Short-Term Modulation of Extracellular Signal-Regulated Kinase 1/2 and Stress-Activated Protein Kinase/c-Jun NH2-Terminal Kinase in Pancreatic Islets by Glucose and Palmitate Possible Involvement of Ceramide

Objectives: The effect of glucose and palmitate on the phosphorylation of proteins associated with cell growth and survival (extracellular signal-regulated kinase 1/2 [ERK1/2] and stress-activated protein kinase/c-Jun NH2-terminal kinase [SAPK/JNK]) and on the expression of immediate early genes was investigated. Methods: Groups of freshly isolated rat pancreatic islets were incubated in 10-mmol/L glucose with palmitate, LY294002, or fumonisin B1 for the measurement of the phosphorylation and the content of ERK1/2, JNK/SAPK, and v-akt murine thymoma viral oncongene (AKT) (serine 473) by immunoblotting. The expressions of the immediate early genes, c-fos and c-jun, were evaluated by reverse transcription-polymerase chain reaction. Results: Glucose at 10 mmol/L induced ERK1/2 and AKT phosphorylations and decreased SAPK/JNK phosphorylation. Palmitate (0.1 mmol/L) abolished the glucose effect on ERK1/2, AKT, and SAPK/JNK phosphorylations. LY294002 caused a similar effect. The inhibitory effect of palmitate on glucose-induced ERK1/2 and AKT phosphorylation changes was not observed in the presence of fumonisin B1. Glucose increased c-fos and decreased c-jun expressions. Palmitate and LY294002 abolished these latter glucose effects. The presence of fumonisin B1 abolished the effect induced by palmitate on c-jun expression. Conclusions: Our results suggest that short-term changes of mitogen-activated protein kinase and AKT signaling pathways and c-fos and c-jun expressions caused by glucose are abolished by palmitate through phosphatidylinositol 3-kinase inhibition via ceramide synthesis.

Involvement of Phosphatidylinositol-3 Kinase/AKT/PKC zeta/lambda Pathway in the Effect of Palmitate on Glucose-Induced Insulin Secretion

Objectives: In the present study, a novel pathway by which palmilate potentiates glucose-induced insulin secretion by pancreatic beta cells was investigated. Methods: Groups of freshly isolated islets were incubated in 10 mM glucose with palmitate, LY294002, wortmannin, and fumonism B I for measurement of insulin secretion by radioimmunoassay (RIA). Also, phosphorylation and content of AKT and PKC proteins were evaluated by immunoblotting. Results: Glucose plus palmitate and glucose plus LY294002 or wortmannin (PI3K inhibitors) increased glucose-induced insulin secretion by isolated pancreatic islets. Glucose at 10 mM induced AKT and PKC zeta/lambda phosphorylation. Palmitate (0.1 mM) abolished glucose stimulation of AKT and PKC zeta/lambda phosphorylation possibly through PI3K inhibition because both LY294002 (50 mu M) and wortmannin (100 nM) caused the same effect. The inhibitory effect of palmitate on glucose-induced AKT and PKC zeta/lambda phosphorylation and the stimulatory effect of palmitate on glucose-induced insulin secretion were not observed in the presence of fumonisin B1, all inhibitor of ceramide synthesis. Conclusions: These findings support the proposition that palmilate increases insulin release in the presence of 10 mM glucose by inhibiting PI3K activity through a mechanism that involves ceramide synthesis.

Alterations of NADPH Oxidase Activity in Rat Pancreatic Islets Induced by a High-Fat Diet

Objective: The aim of this study was to evaluate the effect of a high-fat diet (HFD) on nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in rat pancreatic islets. We investigated if changes in NADPH oxidase are connected to beta cell dysfunction reported in obese animals. Methods: Male Wistar rats were fed a HFD or control diet for 3 months. DNA fragmentation, insulin secretion, and [U-(14)C] glucose oxidation were examined in isolated pancreatic islets. The oxidative stress markers nitrotyrosine and 4-hydroxy-2-nonenal were assessed by immunohistochemistry. The protein content of gp91(phox) and p47(phox) was evaluated by Western blotting. Production of reactive oxygen species (ROS) was determined by a fluorescence assay using hydroethidine. Results: Occurrence of DNA fragmentation was reduced in pancreatic islets from HFD rats. There were no differences in oxidative stress markers between the groups. Glucose oxidation and insulin secretion were elevated due to high glucose in pancreatic islets from HFD rats. Protein concentrations of p47(phox) and gp91(phox) subunits were reduced and ROS production was diminished in pancreatic islets from HFD rats. Conclusions: The diminished content of NADPH oxidase subunits and ROS concentrations may be associated with increased glucose oxidation and insulin secretion in an attempt to compensate for the peripheral insulin resistance elicited by the HFD.

Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats

Ogias D, de Andrade Sa ER, Kasai A, Moisan M, Alvares EP, Gama P. Fasting differentially regulates plasma corticosterone-binding globulin, glucocorticoid receptor, and cell cycle in the gastric mucosa of pups and adult rats. Am J Physiol Gastrointest Liver Physiol 298: G117-G125, 2010. First published October 15, 2009; doi:10.1152/ajpgi.00245.2009.-The nutritional status influences gastric growth, and interestingly, whereas cell proliferation is stimulated by fasting in suckling rats, it is inhibited in adult animals. Corticosterone takes part in the mechanisms that govern development, and its effects are regulated in particular by corticosterone-binding globulin (CBG) and glucocorticoid receptor (GR). To investigate whether corticosterone activity responds to fasting and how possible changes might control gastric epithelial cell cycle, we evaluated different parameters during the progression of fasting in 18- and 40-day-old rats. Food restriction induced higher corticosterone plasma concentration at both ages, but only in pups did CBG binding increase after short-and long-term treatments. Fasting also increased gastric GR at transcriptional and protein levels, but the effect was more pronounced in 40-day-old animals. Moreover, in pups, GR was observed in the cytoplasm, whereas, in adults, it accumulated in the nucleus after the onset of fasting. Heat shock protein (HSP) 70 and HSP 90 were differentially regulated and might contribute to the stability of GR and to the high cytoplasmic levels in pups and elevated shuttling in adult rats. As for gastric epithelial cell cycle, whereas cyclin D1 and p21 increased during fasting in pups, in adults, cyclin E slowly decreased, concomitant with higher p27. In summary, we demonstrated that corticosterone function is differentially regulated by fasting in 18-and 40-day-old rats, and such variation might attenuate any possible suppressive effects during postnatal development. We suggest that this mechanism could ultimately increase cell proliferation and allow regular gastric growth during adverse nutritional conditions.

Determinants of Accelerated Small Intestinal Transit in Alcohol-Related Chronic Pancreatitis

Patients with chronic pancreatitis may have abnormal gastrointestinal transit, but the factors underlying these abnormalities are poorly understood. Gastrointestinal transit was assessed, in 40 male outpatients with alcohol-related chronic pancreatitis and 18 controls, by scintigraphy after a liquid meal labeled with (99m)technetium-phytate. Blood and urinary glucose, fecal fat excretion, nutritional status, and cardiovascular autonomic function were determined in all patients. The influence of diabetes mellitus, malabsorption, malnutrition, and autonomic neuropathy on abnormal gastrointestinal transit was assessed by univariate analysis and Bayesian multiple regression analysis. Accelerated gastrointestinal transit was found in 11 patients who showed abnormally rapid arrival of the meal marker to the cecum. Univariate and Bayesian analysis showed that diabetes mellitus and autonomic neuropathy had significant influences on rapid transit, which was not associated with either malabsorption or malnutrition. In conclusion, rapid gastrointestinal transit in patients with alcohol-related chronic pancreatitis is related to diabetes mellitus and autonomic neuropathy.

Fluorescence spectroscopy to diagnose hepatic steatosis in a rat model of fatty liver

Steatosis is diagnosed on the basis of the macroscopic aspect of the liver evaluated by the surgeon at the time of organ extraction or by means of a frozen biopsy. In the present study, the applicability of laser-induced fluorescence (LIF) spectroscopy was investigated as a method for the diagnosis of different degrees of steatosis experimentally induced in rats. Rats received a high-lipid diet for different periods of time. The animals were divided into groups according to the degree of induced steatosis diagnosis by histology. The concentration of fat in the liver was correlated with LIF by means of the steatosis fluorescence factor (SFF). The histology classification, according to liver fat concentration was, Severe Steatosis, Moderate Steatosis, Mild Steatosis and Control (no liver steatosis). Fluorescence intensity could be directly correlated with fat content. It was possible to estimate an average of fluorescence intensity variable by means of different confidence intervals (P=95%) for each steatosis group. SFF was significantly higher in the Severe Steatosis group (P < 0.001) compared with the Moderate Steatosis, Mild Steatosis and Control groups. The various degrees of steatosis could be directly correlated with SFF. LIF spectroscopy proved to be a method capable of identifying the degree of hepatic steatosis in this animal model, and has the potential of clinical application for non-invasive evaluation of the degree of steatosis.

