Satiety induced by bile acids is mediated via vagal afferent pathways.

The aim of this study was to elucidate the role and the pathways used by bile acid receptor TGR5 in transmitting satiety signals. We showed TGR5 colocalized with cholecystokinin type A (CCK-A) receptors in a subpopulation of rat nodose ganglia (NG) neurons. Intra-arterial injection of deoxycholic acid (DCA) dose-dependently increased firing rate in NG while a subthreshold dose of DCA and CCK-8 increased firing rates synergistically. TGR5-specific agonist oleanolic acid induced NG neuronal firing in a dose-dependent manner. However, the same units did not respond to GW4064, a nuclear receptor-specific agonist. Quantity of DCA-activated neurons in the hypothalamus was determined by c-Fos expression. Combining DCA and CCK-8 caused a 4-fold increase in c-Fos activation. In the arcuate nucleus, c-Fos-positive neurons coexpressed cocaine and amphetamine regulated transcript and proopiomelanocortin. DCA-induced c-Fos expression was eliminated following truncal vagotomy or silencing of TGR5 in the NG. Feeding studies showed intravenous injection of 1 µg/kg of DCA reduced food intake by 12% ± 3%, 24% ± 5%, and 32% ± 6% in the first 3 hours, respectively. Silencing of TGR5 or CCK-A receptor in the NG enhanced spontaneous feeding by 18% ± 2% and 13.5% ± 2.4%, respectively. When both TGR5 and CCK-A receptor were silenced, spontaneous feeding was enhanced by 37% ± 4% in the first 3 hours, suggesting that bile acid may have a physiological role in regulating satiety. Working in concert with CCK, bile acid synergistically enhanced satiety signals to reduce spontaneous feeding.

FODMAP diet modulates visceral nociception by lipopolysaccharide-mediated intestinal inflammation and barrier dysfunction.

Foods high in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) exacerbate symptoms of irritable bowel syndrome (IBS); however, their mechanism of action is unknown. We hypothesized that a high-FODMAP (HFM) diet increases visceral nociception by inducing dysbiosis and that the FODMAP-altered gut microbial community leads to intestinal pathology. We fed rats an HFM and showed that HFM increases rat fecal Gram-negative bacteria, elevates lipopolysaccharides (LPS), and induces intestinal pathology, as indicated by inflammation, barrier dysfunction, and visceral hypersensitivity (VH). These manifestations were prevented by antibiotics and reversed by low-FODMAP (LFM) diet. Additionally, intracolonic administration of LPS or fecal supernatant (FS) from HFM-fed rats caused intestinal barrier dysfunction and VH, which were blocked by the LPS antagonist LPS-RS or by TLR4 knockdown. Fecal LPS was higher in IBS patients than in healthy subjects (HS), and IBS patients on a 4-week LFM diet had improved IBS symptoms and reduced fecal LPS levels. Intracolonic administration of FS from IBS patients, but not FS from HS or LFM-treated IBS patients, induced VH in rats, which was ameliorated by LPS-RS. Our findings indicate that HFM-associated gut dysbiosis and elevated fecal LPS levels induce intestinal pathology, thereby modulating visceral nociception and IBS symptomatology, and might provide an explanation for the success of LFM diet in IBS patients.

KATP channels in the nodose ganglia mediate the orexigenic actions of ghrelin.

