Leptin stimulates neuropeptide Y and cocaine amphetamine-regulated transcript coexpressing neuronal activity in the dorsomedial hypothalamus in diet-induced obese mice.

Neuropeptide Y (NPY) neurons in both the arcuate nucleus of the hypothalamus (ARH) and the dorsomedial hypothalamus (DMH) have been implicated in food intake and obesity. However, while ARH NPY is highly expressed in the lean animal, DMH NPY mRNA expression is observed only after diet-induced obesity (DIO). Furthermore, while ARH NPY neurons are inhibited by leptin, the effect of this adipokine on DMH NPY neurons is unknown. In this study we show that in contrast to the consistent expression in the ARH, DMH NPY mRNA expression was undetectable until after 10 weeks in mice fed a high-fat diet, and peaked at 20 weeks. Surprisingly, electrophysiological experiments demonstrated that leptin directly depolarized and increased the firing rate of DMH NPY neurons in DIO mice. To further differentiate the regulation of DMH and ARH NPY populations, fasting decreased expression of DMH NPY expression, while it increased ARH NPY expression. However, treatment with a leptin receptor antagonist failed to alter DMH NPY expression, indicating that leptin may not be the critical factor regulating mRNA expression. Importantly, we also demonstrated that DMH NPY neurons coexpress cocaine amphetamine-regulated transcript (CART); however, CART mRNA expression in the DMH peaked earlier in the progression of DIO. This study demonstrates novel and important findings. First, NPY and CART are coexpressed in the same neurons within the DMH, and second, leptin stimulates DMH NPY neurons. These studies suggest that during the progression of DIO, there is an unknown signal that drives the expression of the orexigenic NPY signal within the DMH, and that the chronic hyperleptinemia increases the activity of these NPY/CART neurons.

Efferent projections of neuropeptide Y-expressing neurons of the dorsomedial hypothalamus in chronic hyperphagic models.

The dorsomedial hypothalamus (DMH) has long been implicated in feeding behavior and thermogenesis. The DMH contains orexigenic neuropeptide Y (NPY) neurons, but the role of these neurons in the control of energy homeostasis is not well understood. NPY expression in the DMH is low under normal conditions in adult rodents but is significantly increased during chronic hyperphagic conditions such as lactation and diet-induced obesity (DIO). To understand better the role of DMH-NPY neurons, we characterized the efferent projections of DMH-NPY neurons using the anterograde tracer biotinylated dextran amine (BDA) in lactating rats and DIO mice. In both models, BDA- and NPY-colabeled fibers were limited mainly to the hypothalamus, including the paraventricular nucleus of the hypothalamus (PVH), lateral hypothalamus/perifornical area (LH/PFA), and anteroventral periventricular nucleus (AVPV). Specifically in lactating rats, BDA-and NPY-colabeled axonal swellings were in close apposition to cocaine- and amphetamine-regulated transcript (CART)-expressing neurons in the PVH and AVPV. Although the DMH neurons project to the rostral raphe pallidus (rRPa), these projections did not contain NPY immunoreactivity in either the lactating rat or the DIO mouse. Instead, the majority of BDA-labeled fibers in the rRPa were orexin positive. Furthermore, DMH-NPY projections were not observed within the nucleus of the solitary tract (NTS), another brainstem site critical for the regulation of sympathetic outflow. The present data suggest that NPY expression in the DMH during chronic hyperphagic conditions plays important roles in feeding behavior and thermogenesis by modulating neuronal functions within the hypothalamus, but not in the brainstem.

The neuroendocrine basis of lactation-induced suppression of GnRH: role of kisspeptin and leptin.

Lactation is an important physiological model of the integration of energy balance and reproduction, as it involves activation of potent appetitive neuropeptide systems coupled to a profound inhibition of pulsatile GnRH/LH secretion. There are multiple systems that contribute to the chronic hyperphagia of lactation: 1) suppression of the metabolic hormones, leptin and insulin, 2) activation of hypothalamic orexigenic neuropeptide systems NPY, AGRP, orexin (OX) and melanin concentrating hormone (MCH), 3) special induction of NPY expression in the dorsomedial hypothalamus, and 4) suppression of anorexigenic systems POMC and CART. These changes ensure adequate energy intake to meet the metabolic needs of milk production. There is significant overlap in all of the systems that regulate food intake with the regulation of GnRH, suggesting there could be several redundant factors acting to suppress GnRH/LH during lactation. In addition to an overall increase in inhibitory tone acting directly on GnRH cell bodies that is brought about by increases in orexigenic systems, there are also effects at the ARH to disrupt Kiss1/neurokinin B/dynorphin neuronal function through inhibition of Kiss1 and NKB. These changes could lead to an increase in inhibitory auto-regulation of the Kiss1 neurons and a possible disruption of pulsatile GnRH release. While the low levels of leptin and insulin contribute to the changes in ARH appetitive systems, they do not appear to contribute to the suppression of ARH Kiss1 or NKB. The inhibition of Kiss1 may be the key factor in the suppression of GnRH during lactation, although the mechanisms responsible for its inhibition are unknown.

