Estrous Cycle Modulation of Feeding and Relaxin-3/Rxfp3 mRNA Expression: Implications for Estradiol Action.

INTRODUCTION: Food intake varies during the ovarian hormone/estrous cycle in humans and rodents, an effect mediated mainly by estradiol. A potential mediator of the central anorectic effects of estradiol is the neuropeptide relaxin-3 (RLN3) synthetized in the nucleus incertus (NI) and acting via the relaxin family peptide-3 receptor (RXFP3). METHODS: We investigated the relationship between RLN3/RXFP3 signaling and feeding behavior across the female rat estrous cycle. We used in situ hybridization to investigate expression patterns of Rln3 mRNA in NI and Rxfp3 mRNA in the hypothalamic paraventricular nucleus (PVN), lateral hypothalamic area (LHA), medial preoptic area (MPA), and bed nucleus of the stria terminalis (BNST), across the estrous cycle. We identified expression of estrogen receptors (ERs) in the NI using droplet digital PCR and assessed the electrophysiological responsiveness of NI neurons to estradiol in brain slices. RESULTS: Rln3 mRNA reached the lowest levels in the NI pars compacta during proestrus. Rxfp3 mRNA levels varied across the estrous cycle in a region-specific manner, with changes observed in the perifornical LHA, magnocellular PVN, dorsal BNST, and MPA, but not in the parvocellular PVN or lateral LHA. G protein-coupled estrogen receptor 1 (Gper1) mRNA was the most abundant ER transcript in the NI. Estradiol inhibited 33% of type 1 NI neurons, including RLN3-positive cells. CONCLUSION: These findings demonstrate that the RLN3/RXFP3 system is modulated by the estrous cycle, and although further studies are required to better elucidate the cellular and molecular mechanisms of estradiol signaling, current results implicate the involvement of the RLN3/RXFP3 system in food intake fluctuations observed across the estrous cycle in female rats.

Differential effects of relaxin-3 and a selective relaxin-3 receptor agonist on food and water intake and hypothalamic neuronal activity in rats.

The neuropeptide relaxin-3 (RLN3) binds with high affinity to its cognate receptor, relaxin-family peptide receptor 3 (RXFP3), and with lower affinity to RXFP1, the cognate receptor for relaxin. Intracerebroventricular (icv) administration of RLN3 in rats strongly increases food and water intake and alters the activity of the hypothalamic-pituitary-adrenal (HPA) and gonadal (HPG) axes, but the relative involvement of RXFP3 and RXFP1 in these effects is not known. Therefore, the effects of icv administration of equimolar (1.1 nmol) amounts of RLN3 and the RXFP3-selective agonist RXFP3-A2 on food and water intake, plasma levels of corticosterone, testosterone, and oxytocin and c-fos mRNA expression in key hypothalamic regions in male rats were compared. Food intake was increased by both RLN3 and RXFP3-A2, but the orexigenic effects of RXFP3-A2 were significantly stronger than RLN3, 30 and 60min after injection. Water intake and plasma corticosterone and testosterone levels were significantly increased by RLN3, but not by RXFP3-A2. Conversely, RXFP3-A2 but not RLN3 decreased oxytocin plasma levels. RLN3, but not RXFP3-A2, increased c-fos mRNA levels in the parvocellular (PVNp) and magnocellular (PVNm) paraventricular and supraoptic (SON) hypothalamic nuclei, in the ventral medial preoptic area (MPAv), and in the organum vasculosum of the lamina terminalis (OVLT). A significant increase in c-fos mRNA expression was induced in the perifornical lateral hypothalamic area (LHApf) by RLN3 and RXFP3-A2. These results suggest that RXFP1 is involved in the RLN3 stimulation of water intake and activation of the HPA and HPG axes. The reduced food intake stimulation by RLN3 compared to RXFP3-A2 may relate to activation of both orexigenic and anorexigenic circuits by RLN3.

Inhibition in the lateral septum increases sucrose intake and decreases anorectic effects of stress.

