Lower esophageal sphincter relaxation and activation of medullary neurons by subdiaphragmatic vagal stimulation in the mouse

BACKGROUND & AIMS: Isolated lower esophageal sphincter (LES) relaxation associated with belching and vomiting and the transient LES relaxation associated with gastroesophageal reflux are gastric afferent-mediated vagovagal reflexes. We aimed to identify the brain stem vagal subnuclei involved in these reflexes. METHODS: In anesthetized mice, LES pressures were recorded using a manometric technique and response to electrical stimulation of the ventral trunk of subdiaphragmatic vagus was investigated. Anatomy of the vagal subnuclei was defined, and activated subnuclei with ventral subdiaphragmatic vagus stimulation were detected by c-fos immunohistochemical staining. RESULTS: Ventral subdiaphragmatic vagal stimulation elicited frequency-dependent LES relaxation without evoking esophageal contractions and induced c-fos expression in interneurons in medial, dorsomedial, and commissural subnuclei along with outer shell of area postrema and motoneurons in the caudal dorsal motor nucleus of vagus. Brain stem subnuclei including interstitial, intermediate, and central subnuclei, and nucleus ambiguous, which have been reported to be involved in the response to swallowing, were not activated. CONCLUSIONS: Stimulation of the ventral subdiaphragmatic vagus causes isolated LES relaxation and activates neurons in select vagal subnuclei that may represent the brain stem circuit involved in the abdominal vagal-afferent-evoked isolated LES relaxation. These observations suggest that different brain stem circuits are involved in swallow-induced and gastric afferent-mediated isolated LES relaxations.     

Vagotomy dissociates short- and long-term controls of circulating ghrelin

Plasma ghrelin levels are responsive to short- and long-term nutrient fluctuation, rapidly decreasing with food consumption and increasing with food deprivation or weight loss. We hypothesized a vagal contribution to both responses. Nutrient-related ghrelin suppression may be mediated by gastrointestinal load-related vagal afferent activity, or depend upon vagal efferent input to the foregut, where most ghrelin is produced. Similarly, the deprivation-induced ghrelin rise could require state-related vagal afferent or efferent activity. Here, we examined the role of the vagus nerve in the regulation of plasma ghrelin by sampling blood from rats with subdiaphragmatic vagotomy and from sham-operated controls over 48 h of food deprivation, and before and after gastric gavage of liquid diet. Vagotomy affected neither baseline ghrelin levels nor the suppression of ghrelin by a nutrient load. The food deprivation-induced elevation of plasma ghrelin levels ( approximately 160% of baseline), however, was completely prevented by subdiaphragmatic vagotomy. In a separate experiment, the deprivation-related rise in plasma ghrelin was substantially reduced by atropine methyl nitrate treatment, indicating that the response to fasting is driven by increased vagal efferent tone. The dissociation between nutrient load- and deprivation-related ghrelin responses indicates that the regulation of circulating ghrelin levels involves separate mechanisms operating through anatomically distinct pathways.     

Corticotropin-releasing factor-overexpressing mice exhibit reduced neuronal activation in the arcuate nucleus and food intake in response to fasting

Corticotropin-releasing factor (CRF) overexpressing (OE) mice are a genetic model that exhibits features of chronic stress. We investigated whether the adaptive feeding response to a hypocaloric challenge induced by food deprivation is impaired under conditions of chronic CRF overproduction. Food intake response to a 16-h overnight fast and ip injection of gut hormones regulating food intake were compared in CRF-OE and wild type (WT) littermate mice along with brain Fos expression, circulating ghrelin levels, and gastric emptying of a nonnutrient meal. CRF-OE mice injected ip with saline showed a 47 and 44% reduction of 30-min and 4-h cumulative food intake response to an overnight fast, respectively, compared with WT. However, the 30-min food intake decrease induced by ip cholecystokinin (3 microg/kg) and increase by ghrelin (300 microg/kg) were similar in CRF-OE and WT mice. Overnight fasting increased the plasma total ghrelin to similar levels in CRF-OE and WT mice, although CRF-OE mice had a 2-fold reduction of nonfasting ghrelin levels. The number of Fos-immunoreactive cells induced by fasting in the arcuate nucleus was reduced by 5.9-fold in CRF-OE compared with WT mice whereas no significant changes were observed in other hypothalamic nuclei. In contrast, fasted CRF-OE mice displayed a 5.6-fold increase in Fos-immunoreactive cell number in the dorsal motor nucleus of the vagus nerve and a 34% increase in 20-min gastric emptying. These findings indicate that sustained overproduction of hypothalamic CRF in mice interferes with fasting-induced activation of arcuate nucleus neurons and the related hyperphagic response.     

