Dipeptidyl Peptidase IV Inhibition Prevents the Formation and Promotes the Healing of Indomethacin-Induced Intestinal Ulcers in Rats

Backgrounds and Aims

We studied the intestinotrophic hormone glucagon-like peptide-2 (GLP-2) as a possible therapy for non-steroidal anti-inflammatory drug (NSAID)-induced intestinal ulcers. Luminal nutrients release endogenous GLP-2 from enteroendocrine L cells. Since GLP-2 is degraded by dipeptidyl peptidase IV (DPPIV), we hypothesized that DPPIV inhibition combined with luminal administration of nutrients potentiates the effects of endogenous GLP-2 on intestinal injury.

Methods

Intestinal injury was induced by indomethacin (10 mg/kg, sc) in fed rats. The long-acting DPPIV inhibitor K579 was given intragastrically (ig) or intraperitoneally (ip) before or after indomethacin treatment. l-Alanine (l-Ala) and inosine 5′-monophosphate (IMP) were co-administered ig after the treatment.

Results

Indomethacin treatment induced intestinal ulcers that gradually healed after treatment. Pretreatment with ig or ip K579 given at 1 mg/kg reduced total ulcer length, whereas K579 at 3 mg/kg had no effect. Exogenous GLP-2 also reduced intestinal ulcers. The preventive effect of K579 was dose-dependently inhibited by a GLP-2 receptor antagonist. Daily treatment with K579 (1 mg/kg), GLP-2, or l-Ala + IMP after indomethacin treatment reduced total ulcer length. Co-administration (ig) of K579 and l-Ala + IMP further accelerated intestinal ulcer healing.

Conclusion

DPPIV inhibition and exogenous GLP-2 prevented the formation and promoted the healing of indomethacin-induced intestinal ulcers, although high-dose DPPIV inhibition reversed the preventive effect. Umami receptor agonists also enhanced the healing effects of the DPPIV inhibitor. The combination of DPPIV inhibition and luminal nutrient-induced GLP-2 release may be a useful therapeutic tool for the treatment of NSAIDs-induced intestinal ulcers.     

Is l-Glutathione More Effective Than l-Glutamine in Preventing Enteric Diabetic Neuropathy?

Background

Diabetes and its complications appear to be multifactorial. Substances with antioxidant potential have been used to protect enteric neurons in experimental diabetes.

Aim

This study evaluated the effects of supplementation with l-glutamine and l-glutathione on enteric neurons in the jejunum in diabetic rats.

Methods

Rats at 90 days of age were distributed into six groups: normoglycemic, normoglycemic supplemented with 2 % l-glutamine, normoglycemic supplemented with 1 % l-glutathione, diabetic (D), diabetic supplemented with 2 % l-glutamine (DG), and diabetic supplemented with 1 % l-glutathione (DGT). After 120 days, the jejunums were immunohistochemically stained for HuC/D+ neuronal nitric oxide synthase (nNOS) and vasoactive intestinal polypeptide (VIP). Western blot was performed to evaluate nNOS and VIP. Submucosal and myenteric neurons were quantitatively and morphometrically analyzed.

Results

Diabetic neuropathy was observed in myenteric HuC/D, nNOS, and VIP neurons (p < 0.05). In the submucosal plexus, diabetes did not change nitrergic innervation but increased VIPergic neuronal density and body size (p < 0.05). Supplementation with l-glutathione prevented changes in HuC/D neurons in the enteric plexus (p < 0.05), showing that supplementation with l-glutathione was more effective than with l-glutamine. Myenteric nNOS neurons in the DGT group exhibited a reduced density (34.5 %) and reduced area (p < 0.05). Submucosal neurons did not exhibit changes. The increase in VIP-expressing neurons was prevented in the submucosal plexus in the DG and DGT groups (p < 0.05).

Conclusion

Supplementation with l-glutathione exerted a better neuroprotective effect than l-glutamine and may prevent the development of enteric diabetic neuropathy.     

d-dimer as a marker of severity in patients with severe acute pancreatitis

Background/purpose

Coagulative disorder is known to occur in the early phase of severe acute pancreatitis (SAP) and d-dimer is a commonly used clinical parameter of hemostasis. The aim of this study was to assess the value of the plasma d-dimer level as a marker of severity in the first 3?days after admission in patients with SAP.

Methods

From January 2009 to February 2011, 45 patients admitted for SAP were included in this observational study. The d-dimer level was measured on a daily basis during days 1?C3 after admission and the acute physiology and chronic health evaluation (APACHE) II score, sequential organ failure assessment (SOFA) score, and other clinical parameters were recorded at the same time. The maximum and the mean d-dimer values were used for analysis and compared with other prognostic factors of SAP.

