Sleep, brain energy levels, and food intake

Background

The feeling of hunger and feeding, a wake–state-dependent behavior, is regulated by specific centers within the hypothalamus. While paraventricular nucleus (PVN), arcuate nucleus (ARC), and dorso- and ventromedial hypothalamus (DMH/VMH) regulate feeding, the lateral hypothalamus (LH) is associated both with feeding and wake/REM sleep regulation. In order to examine the effects of sleep and wakefulness on food intake and body weight, we also measured hypothalamic ATP concentrations, which are known to be involved in feeding behavior and sleep–wake regulation.

Methods

In rats, food intake and body weight was measured during a 24-h light–dark cycle and during 6 h of sleep deprivation (SD) performed by gentle handling. Tissue samples from the PVN, ARC/DMH/VMH, and LH were collected after 6 h of SD and from time-matched diurnal controls. ATP was measured by luciferin-luciferase bioluminescence assay.

Results

Across the 24-h light–dark period, rats consumed approximately 28.13±4.48 g of food and gained 5.22±1.65 g with a positive correlation between food intake and body weight. During SD, while food intake increased significantly +147.31±6.13%, they lost weight significantly (–93.29±13.64%) when compared to undisturbed controls. SD resulted in a significant decrease in ATP levels only in LH (–44.60±21.13%) with no change in PVN, ARC/DMH/VMH region when compared with undisturbed controls.

Conclusion

The results indicate a strong overall correlation between ATP concentrations in the LH and individual food intake and suggest a sleep–wake dependent neuronal control of food intake and body weight.     

下丘脑室旁核外源性orexin-A对肥胖大鼠摄食的影响及NPY受体信号的调控

目的:探讨饮食诱导肥胖(DIO)大鼠神经肽Y(NPY)受体信号通路是否参与下丘脑室旁核(PVN)外源性注射食欲素A(orexin-A)对摄食和葡萄糖敏感(GS)神经元兴奋性的调控。方法:采用荧光免疫组织化学实验观察PVN中orexin-A受体(即食欲素1型受体,OX1R)和NPY受体Y5(NPY-5R)的表达;采用单细胞外放电记录观察orexin-A对PVN内GS神经元兴奋性的影响;分别于SD大鼠和DIO大鼠PVN埋置套管,经套管注射orexinA、OX1R拮抗剂SB-334867和NPY-5R拮抗剂CGP-71683,观察大鼠0~2 h和0~4 h摄食量。结果:DIO大鼠PVN中OX1R和NPY-5R的表达显著高于SD大鼠。Orexin-A抑制PVN内葡萄糖抑制性(GI)神经元,兴奋葡萄糖兴奋性(GE)神经元,但是orexin-A对GS神经元的兴奋或抑制效应可被NPY-5R拮抗剂CGP-71683部分阻断,且与SD大鼠相比,orexin-A对DIO大鼠PVN内GS神经元兴奋或抑制效应更加明显。PVN内注射orexin-A可增加SD大鼠和DIO大鼠摄食量。但是orexin-A诱导的促摄食效应被NPY-5R拮抗剂CGP-71683部分阻断。与SD大鼠相比,orexin-A诱导的促摄食效应在DIO大鼠中更加明显。结论:PVN内外源性注射orexin-A可能主要通过OX1R信号通路参与大鼠摄食和GS神经元兴奋性调控,NPY-5R信号也参与了该过程调控,在DIO大鼠中更敏感。     

Effects of chronic intrahypothalamic infusion of insulin on food intake and diurnal meal patterning in the rat

In Experiment 1, rats were chronically infused with insulin (2.7, 27, or 270 ng/hr) or 0.9% saline into the ventromedial (VMH), medial perifornical (PF), or lateral (LH) hypothalamus. VMH infusions of insulin caused a significant, dose-dependent decrease in food intake and body weight; PF infusion of insulin was less effective, but significant; whereas LH infusions of insulin were ineffective. In Experiment 2, rats were chronically infused with insulin (0.54 ng/hr) or 0.9% saline into the VMH, paraventricular (PVN), or posterior (PN) hypothalamic nucleus. Subjects that received VMH or PN infusions of insulin failed to regain weight lost as a result of surgery even 2 weeks after infusion; subjects that received PVN infusions of insulin regained their preoperative weights faster than did controls. All of the groups that received insulin significantly increased their daytime food intake during the infusion period and decreased their night food intake slightly; 24-hr food intake remained unchanged.     

