AJP: Gastrointestinal and Liver Physiology current issue

Endocannabinoids and Liver Disease. IV. Endocannabinoid involvement in obesity and hepatic steatosis

Kunos, G., Osei-Hyiaman, D. — 2008-05-06

Endocannabinoids are endogenous lipid mediators that interact with the same receptors as plant-derived cannabinoids to produce similar biological effects. The well-known appetitive effect of smoking marijuana has prompted inquiries into the possible role of endocannabinoids in the control of food intake and body weight. This brief review surveys recent evidence that endocannabinoids and their receptors are involved at multiple levels in the control of energy homeostasis. Endocannabinoids are orexigenic mediators and are part of the leptin-regulated central neural circuitry that controls energy intake. In addition, they act at multiple peripheral sites including adipose tissue, liver, and skeletal muscle to promote lipogenesis and limit fat elimination. Their complex actions could be viewed as anabolic, increasing energy intake and storage and decreasing energy expenditure, as components of an evolutionarily conserved system that has insured survival under conditions of starvation. In the era of plentiful food and limited physical activity, pharmacological inhibition of endocannabinoid activity offers benefits in the treatment of obesity and its hormonal/metabolic consequences.

The Adventures of Sonic Hedgehog in Development and Repair. IV. Sonic hedgehog processing, secretion, and function in the stomach

Zavros, Y. — 2008-05-06

Sonic hedgehog (Shh) is recognized as one of the main morphogens that regulates cell differentiation during early development of the stomach. In the adult stomach, Shh is expressed and secreted from the acid-producing parietal cells, where it is believed to play an essential role in gastric tissue homeostasis and normal differentiation of the epithelium. The present Themes article focuses on reviewing the literature and controversies surrounding the processing and secretion and the role of Shh in the adult stomach.

Personal reminiscences about Morton Grossman and the founding of the Center for Ulcer Research and Education (CURE)

Guth, P. H., Kaunitz, J. D. — 2008-05-06

The Center for Ulcer Research and Education (CURE) from its onset was primarily the work of one man: Professor Morton Grossman, or "Mort" as he was known and called by all. Mort's legacy includes a large body of scientific publications, the first National Institutes of Health Digestive Diseases Center (CURE), and, most importantly, a group of scientists who have become academic leaders and who have made important contributions in the fields of upper gastrointestinal (GI) tract secretion, hormones and receptors, mucosal defense mechanisms, the design and conduct of randomized clinical trials, and ulcer epidemiology. Indeed, Mort is considered to be a founding father of modern academic GI research. I was fortunate to have known and worked with Mort and would like to memorialize his contributions so that his memory can inspire the next generation of academicians.

Dose-response effect of a {beta}3-adrenergic receptor agonist, solabegron, on gastrointestinal transit, bowel function, and somatostatin levels in health

2008-05-06

β₃-Adrenoceptors(β₃-AR) are expressed by cholinergic myenteric neurons and β₃-AR agonists are effective in experimental models of diarrhea. Our aim was to explore the effects of a β₃-AR agonist, solabegron, on gastrointestinal transit, safety, bowel function, plasma somatostatin, and solabegron pharmacokinetics (PK) following single and multiple doses. In a single-center, double-blind, parallel-group trial, 36 healthy volunteers were randomized to oral solabegron (50 or 200 mg twice daily) or placebo. Transit was measured by a validated method (^99mTc-labeled egg meal and ¹¹¹In charcoal delivered to the colon via delayed-release capsule). Stool frequency, form, and ease of passage were measured on a validated daily diary; plasma somatostatin by radioimmunoassay and plasma solabegron and its active metabolite by validated liquid chromatography-tandem mass spectroscopy analysis followed by PK analysis using noncompartmental methods. There were no overall or dose-related effects of solabegron on gastric, small bowel, or colonic transit, plasma somatostatin levels, stool frequency, form, or ease of passage in healthy volunteers. Solabegron and active metabolite exposures (area under the curve and maximum serum concentration) at both dose levels were consistent with PK at similar doses in previous phase I studies. We concluded that 7 days of the β₃-AR agonist, solabegron, 50 or 200 mg twice daily, did not significantly alter gastrointestinal or colonic transit or bowel function. In this study, medication was generally well tolerated with few adverse events reported and no clinically significant changes in vital signs observed. Further studies on clinical efficacy, visceral sensitivity, and gastrointestinal transit are required in irritable bowel syndrome patients.