Melanocortinergic activation by melanotan II inhibits feeding and increases uncoupling protein 1 messenger ribonucleic acid in the developing rat.

The hypothalamic neurocircuitry that regulates energy homeostasis in adult rats is not fully developed until the third postnatal week. In particular, fibers from the hypothalamic arcuate nucleus, including both neuropeptide Y (NPY) and alpha-MSH fibers, do not begin to innervate downstream hypothalamic targets until the second postnatal week. However, alpha-MSH fibers from the brainstem and melanocortin receptors are present in the hypothalamus at birth. The present study investigated the melanocortin system in the early postnatal period by examining effects of the melanocortin receptor agonist melanotan II (MTII) on body weight, energy expenditure, and hypothalamic NPY expression. Rat pups were injected ip with MTII (3 mg/kg body weight) or saline on postnatal day (P) 5 to P6, P10-P11, or P15-P16 at 1700 and 0900 h and then killed at 1300 h. Stomach weight and brown adipose tissue uncoupling protein 1 mRNA were determined. In addition, we assessed central c-Fos activation 90 min after MTII administration and hypothalamic NPY mRNA after twice daily MTII administration from P5-P10 or P10-P15. MTII induced hypothalamic c-Fos activation as well as attenuating body weight gain in rat pups. Stomach weight was significantly decreased and uncoupling protein 1 mRNA was increased at all ages, indicating decreased food intake and increased energy expenditure, respectively. However, MTII had no effect on NPY mRNA levels in any hypothalamic region. These findings demonstrate that MTII can inhibit food intake and stimulate energy expenditure before the full development of hypothalamic feeding neurocircuitry. These effects do not appear to be mediated by changes in NPY expression.

Metabolic adaptations in skeletal muscle during lactation: complementary deoxyribonucleic acid microarray and real-time polymerase chain reaction analysis of gene expression.

Lactation and fasting are two physiological models characterized by negative energy balance. Our previous studies demonstrated that uncoupling protein (UCP) 3 expression in skeletal muscle was down-regulated during lactation and up-regulated during fasting. The present studies used cDNA microarray and real-time PCR to perform a systems and comparative analysis in gene expression in skeletal muscle under conditions of negative energy balance. Gastrocnemius skeletal muscle RNA pools were generated from the following groups of rats: cycling diestrous females, cycling females with 48 h of fasting, lactation, and lactation + leptin. Of those known genes studied, 35 genes were up-regulated and 49 were down-regulated during lactation. Leptin treatment during lactation reversed the differential regulation of about 80% of these genes, demonstrating the importance of the leptin suppression to the changes in skeletal muscle metabolism. GenMAPP analysis revealed a coordinated regulation at key steps in glycolysis/gluconeogenesis, the tricarboxylic acid cycle, and lipid metabolism, indicating an increased rate of lactate production through glycolysis and reduced fatty acid degradation in skeletal muscle during lactation. Particular interest was paid to those genes that changed in a similar manner to UCP3 mRNA. Many of these genes that were decreased during lactation and increased during fasting are involved in fatty acid degradation and transport, including acyl-coenzyme A dehydrogenase for medium chain fatty acid, carnitine palmitoyltransferase 1, and fatty acid translocase. The current studies provide a basis for investigating the mechanisms underlying metabolic adaptations during lactation and fasting and highlight the importance of UCP3 in lipid metabolism.

Melanocortin 4 receptor-mediated hyperphagia and activation of neuropeptide Y expression in the dorsomedial hypothalamus during lactation.