Helicobacter pylori and Iron-deficiency Anemia in Adolescents in Brazil

Aim: The aim of the study was to evaluate the association between Helicobacter pylori infection and iron deficiency (ID) in adolescents attending a public school. Patients and Methods: From March to June 2001, a cross-sectional study was conducted among adolescents (10-16 years) enrolled in a single public school in Sao Paulo, Brazil. Of 400 eligible students, 195 agreed to participate, but 1 was excluded due to sickle cell disease. A blood sample was collected from each subject to measure hemoglobin and ferritin. H pylori status was investigated with the 13 C-urea breath test. All of the subjects with either anemia or ID were given iron therapy. Results: H pylori prevalence was 40.7% (79/194), being higher in male subjects (45/90 vs 34/104, P = 0.014). There was no relation between infection and nutritional status. Abnormally low serum ferritin was observed in 12 subjects, half of whom were positive for H pylori (odds ratio [OR] 1.49, 95% confidence interval [CI] 0.38-5.81). The median serum ferritin was 33.6 ng/mL (interquartile range 23.9-50.9) in infected subjects and 35.1 ng/mL (interquartile range 23.7-53.9) in uninfected subjects. Anemia was detected in 2% (4/194) of the students, half of whom were infected (OR 1.47, 95% CI 0.1-20.6). The mean hemoglobin value in infected subjects was 13.83 g/dL +/- 1.02 versus 14 g/dL +/- 1.06 in uninfected subjects. Conclusions: The study was not able to find a relation between H pylori infection and ID or anemia.

Colestase crônica em crianças e em adolescentes : avaliação nutricional, avaliação neurológica e níveis plasmáticos de vitaminas D e E

A colestase crônica na infância e na adolescência interfere diretamente no cres-cimento e no desenvolvimento do indivíduo e produz conseqüências clínicas relacionadas com a má absorção das vitaminas lipossolúveis da dieta. A vitamina E exerce um importante papel na estrutura e na função dos sistemas nervoso e musculoesquelético. A vitamina D tem reconhecida influência sobre a fisiopatologia da osteopenia colestática que se manifesta como osteoporose, raquitismo ou osteomalácia. A realização de dosagens plasmáticas dessas vitami-nas é essencial para detectar precocemente suas deficiências, bem como para monitorizar uma adequada suplementação. Essas dosagens não são realizadas de rotina no nosso meio. Os objetivos do presente estudo foram verificar os níveis plasmáticos de vitami-nas D e E em uma amostra de crianças e adolescentes com colestase crônica; verificar o esta-do nutricional e a ingestão de macro e micronutrientes desses pacientes; verificar o uso de su-plemento de vitaminas, o tempo de colestase; e realizar avaliação neurológica para estabelecer eventual relação com os níveis plasmáticos de vitamina E. A amostra constou de 22 crianças e adolescentes com colestase crônica que con-sultavam no ambulatório ou estiveram internadas na Unidade de Gastroenterologia Pediátrica do Hospital de Clínicas de Porto Alegre no período de dezembro de 2000 a abril de 2002. Como controles, participaram 17 crianças eutróficas e normais do ponto de vista gastroentero-lógico com faixa etária correspondente. Foram realizadas avaliação nutricional e avaliação neurológica. Foi pesquisado o tempo de colestase e o uso de suplemento de vitaminas lipossolúveis. A técnica utilizada para as dosagens da vitamina E foi a cromatografia líquida de alta precisão (HPLC) e as dosagens plasmáticas de vitamina D pela técnica de radioimunoensaio. A prevalência de desnutrição variou entre 23,8% a 63,0% considerando as diferentes medidas e padrões utilizados. O inquérito alimentar realizado demonstrou uma ingestão calórica média de 89,33 ± 27,4% em relação ao recomendado para idade com uma distribui-ção dos macronutrientes em relação às calorias ingeridas dentro dos valores de referência para o grupo em questão, havendo, porém, uma pobre ingestão de micronutrientes como ferro e zinco. O exame neurológico foi alterado em 43% dos pacientes colestáticos, em que foram constatadas vinte alterações neurológicas em nove pacientes. Não obtivemos resultados con-fiáveis para os níveis plasmáticos de vitamina E, apesar de realizar 3 etapas para validação. O valor médio de vitamina D entre os pacientes foi de 13,7 ± 8,39 ng/ml, enquanto que no grupo controle foi de 25,58 ± 16,73 ng/ml (P = 0,007), havendo uma prevalência de hipovitaminose D entre esses pacientes de 36%. Não foi observada relação entre estado nutricional, tempo de colestase ou uso de suplemento oral de vitaminas lipossolúveis e os níveis plasmáticos refe-ridos. Concluímos que a média de níveis plasmáticos de vitamina D nas crianças e nos adolescentes colestáticos do estudo foi significativamente menor do que nos controles nor-mais sem relação significativa com estado nutricional, tempo de colestase ou uso de suple-mento de vitaminas. As alterações neurológicas foram freqüentes e a prevalência de desnutri-ção nos pacientes foi semelhante à encontrada na literatura. A ingesta calórica foi deficiente havendo porém, um equilíbrio dos macronutrientes e ingestão insuficiente de ferro e zinco.

