Dexamethasone induces posttranslational degradation of GLUT2 and inhibition of insulin secretion in isolated pancreatic beta cells. Comparison with the effects of fatty acids.

GLUT2 expression is strongly decreased in glucose-unresponsive pancreatic beta cells of diabetic rodents. This decreased expression is due to circulating factors distinct from insulin or glucose. Here we evaluated the effect of palmitic acid and the synthetic glucocorticoid dexamethasone on GLUT2 expression by in vitro cultured rat pancreatic islets. Palmitic acid induced a 40% decrease in GLUT2 mRNA levels with, however, no consistent effect on protein expression. Dexamethasone, in contrast, had no effect on GLUT2 mRNA, but decreased GLUT2 protein by about 65%. The effect of dexamethasone was more pronounced at high glucose concentrations and was inhibited by the glucocorticoid antagonist RU-486. Biosynthetic labeling experiments revealed that GLUT2 translation rate was only minimally affected by dexamethasone, but that its half-life was decreased by 50%, indicating that glucocorticoids activated a posttranslational degradation mechanism. This degradation mechanism was not affecting all membrane proteins, since the alpha subunit of the Na+/K+-ATPase was unaffected. Glucose-induced insulin secretion was strongly decreased by treatment with palmitic acid and/or dexamethasone. The insulin content was decreased ( approximately 55 percent) in the presence of palmitic acid, but increased ( approximately 180%) in the presence of dexamethasone. We conclude that a combination of elevated fatty acids and glucocorticoids can induce two common features observed in diabetic beta cells, decreased GLUT2 expression, and loss of glucose-induced insulin secretion.

Alterations of bone microarchitecture in young patients with inflammatory bowel diseases are associated with fracture risk during growth

Aims: Inflammatory bowel diseases (IBD) appearing during childhood and adolescence compromise peak bone mass acquisition and increase fracture risk. The structural determinants of bone fragility in IBD however remain unknown. Methods: We investigated volumetric bone mineral density (vBMD), trabecular and cortical bone microstructure at distal radius and tibia by high-resolution pQCT (XtremeCT, Scanco, Switzerland), aBMD at distal radius, hip and spine and vertebral fracture assessment (VFA) by DXA in 107 young patients (mean age 22.8 yrs, range 12.2-33.7 yrs; 62 females and 45 males) with Crohn's disease (n=75), ulcerative colitis (n=25), undetermined colitis (n=2), and no definitive diagnosis (n=5), and in 389 healthy young individuals. Results: Mean disease duration was 6.1 yrs, 89/107 IBD patients received corticosteroids, 83 other immunomodulators, and 59 vitamin D. Clinical fractures were reported by 38 patients (mean age at 1st fracture, 12.6 yrs), the vast majority of the forearm, arm or hand; 5 had vertebral crush fractures (Grade 1 or 2) and 11 had multiple fractures. As compared to healthy controls (matched 2:1 for age, sex, height and fracture history), the 102 patients with established IBD had similar weight but significantly lower aBMD at all sites, lower trabecular (Tb) BV/TV and number, and greater Tb separation and inhomogeneous Tb distribution (1/SD TbN) at both distal radius and tibia, lower tibia cortical thickness (CTh), but no differences in cortical vBMD nor bone perimeter. Among IBD's, aBMD was not associated with fractures (by logistic regression adjusted for age, age square, sex, height, weight and protein intake). However, radius and tibia Tb BV/TV, thickness and SD 1/TbN, as well as radius Tb separation and perimeter, were significantly associated with fracture risk (fully adjusted as above), whereas cortical vBMD and CTh were not. After adjustment for aBMD at radius, respectively at femur neck, radius SD 1/TbN and tibia BV/TV, TbTh and perimeter remained independently associated with fracture risk. Conclusions: Young subjects with IBD have low bone mass and poor bone microarchitecture compared to healthy controls. Alterations of bone microarchitecture, particularly in the trabecular bone compartment, are specifically associated with increased fracture risk during growth.

