Insulin acts at different CNS sites to decrease acute sucrose intake and sucrose self-administration in rats

Findings from our laboratory and others have demonstrated that the hormone insulin has chronic effects within the CNS to regulate energy homeostasis and to decrease brain reward function. In this study, we compared the acute action of insulin to decrease intake of a palatable food in two different behavioral tasks-progressive ratios sucrose self-administration and micro opioid-stimulated sucrose feeding-when administered into several insulin-receptive sites of the CNS. We tested insulin efficacy within the medial hypothalamic arcuate (ARC) and paraventricular (PVN) nuclei, the nucleus accumbens, and the ventral tegmental area. Administration of insulin at a dose that has no chronic effect on body weight (5 mU) into the ARC significantly suppressed sucrose self-administration (75+/-5% of paired control). However, although the mu opioid DAMGO, [D-Ala2,N-MePhe4,Gly5-ol]-enkephalin acetate salt, stimulated sucrose intake at all four CNS sites, the ventral tegmental area was the only sensitive site for a direct effect of insulin to antagonize acute (60 min) micro opioid-stimulated sucrose feeding: sucrose intake was 53+/-8% of DAMGO-induced feeding, when insulin was coadministered with DAMGO. These findings demonstrate that free feeding of sucrose, and motivated work for sucrose, can be modulated within unique sites of the CNS reward circuitry. Further, they support the interpretation that adiposity signals, such as insulin, can decrease different aspects of ingestion of a palatable food, such as sucrose, in an anatomically specific manner.     

Insulin, leptin, and food reward: update 2008

The hormones insulin and leptin have been demonstrated to act in the central nervous system (CNS) as regulators of energy homeostasis at medial hypothalamic sites. In a previous review, we described new research demonstrating that, in addition to these direct homeostatic actions at the hypothalamus, CNS circuitry that subserves reward and motivation is also a direct and an indirect target for insulin and leptin action. Specifically, insulin and leptin can decrease food reward behaviors and modulate the function of neurotransmitter systems and neural circuitry that mediate food reward, i.e., midbrain dopamine and opioidergic pathways. Here we summarize new behavioral, systems, and cellular evidence in support of this hypothesis and in the context of research into the homeostatic roles of both hormones in the CNS. We discuss some current issues in the field that should provide additional insight into this hypothetical model. The understanding of neuroendocrine modulation of food reward, as well as food reward modulation by diet and obesity, may point to new directions for therapeutic approaches to overeating or eating disorders.     

Apolipoprotein A-IV interacts synergistically with melanocortins to reduce food intake

Apolipoprotein (apo) A-IV is an anorexigenic gastrointestinal peptide that is also synthesized in the hypothalamus. The goal of these experiments was to determine whether apo A-IV interacts with the central melanocortin (MC) system in the control of feeding. The third ventricular (i3vt) administration of a subthreshold dose of apo A-IV (0.5 microg) potentiated i3vt MC-induced (metallothionein-II, 0.03 nmol) suppression of 30-min feeding in Long-Evans rats. A subthreshold dose of the MC antagonist (SHU9119, 0.1 nmol, i3vt) completely attenuated the anorectic effect of i3vt apo A-IV (1.5 microg). The i3vt apo A-IV significantly elevated the expression of c-Fos in neurons of the paraventricular nucleus of the hypothalamus, but not in the arcuate nucleus or median eminence. In addition, c-Fos expression was not colocalized with proopiomelanocortin-positive neurons. These data support a synergistic interaction between apo A-IV and melanocortins that reduces food intake by acting downstream of the arcuate.     

AAV-mediated leptin receptor installation improves energy balance and the reproductive status of obese female Koletsky rats