Tumor necrosis factor-{alpha} directly stimulates the overproduction of hepatic apolipoprotein B100-containing VLDL via impairment of hepatic insulin signaling

Qin, B., Anderson, R. A., Adeli, K. — 2008-05-06

Insulin-resistant states are commonly associated with both increased circulating levels of tumor necrosis factor (TNF)- and hepatic overproduction of very low density lipoproteins (VLDL). Here, we provide evidence that increased TNF- can directly stimulate the hepatic assembly and secretion of apolipoprotein B (apoB) 100-containing VLDL₁, using the Syrian golden hamster, an animal model that closely resembles humans in hepatic VLDL-apoB100 metabolism. In vivo TNF- infusion for 4 h in chow-fed hamsters induced whole-body insulin resistance on the basis of euglycemic hyperinsulinemic clamp studies. Immunoprecipitation and immunoblotting analysis of livers from TNF--treated hamsters indicated decreased tyrosine phosphorylation of insulin receptor (IR)-β, IR substrate-1 (Tyr), Akt (Ser⁴⁷³), p38, ERK1/2, and JNK but increased serine phosphorylation of IRS-1 (Ser³⁰⁷) and Shc. TNF- infusion also significantly increased hepatic production of total circulating apoB100 and VLDL-apoB100 in both fasting and postprandial (fat load) states. Ex vivo experiments, using cultured primary hepatocytes from hamsters, also showed TNF--induced VLDL-apoB100 oversecretion, an effect that was blocked by TNF receptor 2 antibody. Unexpectedly, TNF- decreased the sterol regulatory element-binding protein-1c mass and mRNA levels but significantly increased microsomal triglyceride transfer protein mass and mRNA levels in primary hepatocytes. In summary, these data provide direct evidence that TNF- induces whole-body insulin resistance and impairs hepatic insulin signaling accompanied by overproduction of apoB100-containing VLDL particles, an effect likely mediated via TNF receptor 2.

Using the lymph fistula rat model to study the potentiation of GIP secretion by the ingestion of fat and glucose

Lu, W. J., Yang, Q., Sun, W., Woods, S. C., D'Alessio, D., Tso, P. — 2008-05-06

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin produced in the K cells of the intestine and secreted into the circulating blood following ingestion of carbohydrate- and fat-containing meals. GIP contributes to the regulation of postprandial insulin secretion and is essential for normal glucose tolerance. We have established a method of assaying GIP in response to nutrients using the intestinal lymph fistula model. Administration of Ensure, a mixed-nutrient liquid meal, stimulated a significant increase in intestinal lymphatic GIP levels that were approximately threefold those of portal plasma. Following the meal, lymph GIP peaked at 60 min (P < 0.001) and remained elevated for 4 h. Intraduodenal infusions of isocaloric and isovolumetric lipid emulsions or glucose polymer induced lymph GIP concentrations that were four and seven times the basal levels, respectively. The combination of glucose plus lipid caused an even greater increase of lymph GIP than either nutrient alone. In summary, these findings demonstrated that intestinal lymph contains high concentrations of GIP that respond to both enteral carbohydrate and fat absorption. The change in lymphatic GIP concentration is greater than the change observed in the portal blood. These studies allow the detection of GIP levels at which they exert their local physiological actions. The combination of glucose and lipid has a potentiating effect in the stimulation of GIP secretion. We conclude from these studies that the lymph fistula rat is a novel approach to study in vivo GIP secretion in response to nutrient feeding in conscious rats.