Increases in plasma insulin levels in response to electrical stimulation of the dorsal motor nucleus of the vagus nerve

In order to investigate the physiological counterpart of the anatomical finding showing that the dorsal motor nucleus of the vagus nerve (DMX) is a source of efferent vagal fibers innervating the pancreas, unilateral electrical stimulation using monopolar electrodes (50 microA, 30 Hz, 0.2 msec) at a glycemia of 150 mg/100 ml was performed in normal anesthetized rats. DMX stimulation resulted in rapid (within 1 min) rise in plasma insulin levels (greater than or equal to 200%). Stimulation of the nucleus of tractus solitarius, anatomically connected to DMX, also produced a 50% increase in insulinemia. The effect of DMX stimulation was almost completely abolished by atropine pretreatment or acute bilateral subdiaphragmatic vagotomy. The effect of DMX stimulation was not potentiated by the alpha-adrenergic blocker (infusion of phentolamine) indicating that no inhibitory fiber was recruited during DMX stimulation. It is concluded that DMX is connected to the endocrine pancreas exclusively via vagal fibers and has a role in neurally mediated insulin release.     

Delayed short-term secretory regulation of ghrelin in obese animals: evidenced by a specific RIA for the active form of ghrelin

Ghrelin is an acylated peptide, whose lipid modification is essential for its biological activities. Previous studies demonstrated that it strongly stimulates GH release and has a potent orexigenic action. Meanwhile, there is enough evidence showing that feeding states influence plasma ghrelin levels. Fasting stimulates ghrelin secretion, and feeding reduces plasma ghrelin levels. In this study we examined the regulation of plasma ghrelin by fasting in genetically obese animals considering its molecular forms. Plasma levels of active form of ghrelin as well as those of total ghrelin were reduced in ob/ob and db/db mice compared with those in their control mice. Zucker fatty (fa/fa) rats also showed lower plasma ghrelin levels by fasting than the control rats. Insulin-induced hypoglycemia, however, stimulated ghrelin secretion in the fasted fatty rats. Moreover, glucose injection was revealed to reduce plasma ghrelin levels in rats. The effect of the severity of obesity on secretory regulation of ghrelin was also studied. Older fatty rats showed low plasma ghrelin levels even after 48-h fasting. These data suggest that the short-term secretory regulation of total ghrelin and the active form of ghrelin is delayed in obese animals and that blood glucose levels may be involved in the delayed regulation.     

Vagal stimulation and its role in eliciting gastrin but not glucagon release from the isolated perfused dog stomach.

Electrical stimulation (10 V, 10 Hz, 3 min) of both dorsal and ventral vagal trunks of the isolated canine stomach perfused with whole blood induced strong gastric contractions, transient release of cyclic GMP and marked release of gastrin. No gastric-glucagon release was elicited either at 'normal' (4.8 +/- 0.1 mmol/l) or at low (1.5 +/- 0.1 mmol/l) concentrations of blood glucose. It is concluded that, in conditions effective for the stimulation of gastrin release, electrical stimulation of the vagus nerves does not stimulate glucagon release from the isolated perfused dog stomach. Thus one of the well-accepted mechanisms controlling pancreatic-glucagon secretion, vagal stimulation, is ineffective on gastric-glucagon release.     

