Neuropeptides,food intake and body weight regulation: a hypothalamic focus

Energy homeostasis is controlled by a complex neuroendocrine system consisting of peripheral signals like leptin and central signals, in particular, neuropeptides. Several neuropeptides with anorexigenic (POMC, CART, and CRH) as well as orexigenic (NPY, AgRP, and MCH) actions are involved in this complex (partly redundant) controlling system. Starvation as well as overfeeding lead to changes in expression levels of these neuropeptides, which act downstream of leptin, resulting in a physiological response. In this review the role of several anorexigenic and orexigenic (hypothalamic) neuropeptides on food intake and body weight regulation is summarized.     

Differential expression of hypothalamic neuropeptides in the early phase of diet-induced obesity in mice

Exposure to high-fat diets for prolonged periods results in positive energy balance and obesity, but little is known about the initial physiological and neuroendocrine response of obesity-susceptible strains to high-fat feeding. To assess responses of C57BL/6J mice to high- and low-fat diets, we quantitated the hypothalamic expression of neuropeptides implicated in weight regulation and neuroendocrine function over a 2-wk period. Exposure to high-fat diet increased food consumption over a 2-day period during which leptin levels were increased when assessed by a frequent sampling protocol [area under the curve (AUC): 134.6 +/- 10.3 vs. 100 +/- 12.3, P = 0.03 during first day and 126.5 +/- 8.2 vs. 100 +/- 5.2, P = 0.02 during second day]. During this period, hypothalamic expression of neuropeptide Y (NPY) and agouti-related protein (AgRP) decreased by approximately 30 and 50%, respectively (P < 0.001). After 1 wk, both caloric intake and hypothalamic expression of NPY and AgRP returned toward baseline. After 2 wk, cumulative caloric intake was again higher in the high-fat group, and now proopiomelanocortin (POMC) was elevated by 76% (P = 0.01). This study demonstrates that high-fat feeding induces hyperphagia, hyperleptinemia, and transient suppression of orexigenic neuropeptides during the first 2 days of diet. The subsequent induction of POMC may be a second defense against obesity. Attempts to understand the hypothalamic response to high-fat feeding must examine the changes as they develop over time.     

Neither agouti-related protein nor neuropeptide Y is critically required for the regulation of energy homeostasis in mice

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp(-/-)) mice to examine the physiological role of AgRP. Agrp(-/-) mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp(-/-) mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp(-/-) mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp(-/-);Npy(-/-)) mice to determine whether NPY or AgRP plays a compensatory role in Agrp(-/-) or NPY-deficient (Npy(-/-)) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp(-/-);Npy(-/-) mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.     

Role of neuropeptide Y and its receptors in the progression of endocrine-related cancer

The neuropeptide Y (NPY) family of peptides, in addition to its many physiological actions, has also been involved in the modulation of tumor progression, with specific reference to endocrine-related cancers such as neuroendocrine tumors, breast and prostate cancers. These have been found either to express NPY receptors, or to secrete NPY-related peptides, or both. The study of the role of the NPY family of peptides in the biology of endocrine-related tumors, specifically concerning cell proliferation, angiogenesis, invasion and metastatization, may help to clarify some aspects of tumor pathophysiology, as well as to indicate novel diagnostic markers and therapeutical approaches.     

Hypothalamic gene expression following ghrelin therapy to gastrectomized rodents

We investigated whether ghrelin depletion (by gastrectomy surgery) and/or treatment/replacement with the gastric hormone ghrelin alters the expression of key hypothalamic genes involved in energy balance, in a manner consistent with ghrelin's pro-obesity effects. At 2 weeks after surgery mice were treated with ghrelin (12 nmol/mouse/day, sc) or vehicle for 8 weeks. Gastrectomy had little effect on the expression of these genes, with the exception of NPY mRNA in the arcuate nucleus that was increased. Ghrelin treatment (to gastrectomized and sham mice) increased the mRNA expression of orexigenic peptides NPY and AgRP while decreasing mRNA expression of the anorexigenic peptide POMC. Two weeks gavage treatment with the ghrelin mimetic, MK-0677, to rats increased NPY and POMC mRNA in the arcuate nucleus and MCH mRNA in the lateral hypothalamus. Thus, while predicted pro-obesity ghrelin signalling pathways were activated by ghrelin and ghrelin mimetics, these were largely unaffected by gastrectomy.     