Selective expansion of the {beta}-cell compartment in the pancreas of keratinocyte growth factor transgenic mice

Wagner, M., Koschnick, S., Beilke, S., Frey, M., Adler, G., Schmid, R. M. — 2008-05-06

Epithelial-mesenchymal interactions are essential for growth, differentiation, and regeneration of exocrine and endocrine cells in the pancreas. The keratinocyte growth factor (KGF) is derived from mesenchyme and has been shown to promote epithelial cell differentiation and proliferation in a paracrine fashion. Here, we have examined the effect of ectopic expression of KGF on pancreatic differentiation and proliferation in transgenic mice by using the proximal elastase promoter. KGF transgenic mice were generated following standard procedures and analyzed by histology, morphometry, immunohistochemistry, Western blot analysis, and glucose tolerance testing. In KGF transgenic mice, the number of islets, the average size of islets, and the relation of endocrine to exocrine tissue are increased compared with littermate controls. An expansion of the β-cell population is responsible for the increase in the endocrine compartment. Ectopic expression of KGF results in proliferation of β-cells and pancreatic duct cells most likely through activation of the protein kinase B (PKB)/Akt signaling pathway. Glucose tolerance and insulin secretion are impaired in transgenic animals. These results provide evidence that ectopic expression of KGF in acinar cells promotes the expansion of the β-cell lineage in vivo through activation of the PKB/Akt pathway. Furthermore, the observed phenotype demonstrates that an increase in the β-cell compartment does not necessarily result in an improved glucose tolerance in vivo.

CCK-induced pancreatic growth is not limited by mitogenic capacity in mice

2008-05-06

In mice fed trypsin inhibitor (camostat) to elevate endogenous CCK, pancreatic growth plateaus by 7 days. It is unknown whether this represents the maximum growth capacity of the pancreas. To test the ability of CCK to drive further growth, mice were fed chow containing camostat (0.1%) for 1 wk, then fed standard chow for 1 wk, and finally returned to the camostat diet for a week. Pancreatic mass increased to 245% of initial value (iv) following 1 wk of camostat feeding, decreased to 147% iv following a 1 wk return to normal chow, and increased to 257% iv with subsequent camostat feeding. Camostat feeding was associated with significant increases in circulating CCK and changes in pancreatic mass were paralleled by changes in protein and DNA content. Moreover, regression of the pancreas following camostat feeding was associated with changes in the expression of the autophagosome marker LC3. Pancreatic protein synthetic rates were 130% of control after 2 days on camostat but were equivalent to control after 7 days. Changes in the phosphorylation of 4E-BP1 and S6, downstream effectors of mammalian target of rapamycin (mTOR), paralleled changes in protein synthetic rates. Cellular content of Akt, an upstream activating kinase of mTOR, decreased after 7 days of camostat feeding whereas expression of the E3 ubiquitin-ligases and the cell cycle inhibitor p21 increased after 2 days. These results indicate that CCK-stimulated growth of the pancreas is not limited by acinar cell mitogenic capacity but is due, at least in part, to inhibition of promitogenic Akt signaling.

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

Zhou, S.-Y., Lu, Y.-X., Owyang, C. — 2008-05-06

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-HT₃ 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.

Enteropathogenic E. coli-induced barrier function alteration is not a consequence of host cell apoptosis

Viswanathan, V. K., Weflen, A., Koutsouris, A., Roxas, J. L., Hecht, G. — 2008-05-06

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that perturbs intestinal epithelial function. Many of the alterations in the host cells are mediated by effector molecules that are secreted directly into epithelial cells by the EPEC type III secretion system. The secreted effector molecule EspF plays a key role in redistributing tight junction proteins and altering epithelial barrier function. EspF has also been shown to localize to mitochondria and trigger membrane depolarization and eventual host cell death. The relationship, if any, between EspF-induced host cell death and epithelial barrier disruption is presently not known. Site-directed mutation of leucine 16 (L16E) of EspF impairs both mitochondrial localization and consequent host cell death. Although the mutation lies within a region critical for type III secretion, EspF(L16E) is secreted efficiently from EPEC. Despite its inability to promote cell death, EspF(L16E) was not impaired for tight junction alteration or barrier disruption. Consistent with this, the pan-caspase inhibitor Q-VD-OPH, despite reducing EPEC-induced host cell death, had no effect on infection-mediated barrier function alteration. Thus EPEC alters the epithelial barrier independent of its ability to induce host cell death.