Differential expression of nicotinamide adenine dinucleotide phosphate-diaphorase in hypothalamic areas of obese Zucker rats

Several studies have suggested that the activity of nitric oxide synthase (NOS) may be involved in the regulation of food intake in the genetically obese Zucker rats. In the present study, we investigated the expression of NOS in various hypothalamic regions of obese and lean Zucker rats using nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. Obese Zucker rats showed significantly lower staining intensities of NADPH-diaphorase-positive neurons in the paraventricular nucleus (PVN), lateral hypothalamic area (LHA) and ventromedial hypothalamic nucleus (VMH) than lean Zucker rats did. The differences in staining intensities between obese and lean Zucker rats were large in both the PVN and LHA, but such differences were relatively small in the VMH.     

Prostaglandins and food intake of rats: A component of energy balance regulation?

Modulation of food intake by signals arising in adipose tissue has been an important component of theories concerning energy balance regulation. Our experiments tested the effects of some prostaglandins (PG) which are produced in adipose tissue, on food and water intakes in rats. Rats were injected intrahypothalamically with 1 μg of either PGE₁ or PGB₁ in 1 μl bilaterally prior to a 2-hr daily meal. PGE₁, but not PGB₁, reduced food intake of the rats in groups with lateral hypothalamic (LH) and anterior commissure (AC) cannulas, but did not reduce food intake in groups with perifornical hypothalamic (PFH) or mammillary body (MB) cannulas. Water intake was reduced in all groups injected with PGE₁ except the LH and MB groups. Anterior and medial hypothalamic groups showed a sustained 2°C rise in rectal temperature following injections of PGE₁. Subcutaneous injections of PGE₁ reduced food intake of three different groups of rats at different levels of adiposity. We hypothesize that some PG's may be components of a signal relating fat depots and energy balance regulation.     

Use of peripheral blood transcriptome biomarkers for epilepsy prediction

Fenfluramine reduces hunger and promotes body weight loss by increasing central serotonin (5-HT) signaling. More recently, neuropeptides have been linked to the regulation of feeding behavior, metabolism and body weight. To examine possible interactions between 5-HT and neuropeptides in appetite control, fenfluramine (200 nmol/0.5 μl/side) was administered directly into the hypothalamic paraventricular nuclei (PVN) of male rats. Bilateral fenfluramine produced significant hypophagia and increased expression of PVN corticotropin releasing factor (CRF) mRNA and neuropeptide Y (NPY) mRNA in the arcuate nucleus within the first hour after drug administration. Fenfluramine's effects on feeding behavior and mRNA expression were blocked by PVN injections of a 5-HT(1-2) receptor antagonist, metergoline (15 nmol/0.5 μl/side). These data suggest that 5-HT neurons targeting hypothalamic paraventricular CRF neurons may participate in an appetite control circuit for reducing food intake.     

Deficits in the control of food intake after hypothalamic paraventricular nucleus lesions

Noradrenergic mechanisms of the hypothalamic paraventricular nucleus (PVN) have been shown to play an important role in the stimulation of feeding To determine the influence of this nucleus in monitoring and controlling responses to physiological and pharmacological challenges, PVN electrolytic lesion rats were tested for their behavioral responsiveness to agents known to affect the alpha-2 noradrenergic system as well as release of corticosterone, and to short- and long-term periods of food deprivation. Discrete lesions of the PVN produced enhanced feeding, particularly of carbohydrate, in freely-feeding rats maintained on a macronutrient self-selection paradigm. Lesion rats demonstrated a behavioral deficit in food intake regulation (a decrease in carbohydrate ingestion) in response to 5-hr and 24-hr fasts, showed a disturbance in circadian feeding, and exhibited a dramatic decrease in circulating corticosterone. However, feeding in response to 2-deoxy-D-glucose and insulin remained intact, suggesting that noradrenergic receptors within the PVN are not involved in the mediation of glucoprivic-induced feeding.     