Thyrotropin-releasing hormone in the dorsal vagal complex stimulates hepatic blood flow in rats

Central administration of thyrotropin-releasing hormone (TRH) enhances hepatic blood flow in animal models. TRH nerve fibers and receptors are localized in the dorsal vagal complex (DVC), and retrograde tracing techniques have shown that hepatic vagal nerves arise mainly from the left DVC. However, nothing is known about the central sites of action for TRH to elicit the stimulation of hepatic blood flow. The effect of microinjection of a TRH analogue into the DVC on hepatic blood flow was investigated in urethane-anesthetized rats. After measuring basal flow, a stable TRH analogue (RX-77368) was microinjected into the DVC and hepatic blood flow response was observed for 120 minutes by laser Doppler flowmetry. Either left or right cervical vagotomy or hepatic branch vagotomy was performed 2 hours before the peptide. Microinjection of RX-77368 (0.5-5 ng) into the left DVC dose-dependently increased hepatic blood flow. The stimulation of hepatic blood flow by RX-77368 microinjection into the left DVC was eliminated by left cervical and hepatic branch vagotomy but not by right cervical vagotomy. By contrast, microinjection of RX-77368 into the right DVC did not significantly alter hepatic blood flow. These results suggest that TRH acts in the left DVC to stimulate hepatic blood flow through the left cervical and hepatic vagus, indicating that neuropeptides may act in the specific brain nuclei to regulate hepatic function.     

Site-specific formation of gastric ulcers by the electric stimulation of the left or right gastric branch of the vagus nerve in the rat

The present study was conducted to investigate the role of the parasympathetic nervous system innervating the stomach in gastric ulcer formation, with special reference to its neuroanatomic characteristics in rats. First, the effects of electric vagal stimulation on the gastric mucosa were examined. The electric stimulation of the left or right gastric branch of the vagus nerve caused gastric mucosal lesions to develop. Interestingly, however, gastric lesions were found on the anterior wall in the rats that had received electric stimulation to the left gastric branch of the vagus nerve and on the posterior wall in the rats that had received stimulation to the right gastric branch. Next, the cells of origin projecting to the left or right gastric branch of the vagus nerve were identified by means of a horseradish peroxidase retrograde tracer method. The left and right gastric branches were found to be innervated by the left and right dorsal motor nuclei of the vagus nerve in the medulla oblongata, respectively. It has been reported that the left and right dorsal motor nuclei of the vagus nerve separately innervate the anterior or posterior gastric wall. The present results, therefore, suggest that the long-lasting excitation of neurons in the dorsal motor nucleus facilitates the site-specific formation of gastric ulcers through the left or right gastric branch of the vagus nerve.     

Ghrelin drives GH secretion during fasting in man

OBJECTIVES: In humans, fasting leads to elevated serum GH concentrations. Traditionally, changes in hypothalamic GH-releasing hormone and somatostatin release are considered as the main mechanisms that induce this elevated GH secretion during fasting. Ghrelin is an endogenous ligand of the GH secretagogue receptor and is synthesized in the stomach. As ghrelin administration in man stimulates GH release, while serum ghrelin concentrations are elevated during fasting in man, this increase in ghrelin levels might be another mechanism whereby fasting results in stimulation of GH release. DESIGN AND SUBJECTS: In ten healthy non-obese males we performed a double-blind placebo-controlled crossover study comparing fasting with and fasting without GH receptor blockade. GH, ghrelin, insulin, glucose and free fatty acids were assessed. RESULTS: While ghrelin levels do not vary considerably in the fed state, fasting rapidly induced a diurnal rhythm in ghrelin concentrations. These changes in serum ghrelin concentrations during fasting were followed by similar, profound changes in serum GH levels. The rapid development of a diurnal ghrelin rhythm could not be explained by changes in insulin, glucose, or free fatty acid levels. Compared with fasting without pegvisomant, fasting with pegvisomant did not change the ghrelin rhythm. CONCLUSIONS: These data indicate that ghrelin is the main driving force behind the enhanced GH secretion during fasting.     