A cholesterol-free, high-fat diet suppresses gene expression of cholesterol transporters in murine small intestine

2008-05-06

Transporters present in the epithelium of the small intestine determine the efficiency by which dietary and biliary cholesterol are taken up into the body and thus control whole-body cholesterol balance. Niemann-Pick C1 Like Protein 1 (Npc1l1) transports cholesterol into the enterocyte, whereas ATP-binding cassette transporters Abca1 and Abcg5/Abcg8 are presumed to be involved in cholesterol efflux from the enterocyte toward plasma HDL and back into the intestinal lumen, respectively. Abca1, Abcg5, and Abcg8 are well-established liver X receptor (LXR) target genes. We examined the effects of a high-fat diet on expression and function of cholesterol transporters in the small intestine in mice. Npc1l1, Abca1, Abcg5, and Abcg8 were all downregulated after 2, 4, and 8 wk on a cholesterol-free, high-fat diet. The high-fat diet did not affect biliary cholesterol secretion but diminished fractional cholesterol absorption from 61 to 42% (P < 0.05). In an acute experiment in which triacylglycerols of unsaturated fatty acids were given by gavage, we found that this downregulation occurs within a 6-h time frame. Studies in LXR-null mice, confirmed by in vitro data, showed that fatty acid-induced downregulation of cholesterol transporters is LXR independent and associated with a posttranslational increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity that reflects induction of cholesterol biosynthesis as well as with a doubling of neutral fecal sterol loss. This study highlights the induction of adaptive changes in small intestinal cholesterol metabolism during exposure to dietary fat.

Polyunsaturated fatty acids block platelet-activating factor-induced phosphatidylinositol 3 kinase/Akt-mediated apoptosis in intestinal epithelial cells

Lu, J., Caplan, M. S., Li, D., Jilling, T. — 2008-05-06

We have shown earlier that platelet-activating factor (PAF) causes apoptosis in enterocytes via a mechanism that involves Bax translocation to mitochondria, followed by caspase activation and DNA fragmentation. Herein we report that, in rat small intestinal epithelial cells (IEC-6), these downstream apoptotic effects are mediated by a PAF-induced inhibition of the phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (Akt) signaling pathway. Treatment with PAF results in rapid dephosphorylation of Akt, phosphoinositide-dependent kinase-1, and the YXXM p85 binding motif of several proteins and redistribution of Akt-pleckstrin homology domain-green fluorescent protein, i.e., an in vivo phosphatidylinositol (3,4,5)-trisphosphate sensor, from membrane to cytosol. The proapoptotic effects of PAF were inhibited by both n-3 and n-6 polyunsaturated fatty acids but not by a saturated fatty acid palmitate. Indomethacin, an inhibitor of prostaglandin biosynthesis, did not influence the baseline or PAF-induced apoptosis, but 2-bromopalmitate, an inhibitor of protein palmitoylation, inhibited all of the proapoptotic effects of PAF. Our data strongly suggest that an inhibition of the PI 3-kinase/Akt signaling pathway is the main mechanism of PAF-induced apoptosis in enterocytes and that polyunsaturated fatty acids block this mechanism very early in the signaling cascade independently of any effect on prostaglandin synthesis, and probably directly via an effect on protein palmitoylation.

Platelet-activating factor-induced chloride channel activation is associated with intracellular acidosis and apoptosis of intestinal epithelial cells

