Andrea Heldsinger

Vagal Control of Satiety and Hormonal Regulation of Appetite

The paradigm for the control of feeding behavior has changed significantly. In this review, we present evidence that the separation of function in which cholecystokinin (CCK) controls short-term food intake and leptin regulate long-term eating behavior and body weight become less clear. In addition to the hypothalamus, the vagus nerve is critically involved in the control of feeding by transmitting signals arising from the upper gut to the nucleus of the solitary tract. Among the peripheral mediators, CCK is the key peptide involved in generating the satiety signal via the vagus. Leptin receptors have also been identified in the vagus nerve. Studies in the rodents clearly indicate that leptin and CCK interact synergistically to induce short-term inhibition of food intake and long-term reduction of body weight. The synergistic interaction between vagal CCK-A receptor and leptin is mediated by the phosphorylation of signal transducer and activator of transcription3 (STAT3), which in turn, activates closure of K+ channels, leading to membrane depolarization and neuronal firing. This involves the interaction between CCK/SRC/phosphoinositide 3-kinase cascades and leptin/Janus kinase-2/phosphoinositide 3-kinase/STAT3 signaling pathways. It is conceivable that malfunctioning of these signaling molecules may result in eating disorders.

Synergistic interaction between leptin and cholecystokinin in the rat nodose ganglia is mediated by PI3K and STAT3 signaling pathways: implications for leptin as a regulator of short term satiety.

Research has shown that the synergistic interaction between vagal cholecystokinin-A receptors (CCKARs) and leptin receptors (LRbs) mediates short term satiety. We hypothesize that this synergistic interaction is mediated by cross-talk between signaling cascades used by CCKARs and LRbs, which, in turn, activates closure of K+ channels, leading to membrane depolarization and neuronal firing. Whole cell patch clamp recordings were performed on isolated rat nodose ganglia neurons. Western immunoblots elucidated the intracellular signaling pathways that modulate leptin/CCK synergism. In addition, STAT3, PI3K, Src, and MAPK genes were silenced by lentiviral infection and transient Lipofectamine transfection of cultured rat nodose ganglia to determine the effect of these molecules on leptin/CCK synergism. Patch clamp studies showed that a combination of leptin and CCK-8 caused a significant increase in membrane input resistance compared with leptin or CCK-8 alone. Silencing the STAT3 gene abolished the synergistic action of leptin/CCK-8 on neuronal firing. Leptin/CCK-8 synergistically stimulated a 7.7-fold increase in phosphorylated STAT3 (pSTAT3), which was inhibited by AG490, C3 transferase, PP2, LY294002, and wortmannin, but not PD98059. Silencing the Src and PI3K genes resulted in a loss of leptin/CCK-stimulated pSTAT3. We conclude that the synergistic interaction between vagal CCKARs and LRbs is mediated by the phosphorylation of STAT3, which, in turn, activates closure of K+ channels, leading to membrane depolarization and neuronal firing. This involves the interaction between CCK/Src/PI3K cascades and leptin/JAK2/PI3K/STAT3 signaling pathways. Malfunctioning of these signaling molecules may result in eating disorders.

Diabetic Visceral Hypersensitivity Is Associated With Activation of Mitogen-Activated Kinase in Rat Dorsal Root Ganglia

