Gastroprotective action of orexin-A against stress-induced gastric damage is mediated by endogenous prostaglandins, sensory afferent neuropeptides and nitric oxide

Orexin-A, identified in the neurons and endocrine cells in the gut, has been implicated in control of food intake and sleep behavior but little is known about its influence on gastric secretion and mucosal integrity. The effects of orexin-A on gastric secretion and gastric lesions induced in rats by 3.5 h of water immersion and restraint stress (WRS) or 75% ethanol were determined. Orexin-A (5-80 microg/kg i.p.) increased gastric acid secretion and attenuated gastric lesions induced by WRS and this was accompanied by the significant rise in plasma orexin-A, CGRP and gastrin levels, the gastric mucosal blood flow (GBF), luminal NO concentration and an increase in mRNA for CGRP and overexpression of COX-2 protein and the generation of PGE(2) in the gastric mucosa. Orexin-A-induced protection was abolished by selective OX-1 receptor antagonist, vagotomy and attenuated by suppression of COX-1 and COX-2, deactivation of afferent nerves with neurotoxic dose of capsaicin, pretreatment with CCK(2)/gastrin antagonist, CGRP(8-37) or capsazepine and by inhibition of NOS with L-NNA. This study shows for the first time that orexin-A exerts a potent protective action on the stomach of rats exposed to non-topical ulcerogens such as WRS or topical noxious agents such as ethanol and these effects depend upon hyperemia mediated by COX-PG and NOS-NO systems, activation of vagal nerves and sensory neuropeptides such as CGRP released from sensory nerves probably triggered by an increase in gastric acid secretion induced by this peptide.     

Exogenous and endogenous ghrelin in gastroprotection against stress-induced gastric damage

Ghrelin, identified in the gastric mucosa has been involved in control of food intake and growth hormone (GH) release but little is known about its influence on gastric secretion and mucosal integrity. The effects of ghrelin on gastric secretion, plasma gastrin and gastric lesions induced in rats by 75% ethanol or 3.5 h of water immersion and restraint stress (WRS) were determined. Exogenous ghrelin (5, 10, 20, 40 and 80 microg/kg i.p.) increased gastric acid secretion and attenuated gastric lesions induced by ethanol and WRS and this was accompanied by the significant rise in plasma ghrelin level, gastric mucosal blood flow (GBF) and luminal NO concentrations. Ghrelin-induced protection was abolished by vagotomy and attenuated by suppression of COX, deactivation of afferent nerves with neurotoxic dose of capsaicin or CGRP(8-37) and by inhibition of NOS with L-NNA but not influenced by medullectomy and administration of 6-hydroxydopamine. We conclude that ghrelin exerts a potent protective action on the stomach of rats exposed to ethanol and WRS, and these effects depend upon vagal activity, sensory nerves and hyperemia mediated by NOS-NO and COX-PG systems.     

Association between nesfatin-1 levels and metabolic improvements in severely obese patients who underwent biliopancreatic derivation with duodenal switch

ContextNesfatin-1 is a neuroendocrine peptide with potent anorexigenic activity in rodents. The potential role of nesfatin-1 on the regulation of energy balance, metabolic functions and inflammation is currently debated in obese humans. In the present study, nesfatin-1 fluctuations and their associations with metabolic factors were investigated in severely obese patients who underwent biliopancreatic diversion with duodenal switch (BPD/DS) and severely obese controls (SOC).Basic proceduresSixty severely obese patients who underwent BPD/DS and 15 SOC (matched for BMI and age) were included in the study. Associations between nesfatin-1 levels and body composition, glucose metabolism, lipid profile as well as inflammatory markers were evaluated at baseline and over a post-surgery12-month (12 M) period.Main findingsBody weight was reduced at 6 M and at 12 M in BPD/DS patients (P < 0.001). Nesfatin-1 levels were reduced at 6 M (women: P < 0.05) and at 12 M (men and women; P < 0.001) in BPD/DS patients. At baseline, nesfatin-1 levels negatively correlated with weight, fat (FM) and fat-free mass (FFM) in the whole population (combined BPD/DS and SOC patients). At 12 M, nesfatin-1 concentrations positively correlated with weight, FM, fasting insulin, insulin resistance, total cholesterol, LDL-cholesterol, triglyceride and apoB values. At 12 M, % changes in nesfatin-1 were positively associated with% changes in weight, FM, FFM, fasting insulin, insulin resistance, total cholesterol, LDL-cholesterol, apoB and C-reactive protein.ConclusionNesfatin-1 levels decrease following BPD/DS-induced weight loss and are significantly associated with parameters of metabolic health.     

