Altered insulin secretion dynamics relate to oxidative stress and inflammasome activation in children with obesity and insulin resistance.

BACKGROUND: Insulin resistance (IR) is considered the main driver of obesity related metabolic complications, and is related to oxidative stress and inflammation, which in turn promote each other. There is currently no specific definition of IR in children, rather, that for adult population is used by pediatric endocrinologists instead. Altered insulin secretion dynamics are associated with worse metabolic profiles and type 2 diabetes mellitus development, thus we aimed to test whether insulin response relates to oxidative stress and inflammation in children. METHODS: We conducted a case-control study, including 132 children classified as follows: 33 children without obesity (Lean); 42 with obesity but no IR according to the American Diabetes Association criteria for adults (OBIR-); 25 with obesity and IR and an early insulin response to an oral glucose tolerance test (OGTT) (EP-OBIR +); 32 with obesity, IR, and a late insulin peak (LP-OBIR +); and studied variables associated with lipid and carbohydrate metabolism, oxidative stress, inflammation and inflammasome activation. RESULTS: The measured parameters of children with obesity, IR, and an early insulin response were similar to those of children with obesity but without IR. It was late responders who presented an impaired antioxidant system and elevated oxidative damage in erythrocytes and plasma, and inflammasome activation at their white blood cells, despite lower classical inflammation markers. Increased uric acid levels seems to be one of the underlying mechanisms for inflammasome activation. CONCLUSIONS: It is insulin response to an OGTT that identifies children with obesity suffering oxidative stress and inflammasome activation more specifically. Uric acid could be mediating this pathological inflammatory response by activating NLRP3 in peripheral blood mononuclear cells.

Toxicity Induced by Cytokines, Glucose, and Lipids Increase Apoptosis and Hamper Insulin Secretion in the 1.1E7 Beta Cell-Line.

Basic research on types 1 and 2 diabetes mellitus require early stage studies using beta cells or cell lines, ideally of human origin and with preserved insulin secretion in response to glucose. The 1.1E7 cells are a hybrid cell line resulting from the electrofusion of dispersed human islets and PANC-1 cells, capable of secreting insulin in response to glucose, but their survival and function under toxic conditions remains untested. This characterization is the purpose of the present study. We treated these cells with a cytokine mix, high glucose, palmitate, and the latter two combined. Under these conditions, we measured cell viability and apoptosis (MTT, Caspase Glo and TUNEL assays, as well as caspase-8 and -9 levels by Western blotting), endoplasmic reticulum stress markers (EIF2AK3, HSPA4, EIF2a, and HSPA5) by real-time PCR, and insulin secretion with a glucose challenge. All of these stimuli (i) induce apoptosis and ER stress markers expression, (ii) reduce mRNA amounts of 2-5 components of genes involved in the insulin secretory pathway, and (iii) abrogate the insulin release capability of 1.1E7 cells in response to glucose. The most pronounced effects were observed with cytokines and with palmitate and high glucose combined. This characterization may well serve as the starting point for those choosing this cell line for future basic research on certain aspects of diabetes.

Iron Metabolism in Obesity and Metabolic Syndrome.

Obesity is an excessive adipose tissue accumulation that may have detrimental effects on health. Particularly, childhood obesity has become one of the main public health problems in the 21st century, since its prevalence has widely increased in recent years. Childhood obesity is intimately related to the development of several comorbidities such as nonalcoholic fatty liver disease, dyslipidemia, type 2 diabetes mellitus, non-congenital cardiovascular disease, chronic inflammation and anemia, among others. Within this tangled interplay between these comorbidities and associated pathological conditions, obesity has been closely linked to important perturbations in iron metabolism. Iron is the second most abundant metal on Earth, but its bioavailability is hampered by its ability to form highly insoluble oxides, with iron deficiency being the most common nutritional disorder. Although every living organism requires iron, it may also cause toxic oxygen damage by generating oxygen free radicals through the Fenton reaction. Thus, iron homeostasis and metabolism must be tightly regulated in humans at every level (i.e., absorption, storage, transport, recycling). Dysregulation of any step involved in iron metabolism may lead to iron deficiencies and, eventually, to the anemic state related to obesity. In this review article, we summarize the existent evidence on the role of the most recently described components of iron metabolism and their alterations in obesity.