Role of the AMP-activated protein kinase (AMPK) signaling pathway in the orexigenic effects of endogenous ghrelin

Ghrelin, released from the stomach, stimulates food intake through activation of the ghrelin receptor (GHS-R) located on neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons in the hypothalamus. A role for the energy sensor AMP-activated protein kinase (AMPK) and its downstream effector uncoupling protein 2 (UCP2) in the stimulatory effect of exogenous ghrelin on NPY/AgRP expression and food intake has been suggested. This study aimed to investigate whether a rise in endogenous ghrelin levels is able to influence hypothalamic AMPK activity, pACC, UCP2 and NPY/AgRP expression through activation of GHS-R. An increase in endogenous ghrelin levels was established by fasting (24h) or by induction of streptozotocin(STZ)-diabetes (15 days) in GHS-R(+/+) and GHS-R(-/-) mice. GHS-R(+/+) mice showed a significant increase in AgRP and NPY mRNA expression after fasting, which was not observed in GHS-R(-/-) mice. Fasting did not affect AMPK activity nor ACC phosphorylation in both genotypes and increased UCP2 mRNA expression. The hyperghrelinemia associated with STZ-induced diabetes was accompanied by an increased NPY and AgRP expression in GHS-R(+/+) but not in GHS-R(-/-) mice. AMPK activity and UCP2 expression in GHS-R(+/+) mice after induction of diabetes were decreased to a similar extent in both genotypes. Exogenous ghrelin administration tended to decrease hypothalamic AMPK activity. In conclusion, an increase in endogenous ghrelin levels triggered by fasting or STZ-induced diabetes stimulates the expression of AgRP and NPY via interaction with the GHS-R. The changes in AMPK activity, pACC and UCP2 occur independently from GHS-R suggesting that they do not play a major role in the orexigenic effect of endogenous ghrelin.     

Acute food deprivation and chronic food restriction differentially affect hypothalamic NPY mRNA expression

Although acute food deprivation and chronic food restriction both result in body weight loss, they produce different metabolic states. To evaluate how these two treatments affect hypothalamic peptide systems involved in energy homeostasis, we compared patterns of hypothalamic neuropeptide Y (NPY), agouti-related protein (AgRP), proopiomelanocotin (POMC), and leptin receptor gene expression in acutely food-deprived and chronically food-restricted rats. Both acute food deprivation and chronic food restriction reduced body weight and circulating leptin levels and resulted in increased arcuate NPY and decreased arcuate POMC gene expression. Arcuate AgRP mRNA levels were only elevated in acutely deprived rats. NPY gene expression was increased in the compact subregion of the dorsomedial hypothalamus (DMH) in response to chronic food restriction, but not in response to acute food deprivation. Leptin receptor expression was not affected by either treatment. Double in situ hybridization histochemistry revealed that, in contrast to the situation in the arcuate nucleus, NPY and leptin receptor mRNA-expressing neurons were not colocalized in the DMH. Together, these data suggest that arcuate and DMH NPY gene expression are differentially regulated. DMH NPY-expressing neurons do not appear to be under the direct control of leptin signaling.     

Expression of neuropeptides and their receptors in the developing retina of mammals

The present review examines various aspects of the developmental expression of neuropeptides and of their receptors in mammalian retinas, emphasizing their possible roles in retinal maturation. Different peptidergic systems have been investigated with some detail during retinal development, including substance P (SP), somatostatin (SRIF), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), neuropeptide Y (NPY), opioid peptides and corticotrophin-releasing factor (CRF). Overall, the developmental expression of most peptides is characterized by early appearance, transient features and achievement of the mature pattern at the time of eye opening. Concerning possible developmental actions of neuropeptides, recent studies imply a role of SP in the modulation of cholinergic neurotransmission in early postnatal rabbit retinas, when cholinergic cells participate in the retinal spontaneous waves of activity. In addition, the presence of transient SRIF expressing ganglion cells and recent observations in SRIF receptor knock-out mice indicate variegated roles of this peptide in the development of the retina and of retinofugal projections. Furthermore, VIP and PACAP exert protective and growth-promoting actions that may sustain retinal neurons during their development, and opioid peptides may control cell proliferation in the developing retina. Finally, a peak in the expression of certain peptides, including VIP, NPY and CRF, is present around the time of eye opening, when the retina begins the analysis of structured visual information, suggesting important roles of these peptides during this delicate phase of retinal development. In summary, although the physiological actions of peptides during retinal development are far from being clarified, the data reviewed herein indicate promising perspectives in this field of study.     