OBJECTIVE Diabetic patients often experience visceral hypersensitivity and anorectal dysfunction. We hypothesize that the enhanced excitability of colon projecting dorsal root ganglia (DRG) neurons observed in diabetes is caused by a decrease in the amplitude of the transient A-type K+ (IA) currents resulting from increased phosphorylation of mitogen-activated protein kinases (MAPK) and reduced opening of Kv4.2 channels. RESEARCH DESIGN AND METHODS We performed patch-clamp recordings of colon projecting DRG neurons from control and streptozotocin-induced diabetic (STZ-D) rats. Western blot analyses and immunocytochemistry studies were used to elucidate the intracellular signaling pathways that modulate the IA current. In vivo studies were performed to demonstrate that abnormal MAPK signaling is responsible for the enhanced visceromotor response to colorectal distention in STZ-D rats. RESULTS Patch-clamp studies demonstrated that IA current was diminished in the colon projecting DRG neurons of STZ-D rats. Western blot analysis of STZ-D DRG neurons revealed increases in phosphorylated MAPK and KV4.2. In diabetic DRG neurons, increased intracellular Ca2+ ([Ca2+]i), protein kinase C (PKC), and MAPK were involved in the regulation of IA current through modulation of Kv4.2. Hypersensitive visceromotor responses to colorectal distention in STZ-D rats were normalized by administration of MAPK inhibitor U0126. CONCLUSIONS We demonstrated that reduction of the IA current in STZ-D DRG neurons is triggered by impaired [Ca2+]i ion homeostasis, and this in turn activates the PKC-MAPK pathways, resulting in decreased opening of the Kv4.2 channels. Hence, the PKC-MAPK–Kv4.2 pathways represent a potential therapeutic target for treating visceral hypersensitivity in diabetes.

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.

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.

Intraluminal acid activates esophageal nodose C fibers after mast cell activation

Acid reflux in the esophagus can induce esophageal painful sensations such as heartburn and noncardiac chest pain. The mechanisms underlying acid-induced esophageal nociception are not clearly understood. In our previous studies, we characterized esophageal vagal nociceptive afferents and defined their responses to noxious mechanical and chemical stimulation. In the present study, we aim to determine their responses to intraluminal acid infusion. Extracellular single-unit recordings were performed in nodose ganglion neurons with intact nerve endings in the esophagus using ex vivo esophageal-vagal preparations. Action potentials evoked by esophageal intraluminal acid perfusion were compared in naive and ovalbumin (OVA)-challenged animals, followed by measurements of transepithelial electrical resistance (TEER) and the expression of tight junction proteins (zona occludens-1 and occludin). In naive guinea pigs, intraluminal infusion with either acid (pH = 2-3) or capsaicin did not evoke an action potential discharge in esophageal nodose C fibers. In OVA-sensitized animals, following esophageal mast cell activation by in vivo OVA inhalation, intraluminal acid infusion for about 20 min started to evoke action potential discharges. This effect is further confirmed by selective mast cell activation using in vitro tissue OVA challenge in esophageal-vagal preparations. OVA inhalation leads to decreased TEER and zona occludens-1 expression, suggesting an impaired esophageal epithelial barrier function after mast cell activation. These data for the first time provide direct evidence of intraluminal acid-induced activation of esophageal nociceptive C fibers and suggest that mast cell activation may make esophageal epithelium more permeable to acid, which subsequently may increase esophageal vagal nociceptive C fiber activation.

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 ghrelins inhibitory effect on STAT3 phosphorylation, neuronal firing, and feeding behavior.

Regulation and Actions of Gastrointestinal Somatostatin

Somatostatin (SS), first isolated from ovine hypothalami (1), is also present in large amounts in the gut in several species including man (2–5), providing the second largest store of tissue SS in the body. In the gut the peptide is found in neural structures in the deep submucosa and muscularis and muscularis mucosae (6), as well as in endocrine cells located in the mucosal layer and the epithelia of gut segments (7, 8). The SS containing nerve terminals are spread diffusely along the gatrointestinal tract; the endocrine cells are found concentrated in the fundus and antrum of the stomach, the pancreas, and spread diffusely throughout the rest of gastrointestinal tract (2, 6, 7). The functional correlate of this distribution has not been established. It does appear that circulating SS originates predominantly from the stomach, which may prove to be the primary source for the systemic hormonal form of the peptide (9). Percutaneous transhepatic venous sampling in humans for estimation of SS-like immunoreactivity (SS-LI) levels reveals the highest concentrations in the gastroepiploic vein and the gastrocolic trunk without a positive gradient in the splenic or mesenteric veins (10). Thus, the stomach is likely to be the major source of circulating SS and it is unlikely that the pancreas, small, or large bowel contribute in any measure.