Sucrose-overeating rats with decreased anorectic response to stress showed lower stress-induced activation of c-fos expression in the lateral septum (LS). The present study tested a hypothesis that neuronal inhibition in the LS is important for the development and maintenance of the sucrose-overeating phenotype. Sucrose overeating was developed with weekly episodes of food restriction (21 h per day, 4 days per week) followed by 1-h access to sucrose. The anorectic effects of stress on 1-h sucrose intake were estimated using weekly foot shock stress sessions. The development of the sucrose-overeating phenotype was accompanied by a decrease in the anorectic effects of stress and by an increase in LS mRNA expression of a γ-aminobutyric acid (GABA) synthesising enzyme, glutamic acid decarboxylase 67 in stressed rats. Direct recordings of neuronal firing in the LS in rats submitted to repeated weekly cycles of food restriction, sucrose refeeding and stress showed that the development of sucrose overeating increased the percentage of LS neurons inhibited during anticipation and at the start of clusters (CS) of sucrose licking. In addition, the CS-excited LS neurons showed a decrease in responsiveness to sucrose during the development of sucrose overeating. Direct injection of baclofen, an agonist of the GABAB receptor, into the LS decreased the anorectic effects of stress and increased sucrose intake. These results suggest that an increase in inhibitory effects in the LS is important for the development of sucrose overeating and the decreased anorectic effects of stress.

Sex-specific effects of relaxin-3 on food intake and brain expression of corticotropin-releasing factor in rats.

This study compared the effects of relaxin-3 (RLN3) on food intake, plasma corticosterone, and the expression of corticotropin-releasing factor (CRF) in male and female rats. RLN3 was injected into the lateral ventricle at 25, 200, and 800 pmol concentrations. RLN3 at 25 pmol increased food intake (grams) at 30 and 60 minutes after injection in female but not male rats. Female rats also showed higher increase in relative to body weight (BW) food intake (mg/g BW) for all RLN3 concentrations at 30 minutes and for 800 pmol of RLN3 at 60 minutes. Moreover, RLN3 at 800 pmol significantly increased 24-hour BW gain in female but not male rats. At 60 minutes after administration, 800 pmol of RLN3 produced a significant increase in plasma corticosterone and in the expression of CRF and c-fos mRNAs in the parvocellular paraventricular hypothalamic nucleus (PVN) in male but not female rats. The levels of c-fos mRNA in the magnocellular PVN were increased by RLN3 but did not differ between the sexes. Conversely, expression of CRF mRNA in the medial preoptic area was increased in female rats but was not sensitive to 800 pmol of RLN3. In the bed nucleus of the stria terminalis, 800 pmol of RLN3 significantly increased CRF mRNA expression in female but not male rats. Therefore, female rats showed more sensitivity and stronger food intake increase in response to RLN3. The differential effects of RLN3 on CRF expression in the PVN and bed nucleus of the stria terminalis may contribute to the sex-specific difference in the behavioral response.

Neuroactive peptides as putative mediators of antiepileptic ketogenic diets.