2008-05-06

Platelet-activating factor (PAF) is a phospholipid inter- and intracellular mediator implicated in intestinal injury primarily via induction of an inflammatory cascade. We find that PAF also has direct pathological effects on intestinal epithelial cells (IEC). PAF induces Cl^– channel activation, which is associated with intracellular acidosis and apoptosis. Using the rat small IEC line IEC-6, electrophysiological experiments demonstrated that PAF induces Cl^– channel activation. This PAF-activated Cl^– current was inhibited by Ca²⁺ chelation and a calcium calmodulin kinase II inhibitor, suggesting PAF activation of a Ca²⁺-activated Cl^– channel. To determine the pathological consequences of Cl^– channel activation, microfluorimetry experiments were performed, which revealed PAF-induced intracellular acidosis, which is also inhibited by the Cl^– channel inhibitor 4,4'diisothiocyanostilbene-2,2'disulfonic acid and Ca²⁺ chelation. PAF-induced intracellular acidosis is associated with caspase 3 activation and DNA fragmentation. PAF-induced caspase activation was abolished in cells transfected with a pH compensatory Na/H exchanger construct to enhance H⁺ extruding ability and prevent intracellular acidosis. As ClC-3 is a known intestinal Cl^– channel dependent on both Ca²⁺ and calcium calmodulin kinase II phosphorylation, we generated ClC-3 knockdown cells using short hairpin RNA. PAF induced Cl^– current; acidosis and apoptosis were all significantly decreased in ClC-3 knockdown cells. Our data suggest a novel mechanism of PAF-induced injury by which PAF induces intracellular acidosis via activation of the Ca²⁺-dependent Cl^– channel ClC-3, resulting in apoptosis of IEC.

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

Zhou, S.-Y., Lu, Y.-X., Yao, H., Owyang, C. — 2008-05-06

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.

Obestatin inhibits motor activity in the antrum and duodenum in the fed state of conscious rats

Ataka, K., Inui, A., Asakawa, A., Kato, I., Fujimiya, M. — 2008-05-06

Obestatin is a novel peptide encoded by the ghrelin precursor gene; however, its effects on gastrointestinal motility remain controversial. Here we have examined the effects of obestatin on fed and fasted motor activities in the stomach and duodenum of freely moving conscious rats. We examined the effects of intravenous (IV) injection of obestatin on the percentage motor index (%MI) and phase III-like contractions in the antrum and duodenum. The brain mechanism mediating the action of obestatin on gastroduodenal motility and the involvement of vagal afferent pathway were also examined. Between 30 and 90 min after IV injection, obestatin decreased the %MI in the antrum and prolonged the time taken to return to fasted motility in the duodenum in fed rats given 3 g of chow after 18 h of fasting. Immunohistochemical analysis demonstrated that corticotropin-releasing factor- and urocortin-2-containing neurons in the paraventricular nucleus in the hypothalamus were activated by IV injection of obestatin. Intracerebroventricular injection of CRF type 1 and type 2 receptor antagonists prevented the effects of obestatin on gastroduodenal motility. Capsaicin treatment blocked the effects of obestatin on duodenal motility but not on antral motility. Obestatin failed to antagonize ghrelin-induced stimulation of gastroduodenal motility. These results suggest that, in the fed state, obestatin inhibits motor activity in the antrum and duodenum and that CRF type 1 and type 2 receptors in the brain might be involved in these effects of obestatin on gastroduodenal motility.

Differential mechanisms of hepatic vascular dysregulation with mild vs. moderate ischemia-reperfusion

Lee, S. H., Culberson, C., Korneszczuk, K., Clemens, M. G. — 2008-05-06

Endotoxemia produces hepatic vascular dysregulation resulting from inhibition of endothelin (ET)-stimulated NO production. Mechanisms include overexpression of caveolin-1 (Cav-1) and altered phosphorylation of endothelial nitric oxide (NO) synthase (NOS; eNOS) in sinusoidal endothelial cells. Since ischemia-reperfusion (I/R) also causes vascular dysregulation, we tested whether the mechanisms are the same. Rats were exposed to either mild (30 min) or moderate (60 min) hepatic ischemia in vivo followed by reperfusion (6 h). Livers were harvested and prepared into precision-cut liver slices for in vitro analysis of NOS activity and regulation. Both I/R injuries significantly abrogated both the ET-1 (1 µM) and the ET_B receptor agonist (IRL-1620, 0.5 µM)-mediated stimulation of NOS activity. 30 min I/R resulted in overexpression of Cav-1 and loss of ET-stimulated phosphorylation of Ser1177 on eNOS, consistent with an inflammatory response. Sixty-minute I/R also resulted in loss of ET-stimulated Ser1177 phosphorylation, but Cav-1 expression was not altered. Moreover, expression of ET_B receptors was significantly decreased. This suggests that the failure of ET to activate eNOS following 60-min I/R is associated with decreased protein expression consistent with ischemic injury. Thus hepatic vascular dysregulation following I/R is mediated by inflammatory mechanisms with mild I/R whereas ischemic mechanisms dominate following more severe I/R stress.