Nesfatin-1 improves oxidative skin injury in normoglycemic or hyperglycemic rats

Hyperglycemia is one of the major causes of suppressed angiogenesis and impaired wound healing leading to chronic wounds. Nesfatin-1 a novel peptide was reported to have antioxidant and anti-apoptotic properties. This study is aimed to investigate the potential healing-promoting effects of nesfatin-1 in non-diabetic or diabetic rats with surgical wounds. In male Sprague-Dawley rats, hyperglycemia was induced by intraperitoneal (ip) injection of streptozotocin (55 mg/kg). Under anesthesia, dorsum skin tissues of normoglycemic (n = 16) and hyperglycemic rats were excised (2 × 2 cm, full-thickness), while control rats (n = 16) had neither hyperglycemia nor wounds. Half of the rats in each group were treated ip with saline, while the others were treated with nesfatin-1 (2 μg/kg/day) for 3 days until they were decapitated. Plasma interleukin-1-beta (IL-1β), transforming growth factor-beta (TGF-β-1), IL–6 levels, and dermal tissue malondialdehyde (MDA), glutathione (GSH) levels, myeloperoxidase (MPO) and caspase-3 activity were measured. For histological examination, paraffin sections were stained with hematoxylin-eosin or Masson’s trichrome and immunohistochemistry for vascular endothelial growth factor (VEGF) was applied. ANOVA and Student’s t-tests were used for statistical analysis. Compared to control rats, skin MPO activity, MDA and caspase-3 levels were increased similarly in saline-treated normo- and hyperglycemic rats. Nesfatin-1 depressed MDA, caspase-3, MPO activity and IL-1β with concomitant elevations in dermal GSH and plasma TGF-β-1 levels. Histopathological examination revealed regeneration of epidermis, regular arrangement of collagen fibers in the dermis and a decrease in VEGF immunoreactivity in the epidermal keratinocytes of nesfatin-1-treated groups. Nesfatin-1 improved surgical wound healing in both normo- and hyperglycemic rats via the suppression of neutrophil recruitment, apoptosis and VEGF activation.     

Nesfatin-1对大鼠摄食、胃酸分泌、胃运动及胃排空的影响

目的:观察Nesfatin-1对大鼠摄食、胃酸分泌、胃运动及胃排空的影响并探究其可能机制。方法:将大鼠随机分为摄食实验组、胃酸实验组、胃运动实验组以及胃排空实验组。大鼠经腹内侧核置管后给予nasfatin-1,检测大鼠摄食量,使用Na OH滴定法测定大鼠胃酸分泌,记录清醒大鼠胃运动,以比色法测定大鼠胃排空。结果:低剂量和高剂量nesfatin-1均减少2小时累积食物摄入量;高剂量组4小时累积食物摄入量仍显著低于NS对照组。Nesfatin-1能够抑制2-DG对胃酸分泌的促进作用。SHU9119能够部分阻断nesfatin-1对2-DG的抑制作用。Nesfatin-1能够抑制胃运动及胃排空,SHU9119可部分阻断nesfatin-1对胃运动及胃排空的抑制作用。结论:Nesfatin-1能够调控大鼠摄食、胃酸分泌、胃运动及胃排空,黑皮质素信号通路可能也参与该调控过程。     

