Acute and chronic effects of the organophosphate malathion on the pancreatic α and ß cell viability, cell structure, and voltage-gated K+ currents.

Studies indicate that the pesticide malathion may have a role in diabetes. Herein, we determined the effects of different concentrations of malathion on survival, ultrastructure, and electrophysiologic islet cell parameters. Acutely, high concentrations of malathion (0.5 or 1 mM) increased cell death in rat islet cells, while low concentrations (0.1 mM) caused signs of cell damage in pancreatic α and ß cells. Exposure of RINm5F cells to malathion for 24 or 48 h confirmed the reduction in ß-cell viability at lower concentrations (0.001-100 µM). Chronic exposure of mouse pancreatic α and ß cells to 3 nM of malathion led to increased voltage-gated K+ (Kv) currents in α-cells. Our findings show a time and concentration dependency for the malathion effect on the reduction of islet cell viability and indicate that pancreatic α cells are more sensitive to malathion effects on Kv currents and cell death.

Role of the clock gene Rev-erbα in metabolism and in the endocrine pancreas.

Several hormones are regulated by circadian rhythms to adjust the metabolism to the light/dark cycles and feeding/activity patterns throughout the day. Circadian rhythms are mainly governed by the central clock located in the suprachiasmatic nucleus but also by clocks present in peripheral organs, like the endocrine pancreas. Plasma glucose levels and the main pancreatic hormones insulin and glucagon also exhibit daily variations. Alterations in circadian rhythms are associated with metabolic disturbances and pathologies such as obesity and diabetes. The molecular components of central and peripheral clocks and their regulatory mechanisms are well established. Among the different clock genes, Rev-erbα is considered one of the key links between circadian rhythms and metabolism. Rev-erbα is a critical part of a negative feedback loop in the core circadian clock and modulates the clock oscillatory properties. In addition, Rev-erbα plays an important role in the regulation of lipid and glucose metabolism, thermogenesis, adipocyte and muscle differentiation as well as mitochondrial function. In the endocrine pancreas, Rev-erbα regulates insulin and glucagon secretion and pancreatic ß-cell proliferation. In the present review, we discuss all these subjects and, particularly, the role of the clock gene Rev-erbα in the endocrine pancreas.

Selective inactivation of NADPH oxidase 2 causes regression of vascularization and the size and stability of atherosclerotic plaques.

BACKGROUND: A variety of NADPH oxidase (Nox) isoforms including Noxs 1, 2, 4 and 5 catalyze the formation of reactive oxygen species (ROS) in the vascular wall. The Nox2 isoform complex has arguably received the greatest attention in the progression of atherogenesis in animal models. Thus, in the current study we postulated that specific Nox2 oxidase inhibition could reverse or attenuate atherosclerosis in mice fed a high-fat diet. METHODS: We evaluated the effect of isoform-selective Nox2 assembly inhibitor on the progression and vascularization of atheromatous plaques. Apolipoprotein E-deficient mice (ApoE-/-) were fed a high fat diet for two months and treated over 15 days with Nox2ds-tat or control sequence (scrambled); 10 mg/kg/day, i.p. Mice were sacrificed and superoxide production in arterial tissue was detected by cytochrome C reduction assay and dihydroethidium staining. Plaque development was evaluated and the angiogenic markers VEGF, HIF1-α and visfatin were quantified by real time qRT-PCR. MMP-9 protein release and gelatinolytic activity was determined as a marker for vascularization. RESULTS: Nox2ds-tat inhibited Nox-derived superoxide determined by cytochrome C in carotid arteries (2.3 ± 0.1 vs 1.7 ± 0.1 O2(•-) nmol/min*mg protein; P < 0.01) and caused a significant regression in atherosclerotic plaques in aorta (66 ± 6 µm(2) vs 37 ± 1 µm(2); scrmb vs. Nox2ds-tat; P < 0.001). Increased VEGF, HIF-1α, MMP-9 and visfatin expression in arterial tissue in response to high-fat diet were significantly attenuated by Nox2ds-tat which in turn impaired both MMP-9 protein expression and activity. CONCLUSION: Given these results, it is quite evident that selective Nox inhibitors can reverse vascular pathology arising with atherosclerosis.