Contribution of the sympathetic hormone epinephrine to the sensitizing effect of ethanol on LPS-induced liver damage in mice

von Montfort, C., Beier, J. I., Guo, L., Kaiser, J. P., Arteel, G. E. — 2008-05-06

It is well known that ethanol preexposure sensitizes the liver to LPS hepatotoxicity. The mechanisms by which ethanol enhances LPS-induced liver injury are not completely elucidated but are known to involve an enhanced inflammatory response. Ethanol exposure also increases the metabolic rate of the liver, and this effect of ethanol on liver is mediated, at least in part, by the sympathetic hormone, epinephrine. However, whether or not the sympathetic nervous system also contributes to the sensitizing effect of ethanol preexposure on LPS-induced liver damage has not been determined. The purpose of this study was therefore to test the hypotheses that 1) epinephrine preexposure enhances LPS-induced liver damage (comparable to that of ethanol preexposure) and that 2) the sympathetic nervous system contributes to the sensitizing effect of ethanol. Accordingly, male C57BL/6J mice were administered epinephrine for 5 days (2 mg/kg per day) via osmotic pumps or bolus ethanol for 3 days (6 g/kg per day) by gavage. Twenty-four hours later, mice were injected with LPS (10 mg/kg ip). Both epinephrine and ethanol preexposure exacerbated LPS-induced liver damage and inflammation. Concomitant administration of propranolol with ethanol significantly attenuated the sensitizing effect of ethanol on LPS-induced liver damage. These data support the hypothesis that the sympathetic nervous system contributes, at least in part, to the mechanism of the sensitizing effect of ethanol. These results also suggest that sympathetic tone may contribute to the initiation and progression of alcoholic liver disease.

CUGBP2 downregulation by prostaglandin E2 protects colon cancer cells from radiation-induced mitotic catastrophe

2008-05-06

Prostaglandin E₂ (PGE₂) is a potent inhibitor of ionizing radiation (IR)-induced cell death. Exposure of colon cancer cells to IR leads to increased CUGBP2 expression. Therefore, we tested the hypothesis that PGE₂ radioprotects colon cancer cells by inhibiting CUGBP2 expression. Exposure of HCT-116 cells to -IR (0–12 Gy) resulted in a dose-dependent reduction in cell growth and an increase in the G₂-M phase of the cell cycle. Western blot analyses demonstrated increased levels of activated caspase 9 and caspase 3. In addition, whereas Bax expression is increased, that of Bcl-2 and Bcl-x_L was reduced. Further analyses demonstrated increased activation of Chk1 and Chk2 kinases, coupled with higher levels of nuclear cyclin B1 and Cdc2. Pretreatment with PGE₂ suppressed the activation of caspase 3 and caspase 7 and inhibited Bax expression. In addition, PGE₂ treatment restored growth and colony formation to control levels. IR significantly upregulated the expression of CUGBP2 in the cells, which was suppressed when cells were pretreated with PGE₂. Ectopic overexpression of CUGBP2 also induced apoptosis. Furthermore, it reversed the PGE₂-mediated protection from IR-induced mitotic catastrophe. Furthermore, there was an increase in nuclear localization of cyclin B1 and Cdc2 coupled with increased phosphorylation of p53, Chk1, Chk2, and Cdc25c proteins. Cell cycle analysis also demonstrated increased G₂-M transition. In contrast, siRNA-mediated suppression of CUGBP2 expression restored normal cell cycle progression and decreased IR-induced apoptosis. Taken together, these data demonstrate that PGE₂ protects colon cancer cells from IR-induced mitotic catastrophe in part through suppression of CUGBP2 expression.