In several hyperphagic models, including lactation, in which hypothalamic melanocortin signaling is reduced, a novel expression of NPY mRNA in the dorsomedial hypothalamus (DMH) has been observed, suggesting that melanocortin signaling and the induced NPY in the DMH may constitute unique neurocircuitry in mediating energy balance. Using lactating rats as a model, the present study first showed that in the DMH abundant alpha-MSH and agouti-related protein fibers are in close apposition to NPY-positive cells. However, no NPY and MC4R (a melanocortin receptor) double-labeled neurons were observed. These data suggested that melanocortin input may synapse on presynaptic terminals that then synapse on DMH NPY cells. To study the function of DMH MC4Rs in energy balance, an MC3/4R-selective agonist, melanotan II (MTII), was injected bilaterally into the DMH. MTII injection significantly suppressed feeding induced by 24 hr fasting or suckling-induced hyperphagia. Furthermore, MTII treatment greatly attenuated suckling-induced NPY expression in the DMH. MTII treatment also stimulated uncoupling protein 1 activity in the brown adipose tissue of suckling female rats, indicative of increased sympathetic outflow. In summary, the present study demonstrated that the melanocortin system in the DMH not only plays an important role in inducing NPY expression in the DMH of lactating rats but also in regulating energy homeostasis, at least in part, by modulating appetite and energy expenditure.

Regulation of hypothalamic neuropeptide Y messenger ribonucleic acid expression during lactation: role of prolactin.

In the present study, we investigated the role of prolactin (PRL) in the suckling-induced increase in hypothalamic neuropeptide Y (NPY) gene expression in the dorsomedial nucleus of the hypothalamus (DMH) and the caudal portion of the arcuate nucleus of the hypothalamus (ARH-C). Lactating rats were deprived of their eight-pup litters on d 9 postpartum. After 48 h, the animals were randomly divided into two groups: nonsuckled controls and eight pups suckling for 24 h. In addition, some of the suckled animals received two injections of bromocriptine (0.5 mg/rat per injection) to inhibit suckling-induced PRL secretion. Some bromocriptine-treated rats also received ovine PRL (1 mg/rat per injection). In situ hybridization was performed to measure NPY mRNA levels. Suckling for 24 h induced a significant increase in NPY mRNA levels in the DMH and ARH-C. Bromocriptine treatment greatly attenuated the increase of NPY mRNA in the DMH but not in the ARH. Injections of ovine PRL in bromocriptine-treated rats greatly restored DMH NPY mRNA levels but had no additional effects on the ARH NPY expression. Double-label in situ hybridization for NPY and PRL receptor (PRL-R) in the lactating rat brains showed that NPY-positive neurons in the DMH also express PRL-R mRNA. On the contrary, few ARH NPY neurons expressed PRL-R. These data suggest that PRL could act directly on DMH NPY neurons to modulate NPY gene expression during lactation. Thus, the results from the present study demonstrate that NPY neurons in the DMH and ARH are differentially regulated by PRL during lactation.

Ontogeny of the hypothalamic neuropeptide Y system.

Early onset obesity and type II diabetes is rapidly becoming an epidemic, especially within the United States. This dramatic increase is likely due to many factors including both prenatal and postnatal environmental cues. The purpose of this review is to highlight some of the recent advances in our knowledge of the development of the hypothalamic circuits involved in the regulation of energy balance, with a focus on the neuropeptide Y (NPY) system. Unlike the adult rat, during the postnatal period NPY is transiently expressed in several hypothalamic regions, along with the expected expression within the arcuate nucleus (ARH). These transient populations of NPY neurons during the postnatal period may provide local NPY production to sustain the necessary energy intake during this critical growth phase. This may be physiologically important since ARH-NPY projections do not fully develop until the 3rd postnatal week. The significance of this ontogeny is that many peripheral metabolic signals have little effect of feeding prior to the development of the ARH projections. The essential questions now are whether prenatal and/or postnatal exposure to high levels of insulin or leptin during development can cause permanent changes in the function of hypothalamic circuits. It is vital to understand not only the natural development of the hypothalamic circuits that regulate energy homeostasis, but also their abnormal development caused by maternal and postnatal environmental cues. This will be pivotal for designing intervention and therapeutics to treat early onset obesity/type II diabetes, which may very well need to be different from those designed to prevent/treat adult onset obesity/type II diabetes.

Distribution of corticotropin releasing hormone receptor immunoreactivity in the rat hypothalamus: coexpression in neuropeptide Y and dopamine neurons in the arcuate nucleus.