Various ketogenic diet (KD) therapies, including classic KD, medium chain triglyceride administration, low glycemic index treatment, and a modified Atkins diet, have been suggested as useful in patients affected by pharmacoresistant epilepsy. A common goal of these approaches is to achieve an adequate decrease in the plasma glucose level combined with ketogenesis, in order to mimic the metabolic state of fasting. Although several metabolic hypotheses have been advanced to explain the anticonvulsant effect of KDs, including changes in the plasma levels of ketone bodies, polyunsaturated fatty acids, and brain pH, direct modulation of neurotransmitter release, especially purinergic (i.e., adenosine) and γ-aminobutyric acidergic neurotransmission, was also postulated. Neuropeptides and peptide hormones are potent modulators of synaptic activity, and their levels are regulated by metabolic states. This is the case for neuroactive peptides such as neuropeptide Y, galanin, cholecystokinin, and peptide hormones such as leptin, adiponectin, and growth hormone-releasing peptides (GHRPs). In particular, the GHRP ghrelin and its related peptide des-acyl ghrelin are well-known controllers of energy homeostasis, food intake, and lipid metabolism. Notably, ghrelin has also been shown to regulate the neuronal excitability and epileptic activation of neuronal networks. Several lines of evidence suggest that GHRPs are upregulated in response to starvation and, particularly, in patients affected by anorexia and cachexia, all conditions in which also ketone bodies are upregulated. Moreover, starvation and anorexia nervosa are accompanied by changes in other peptide hormones such as adiponectin, which has received less attention. Adipocytokines such as adiponectin have also been involved in modulating epileptic activity. Thus, neuroactive peptides whose plasma levels and activity change in the presence of ketogenesis might be potential candidates for elucidating the neurohormonal mechanisms involved in the beneficial effects of KDs. In this review, we summarize the current evidence for altered regulation of the synthesis of neuropeptides and peripheral hormones in response to KDs, and we try to define a possible role for specific neuroactive peptides in mediating the antiepileptic properties of diet-induced ketogenesis.

Improving the heat transfer efficiency of synthetic oil with silica nanoparticles.

The heat transfer properties of synthetic oil (Therminol 66) used for high temperature applications was improved by introducing 15 nm silicon dioxide nanoparticles. Stable suspensions of inorganic nanoparticles in the non-polar fluid were prepared using a cationic surfactant (benzalkonium chloride). The effects of nanoparticle and surfactant concentrations on thermo-physical properties (viscosity, thermal conductivity and total heat absorption) of these nanofluids were investigated in a wide temperature range. The surfactant-to-nanoparticle (SN) ratio was optimized for higher thermal conductivity and lower viscosity, which are both critical for the efficiency of heat transfer. The rheological behavior of SiO(2)/TH66 nanofluids was correlated to average agglomerate sizes, which were shown to vary with SN ratio and temperature. The conditions of ultrasonic treatment were studied and the temporary decrease of agglomerate size from an equilibrium size (characteristic to SN ratio) was demonstrated. The heat transfer efficiencies were estimated for the formulated nanofluids for both turbulent and laminar flow regimes and were compared to the performance of the base fluid.

Brain activation following peripheral administration of the GLP-1 receptor agonist exendin-4.

The aim of our study was to investigate the anorectic and brain stimulatory effects of various doses of exendin-4 (Ex-4) and to investigate the role of the vagus nerve in Ex-4-induced brain activation. A dose-related increase in c-fos mRNA expression was observed following Ex-4 administration (0.155-15.5 µg/kg). Doses of Ex-4 that caused anorexia without aversive effects (0.155, 0.775 µg/kg) induced c-fos expression in the hypothalamic arcuate and paraventricular (PVH; parvocellular) nuclei as well as in the limbic and brainstem structures. Doses of Ex-4 that caused aversion (1.55, 15.5 µg/kg) stimulated the same regions (in a more intense way) and additionally activated the magnocellular hypothalamic structures (supraoptic nucleus and PVH magnocellular). The brain c-fos pattern induced by Ex-4 showed both similarities and differences with that induced by refeeding. Subdiaphragmatic vagotomy significantly blunted the stimulation of c-fos mRNA expression induced by Ex-4 in the nodose ganglion, the medial part of nucleus of the solitary tract, and the parvocellular division of the PVH. Pretreatment with Ex-9-39 (330 µg/kg ip) impaired the neuronal activation evoked by Ex-4 in all brain regions and in the nodose ganglion. Effects of Ex-4 on hypothalamic-pituitary-adrenal axis activity were not altered by vagotomy. Results of this study demonstrate and relate the anorectic and brain stimulatory effects of aversive and nonaversive doses of Ex-4 and indicate that the activation of specific central regions induced by the peripheral administration of Ex-4 is, at least in part, dependent on the integrity of the vagus nerve.

Acute injection of ASP in the third ventricle inhibits food intake and locomotor activity in rats.