Repeated hypothalamic stimulation with neuropeptide Y increases daily carbohydrate and fat intake and body weight gain in female rats

Neuropeptide Y (NPY), repeatedly injected in the hypothalamic paraventricular nucleus (PVN), produces dramatic obesity and overeating in female rats maintained on a single nutritionally complete diet. In the present study, we investigated whether these effects could also be obtained in animals with a choice of three pure macronutrients: protein, carbohydrate, and fat. Female rats with indwelling PVN cannulas were injected with NPY (235 pmol) or its saline vehicle every 8 hr for 6 days. A third group was left undisturbed. Consumption of each macronutrient and body weight were measured every 24 hr for 6 days preinjection, 6 days during injections, and 21 days after the injections were terminated. Relative to vehicle or preinjection rates of body weight gain (approximately 1.5 g/day), NPY dramatically enhanced weight gain to a rate of 9.3 g/day and more than doubled total daily food intake. This augmentation was accounted for by increases in carbohydrate intake (+26.4 kcal/day) and fat intake (+48.5 kcal/day), with no significant potentiation of protein consumption. When the NPY injections were terminated, body weight and macronutrient intake returned to control levels within 1 or 2 weeks. These findings are consistent with a role for NPY in hypothalamic mechanisms of macronutrient intake and body weight regulation and suggest that disturbances in brain NPY may contribute to the development of eating and weight disorders.     

Ghrelin、melanotan-Ⅱ以及胆囊收缩素对下丘脑双侧室旁核损伤大鼠摄食的影响

目的探讨大鼠下丘脑双侧室旁核(PVN)损伤引起的过食和肥胖的产生机制。方法 Wistar雄性大鼠36只,通过电损伤下丘脑双侧PVN使大鼠产生过食和肥胖,检测ghrelin、melanotan-II(MT-II,一种合成的α-黑色素细胞刺激素——α-MSH的结构同源体)以及胆囊收缩素-8(CCK-8)摄食作用的改变。结果双侧PVN损伤后大鼠的体重和摄食量明显增加,显示出过食和肥胖,手术后1周,外周给与Ghrelin,3～4h后PVN损伤组比伪损伤组的摄食量增加明显,Ghrelin显示出更强的刺激摄食的作用;中枢给予MT-Ⅱ可明显抑制24h节食大鼠的食物摄取,然而PVN损伤后MT-II对24h节食大鼠摄食的抑制作用减弱甚至消失;外周给予CCK-8可明显抑制大鼠各时间段的摄食作用,PVN损伤组和伪损伤组之间未见明显差异。结论大鼠下丘脑双侧PVN损伤引起的过食和肥胖可能与增强的ghrelin对摄食的刺激作用和(或)α-MSH受体的破坏有关。     

Kisspeptin signaling in the amygdala modulates reproductive hormone secretion

Kisspeptin (encoded by KISS1) is a crucial activator of reproductive function. The role of kisspeptin has been studied extensively within the hypothalamus but little is known about its significance in other areas of the brain. KISS1 and its cognate receptor are expressed in the amygdala, a key limbic brain structure with inhibitory projections to hypothalamic centers involved in gonadotropin secretion. We therefore hypothesized that kisspeptin has effects on neuronal activation and reproductive pathways beyond the hypothalamus and particularly within the amygdala. To test this, we mapped brain neuronal activity (using manganese-enhanced MRI) associated with peripheral kisspeptin administration in rodents. We also investigated functional relevance by measuring the gonadotropin response to direct intra-medial amygdala (MeA) administration of kisspeptin and kisspeptin antagonist. Peripheral kisspeptin administration resulted in a marked decrease in signal intensity in the amygdala compared to vehicle alone. This was associated with an increase in luteinizing hormone (LH) secretion. In addition, intra-MeA administration of kisspeptin resulted in increased LH secretion, while blocking endogenous kisspeptin signaling within the amygdala by administering intra-MeA kisspeptin antagonist decreased both LH secretion and LH pulse frequency. We provide evidence for the first time that neuronal activity within the amygdala is decreased by peripheral kisspeptin administration and that kisspeptin signaling within the amygdala contributes to the modulation of gonadotropin release and pulsatility. Our data suggest that kisspeptin is a ‘master regulator’ of reproductive physiology, integrating limbic circuits with the regulation of gonadotropin-releasing hormone neurons and reproductive hormone secretion.     