Decreased plasma nesfatin-1 levels in patients with acute myocardial infarction

Nesfatin-1 is a novel anorexigenic hormone which has close relationship with diabetes, obese, anorexia nervosa, psychiatric disorders and neurogenic diseases. The aim of our study was to evaluate levels of plasma nesfatin-1 among patients presenting with coronary artery disease and the correlation between nesfatin-1 levels and other clinical parameters. Fasting plasma levels of nesfatin-1 were tested in 48 acute myocardial infarction (AMI) patients, 74 stable angina pectoris (SAP) patients and 34 control subjects. All of them were examined by coronary angiography. The severity of coronary atherosclerosis was assessed using the Gensini score. Plasma nesfatin-1 levels were significantly lower in AMI group than SAP group or control group (0.91 ± 0.08 ng/mL vs. 0.98 ± 0.19 ng/mL and 1.09 ± 0.39 ng/mL, respectively, P < 0.05). In AMI patients, plasma nesfatin-1 levels were negatively correlated with high-sensitivity C-reactive protein, neutrophil% or Gensini scores. Such information implies that lower nesfatin-1 concentration may play a very important role in the development of AMI.     

Fasting plasma levels of nesfatin-1 in patients with type 1 and type 2 diabetes mellitus and the nutrient-related fluctuation of nesfatin-1 level in normal humans

The novel satiety factor nesfatin-1 has been shown to decrease food intake and body weight in rodents after i.c.v. injection. However, no further developments regarding the true patho-physiological relevance of nesfatin-1 in obesity and type 1 diabetes mellitus (T1 DM) and type 2 diabetes mellitus (T2 DM) have been reported. A recent study by Stengel et al. demonstrated that a down-regulation of NUCB2 mRNA in gastric endocrine cells was observed after 24-h fasting. They raised the possibility that nesfatin/NUCB2 gene expression may be regulated by nutritional status, suggesting that nesfatin-1 in the stomach might play a role in satiety. In the present study, fasting levels in plasma nesfatin-1, insulin and glucose were measured and analyzed in healthy subjects and in patients with T1 DM and T2 DM. Plasma nesfatin-1 levels were measured 6 times before and after oral glucose ingestion in healthy subjects. No sex differences in plasma nesfatin-1 were found. The mean fasting plasma nesfatin-1 levels were slightly but not significantly higher in T1 DM patients compared to healthy subjects. However, fasting plasma nesfatin-1 levels were significantly lower in T2 DM patients compared to healthy subjects and T1 DM patients. Plasma nesfatin-1 did not change acutely, although a small rise in circulating nesfatin-1 occurred within 30 min after the beginning of an oral glucose ingestion (from a mean basal value of 0.99 ± 0.23 ng/ml to a maximum of 1.08 ± 0.24 ng/ml). No significant difference in plasma nesfatin-1 before and after an oral glucose was observed. In conclusion, we showed that fasting nesfatin-1 was significantly lower in T2 DM patients compared to healthy subjects and T1 DM patients. The significance of this result is unclear but the reduction in fasting nesfatin-1 may be one of the appetite-related hormones involved in diabetic hyperphagia. In addition, neither glucose nor saline ingestions affected plasma nesfatin-1, suggesting that gastric chemosensation is not sufficient for the nesfatin-1 response under the present conditions.     