Acylation-stimulating protein (ASP; also known as C3adesArg) stimulates triglyceride synthesis and glucose transport via interaction with its receptor C5L2, which is expressed peripherally (adipose tissue, muscle) and centrally. Previous studies have shown that ASP-deficient mice (C3KO) and C5L2-deficient mice (C5L2KO) are hyperphagic (59 to 229% increase, P < 0.0001), which is counterbalanced by increased energy expenditure measured as oxygen consumption (Vo(2)) and a lower RQ. The aim of the present study was to evaluate ASP's effect on food intake, energy expenditure, and neuropeptide expression. Male rats were surgically implanted with intracerebroventricular (icv) cannulas directed toward the third ventricle. After a 5-h fast, rats were injected, and food intake was assessed at 0.5, 1, 2, 4, 16, 24, and 48 h, with a 5- to 7-day washout period between each injection. Acute icv injections of ASP (0.3-1,065 pmol) had a time-dependent effect on decreasing food intake by 20 to 57% (P < 0.05). Decreases were detected by 30 min (maximum 57%, P < 0.01) and at the highest dose effects extended to 48 h (19%, P < 0.05, 24- to 48-h period). Daily body weight gain was decreased by 131% over the first 24 h and 29% over the second 24 h (P < 0.05). A conditioned taste aversion test indicated that there was no malaise. Furthermore, acute ASP injection affected energy substrate usage, demonstrated by decreased Vo(2) and RQ (P < 0.05; implicating greater fatty acid usage), with a 49% decrease in total activity over 24 h (P < 0.05). ASP administration also increased anorexic neuropeptide POMC expression (44%) in the arcuate nucleus, with no change in NPY. Altogether ASP may have central in addition to peripheral effects.

Intermittent access to sucrose increases sucrose-licking activity and attenuates restraint stress-induced activation of the lateral septum.

Intermittent access to palatable food can attenuate anorectic and hormonal responses to stress in rats. The neuronal mechanisms of modulation of stress response by diets are not fully understood. The present study was conducted to create rat models with intermittent access to sucrose that demonstrate resistance to stress-induced hypophagia, to study the pattern of sucrose consumption by these rat models, and to investigate in which brain structures intermittent sucrose regimens modify stress-induced neuronal activation. The obtained results demonstrate that 6-wk intermittent access to sucrose without food restriction (4 day/wk ad libitum access to sucrose in addition to chow, and following 3 day/wk exclusive feeding of chow; SIA rats) and combined with food restriction (4 day/wk access to chow and sucrose restricted to 2 h/day, and following 3 days/wk on unrestricted chow; SIR rats) increased sucrose-licking activity. The alterations in the rats' feeding behavior were accompanied by a resistance of their body weight gain and food intake to 1-h restraint stress applied once per week. The chronic intermittent sucrose consumption significantly lowered, in the SIA and SIR rats, the levels of expression of corticotropin-releasing factor type 2 receptor and restraint stress-induced expression of c-fos mRNA in the medioventral part of the lateral septum. Conversely, the levels of the corticotropin-releasing factor type 2 receptor transcript in the ventromedial hypothalamic nucleus were decreased only in the food-restricted SIR rats. The lower stress-induced neuronal activation in the medioventral part of the lateral septum may contribute to the attenuated anorectic stress response in the rats maintained on intermittent sucrose regimens.

The brain endocannabinoid system in the regulation of energy balance.

The role played by the endocannabinoid system in the regulation of energy balance is currently generating a great amount of interest among several groups of investigators. This interest in large part comes from the urgent need to develop anti-obesity and anti-cachexia drugs around target systems (such as the endocannabinoid system), which appears to be genuinely involved in energy balance regulation. When activated, the endocannabinoid system favors energy deposition through increasing energy intake and reducing energy expenditure. This system is activated in obesity and following food deprivation, which further supports its authentic function in energy balance regulation. The cannabinoid receptor type 1 (CB1), one of the two identified cannabinoid receptors, is expressed in energy-balance brain structures that are also able to readily produce or inactivate N-arachidonoyl ethanolamine (anandamide) and 2-arachidonoylglycerol (2AG), the most abundantly formed and released endocannabinoids. The brain action of endocannabinoid system on energy balance seems crucial and needs to be delineated in the context of the homeostatic and hedonic controls of food intake and energy expenditure. These controls require the coordinated interaction of the hypothalamus, brainstem and limbic system and it appears imperative to unravel those interplays. It is also critical to investigate the metabolic endocannabinoid system while considering the panoply of functions that the endocannabinoid system fulfills in the brain and other tissues. This article aims at reviewing the potential mechanisms whereby the brain endocannabinoid system influences the regulation energy balance.