Helicobacter pylori and EBV in gastric carcinomas: Methylation status and microsatellite instability

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Upper gastrointestinal histopathological findings in children and adolescents with nonulcer dyspepsia with helicobacter pylori infection

Objectives: The aim of the study was to investigate the histopathological lesions in the upper gastrointestinal mucosa associated with Helicobacter pylori infection in children with nonulcer dyspepsia.Methods: A cross-sectional case-control study was performed on 185 Brazilian children and adolescents (4-17 years, mean 9.5 +/- 2.7 years), 63.2% girls, submitted to upper gastrointestinal endoscopy. The histopathological lesions of the esophageal and gastric mucosa were analyzed in biopsy samples.Results: H pylori infection was identified in 96 children (51.8%). Moderate to severe chronic active gastritis was present in antrum (70.5%) and corpus (45.2%), with higher grading in antrum than in corpus (P<0.05). The topographic distribution of inflammation was pangastritis (61.9%), followed by antral (32.1%) and corpus (5.9%). H pylori density was higher in antrum than in corpus. Intestinal metaplasia was not found in the H pylori-infected group, nor was significant gastric atrophy. The scores for esophagitis were significantly higher (P<0.05) in the noninfected group (1.4 +/- 0.8) than in the H pylori-infected group (1.07 +/- 0.9), with significant negative correlation (r = 0.29; P<0.05) with the scores of gastric inflammation.Conclusions: The prevalence of H pylori infection was high among children with dyspepsia and associated with moderate/severe degrees of gastric inflammation. The high scores of esophagitis in the noninfected group point to 2 distinct groups of pathological conditions sharing similar clinical patterns.

A novel biomagnetic approach to study caecocolonic motility in humans

Motility patterns play a major role in human colonic functions; however, its physiological significance is poorly understood. Several studies have been introducing the Alternating Current Biosusceptometry (ACB) as a valuable tool in gastroenterology and pharmaceutical research. Using gold standard techniques, great effort has been made to validate ACB as a method for measuring gastrointestinal motility in humans and animals. The aim of this study was to evaluate caecocolonic motility and its response to a meal in healthy volunteers. The results showed a dominant frequency of 3.17 +/- 0.13 cycles per minute (mean +/- SD) that remained unchanged even after a standardized meal (P > 0.01). The colonic response to a meal was recorded as a considerable increase in amplitude, reflected by motility index (P < 0.01) and was observed for all the volunteers. The caecocolonic motility could be assessed by the ACB providing new insights into physiological patterns of motility. Moreover, the method is non-invasive, radiation-free, cost-effective and independent of bowel preparation.

Effect of Helicobacter pylori infection on IL-8, IL-1 and COX-2 expression in patients with chronic gastritis and gastric cancer

Objective. Helicobacter pylori infection is related to gastric cancer development, and chronic inflammation is presumed to be the main cause. The aim of the present study was to evaluate the influence of H. pylori cagA, vacA, iceA, and babA genotypes on COX-2, IL-1, and IL-8 expression. Material and methods. of the 217 patients included in the study, 26 were uninfected, 127 had chronic gastritis and were H. pylori-positive, and 64 had gastric cancer. Bacterial genotypes were evaluated by polymerase chain reaction (PCR), and the expression values were determined by quantitative real-time PCR and immunohistochemistry. Results. An association was found between the infection with cagA, vacA s1m1 strains and gastric cancer development. Regarding the 3' region of the cagA gene, we also found an association between the infection with cagA EPIYA-ABCCC strains and clinical outcome. Higher levels of IL-8, IL-1, and COX-2 were detected in gastric mucosa from infected patients with chronic gastritis, and they were also associated with the infection by cagA, vacA s1m1 strains. The IL-8 and IL-1 levels decrease significantly from chronic gastritis to gastric cancer, while the relative expression remained unaltered when COX-2 expression was analyzed among patients with gastritis and cancer. Conclusions. Since inflammatory response to H. pylori infection plays an important role in cellular proliferation and gastric mucosal damage, the up-regulation of IL-1, IL-8, and COX-2 in patients with chronic gastritis has an important clinical implication in gastric carcinogenesis.

