Estradiol treatment increases feeding-induced c-Fos expression in the brains of ovariectomized rats

The steroid hormone estradiol decreases meal size by increasing the potency of negative-feedback signals involved in meal termination. We used c-Fos immunohistochemistry, a marker of neuronal activation, to investigate the hypothesis that estradiol modulates the processing of feeding-induced negative-feedback signals within the nucleus of the solitary tract (NTS), the first central relay of the neuronal network controlling food intake, and within other brain regions related to the control of food intake. Chow-fed, ovariectomized rats were injected subcutaneously with 10 microg 17-beta estradiol benzoate or sesame oil vehicle on 2 consecutive days. Forty-eight hours after the second injections, 0, 5, or 10 ml of a familiar sweet milk diet were presented for 20 min at dark onset. Rats were perfused 100 min later, and brain tissue was collected and processed for c-Fos-like immunoreactivity. Feeding increased the number of c-Fos-positive cells in the NTS, the paraventricular nucleus of the hypothalamus (PVN), and the central nucleus of the amygdala (CeA) in oil-treated rats. Estradiol treatment further increased this response in the caudal, subpostremal, and intermediate NTS, which process negative-feedback satiation signals, but not in the rostral NTS, which processes positive-feedback gustatory signals controlling meal size. Estradiol treatment also increased feeding-induced c-Fos in the PVN and CeA. These results indicate that modest amounts of food increase neuronal activity within brain regions implicated in the control of meal size in ovariectomized rats and that estradiol treatment selectively increases this activation. They also suggest that estradiol decreases meal size by increasing feeding-related neuronal activity in multiple regions of the distributed neural network controlling meal size.     

Estradiol treatment increases CCK-induced c-Fos expression in the brains of ovariectomized rats

The ovarian hormone estradiol reduces meal size and food intake in female rats, at least in part by increasing the satiating potency of CCK. Here we used c-Fos immunohistochemistry to determine whether estradiol increases CCK-induced neuronal activation in several brain regions implicated in the control of feeding. Because the adiposity signals leptin and insulin appear to control feeding in part by increasing the satiating potency of CCK, we also examined whether increased adiposity after ovariectomy influences estradiol's effects on CCK-induced c-Fos expression. Ovariectomized rats were injected subcutaneously with 10 microg 17beta-estradiol benzoate (estradiol) or vehicle once each on Monday and Tuesday for 1 wk (experiment 1) or for 5 wk (experiment 2). Two days after the final injection of estradiol or vehicle, rats were injected intraperitoneally with 4 microg/kg CCK in 1 ml/kg 0.9 M NaCl or with vehicle alone. Rats were perfused 60 min later, and brain tissue was collected and processed for c-Fos immunoreactivity. CCK induced c-Fos expression in the nucleus of the solitary tract (NTS), area postrema (AP), paraventricular nucleus of the hypothalamus (PVN), and central nucleus of the amygdala (CeA) in vehicle- and estradiol-treated ovariectomized rats. Estradiol treatment further increased this response in the caudal, subpostremal, and intermediate NTS, the PVN, and the CeA, but not in the rostral NTS or AP. This action of estradiol was very similar in rats tested before (experiment 1) and after (experiment 2) significant body weight gain, suggesting that adiposity does not modulate CCK-induced c-Fos expression or interact with estradiol's ability to modulate CCK-induced c-Fos expression. These findings suggest that estradiol inhibits meal size and food intake by increasing the central processing of the vagal CCK satiation signal.     

Structural aspects of agonism and antagonism in the oestrogen receptor

We have determined the three-dimensional structures of both alpha- and beta-forms of the ligand-binding domain of the oestrogen receptor (ER) in complexes with a range of receptor agonists and antagonists. Here, we summarize how these structures provide both an understanding of the ER's distinctive pharmacophore and a rationale for its ability to bind a diverse range of chemically distinct compounds. In addition, these studies provide a unique insight into the mechanisms that underlie receptor activation, as well as providing a structural basis for the antagonist action of molecules, such as raloxifene.     

Estradiol mitigates ischemia reperfusion-induced acute renal failure through NMDA receptor antagonism in rats

Understanding the molecular mechanism of gastric cancer cell apoptosis is pivotal for the development of precise therapies targeting this disease. In the present study, we examined the effects of annexin A7 inhibition on the apoptosis of gastric cancer cells and the growth of tumour xenografts in vivo. Expression of annexin A7 in BGC823 cells was suppressed by small interference RNA, and cells apoptosis was assessed by flow cytometry. The mechanism by which annexin A7 mediates apoptosis in BGC823 cells was explored by determining the expression of key apoptosis regulators. In addition, by suppressing annexin A7 in BGC823 cells with small hairpin RNA, we studied the effects of annexin A7 inhibition on in vivo tumour growth. Our results showed that inhibiting annexin A7 expression induced more than fivefold increase in BGC823 cell apoptosis in vitro. This was in concord with a significant decrease of Bcl-2 expression and increases of Bax, Caspase-3, and Caspase-9. The activities of caspase-3 and caspase-9 were increased by 2.95?±?0.18 and 3.70?±?0.33 times, respectively, upon the annexin A7 downregulation in BGC823 cells. Importantly, suppressing annexin A7 showed the same apoptotic mechanism in vivo and significantly inhibited the growth of BGC823 xenografts in mice. These data suggest that annexin A7 likely protects gastric cells from apoptosis and targeting it may represent a valuable strategy in future therapeutic development.     

Antibodies raised against different extracellular loops of the melanocortin-3 receptor affect energy balance and autonomic function in rats

Melanocortin receptors (MCR) play an important role in the regulation of energy balance and autonomic function. In the present studies, we used active immunization against peptide sequences from the first and the third extracellular loop (EL1 and EL3) of the MC3R to generate selective antibodies (Abs) against this MCR subtype in rats. Immunization with the EL1 peptide resulted in Abs that enhanced the effects of the endogenous ligand α-melanocyte-stimulating hormone (α-MSH), whereas immunization with the EL3 peptide resulted in Abs acting as non-competitive antagonists. The phenotype of immunized rats chronically instrumented with telemetry transducers was studied under four different conditions: a high-fat diet was followed by standard lab chow, by fasting, and finally by an intraperitoneal injection of lipopolysaccharide (LPS). Under high-fat diet, food intake and body weight were higher in the EL3 than in the EL1 or the control group. Blood pressure was increased in EL3 rats and locomotor activity was reduced. Plasma concentrations of triglycerides, insulin, and leptin tended to rise in the EL3 group. After switching to standard lab chow, the EL1 group showed a small significant increase in blood pressure that was more pronounced and associated with an increase in heart rate during food restriction. No differences between the EL1 or the EL3 group were observed after LPS injection. These results show that immunization against the MC3R resulted in the production of Abs with positive or negative allosteric properties. The presence of such Abs induced small changes in metabolic and cardiovascular parameters.     

Antibodies raised against different extracellular loops of the melanocortin-3 receptor affect energy balance and autonomic function in rats

Melanocortin receptors (MCR) play an important role in the regulation of energy balance and autonomic function. In the present studies, we used active immunization against peptide sequences from the first and the third extracellular loop (EL1 and EL3) of the MC3R to generate selective antibodies (Abs) against this MCR subtype in rats. Immunization with the EL1 peptide resulted in Abs that enhanced the effects of the endogenous ligand α-melanocyte-stimulating hormone (α-MSH), whereas immunization with the EL3 peptide resulted in Abs acting as non-competitive antagonists. The phenotype of immunized rats chronically instrumented with telemetry transducers was studied under four different conditions: a high-fat diet was followed by standard lab chow, by fasting, and finally by an intraperitoneal injection of lipopolysaccharide (LPS). Under high-fat diet, food intake and body weight were higher in the EL3 than in the EL1 or the control group. Blood pressure was increased in EL3 rats and locomotor activity was reduced. Plasma concentrations of triglycerides, insulin, and leptin tended to rise in the EL3 group. After switching to standard lab chow, the EL1 group showed a small significant increase in blood pressure that was more pronounced and associated with an increase in heart rate during food restriction. No differences between the EL1 or the EL3 group were observed after LPS injection. These results show that immunization against the MC3R resulted in the production of Abs with positive or negative allosteric properties. The presence of such Abs induced small changes in metabolic and cardiovascular parameters.     

Activation of central melanocortin-4 receptor suppresses lipopolysaccharide-induced fever in rats

Activation of central melanocortin receptors (MCR) inhibits fever, but the identity of the MCR subtype(s) mediating this antipyretic effect is unknown. To determine whether selective central melanocortin receptor-4 (MC4R) activation produces antipyretic effects, the MC4R selective agonist MRLOB-0001 (CO-His-d-Phe-Arg-Trp-Dab-NH(2)) was administered intracerebroventricularly to rats treated with Escherichia coli lipopolysaccharide (LPS, 30 microg/kg ip). Treatment with MRLOB-0001 (150 ng icv) did not lower core body temperature (T(c)) in afebrile rats but did suppress LPS-induced increases in T(c) and associated decreases in tail skin temperature (T(sk)), an indicator of vasomotor thermoeffector function. In contrast, systemic treatment with MRLOB-0001 (150 ng iv) did not produce similar antipyretic effects. Coadministration of the selective MC4R antagonist HS014 (1 microg icv) blocked the antipyretic effects of MRLOB-0001. HS014 alone (1 microg icv) had no significant effect on LPS-induced increases in T(c) or decreases in T(sk) and in afebrile rats had no significant effects on T(c) or T(sk). We conclude that pharmacological activation of central MC4R suppresses febrile increases in T(c) and that inhibition of heat conservation pathways may contribute to this effect. These findings suggest that the central MC4R may mediate the long-recognized antipyretic effects of centrally administered melanocortins.     

Acute arginine supplementation fails to improve muscle endurance or affect blood pressure responses to resistance training

Dietary supplement companies claim that arginine supplements acutely enhance skeletal muscular endurance. The purpose of this study was to determine whether acute arginine α-ketoglutarate supplementation (AAKG) will affect local muscle endurance of the arm and shoulder girdle or the blood pressure (BP) response to anaerobic exercise. Twelve trained college-aged men (22.6 ± 3.8 years) performed 2 trials of exercise separated by at least 1 week. At 4 hours before, and 30 minutes before exercise, a serving of an AAKG supplement (3,700 mg arginine alpha-ketoglutarate per serving) or placebo was administered. Resting BP was assessed pre-exercise after 16 minutes of seated rest, and 5 and 10 minutes postexercise. Three sets each of chin-ups, reverse chin-ups, and push-ups were performed to exhaustion with 3 minutes of rest between each set. Data were analyzed using repeated-measures analysis of variance and paired t-tests. The AAKG supplementation did not improve muscle endurance or significantly affect the BP response to anaerobic work. Subjects performed fewer total chin-ups (23.75 ± 6.38 vs. 25.58 ± 7.18) and total trial repetitions (137.92 ± 28.18 vs. 141.08 ± 28.57) in the supplement trial (p ≤ 0.05). Subjects executed fewer reverse chin-ups (5.83 ± 1.85 vs. 6.75 ± 2.09) during set 2 after receiving the supplement as compared to the placebo (p < 0.05). Because AAKG supplementation may hinder muscular endurance, the use of these supplements before resistance training should be questioned.     

Ablation of eosinophil and IgE responses with anti-IL-5 or anti-IL-4 antibodies fails to affect immunity against Schistosoma mansoni in the mouse

To investigate the role of anaphylactic immune responses in protective immunity against schistosomiasis, mice vaccinated with irradiated cercariae of Schistosoma mansoni were treated with neutralizing mAb antibodies against either IL-5 or IL-4 before and during challenge infection. Anti-IL-5-treated vaccinated mice showed a complete ablation of circulating as well as tissue eosinophils present in inflammatory reactions to migrating schistosomula in the skin and lungs but nevertheless eliminated challenge infections as effectively as vaccinated animals treated with a control mAb. Similarly, treatment of vaccinated mice with an anti-IL-4 mAb markedly reduced serum IgE although failing to diminish immunity. The effect of anti-IL-5 mediated eosinophil depletion was also assessed in a second model in which resistance is induced by concomitant chronic infection. Again, normal, unaltered protection was observed in the absence of circulating and tissue eosinophils. In contrast to the above findings, treatment with anti-IFN-gamma was found to cause a partial depletion of immunity in vaccinated mice whereas, paradoxically, increasing the numbers of inflammatory reactions against invading schistosomula in the lungs. These observations argue against a requirement for either eosinophils or IgE in the anti-schistosome immunity induced by vaccination with irradiated cercariae or for eosinophils in the resistance resulting from previous infection in mice and support previous data suggesting a role for an IFN-gamma dependent cell-mediated effector mechanism in vaccine-induced resistance.     

Estradiol plus progesterone treatment and precopulatory behavior in prepubertally ovariectomized female rats: dose-response relationships

The copulatory and precopulatory behavioral repertoire expressed in terms of the intensity scale of sexual responsiveness was investigated in female rats ovariectomized on the 30th day after birth and treated for 9 weeks, once weekly, with estradiol dipropionate (5, 10, 15, 20, and 30 micrograms per animal) plus progesterone (in the range from 0.0 to 2.4 mg per animal). It was found that the higher the dosage of estradiol and progesterone used, the more complete the precopulatory behavioral pattern that was induced. Further, the same level of sexual responsiveness was achieved by various combinations of both hormones used. The dose-response relationships are less complicated in prepubertally ovariectomized females in comparison with those spayed as adults. Finally, the most complete pattern, characterized by ritualized darting, was not achieved at all.     

Obesity-associated mutations in the human melanocortin-4 receptor gene

Mutations in the human melanocortin-4 receptor (MC4R) gene have been associated with severe obesity. Many of the mutations result in partial or complete loss-of-function based on the nature of the mutation or the function of mutated receptors when tested in heterologous expression systems. This review discusses the role of MC4R in the central regulation of body weight, the pathogenic mechanisms of the mutations, and the validity of MC4R as an anti-obesity drug target.     

Molecular determinants of ligand binding to the human melanocortin-4 receptor

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.     

Estradiol increases growth hormone secretion in rats exposed to swimming stress or reserpine treatment

The secretory pattern of growth hormone (GH) in female rats differs from that in males with respect to e.g. the inter-peak baseline levels being higher in females. In the present study the influence of sex steroids on plasma GH levels was investigated in male rats under various conditions. Administration of estradiol, but not testosterone, was found to increase GH release in rats with suppressed levels induced by exposure to swimming stress or by treatment with the monoamine depleting agent reserpine. In line with previous studies, administration of estradiol was found to increase also inter-peak GH levels in adult male rats; i.e. to cause a feminization of the secretory pattern. In stressed and in reserpinized animals as well as in normal male rats, the effect of estradiol is similar to that earlier demonstrated for somatostatin antiserum, and hence it is suggested that estradiol may act antagonistic to the GH inhibiting factor.     

Downregulation of melanocortin-4 receptor during refeeding and its modulation by adrenalectomy in rats