Plant catalases as NO and H2S targets.

Catalase is a powerful antioxidant metalloenzyme located in peroxisomes which also plays a central role in signaling processes under physiological and adverse situations. Whereas animals contain a single catalase gene, in plants this enzyme is encoded by a multigene family providing multiple isoenzymes whose number varies depending on the species, and their expression is regulated according to their tissue/organ distribution and the environmental conditions. This enzyme can be modulated by reactive oxygen and nitrogen species (ROS/RNS) as well as by hydrogen sulfide (H2S). Catalase is the major protein undergoing Tyr-nitration [post-translational modification (PTM) promoted by RNS] during fruit ripening, but the enzyme from diverse sources is also susceptible to undergo other activity-modifying PTMs. Data on S-nitrosation and persulfidation of catalase from different plant origins are given and compared here with results from obese children where S-nitrosation of catalase occurs. The cysteine residues prone to be S-nitrosated in catalase from plants and from bovine liver have been identified. These evidences assign to peroxisomes a crucial statement in the signaling crossroads among relevant molecules (NO and H2S), since catalase is allocated in these organelles. This review depicts a scenario where the regulation of catalase through PTMs, especially S-nitrosation and persulfidation, is highlighted.

Higher levels of serum uric acid influences hepatic damage in patients with non-alcoholic fatty liver disease (NAFLD)

Background: recent evidence suggests a causal link between serum uric acid and the metabolic syndrome, diabetes mellitus, arterial hypertension, and renal and cardiac disease. Uric acid is an endogenous danger signal and activator of the inflammasome, and has been independently associated with an increased risk of cirrhosis. Aim and methods: six hundred and thirty-four patients from the nation-wide HEPAMET registry with biopsy-proven NAFLD (53% NASH) were analyzed to determine whether hyperuricemia is related with advanced liver damage in patients with non-alcoholic fatty liver disease (NAFLD). Patients were divided into three groups according to the tertile levels of serum uric acid and gender. Results: the cohort was composed of 50% females, with a mean age of 49 years (range 19-80). Patients in the top third of serum uric acid levels were older (p = 0.017); they had a higher body mass index (p < 0.01), arterial blood pressure (p = 0.05), triglyceridemia (p = 0.012), serum creatinine (p < 0.001) and total cholesterol (p = 0.016) and lower HDL-cholesterol (p = 0.004). According to the univariate analysis, the variables associated with patients in the top third were more advanced steatosis (p = 0.02), liver fibrosis (F2-F4 vs F0-1; p = 0.011), NASH (p = 0.002) and NAS score (p = 0.05). According to the multivariate logistic regression analysis, the top third of uric acid level was independently associated with steatosis (adjusted hazard ratio 1.7; CI 95%: 1.05-2.8) and NASH (adjusted hazard ratio 1.8; CI 95%: 1.08-3.0) but not with advanced fibrosis (F2-F4) (adjusted hazard ratio 1.09; CI 95%: 0.63-1.87). Conclusion: higher levels of serum uric acid were independently associated with hepatocellular steatosis and NASH in a cohort of patients with NAFLD. Serum uric acid levels warrants further evaluation as a component of the current non-invasive NAFLD scores of histopathological damage

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Higher levels of serum uric acid influences hepatic damage in patients with non-alcoholic fatty liver disease (NAFLD).