Ghrelin, a hunger signalling peptide derived from the peripheral tissues, overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. Using in vivo and in vitro electrophysiological techniques, we show that ghrelin hyperpolarizes neurons and inhibits currents evoked by leptin and CCK-8. Administering a KATP channel antagonist or silencing Kir6.2, a major subunit of the KATP channel, abolished ghrelin inhibition. The inhibitory actions of ghrelin were also abolished by treating the vagal ganglia neurons with pertussis toxin, as well as phosphatidylinositol 3-kinase (PI3K) or extracellular signal-regulated kinase 1 and 2 (Erk1/2) small interfering RNA. Feeding experiments showed that silencing Kir6.2 in the vagal ganglia abolished the orexigenic actions of ghrelin. These data indicate that ghrelin modulates vagal ganglia neuron excitability by activating KATP conductance via the growth hormone secretagogue receptor subtype 1a-Gαi -PI3K-Erk1/2-KATP pathway. This provides a mechanism to explain the actions of ghrelin with respect to overcoming anorexigenic signals that act via the vagal afferent pathways. Ghrelin is the only known hunger signal derived from the peripheral tissues. Ghrelin overcomes the satiety signals evoked by anorexigenic molecules, such as cholecystokinin (CCK) and leptin, to stimulate feeding. The mechanisms by which ghrelin reduces the sensory signals evoked by anorexigenic hormones, which act via the vagus nerve to stimulate feeding, are unknown. Patch clamp recordings of isolated rat vagal neurons show that ghrelin hyperpolarizes neurons by activating K(+) conductance. Administering a KATP channel antagonist or silencing Kir6.2, a major subunit of the KATP channel, abolished ghrelin inhibition in vitro and in vivo. Patch clamp studies show that ghrelin inhibits currents evoked by leptin and CCK-8, which operate through independent ionic channels. The inhibitory actions of ghrelin were abolished by treating the vagal ganglia neurons with pertussis toxin, as well as phosphatidylinositol 3-kinase (PI3K) or extracellular signal-regulated kinase 1 and 2 (Erk1/2) small interfering RNA. In vivo gene silencing of PI3K and Erk1/2 in the nodose ganglia prevented ghrelin inhibition of leptin- or CCK-8-evoked vagal firing. Feeding experiments showed that silencing Kir6.2 in the vagal ganglia abolished the orexigenic actions of ghrelin. These data indicate that ghrelin modulates vagal ganglia neuron excitability by activating KATP conductance via the growth hormone secretagogue receptor subtype 1a-Gαi -PI3K-Erk1/2-KATP pathway. The resulting hyperpolarization renders the neurons less responsive to signals evoked by anorexigenic hormones. This provides a mechanism to explain the actions of ghrelin with respect to overcoming anorexigenic signals that act via the vagal afferent pathways.

Upregulation of bile acid receptor TGR5 and nNOS in gastric myenteric plexus is responsible for delayed gastric emptying after chronic high-fat feeding in rats.

Chronic high-fat feeding is associated with functional dyspepsia and delayed gastric emptying. We hypothesize that high-fat feeding upregulates gastric neuronal nitric oxide synthase (nNOS) expression, resulting in delayed gastric emptying. We propose this is mediated by increased bile acid action on bile acid receptor 1 (TGR5) located on nNOS gastric neurons. To test this hypothesis, rats were fed regular chow or a high-fat diet for 2 wk. Rats fed the high-fat diet were subjected to concurrent feeding with oral cholestyramine or terminal ileum resection. TGR5 and nNOS expression in gastric tissue was measured by immunohistochemistry, PCR, and Western blot. Gastric motility was assessed by organ bath and solid-phase gastric emptying studies. The 2-wk high-fat diet caused a significant increase in neurons coexpressing nNOS and TGR5 in the gastric myenteric plexus and an increase in nNOS and TGR5 gene expression, 67 and 111%, respectively. Enhanced nonadrenergic, noncholinergic (NANC) relaxation, deoxycholic acid (DCA)-induced inhibition in fundic tissue, and a 26% delay in gastric emptying accompanied these changes. A 24-h incubation of whole-mount gastric fundus with DCA resulted in increased nNOS and TGR5 protein expression, 41 and 37%, respectively. Oral cholestyramine and terminal ileum resection restored the enhanced gastric relaxation, as well as the elevated nNOS and TGR5 expression evoked by high-fat feeding. Cholestyramine also prevented the delay in gastric emptying. We conclude that increased levels of circulatory bile acids induced by high-fat feeding upregulate nNOS and TGR5 expression in the gastric myenteric plexus, resulting in enhanced NANC relaxation and delayed gastric emptying.

Ghrelin induces leptin resistance by activation of suppressor of cytokine signaling 3 expression in male rats: implications in satiety regulation.