Third ventricular coinjection of subthreshold doses of NPY and AgRP stimulate food hoarding and intake and neural activation

We previously demonstrated that 3rd ventricular (3V) neuropeptide Y (NPY) or agouti-related protein (AgRP) injection potently stimulates food foraging/hoarding/intake in Siberian hamsters. Because NPY and AgRP are highly colocalized in arcuate nucleus neurons in this and other species, we tested whether subthreshold doses of NPY and AgRP coinjected into the 3V stimulates food foraging, hoarding, and intake, and/or neural activation [c-Fos immunoreactivity (c-Fos-ir)] in hamsters housed in a foraging/hoarding apparatus. In the behavioral experiment, each hamster received four 3V treatments by using subthreshold doses of NPY and AgRP for all behaviors: 1) NPY, 2) AgRP, 3) NPY+AgRP, and 4) saline with a 7-day washout period between treatments. Food foraging, intake, and hoarding were measured 1, 2, 4, and 24 h and 2 and 3 days postinjection. Only when NPY and AgRP were coinjected was food intake and hoarding increased. After identical treatment in separate animals, c-Fos-ir was assessed at 90 min and 14 h postinjection, times when food intake (0-1 h) and hoarding (4-24 h) were uniquely stimulated. c-Fos-ir was increased in several hypothalamic nuclei previously shown to be involved in ingestive behaviors and the central nucleus of the amygdala (CeA), but only in NPY+AgRP-treated animals (90 min and 14 h: magno- and parvocellular regions of the hypothalamic paraventricular nucleus and perifornical area; 14 h only: CeA and sub-zona incerta). These results suggest that NPY and AgRP interact to stimulate food hoarding and intake at distinct times, perhaps released as a cocktail naturally with food deprivation to stimulate these behaviors.     

Effect of dietary fatty acid composition on food intake, triglycerides, and hypothalamic peptides

While a high-fat diet when compared to low-fat diet is known to produce overeating and health complications, less is known about the effects produced by fat-rich diets differing in their specific composition of fat. This study examined the effects of a high-fat diet containing relatively high levels of saturated compared to unsaturated fatty acids (HiSat) to a high-fat diet with higher levels of unsaturated fatty acids (USat). A HiSat compared to USat meal caused rats to consume more calories in a subsequent chow test meal. The HiSat meal also increased circulating levels of triglycerides (TG) and expression of the orexigenic peptides, galanin (GAL) in the hypothalamic paraventricular nucleus (PVN) and orexin (OX) in the perifornical lateral hypothalamus (PFLH). A similar increase in TG levels and PVN GAL and PFLH OX was also seen in rats given chronic access to the HiSat compared to USat diet, while neuropeptide Y (NPY) and agouti-related protein (AgRP) in the arcuate nucleus showed decreased expression. The importance of TG in producing these changes was supported by the finding that the TG-lowering medication gemfibrozil as compared to vehicle, when peripherally administered before consumption of a HiSat meal, significantly decreased the expression of OX, while increasing the expression of NPY and AgRP. These findings substantiate the importance of the fat composition in a diet, indicating that those rich in saturated compared to unsaturated fatty acids may promote overeating by increasing circulating lipids and specific hypothalamic peptides, GAL and OX, known to preferentially stimulate the consumption of a fat-rich diet.     

Pregnancy-induced hyperphagia is associated with increased gene expression of hypothalamic agouti-related peptide in rats

Pregnancy is characterized by an increase in food intake that, in turn, produce a positive energy balance in order to face the considerable metabolic demands associated with the challenge of reproduction. Since hypothalamus is a key brain region involved in many peripheral signals and neuronal pathways that control energy homeostasis and food intake, we investigated if during pregnancy the increase in food intake is mediated by stimulating orexigenic and/or inhibiting anorexigenic neural pathways. We examined hypothalamic gene expressions of Ob-Rb, NPY, AgRP, POMC, MC4-R, and preproorexins in pregnant Wistar rats at day 19 of gestation. Food intake and body weight were increased progressively during the pregnancy. Visceral fat mass depots and serum leptin levels were also increased when compared with virgin animals. No differences were found in mRNA expression of Ob-Rb, POMC, MC4-R, NPY or preproorexin between virgin and pregnant animals. However, pregnancy produced a selective increase in AgRP mRNA levels. These results indicate that the positive energy balance that occurred during pregnancy can hardly be explained by changes in Ob-Rb despite hyperleptinemia associated with pregnancy. The enhanced expression of AgRP suggests the involvement of this neuropeptide in mediating pregnancy-associated hyperphagia.     