BACKGROUND: recent evidence suggests a causal link between serum uric acid and the metabolic syndrome, diabetes mellitus, arterial hypertension, and renal and cardiac disease. Uric acid is an endogenous danger signal and activator of the inflammasome, and has been independently associated with an increased risk of cirrhosis. AIM AND METHODS: six hundred and thirty-four patients from the nation-wide HEPAMET registry with biopsy-proven NAFLD (53% NASH) were analyzed to determine whether hyperuricemia is related with advanced liver damage in patients with non-alcoholic fatty liver disease (NAFLD). Patients were divided into three groups according to the tertile levels of serum uric acid and gender. RESULTS: the cohort was composed of 50% females, with a mean age of 49 years (range 19-80). Patients in the top third of serum uric acid levels were older (p = 0.017); they had a higher body mass index (p < 0.01), arterial blood pressure (p = 0.05), triglyceridemia (p = 0.012), serum creatinine (p < 0.001) and total cholesterol (p = 0.016) and lower HDL-cholesterol (p = 0.004). According to the univariate analysis, the variables associated with patients in the top third were more advanced steatosis (p = 0.02), liver fibrosis (F2-F4 vs F0-1; p = 0.011), NASH (p = 0.002) and NAS score (p = 0.05). According to the multivariate logistic regression analysis, the top third of uric acid level was independently associated with steatosis (adjusted hazard ratio 1.7; CI 95%: 1.05-2.8) and NASH (adjusted hazard ratio 1.8; CI 95%: 1.08-3.0) but not with advanced fibrosis (F2-F4) (adjusted hazard ratio 1.09; CI 95%: 0.63-1.87). CONCLUSION: higher levels of serum uric acid were independently associated with hepatocellular steatosis and NASH in a cohort of patients with NAFLD. Serum uric acid levels warrants further evaluation as a component of the current non-invasive NAFLD scores of histopathological damage.

Obesity induced alterations in redox homeostasis and oxidative stress are present from an early age.

OBJECTIVES: Oxidative stress and inflammation have been postulated as underlying mechanisms for the development of obesity-related insulin resistance. This association however, remains elusive especially in childhood. We sought to investigate this relation by measuring oxidative stress and antioxidant response biomarkers, before and during an oral glucose tolerance test (OGTT), in different biological samples from obese children. SUBJECTS: 24 children were recruited for the study, (18 obese and 6 controls). After OGTT, the obese group was subdivided in two, according to whether or not carbohydrate metabolic impairment (Ob.IR+, Ob.IR-; respectively) was found. Different biomarkers were analyzed after fasting (T = 0) and during an OGTT (T = 60 and 120 min). Lipoperoxides were measured in plasma, erythrocytes, and urine; while advanced glycation end products were determined in plasma, and redox status (GSH/GSSG ratio) in erythrocytes. RESULTS: We found marked differences in the characterization of the oxidative status in urine and erythrocytes, and in the dynamics of the antioxidant response during OGTT. Specifically, Ob.IR+ children show increased oxidative stress, deficient antioxidant response and a significant imbalance in redox status, in comparison to controls and Ob.IR- children. CONCLUSION: Obese children with insulin resistance show increased levels of oxidative stress biomarkers, and a stunted antioxidant response to an OGTT leading to increased oxidative stress after a single glucose load, as detected in erythrocytes, but not in plasma. We propose erythrocytes as sensors of early and acute changes in oxidative stress associated with insulin resistance in childhood obesity. This is a pilot study, performed with a limited sample size, so data should be interpreted with caution until reproduced.

Gut-liver axis, cirrhosis and portal hypertension: the chicken and the egg.

The term gut-liver axis is used to highlight the close anatomical and functional relationship between the intestine and the liver. The intestine has a highly specialized epithelial membrane which regulates transport across the mucosa. Due to dysbiosis, impairment of the intestinal barrier and altered immunity status, bacterial products can reach the liver through the portal vein, where they are recognized by specific receptors, activate the immune system and lead to a proinflammatory response. Gut microbiota and bacterial translocation play an important role in the pathogenesis of chronic liver diseases, including alcoholic and non-alcoholic fatty liver disease, cirrhosis, and its complications, such as portal hypertension, spontaneous bacterial peritonitis and hepatic encephalopaty. The gut microbiota also plays a critical role as a modulator of bile acid metabolism which can also influence intestinal permeability and portal hypertension through the farnesoid-X receptor. On the other hand, cirrhosis and portal hypertension affect the microbiota and increase translocation, leading to a "chicken and egg" situation, where translocation increases portal pressure, and vice versa. A myriad of therapies targeting gut microbiota have been evaluated specifically in patients with chronic liver disease. Further studies targeting intestinal microbiota and its possible hemodynamic and metabolic effects are needed. This review summarizes the current knowledge about the role of gut microbiota in the pathogenesis of chronic liver diseases and portal hypertension.