The dynamics of neuronal activation during food anticipation and feeding in the brain of food-entrained rats.

Rats can anticipate a daily mealtime when they are maintained on restricted feeding schedules (RFS). Neural substrates of the food-entrainable oscillator (FEO) are not yet fully understood. The numerous lesions of a single brain region failed to abolish the behavioral anticipation of a daily meal, suggesting that the FEO may be represented by a distributed neuronal network. The present study was designed to detect the dynamics of neuronal activation, using as a marker the expression of c-fos mRNA in the brain of rats subjected to 2-hour daily RFS, 3, 2 and 1 h before the expected meal, at the time of the usual feeding, and 1 h after feeding. We also aimed to clarify whether the increase in plasma corticosterone in food-anticipating rats coincides with the increase in expression of corticotropin releasing factor (CRF) mRNA in the paraventricular hypothalamic nucleus (PVH). The obtained results revealed that the neuronal activation occurring 3 h before the expected meal was not confined to one brain structure, but was evident in the anterior hippocampal continuation and septohippocampal nucleus (AH/SHi), the anterior part of the paraventricular thalamic nucleus (PVTa), and the dorsomedial hypothalamic nucleus (DMH), thus representing distributed septohippocampal-thalamo-hypothalamic circuitry that may act as the FEO. The pattern of neuronal activation after feeding was different from that detected during food anticipation for some specific nucleus or subregions. The increase in plasma corticosterone during food anticipation was not accompanied by an increase in CRF mRNA levels, suggesting that factors other than CRF are involved in the control of adrenocortical secretion under RFS.

Contribution of the vagus nerve and lamina terminalis to brain activation induced by refeeding.

Following refeeding, c-fos expression is induced in a particular set of brain regions that include the nucleus of the solitary tract (NTS), parabrachial nucleus (PB), central amygdala (CeA), paraventricular hypothalamic nucleus (PVH), supraoptic nucleus (SON) and the circumventricular organs. Within the PVH, the expression is particularly intense in the magnocellular division of the nucleus and it is as yet not clear how this activation occurs. The respective contribution of the vagus afferents and lamina terminalis, which conveys signals entering the brain through the forebrain circumventricular organs, has been investigated in rats subjected to a unilateral cervical vagotomy (UCV) or a unilateral lesion of the fibres running within the lamina terminalis (ULT) and projecting to the neuroendocrine hypothalamus. UCV significantly decreased postprandial c-fos expression in the NTS, PB, CeA and parvocellular division of the PVH. In contrast, ULT impaired postprandial activation of the magnocellular neurons in the PVH and SON. The present study also characterized the types of neurons activated in the PVH and SON during refeeding. In the magnocellular regions, arginine-vasopressin (AVP) neurons were activated upon refeeding whereas there was no apparent induction of Fos expression in oxytocin cells. In the parvocellular PVH, postprandial Fos was induced only in 30% of the corticotrophin-releasing factor (CRF) and AVP neurons. The results of the present study suggest that the postprandial activation of the brain requires the integrity of both the vagal- and lamina terminalis-associated pathways.

Effects of treadmill running on brain activation and the corticotropin-releasing hormone system.