Results

Both the maximum and mean levels of d-dimer were significantly different between patients with and without clinical variables such as multiple-organ dysfunction syndrome (MODS), need for surgical intervention, and the presence of pancreatic infection. The d-dimer level also showed great precision for the prediction of MODS and secondary infection. Additionally, the d-dimer level correlated well with two usual markers of SAP severity?Cthe APACHE II score and the C-reactive protein level.

Conclusion

d-dimer measurement is a useful, easy, and inexpensive early prognostic marker of the evolution and complications of SAP.     

L-citrulline Prevents Alveolar and Vascular Derangement in a Rat Model of Moderate Hyperoxia-induced Lung Injury

Background

Moderate normobaric hyperoxia causes alveolar and vascular lung derangement in the newborn rat. Endogenous nitric oxide (NO), which promotes lung growth, is produced from the metabolism of l-arginine to l-citrulline in endothelial cells. We investigated whether administering l-citrulline by raising the serum levels of l-arginine and enhancing NO endogenous synthesis attenuates moderate hyperoxia-induced lung injury.

Methods

Newborn rats were exposed to FiO₂?=?0.6 or room air for 14?days to induce lung derangement and then were administered l-citrulline or a vehicle (sham). Lung histopathology was studied with morphometric features. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis. Lung vascular endothelial growth factor (VEGF), nitric oxide synthase (eNOS), and matrix metalloproteinase 2 (MMP2) gene and protein expressions were assessed.

Results

Serum l-arginine rose in the L-citr?+?hyperoxia group (p?=?0.05), as well as the Von Willebrand factor stained vessels count (p?=?0.0008). Lung VEGF immune staining, localized on endothelial cells, was weaker in the sections under hyperoxia than the l-citr?+?hyperoxia and room air groups. This pattern was comparable with the VEGF gene and protein expression profiles. Mean alveolar size increased in the untreated hyperoxia and sham-treated groups compared with the groups reared in room air or treated with l-citrulline under exposure to hyperoxia (p?=?0.0001). Lung VEGF and eNOS increased in the l-citrulline-treated rats, though this treatment did not change MMP2 gene expression but regulated the MMP2 active protein, which rose in BALF (p?=?0.003).

Conclusions

We conclude that administering l-citrulline proved effective in improving alveolar and vascular growth in a model of oxygen-induced pulmonary damage, suggesting better lung growth and matrix regulation than in untreated groups.     

Intraperitoneal Administration of Fetuin-A Attenuates d-Galactosamine/Lipopolysaccharide-Induced Liver Failure in Mouse

Background

Fulminant hepatic failure (FHF) is a devastating syndrome, which sometimes results in death or liver transplantation, in which inflammation would aggravate the development of fetuin-A which would act as an anti-inflammatory factor and may be an available approach to attenuate FHF.

Aims

The purpose of this study was to investigate the effects of fetuin-A on d-galactosamine/lipopolysaccharide (d-GalN/LPS)-induced liver failure in mice.

Methods

A mouse model of FHF induced by d-GalN/LPS was established and fetuin-A was injected intraperitoneally prior to d-GalN/LPS treatment. At different time points after d-GalN/LPS intervention, serum TNF-α and IL-6 levels were measured by ELISA. Fetuin-A mRNA and protein expression in liver tissues was assessed by RT-PCR, Western blotting and immunohistochemical staining. Besides, an observation of liver tissue injury, the apoptosis of hepatocytes, was analyzed by TUNEL assay.

Results

Expression of fetuin-A mRNA and protein in liver tissue were significantly and gradually decreased after d-GalN/LPS administration. A pre-intervention of exogenous fetuin-A significantly improved the liver function, decreased TNF-α and IL-6 expression in peripheral blood, and liver tissue inhibited hepatocyte apoptosis responded to d-GalN/LPS induction so as to decrease the mortality rates of FHF mouse. Meanwhile, fetuin-A was negatively correlated with the hepatic pathological score and TNF-α protein staining in FHF mouse.

Conclusions

An intraperitoneal injection of fetuin-A attenuates d-GalN/LPS-induced FHF in mice. Fetuin-A might be a protective agent of liver damage partly through inhibiting liver inflammatory response and hepatocyte apoptosis.     