Differential Responses of Orexigenic Neuropeptides to Fasting in Offspring of Obese Mothers

Maternal obesity due to long‐term high‐fat diet (HFD) consumption leads to faster growth in offspring during suckling, and increased adiposity at 20 days of age. Decreased expression of the orexigenic neuropeptide Y (NPY) and increased anorexigenic proopiomelanocortin (POMC) mRNA expression were observed in the fed state. However, hunger is the major drive to eat and hypothalamic appetite regulators change in response to meals. Therefore, it is important to compare both satiated and fasting states. Female Sprague–Dawley rats (8 weeks old) were fed a cafeteria‐style HFD (15.33 kJ/g) or chow for 5 weeks before mating, with the same diet continuing throughout gestation and lactation. At postnatal day 20, male pups were killed either after overnight fasting or in the fed state. Pups from obese dams were hyperphagic during both pre‐ and postweaning periods. Pups from obese dams had higher hypothalamic mRNA expression of POMC and NPY Y1 receptor, but lower hypothalamic melanocortin‐4 receptor (MC4R) and its downstream target single‐minded gene 1 (Sim1), in the fed state. Overnight fasting reduced circulating glucose, insulin, and leptin and increased hypothalamic NPY Y1 receptor mRNA in pups from both lean and obese dams. Hypothalamic NPY and agouti‐related protein (AgRP) were only increased by fasting in pups from obese dams; reductions in MC4R and Sim1 were only seen in pups from lean dams. At weaning, the suppressed orexigenic signals in offspring from obese dams were normalized after overnight fasting, although anorexigenic signaling appeared impaired in these animals. This may contribute to their hyperphagia and faster growth.     

A central thermogenic-like mechanism in feeding regulation: an interplay between arcuate nucleus T3 and UCP2

The active thyroid hormone, triiodothyronine (T3), regulates mitochondrial uncoupling protein activity and related thermogenesis in peripheral tissues. Type 2 deiodinase (DII), an enzyme that catalyzes active thyroid hormone production, and mitochondrial uncoupling protein 2 (UCP2) are also present in the hypothalamic arcuate nucleus, where their interaction and physiological significance have not been explored. Here, we report that DII-producing glial cells are in direct apposition to neurons coexpressing neuropeptide Y (NPY), agouti-related protein (AgRP), and UCP2. Fasting increased DII activity and local thyroid hormone production in the arcuate nucleus in parallel with increased GDP-regulated UCP2-dependent mitochondrial uncoupling. Fasting-induced T3-mediated UCP2 activation resulted in mitochondrial proliferation in NPY/AgRP neurons, an event that was critical for increased excitability of these orexigenic neurons and consequent rebound feeding following food deprivation. These results reveal a physiological role for a thyroid-hormone-regulated mitochondrial uncoupling in hypothalamic neuronal networks.     

Peptide YY and neuropeptide Y induce villin expression, reduce adhesion, and enhance migration in small intestinal cells through the regulation of CD63, matrix metalloproteinase-3, and Cdc42 activity

Peptide YY (PYY) and neuropeptide Y (NPY) are regulatory peptides synthesized in the intestine and brain, respectively, that modify physiological functions affecting nutrient assimilation and feeding behavior. Because PYY and NPY also alter the expression of intestine-specific differentiation marker proteins and the tetraspanin CD63, which is involved in cell adhesion, we investigated whether intestinal cell differentiation could be linked to mucosal cell adhesion and migration through these peptides. PYY and NPY significantly decreased cell adhesion and increased cell migration in a dose-dependent manner prior to cell confluency in our model system, non-tumorigenic small intestinal hBRIE 380i cells. Both peptides reduced CD63 expression and CD63-dependent cell adhesion. CD63 overexpression increased and antisense CD63 cDNA decreased intestinal cell adhesion. In parallel, both PYY and NPY increased expression of matrix metalloproteinase-3 (MMP-3) to a level sufficient to induce cell migration by activating the Rho GTPase Cdc42. The effects of both peptides on cell migration were blocked in cells constitutively overexpressing dominant-negative Cdc42. PYY and NPY also significantly induced the expression of the differentiation marker villin, which could be eliminated by an MMP inhibitor at a concentration that inhibits cell migration. Increased MMP-3 activity, which enhanced cell migration, also induced villin mRNA levels. Therefore, these data indicate that the alteration of adhesion and migration by PYY and NPY occurs in part by synchronous modulation of three proteins that are involved in extracellular matrix-basolateral membrane interactions, CD63, MMP-3 and Cdc42, and that PYY/NPY regulation of expression of mucosal proteins such as villin is linked to the process of cell migration and adhesion.     

Somato-Dendritic Localization and Signaling by Leptin Receptors in Hypothalamic POMC and AgRP Neurons

Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP)/Neuropeptide Y (NPY)/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb). Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM), confocal-laser scanning microscopy (CLSM), and electron microscopy (EM) to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb ^+/+ mice and in Leprb ^db/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin’s central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms.