The Combination of Cholecystokinin and Stress Amplifies an Inhibition of Appetite, Gastric Emptying, and an Increase in c-Fos Expression in Neurons of the Hypothalamus and the Medulla Oblongata.

Cholecystokinin (CCK) had been the first gastrointestinal hormone known to exert anorexic effects. CCK had been inferred to contribute to the onset of functional dyspepsia (FD) symptoms. To understand the pathophysiology of FD, the roles of stress have to be clarified. In this study, we aimed to clarify the influence of stress on the action of cholecystokinin (CCK) on appetite and gastric emptying. Using rats, stress was simulated by giving restraint stress or intraperitoneal injection of the stress-related peptide hormone urocortin 1 (UCN1). The effects of CCK and restraint stress, alone or in combination, on food intake and gastric motility were examined, and c-Fos expression in the neurons of appetite control network in the central nervous system was assessed by immunohistochemical staining. CCK inhibited food intake and gastric emptying in a dose-dependent manner. Food intake for 1 h was significantly lower with UCN1 (2 nmol/kg) than with the saline control. Restraint stress amplified the suppressive effects of CCK on food intake for 1 h and on gastric emptying. With regard to brain function, the CCK induced c-Fos expression in the neurons of the nucleus tractus solitarius and paraventricular nucleus of the hypothalamus was markedly and significantly amplified by the addition of restraint stress with CCK. The results suggested that stress might amplify the anorexic effects of CCK through activation of the nuclei that comprise the brain neuronal network for satiation; this might play a role in the pathogenesis of the postprandial distress syndromes of functional dyspepsia.

Peripheral α2-ß1 adrenergic interactions mediate the ghrelin response to brain urocortin 1 in rats.

The autonomic nervous system (ANS) conveys neuronal input from the brain to the stomach. We investigated mechanisms through which urocortin 1 (UCN1) injected intracerebroventricularly (ICV, 300 pmol/rat) inhibits circulating ghrelin in rats. This was achieved by assessing (1) the induction of c-fos gene expression as a marker of neuronal activation in specific hypothalamic and caudal brainstem regulating ANS; (2) the influence of vagotomy and pharmacological blockade of central and peripheral α- and ß-adrenergic receptor (AR) on ICV UCN1-induced reduction of plasma ghrelin levels (determined by ELISA); and (3) the relevance of this pathway in the feeding response to a fast in rats. UCN1 increased c-fos mRNA expression in key brain sites influencing sympathetic activity namely the hypothalamic paraventricular and ventromedial nuclei, locus coeruleus, nucleus of the solitary tract, and rostral ventrolateral medulla, by 16-, 29-, 6-, 37-, and 13-fold, respectively. In contrast, the dorsal motor nucleus of the vagus had little c-fos mRNA expression and ICV UCN1 induced a similar reduction in acylated ghrelin in the sham-operated (31%) and vagotomized (41%) rats. An intraperitoneal (IP) injection of either a non-selective α- or selective α2-AR antagonist reduced, while a selective α2-AR agonist enhanced ICV UCN1-induced suppression of plasma acylated ghrelin levels. In addition, IP injection of a non-selective ß- or selective ß1-AR agonist blocked, and selective ß1-AR antagonist augmented, the ghrelin response to ICV UCN1. The IP injections of a selective α1- or non-selective ß or ß2-AR antagonists, or any of the pretreatments given ICV had no effect. ICV UCN1 reduced the 2-h food intake in response to a fast by 80%, and this effect was partially prevented by a selective α2-AR antagonist. These data suggest that ICV UCN1 reduces plasma ghrelin mainly through the brain sympathetic component of the ANS and peripheral AR specifically α2-AR activation and inactivation of ß1-AR. The α2-AR pathway contributes to the associated reduction in food intake.

Urocortin1-induced anorexia is regulated by activation of the serotonin 2C receptor in the brain.

This study was conducted to determine the mechanisms by which serotonin (5-hydroxytryptamine, 5-HT) receptors are involved in the suppression of food intake in a rat stress model and to observe the degree of activation in the areas of the brain involved in feeding. In the stress model, male Sprague-Dawley rats (8 weeks old) were given intracerebroventricular injections of urocortin (UCN) 1. To determine the role of the 5-HT2c receptor (5-HT2cR) in the decreased food intake in UCN1-treated rats, specific 5-HT2cR or 5-HT2b receptor (5-HT2bR) antagonists were administered. Food intake was markedly reduced in UCN1-injected rats compared with phosphate buffered saline treated control rats. Intraperitoneal administration of a 5-HT2cR antagonist, but not a 5-HT2bR antagonist, significantly inhibited the decreased food intake. To assess the involvement of neural activation, we tracked the expression of c-fos mRNA as a neuronal activation marker. Expression of the c-fos mRNA in the arcuate nucleus, ventromedial hypothalamic nucleus (VMH) and rostral ventrolateral medulla (RVLM) in UNC1-injected rats showed significantly higher expression than in the PBS-injected rats. Increased c-fos mRNA was also observed in the paraventricular nucleus (PVN), the nucleus of the solitary tract (NTS), and the amygdala (AMG) after injection of UCN1. Increased 5-HT2cR protein expression was also observed in several areas. However, increased coexpression of 5-HT2cR and c-fos was observed in the PVN, VMH, NTS, RVLM and AMG. Whereas, pro-opiomelanocortin mRNA expression was not changed. In an UNC1-induced stress model, 5-HT2cR expression and activation was found in brain areas involved in feeding control.