Colonic lactate metabolism andd-lactic acidosis

d-Lactic acidosis is seen in patients with intestinal bypass or short bowels in whom colonic producedd-lactate accumulates. An intestinal bypassed patient withd-lactic acidosis had higher fecald-lactate (122.4 mmol/liter) andl-lactate (90.1 mmol/liter) than described before in humans.d-Lactate fluctuated between 0.5 and 3.1 mmol/liter in plasma (normal<0.1 mmol/liter) and between 1.1 and 52.8 mmol/liter in urine (normal<0.7 mmol/liter) within a few hours, indicating that the human organism do metabolize and excreted-lactate. The patient withd-lactic acidosis had a 10-fold increasedDl-lactate production from glucose in fecal homogenates compared to 14 healthy controls and a patient with intestinal bypass, who did not haved-lactic acidosis. A 67% carbohydrate (starch)-enriched diet resulted in a minor elevation of fecal and plasma lactate, whereas 50 + 100+150 g of ingested lactose increasedd-lactate in feces (84.0 mmol/liter) and plasma (2.3 mmol/liter) considerably in the patient withd-lactic acidosis. Intestinal prolongation (22 cm ileum) had a temporary effect on fecal and plasmad-lactate, but intestinal continuity was reestablished 26 months later becaused-lactic acidosis recurred (plasma 8.6 mmol/liter, urine 101.3 mmol/liter). Large amounts of lactulose (160 g/day) to 12 normal individuals increasedd-lactate to 13.6±3.5 mmol/liter in feces, but never increasedd-lactate in plasma or urine. Thein vitro fermentation of glucose in fecal homogenates increasedDl-lactate, which disappeared after complete metabolization of the glucose.l-Lactate was converted tod-lactate andvice versa, and both were degraded to the short-chain fatty acids acetate, propionate, and butyrate. An infrequent, but elevated ability of the colonic flora to produce lactate may be a prerequisite ford-lactic acidosis to occur and may explain why the syndrome is so seldom seen even in patients with intestinal bypass or short bowels. The suggestion thatd-lactate is not metabolized and hence accumulates is probably not valid.     

Increased Gap Density Predicts Weakness of the Epithelial Barrier In Vivo by Confocal Laser Endomicroscopy in Indomethacin-Induced Enteropathy

Background and Aims

The intestinal epithelial barrier plays an important role in the pathogenesis of non-steroidal anti-inflammatory drug-induced enteropathy, and its disruption is often associated with increased cell shedding. The purpose of this report is to observe the gap density in indomethacin-induced small intestinal damage by confocal laser endomicroscopy (CLE) and to investigate the mechanisms involved in this process and how mucosal protectants improve intestinal epithelial barrier dysfunction. CLE is expected to provide a new way for evaluating non-steroidal anti-inflammatory drugs-induced enteropathy in humans and assessing drug efficacy.

Methods

Using the new technique of CLE, we established a method to evaluate, in real time, intestinal damage after the administration of indomethacin in Wistar rats by investigating the gap density in the small intestine. The mucosal protectant teprenone and acid-suppressant rabeprazole were then given by gavage before and after the administration of indomethacin, and the mechanisms affecting the intestinal epithelial barrier were investigated.

Results

Using CLE, gaps could be clearly observed and easily distinguished from goblet cells. Gap density was increased after the administration of indomethacin. During this process, the expression of tumor necrosis factor-α, nuclear factor-κB, and caspase-3 was up-regulated and the expression of tight junctions was down-regulated, which led to the damage of the epithelial barrier. Teprenone and rabeprazole could intervene in this pathway and protect the integrity of the epithelial barrier.

Conclusions

CLE can be objective, accurate, and real time in investigating gap density. Teprenone and rabeprazole can prevent indomethacin-induced intestinal lesions and protect the epithelial barrier by intervening in the tumor necrosis factor-α pathway. Gap density was expected to be an indicator of evaluating intestinal inflammation and drug efficacy.     

Significance of serum ��-d-glucan levels in recipients of living donor liver transplantation

Background/purpose

Early identification and treatment of fungal infections is essential for recipients of liver transplants, but the sensitivity of surveillance culture is insufficient. Measurement of the serum level of ??-d-glucan is a rapid diagnostic strategy for invasive fungal infection. We aimed to evaluate the significance of serum ??-d-glucan levels in transplant recipients after living donor liver transplantation (LDLT).

Methods

We retrospectively analyzed the clinical and laboratory data of 100 consecutive adult transplant recipients after LDLT performed between August 1997 and August 2009.