Leptin is a hormone secreted primarily by white adipocytes that regulates energy homeostasis and reproduction via CNS receptors. Koletsky (f/f) rats with a leptin receptor (OB-Rb) gene mutation are obese, diabetic and infertile. We employed recombinant adeno-associated viral (rAAV) vectors to transfer the human OB-Rb gene into the brains of female Koletsky rats to identify sites of leptin action in the brain. rAAV-OB-Rb was microinjected into the medial preoptic area (MPOA), the paraventricular nucleus (PVN), the ventromedial hypothalamus, the arcuate nucleus (ARC), or the dorsal vagal complex in the brainstem. Food intake and body weight were monitored bi-weekly for 55 days. Vaginal cytology was examined daily to assess estrous cyclicity. After sacrifice, uncoupling protein-1 (UCP-1) mRNA in brown adipose tissue and serum concentrations of leptin, insulin, glucose, estradiol and progesterone were measured. Expression of OB-Rb was documented by RT-PCR and site specificity of microinjection was verified by immunohistochemical detection of green fluorescent protein following a control microinjection of rAAV-GFP. OB-Rb installation in the ARC reduced food intake, however, energy expenditure, assessed by UCP-1 mRNA expression, was increased by OB-Rb installation in all sites except the PVN. When injected into the MPOA and ARC, rAAV-OB-Rb stimulated the reproductive axis as evidenced by normalization of estrous cycle length and increased luteinizing hormone releasing hormone concentrations in the hypothalamus. These studies show that long-term installation of a functional leptin receptor in the CNS is achievable using rAAV vectors and further show that leptin acts on specific sites in the brain to produce differential effects on food intake, energy expenditure and reproduction.     

Neuronal Expression of Glucosylceramide Synthase in Central Nervous System Regulates Body Weight and Energy Homeostasis

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.     

Nicotine modulates multiple regions in the limbic stress network regulating activation of hypophysiotrophic neurons in hypothalamic paraventricular nucleus

Nicotine intake affects CNS responses to stressors. We reported that nicotine self-administration (SA) augmented the hypothalamo-pituitary-adrenal (HPA) stress response, in part because of the altered neurotransmission and neuropeptide expression within hypothalamic paraventricular nucleus (PVN). Limbic-PVN interactions involving medial prefrontal cortex, amygdala, and bed nucleus of the stria terminalis (BST) greatly impact the HPA stress response. Therefore, we investigated the effects of nicotine SA on stress-induced neuronal activation in limbic-PVN network, using c-Fos protein immunohistochemistry and retrograde tracing. Nicotine decreased stress-induced c-Fos in prelimbic cortex (PrL), anteroventral BST (avBST), and peri-PVN, but increased c-Fos induction in medial amygdala (MeA), locus coeruleus, and PVN. Fluoro-gold (FG) was injected into avBST or PVN, as GABAergic neurons in avBST projecting to PVN corticotrophin-releasing factor neurons relay information from both PrL glutamatergic and MeA GABAergic neurons. The stress-induced c-Fos expression in retrograde-labeled FG+ neurons was decreased in PrL by nicotine, but increased in MeA, and also reduced in avBST. Therefore, within limbic-PVN network, nicotine SA exerts selective regional effects on neuronal activation by stress. These findings expand the mechanistic framework by demonstrating altered limbic-BST-PVN interactions underlying the disinhibition of PVN corticotrophin-releasing factor neurons, an essential component of the amplified HPA response to stress by nicotine.     

Wired on hormones: endocrine regulation of hypothalamic development

The hormones that regulate the hypothalamic circuits that control essential functions, such as reproduction and energy homeostasis, also specify brain architecture by regulating key developmental events. The cellular mechanisms underlying the developmental actions of testosterone and estrogen to determine patterns of neuronal cell death, synaptogenesis and axon guidance are being identified. Recent neuroanatomical evidence indicates that the adipocyte-derived hormone leptin may direct the development of hypothalamic pathways involved in energy homeostasis by promoting axonal projections from the arcuate nucleus of the hypothalamus to other hypothalamic sites that mediate the effects of leptin on food intake and body weight. Understanding how sex steroids and leptin regulate hypothalamic development will enable us to identify hormonally directed signaling events essential to the specification of neural circuitry that is optimized for sustained homeostasis.     

Unacylated Ghrelin Suppresses Ghrelin-Induced Neuronal Activity in the Hypothalamus and Brainstem of Male Rats

Ghrelin, the endogenous growth hormone secretagogue, has an important role in metabolic homeostasis. It exists in two major molecular forms: acylated (AG) and unacylated (UAG). Many studies suggest different roles for these two forms of ghrelin in energy balance regulation. In the present study, we compared the effects of acute intracerebroventricular administration of AG, UAG and their combination (AG+UAG) to young adult Wistar rats on food intake and central melanocortin system modulation. Although UAG did not affect food intake it significantly increased the number of c-Fos positive neurons in the arcuate (ARC), paraventricular (PVN) and solitary tract (NTS) nuclei. In contrast, UAG suppressed AG-induced neuronal activity in PVN and NTS. Central UAG also modulated hypothalamic expression of Mc4r and Bmp8b, which were increased and Mc3r, Pomc, Agrp and Ucp2, which were decreased. Finally, UAG, AG and combination treatments caused activation of c-Fos in POMC expressing neurons in the arcuate, substantiating a physiologic effect of these peptides on the central melanocortin system. Together, these results demonstrate that UAG can act directly to increase neuronal activity in the hypothalamus and is able to counteract AG-induced neuronal activity in the PVN and NTS. UAG also modulates expression of members of the melanocortin signaling system in the hypothalamus. In the absence of an effect on energy intake, these findings indicate that UAG could affect energy homeostasis by modulation of the central melanocortin system.     