Adrenalectomy abolishes fasting-induced down-regulation of NADPH-diaphorase in the rat paraventricular nucleus

This study was conducted to define the molecular mechanism of fasting-induced down-regulation of neuronal nitric oxide synthase (nNOS) expression in the hypothalamic paraventricular nucleus (PVN). Rats were adrenalectomized (ADX), and then either underwent food deprivation or received varying doses of dexamethasone for 48 h. The brain tissues were processed for NADPH-diaphorase (NADPH-d) staining, a histochemical marker of nNOS enzyme activity. Both the ADX and the sham operated rats showed a significant weight loss after 48 h of food deprivation. Food deprivation decreased the number of NADPH-d containing cells in the PVN of sham rats, however, not in the ADX rats. Dexamethasone dose- dependently decreased NADPH-d cells in the PVN of ADX rats. The effect of ADX or dexamethasone was limited to the parvocellular subdivision of PVN. These results suggest that the adrenal glucocorticoids may down-regulate nNOS expression in the PVN during food deprivation.     

Melatonin enhances NADPH-diaphorase activities in the hypothalamus of maternally-separated rats

Maternal separation or social isolation is a risk factor in the development of mammalian species affecting both physical and mental growth, and food intake regulation. Melatonin has been known to regulate body weight on various species including rodents. We investigated the effect of melatonin treatment on the expression of nitric oxide synthase, which may involved in food intake regulation, in the brain of maternally separated-rats using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. Melatonin (10 mg/kg i.p.) was injected to 14-day-old maternally-separated rats for 7 days. Maternally-separated rats with melatonin administration showed significantly higher staining intensities of NADPH-d-positive neurons in the paraventricular nucleus (PVN) and in lateral hypothalamic area (LHA) than maternally-separated without melatonin administration (P < 0.05). Body weight of melatonin treated rats significantly increased at the 6th and 7th day compared to that of rats without melatonin treatment (P < 0.05). These results indicate that melatonin may be associated with increase body weight via NOS in the hypothalamic areas in maternally-separated or socially isolated rats.     

Amygdalar opioids modulate hypothalamic melanocortin-induced anorexia

We wanted to assess the possibility that opioid activity in the central amygdala (CeA) could modulate the feeding inhibition of melanocortin stimulation of the paraventricular hypothalamus (PVN). The melanocortin system is important in both the acute regulation of satiety and feeding behavior and in the integration of long-term appetite signals. Melanotan II (MTII) is a synthetic MC3R and MC4R agonist which reduces food intake when given intracerebroventricularly (ICV) and into the PVN. Tyr-D-Ala-Gly-(me) Phe-Gly-ol (DAMGO), a µ-opioid receptor agonist, increases food intake, while opioid antagonists, like naltrexone (NTX), inhibit food intake after injection into many brain sites involved in appetite regulation, including the CeA. In food-deprived male Sprague-Dawley rats, co-injected intra-PVN MTII partially blocked the orexigenic effect of co-injected intra-CeA DAMGO. Intra-CeA NTX co-injected with intra-PVN MTII reduced food intake significantly more than either alone. NTX administered intra-CeA reduced c-Fos-immunoreactivity (IR) in nucleus accumbens neurons significantly compared to the intra-PVN MTII treated animals, animals co-injected intra-PVN with MTII and intra-CeA with NTX animals, and control animals. Intra-PVN MTII induced c-Fos-IR in significantly more PVN neurons than observed in control animals. Intra-CeA NTX co-injected with intra-PVN MTII induced c-Fos-IR significantly in PVN neurons relative to control and intra-CeA NTX animals. Such data support the significance of opioid action within the CeA as a modulator of the feeding regulation action of melanocortins within the PVN, occurring within the context of a larger appetitive network.     

