Orexigenic peptides

Neuropeptide Y (NPY) is a 36 aminoacid peptide belonging to the same family as pancreatic polypeptide. It is the most abundant peptide found in the hypothalamus. NPY is mainly synthetized in the ARC nucleus, but it is also found in the PVN, dorsomedial nucleus (DMN) and perifornical hypothalamus (PFH). NPY receptors are G-protein coupled receptors and are known as Y1, Y2, Y4, Y5, Y6 receptors.

In 1984, Clark JT et al showed for the first time that NPY infused centrally increases appetite. This effect was blocked by the opiate antagonist, naloxone and the dopamine antagonist, haloperidol. NPY-infused animals preferred high carbohydrate over high fat or high protein-containing diets (Stanley, Daniel et al. 1985; Morley, Levine et al. 1987).

It was then showed that semi-chronic (6 days) intracerebroventricular NPY infusion in normal rats leads to a marked increase in food intake and body weight gain (Sainsbury, Rohner-Jeanrenaud et al. 1997).

NPY plays a role not only in energy intake, but also in modulating the activity of the sympathetic nervous system since central administration of NPY suppresses the activity of sympathetic nerves innervating BAT in a dose-dependent manner (Egawa, Yoshimatsu et al. 1990; Egawa, Yoshimatsu et al. 1991). Similar results were found by Billintong et al who demonstrated that centrally injected NPY leads to a decrease in guanosine diphosphate (GDP)-binding, used as a marker of brown fat thermogenic activity (Billington, Briggs et al. 1994). NPY infusion into the PVN decreases uncoupling protein 1 (UCP1) mRNA expression in BAT. No effect was observed on uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3) mRNA expression in white adipose tissue (WAT) and muscle, respectively (Kotz, Wang et al. 2000).

The ob mRNA expression in inguinal white adipose tissue (WATi) is higher in NPY-infused rats compared to controls (Sainsbury, Cusin et al. 1996). Moreover, intracerebroventricular injection of NPY leads to an increase in the activity and mRNA expression of lipoprotein lipase (LPL) in WAT (Billington, Briggs et al. 1994).

Rats intracerebroventricularly infused with NPY for 7 days exhibit a series of metabolic alterations that are similar to those encountered during the development of obesity. Thus, centrally infused NPY leads to basal hyperinsulinemia, to an enhanced insulin response to meal feeding and to an increase in insulin-stimulated glucose uptake by adipose tissue.

Such increase in glucose uptake may be due to an increase in glucose transporter 4 (GLUT-4) mRNA and protein levels in adipocytes (Zarjevski, Cusin et al. 1994).

Furthermore, the NPY-induced hyperinsulinemia is accompanied by muscle insulin resistance (Zarjevski, Cusin et al. 1994). These effects are prevented by adrenalectomy (ADX). Adrenal glands and corticosterone are thus necessary for the establishment of the anabolic defects induced by NPY (Sainsbury, Cusin et al. 1997). Interestingly, in ADX rats, central glucocorticoid supplementation is sufficient to allow for the obesity-inducing effects of NPY to occur. This was observed for most of the NPY effects, except for the expression of UCP1 and UCP3 in BAT which is decreased in NPY-infused rats, an effect that is not observed in ADX rats supplemented with central dexametasone infusion. It can be concluded that centrally infused NPY has obesity-promoting effects which require the presence of glucocorticoids, while the NPY effect on thermogenesis appears to be independent from these hormones (Zakrzewska, Sainsbury et al. 1999).

Other actions of NPY are to stimulate the hypothalamo-pituitary-adrenal-axis (HPA) and to inhibit the gonadotropic axis. Central NPY also leads to decreases in plasma levels of thriiodothyronine (T3) and thyroxine (T4), accompanied by abnormal or low thyroid stimulating hormone (TSH) levels and reduced prothyrotropin releasing hormone (proTRH) mRNA in the PVN of the hypothalamus. Thus, centrally infused NPY results in a state of central hypothyroidism (Fekete, Sarkar et al. 2002).

Fasting or diet induces weight loss and decreases fat stores (low leptin) in type 1 diabetes (low insulin). Both low leptin and insulin lead to an increase in NPY synthesis and secretion. This effect is reversible, since a decreased expression is seen after insulin treatment of type 1 diabetic animals. Similarly, an increase in leptin leads to a decrease in NPY expression. In animal models of energy deficiency, NPY synthesis and secretion are up-regulated. Thus, the adiposity signals insulin and leptin control the expression of hypothalamic NPY (Ramos, Meguid et al. 2005; Woods 2005).