Peripheral functional organisation of vagally evoked gastric motor responses in the ferret.

The aims of the present study were to determine the relative amplitudes of intragastric motor responses evoked by different vagal branches and to establish whether the effects of acute or chronic vagotomy could be predicted from these data. Intragastric pressure responses to electrical stimulation of the vagus were measured in urethane-anaesthetised ferrets and acute or chronic vagotomies were performed. The results show that the left and right cervical vagi were equipotential and fully overlaped each other. Their contributions to the dorsal trunk were equipotential and fully overlapping and so were their contributions to the ventral trunk. The dorsal trunk was more effective than the ventral trunk and there was total functional overlap between these two trunks. Vagal evoked gastric motor responses of the ferret are apparently organised in a different way from vagally induced acid secretion or hormone release in the cat. Acute removal of a trunk led to a reduction in evoked responses that was not linear function of the effect of stimulation of that trunk. In contrast, chronic removal caused a relative increase in evoked responses that ws inversely related to the decrease caused by acute removal. The implications of total functional overlap and neuromuscular reorganisation after chronic vagotomy are discussed.     

Acetylcholine regulates ghrelin secretion in humans

Ghrelin secretion has been reportedly increased by fasting and energy restriction but decreased by food intake, glucose, insulin, and somatostatin. However, its regulation is still far from clarified. The cholinergic system mediates some ghrelin actions, e.g. stimulation of gastric contractility and acid secretion and its orexigenic activity. To clarify whether ghrelin secretion undergoes cholinergic control in humans, we studied the effects of pirenzepine [PZ, 100 mg per os (by mouth)], a muscarinic antagonist, or pyridostigmine (PD, 120 mg per os), an indirect cholinergic agonist, on ghrelin, GH, insulin, and glucose levels in six normal subjects. PD increased (P < 0.05) GH (change in area under curves, mean +/- SEM, 790.9 +/- 229.3 microg(*)min/liter) but did not modify insulin and glucose levels. PZ did not significantly modify GH, insulin, and glucose levels. Circulating ghrelin levels were increased by PD (11290.5 +/- 6688.7 pg(*)min/ml; P < 0.05) and reduced by PZ (-23205.0 +/- 8959.5 pg(*)min/ml; P < 0.01). The PD-induced ghrelin peak did not precede that of GH. In conclusion, circulating ghrelin levels in humans are increased and reduced by cholinergic agonists and antagonists, respectively. Thus, ghrelin secretion is under cholinergic, namely muscarinic, control in humans. The variations in circulating ghrelin levels induced by PD and PZ are unlikely to mediate the cholinergic influence on GH secretion.     

Effect of electrical stimulation of the vagus nerve on insulinemia and glycemia in Acomys cahirinus mice

To investigate the parasympathetic regulation of the endocrine pancreas in spiny mice (Acomys cahirinus), unilateral electrical stimulations of the left cervical vagus nerve were performed in these animals and their controls, the albino mice. Plasma insulin and glucose levels were measured before and after the stimulation. The stimulation parameters were: 2-2.5 V, 14 Hz, 1 msec for the albino mice and 3 V, 14 Hz, 1 msec or 15-20 V, 20 Hz, 1 msec for the spiny mice. Already 2 min after the start of the stimulation, the acomys as well as the albino mice showed a significant increase in plasma insulin levels which was accompanied by a weak but significant increase in glycemia. However, the total insulin output in the acomys mice was half than that of the albino mice. Carbachol administration had no effect on insulin secretion in the acomys mice, while it increased that of the controls. Atropine pretreatment failed to abolish the insulin release elicited by electrical stimulation of the vagus nerve in the acomys mice, while it abolished it in the albino ones. It is proposed that the vagus-nerve mediated insulin release that is present in the acomys mice is exerted, not via muscarinic receptors as in controls, but possibly via other neurotransmitter(s).     