An abundance of physiological data suggests an interaction between neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) in the regulation of endocrine and autonomic functions. Previously, studies in our laboratory have indicated that NPY neurons in the arcuate nucleus of the hypothalamus (ARH) project to and come in close contact with CRH neurons in the paraventricular nucleus of the hypothalamus (PVH). Conversely, it has been demonstrated that the ventromedial portion of the ARH, an area containing NPY neurons, displays CRH receptor binding and CRH receptor mRNA. These data suggest a possible reciprocal feedback regulation between NPY and CRH neurons. The ARH also contains several other populations of neurons that may be targets of the CRH system and express CRH receptors; most notable are tuberoinfundibular dopaminergic neurons (TIDA). The PVH is an important component in the regulation of prolactin secretion and may play a role in the suppression of TIDA activity, which is a critical step in the prolactin stress response. The purpose of the present study was to characterize the distribution and cellular localization of CRH R(1) receptor-like immunoreactivity (CRH R(1)-ir) in the rat hypothalamus and to determine the phenotype of neurons in the ARH that contain CRH R(1)-ir. CRH R(1)-ir was present throughout the rat brain. Hypothalamic regions with the highest levels of immunostaining were the supraoptic nucleus, magnocellular PVH, ARH, and suprachiasmatic nucleus. Double label immunofluorescence was used to demonstrate that CRH R(1)-ir in the ARH was localized to NPY cell bodies. Furthermore, TIDA neurons in the ARH also displayed CRH R(1)-ir. However, despite an abundance of CRH R(1)-ir cells in the ARH, CRH-ir fiber innervation to the ARH was extremely sparse. Therefore, although this study provides neuroanatomical evidence for direct CRH R(1) regulation of ARH NPY and TIDA neurons in the rat, it is not consistent with the idea of a reciprocal feedback loop and suggests the involvement of other CRH-like ligands, such as urocortin.

Orexin neurons express a functional pancreatic polypeptide Y4 receptor.

The receptor subtypes that mediate the effects of neuropeptide Y (NPY) on food intake have not been clearly defined. The NPY Y4 receptor has been identified recently as a potential mediator of the regulation of food intake. The purpose of the present study was to characterize the central site of action of the Y4 receptor using a combination of neuroanatomical and physiological approaches. Using immunocytochemistry, Y4-like immunoreactivity was found to be colocalized with orexin cell bodies in the lateral hypothalamic area (LHA) and orexin fibers throughout the brain. In situ hybridization confirmed the expression of Y4 mRNA in orexin neurons. To determine the functional interaction between Y4 receptors and orexin neurons, we examined the effects of rat pancreatic polypeptide (rPP), a Y4-selective ligand, or NPY, a nonselective ligand, administered directly into the LHA on the stimulation of food and water intake and c-Fos expression. Both rPP and NPY significantly increased food and water intake when they were administered into the LHA, although NPY was a more potent stimulator of food intake. Furthermore, both NPY and rPP significantly stimulated c-Fos expression in the LHA. However, whereas rPP stimulated c-Fos expression in orexin neurons, NPY did not. Neither rPP nor NPY stimulated c-Fos in melanin-concentrating hormone neurons, but both activated neurons of an unknown phenotype in the LHA. These results suggest that a functional Y4 receptor is expressed on orexin neurons and that these neurons are activated in response to a ligand with high affinity for the Y4 receptor (rPP). Although these data suggest a role for central Y4 receptors, the endogenous ligand for this receptor has yet to be clearly established.

Integration of the regulation of reproductive function and energy balance: lactation as a model.

Lactation is a physiological model for studying how the hypothalamus integrates peripheral signals, such as sensory signals (suckling stimulus) and those denoting energy balance (leptin), to alter hypothalamic function regulating food intake/energy balance and reproduction. The characteristics of food intake/energy balance during lactation are extreme hyperphagia, coupled with negative energy balance. The arcuate nucleus Neuropeptide Y (ARH-NPY) system is activated by: (1) brainstem projections specifically activated by the suckling stimulus, and (2) the decrease in leptin in response to the metabolic drain of milk production. NPY neurons from the ARH make direct contact with GnRH neurons and with CRH neurons in the PVH. NPY neurons also make contact with orexin and MCH neurons in the LHA, which, in turn, make contacts with GnRH neurons. Thus, the ARH-NPY system provides a neuroanatomical framework by which to integrate changes in food intake/energy with the regulation of cyclic reproductive function.