Melanocortin system and corticotropin releasing hormone (CRH) are implicated in the control of feeding behavior. Besides its anorexigenic effect on food intake, CRH is one of the most important regulators of hypothalamic-pituitary-adrenal (HPA) axis activity. Therefore, there could be an interplay between HPA axis activity and melanocortin system. We investigated the expression of melanocortin-4 receptor (MC4-R) mRNA in the hypothalamus of rats after 14 days of food restriction or after a fasting-refeeding regimen, in sham or adrenalectomized rats. Male Wistar rats were subjected to free access to food or food ingestion restricted for 2 h a day (8-10 AM) during 14 d, when plasma corticosterone, ACTH, insulin, leptin concentrations, and MC4-R mRNA expression were determined before and after refeeding. Another set of rats was fasted for 48 h, followed by refeeding during 2 or 4 h on the seventh day after adrenalectomy (ADX) or sham surgery. On the day of the experiment, rats were anesthetized and perfused and the brain processed for MC4-R mRNA by in situ hybridization. Long-term reduction of food intake, either secondary to food restriction or adrenalectomy, reduced body weight gain and also leptin and insulin plasma concentrations. Food ingestion reduced MC4-R expression in the paraventricular nucleus in naive rats subjected to food restriction and also in sham rats fasted for 48 h. However, after ADX, MC4-R expression was not changed by refeeding. In conclusion, the present data indicate that MC4-R expression is downregulated by food ingestion and this response could be modulated by glucocorticoid withdrawal.     

Behavioral and IL-2 responses to diosgenin in ovariectomized rats

Neuroimmune system is involved in communication between the endocrine and nervous systems, which may take part in the effects of dioscorea, reversing changes of anxiety-like behavior and interleukin (IL)-2 levels in the brains of ovariectomized (OVX) rats. This study was aimed at evaluating administration of diosgenin, an ingredient of dioscorea, on neuroimmune and behavioral functions in OVX animals. One month after ovariectomy, female Wistar rats were fed daily with diosgenin (0, 10, 50 or 100 mg/kg/day) and the elevated plus-maze and learned helplessness tests were used to measure anxiety-like and depressive behaviors after 23 and 24 days of diosgenin treatment, respectively. In the learned helplessness test, the rats needed to cross from one compartment of the shuttle box to the opposite compartment to avoid or escape the shock. If the rat failed to escape the shock in 10 sec, a "failure" was recorded. Two days after the behavioral tests, the brain was removed to measure levels of IL-2 which was used as an indicator of neuroimmune function. Anxiety-like behavior in the OVX rats was not affected by diosgenin treatment. However, avoidance behavior in the learned helplessness test in the OVX rats with high anxiety (HA) levels was improved by treatment with diosgenin at the dosage of 10mg/kg/day. Interestingly, the number of failures in the same test was increased when the dosage of diosgenin was increased to 50 mg/kg/day, and this was accompanied by an increase in IL-2 levels in the pituitary gland. In addition, treatment with 100 mg/kg/day of diosgenin resulted in decreased IL-2 levels in the amygdala and prefrontal cortex of the OVX rats with low anxiety levels, and in increased IL-2 levels in the amygdala of OVX HA rats. These results show that chronic diosgenin treatment influences IL-2 levels in the brain of OVX rats and affects depressive behavior in OVX HA rats, but not OVX low anxiety rats.     

Activation of the melanocortin-4 receptor causes enhanced excitation in presympathetic paraventricular neurons in obese Zucker rats

Sympathetic nerve activity is increased in obesity-related hypertension. However, the central mechanisms involved in the increased sympathetic outflow remain unclear. The hypothalamic melanocortin system is important for regulating energy balance and sympathetic outflow. To understand the mechanisms by which the melanocortin systems regulates sympathetic outflow, we investigated the role of melanocortin 4 receptors (MC4R) in regulating presympathetic paraventricular nucleus (PVN) neurons. We performed whole-cell patch-clamp recordings on retrogradely labeled PVN neurons projecting to the rostral ventrolateral medulla in brain slices from obese zucker rats (OZRs) and lean zucker rats (LZRs). The MC4R agonists melanotan II (MTII) and α-melanocyte-stimulating hormone (α-MSH) increased the firing activity and depolarized the labeled PVN neurons from both LZRs and OZRs in a concentration-dependent manner. MTII produced significant greater increase in the firing activity in OZRs than in LZRs. Blocking MC4R with the specific antagonist SHU9119 had no effect on the basal firing rate but abolished the MTII-induced increase in the firing rate in both OZRs and LZRs. Furthermore, intracellular dialysis of guanosine 5'-O-(2-thodiphosphate), but not bath application of kynurenic acid and bicuculline, eliminated the MTII-induced increase in firing activity. In addition, MTII had no effect on the frequency and amplitude of glutamatergic excitatory postsynaptic currents and GABAergic inhibitory postsynaptic currents in labeled PVN neurons. Collectively, our findings suggest that MC4R contributes to the elevated excitability of PVN presympathetic neurons, which may be involved in obesity-related hypertension.     