To study the in vivo physiological effects of NPY, rodent models of NPY overexpression or deletion were constructed and their metabolic profile was analyzed. Inui et al demonstrated that transgenic mice overexpressing NPY by 18% in the arcuate nucleus have no increase in food intake and body weight, but show more signs of anxiety that controls (Inui, Okita et al. 1998). Later it was observed that these animals, when given a 50% sucrose-rich diet, have a significant increase in body weight compared to controls, accompanied by hyperinsulinemia and hyperglycemia. NPY-overexpressing animals also increase their food intake, an effect that lasts several weeks after termination of the 50%

sucrose diet. The effects on food intake could be significantly inhibited by antagonists of NPY Y-1 but not of Y-5 receptors (Kaga, Inui et al. 2001).

Initial studies on NPY knockout mice did not show any effect on food intake and body weight, whether the animals were fed a standard or a high fat diet (Erickson, Clegg et al.

1996). However, peripheral treatment with leptin leads to a more pronounced reduction of food intake in NPY^-/- animals compared to controls, demonstrating that leptin action is enhanced in NPY knockout animals and therefore suggesting that NPY may antagonize leptin (Hollopeter, Erickson et al. 1998). Further investigations on the relationship between leptin and NPY were published by Erickson et al, demonstrating that NPY^-/- mice crossed with the leptin knockout mice (NPY^-/-/ob/ob) present a reduction in food intake and body weight accompanied by an increase in energy expenditure. Thus, leptin-deficient mice require the presence of NPY to develop an obese phenotype (Erickson, Hollopeter et al. 1996). Bannon et al also demonstrated that mice lacking the NPY gene (NPY^-/-) have a reduction in food intake after 1, 2 and 4h after re-feeding following 24h and 48h of food deprivation (Bannon, Seda et al. 2000).

A very convincing demonstration of the importance of NPY/AGPR pathway for food intake was given by Luquet et al who ablated the NPY/AGRP neurons in adult mice by targeting the human diphtheria toxin receptor to the locus of AGRP. Injection of diphtheria toxin led to a more than 80% destruction of NPY neurons accompanied by a sharp drop in food intake and body weight, the latter decreasing by 20% in two days in all animals (Luquet, Perez et al. 2005). Similar results were published simultaneously by Gropp et al (Gropp, Shanabrough et al. 2005). In performing the same ablation on neonate mice, however, only a minor reduction of body weight and no effect on food intake were observed. Furthermore, these mice were near normal with respect to body

demonstrate that compensatory mechanisms can restore a normal feeding behaviour and, importantly, they offer a possible explanation for the lack of effect of NPY gene deletion on food intake obtained by Erickson et al.

Models of NPY receptor (Y1, Y2, Y4 and Y5) knockout mice have been constructed, most of them on a mixed 129Sv/C57BL/6 or 129Sv/Balb/c background. Y1-/- mice display only a slight reduction in food intake, while fasting-induced re-feeding is more diminished (Pedrazzini 2004). Kushi et al, however, found that these mice are slightly hyperinsulinemic and, quite contrary to expectations, develop obesity in later life. They also display increased levels of UCP1 in BAT, suggesting increased energy expenditure (Kushi, Sasai et al. 1998). In the ARC, CART as well as POMC mRNA were strongly decreased, whereas no effects were observed on NPY or AGRP mRNA levels (Herzog 2003). A more distinct phenotype with respect to body weight was produced by crossing Y1-/- and ob/ob mice. This double knockout has a significantly lower body weight compared to ob/ob mice (Pralong, Gonzales et al. 2002).

Sainsbury et al generated both a conventional germ-line Y2 knockout and a conditional hypothalamic Y2 knockout and compared their phenotypes. For the germ-line knockout, a reduction in body weight gain was observed despite the fact that food intake was increased (females) or unchanged (males). Re-feeding after starvation was, furthermore, substantially increased in Y2-/- mice. These mice also displayed significant changes in neuropeptides related to energy balance. Thus, POMC and CART in the ARC and CRF in the PVN were decreased, whereas hypothalamic NPY and AGRP were increased at the mRNA level (Sainsbury, Cooney et al. 2002). In the conditional hypothalamic Y2

knockout model, significant decreases in body weight associated with significant increases in food intake were observed. Interestingly, the effects on food intake and body weight in this model were transient and fully overcome after ~12 and ~24 days after gene deletion, respectively, emphasising the capacity of the organism to compensate for a missing gene. Furthermore, mRNA levels of NPY, AGRP, POMC and CART were all increased in knockout animals in this model (Sainsbury, Schwarzer et al. 2002).