Lateral hypothalamic lesions and fenfluramine increase thermogenesis in brown adipose tissue

The effects of treatment with fenfluramine or electrolytic lesions in the lateral hypothalamus (LH) on binding of guanosine 5-diphosphate (GDP) by mitochondria from brown adipose tissue have been compared in 4 experiments. In 2 experiments the lesions were lateral to the anterior hypothalamic nucleus and in the other two they were lateral to the ventromedial hypothalamic nucleus. Binding of GDP to mitochondria was significantly increased 18 hours after an electrolytic lesion in either LH site. d,1-Fenfluramine, 20 mg/kg, also increased GDP binding in both acute experiments. In the other 2 experiments GDP binding was measured 11 days after the LH lesion or after 11 daily injections of fenfluramine. When the chronic lesions were lateral to the VMN, there was a transient drop in food intake and body weight. With more anterior lesions, body weight remained significantly lower than in sham-operated rats although food intake returned slowly to control levels. Fenfluramine-treated rats had lower body weights in both chronic experiments even after food intake returned to normal. GDP-binding to mitochondria from interscapular brown adipose tissue was elevated in both of the chronically-treated fenfluramine groups but was only increased in the LH-lesioned rats whose body weight remained below normal.     

Paraventricular hypothalamic clonidine increases rather than decreases susceptibility to activity-based anorexia in the rat.

Anorexia has been related to reduced activity of the paraventricular hypothalamic (PVN) noradrenergic-feeding system. In this study we determined whether clonidine (an alpha 2-adrenergic agonist) infused into the PVN reduced susceptibility to activity-based anorexia (ABA) in the rat. In Experiment 1, clonidine (6 doses) was chronically infused into the PVN of male Sprague-Dawley rats. All animals were exposed to ABA (1.5 hr/day food access; 22.5 hr/day running wheel access) until a 25% body weight loss was reached. Dose-related increases in susceptibility to ABA and decreases in food intake were observed. In Experiment 2, for which heavier animals and 3 doses of clonidine were used, we found no difference in food intake and wheel activity but increased susceptibility to ABA. Chronic clonidine infused into the PVN does not produce hyperphagia and exacerbates rather than attenuates susceptibility to ABA.     

Cholinergic input to the supraoptic nucleus increases Fos expression and body temperature in rats

To examine the role played by cholinergic input and processes in the supraoptic nucleus (SON) in the control of body temperature and water intake in rats, we used microdialysis to stimulate and analyze SON without disturbing the behavior of unanesthetized rats. After microdialysis, we also investigated immunoreactivity for c-Fos protein in the brain as an index of neuronal activation. Stimulation with neostigmine, an acetylcholine esterase inhibitor, through the microdialysis probe increased the extracellular concentration of acetylcholine in the SON. This cholinergic stimulation dose-dependently increased body temperature but did not significantly change the water intake. The stimulation markedly increased c-Fos-like immunoreactivity (Fos-IR) in the SON and certain hypothalamic areas, including the paraventricular nucleus (PVN) and median preoptic nucleus (MnPO). Fos-IR was also evident in certain regions of the pons and brainstem, including the locus ceruleus (LC), area postrema (AP), and nucleus of the solitary tract (NTS). Addition of atropine, a muscarinic receptor antagonist, to the dialysis medium containing neostigmine attenuated the increase of Fos-IR and suppressed the neostigmine-induced responses in body temperature. These results suggest that cholinergic input and activation of the muscarinic cholinoceptive neurons in the SON contribute to the regulation of body temperature. Activation of noradrenergic pathways in the brainstem including LC and NTS may be involved in the thermoregulation mechanism.     

The effect of leptin receptor deficiency and fasting on cannabinoid receptor 1 mRNA expression in the rat hypothalamus, brainstem and nodose ganglion

Despite ample evidence for the involvement of the endocannabinoid system in the control of appetite, food intake and energy balance, relatively little is known about the regulation of cannabinoid receptor 1 (CB₁R) expression in respect to leptin signalling and fasting. In the present study, we examined CB₁R mRNA levels in lean (Fa/?) and obese (fa/fa) male Zucker rats under basal and food-restricted conditions. Using stereological sampling principles coupled with semi-quantitative radioactive in situ hybridization we provide semi-quantitative estimates of CB₁R mRNA expression in key appetite regulatory hypothalamic and brainstem areas, as well as in the nodose ganglia. Whereas no effect of fasting were determined on CB₁R mRNA levels in the paraventricular (PVN) and ventromedial hypothalamic (VMH) nucleus, in the brainstem dorsal vagal complex or nodose ganglion of lean Zucker rats, CB₁R mRNA levels were consistently elevated in obese Zucker rats pointing to a direct influence of disrupted leptin signalling on CB₁R mRNA regulation.     