The anorexigenic adipocyte-derived hormone leptin and the orexigenic hormone ghrelin act in opposition to regulate feeding behavior via the vagal afferent pathways. The mechanisms by which ghrelin exerts its inhibitory effects on leptin are unknown. We hypothesized that ghrelin activates the exchange protein activated by cAMP (Epac), inducing increased SOCS3 expression, which negatively affects leptin signal transduction and neuronal firing in nodose ganglia (NG) neurons. We showed that 91 ± 3% of leptin receptor (LRb) -bearing neurons contained ghrelin receptors (GHS-R1a) and that ghrelin significantly inhibited leptin-stimulated STAT3 phosphorylation in rat NG neurons. Studies of the signaling cascades used by ghrelin showed that ghrelin caused a significant increase in Epac and suppressor of cytokine signaling 3 (SOCS3) expression in cultured rat NG neurons. Transient transfection of cultured NG neurons to silence SOCS3 and Epac genes reversed the inhibitory effects of ghrelin on leptin-stimulated STAT3 phosphorylation. Patch-clamp studies and recordings of single neuronal discharges of vagal primary afferent neurons showed that ghrelin markedly inhibited leptin-stimulated neuronal firing, an action abolished by silencing SOCS3 expression in NG. Plasma ghrelin levels increased significantly during fasting. This was accompanied by enhanced SOCS3 expression in the NG and prevented by treatment with a ghrelin antagonist. Feeding studies showed that silencing SOCS3 expression in the NG reduced food intake evoked by endogenous leptin. We conclude that ghrelin exerts its inhibitory effects on leptin-stimulated neuronal firing by increasing SOCS3 expression. The SOCS3 signaling pathway plays a pivotal role in ghrelin's inhibitory effect on STAT3 phosphorylation, neuronal firing, and feeding behavior.

Essential elements for glucosensing by gastric vagal afferents: immunocytochemistry and electrophysiology studies in the rat.

Glucosensing nodose ganglia neurons mediate the effects of hyperglycemia on gastrointestinal motility. We hypothesized that the glucose-sensing mechanisms in the nodose ganglia are similar to those of hypothalamic glucose excited neurons, which sense glucose through glycolysis. Glucose metabolism leads to ATP-sensitive potassium channel (K(ATP)) channel closure and membrane depolarization. We identified glucosensing elements in the form of glucose transporters (GLUTs), glucokinase (GK), and K(ATP) channels in rat nodose ganglia and evaluated their physiological significance. In vitro stomach-vagus nerve preparations demonstrated the gastric vagal afferent response to elevated glucose. Western blots and RT-PCR revealed the presence of GLUT1, GLUT3, GLUT4, GK, and Kir6.2 in nodose ganglia neurons and gastric branches of the vagus nerve. Immunocytochemistry confirmed the expression of GLUT3, GK, and Kir6.2 in nodose ganglia neurons (46.3 ± 3%). Patch-clamp studies detected glucose excitation in 30% (25 of 83) of gastric-projecting nodose ganglia neurons, which was abolished by GLUT3 or GK short hairpin RNA transfections. Silencing GLUT1 or GLUT4 in nodose ganglia neurons did not prevent the excitatory response to glucose. Elevated glucose elicited a response from 43% of in vitro nerve preparations. A dose-dependent response was observed, reaching maximum at a glucose level of 250 mg/dl. The gastric vagal afferent responses to glucose were inhibited by diazoxide, a K(ATP) channel opener. In conclusion, a subset of neurons in the nodose ganglia and gastric vagal afferents are glucoresponsive. Glucosensing requires a GLUT, GK, and K(ATP) channels. These elements are transported axonally to the gastric vagal afferents, which can be activated by elevated glucose through modulation of K(ATP) channels.

Cocaine- and amphetamine-regulated transcript is the neurotransmitter regulating the action of cholecystokinin and leptin on short-term satiety in rats.