Recalled and recorded bowel habits confirm early onset and high frequency of constipation in day-care nursery children

CONTEXTO: Mães relatam início precoce de constipação em crianças atendidas em clínicas de gastroenterologia. OBJETIVOS: Estudar o hábito intestinal em crianças de baixa idade na comunidade, para avaliar se início precoce da constipação é confirmado neste contexto e se há concordância entre o hábito intestinal relatado e o registrado prospectivamente. MÉTODOS: Obtiveram-se dados sobre evacuações de 57 crianças com idade 6.0-40.7 meses, mediante relato materno (questionário sobre características fecais predominantes) e, a seguir, foram registradas 1.934 evacuações em casa e na creche. O hábito intestinal foi classificado como adequado, constipação, diarréia funcional, outro hábito intestinal, conforme frequência evacuatória e proporção das características fecais (macias, duras e/ou que escorrem). Usaram-se dois critérios para classificar o hábito intestinal registrado, devido indefinição no ponto de corte para fezes duras na identificação de constipação em crianças: critério predominante e critério adulto, respectivamente com >50% e >25% de evacuações com consistência alterada. Usou-se estatística não-paramétrica e, para concordância entre hábito intestinal relatado e registrado, o índice Kappa. RESULTADOS: Constipação ocorreu em 17.5%, 10.5%, 19.3% das crianças, respectivamente pelo relato e pelo registro segundo critérios predominante e adulto. Constipação foi o hábito intestinal mais frequentemente relatado, versus 12.3% outro hábito intestinal. Só uma criança se classificou como tendo diarréia funcional (pelo critério adulto). Concordância entre o hábito intestinal relatado e o registrado foi razoável (fair) para constipação, pelos critérios predominante e adulto (K=0.28 e 0.24, respectivamente), mas apenas leve (slight) para os demais hábito intestinal (K <0.16). Entretanto, dados individuais indicaram melhor relação entre relato de constipação com o critério adulto do que com o critério predominante. CONCLUSÕES: Confirmou-se que constipação é frequente e de início precoce. Concordância razoável entre o hábito intestinal relatado e o registrado segundo os dois critérios indica que o relato materno é razoavelmente confiável para detectar constipação.

Prospective Evaluation of Dietary Treatment in Childhood Constipation: High Dietary Fiber and Wheat Bran Intake Are Associated With Constipation Amelioration

Objectives: The aim of the study was to evaluate, over 24 months, the intake of dietary fiber (DF) and the bowel habit (BH) of constipated children advised a DF-rich diet containing wheat bran.Patients and Methods: BH and dietary data of 28 children with functional constipation defined by the Boston criteria were obtained at visit 1 (V1, n = 28) and at 4 follow-up visits (V2-V5, n = 80). At each visit the BH was rated BAD (worse/unaltered; improved but still complications) or RECOVERY (REC) (improved, no complications; asymptomatic), and a food intake questionnaire was applied. DF intake was calculated according to age (year)+5 to 10 g/day and bran intake according to international tables. Nonparametric statistics were used.Results: Median age (range) was 7.25 years (0.25-15.6 years); 21 children underwent bowel washout (most before V1/V2), and 14 had the last visit at V3/V4. DF intake, bran intake, and the BH rate significantly increased at V2 and remained higher than at V1 through V2 to V5. At V1, median DF intake was 29.9% below the minimum recommended and at the last visit 49.9% above it. Twenty-four children accepted bran at 60 visits, at which median bran intake was 20 g/day and median proportion of DF due to bran 26.9%. Children had significantly higher DF and higher bran intake at V2 to V5 at which they had REC than at those at which they presented BAD BH. DF intake > age+10 g/day was associated with bran acceptance and REC. At the last visit 21 children presented REC (75%); 20 of them were asymptomatic and 18 were off washout/laxatives.Conclusions: High DF and bran intake are feasible in constipated children and contribute to amelioration of constipation.