Role of melanin-concentrating hormone in the control of ethanol consumption: Region-specific effects revealed by expression and injection studies

The peptide melanin-concentrating hormone (MCH), produced mainly by cells in the lateral hypothalamus (LH), perifornical area (PF) and zona incerta (ZI), is suggested to have a role in the consumption of rewarding substances, such as ethanol, sucrose and palatable food. However, there is limited information on the specific brain sites where MCH acts to stimulate intake of these rewarding substances and on the feedback effects that their consumption has on the expression of endogenous MCH. The current study investigated MCH in relation to ethanol consumption, in Sprague-Dawley rats. In Experiment 1, chronic consumption of ethanol (from 0.70 to 2.7 g/kg/day) dose-dependently reduced MCH gene expression in the LH. In Experiments 2-4, the opposite effect was observed with acute oral ethanol, which stimulated MCH expression specifically in the LH but not the ZI. In Experiment 5, the effect of MCH injection in brain-cannulated rats on ethanol consumption was examined. Compared to saline, MCH injected in the paraventricular nucleus (PVN) and nucleus accumbens (NAc) selectively stimulated ethanol consumption without affecting food or water intake. In contrast, it reduced ethanol intake when administered into the LH, while having no effect in the ZI. These results demonstrate that voluntary, chronic consumption of ethanol leads to local negative feedback control of MCH expression in the LH. However, with a brief exposure, ethanol stimulates MCH-expressing neurons in this region, which through projections to the feeding-related PVN and reward-related NAc can promote further drinking behavior.     

Perfluorooctane sulfonate influences feeding behavior and gut motility via the hypothalamus

Perfluorinated compounds (PFCs) have been employed as surface treatment agents in a variety of products. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are the two most commonly found PFCs in the environment and human blood. We investigated the effects of PFOS and PFOA on feeding behavior. PFOS or PFOA was administered intracerebroventricularly in mice or rats. Following administration, food intake, gastroduodenal motility, gastric emptying, gene expression of hypothalamic neuropeptides, and c-Fos expression along with immunoreaction for urocortin 2 in the paraventricular nucleus (PVN) were determined. Centrally administered PFOS and PFOA decreased food intake. Administration of PFOS decreased gastric emptying and disrupted the fasted motor activity in the antrum and duodenum. The gene expression of urocortin 2 in the hypothalamus and c-Fos expression and immunoreaction for urocortin 2 in the PVN were increased by the action of PFOS. A centrally administered corticotropin-releasing factor type 2 receptor (CRFR2) antagonist blocked PFOS-induced anorexia. These findings indicate that PFOS and PFOA influence feeding behavior. This effect is mediated via the activation of hypothalamic urocortin 2 and CRFR2, and the suppression of gastroduodenal motor activity. These observations indicate that PFCs may act centrally to influence behavior and physiological functions in humans.     

Materno-fetal coordination of stress-induced Fos expression in the hypothalamic paraventricular nucleus during pregnancy

This study investigates whether maternal stress during pregnancy induces maternal and fetal hypothalamic paraventricular nucleus (PVN) neuronal activation and the effects of maternal stress on fetal hypothalamic and PVN brain-derived neurotrophic factor (BDNF) expression. Pregnant rats were exposed to three types of maternal stress with varying severity (restraint, forced walking and immobilization) for 30 min on gestational day 21. Severity of stress was assessed by measurement of maternal plasma corticosterone 30 min following the stimulus. Maternal plasma corticosterone increased in each stress response group (immobilization>forced walking>restraint). Further, the expression of Fos protein, a marker of neuronal activation, increased in the fetal and maternal PVN in direct relation to the severity of stress treatments. Forced walking and immobilized stress, but not restraint stress, significantly increased BDNF expression in the fetal hypothalamus.These findings suggest that the fetal hypothalamic-pituitary-adrenal (HPA) response following maternal stress mirrors maternal HPA activation. In addition, BDNF may play a role in protecting fetal brain neurons from damage caused by severe stress.