Vagal CCK-A receptors (CCKARs) and leptin receptors (LRbs) interact synergistically to mediate short-term satiety. Cocaine- and amphetamine-regulated transcript (CART) peptide is expressed by vagal afferent neurons. We sought to demonstrate that this neurotransmitter regulates CCK and leptin actions on short-term satiety. We also examined the signal transduction pathways responsible for mediating the CART release from the nodose ganglia (NG). ELISA studies coupled with gene silencing of NG neurons by RNA interference elucidated intracellular signaling pathways responsible for CCK/leptin-stimulated CART release. Feeding studies followed by gene silencing of CART in NG established the role of CART in mediating short-term satiety. Immunohistochemistry was performed on rat NG neurons to confirm colocalization of CCKARs and LRbs; 63% of these neurons contained CART. Coadministration of CCK-8 and leptin caused a 2.2-fold increase in CART release that was inhibited by CCK-OPE, a low-affinity CCKAR antagonist. Transfection of cultured NG neurons with steroid receptor coactivator (SRC) or phosphatidylinositol 3-kinase (PI3K) small-interfering RNA (siRNA) or STAT3 lentiviral short hairpin RNA inhibited CCK/leptin-stimulated CART release. Silencing the expression of the EGR-1 gene inhibited the CCK/leptin-stimulated CART release but had no effect on CCK/leptin-stimulated neuronal firing. Electroporation of NG with CART siRNA inhibited CCK/leptin stimulated c-Fos expression in rat hypothalamus. Feeding studies following electroporation of the NG with CART or STAT3 siRNA abolished the effects of CCK/leptin on short-term satiety. We conclude that the synergistic interaction of low-affinity vagal CCKARs and LRbs mediates CART release from the NG, and CART is the principal neurotransmitter mediating short-term satiety. CART release from the NG involves interaction between CCK/SRC/PI3K cascades and leptin/JAK2/PI3K/STAT3 signaling pathways.

Inhibition of gastric motility by hyperglycemia is mediated by nodose ganglia KATP channels.

The inhibitory action of hyperglycemia is mediated by vagal afferent fibers innervating the stomach and duodenum. Our in vitro studies showed that a subset of nodose ganglia neurons is excited by rising ambient glucose, involving inactivation of ATP-sensitive K(+) (K(ATP)) channels and leading to membrane depolarization and neuronal firing. To investigate whether nodose ganglia K(ATP) channels mediate gastric relaxation induced by hyperglycemia, we performed in vivo gastric motility studies to examine the effects of K(ATP) channel activators and inactivators. Intravenous infusion of 20% dextrose induced gastric relaxation in a dose-dependent manner. This inhibitory effect of hyperglycemia was blocked by diazoxide, a K(ATP) channel activator. Conversely, tolbutamide, a K(ATP) channel inactivator, induced dose-dependent gastric relaxation, an effect similar to hyperglycemia. Vagotomy, perivagal capsaicin treatment, and hexamethonium each prevented the inhibitory action of tolbutamide. Similarly, N(G)-nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase, also blocked tolbutamide's inhibitory effect. To show that K(ATP) channel inactivation at the level of the nodose ganglia induces gastric relaxation, we performed electroporation of the nodose ganglia with small interfering RNA of Kir6.2 (a subunit of K(ATP)) and plasmid pEGFP-N1 carrying the green fluorescent protein gene. The gastric responses to hyperglycemia and tolbutamide were not observed in rats with Kir6.2 small interfering RNA-treated nodose ganglia. However, these rats responded to secretin, which acts via the vagal afferent pathway, independently of K(ATP) channels. These studies provide in vivo evidence that hyperglycemia induces gastric relaxation via the vagal afferent pathway. This action is mediated through inactivation of nodose ganglia K(ATP) channels.

Spatial organization of neurons in the dorsal motor nucleus of the vagus synapsing with intragastric cholinergic and nitric oxide/VIP neurons in the rat.