Results

Seventy-one had high serum ??-d-glucan levels (>20?pg/ml) after LDLT. Nearly half (47.2%) of the episodes of increase occurred within the first 5?days after surgery. The mortality rate of the recipients with high serum ??-d-glucan levels was similar to that of the recipients without high levels. However, in terms of the time line of increase, the recipients with high serum ??-d-glucan levels from 15?days onward after surgery showed a significantly higher mortality rate than those with high levels before 15?days after surgery (33.3 and 4.3%, respectively; p?Conclusions High serum levels of ??-d-glucan at late time points after LDLT indicate established fungal infection and higher mortality.     

Injection of l-glutamate into the insular cortex produces sleep apnea and serotonin reduction in rats

Purpose

Obstructive sleep apnea (OSA) is primarily characterized by repetitive episodes of complete or partial obstruction of airflow during sleep. The neuronal and cellular mechanisms underlying this process are not fully understood, although the focus of some studies is on putative serotonin (5-HT) mechanisms, and serotonergic therapy may be beneficial to OSA patients. This study aimed to demonstrate possible changes in 5-HT associated with induction of OSA in a rat model.

Methods

Apnea was induced in rats by injection of l-glutamate (l-Glu) into the insular cortex. We examined changes in: (1) simultaneous genioglossus and diaphragm EMG activity; and (2) peripheral and cerebral levels of 5-HT, by histology.

Results

Injection of l-glutamate (l-Glu) into the insular cortex induced apnea in the rats. l-Glu stimulation of the insular cortex also produced significant reductions in plasma 5-HT levels and the expression of 5-HT in the brainstem. In addition, lower activity was observed in the GG and a higher activity was observed in the diaphragm, as compared to controls.

Conclusion

Data indicate that l-Glu stimulation of the insular cortex simulates the electrical activity of the genioglossus muscle and diaphragm in sleep apnea, and contributes to reduced peripheral and cerebral 5-HT levels in rats. The results of our study suggest that 5-HT may play a role in the pathogenesis of OSA.     

Prevention of colonic spasm using l-menthol in colonoscopic examination

Purpose

The detailed efficacy of intraluminal l-menthol for preventing colonic spasm is not known. The aim of this study was to evaluate the effectiveness of l-menthol in preventing colonic spasm during colonoscopy.

Methods

We analyzed 65 patients (mean age: 71.7 years; 49 men and 16 women) who were administered 0.8 % l-menthol (MINCLEA, Nihon Seiyaku, Tokyo, Japan) intraluminally for severe colonic spasm during colonoscopic examination at Kyoto Prefectural University of Medicine between February 2012 and May 2013. The efficacy of l-menthol was defined as the absence of colonic spasm during a period of 30 s, and its effect was evaluated at 30 s, 1 min, and 5 min after administration. Additionally, various characteristics of these patients were analyzed. Twenty-seven patients with severe colonic spasm were administered intraluminal water and assessed as controls.

Results

l-Menthol was effective in preventing colonic spasms in 60.0 %, 70.8 %, and 46.5 % of patients at 30 s, 1 min, and 5 min, respectively. In contrast, water was effective in 22.2 %, 29.6 %, and 48.1 % of patients at 30 s, 1 min, and 5 min, respectively. There was a significant difference about the efficacy at 30 s and 1 min between l-menthol and water (P?=?0.0009, P?=?0.0006).

Conclusions

l-Menthol (0.8 %) was effective in preventing colonic spasm during colonoscopic examination.     

Inhibition of atrial fibrillation by low-level vagus nerve stimulation: the role of the nitric oxide signaling pathway

Purpose

We examined the role of the phosphatidylinositol-3 kinase (PI3K)/nitric oxide (NO) signaling pathway in low-level vagus nerve stimulation (LLVNS)-mediated inhibition of atrial fibrillation (AF).

Methods

In 17 pentobarbital anesthetized dogs, bilateral thoracotomies allowed the attachment of electrode catheters to the superior and inferior pulmonary veins and atrial appendages. Rapid atrial pacing (RAP) was maintained for 6 h. Each hour, programmed stimulation was used to determine the window of vulnerability (WOV), a measure of AF inducibility, at all sites. During the last 3 h, RAP was overlapped with right LLVNS (50 % below that which slows the sinus rate). In group 1 (n?=?7), LLVNS was the only intervention, whereas in groups 2 (n?=?6) and 3 (n?=?4), the NO synthase inhibitor N ^G-nitro-l-arginine methyl ester (l-NAME) and the PI3K inhibitor wortmannin, respectively, were injected in the right-sided ganglionated plexi (GP) during the last 3 h. The duration of acetylcholine-induced AF was determined at baseline and at 6 h. Voltage–sinus rate curves were constructed to assess GP function.