Nesfatin-1 inhibits gastric acid secretion via a central vagal mechanism in rats

Nesfatin-1, a novel hypothalamic peptide, inhibits nocturnal feeding behavior and gastrointestinal motility in rodents. The effects of nesfatin-1 on gastrointestinal secretory function, including gastric acid production, have not been evaluated. Nesfatin-1 was injected into the fourth intracerebral ventricle (4V) of chronically cannulated rats to identify a nesfatin dose sufficient to inhibit food intake. Nesfatin-1 (2 μg) inhibited dark-phase food intake, in a dose-dependent fashion, for >3 h. Gastric acid production was evaluated in urethane-anesthetized rats. Nesfatin-1 (2 μg) was introduced via the 4V following endocrine stimulation of gastric acid secretion by pentagastrin (2 μg·kg(-1)·h(-1) iv), vagal stimulation with 2-deoxy-d-glucose (200 mg/kg sc), or no stimulus. Gastric secretions were collected via gastric cannula and neutralized by titration to determine acid content. Nesfatin-1 did not affect basal and pentagastrin-stimulated gastric acid secretion, whereas 2-deoxy-d-glucose-stimulated gastric acid production was inhibited by nesfatin-1 in a dose-dependent manner. c-Fos immunofluorescence in brain sections was used to evaluate in vivo neuronal activation by nesfatin-1 administered via the 4V. Nesfatin-1 caused activation of efferent vagal neurons, as evidenced by a 16-fold increase in the mean number of c-Fos-positive neurons in the dorsal motor nucleus of the vagus (DMNV) in nesfatin-1-treated animals vs. controls (P < 0.01). Finally, nesfatin-induced Ca(2+) signaling was evaluated in primary cultured DMNV neurons from neonatal rats. Nesfatin-1 caused dose-dependent Ca(2+) increments in 95% of cultured DMNV neurons. These studies demonstrate that central administration of nesfatin-1, at doses sufficient to inhibit food intake, results in inhibition of vagally stimulated secretion of gastric acid. Nesfatin-1 activates DMNV efferent vagal neurons in vivo and triggers Ca(2+) signaling in cultured DMNV neurons.     

Expression of cyclooxygenase (COX)-1 and COX-2 in adaptive cytoprotection induced by mild stress.

Prostaglandins (PG) derived from COX-1 play an important role in the maintenance of mucosal integrity but the role of COX-2-derived products in mucosal defence mechanism has not been fully explained. Mild stress is known to prevent gastric mucosal lesions induced by severe stress via the phenomenon of adaptive cytoprotection but it remains unknown which COX is involved in this adaptation. In this study, the mucosal expression of COX-1 and COX-2 was examined and the inhibitors of these enzymes were used to determine the contribution of these enzymes in adaptive cytoprotection induced by mild stress. Male Wistar rats were exposed to mild water immersion and restraint stress (WRS) at various time intervals ranging from 5 min up to 2 h followed 1 h later by exposure to severe 3.5 h WRS with or without pretreatment with: 1) NS-398 (10 mg x kg(-1) i.g.), a selective COX-2 inhibitor; 2) resveratrol (5 mg x kg(-1) i.g.), a selective COX-1 inhibitor; 3) meloxicam (2 mg x kg(-1) i.g.), preferential COX-2 inhibitor; and 4) indomethacin (5 mg x kg(-1) i.p), non-selective inhibitor of COX. The number of WRS lesions was counted, gastric blood flow (GBF) was measured by H2-gas clearance technique, mucosal biopsy samples were taken for the assessment of PGE2 by radioimmunoassay, and the expression of COX-1 and COX-2 mRNA by RT-PCR. WRS for 3.5 h produced numerous gastric lesions, decreased GBF by 48% and inhibited formation of PGE2 by 68% as compared to intact mucosa. Exposure to mild WRS during 5-30 min by itself failed to affect mucosal integrity but significantly attenuated gastric lesions induced by exposure to severe 3.5 h stress; the maximal protective effect being achieved with mild WRS during 15 min. This protective effect was accompanied by the rise in GBF and the generation of PGE2 in the gastric mucosa. After extension of mild WRS from 15 min up to 1 or 2 h before more severe 3.5 h WRS, the loss of cytoprotective effect of mild WRS against severe stress accompanied by significant fall in the GBF were observed. Pretreatment with NS-398 (10 mg x kg(-1) i.g.) that failed to affect mucosal PGE2 generation, reduced significantly the protection and accompanying rise in GBF produced by mild WRS whereas resveratrol partly reduced the protection and the rise in GBF induced by mild WRS. Meloxicam or indomethacin significantly inhibited PGE2 generation and completely abolished the hyperemia and protection induced by mild WRS against more severe stress. The protective and hyperemic effects of mild WRS were completely restored by the addition of 16,16 dm PGE2 (5 microg x kg(-1) i.g.) to NS-398 or resveratrol, while the deleterious effects of meloxicam and indomethacin were significantly attenuated by the concomitant treatment with this PGE2 analogue. We conclude that PG derived from both, COX-1 and COX-2 appear to be involved in adaptive cytoprotection developed in response to mild stressors.     