Effects of peripheral CCK receptor blockade on feeding responses to duodenal nutrient infusions in rats

Type A cholecystokinin receptor (CCKAR) antagonists differing in blood-brain barrier permeability were used to test the hypothesis that duodenal delivery of protein, carbohydrate, and fat produces satiety in part by an essential CCK action at CCKARs located peripheral to the blood-brain barrier. Fasted rats with open gastric fistulas received devazepide (1 mg/kg iv) or A-70104 (700 nmol. kg(-1). h(-1) iv) and either a 30-min intravenous infusion of CCK-8 (10 nmol. kg(-1). h(-1)) or duodenal infusion of peptone, maltose, or Intralipid beginning 10 min before 30-min access to 15% sucrose. Devazepide penetrates the blood-brain barrier; A-70104, the dicyclohexylammonium salt of Nalpha-3-quinolinoyl-d-Glu-N,N-dipentylamide, does not. CCK-8 inhibited sham feeding by approximately 50%, and both A-70104 and devazepide abolished this response. Duodenal infusion of each of the macronutrients dose dependently inhibited sham feeding. A-70104 and devazepide attenuated inhibitory responses to each macronutrient. Thus endogenous CCK appears to act in part at CCKARs peripheral to the blood-brain barrier to inhibit food intake.     

Glutamate antagonism fails to reverse mitochondrial dysfunction in late phase of experimental neonatal asphyxia in rats

Because estrogen plays important neurotrophic and neuroprotective roles in the brain by activating estrogen receptors (ERs), disruption of normal estrogen signaling can leave neurons vulnerable to a variety of insults, including β-amyloid peptide (Aβ). Aroclor1254 (A1254) belongs to the endocrine-disrupting chemical (EDC) polychlorinated biphenyls and has anti-estrogenic properties. In the present study, we evaluated the effect of A1254 on the protective activity of estrogen against Aβ toxicity in differentiated cholinergic SN56 cells. Aged Aβ25-35 causes apoptotic cell death in differentiated SN56 cells, and the cytotoxic evidences are effectively rescued by estrogen. We found that A1254 abolishes the neuroprotective activity of estrogen against Aβ toxicity, and attenuates the suppressive effect of estrogen on Aβ-induced tau phosphorylation and JNK activation. The effects of A1254 on the neuroprotective effects of estrogen in Aβ toxicity are very similar to the effects of the estrogen receptor antagonist ICI182,780. Thus, exposure to EDCs that have anti-estrogenic activity might interfere with normal estrogen-activated neuroprotective signaling events and leave neurons more vulnerable to dangerous stimuli. Our present results provide new understanding of the mechanisms contributing to the harmful effects of EDCs on the function and viability of neurons, and the possible relevance of EDCs in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease.     

Structure-activity relationship of linear tetrapeptides Tic-DPhe-Arg-Trp-NH2 at the human melanocortin-4 receptor and effects on feeding behaviors in rat

The melanocortin subtype-4 receptor (MC4R) has been implicated in the control of feeding behavior and body weight regulation. A series of tetrapeptides, based on Tic-DPhe-Arg-Trp-NH2-a mimic of the putative message sequence "His-Phe-Arg-Trp" and modified at the DPhe position, were prepared and pharmacologically characterized for potency and selectivity. Substitution of His with Tic gave peptides with significant increases in selectivity. The effects of the substitution pattern of DPhe were investigated and it has significant influences on potency and the level of the maximum cAMP accumulation. Intracerebroventricular administration of peptide 10 induced significant inhibition of cumulative overnight food intake and feeding duration in rats.