Study on the expression of c-Fos protein in the brain of rats after ingestion of food rich in lycopene.

Lycopene, a reddish pigment contained in tomato, belongs to the carotenoid family along with beta-carotene and rutein. This study examined whether administration of lycopene to rats would induce excitation of neurons in the central nervous system. Continuous intake of lycopene-rich food was found to induce accumulation of lycopene in the plasma and liver, and stimulated central neurons in the paraventricular nucleus, ventromedial nucleus and supraoptic nucleus of the hypothalamus, which are known to be involved in the functions of athrocytosis and water drinking. These findings suggest that lycopene may have some influence on feeding and water-drinking behaviors.

Apelin-12 stimulates acid secretion through an increase of histamine release in rat stomachs.

BACKGROUND: Apelin is a peptide that was originally isolated from bovine stomach extract and has been demonstrated to be an endogenous ligand for orphan receptor APJ. Both apelin and the APJ receptor are widely distributed in the whole body. Apelin is supposed to have important regulatory roles in the function of many organs such as in the cardiovascular system; however, the mechanism of apelin function has not been elucidated. In this study, we studied the action of apelin in acid secretion and demonstrated its mechanism of action. METHODS: Gastric lumen-perfused rats were prepared and their stomachs were perfused with a saline solution using a peristaltic pump. Apelin-12, 36 or Pyr(1)-apelin-13, were intravenously injected to examine their effects on acid secretion in rats. In some experiments, rats were pretreated with famotidine (0.33 mg/kg) or atropine sulfate (0.1mg/kg) intravenously injected 5 or 15 min before apelin injection. Furthermore, isolated vascularly perfused rat stomachs were prepared to examine the effect of apelin on histamine release, which was assayed in the effluent by radioimmunoassay. Messenger RNA of histidine decarboxylase (HDC) in gastric mucosa of isolated stomach was measured by real-time RT-PCR. RESULTS: Apelin-12 (20-100 µg/kg) dose-dependently increased gastric acid secretion, with a maximum of 203% at 100 µg/kg (n=5). Neither Pyr(1)-apelin-13 nor apelin-36 caused a significant increase in acid secretion. Famotidine completely blocked the stimulatory action of apelin on acid secretion. Apelin-12 (100 µg/20 ml/10 min) markedly increased histamine release from isolated vascularly perfused rat stomachs by 278%, and also increased the mRNA of HDC by 480% of the control. Atropine sulfate did not abolish the effect of apelin on the secretion of gastric acid. Apelin-12 amplified an increase of acid secretion stimulated by gastrin injection. CONCLUSION: These results indicate that apelin-12 stimulates gastric acid secretion through an increase in histamine release and synthesis from gastric mucosa, suggesting that apelin might play a role in the secretion of gastric acid or serve as a regulating factor of the secretion of gastric acid.

Urocortin 1 reduces food intake and ghrelin secretion via CRF(2) receptors.

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.

Comparison of the actions of acylated and desacylated ghrelin on acid secretion in the rat stomach.

BACKGROUND: Ghrelin, a growth-hormone-releasing peptide, has two major molecular forms: acylated (acyl) and desacylated (desacyl). Recent studies suggest different roles for these two forms. In the present study, we compared desacyl and acyl ghrelin with regard to acid secretion and histamine production in the rat stomach. METHODS: We performed in vivo experiments using gastric lumen-perfused rats. The effects of the two forms of ghrelin on gastrin (gastrin-17)-stimulated acid secretion were also examined. Furthermore, to examine the effects of ghrelin on histamine production, histidine decarboxylase messenger ribonucleic acid in the gastric corpus mucosa was measured by reverse transcription-polymerase chain reaction. RESULTS: Intravenous administration of acyl ghrelin at 20 µg/kg increased gastric acid secretion to 4.8 times greater than control levels. However, desacyl ghrelin had no effect on acid secretion, even at 200 µg/kg. Acyl ghrelin enhanced gastrin-stimulated acid secretion while desacyl ghrelin did not. Vagotomy markedly inhibited the enhancement of gastrin-stimulated acid secretion by acyl ghrelin. Acyl ghrelin increased histidine decarboxylase messenger ribonucleic acid concentration by 2.3 times compared with basal levels at 1 h after administration and by 2.7 times at 2 h after administration; desacyl ghrelin had no such effect. Synergism between acyl ghrelin and gastrin was seen regarding histidine decarboxylase messenger ribonucleic acid concentration. CONCLUSIONS: The results indicate that acyl ghrelin stimulates gastric acid secretion via a mechanism involving activation of the vagus nerve and histamine release and synthesis and that desacyl ghrelin has no action on gastric acid secretion. Furthermore, the results demonstrate synergism between gastrin and acyl ghrelin in terms of gastric acid secretion via a mechanism involving histamine release and synthesis.

Effects of treadmill running exercise on neuronal expression of c-Fos protein in the medulla oblongata after unilateral phrenicotomy in Wistar rats.

The purpose of this study was to examine whether treadmill running exercise after phrenicotomy resulted in change in neuronal expression of c-Fos protein in the medulla oblongata in Wistar rats. All rats were subjected to left-sided unilateral phrenicotomy. In test experiments, rats were trained with treadmill running for 4 weeks, while in control experiments rats were not provided with the opportunity to exercise. c-Fos protein, a transient transcription factor, was immunohistochemically stained by the ABC method, and surveyed in the medulla oblongata. Numbers of c-Fos-immunoreactive (c-Fos-ir) neurons were counted microscopically, and their distribution in the medulla oblongata was mapped. In the dorsal respiratory group (DRG), numbers of c-Fos-ir neurons were found to be significantly increased in the test rats compared with control rats, while in the ventral respiratory group (VRG) no significant differences were found in numbers of c-Fos-ir neurons between test and control rats. These findings suggest that DRG neurons may play more important roles than VRG neurons in recovery of respiration in treadmill exercise after unilateral phrenicotomy in Wistar rats.

Distribution of c-Fos immunoreactive neurons in the brain after intraperitoneal injection of apelin-12 in Wistar rats.

The present study surveyed activation of central neurons following peripheral administration of apelin-12 (AP12), an apelin peptide homologue, by examining the distribution of neurons expressing c-Fos protein. AP12 is known to induce gastric acid secretion among other physiological functions such as regulation of circulation. It was recently reported that apelin counteracted the effect of arginine vasopressin (AVP) in the maintenance of body fluid homeostasis. We attempted to clarify which neurons in the central nervous system express c-Fos protein after intraperitoneal injection of AP12. Intraperitoneally administered AP12 induced expression of c-Fos protein in several nuclei throughout the brain. In the paraventricular nucleus of the hypothalamus (PAH), lateral hypothalamic area (LH), paraventricular nucleus of the thalamus (PVT), periaqueductal gray matter (PAG), bed nucleus of the stria terminalis (BNST), locus coeruleus (LC), lateral parabrachial nucleus (Pbl), the complex of the solitary tract nucleus (NTS) and dorsal motor nucleus of the vagus nerve (DMX), numbers of neurons expressing c-Fos protein were much higher in test than in control experiments. These findings suggest that AP12 stimulates central neurons that may play roles in the regulation of gastric acid, and hypothalamic neurons that may play roles in the maintenance of body fluid homeostasis as well as other physiological functions.

Expression of c-Fos protein in the brain after intravenous injection of ghrelin in rats.

In this study, we surveyed central neurons that might be activated after peripheral administration of a gut-brain peptide ghrelin, by examining neurons expressing c-Fos protein. First, we examined the relationship between the dose of ghrelin and the amount of gastric acid secreted. Ghrelin induced a significant increase in the amount of gastric acid secretion in a dose-dependent manner. Secondly, we examined central neurons that expressed c-Fos protein after intravenous injection of ghrelin. We found that intravenously injected ghrelin induced the neural expression of c-Fos protein in several nuclei and circumventricular organs in the brain. These results suggest that ghrelin released into the circulation may stimulate central neurons that have some role in the control mechanism for gastric acid secretion.

Histamine mediates the stimulatory action of ghrelin on acid secretion in rat stomach.

Ghrelin, a novel growth hormone-releasing peptide, is present in the rat and human stomach and is known to stimulate acid secretion and stomach motility. However, the mechanism of action of ghrelin is not fully understood. In the present study, we attempted to elucidate the role of histamine in ghrelin-induced acid secretion in rat stomach. Intravenous administration of ghrelin at 0.8 to 20 microg/kg dose dependently increased gastric acid secretion, as measured by the gastric lumen perfusion method. The maximum response was almost equal to that of gastrin (20 microg/kg). These actions were abolished by bilateral subdiaphragmatic vagotomy. Famotidine (0.33 mg/kg) also completely inhibited the effects of ghrelin. Furthermore, ghrelin increased histidine decarboxylase (HDC) messenger RNA (mRNA) levels, as measured by real-time reverse transcription-polymerase chain reaction using LightCycler. The action of ghrelin on HDC mRNA was abolished by vagotomy. Ghrelin did not affect histamine release from isolated vascularly perfused rat stomach. Taken together, these results suggest that ghrelin stimulates gastric acid secretion via a mechanism involving activation of vagal efferent nerve and histamine release from gastric enterochromaffin-like cells.

Neuronal expression of nuclear transcription factor MafG in the rat medulla oblongata after baroreceptor stimulation.

The medulla oblongata is the site of central baroreceptive neurons in mammals. These neurons express specific basic-leucine zipper transcription factors (bZIP) after baroreceptor stimulation. Previously we showed that activation of baroreceptors induced expression of nuclear transcription factors c-Fos and FosB in central baroreceptive neurons. Here we studied the effects of baroreceptor stimulation on induction of MafG, a member of small Maf protein family that functions as dimeric partners for various bZIP transcription factors by forming transcription-regulating complexes, in the rat medulla oblongata. To determine whether gene expression of MafG is induced by stimulation of arterial baroreceptors, we examined the expression of its mRNA by semi-quantitative reverse transcription-PCR method and its gene product by immunohistochemistry. We found that the number of MafG transcripts increased significantly in the medulla oblongata after baroreceptor stimulation. MafG-immunoreactive neurons were distributed in the nucleus tractus solitarii, the dorsal motor nucleus of the vagus nerve, the ambiguous nucleus and the ventrolateral medulla. The numbers of MafG-immunoreactive neurons in these nuclei were significantly greater in test rats than in saline-injected control rats. We also found approximately 20% of MafG-immunoreactive neurons coexpress FosB after baroreceptor stimulation. Our results suggest that MafG cooperates with FosB to play critical roles as an immediate early gene in the signal transduction of cardiovascular regulation mediated by baroreceptive signals in the medulla oblongata.

Extracellular acidification enhances DNA binding activity of MafG-FosB heterodimer.

Cells are quite sensitive to a change of the extracellular pH and respond to it through detection of the H+/HCO3- level in extracellular fluid. However, little is known about molecular details induced by acidosis, such as intracellular pathways and gene expression. Here we describe properties of gene expression, protein interaction, and DNA binding activity of basic region leucine zipper (bZIP) transcription factor Maf and FosB during extracellular acidification. When cells were incubated with low pH medium, the expressions of small Maf proteins (MafG, MafK, and MafF) and FosB were clearly increased in an extracellular pH-dependent manner and expressed transiently with a peak after 1-2 h after stimulation. Immunofluorescence and protein binding studies indicated that MafG was partially co-localized with FosB in the nucleus and MafG can form heterodimers with FosB at extracellular pH 7.40. Moreover, we found that MafG-FosB complexes are able to bind to AP-1 consensus sequence, TGACTCA. To investigate whether extracellular acidification influences to dimerization and DNA binding activity of MafG and FosB, extracellular pH of cultured cells was decreased from 7.40 to 6.80. The decrease in extracellular pH led to enhanced dimerization of MafG with FosB leading to augmentation of the DNA binding activity of the heterodimer to AP-1 consensus sequence. Moreover, extracellular acidification induces mRNA expression of matrix metalloproteinase-1, one of the genes that are regulated by AP-1. These results suggest that MafG-FosB complexes are involved in transcriptional regulation in response to extracellular acidification.

Neuronal expression of Fos protein in the brain after intravenous injection of gastrin in rats.

The present study examined whether the central neurons are involved in the stimulatory action of gastrin on the secretion of gastric acid. Gastrin (20 microg), which was examined and ascertained to induce a marked increase in gastric acid secretion in gastric-lumen perfused rats, was intravenously injected in Wistar rats under anesthesia with pentobarbital sodium. In the experiments, 1 h after injecting gastrin, rats were perfused and fixed, the brain was removed and sectioned at 40 microm thickness. Every fourth section was treated with anti-c-Fos antiserum, and c-Fos protein was immunohistochemically stained using the avidin-biotin complex method. It was found that c-Fos protein was expressed in neurons of the lateral habenular nucleus, the central nucleus amygdala, the lateral parabrachial nucleus in the pons, and the complex area of the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve in the medulla oblongata. The control rats were injected with saline solution, and the brain sections were processed similarly as described above. c-Fos protein was expressed in few neurons in the nuclei above in the control rats. These results suggest that gastrin released into the circulation might stimulate central neurons which, in turn, may relate to the control mechanism for the secretion of gastric acid.