Nesfatin-1及MCH对大鼠胃酸分泌和胃运动的影响

目的:本实验主要探究nesfatin-1对胃运动和胃酸分泌的影响,以及弓状核(ARC)-下丘脑外侧区(LHA)nesfatin-1神经通路在该过程中的作用。方法:采用逆行追踪和免疫组织化学染色实验观察ARC-LHA nesfatin-1神经通路的构成;在体胃运动实验观察nesfatin-1对胃运动的影响以电刺激ARC对胃运动的影响;采用幽门结扎法测量胃液和胃酸分泌量。结果:LHA微量注射nesfatin-1抑制胃运动和胃酸分泌,但是预先注射黑色素浓集激素(MCH)受体拮抗剂PMC-3881-PI减弱nesfatin-1对胃运动和胃酸分泌的抑制作用。电刺激ARC后,胃收缩幅度和频率显著增强,胃酸分泌明显增多。nesfatin-1抗体或PMC-3881-PI对电刺激ARC诱导的胃运动没有显著影响,但是能够改变电刺激ARC诱导的胃酸分泌。结论:ARC-LHA间nesfafin-1通路可调控大鼠胃运动和胃酸分泌,并且黑色素浓集激素也参与调节该过程。     

Cellular and molecular mechanisms of 17β-estradiol postconditioning protection against gastric mucosal injury induced by ischemia/reperfusion in rats

AimsTo investigate the protective effects of 17β-estradiol postconditioning against ischemia/reperfusion (I–R)-induced gastric mucosal injury in rats.Main methodsThe animal model of gastric ischemia/reperfusion was established by clamping of the celiac artery for 30 min and reperfusion for 30 min, 1 h, 3 h, 6 h, 12 h or 24 h. 17β-estradiol at doses of 5, 50 or 100 μg/kg (rat) was administered via peripheral veins 2 min before reperfusion. In a subgroup of rats, the estrogen receptor antagonist fulvestrant (Ful, 2 mg/kg) was intravenously injected prior to 17β-estradiol administration. Histological and immunohistochemical methods were employed to assess the gastric mucosal injury index and gastric mucosal cell apoptosis and proliferation. The malondialdehyde (MDA) concentration, superoxide dismutase (SOD) activity, xanthine oxidase (XOD) activity and hydroxyl free radical (–OH) inhibitory ability were determined by colorimetric assays. Subsequently, the expression of Bcl-2 and Bax in rat gastric mucosa was examined by western blotting.Key findings17β-estradiol dose-dependently inhibited gastric I–R (GI–R) injury, and 17β-estradiol (50 μg/kg) markedly attenuated GI–R injury 1 h after reperfusion. 17β-estradiol inhibited gastric mucosal cell apoptosis and promoted gastric mucosal cell proliferation in addition to increasing SOD activity and –OH inhibitory ability and decreasing the MDA content and XOD activity. The Bax protein level increased 1 h after GI–R and was markedly reduced by intravenous administration of 17β-estradiol. In contrast, the level of Bcl-2 protein decreased 1 h after GI–R and was restored to normal levels by intravenous administration of 17β-estradiol. These effects of 17β-estradiol were inhibited by pretreatment with fulvestrant.Significance17β-estradiol postconditioning should be investigated further as a possible strategy against gastric mucosal injury.     

Presence of adropin,nesfatin-1, apelin-12, ghrelins and salusins peptides in the milk,cheese whey and plasma of dairy cows

Biological fluids (milk and serum/plasma) and cheese whey milk-derived fluid contain numerous molecules, especially amino acids and proteins. Therefore, the purpose of this study was to find out whether cheese whey (n:6), cow milk (n:6) and its blood (n = 6) have adropin, nesfatin-1, apelin-12, ghrelins and salusin peptides. Adropin, nesfatin-1, apelin-12 concentrations were measured by ELISA, whereas ghrelin and salusin concentrations were measured by EIA methods. It was found that adropin, nesfatin-1, apelin-12, des-acylated ghrelin and salusins in cheese whey were higher than in the corresponding milk peptides and plasma of dairy cows, with the exception of salusin alpha and acylated ghrelin in milk being the same than that of the corresponding cheese whey concentration and plasma of dairy cows. A correlation was also found between milk peptides and cheese whey, as also with plasma of dairy cows. The data suggest that peptides in cow milk might be an important and nutritious food for (neonatal) calves and human diet due to their biological and physiological properties.     