Pancreatic Trauma in Children

Objectives and Study: To document the demographics, mechanisms and outcome of traumatic pancreatitis in children at a single large tertiary referral centre in Australia. Methods: We undertook a 10-year retrospective audit of children admitted to the Royal Children's Hospital [RCH], Melbourne, Australia with a hospital coded diagnosis which included pancreatic injury between 1993 and 2002. Data included patient demographics, source of admission, mechanism of injury, pancreatic complications, associated injuries, Intensive Care Unit [ICU] admission, results of any operative findings, results of any acute computed tomography (CT) and/or ultrasound (US) imaging of pancreas, selected laboratory findings and length of stay. Results: We identified two distinct groups of patients in the 91 documented cases of pancreatic trauma (median age 8.0 yr, range 0.6-15.8 yr; M:F 2.5:1.0). Fifty-nine had a history of abdominal trauma and elevated serum lipase but no CT or ultrasound evidence of pancreatic injury (Group A). Thirty-two had a history of abdominal trauma, elevated serum lipase but also had CT scan and/or ultrasound evidence of pancreatic injury[Group B]. Patients with ''less severe'' injury based on normal imaging had a lower initial lipase level [Group A, median 651 U/L (interquartile range 520 - 1324) vs, Group B, 1608 U/L (interquartile range 680-3526); P = 0.005] and shorter admission time [Group A, 9.0 days (interquartile range 5.5-15.5) vs Group B, 13.4 days (interquartile range 6.8 - 23.8), P = 0.04]. There were no differences with respect to mortality [Group A, 13.5 % vs Group B, 12.5 %] but patients with evidence of injury on imaging were more likely to have surgical intervention [P = 0.0001]. The single most important overall cause of pancreatic trauma was involvement in a motor vehicle accident as a passenger or pedestrian. However, in children with high-grade ductal injury, bicycle handlebar injuries were most common. Associated injuries were common in both groups. Conclusion: Significant pancreatic injury can occur in the absence of abnormality on medical imaging. Pancreatic trauma commonly occurs in the context of multiple injuries after motor vehicle accidents in children and bicycle handlebar injuries, especially in boys. Most children can be treated conservatively, with surgical intervention being limited to high-grade ductal injury.

Characterization of pancreatic NMDA receptors as possible drug targets for diabetes treatment.

In the nervous system, NMDA receptors (NMDARs) participate in neurotransmission and modulate the viability of neurons. In contrast, little is known about the role of NMDARs in pancreatic islets and the insulin-secreting beta cells whose functional impairment contributes to diabetes mellitus. Here we found that inhibition of NMDARs in mouse and human islets enhanced their glucose-stimulated insulin secretion (GSIS) and survival of islet cells. Further, NMDAR inhibition prolonged the amount of time that glucose-stimulated beta cells spent in a depolarized state with high cytosolic Ca(2+) concentrations. We also noticed that, in vivo, the NMDAR antagonist dextromethorphan (DXM) enhanced glucose tolerance in mice, and that in vitro dextrorphan, the main metabolite of DXM, amplified the stimulatory effect of exendin-4 on GSIS. In a mouse model of type 2 diabetes mellitus (T2DM), long-term treatment with DXM improved islet insulin content, islet cell mass and blood glucose control. Further, in a small clinical trial we found that individuals with T2DM treated with DXM showed enhanced serum insulin concentrations and glucose tolerance. Our data highlight the possibility that antagonists of NMDARs may provide a useful adjunct treatment for diabetes.

Tomosyn-1 is involved in a post-docking event required for pancreatic beta-cell exocytosis.

Although the assembly of a ternary complex between the SNARE proteins syntaxin-1, SNAP25 and VAMP2 is known to be crucial for insulin exocytosis, the mechanisms controlling this key event are poorly understood. We found that pancreatic beta-cells express different isoforms of tomosyn-1, a syntaxin-1-binding protein possessing a SNARE-like motif. Using atomic force microscopy we show that the SNARE-like domain of tomosyn-1 can form a complex with syntaxin-1 and SNAP25 but displays binding forces that are weaker than those observed for VAMP2 (237+/-13 versus 279+/-3 pN). In pancreatic beta-cells tomosyn-1 was found to be concentrated in cellular compartments enriched in insulin-containing secretory granules. Silencing of tomosyn-1 in the rat beta-cell line INS-1E by RNA interference did not affect the number of secretory granules docked at the plasma membrane but led to a reduction in stimulus-induced exocytosis. Replacement of endogenous tomosyn-1 with mouse tomosyn-1, which differs in the nucleotide sequence from its rat homologue and escapes silencing, restored a normal secretory rate. Taken together, our data suggest that tomosyn-1 is involved in a post-docking event that prepares secretory granules for fusion and is necessary to sustain exocytosis of pancreatic beta-cells in response to insulin secretagogues.

