be identical to those of angiotensin-converting enzyme inhibitors. The two classes of agents also have similar antihypertensive effects in various forms of experimental renal hypertension. However, it is not clear whether then efficacy will be comparable in patients with essential hypertension. The main shortcoming of the currently available renin inhibitors is their low bioavailability after oral administration and their short duration of action. The improvement of these pharmacokinetic properties is the main challenge for future research in this area.

Biologic response to chronic blockade of vasopressin receptors in Sprague-Dawley rats.

The renal tubular arginine vasopressin receptor antagonist, d-(CH2)5-D-Tyr(Et)VAVP, is a potent inhibitor of the vasopressin-induced stimulation of adenylate cyclase in rat renal medullary homogenates in vitro. In acute experiments in vivo, this antagonist increased urine volume and decreased urine osmolality after i.v. or s.c. administration in normally hydrated or dehydrated Sprague-Dawley rats. It did not show any effects in water-loaded rats. The duration of action of the antagonist was between 3 to 4 hr. Chronic i.v. infusion or repeated s.c. injections did not result in a persistent diabetes insipidus. A transient rise in water excretion was followed by a progressive normalization. The marked initial water loss was fully compensated for by an increased water intake so that plasma volume and extracellular fluid volume remained unchanged. After 1 week of treatment with the antagonist, glomerular filtration rate and plasma renin activity were not significantly different from base-line values. Only small functional deficits in renal concentrating capacity became manifest when drinking water was withheld. It is possible that the activation of endogenous compensatory mechanisms restored water balance during chronic arginine vasopressin receptor blockade. An intrinsic agonism of this antagonist, which was not detectable in acute experiments, might have contributed to the normalization of water balance by limiting the maximum anti-antidiuretic effects of renal tubular arginine vasopressin receptor blockade.

Evaluation of the pharmacologic properties of a vasopressin antagonist in Brattleboro rats.

The arginine vasopressin (AVP) analog d-(CH2)5-D-Tyr(Et)VAVP is a potent competitive antagonist of AVP at renal tubular AVP receptors. In Sprague-Dawley rats, this compound induces diuresis after single injections but only a transient diabetes insipidus-like state during continuous infusion. To further evaluate the pharmacologic profile of d-(CH2)5-D-Tyr(Et)VAVP, the present experiments were performed in Brattleboro rats homozygous for hereditary hypothalamic diabetes insipidus. In these rats, acute and chronic administration of the antagonist induced significant antidiuretic effects. These agonistic effects persisted for up to 4 days after single injections and for more than 2 weeks after stopping continuous infusions. The antidiuretic effects of the antagonist during chronic administration were indistinguishable from those of AVP replacement. When the renal tubular AVP receptor antagonist was infused into diabetes insipidus rats that had received AVP for 1 week, it induced a transient rise in water intake. However, the peak values after administration of the antagonist were much lower than after AVP withdrawal. These observations suggest that d-(CH2)5-D-Tyr(Et)VAVP has substantial agonistic properties that are not detectable in Sprague-Dawley rats except for limiting the compound's maximum anti-antidiuretic efficacy. These agonistic effects together with endogenous compensatory mechanisms may allow Sprague-Dawley rats to maintain a normal water balance during the continuous administration of d-(CH2)5-D-Tyr(Et)VAVP.

[3H]AVP binding to rat renal tubular receptors during long-term treatment with an antagonist of arginine vasopressin.

The interaction of an antagonist of arginine vasopressin (AVP), d(CH2)5-D-Tyr(Et)VAVP, with renal tubular V2 receptors were studied in medullary membrane preparations from kidneys of Sprague-Dawley and Brattleboro rats. In both rat strains, V2 receptors had comparable KD and Bmax values for binding of [3H]AVP. In vitro studies revealed that the V2-antagonist was more potent than cold AVP in displacing [3H]AVP. In vivo treatment of Sprague-Dawley rats with the antagonist over one week resulted only in a transient state of diabetes insipidus (DI). No specific [3H]AVP binding was detectable throughout the period of administration. Chronic treatment of Brattleboro rats resulted in a complete normalization of water intake. This agonistic effect was also associated with undetectable [3H]AVP binding. After stopping the infusion of d(CH2)5-D-Tyr(Et)VAVP, Bmax values tended to rise but had still not reached base line values after 6 days. In contrast, the chronic infusion of AVP in Brattleboro rats resulted in a reduction in water intake which was accompanied by a decreased Bmax. [3H]AVP binding remained detectable during the entire treatment period. Thereafter Bmax was restored to base line values within 2 days of stopping the infusion. These results suggest that d(CH2)5-D-Tyr(Et)VAVP has a high affinity for V2 receptors in both Sprague-Dawley and Brattleboro rats. Its rate of dissociation from the receptor appears to be much slower than that of AVP. In Brattleboro rats, the binding of d(CH2)5-D-Tyr(Et)VAVP leads to an antidiuretic response. In Sprague-Dawley rats, a transient diuretic response is followed by a progressive normalization in water intake. This occurs despite persistent and complete blockade of renal medullary V2 receptors.

Responsiveness of locus ceruleus neurons in hypertensive rats to vasopressin.

We studied the actions of vasopressin administered microiontophoretically onto neurons of the locus ceruleus in rats with deoxycorticosterone-acetate (DOCA)-salt hypertension and in control (normotensive) rats. Rats were studied at 3 days (prehypertensive stage) and 4 to 6 weeks after DOCA-salt treatment (chronic hypertensive stage). Experiments were performed in anesthetized rats using conventional microiontophoretic and single-cell recording techniques. Three days after DOCA-salt administration, the treated rats showed no rise in arterial pressure in comparison with control rats, but 4 to 6 weeks later, the treated rats had significantly greater pressures (p less than 0.01) than controls. Vasopressin administered with currents of 10 to 90 nA for 1 minute produced a current-dependent increase in the firing rate of noradrenergic neurons in all rats. Increases in the firing rate of noradrenergic neurons in DOCA-salt-treated rats, whether in the prehypertensive or the chronic stage, were significantly greater than increases in control rats. These findings indicate that 1) vasopressin can affect neuronal activity in the locus ceruleus and 2) noradrenergic neurons in the locus ceruleus of DOCA-salt-treated rats have an increased responsiveness to the excitatory effects of vasopressin in both prehypertensive and chronic stages of hypertension.