Y2 receptors are highly expressed on NPY neurons in the ARC (Batterham, Cowley et al.

2002). A specific (Dumont, Cadieux et al. 2000) and endogenous ligand of Y2 is the gut-derived anorexigenic peptide, PYY(3-36). Peripheral administration of PYY(3-36) decreases hypothalamic NPY mRNA levels, electrical activity of ARC NPY neurons and inhibits food intake in wild-type but not in Y2-/- mice, suggesting that Y2 receptors

mediate the autoinhibition of NPY synthesis (Batterham, Cowley et al. 2002; Acuna-Goycolea and van den Pol 2005).

Y4-/--mice display a small but significant reduction in body weight gain and a reduction in food intake. These mice did not display any change in neuropeptides in the ARC.

However, such changes appeared when crossed with ob/ob mice. Thus Y4-/-, ob/ob mice displayed significant reductions in both NPY and AGRP mRNA levels compared to ob/ob mice (Sainsbury, Schwarzer et al. 2002). Furthermore, a double knockout of Y2

and Y4 receptors has recently been shown to provide a substantial protection against diet-induced obesity (Sainsbury, Bergen et al. 2006).Y5 receptor knockout mice, finally, display a late onset obesity with increases in food intake and body weight. These mice are normal with respect to fasting-induced re-feeding (Marsh, Hollopeter et al. 1998;

Kanatani, Mashiko et al. 2000).

In conclusion, centrally infused NPY can control food intake and body weight. NPY also exerts a negative control on the sympathetic nervous system, decreasing thermogenesis.

NPY infusion furthermore leads to an increase in insulin-stimulated glucose uptake in WAT accompanied by an increase in ob and LPL mRNA expression and in LPL activity.

NPY-infused animals develop an obesity syndrome in which glucocorticoid contribution is essential. NPY also induces hypothyroidism.

Leptin and insulin can regulate the hypothalamic NPY expression. Overexpression of NPY does not have any effect on food intake and body weight. The lack of a strong phenotype of NPY knockout mice has been given a satisfactory explanation due to the works of Luquet et al and Gropp et al. Studies of double knockout (NPY^-/-/ob/ob^-/-) animals show a decrease in the obese phenotype, demonstrating that NPY acts downstream of leptin and, moreover, that the presence of NPY is essential for the development of obesity in leptin-deficient mice. The results from knockouts of the NPY receptors are in part contrary to expected results and age-related compensatory mechanisms, as demonstrated by Luquet et al, may have occured. Another possibility is that hypothalamic and non-hypothalamic NPY receptors affect energy homeostasis differently (Baldock, Allison et al. 2007).

Agouti and AGRP

The agouti protein was identified in agouti (A^y/a) mice which is an autosomal dominant model of genetic obesity. The protein is constitutively expressed throughout the body of the yellow agouti A^y mice. These mice have a characteristic yellow coat colour, an increase in body length and an obese phenotype, accompanied by insulin resistance and hyperglycemia. The agouti molecule is expressed in the skin of these mice and is an antagonist of alpha-melanocyte stimulating hormone (MSH) on MC-1 receptors.

Agouti-related peptide (AGRP), a 132 amino acid peptide has sequence similarity to agouti. It is synthetized mainly in the ARC nucleus in neurons which co-express NPY.

AGRP is an antagonist of MSH on the melanocortin receptors 3 and 4 (MC3-R and MC4-R) which are expressed in the brain (Arora and Anubhuti 2006).

Inhibition of these receptors by AGRP leads to an increase in food intake and caloric efficiency and to an impaired thermogenesis, leading to an obese phenotype. Central AGRP administration has a surprisingly long-lasting effect. Thus, acute intracerebroventricular injection of a single bolus of AGRP causes an increase in food intake that lasts for at least one week. Furthermore, AGRP expression changes with diet and the nutritional state. Thus, rats fed on a high fat diet for 22 weeks exhibit reduced hypothalamic AGRP mRNA expression with a concomitant increase in MC4-R mRNA expression (Ramos, Meguid et al. 2005).