The role of the dorsal vagal complex and the vagus nerve in feeding effects of melanocortin-3/4 receptor stimulation

Fourth intracerebroventricular (4th-icv) administration of the melanocortin-3/4 receptor (MC3/4-R) agonist, MTII, reduces food intake; the antagonist, SHU9119, increases feeding. The dorsal motor nucleus of the vagus nerve (DMX) contains the highest density of MC4-R messenger RNA in the brain. To explore the possibility that the DMX contributes to 4th-icv MC4-R effects, we delivered doses of MTII and SHU9119 that are subthreshold for ventricular response unilaterally through a cannula centered above the DMX. MTII markedly suppressed 2-h (50%), 4-h (50%), and 24-h (33%) intake. Feeding was significantly increased 4 h (50%) and 24 h (20%) after SHU9119 injections. These results suggest that receptors in the DMX, or the dorsal vagal complex more generally, underlie effects obtained with 4th-icv administration of these ligands. We investigated possible vagal mediation of 4th-icv MTII effects by giving the agonist to rats with subdiaphragmatic vagotomy. MTII suppressed 2-, 4-, and 24-h liquid diet intake (approximately 80%) to the same extent in vagotomized and surgical control rats. We conclude that stimulation or antagonism of MC3/4-Rs in the dorsal vagal complex yields effects on food intake that do not require an intact vagus nerve.     

胃电刺激诱导大鼠延髓孤束核和迷走神经运动背核c-fos蛋白表达

背景：胃电刺激（GES）可以调控胃慢波，但其作用机制尚不完全清楚。原癌基因蛋白c—fos的表达可作为神经元功能活动的标志物。目的：以c-fos蛋白的表达为观察指标，探讨GES调控胃肌电慢波的神经机制。方法：将雌性Wistar大鼠随机分为对照组和GES组。GES以控制胃慢波为准，持续1h。分别于刺激后0．5、1、2和5h处死大鼠，以免疫组化方法观察c—fos蛋白在延髓孤束核和迷走神经运动背核中的表达。结果：对照组延髓孤束核和迷走神经运动背核仅见微量c—fos蛋白表达，GES后0．5h其表达开始增强，1h时达高峰，以后逐渐减弱。结论：GES可以调控胃肌电慢波，GES后延髓孤束核和迷走神经运动背核神经元c-fos蛋白表达阳性提示迷走神经可能参与了该调控作用。     

Inhibitory and facilitatory areas of the dorsal medulla mediating ACTH release in the cat.

To define the role of the dorsal medulla in the control of release of ACTH, the authors stimulated electrically (30 sec, 100 muA, 50 Hz) 50 sites in the vicinity of the solitary nuclei of 11 cats anesthetized with chloralose/urethane. Responses of arterial pressure to electrical stimulation were not correlated significantly with release of ACTH. Indirect effects of changes in arterial pressure could not explain changes in release of ACTH. Concentrations of ACTH were measured by radioimmunoassay. Active areas associated with the solitary nucleus were : 1) lateral inhibitory: ventral and lateral to the solitary tract (mean delta ACTH:-153, -86, -97 pg/ml at 1.5, 3.0 and 6.0 min respectively; P less than 0.01); 2) medial inhibitory: medial dorsal motor nucleus of the vagus and extending to the midline (mean delta ACTH: -81, -107, -67 pg/ml; P less than 0.01); and 3) intermediate facilitatory: lateral nucleus intercalatus and adjacent reticular formation (mean delta ACTH: +105, +158, +4 pg/ml; P less than 0.01). The former two areas contain neurons activated by atrial stretch, and the latter area contains neurons inhibited by atrial stretch. Since changes in ACTH levels are inversely correlated with atrial stretch, the results suggest that the changes in release of ACTH are the result of direct stimulation of neural systems of the solitary nuclei mediating release of ACTH in response to hemodynamic changes.     