Results

LLVNS significantly decreased the acetylcholine-induced AF duration by 8.2?±?0.9 min (p?<?0.0001). Both l-NAME and wortmannin abrogated this effect. The cumulative WOV (the sum of the individual WOVs) decreased toward baseline with LLVNS (p?<?0.0001). l-NAME and wortmannin blunted this effect during the fifth (l-NAME only, p?<?0.05) and the sixth hour (l-NAME and wortmannin, p?<?0.05). LLVNS suppressed the ability of GP stimulation to slow the sinus rate, whereas l-NAME and wortmannin abolished this effect.

Conclusion

The anti-arrhythmic effects of LLVNS involve the PI3K/NO signaling pathway.     

L-arginine and glycine supplementation in the repair of the irradiated colonic wall of rats

Purpose

Radiotherapy is widely used for cancer treatment but has harmful effects. This study aimed to assess the effects of L-arginine and glycine supplementation on the colon wall of rats submitted to abdominal irradiation.

Methods

Forty male Wistar rats were randomly divided into four groups: I??healthy, II??irradiated with no amino acid supplementation, III??irradiated and supplemented with L-arginine, and IV??irradiated and supplemented with glycine. The animals received supplementation for 14?days, with irradiation being applied on the eighth day of the experiment. All animals underwent laparotomy on the 15th day for resection of a colonic segment for stereologic analysis. Parametric and nonparametric tests were used for statistical analysis, with the level of significance set at p ??0.05.

Results

Stereologic analysis showed that irradiation induced a reduction of the total volume of the colon wall of group II and III animals compared to healthy controls, but not of group IV animals supplemented with glycine. The mucosal layer of the irradiated animals of all groups was reduced compared to healthy group I animals, but supplementation with L-arginine and glycine was effective in maintaining the epithelial surface of the mucosal layer.

Conclusion

The present results suggest that glycine supplementation had a superior effect on the irradiated colon wall compared to L-arginine supplementation since it was able to maintain the thickness of the wall and the epithelial surface of the mucosa, whereas L-arginine maintained the partial volume of the epithelium and the epithelial surface, but not the total volume of the intestinal wall.     

Adiponectin Modulates DCA-Induced Inflammation via the ROS/NF-Kappa B Signaling Pathway in Esophageal Adenocarcinoma Cells

Background

Deoxycholic acid (DCA) promotes the development and progression of esophageal adenocarcinoma (EAC) by inducing inflammation. Adiponectin is reported to have anti-inflammatory and anti-tumor effects.

Purpose

This study investigated the effects of two types of adiponectin, full-length adiponectin (f-Ad) and globular adiponectin (g-Ad), on DCA-induced inflammation, and investigated the involvement of the reactive oxygen species (ROS)/NF-κB signaling pathway in inflammation in EAC.

Methods

OE19 cells were treated with DCA (50–300 μM) and/or f-Ad/g-Ad (10.0 μg/ml) or N-acetylcysteine (NAC). The viability of cells exposed to DCA was measured by use of the MTT assay. mRNA and protein levels of the inflammatory factors were examined by real-time PCR and ELISA. Intra-cellular ROS levels were determined by use of flow cytometry. Protein levels of total and p-NF-κB p65 were measured by western blot.

Results

DCA induced dose and time-dependent cytotoxicity. mRNA and protein expression of TNF-α, IL-8, and IL-6 in cells treated with DCA alone were up-regulated, and intra-cellular ROS and p-NF-κB p65 protein levels were also increased. g-Ad promoted inflammatory factor production, ROS levels, and p-NF-κB p65 protein expression whereas f-Ad had a suppressive effect. When combined with DCA, g-Ad enhanced the pro-inflammatory effect of DCA whereas f-Ad, similar to NAC, suppressed the effect.

Conclusion

DCA has a pro-inflammatory effect in EAC. f-Ad has an anti-inflammatory effect whereas g-Ad seems to have a pro-inflammatory effect in an ROS/NF-κB p65-dependent manner. This indicates that f-Ad could be a potential anti-inflammatory reagent for cancer therapy.     

A novel DPP IV-resistant C-terminally extended glucagon analogue exhibits weight-lowering and diabetes-protective effects in high-fat-fed mice mediated through glucagon and GLP-1 receptor activation

Aims/hypothesis

Modification of the structure of glucagon could provide useful compounds for the potential treatment of obesity-related diabetes.

Methods

This study evaluated N-acetyl-glucagon, (d-Ser²)glucagon and an analogue of (d-Ser²)glucagon with the addition of nine amino acids from the C-terminal of exendin(1-39), namely (d-Ser²)glucagon-exe.

Results

All analogues were resistant to dipeptidyl peptidase IV degradation. N-Acetyl-glucagon lacked acute insulinotropic effects in BRIN BD11 cells, whereas (d-Ser²)glucagon and (d-Ser²)glucagon-exe evoked significant (p?d-Ser²)glucagon-exe stimulated cAMP production (p?GLP-1-receptor (GLP-1R)-transfected cells but not in glucose-dependent insulinotropic polypeptide-receptor-transfected cells. In normal mice, N-acetyl-glucagon and (d-Ser²)glucagon retained glucagon-like effects of increasing (p?d-Ser²)glucagon-exe was devoid of hyperglycaemic actions but substantially (p?d-Ser²)glucagon-exe reduced the glycaemic excursion (p?p?d-Ser²)glucagon-exe. Twice-daily administration of (d-Ser²)glucagon-exe to high-fat-fed mice for 28 days significantly (p?p?p?2 consumption and locomotor activity were (p?p?p?p?p?Conclusions/interpretation This study emphasises the potential of (d-Ser²)glucagon-exe for the treatment of obesity-related diabetes.     

Potential usefulness of hydrogen breath test withd-xylose in clinical management of intestinal malabsorption

Hydrogen breath tests (H₂ BT) have been used extensively to investigate intestinal dissacharidase deficiencies. A potentially useful test for assessing intestinal absorptive function, the H₂ BT withd-xylose (H₂ BT-d-xylose), has received scant attention. We report here the results of our investigation of this test in 45 patients. Fifteen patients had proved malabsorption that was due to nontropical sprue in nine, and to lymphoma, Whipple's disease, or giardiasis in the remainder. Nine patients had small-bowel bacterial overgrowth secondary to either postsurgical sequelae or intestinal dysmotility. Twenty-one patients with irritable bowel syndrome and 21 healthy individuals served as control groups. All participants ingested 25 g ofd-xylose, and alveolar breath samples were obtained thereafter at 30 min intervals for 5 hr. Breath H₂ was measured by chromatography. Basal H₂ production, peak change (Δ) and area under the curve (AUC) were calculated. Simultaneously, 5-hr urinary excretion ofd-xylose was measured by colorimetry and served as the reference test. In healthy individuals,d-xylose ingestion increased H₂ production (Δ=5.8±1.4 ppm,P<0.001). Changes were similar in patients with the irritable bowel syndrome. In contrast, the increase was of a much greater magnitude in the malabsorption group (Δ=49.9±7.2 ppm,P<0.001 vs healthy controls). AUC analysis yielded comparable results. Test performance analysis showed that, in malabsorption the H₂ BT-d-xylose had a sensitivity index of 0.86, which was identical to that of the urinaryd-xylose test. Specificity was 1 and 0.95, respectively; and predictability 1 and 0.93, respectively. All patients who responded to treatment normalized their H₂ production, whereas those who did not respond maintained their high H₂ production. In the bacterial-overgrowth group, the H₂ BT-d-xylose was only positive when the urinary excretion ofd-xylose was positive (five patients), whereas that three of the remaining four patients with normal urinary excretion ofd-xylose also had a normal breath test. We conclude that the hydrogen breath test withd-xylose is a useful, valid, and practical test for the diagnosis and follow-up of malabsorption.     

Expression levels of the mitochondrial IAP antagonists Smac/DIABLO and Omi/HtrA2 in clear-cell renal cell carcinomas and their prognostic value

Purpose

Numerous molecular parameters are thought to be implicated in renal cell carcinoma (RCC) tumor biology and may therefore reflect the malignant potential of individual tumors. Their investigation may thus help to improve the postoperative management of RCC patients. This study characterized the mRNA expression levels and evaluated the prognostic effect of the mitochondrial inhibitor of apoptosis antagonists Smac/DIABLO and Omi/HtrA2 in tumor tissue from clear-cell RCC patients.

Methods

The relative gene expression (RGE) was analyzed by real-time RT-PCR in tumor tissue obtained from 85 patients (median follow-up: 47 months) following surgical treatment. Expression data was correlated to clinico-pathological variables and outcome.