Characterization of the galactono-1,4-lactone dehydrogenase from pepper fruits and its modulation in the ascorbate biosynthesis. Role of nitric oxide.

Pepper fruit is one of the highest vitamin C sources of plant origin for our diet. In plants, ascorbic acid is mainly synthesized through the L-galactose pathway, being the L-galactono-1,4-lactone dehydrogenase (GalLDH) the last step. Using pepper fruits, the full GalLDH gene was cloned and the protein molecular characterization accomplished. GalLDH protein sequence (586 residues) showed a 37 amino acids signal peptide at the N-terminus, characteristic of mitochondria. The hydrophobic analysis of the mature protein displayed one transmembrane helix comprising 20 amino acids at the N-terminus. By using a polyclonal antibody raised against a GalLDH internal sequence and immunoblotting analysis, a 56kDa polypeptide cross-reacted with pepper fruit samples. Using leaves, flowers, stems and fruits, the expression of GalLDH by qRT-PCR and the enzyme activity were analyzed, and results indicate that GalLDH is a key player in the physiology of pepper plants, being possibly involved in the processes which undertake the transport of ascorbate among different organs. We also report that an NO (nitric oxide)-enriched atmosphere enhanced ascorbate content in pepper fruits about 40% parallel to increased GalLDH gene expression and enzyme activity. This is the first report on the stimulating effect of NO treatment on the vitamin C concentration in plants. Accordingly, the modulation by NO of GalLDH was addressed. In vitro enzymatic assays of GalLDH were performed in the presence of SIN-1 (peroxynitrite donor) and S-nitrosoglutahione (NO donor). Combined results of in vivo NO treatment and in vitro assays showed that NO provoked the regulation of GalLDH at transcriptional and post-transcriptional levels, but not post-translational modifications through nitration or S-nitrosylation events promoted by reactive nitrogen species (RNS) took place. These results suggest that this modulation point of the ascorbate biosynthesis could be potentially used for biotechnological purposes to increase the vitamin C levels in pepper fruits.

Ghrelin mitigates ß-cell mass loss during insulitis in an animal model of autoimmune diabetes mellitus, the BioBreeding/Worcester rat.

BACKGROUND: Ghrelin is a peptide hormone with pleiotropic effects. It stimulates cell proliferation and inhibits apoptosis-mediated cell death. It prevents diabetes mellitus in several models of chemical, surgical and biological toxic insults to pancreas in both in vivo and in vitro models and promotes glucose-stimulated insulin secretion under cytotoxic conditions. It has not yet been tested in vivo in an autoimmune model of diabetes with a persistent insult to the ß-cell. Given the immunomodulating effects of ghrelin and its trophic effects on ß-cells, we hypothesized that ghrelin treatment during the early stages of insulitis would delay diabetes onset. METHODS: BioBreeding/Worcester male rats received ghrelin (10 ng/kg/day) before insulitis development. Glucose metabolism was characterized by glucose and insulin tolerance tests. ß-cell mass, islet area, islet number, ß-cell clusters, proliferation and apoptosis and degree of insulitis were analysed by histomorphometry. A Kaplan-Meier survival curve was plotted and analysed applying the log-rank (Mantel-Cox) test. RESULTS: Ghrelin treatment significantly reduced the probability of developing diabetes in our model (p < 0.0001). It decreased islet infiltration and partially prevented ß-cell mass loss, enabling the maintenance of ß-cell neogenesis and proliferation rates. Furthermore, ghrelin treatment did not induce any metabolic perturbations. CONCLUSIONS: These findings support the hypothesis that ghrelin delays the development of autoimmune diabetes by attenuating insulitis and supporting ß-cell mass. GENERAL SIGNIFICANCE: Ghrelin promotes ß-cell viability and function through diverse mechanisms that may have significant implications for diabetes prevention, therapy and also transplant success of both islets and complete pancreas. Copyright © 2016 John Wiley & Sons, Ltd.