Enterooxyntin Release from Isolated Perfused Canine Jejunum

UNLABELLED A humoral factor may mediate the intestinal phase of gastric acid secretion. An ex vivo perfused segment of canine jejunum maintained by an oxygenated asanguinous physiologic perfusate was used to test for release of an enterooxyntin (EO) in response to balloon distention at 30 mm Hg for 15 min. Gastric acid secretion in guinea pig fundic mucosa was determined indirectly by a quantitative cytochemical bioassay (CBA) of oxyntic cell hydroxyl ion production (HIP). An increase in the optical density (OD) caused by the cytochemical stain in the oxyntic cells reflects HIP, an index of acid secretion. Basal OD for segments with distention was 16.6 +/- 0.53 and for those without 15.5 +/- 0.68 (NS). Results are expressed as mean change of OD from basal (mean delta OD +/- SEM). (Table-see text) EO caused greater stimulation of HIP than gastrin or histamine. EO was heat stable. Trichloroacetic acid treatment decreased EO activity as did pronase digestion suggesting that EO is composed of one or more peptides. CONCLUSION EO, an acid secretagogue, is a humoral agent probably composed of one or more peptides and is released by small bowel distention. Mechanical distention of the small bowel may be an important mechanism for the perpetuation of gastric acid secretion. The ex vivo perfused jejunal segment in conjunction with the CBA are ideal tools with which to study mechanisms of release of EO and the mechanism of action of EO on the oxyntic cell.

887 Analgesic Action of Resolvin D1 to Combat Visceral Pain in a Rat Model of Visceral Hypersensitivity

G A A b st ra ct s root ganglia (DRG) neurons. The aim of this study was to examine the downstream signaling pathways mediating these effects, with the focus on Smad 3 and TRPV1. Methods: In vitro patch clamp studies were performed on dissociated DRG neurons. For in vivo studies, CP was induced by intraductal injection of trinitrobenzene sulfonic acid (TNBS) in adult male rats, with controls receiving the same volume of vehicle, followed by intrapancreatic injection of DiI, a retrograde neuronal tracer, to label the DRG neurons that project to pancreas. Three weeks later, SIS3 (2.5mg/kg i.p. daily) was administered for 1 week. Subsequently, thoracic DRGs were harvested and stained for pSmad3 using immunohistochemistry. Phosphorylated Smad3 (pSmad3), Smad3 and TRPV1 protein levels in the thoracic DRGs (T8T12) were measured using immunoblot assay. Results: DRG cells were cultured with vehicle (1μg/ml DMSO-solvent for SIS3), TGF β1(10ng/ml), SIS3-TGF β1(10ng/ml-0.3μM), respectively for 48 hours and then TRPV1 currents were determined by the response to 1μM of capsaicin. TGFβ1 significantly enhanced TRPV1 currents in DRG neurons in culture (99.06±24 pA/pF; n=6) compared to controls (44.77±14 pA/pF; n=8), an effect that was blocked by SIS3 (26.23±4.4 pA/pF; n=6) (p<0.01 by ANOVA). However, sis3 was unable to block TRPA1 currents induced by mustard oil, attesting to the relative specificity of this effect. In vivo, as shown previously, SIS3 significantly attenuates both somatic referred hypersensitivity (measured by VFF) and pancreatic hyperalgesia (measured by behavioral response to pancreatic electrical stimulation) in rats with CP. pSmad3 positive neurons in the DiI-labeled thoracic neurons were significantly increased in CP rats (79.06±4.72% compare with control 67.15±1.53%, p<0.05), which can be reduced by treatment with SIS3 (63.64±2.21%, P<0.05). Immunoblot confirmed that pSmad3 levels, but not total Smad3, were significantly reduced in the DRGs (T8-T12) of rats treated with SIS3 relative to vehicle treated rats (P<0.05). Finally, compared with vehicle treatment we found that SIS3 significantly reduced TRPV1 protein levels in rats with CP (106.11±9.44 vs 70.32±6.17, P<0.05). Conclusions: The present results demonstrate a novel link between TGFβ1 signaling and TRPV1 function and expression, an effect that appears to be mediated by Smad3. These results provide the fundamental basis for developing novel therapeutic approaches for CPinduced pain.