Effects of sleeve gastrectomy plus trunk vagotomy compared with sleeve gastrectomy on glucose metabolism in diabetic rats

AIM To investigate the effects of sleeve gastrectomy plus trunk vagotomy(SGTV) compared with sleeve gastrectomy(SG) in a diabetic rat model.METHODS SGTV, SG, TV and Sham operations were performed on rats with diabetes induced by high-fat diet and streptozotocin. Body weight, food intake, oral glucose tolerance test, homeostasis model assessment of insulin resistance(HOMA-IR), hepatic insulin signaling(IR, IRS1, IRS2, PI3 K and AKT), oral glucose stimulatedinsulin secretion, GLP-1 and ghrelin were compared at various postoperative times.RESULTS Both SG and SGTV resulted in better glucose tolerance, lower HOMA-IR, up-regulated hepatic insulin signaling, higher levels of oral glucose-stimulated insulin secretion, higher postprandial GLP-1 and lower fasting ghrelin levels than the TV and Sham groups. No significant differences were observed between the SG and SGTV groups. In addition, no significant differences were found between the TV and Sham groups in terms of glucose tolerance, HOMA-IR, hepatic insulin signaling, oral glucose-stimulated insulin secretion, postprandial GLP-1 and fasting ghrelin levels. No differences in body weight and food intake were noted between the four groups.CONCLUSION SGTV is feasible for diabetes control and is independent of weight loss. However, SGTV did not result in a better improvement in diabetes than SG alone.     

The use of glucagon challenge tests in the diagnostic evaluation of hypoglycemia due to hepatoma and insulinoma

We previously found that patients with hypoglycemia due to chronic renal and liver disease had anomalous metabolic responses to glucose and glucagon stimulation. In this study we evaluated the use of glucagon (2 mg, iv) tests in the diagnosis of spontaneous hypoglycemia secondary to hepatocellular carcinoma (HCC) and insulinoma. Twenty-one normal subjects, 45 patients with HCC (11 with hypoglycemia), and 14 patients with insulinoma (all with hypoglycemia) were studied. The fasting blood glucose level was low in all patients with hypoglycemia. The fasting plasma insulin and C-peptide concentrations were high in patients with insulinoma and low in patients with HCC and hypoglycemia. The blood glucose responses to glucagon administration were less than normal in patients with HCC and hypoglycemia and within normal limits in patients with insulinoma. The insulinoma patients had increased plasma insulin and C-peptide responses to glucagon despite having low blood glucose levels. Compared with the HCC patients without hypoglycemia, HCC patients with hypoglycemia had impaired plasma insulin and C-peptide responses. The fasting hypoglycemia, hypoinsulinemia, and impaired insulin/C-peptide responses to glucagon in patients with hepatoma and hypoglycemia presumably reflect the production of insulin-like substances by the hepatoma. We conclude that glucagon administration results in characteristic responses in these groups of patients and can be of use in the diagnosis of spontaneous hypoglycemia secondary to hepatoma or insulinoma.     

Neurophysiological and behavioral evidence for selective reinnervation in skin-grafted Rana pipiens

Rana pipiens with skin dorosoventrally reversed can respond to stimulation of the back with forelimb wipes to the belly and to stimulation of the belly with hindlimb wipes to the back. These "misdirected wiping responses" have been explained in terms of two alternative hypotheses of nerve regeneration: nerve respecification or selective reinnervation. Experimental behavioral and neurophysiological experiments reported here support the selective reinnervation hypothesis. Severing ventral nerves, which normally innervate the belly, greatly reduced the percentage of misdirected responses on stimulation of belly skin grafted to the back, while severing dorsal nerves, which normally innervate the back, increased the percentage of misdirected responses elicited under the same circumstances. Moreover, neurophysiological recordings of grafted animals showed three effects of skin grafting on nerve distributions: (i) termination of dorsal and ventral nerve receptive field at graft edges; (ii) overlap of nonadjacent ventral nerve receptive fields; and (iii) dorsal coursing of ventral nerves to reinnervate target belly skin displaced to the back. These neurophysiological observations, and particularly the third effect, also support selective reinnervation as the mechanism of nerve regeneration in skin-grafted Rana pipiens.