¿Es posible suspender el tratamiento con antivirales orales en los pacientes con hepatitis crónica B antígeno e negativa? Experiencia y nuevas expectativas / Is it possible to stop treatment with nucleos(t)ide analogs in patients with e-antigen negative chronic hepatitis B? Experience and new expectations

INTRODUCCIÓN: El tratamiento de la hepatitis crónica B antígeno e negativa (HCB HBeAg negativa) con antivíricos orales (AO) suele prolongarse de forma indefinida debido a que la pérdida del antígeno de superficie como objetivo para su suspensión es un hecho infrecuente. Recientemente han aparecido las primeras evidencias que sugieren finalizar la terapia con AO en casos seleccionados. OBJETIVOS: Analizar la tasa de rebote virológico en pacientes con HCB Age negativa que suspendieron el tratamiento con AO. MATERIAL Y MÉTODOS: Estudio retrospectivo observacional que incluyó 140 casos de HCB HBeAg negativa. Veintidós pacientes, que recibieron exclusivamente AO, los suspendieron por diversos motivos realizándose un seguimiento posterior. Todos presentaban transaminasas normales, ADN indetectable y ausencia de cirrosis o comorbilidades importantes al finalizar el tratamiento. RESULTADOS: Doce pacientes presentaron rebote virológico (54,54%), transcurriendo una media de 6,38 meses (± 1,9) desde la suspensión hasta el rebote (el 75% dentro de los 12 primeros meses tras la suspensión). Cinco recibieron adefovir, uno lamivudina más adefovir, uno tenofovir y 5 lamivudina. La duración media del tratamiento, desde el inicio hasta la suspensión, fue de 38,5 meses (± 4,5). El grupo con respuesta sostenida presentaba una edad media y duración del tratamiento superior a los sujetos con rebote, si bien estas diferencias no resultaron estadísticamente significativas. CONCLUSIONES: Los resultados sugieren que es posible suspender la terapia con AO en casos seleccionados de HCB Age negativa, siempre que no exista cirrosis, se cumpla un tiempo mínimo de tratamiento, las transaminasas sean normales y el ADN indetectable de forma mantenida. En estos casos, se debe realizar un seguimiento estrecho durante el primer año y posteriormente de forma indefinida

BACKGROUND: Treatment of HBeAg-negative chronic hepatitis B (CHB) with nucleos(t)ide analogues (NA) is usually indefinite, since the loss of HBsAg, as a criterion for its discontinuation, is a rare event. Recent evidence suggests that discontinuing NA therapy may be feasible in selected patients. OBJECTIVES: To analyze the rate of virological relapse in patients with HBeAg-negative CHB who discontinued treatment with NAs. METHODS: We performed a single-center observational study that included 140 patients with HBsAg-negative CHB. Twenty-two patients, who received only NAs, discontinued treatment for different reasons and were subsequently monitored. All had normal ALT and AST, undetectable DNA and absence of cirrhosis or significant comorbidities before stopping treatment. RESULTS: Twelve patients showed virologic relapse (54.54%). The mean interval between discontinuation and relapse was 6.38 months (± 1.9) (75% relapsed during the first 12 months after discontinuation). Five received adefovir, 1 lamivudine and adefovir, 1 tenofovir and 5 lamivudine alone. The mean treatment duration in this group was 38.5 months (± 4.5). The sustained response group had a higher mean age and longer treatment duration than patients with virologic relapse but these differences were not statistically significant. CONCLUSIONS: The results suggest that NA treatment can be stopped in selected patients with CHB as long as they are not cirrhotic, have completed a minimum period of treatment, have normal ALT and sustained undetectable DNA. These patients should be closely monitored during the first year and then indefinitely

[Is it possible to stop treatment with nucleos(t)ide analogs in patients with e-antigen negative chronic hepatitis B? Experience and new expectations]. / ¿Es posible suspender el tratamiento con antivirales orales en los pacientes con hepatitis crónica B antígeno e negativa? Experiencia y nuevas expectativas.