Cord blood nesfatin-1 in large for gestational age pregnancies

ObjectiveTo investigate possible alterations in cord blood levels of adipokine nesfatin-1 (secreted by adipose tissue and pancreatic β-cells and implicated in glucose metabolism and insulin resistance), as well as insulin, in large (LGA) and appropriate for gestational age (AGA) pregnancies, granted that these groups differ in body fat mass and metabolic/endocrine mechanisms.Materials and methodsCord blood nesfatin-1 and insulin concentrations were prospectively measured in 40 LGA (9 born from diabetic and 31 from non-diabetic mothers) and 20 AGA singleton full-term infants as well as their mothers.ResultsCord blood nesfatin-1 concentrations were significantly lower in LGA compared to AGA neonates (b = ?0.206, SE 0.07, p = 0.005). However, cord blood nesfatin-1 concentrations were elevated in infants born from mothers with gestational diabetes mellitus (GDM), compared to those born from non-diabetic mothers, after controlling for group (b = 0.190, SE 0.10, p = 0.05). Finally, cord blood nesfatin-1 concentrations were lower in cases of vaginal delivery (b = 0.11, SE 0.05, p = 0.042). Insulin levels were significantly elevated, as customized centiles increased (b = 0.004, SE = 0.002, p = 0.016). No significant correlation was found between insulin and nesfatin-1 in maternal and umbilical cord levels.ConclusionsIn this study nesfatin-1 levels are decreased in LGA compared to AGA fetuses. Fetal nesfatin-1 concentrations are higher in cases of GDM and cord blood nesfatin-1 concentrations are lower in cases of vaginal delivery.     

Novel quinoline incorporating 1,2,4-triazole/oxime hybrids: Synthesis,molecular docking,anti-inflammatory,COX inhibition,ulceroginicity and histopathological investigations

A series of novel quinolines incorporating 1,2,4-triazole/oxime hybrids were prepared. They showed remarkable anti-inflammatory activity and exhibited very low incidence of gastric ulceration, compared to indomethacin. Most of the compounds tested showed remarkable inhibition of the COX-1 isozyme, with IC₅₀’s ranging from 0.48 to 28 µM. Compounds 7c and 9g showed high safety profiles with normal stomach tissue integrity. Docking studies supported the observed in vitro inhibitory activity towards the COX enzymes that may explain their promising anti-inflammatory activity relative to indomethacin. Moreover, differences between the COX-1 and COX-2 isozymes in observed energy scores, as well as in the number of interactions with some of the compounds tested, might predict their higher selectivity towards COX-1 rather than COX-2. Compound 9e was found to inhibit both COXs non-competitively with K_i values of 81 µM and 94.6 µM.     

Synthesis,biological evaluation,and docking studies of novel heterocyclic diaryl compounds as selective COX-2 inhibitors

Three novel series of diaryl heterocyclic derivatives bearing the 2-oxo-5H-furan, 2-oxo-3H-1,3-oxazole, and 1H-pyrazole moieties as the central heterocyclic ring were synthesized and their in vitro inhibitory activities on COX-1 and COX-2 isoforms were evaluated using a purified enzyme assay. The 2-oxo-5H-furan derivative 6b was identified as potent COX inhibitor with selectivity toward COX-1 (COX-1 IC₅₀ = 0.061 μM and COX-2 IC₅₀ = 0.325 μM; selectivity index (SI) = 0.19). Among the 1H-pyrazole derivatives, 11b was found to be a potent COX-2 inhibitor, about 38 times more potent than Rofecoxib (COX-2 IC₅₀ = 0.011 μM and 0.398 μM, respectively), but showed no selectivity for COX-2 isoform. Compound 11c demonstrated strong and selective COX-2 inhibitory activity (COX-1 IC₅₀ = 1 μM, COX-2 IC₅₀ = 0.011 μM; SI = ～92). Molecular docking studies of compounds 6b and 11b–d into the binding sites of COX-1 and COX-2 allowed to shed light on the binding mode of these novel COX inhibitors.     