The dorsal motor nucleus of the vagus (DMV) contains preganglionic neurons that control gastric motility and secretion. Stimulation of different parts of the DMV results in a decrease or an increase in gastric motor activities, suggesting a spatial organization of vagal preganglionic neurons in the DMV. Little is known about how these preganglionic neurons in the DMV synapse with different groups of intragastric motor neurons to mediate contraction or relaxation of the stomach. We used pharmacological and immunohistochemical methods to characterize intragastric neural pathways involved in mediating gastric contraction and relaxation in rats. Microinjections of L-glutamate (L-Glu) into the rostral or caudal DMV produced gastric contraction and relaxation, respectively, in a dose-related manner. Intravenous infusion of hexamethonium blocked these actions, suggesting mediation via preganglionic cholinergic pathways. Atropine inhibited gastric contraction by 85.5 +/- 4.5%. Gastric relaxation was reduced by intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 52.5 +/- 11.9%) or VIP antagonist (56.3 +/- 14.9%). Combined administration of L-NAME and VIP antagonist inhibited gastric relaxation evoked by L-Glu (87.8 +/- 4.3%). Immunohistochemical studies demonstrated choline acetyltransferase immunoreactivity in response to L-Glu microinjection into the rostral DMV in 88% of c-Fos-positive intragastric myenteric neurons. Microinjection of L-Glu into the caudal DMV evoked expression of nitric oxide (NO) synthase and VIP immunoreactivity in 81 and 39%, respectively, of all c-Fos-positive intragastric myenteric neurons. These data indicate spatial organization of the DMV. Depending on the location, microinjection of L-Glu into the DMV may stimulate intragastric myenteric cholinergic neurons or NO/VIP neurons to mediate gastric contraction and relaxation.

Gastric relaxation induced by hyperglycemia is mediated by vagal afferent pathways in the rat.

Hyperglycemia has a profound effect on gastric motility. However, little is known about the site and mechanism that sense alteration in blood glucose level. The identification of glucose-sensing neurons in the nodose ganglia led us to hypothesize that hyperglycemia acts through vagal afferent pathways to inhibit gastric motility. With the use of a glucose-clamp rat model, we showed that glucose decreased intragastric pressure in a dose-dependent manner. In contrast to intravenous infusion of glucose, intracisternal injection of glucose at 250 and 500 mg/dl had little effect on intragastric pressure. Pretreatment with hexamethonium, as well as truncal vagotomy, abolished the gastric motor responses to hyperglycemia (250 mg/dl), and perivagal and gastroduodenal applications of capsaicin significantly reduced the gastric responses to hyperglycemia. In contrast, hyperglycemia had no effect on the gastric contraction induced by electrical field stimulation or carbachol (10(-5) M). To rule out involvement of serotonergic pathways, we showed that neither granisetron (5-HT(3) antagonist, 0.5 g/kg) nor pharmacological depletion of 5-HT using p-chlorophenylalanine (5-HT synthesis inhibitor) affected gastric relaxation induced by hyperglycemia. Lastly, N(G)-nitro-L-arginine methyl ester (L-NAME) and a VIP antagonist each partially reduced gastric relaxation induced by hyperglycemia and, in combination, completely abolished gastric responses. In conclusion, hyperglycemia inhibits gastric motility through a capsaicin-sensitive vagal afferent pathway originating from the gastroduodenal mucosa. Hyperglycemia stimulates vagal afferents, which, in turn, activate vagal efferent cholinergic pathways synapsing with intragastric nitric oxide- and VIP-containing neurons to mediate gastric relaxation.

Prolactin-releasing peptide affects gastric motor function in rat by modulating synaptic transmission in the dorsal vagal complex.