BACKGROUND: Treatment of HBeAg-negative chronic hepatitis B (CHB) with nucleos(t)ide analogues (NA) is usually indefinite, since the loss of HBsAg, as a criterion for its discontinuation, is a rare event. Recent evidence suggests that discontinuing NA therapy may be feasible in selected patients. OBJECTIVES: To analyze the rate of virological relapse in patients with HBeAg-negative CHB who discontinued treatment with NAs. METHODS: We performed a single-center observational study that included 140 patients with HBsAg-negative CHB. Twenty-two patients, who received only NAs, discontinued treatment for different reasons and were subsequently monitored. All had normal ALT and AST, undetectable DNA and absence of cirrhosis or significant comorbidities before stopping treatment. RESULTS: Twelve patients showed virologic relapse (54.54%). The mean interval between discontinuation and relapse was 6.38 months (± 1.9) (75% relapsed during the first 12 months after discontinuation). Five received adefovir, 1 lamivudine and adefovir, 1 tenofovir and 5 lamivudine alone. The mean treatment duration in this group was 38.5 months (± 4.5). The sustained response group had a higher mean age and longer treatment duration than patients with virologic relapse but these differences were not statistically significant. CONCLUSIONS: The results suggest that NA treatment can be stopped in selected patients with CHB as long as they are not cirrhotic, have completed a minimum period of treatment, have normal ALT and sustained undetectable DNA. These patients should be closely monitored during the first year and then indefinitely.

Paniculitis mesentérica como causa de dolor abdominal crónico / Mesenteric panniculitis as a cause of chronic abdominal pain

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Syntaxin-4 is implicated in the secretion of antibodies by human plasma cells.

PCs are responsible for the production and secretion of antibodies, the effector molecules of the humoral immune response. The molecular mechanisms responsible for vesicle docking and secretion implicated in the antibody-secretion process are not well-known, as they have not been studied, but it is known that SNARE proteins are responsible for many membrane-fusion processes in the cell. We show here that freshly isolated human colon LP-PCs and T-PCs from MM-PC patients and the U266 cell line, as a model for PC secretion, contain a set of these proteins. SNAP23, STX3, and STX4 were localized mainly in the plasma membrane of PCs, and interactions of SNAP23 with STX3 and with STX4 were proven by IP. Interaction between SNAP23 and STX4 was also confirmed in situ. With the use of siRNA, as well as shRNA, the functional role of SNAP23, STX3, and STX4 in antibody secretion was also examined. The findings demonstrate that in addition to SNAP23, STX4 is implicated in the antibody secretion by a myeloma cell line and by normal human colon LP-PCs.

Antioxidant systems from Pepper (Capsicum annuum L.): involvement in the response to temperature changes in ripe fruits.

Sweet pepper is susceptible to changes in the environmental conditions, especially temperatures below 15 °C. In this work, two sets of pepper fruits (Capsicum annuum L.) which underwent distinct temperature profiles in planta were investigated. Accordingly, two harvesting times corresponding to each set were established: Harvest 1, whose fruits developed and ripened at 14.9 °C as average temperature; and Harvest 2, with average temperature of 12.4 °C. The oxidative metabolism was analyzed in all fruits. Although total ascorbate content did not vary between Harvests, a shift from the reduced to the oxidized form (dehydroascorbate), accompanied by a higher ascorbate peroxidase activity, was observed in Harvest 2 with respect to Harvest 1. Moreover, a decrease of the ascorbate-generating enzymatic system, the γ-galactono-lactone dehydrogenase, was found at Harvest 2. The activity values of the NADP-dependent dehydrogenases analyzed seem to indicate that a lower NADPH synthesis may occur in fruits which underwent lower temperature conditions. In spite of the important changes observed in the oxidative metabolism in fruits subjected to lower temperature, no oxidative stress appears to occur, as indicated by the lipid peroxidation and protein oxidation profiles. Thus, the antioxidative systems of pepper fruits seem to be involved in the response against temperature changes.