Stimulation of the neurokinin 3 receptor activates protein kinase C{varepsilon} and protein kinase D in enteric neurons

2008-05-06

Tachykinins, acting through NK₃ receptors (NK₃R), contribute to excitatory transmission to intrinsic primary afferent neurons (IPANs) of the small intestine. Although this transmission is dependent on protein kinase C (PKC), its maintenance could depend on protein kinase D (PKD), a downstream target of PKC. Here we show that PKD1/2-immunoreactivity occurred exclusively in IPANs of the guinea pig ileum, demonstrated by double staining with the IPAN marker NeuN. PKC was also colocalized with PKD1/2 in IPANs. PKC and PKD1/2 trafficking was studied in enteric neurons within whole mounts of the ileal wall. In untreated preparations, PKC and PKD1/2 were cytosolic and no signal for activated (phosphorylated) PKD was detected. The NK₃R agonist senktide evoked a transient translocation of PKC and PKD1/2 from the cytosol to the plasma membrane and induced PKD1/2 phosphorylation at the plasma membrane. PKC translocation was maximal at 10 s and returned to the cytosol within 2 min. Phosphorylated-PKD1/2 was detected at the plasma membrane within 15 s and translocated to the cytosol by 2 min, where it remained active up to 30 min after NK₃R stimulation. PKD1/2 activation was reduced by a PKC inhibitor and prevented by NK₃R inhibition. NK₃R-mediated PKC and PKD activation was confirmed in HEK293 cells transiently expressing NK₃R and green fluorescent protein-tagged PKC, PKD1, PKD2, or PKD3. Senktide caused membrane translocation and activation of kinases within 30 s. After 15 min, phosphorylated PKD had returned to the cytosol. PKD activation was confirmed through Western blotting. Thus stimulation of NK₃R activates PKC and PKD in sequence, and sequential activation of these kinases may account for rapid and prolonged modulation of IPAN function.

CCL25 and CCL28 promote {alpha}4{beta}7-integrin-dependent adhesion of lymphocytes to MAdCAM-1 under shear flow

Miles, A., Liaskou, E., Eksteen, B., Lalor, P. F., Adams, D. H. — 2008-05-06

Inflammatory bowel disease is characterized by the recruitment of lymphocytes to the gut via mucosal vessels. Chemokines are believed to trigger ₄β₁- and ₄β₇-integrin-mediated adhesion to vascular cell adhesion molecule-1 (VCAM-1) and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on mucosal vessels, although the contribution of each pathway and the chemokines involved are not well characterized. These interactions occur under conditions of hemodynamic shear, which is critical in determining how lymphocytes integrate chemokine signals to promote transmigration. To define the role of specific chemokines in mediating lymphocyte adhesion to VCAM-1 and MAdCAM-1, we studied the ability of immobilized chemokines to activate adhesion of human lymphocytes in a flow-based adhesion assay. Adhesion to immobilized MAdCAM-1 was ₄β₇ dependent, with no contribution from ₄β₁, whereas ₄β₁ mediated rolling and static adhesion on VCAM-1. Immobilized CC-chemokine ligand (CCL) 25 and CCL28 were both able to trigger ₄β₇-dependent lymphocyte arrest on MAdCAM-1 under shear, highlighting a potential role for these chemokines in the arrest of lymphocytes on postcapillary venules in the gut. Neither had any effect on adhesion to VCAM-1, suggesting that they selectively trigger ₄β₇-mediated adhesion. Immobilized CCL21, CCL25, CCL28, and CXC-chemokine ligand (CXCL) 12 all converted rolling adhesion to static arrest on MAdCAM-1 by activating lymphocyte integrins, but only CCL21 and CXCL12 also triggered a motile phenotype characterized by lamelipodia and uropod formation. Thus ₄β₁/VCAM-1 and ₄β₇/MAdCAM-1 operate independently to support lymphocyte adhesion from flow, and chemokines may act in concert with one chemokine triggering integrin-mediated arrest and a second chemokine promoting motility and transendothelial migration.