Prolactin-releasing peptide (PrRP) is a recently discovered neuropeptide implicated in the central control of feeding behaviour and autonomic homeostasis. PrRP-containing neurones and PrRP receptor mRNA are found in abundance in the caudal portion of the nucleus tractus solitarius (NTS), an area which together with the dorsal motor nucleus of the vagus (DMV) comprises an integrated structure, the dorsal vagal complex (DVC) that processes visceral afferent signals from and provides parasympathetic motor innervation to the gastrointestinal tract. In this study, microinjection experiments were conducted in vivo in combination with whole-cell recording from neurones in rat medullary slices to test the hypothesis that PrRP plays a role in the central control of gastric motor function, acting within the DVC to modulate the activity of preganglionic vagal motor neurones that supply the stomach. Microinjection of PrRP (0.2 pmol (20 nl)(-1)) into the DMV at the level of the area postrema (+0.2 to +0.6 mm from the calamus scriptorius, CS) markedly stimulated gastric contractions and increased intragastric pressure (IGP). Conversely, administration of peptide into the DMV at sites caudal to the obex (0.0 to -0.3 mm from the CS) decreased IGP and reduced phasic contractions. These effects occurred without change in mean arterial pressure and were abolished by ipsilateral vagotomy, indicating mediation via a vagal-dependent mechanism(s). The pattern of gastric motor responses evoked by PrRP mimicked that produced by administration of L-glutamate at the same sites, and both the effects of L-glutamate and PrRP were abolished following local administration of NMDA and non-NMDA-type glutamate receptor antagonists. On the other hand, microinjection of PrRP into the medial or comissural nucleus of the solitary tract (mNTS and comNTS, respectively) resulted in less robust changes in IGP in a smaller percentage of animals, accompanied by marked alterations in arterial pressure. Superfusion of brain slices with PrRP (100-300 nm) produced a small depolarization and increased spontaneous firing in 10 of 30 retrogradely labelled gastric-projecting DMV neurones. The excitatory effects were blocked by administration of TTX (2 mum) or specific glutamate receptor antagonists, indicating that they resulted from interactions of PrRP at a presynaptic site. Congruent with this, PrRP increased the amplitude of excitatory postsynaptic currents (EPSCs, 154 +/- 33%, 12 of 25 neurones) evoked by electrical stimulation in mNTS or comNTS. In addition, administration of PrRP decreased the paired-pulse ratio of EPSCs evoked by two identical stimuli delivered 100 ms apart (from 0.95 +/- 0.08 to 0.71 +/- 0.11, P < 0.05), whereas it did not affect the amplitude of inward currents evoked by exogenous application of L-glutamate to the slice. The frequency, but not amplitude of spontaneous EPSCs and action potential-independent miniature EPSCs was also increased by administration of PrRP, suggesting that the peptide was acting at least in part at receptors on presynaptic nerve terminals to enhance glutamatergic transmission. In recordings obtained from a separate group of slices, we did not observe any direct effects of PrRP on spontaneous discharge or postsynaptic excitability in either mNTS or comNTS neurones (n = 31). These data indicate that PrRP may act within the DVC to regulate gastric motor function by modulating the efficacy of conventional excitatory synaptic inputs from the NTS onto gastric-projecting vagal motor neurones.

Characterization of CCK(A) receptor affinity states and Ca(2+) signal transduction in vagal nodose ganglia.

CCK(A) receptors are present on vagal afferent fibers. The objectives of this study were to identify the presence of high- and low-affinity CCK(A) receptors on nodose ganglia and to characterize the intracellular calcium signal transduction activated by CCK. Stimulation of acutely isolated nodose ganglion cells from rats with 1 nM CCK-8 primarily evoked a Ca(2+) transient followed by a sustained Ca(2+) plateau (45% of cells responded), whereas 10 pM CCK-8 evoked Ca(2+) oscillations (37% of cells responded). CCK-OPE, a high-affinity agonist and low-affinity antagonist of CCK(A) receptors, primarily elicited Ca(2+) oscillations (29% of cells responded). CCK-OPE inhibited the Ca(2+) transient induced by 1 nM CCK-8 but not by carbachol and high K(+). This result suggests the presence of high- and low-affinity states of CCK(A) receptors on nodose ganglia. We further demonstrated that nicardipine (10 microM) but not omega-conotoxins GVIA and MVIIC (10-100 nM) abolished Ca(2+) signaling induced by CCK-8, indicating that an L-type voltage-dependent Ca(2+) channel and not an N- or Q-type Ca(2+) channel is coupled to CCK(A) receptors. In a separate study, we showed that the G protein activator NaF (10 mM) elicited a Ca(2+) transient and inhibited CCK-8-evoked Ca(2+) signaling, indicative of G protein(s) involvement in CCK(A) receptor activity. The G(q) protein antagonist Gp antagonist-2A (10 microM) also abolished the action of CCK-8. This study indicates that CCK(A) receptors exist in both high- and low-affinity states in the nodose ganglia. Activation of high-affinity CCK(A) receptors elicits Ca(2+) oscillations, whereas stimulation of low-affinity CCK(A) receptors evokes a sustained Ca(2+) plateau. These Ca(2+)-signaling modes are mediated through the L-type Ca(2+) channel and involve the participation of G(q) protein.