Endogenous Prostaglandins and Afferent Sensory Nerves in Gastroprotective Effect of Hydrogen Sulfide against Stress-Induced Gastric Lesions

Hydrogen sulfide (H₂S) plays an important role in human physiology, exerting vasodilatory, neuromodulatory and anti-inflammatory effects. H₂S has been implicated in the mechanism of gastrointestinal integrity but whether this gaseous mediator can affect hemorrhagic lesions induced by stress has been little elucidated. We studied the effect of the H₂S precursor L-cysteine, H₂S-donor NaHS, the H₂S synthesizing enzyme (CSE) activity inhibitor- D,L-propargylglycine (PAG) and the gastric H₂S production by CSE/CBS/3-MST activity in water immersion and restraint stress (WRS) ulcerogenesis and the accompanying changes in gastric blood flow (GBF). The role of endogenous prostaglandins (PGs) and sensory afferent nerves releasing calcitonin gene-related peptide (CGRP) in the mechanism of gastroprotection induced by H₂S was examined in capsaicin-denervated rats and those pretreated with capsazepine to inhibit activity of vanilloid receptors (VR-1). Rats were pretreated with vehicle, NaHS, the donor of H₂S and or L-cysteine, the H₂S precursor, with or without the concurrent treatment with 1) nonselective (indomethacin) and selective cyclooxygenase (COX)-1 (SC-560) or COX-2 (rofecoxib) inhibitors. The expression of mRNA and protein for COX-1 and COX-2 were analyzed in gastric mucosa pretreated with NaHS with or without PAG. Both NaHS and L-cysteine dose-dependently attenuated severity of WRS-induced gastric lesions and significantly increased GBF. These effects were significantly reduced by pretreatment with PAG and capsaicin denervation. NaHS increased gastric H₂S production via CSE/CBS but not 3-MST activity. Inhibition of COX-1 and COX-2 activity significantly diminished NaHS- and L-cysteine-induced protection and hyperemia. NaHS increased expression of COX-1, COX-2 mRNAs and proteins and raised CGRP mRNA expression. These effects of NaHS on COX-1 and COX-2 protein contents were reversed by PAG and capsaicin denervation. We conclude that H₂S exerts gastroprotection against WRS-induced gastric lesions by the mechanism involving enhancement in gastric microcirculation mediated by endogenous PGs, sensory afferent nerves releasing CGRP and the activation of VR-1 receptors.     

Nesfatin-1 stimulates glucagon and insulin secretion and beta cell NUCB2 is reduced in human type 2 diabetic subjects

Nesfatin-1 is a novel anorexigenic regulatory peptide. The peptide is the N-terminal part of nucleobindin 2 (NUCB2) and is expressed in brain areas regulating feeding. Outside the brain, nesfatin-1 expression has been reported in adipocytes, gastric endocrine cells and islet cells. We studied NUCB2 expression in human and rodent islets using immunocytochemistry, in situ hybridization and western blot. Furthermore, we investigated the potential influence of nesfatin-1 on secretion of insulin and glucagon in vitro and in vivo in mice and in INS-1 (832/13) cells. The impact of type 2 diabetes (T2D) and glucolipotoxicity on NUCB2 gene expression in human islets and its relationship to insulin secretory capacity and islet gene expression was studied using microarray. Nesfatin-1 immunoreactivity (IR) was abundant in human and rodent beta cells but absent in alpha, delta, PP and ghrelin cells. Importantly, in situ hybridization showed that NUCB2 mRNA is expressed in human and rat islets. Western blot analysis showed that nesfatin-1 IR represented full length NUCB2 in rodent islets. Human islet NUCB2 mRNA was reduced in T2D subjects but upregulated after culture in glucolipotoxic conditions. Furthermore, a positive correlation between NUCB2 and glucagon and insulin gene expression, as well as insulin secretory capacity, was evident. Nesfatin-1 enhanced glucagon secretion but had no effect on insulin secretion from mouse islets or INS-1 (832/13) cells. On the other hand, nesfatin-1 caused a small increase in insulin secretion and reduced glucose during IVGTT in mice. We conclude that nesfatin-1 is a novel glucagon-stimulatory peptide expressed in the beta cell and that its expression is decreased in T2D islets.     