Role of TNF-{alpha} in ileum tight junction alteration in mouse model of restraint stress

Mazzon, E., Cuzzocrea, S. — 2008-05-06

Restraint stress induces permeability changes in the small intestine, but little is known about the role of tumor necrosis factor (TNF)- in the defects of the TJ function. In the present study, we used tumor necrosis factor-R1 knockout mice (TNF--R1KO) to understand the roles of TNF- on ileum altered permeability function in models of immobilization stress. The genetic TNF- inhibition significantly reduced the degree of 1) TNF- production in ileum tissues; 2) the alteration of zonula occludens-1 (ZO-1), claudin-2, claudin-4, claudin-5, and β-catenin (immunohistochemistry); and 3) apoptosis (TUNEL staining, Bax, Bcl-2 expression). Taken together, our results demonstrate that inhibition of TNF- reduces the tight junction permeability in the ileum tissues associated with immobilization stress, suggesting a possible role of TNF- on ileum barrier dysfunction.

Gene expression in human NAFLD

2008-05-06

Despite the high prevalence of nonalcoholic fatty liver disease (NAFLD), little is known of its pathogenesis based on study of human liver samples. By the use of Affymetrix GeneChips (17,601 genes), we investigated gene expression in the human liver of subjects with extreme steatosis due to NAFLD without histological signs of inflammation (liver fat 66.0 ± 6.8%) and in subjects with low liver fat content (6.4 ± 2.7%). The data were analyzed by using sequence-based reannotation of Affymetrix probes and a robust model-based normalization method. We identified genes involved in hepatic glucose and lipid metabolism, insulin signaling, inflammation, coagulation, and cell adhesion to be significantly associated with liver fat content. In addition, genes involved in ceramide signaling (MAP2K4) and metabolism (UGCG) were found to be positively associated with liver fat content. Genes involved in lipid metabolism (PLIN, ACADM), fatty acid transport (FABP4, CD36), amino acid catabolism (BCAT1), and inflammation (CCL2) were validated by real-time PCR and were found to be upregulated in subjects with high liver fat content. The data show that multiple changes in gene expression characterize simple steatosis.

Transient receptor potential vanilloid 4 mediates protease activated receptor 2-induced sensitization of colonic afferent nerves and visceral hyperalgesia

2008-05-06

Protease-activated receptor (PAR₂) is expressed by nociceptive neurons and activated during inflammation by proteases from mast cells, the intestinal lumen, and the circulation. Agonists of PAR₂ cause hyperexcitability of intestinal sensory neurons and hyperalgesia to distensive stimuli by unknown mechanisms. We evaluated the role of the transient receptor potential vanilloid 4 (TRPV4) in PAR₂-induced mechanical hyperalgesia of the mouse colon. Colonic sensory neurons, identified by retrograde tracing, expressed immunoreactive TRPV4, PAR₂, and calcitonin gene-related peptide and are thus implicated in nociception. To assess nociception, visceromotor responses (VMR) to colorectal distension (CRD) were measured by electromyography of abdominal muscles. In TRPV4^+/+ mice, intraluminal PAR₂ activating peptide (PAR₂-AP) exacerbated VMR to graded CRD from 6–24 h, indicative of mechanical hyperalgesia. PAR₂-induced hyperalgesia was not observed in TRPV4^–/– mice. PAR₂-AP evoked discharge of action potentials from colonic afferent neurons in TRPV4^+/+ mice, but not from TRPV4^–/– mice. The TRPV4 agonists 5',6'-epoxyeicosatrienoic acid and 4-phorbol 12,13-didecanoate stimulated discharge of action potentials in colonic afferent fibers and enhanced current responses recorded from retrogradely labeled colonic dorsal root ganglia neurons, confirming expression of functional TRPV4. PAR₂-AP enhanced these responses, indicating sensitization of TRPV4. Thus TRPV4 is expressed by primary spinal afferent neurons innervating the colon. Activation of PAR₂ increases currents in these neurons, evokes discharge of action potentials from colonic afferent fibers, and induces mechanical hyperalgesia. These responses require the presence of functional TRPV4. Therefore, TRPV4 is required for PAR₂-induced mechanical hyperalgesia and excitation of colonic afferent neurons.

Corrigendum for Sarna, Am J Physiol Gastrointest Liver Physiol 294(2): G372-G390, 2008.

2008-05-06