Tempol attenuates atherosclerosis associated with metabolic syndrome via decreased vascular inflammation and NADPH-2 oxidase expression.

Oxidative stress is an important factor in the generation of vascular injury in atherosclerosis. Chronic administration of fructose in rodents is able to facilitate oxidative damage. In the present study we evaluated the role of Tempol, a superoxide dismutase mimetic, on the effect of high fructose intake in apolipoprotein E-deficient (ApoE-KO) mice. Rodents were fed with fructose overload (FF, 10% w/v) for 8 weeks and treated with Tempol 1 mg/kg/day the latest 4 weeks. Tempol revert the pro-oxidant effects caused by FF, diminished lipid peroxidation and impaired vascular NADPH oxidase system through the downregulation of p47phox expression in the vascular wall. Tempol inhibited the expression of vascular adhesion molecule 1 (VCAM-1) in aorta and reduced the development of atheroma plaques. Our results indicate that tempol attenuates oxidative stress by interfering with the correct assembly of Nox2 oxidase complex in the vascular wall and is able to reduce atherosclerosis. Thus tempol represents a potential therapeutic target for preventing risk factors associated with metabolic syndrome.

Leptin downregulates expression of the gene encoding glucagon in alphaTC1-9 cells and mouse islets.

AIMS/HYPOTHESIS: Leptin, released by adipocytes, can modulate glucose homeostasis through direct actions on pancreatic alpha and beta cells. Although this hormone rapidly regulates alpha cell exocytosis, its long-term effects on glucagon gene expression are currently unknown. METHODS: We analysed glucagon mRNA levels and protein content in alphaTC1-9 cells and isolated mouse islets cultured with leptin, as well as in islets from mice treated in vivo with leptin. We also studied the involvement of the signal transducers and activators of transcription (STAT) pathway by western blot, immunofluorescence and interference RNA. RESULTS: Leptin incubation (0.0625-18.75 nmol/l) for 24 h inhibited glucagon gene expression in alphaTC1-9 cells. This inhibitory effect was also observed in isolated mouse islets cultured with leptin, as well as in islets from mice treated with leptin for 5 days. In contrast, no changes were detected in islets from db/db mice, which lack leptin receptors. Leptin treatment also reduced the glucagon protein content in alphaTC1-9 cells and mouse islets. Moreover, leptin induced phosphorylation of STAT3 and its translocation to the nucleus, which was confirmed by western blot analysis in alphaTC1-9 cells and by immunofluorescence in isolated alpha cells. Interestingly, the effect of leptin on glucagon mRNA levels was significantly reduced by Stat3 knockdown. In contrast, pharmacological inhibition of the phosphoinositide 3-kinase pathway did not affect leptin actions. CONCLUSIONS/INTERPRETATION: Our results demonstrate that leptin can regulate glucagon gene expression in alpha cells via a STAT3 pathway, and are important for understanding the role of leptin in glucose homeostasis.

Role of iduronate-2-sulfatase in glucose-stimulated insulin secretion by activation of exocytosis.

Iduronate-2-sulfatase (IDS) is a lysosomal enzyme expressed in pancreatic islets responsible for the degradation of proteoglycans such as perlecan and dermatan sulfate. Previous findings of our group demonstrated the involvement of IDS in the normal pathway of lysosomal degradation of secretory peptides, suggesting a role of this enzyme in beta-cell secretory functionality. The present study was undertaken to characterize the effect of IDS overexpression on insulin release. INS1E cells were transiently transfected with a construct encoding human IDS (hIDS). hIDS overexpression was associated with a gain of function detected by a reduction in heparan sulfate content. hIDS potentiated the glucose-stimulated insulin secretory response compared with controls (61%) with no changes in insulin mRNA levels or insulin peptide content. Results on quantification of the exocytotic process showed a significant increase in hIDS-transfected cells compared with controls. Furthermore, ultramorphological analysis demonstrated an increase in the number of granules in the immediate vicinity of the plasma membrane in hIDS-transfected cells and a decrease in total vesicles per square micrometer. hIDS overexpression induced phosphorylation of protein kinase C (PKC) alpha and its newly myristoylated alanine-rich C kinase substrate, MARCKS. We conclude that IDS has a role in glucose-stimulated insulin secretion via a mechanism that involves the activation of exocytosis through phosphorylation of PKCalpha and MARCKS.