Nesfatin-1 inhibits voltage gated K+ channels in pancreatic beta cells

The anorexigenic neuropeptide NEFA/nucleobindin 2 (NUCB2)/nesfatin-1-containing neurons are distributed in the brain regions involved in feeding regulation. In spite of the growing knowledge of its physiological functions through extensive studies, its molecular mechanism of reaction, including its receptor, remains unknown. NUCB2/nesfatin-1 is also involved in various peripheral regulations, including glucose homeostasis. In pancreatic beta-cells, NUCB2/nesfatin-1 is reported to enhance glucose-stimulated insulin secretion (GSIS) but its exact mechanism remains unknown.To clarify this mechanism, we measured the effect of nesfatin-1 on the electrical activity of pancreatic beta-cells. Using mouse primary beta cells, we measured changes in the ATP-sensitive K⁺ (K_ATP) channel current, the voltage-gated K⁺ (Kv) channel current, and insulin secretion upon application of nesfatin-1.Nesfatin-1 inhibited the Kv channel, but K_ATP channel activity was unaffected. Nesfatin-1 enhanced insulin secretion to a same level as Kv channel blocker tetraethylammonium (TEA). The effect was not further enhanced when nesfatin-1 and TEA were applied simultaneously. The inhibition binding assay with [¹²⁵I]nesfatin-1 in Kv2.1 channels, major contributor of Kv current in beta cell, expressing HEK239 cells indicated the binding of nesfatin-1 on Kv2.1 channel.Because Kv channel inhibition enhances insulin secretion under high glucose conditions, our present data suggest a possible mechanism of nesfatin-1 on enhancing GSIS through regulation of ion channels rather than its unidentified receptor.     

Adaptation of rat gastric tissue against indomethacin toxicity

Indomethacin is used in the treatment of inflammatory diseases. But the drug toxicity limits its usage. This study investigated whether adaptation occurred after various dosages of repeated (chronic) indomethacin in rats to the gastro-toxic effects of indomethacin. It also examined whether the adaptation was related to oxidant–antioxidant mechanisms and oxidative DNA damage in gastric tissue. To illuminate the adaptation mechanism in the gastric tissue of rats given various dosages of chronic indomethacin, the levels of oxidants and antioxidants (GSH, MDA, NO, SOD and MPO), activities of COX-1 and COX-2 enzymes and oxidative DNA damage (8-OHd Gua/10⁵ Gua) were measured. Results were compared to 25-mg/kg single-dose indomethacin group, and the role of oxidant and antioxidant parameters and oxidative DNA damage in the adaptation mechanism was evaluated. The average ulcer areas of gastric tissue of the 0.5-, 1-, 2-, 3-, 4-, and 5-mg/kg dosages of chronic indomethacin given to rats were 19.5 ± 3.7, 12.5 ± 3.3, 10 ± 5.2, 4.5 ± 3.6, 8.6 ± 2.4, and 9.5 ± 2.1 mm², respectively. This rate was measured as 21.3 ± 2.6 mm² in the single-dose indomethacin group. Consequently, after various dosages of repeated (chronic) indomethacin administration in rats, it was observed that a clear adaptation developed against gastric damage and that gastric damage was reduced. The best adaptation was observed in the gastric tissue of the 3-mg/kg chronic indomethacin group. In parallel with the damage reduction, the oxidant parameters (MDA and MPO) and oxidative DNA damage (8-OHd Gua/10⁵ Gua) were reduced, and the antioxidant parameters (GSH, NO and SOD) were increased. There is no relation between COX enzymes and adaptation mechanism. This circumstance shows that not COX-1 and COX-2 enzymes, oxidant and antioxidant parameters may play a role in the adaptation mechanism.