The present study was conducted to investigate the effects of treadmill running on the corticotropin-releasing hormone (CRH), CRH receptor type 1 (CRH-R1) and CRH-binding protein (CRH-BP) in the brain of rats that were killed either at rest, immediately after 60 min of treadmill running, or 180 min following a 60-min session of intensive exercise. The expression of the neuronal activity marker c-FOS was also determined in the three conditions of this study. The levels of c-FOS mRNA immediately following running were high in the cortex, caudate-putamen, lateral septum, bed nucleus of the stria terminalis, dorsal and medial thalamus, hypothalamus, pontine nuclei, locus coeruleus and hypoglossal nucleus. In most brain regions investigated, excluding the locus coeruleus and the cingulate cortex, c-FOS mRNA expression returned to control levels after 2 h of recovery. The highest concentration of cells co-expressing the protein Fos and CRH mRNA neurons was found in the parvocellular part of the paraventricular nucleus, which also expressed CRH heteronuclear RNA and CRH-R1 mRNA. The medial preoptic area (MPOA), the medial mammillary nucleus and the posterior hypothalamic as well as the somatosensory cortex, the medial geniculate nucleus, the reticulotegmental nucleus, and Barrington's nucleus also co-expressed Fos and CRH mRNA. The expression of CRH-BP gene was induced in the MPOA following running. In summary, the present study demonstrates that treadmill running leads to a strong expression of c-FOS mRNA that is widely distributed throughout the brain. c-FOS mRNA was found in structures of the somatosensory and somatomotor systems, indicating that these regions were activated during exercise. The pattern of distribution of c-FOS mRNA showed similarities with that triggered by neurogenic and systemic stresses. The present results also indicate that treadmill running can strongly activate the hypophysiotropic CRH system, which suggests, in agreement with the pattern of c-FOS mRNA distribution, that treadmill running has a strong stress component.

Neuronal activation and corticotropin-releasing hormone expression in the brain of obese (fa/fa) and lean (fa/?) Zucker rats in response to refeeding.

The present study was conducted to investigate the pattern of neuronal activation and corticotropin-releasing hormone (CRH) expression in fed, food deprived and refed lean (Fa/?) and obese (fa/fa) Zucker rats. The pattern of neuronal activation was studied by measuring the expression of the immediate-early gene c-fos. Expression of c-fos and CRH mRNA was determined by in situ hybridization histochemistry. In both lean and obese rats, one hour of refeeding led to a transient increase in c-fos mRNA levels which was detected in the paraventricular hypothalamic nucleus (PVH), the dorsomedial hypothalamic nucleus, the supraoptic nucleus, the paraventricular thalamic nucleus, the central nucleus of amygdala (CeA), the lateral and medial parabrachial nuclei, the nucleus of the solitary tract, and the area postrema. In addition, refeeding led to strong activation of the arginine-vasopressin neurons located in the magnocellular part of the PVH. Following 24 h of food deprivation, CRH expression in the parvocellular division of the PVH was significantly higher in obese rats compared to lean animals. During refeeding, PVH CRH mRNA levels in obese rats decreased to reach control values. The decrease in CRH expression in obese rats was accompanied by the alleviation of the hypercorticosteronemia that characterized obese Zucker rats. CRH mRNA levels in the central nucleus of the amygdala were significantly higher in lean rats than in obese animals, when the rats were fed ad libitum During food deprivation, CeA CRH mRNA levels decreased in lean rats and gradually returned to predeprivation values during refeeding. In refed obese rats, CeA levels of CRH mRNA were higher than those of ad libitum fed or food-deprived obese mutants. In the perifornical region of the lateral hypothalamic area (LHA), the expression of CRH mRNA rose significantly in response to refeeding in lean rats, but not in obese animals. Following the first hour of refeeding, the number of neurons expressing CRH mRNA in the LHA in lean rats almost doubled. The present results demonstrate that refeeding has a stimulating effect in obese Zucker rats in a pattern of activation similar to that seen in lean Fa/? rats. They also demonstrate differences in CRH expression between Fa/? and fa/fa rats after refeeding. The most apparent of these differences was seen in the lateral hypothalamus in which refeeding failed to up-regulate CRH expression in obese rats.