Neuropeptide-Y in the paraventricular nucleus increases ethanol self-administration

The paraventricular nucleus (PVN) of the hypothalamus is known to modulate feeding, obesity, and ethanol intake. Neuropeptide-Y (NPY), which is released endogenously by neurons projecting from the arcuate nucleus to the PVN, is one of the most potent stimulants of feeding behavior known. The role of NPY in the PVN on ethanol self-administration is unknown. To address this issue, rats were trained to self-administer ethanol via a sucrose fading procedure and injector guide cannulae aimed at the PVN were surgically implanted. Microinjections of NPY and NPY antagonists in the PVN were conducted prior to ethanol self-administration sessions. All doses of NPY significantly increased ethanol self-administration and preference, and decreased water intake. The NPY antagonist D-NPY partially reduced ethanol self-administration and completely blocked the effects of an intermediate dose of NPY (10 fmol) on ethanol intake, preference, and water intake. The competitive non-peptide Y1 receptor antagonist BIBP 3226 did not significantly alter ethanol self-administration or water intake when administered alone in the PVN but it completely blocked the effect of NPY (10 fmol) on ethanol intake. NPY infused in the PVN had no effect on ethanol self-administration when tested in rats that did not have a long history of ethanol self-administration. The doses of NPY tested produced no effect on food intake or body weight measured during the 24-h period after infusion in either ethanol-experienced or ethanol-inexperienced rats. These results indicate that elevation of NPY levels in the PVN potently increases ethanol self-administration and that this effect is mediated through NPY Y1 receptors.     

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.     

Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (α-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-α-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POMC mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 µg/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POMC neurons in the hypothalamus and an increased mRNA expression of these neuropeptides.     

Obese OLETF rats exhibit increased operant performance for palatable sucrose solutions and differential sensitivity to D2 receptor antagonism

CCK-1-receptor-deficient Otsuka Long-Evans Tokushima fatty (OLETF) rats are hyperphagic and exhibit a greater preference for sucrose compared with lean controls [Long-Evans Tokushima Otsuka (LETO)]. To directly assess motivation to work for sucrose reward in this model of obesity and type 2 diabetes, we examined the operant performance of OLETF rats at nondiabetic and prediabetic stages (14 and 24 wk of age, respectively) on fixed-ratio (FR) and progressive-ratio (PR) schedules of reinforcement. To evaluate the involvement of dopamine systems, the effects of the D1 receptor antagonist SCH23390 (100 and 200 nmol/kg ip) and the D2 receptor antagonist raclopride (200 and 400 nmol/kg ip), were also tested on PR responding for sucrose. Compared with age-matched LETO rats, 14-wk-old OLETF rats emitted more licks on the "active" empty spout operant on the FR-10 schedule of reinforcement to obtain 0.01 M and 0.3 M sucrose and completed higher ratio requirements on the PR schedule to gain access to 0.3 M and 1.0 M sucrose. At 24 wk, this effect was limited to 1.0 M sucrose. Both antagonists were potent in reducing operant responding to 0.3 M sucrose in both strains at both ages, and there was no strain effect to SCH23390 at either age. OLETF rats, on the other hand, showed an increased sensitivity to the higher dose of raclopride, resulting in reduced responding to sucrose reinforcement at 24 wk. Taken together, these findings provide the first direct evidence for an increased motivation for sucrose reward in the OLETF rats and suggest altered D2 receptor regulation with the progression of obesity and prediabetes.     