Results

The RGE of Smac/DIABLO was lowest in patients with primary metastases, intermediate in those who progressed to metastatic disease, and highest in those who did not develop metastases during follow-up (P = 0.006). Expression levels of Smac/DIABLO and Omi/HtrA2 were strongly correlated with each other (Pearson coefficient 0.90). Recurrence-free and tumor-specific survival was shorter in patients with low Smac/DIABLO levels (P = 0.019 and P = 0.001) as well as in those with low Omi/HtrA2 tumor expression (P = 0.033 and P = 0.032). Contrary to Omi/HtrA2, low Smac/DIABLO levels were still predictive of a reduced time to recurrence (hazard rate 5.31; 95% CI: 1.16–24.21) and tumor-specific survival (hazard rate 4.24; 95% CI: 1.22–14.77) in explorative multivariate analysis.

Conclusions

The mRNA expression levels of the mitochondrial IAP antagonists Smac/DIABLO and Omi/HtrA2 are strongly inter-correlated, but do not relate to tumor stage or grade of RCC. Our data suggest that expression of Smac/DIABLO, but not Omi/HtrA2, is inversely associated with outcome of RCC patients.     

Comparison of the metabolic effects of sustained CCK1 receptor activation alone and in combination with upregulated leptin signalling in high-fat-fed mice

Aims/hypothesis

Cholecystokinin (CCK) and leptin are important hormones with effects on energy balance. The present study assessed the biological effects of (pGlu-Gln)-CCK-8 and [d-Leu-4]-OB3, smaller isoforms of CCK and leptin, respectively.

Methods

The actions and overall therapeutic use of (pGlu-Gln)-CCK-8 and [d-Leu-4]-OB3, alone and in combination, were evaluated in normal and high-fat-fed mice.

Results

(pGlu-Gln)-CCK-8 had prominent (p?<?0.01 to p?<?0.001), acute feeding-suppressive effects, which were significantly augmented (p?<?0.05 to p?<?0.01) by [d-Leu-4]-OB3. In agreement, the acute dose-dependent glucose-lowering and insulinotropic actions of (pGlu-Gln)-CCK-8 were significantly enhanced by concurrent administration of [d-Leu-4]-OB3. Twice daily injection of (pGlu-Gln)-CCK-8 alone and in combination with [d-Leu-4]-OB3 in high-fat-fed mice for 18 days decreased body weight (p?<?0.05 to p?<?0.001), energy intake (p?<?0.01), circulating triacylglycerol (p?<?0.01), non-fasting glucose (p?<?0.05 to p?<?0.001) and triacylglycerol deposition in liver and adipose tissue (p?<?0.001). All treatment regimens improved glucose tolerance (p?<?0.05 to p?<?0.001) and insulin sensitivity (p?<?0.001). Combined treatment with (pGlu-Gln)-CCK-8 and [d-Leu-4]-OB3 resulted in significantly lowered plasma insulin levels, normalisation of circulating LDL-cholesterol and decreased triacylglycerol deposition in muscle. These effects were superior to either treatment regimen alone. There were no changes in overall locomotor activity or respiratory exchange ratio, but treatment with (pGlu-Gln)-CCK-8 significantly reduced (p?<?0.001) energy expenditure.

Conclusions/interpretation

These studies highlight the potential of (pGlu-Gln)-CCK-8 alone and in combination with [d-Leu-4]-OB3 in the treatment of obesity and diabetes.     

The influence of pioglitazone on the plasma amino acid profile in patients with nonalcoholic steatohepatitis (NASH)

Background and aims

The peroxisome proliferator-activated receptor-γ (PPAR-γ) ligand, piglitazone, enhances the degradation of branched-chain amino acids (BCAAs) in adipose tissue. However, it remains unknown whether pioglitazone influences the plasma amino acids (AA) profile in patients with nonalcoholic steatohepatitis (NASH). Thus, we investigated the relation between the therapeutic effect and the AA profile in NASH patients with a prospective study.

Methods

We randomized 25 histologically proven NASH patients to diet treatment only or diet treatment plus pioglitazone (15 mg/day), and investigated the biological data for 24 months. We measured the concentrations of AAs and compared them between the beginning and the end of the study.

Results

Compared with the diet only group, pioglitazone therapy was associated with an increase in body weight (mean change ?1.03 vs. +3.8 kg; p = 0.027) and subcutaneous fat (?3.7 vs. +45.7 cm²; p = 0.056), and decreased ALT levels (?0.6 vs. ?38.4 IU/L; p = 0.029) and HbA1c (0.33 vs. ?0.29 %; p = 0.016). Regarding the AA profile, l-isoleucine, l-leucine, l-histidine, and l-lysine were significantly reduced in patients treated with pioglitazone. Furthermore, l-leucine was significantly reduced compared with those in the diet only group (mean change ?34.8 vs. +4.12 nmol/mL; p = 0.032). Interestingly, there was a significant correlation between the changes in BCAAs, especially l-leucine, and those in ALT regardless of treatment with pioglitazone.