Aspectos teóricos en torno a la gestión del conocimiento en la medicina basada en evidencias / Theoretical aspects of knowledge management in evidence-based medicine

Se precisa el peso de los conceptos datos, información y conocimiento como nociones de alcance primordial en el contexto de las tendencias que se imponen en la aplicación de modernos procedimientos en la práctica médica. Se revela la esencia del nuevo paradigma en la asistencia sanitaria, conocido desde el punto de vista conceptual como medicina basada en evidencias, y se enuncian los retos que debe enfrentar el profesional de la información como gestor de esta y de conocimiento, al efecto de su acertada aplicación por parte del profesional de la salud. Se identifican los pasos necesarios para realizar una eficaz gestión del conocimiento y lograr su socialización en este ámbito, así como ciertas particularidades de los sistemas de salud que facilitan dicho proceso. Por último se pasa revista a la medicina basada en evidencias como expresión de la gestión del conocimiento en el área de la salud, donde se demuestra su correspondencia con las exigencias actuales, resultantes del desarrollo actual en materia informacional.

The importance of data, information and knowledge concepts is defined as notions of fundamental scope in the context of the tendencies imposed in the application of modern procedures in medical practice. The essence of the new paradigm in the health care is revealed, known from the conceptual point of view as evidence based medicine, and the challenges faced by the information professional as information and knowledge manager are dealt with, so that the health professional may apply them appropriately. The necessary steps to carry out an efficient knowledge management and to attain its socialization in this setting are identified as well as certain particularities of the health system that facilitate such process. Finally, a review is made of evidence based medicine as an expression of knowledge management in the health field, where its correspondence with the current demands, resulting from the present development in the informational sphere, was proved.

Aspectos teóricos en torno a la gestión del conocimiento en la medicina basada en evidencias / Theoretical aspects of knowledge management in evidence-based medicine

Se precisa el peso de los conceptos datos, información y conocimiento como nociones de alcance primordial en el contexto de las tendencias que se imponen en la aplicación de modernos procedimientos en la práctica médica. Se revela la esencia del nuevo paradigma en la asistencia sanitaria, conocido desde el punto de vista conceptual como medicina basada en evidencias, y se enuncian los retos que debe enfrentar el profesional de la información como gestor de esta y de conocimiento, al efecto de su acertada aplicación por parte del profesional de la salud. Se identifican los pasos necesarios para realizar una eficaz gestión del conocimiento y lograr su socialización en este ámbito, así como ciertas particularidades de los sistemas de salud que facilitan dicho proceso. Por último se pasa revista a la medicina basada en evidencias como expresión de la gestión del conocimiento en el área de la salud, donde se demuestra su correspondencia con las exigencias actuales, resultantes del desarrollo actual en materia informacional(AU)

The importance of data, information and knowledge concepts is defined as notions of fundamental scope in the context of the tendencies imposed in the application of modern procedures in medical practice. The essence of the new paradigm in the health care is revealed, known from the conceptual point of view as evidence based medicine, and the challenges faced by the information professional as information and knowledge manager are dealt with, so that the health professional may apply them appropriately. The necessary steps to carry out an efficient knowledge management and to attain its socialization in this setting are identified as well as certain particularities of the health system that facilitate such process. Finally, a review is made of evidence based medicine as an expression of knowledge management in the health field, where its correspondence with the current demands, resulting from the present development in the informational sphere, was proved(AU)

Intracellular location of KATP channels and sulphonylurea receptors in the pancreatic beta-cell: new targets for oral antidiabetic agents.