In a rat model of night work, activity during the normal resting phase produces desynchrony in the hypothalamus

Internal synchrony among external cycles and internal oscillators allows adaptation of physiology to cyclic demands for homeostasis. Night work and shift work lead to a disrupted phase relationship between external time cues and internal rhythms, also losing internal coherence among oscillations. This process results in internal desynchrony (ID) in which behavioral, hormonal, and metabolic variables cycle out of phase. It is still not clear whether ID originates at a peripheral or at a central level. In order to determine the possible role of hypothalamic oscillators in ID, we explored with a rat model of "night work" daily rhythms of activity and clock gene expression in the hypothalamus. This study provides evidence that wakefulness and activity during the normal resting phase lead to a shift in the diurnal rhythms of c-Fos and induce a rhythm of PER1 in the arcuate and dorsomedial nucleus of the hypothalamus, both associated with metabolism and regulation of the sleep/wake cycle. Moreover, the number of orexin (ORX)-positive neurons and c-Fos in the perifornical area increased during the working period, suggesting a relevant switch of activity in this brain region induced by the scheduled activity; however, the colocalization of c-Fos in ORX-positive cells was not increased. In contrast, the suprachiasmatic nucleus and the paraventricular nucleus remained locked to the light/dark cycle, resulting in ID in the hypothalamus. Present data suggest that ID occurs already at the level of the first output projections from the SCN, relaying nuclei that transmit temporal signals to other brain areas and to the periphery.     

Identification of a histaminergic circuit in the caudal hypothalamus: An evidence for functional heterogeneity of histaminergic neurons

It is well established that histaminergic neurons in the posterior hypothalamus make connections with whole brain areas and regulate several functions. Recent evidence indicates that histaminergic neurons are heterogeneous cell group and organized into distinct circuits. However, functional circuits of histaminergic neurons have not been fully mapped so far. To address this issue, we have investigated antihistamine-sensitive neuronal activation in the hypothalamus to determine the hypothalamic region primarily innervated by histaminergic neurons. Here we review our recent findings showing the existence of the heterogeneous subpopulations of histaminergic neurons in the TMN that innervated distinct regions to regulate particular functions. We have identified the caudal part of the arcuate nucleus of hypothalamus (cARC) as a target region of histaminergic neurons in food-restricted rats by assessing suppression of c-Fos expression by pretreatment with antihistamines. Histaminergic neurons in the tuberomammillary nucleus (TMN) are morphologically subdivided into five groups (E1–E5). Among the subdivisions, the E3 group was found to be activated corresponding to the activation of cARC neurons. Our findings suggest that this subpopulation selectively innervate cARC neurons. Accumulating reports have also described c-Fos expression in other TMN subpopulations. Various stress challenge induced c-Fos expression primarily in E4 and E5 subpopulations. Motivation- and drug-induced arousal elicited in common activation of ventrolateral part of the TMN containing E1 and E2 subdivisions, which receive projections from wake-active orexin neurons and sleep-active GABA neurons. These lines of evidence support the hypothesis that there are heterogeneous subpopulations in the TMN that innervated distinct regions to regulate particular functions.     

Gene c-Fos expression in brain of rats resistant and predisposed to emotional stress after intraperitoneal injection of the ACTH(4-10)analog--semax

The effect of the ACTH(4-10) analog Semax on immediate early gene c-Fos expression was studied in Wistar rats with high and low resistance to emotional stress under the usual conditions and during psychoemotional loading. Fos-immunoreactive cells in the were counted automatically with the help of a computer. It was shown that under the usual conditions the intraperitoneal Semax injection induced immediate early gene c-Fos expression in the lateral septal region in rats predisposed to emotional stress and in the paraventricular hypothalamus in rats of both groups. Preliminary Semax injection decreased the stress-induced c-Fos expression in the paraventricular hypothalamus and medial septum in rats predisposed to emotional stress and tended to reduce the number of stress-induced c-Fos-immunopositive cells in the lateral septum and basolateral amygdala in both groups of animals. The obtained data suggest that Semax differently affects the immediate early c-Fos gene expression in the brain of rats resistant and predisposed to emotional stress and this effect reflects the antistressor properties of the regulatory peptide.     

Neuronal activation in the hypothalamus and brainstem during feeding in rats

We trained rats to a regime of scheduled feeding, in which food was available for only 2 hr each day. After 10 days, rats were euthanized at defined times relative to food availability, and their brains were analyzed to map Fos expression in neuronal populations to test the hypothesis that some populations are activated by hunger whereas others are activated by satiety signals. Fos expression accompanied feeding in several hypothalamic and brainstem nuclei. Food ingestion was critical for Fos expression in noradrenergic and non-noradrenergic cells in the nucleus tractus solitarii and area postrema and in the supraoptic nucleus, as well as in melanocortin-containing cells of the arcuate nucleus. However, anticipation of food alone activated other neurons in the arcuate nucleus and in the lateral and ventromedial hypothalamus, including orexin neurons. Thus orexigenic populations are strongly and rapidly activated at the onset of food presentation, followed rapidly by activity in anorexigenic populations when food is ingested.