Conclusions

Pioglitazone therapy in NASH subjects significantly reduced the plasma BCAA level and the degradation was closely related to the improvement of the ALT levels. These results suggest that pioglitazone improves insulin resistance and BCAA metabolism in NASH patients.     

Attenuation of indomethacin-induced gastric mucosal injury by prophylactic administration of sake yeast-derived thioredoxin

Background

Indomethacin is one of the group of nonsteroidal anti-inflammatory drugs, which often cause gastric mucosal injury as a side effect. Infiltration and activation of inflammatory cells, production of proinflammatory cytokines and chemokines, generation of reactive oxygen species, and activation of apoptotic signaling are involved in the pathogenesis of indomethacin-induced gastric injury. We examined whether sake yeast-derived thioredoxin (a small redox-active protein with anti-oxidative activity and various redox-regulating functions) reduced indomethacin-induced gastric injury.

Methods

Gastric injury was produced by the intraperitoneal administration of indomethacin (40?mg/kg body weight) to C57BL/6 mice. Prior to the administration of indomethacin, the mice were offered food pellets containing non-genetically modified sake yeast-derived thioredoxin (thioredoxin 200?μg/g) for 3?days. Histological examinations, assessment of myeloperoxidase activity, and analysis of the gene expressions of proinflammatory cytokines and a chemokine (interleukin [IL]-1β, IL-6, and CXCL1) were statistically evaluated. Indomethacin cytotoxicity was determined by lactate dehydrogenase release from murine gastric epithelial GSM06 cells induced by 24-h treatment with 200 and 400?μM indomethacin after 1-h preincubation with 100?μg/ml sake yeast-derived thioredoxin.

Results

Macroscopic (edema, hemorrhage, and ulcers) and histological (necrosis, submucosal edema, neutrophil infiltration) findings induced by indomethacin were significantly reduced by pretreatment with food pellets containing thioredoxin. Gastric myeloperoxidase activity and the gene expressions of proinflammatory cytokines (IL-1β and IL-6) were also significantly reduced by this pretreatment compared with findings in the mice not pretreated with thioredoxin-containing food pellets. The administration of sake yeast-derived thioredoxin significantly reduced indomethacin-induced cytotoxicity in GSM06 cells.

Conclusions

We conclude that oral administration of sake yeast-derived thioredoxin reduces indomethacin-induced gastric injury. Sake yeast-derived thioredoxin may have therapeutic potential against indomethacin-induced gastric injury.     

Effect of Biliary Cirrhosis on Neurogenic Relaxation of Rat Gastric Fundus and Anococcygeus Muscle: Role of Nitric Oxide Pathway

Background

Cirrhosis, associated with a host of hemodynamic abnormalities, could affect the gastrointestinal (GI) tract motility. On the other hand, the nonadrenergic noncholinergic (NANC) neurotransmission has been shown to play a pivotal role in GI tract motility and has been linked with release of nitric oxide (NO) on electrical stimulation. In this study, we investigated the effect of biliary cirrhosis on the neurogenic relaxation of rat gastric fundus and anococcygeus muscle and also the possible role of nitric oxide system in this manner.

Methods

Isolated gastric fundus and anococcygeus strips of sham-operated and biliary cirrhotic (4 weeks after bile duct ligation) rats were mounted under tension in a standard organ bath. Electrical stimulation was applied to obtain NANC-mediated relaxations in precontracted gastric fundus and anococcygeus muscle. The neurogenic relaxations were examined in the presence of different doses of NO synthase inhibitor, N ^w-Nitro-l-Arginine Methyl Ester (l-NAME). The concentration-dependent relaxant responses to the NO donor sodium nitroprusside were also evaluated.

Results

The neurogenic relaxation of both gastric fundus and anococcygeus muscle was significantly (P < 0.001) increased in cirrhotic animals. l-NAME (0.03–1,000 µM) inhibited relaxations in both groups in a dose-dependent manner (P < 0.001), but cirrhotic groups were more resistant to the inhibitory effects of l-NAME (P < 0.01). Sodium nitroprusside-mediated relaxations were similar in two groups.

Conclusions

This study for the first time demonstrated that cirrhosis increases the NO-mediated neurogenic relaxation of both rat gastric fundus and anococcygeus muscle, suggesting a crucial role for the neurogenic NO in the pathophysiology of disturbed GI motility in cirrhosis.