Diabetes Mellitus is by far one of the most propagated chronic diseases, affecting 150 million people worldwide. This affliction is caused by a malfunction of pancreatic endocrine cells, which provokes a failure in the insulin release and glucose homeostasis. Plasma membrane K(ATP) channels have a key role in the stimulus-secretion coupling of pancreatic beta-cells. Consequently, many investigations have developed efficient drugs for the treatment of diabetes, such as sulphonylureas, which specifically close K(ATP) channels leading to an enhanced insulin secretion. Recent studies show that, in addition to its well-known plasma membrane location, sulphonylurea receptors and sulphonylurea-sensitive K(ATP) channels are also present in various intracellular sites including secretory granules, mitochondria, endoplasmic reticulum and more recently, the nucleus. What roles do they play in these organelles? Intracellular K(ATP) channels and sulphonylurea receptors, which operate in conjunction with classical pathways, can provide specific signaling circuits to establish direct links between extracellular signals and different cell functions, such as secretion or gene expression. The study of these intracellular channels provides novel perspectives in the signal transduction of the pancreatic beta-cell, and may offer clues for the development of new strategies in diabetes therapy. In this review we will address this topic with special emphasis on the biophysical basis and functional implications in the pancreatic beta-cell.

A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues.

In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, beta-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates.

Mouse mast cell secretory granules can function as intracellular ionic oscillators.

Fluorescent Ca2+ probes and digital photo-sectioning techniques were used to directly study the dynamics of Ca2+ in isolated mast cell granules of normal (CB/J) and beige (Bg(j)/Bg(j)) mice. The resting intraluminal free Ca2+ concentration ([Ca2+]L) is 25 +/- 4.2 microM (mean +/- SD, n = 68). Exposure to 3 microM inositol 1,4,5-trisphosphate (InsP3) induced periodic oscillations of luminal Ca2+ ([Ca2+]L) of approximately 10 microM amplitude and a period around 8-10 s. The [Ca2+]L oscillations were accompanied by a corresponding oscillatory release of [Ca2+]L to the extraluminal space. Control experiments using ruthenium red (2 microM) and thapsigargin (100 nM) ruled out artifacts derived from the eventual presence of mitochondria or endoplasmic reticulum in the isolated granule preparation. Oscillations of [Ca2+]L and Ca2+ release result from a Ca2+/K+ exchange process whereby bound Ca is displaced from the heparin polyanionic matrix by inflow of K+ into the granular lumen via an apamin-sensitive Ca2+-sensitive K+ channel (ASK(Ca)), whereas Ca2+ release takes place via an InsP3-receptor-Ca2+ (InsP3-R) channel. These results are consistent with previous observations of [Ca2+]L oscillations and release in/from the endoplasmic reticulum and mucin granules, and suggest that a highly conserved common mechanism might be responsible for [Ca2+]L oscillations and quantal periodic Ca2+ release in/from intracellular Ca2+ storage compartments.

Nutrient modulation of polarized and sustained submembrane Ca2+ microgradients in mouse pancreatic islet cells.

The intracellular calcium concentration ([Ca2+]i) near the plasma membrane was measured in mouse pancreatic islet cells using confocal spot detection methods. Whereas small cytosolic Ca2+ gradients were observed with 3 mM glucose, a steeper sustained gradient restricted to domains beneath the plasma membrane (space constant, 0.67 micrometer) appeared with 16.7 mM glucose. When the membrane potential was clamped with increasing K+ concentrations (5, 20 and 40 mM), no [Ca2+]i gradients were observed in any case. Increasing glucose concentration (0, 5 and 16.7 mM) in the presence of 100 microM diazoxide, a K+ channel opener, plus 40 mM K+ induced steeper [Ca2+]i gradients, confirming the role of membrane potential-independent effects of glucose. Prevention of Ca2+ store refilling with 30 microM cyclopiazonic acid (CPA) or blockade of uniporter-mediated Ca2+ influx into the mitochondria with 1 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or 1 microM Ru-360 significantly reduced the steepness of the 16.7 mM glucose-induced [Ca2+]i gradients. Measured values of [Ca2+]i reached 6.74 +/- 0.67 microM at a distance of 0.5 micrometer from the plasma membrane and decayed to 0.27 +/- 0.03 microM at a distance of 2 micrometer. Mathematically processed values at 0.25 and 0 micrometer gave a higher [Ca2+]i, reaching 8.18 +/- 0.86 and 10.05 +/- 0.98 microM, respectively. The results presented indicate that glucose metabolism generates [Ca2+]i microgradients, which reach values of around 10 microM, and whose regulation requires the involvement of both mitochondrial Ca2+ uptake and endoplasmic reticulum Ca2+ stores.

Different effects of tolbutamide and diazoxide in alpha, beta-, and delta-cells within intact islets of Langerhans.

Interaction between the different types of cells within the islet of Langerhans is vital for adequate control of insulin release. Once insulin secretion becomes defective, as in type 2 diabetes, the most useful drugs to increase insulin release are sulfonylureas. It is well-known that sulfonylureas block K(ATP) channels, which results in depolarization of the membrane that provokes calcium influx and increases intracellular calcium concentration ([Ca2+]i), which thereby triggers insulin secretion. The sulfonamide diazoxide produces the opposite effect: it activates K(ATP) channels, resulting in a decreased insulin secretion. Despite such evidence, little is known about the effect of sulfonylureas and sulfonamides in non-beta-cells of the islet of Langerhans. In this article, we describe the effects of tolbutamide and diazoxide on [Ca2+]i in alpha-, beta-, and delta-cells within intact islets of Langerhans. Tolbutamide elicits an increase in [Ca2+li in beta- and delta-cells, regardless of glucose concentrations. Remarkably, tolbutamide is without effect in alpha-cells. When diazoxide is applied, glucose-induced [Ca2+]i oscillations in beta- and delta-cells are abolished, whereas [Ca2+]i oscillations in alpha-cells remain unaltered. Furthermore, the existence of sulfonylurea receptors is demonstrated in beta-cells but not in alpha-cells by using binding of glybenclamide-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) combined with immunostaining for insulin and glucagon.

Homologous and heterologous asynchronicity between identified alpha-, beta- and delta-cells within intact islets of Langerhans in the mouse.

1. Using laser scanning confocal microscopy to image [Ca2+]i within intact murine islets of Langerhans, we analysed the [Ca2+]i signals generated by glucose in immunocytochemically identified alpha-, beta- and delta-cells. 2. Glucagon-containing alpha-cells exhibited [Ca2+]i oscillations in the absence of glucose, which petered out when islets were exposed to high glucose concentrations. 3. Somatostatin-containing delta-cells were silent in the absence of glucose but concentrations of glucose as low as 3 mM elicited oscillations. 4. In pancreatic beta-cells, a characteristic oscillatory calcium pattern was evoked when glucose levels were raised from 3 to 11 mM and this was synchronized throughout the beta-cell population. Remarkably, [Ca2+]i oscillations in non-beta-cells were completely asynchronous, both with respect to each other and to beta-cells. 5. These results demonstrate that the islet of Langerhans behaves as a functional syncytium only in terms of beta-cells, implying a pulsatile secretion of insulin. However, the lack of a co-ordinated calcium signal in alpha- and delta-cells implies that each cell acts as an independent functional unit and the concerted activity of these units results in a smoothly graded secretion of glucagon and somatostatin. Understanding the calcium signals underlying glucagon and somatostatin secretion may be of importance in the treatment of non-insulin-dependent diabetes mellitus since both glucagon and somatostatin appear to regulate insulin release in a paracrine fashion.