Genetic Variations and Diseases in UniProtKB/Swiss-Prot: The Ins and Outs of Expert Manual Curation.

During the last few years, next-generation sequencing (NGS) technologies have accelerated the detection of genetic variants resulting in the rapid discovery of new disease-associated genes. However, the wealth of variation data made available by NGS alone is not sufficient to understand the mechanisms underlying disease pathogenesis and manifestation. Multidisciplinary approaches combining sequence and clinical data with prior biological knowledge are needed to unravel the role of genetic variants in human health and disease. In this context, it is crucial that these data are linked, organized, and made readily available through reliable online resources. The Swiss-Prot section of the Universal Protein Knowledgebase (UniProtKB/Swiss-Prot) provides the scientific community with a collection of information on protein functions, interactions, biological pathways, as well as human genetic diseases and variants, all manually reviewed by experts. In this article, we present an overview of the information content of UniProtKB/Swiss-Prot to show how this knowledgebase can support researchers in the elucidation of the mechanisms leading from a molecular defect to a disease phenotype.

Application of the 2008 definitions for invasive fungal diseases to the trial comparing voriconazole versus amphotericin B for therapy of invasive aspergillosis: a collaborative study of the Mycoses Study Group (MSG 05) and the European Organization for Research and Treatment of Cancer Infectious Diseases Group.

BACKGROUND: Strict definition of invasive aspergillosis (IA) cases is required to allow precise conclusions about the efficacy of antifungal therapy. The Global Comparative Aspergillus Study (GCAS) compared voriconazole to amphotericin B (AmB) deoxycholate for the primary therapy of IA. Because predefined definitions used for this trial were substantially different from the consensus definitions proposed by the European Organization for Research and Treatment of Cancer/Mycoses Study Group in 2008, we recategorized the 379 episodes of the GCAS according to the later definitions. METHODS: The objectives were to assess the impact of the current definitions on the classification of the episodes and to provide comparative efficacy for probable/proven and possible IA in patients treated with either voriconazole or AmB. In addition to original data, we integrated the results of baseline galactomannan serum levels obtained from 249 (65.7%) frozen samples. The original response assessment was accepted unchanged. RESULTS: Recategorization allowed 59 proven, 178 probable, and 106 possible IA cases to be identified. A higher favorable 12-week response rate was obtained with voriconazole (54.7%) than with AmB (29.9%) (P < .0001). Survival was higher for voriconazole for mycologically documented (probable/proven) IA (70.2%) than with AmB (54.9%) (P = .010). Higher response rates were obtained in possible IA treated with voriconazole vs AmB with the same magnitude of difference (26.2%; 95% confidence interval [CI], 7.2%-45.3%) as in mycologically documented episodes (24.3%; 95% CI, 11.9%-36.7%), suggesting that possible cases are true IA. CONCLUSIONS: Recategorization resulted in a better identification of the episodes and confirmed the higher efficacy of voriconazole over AmB deoxycholate in mycologically documented IA.

Estimating uncertainty of alcohol-attributable fractions for infectious and chronic diseases.

Background: Alcohol is a major risk factor for burden of disease and injuries globally. This paper presents a systematic method to compute the 95% confidence intervals of alcohol-attributable fractions (AAFs) with exposure and risk relations stemming from different sources.Methods: The computation was based on previous work done on modelling drinking prevalence using the gamma distribution and the inherent properties of this distribution. The Monte Carlo approach was applied to derive the variance for each AAF by generating random sets of all the parameters. A large number of random samples were thus created for each AAF to estimate variances. The derivation of the distributions of the different parameters is presented as well as sensitivity analyses which give an estimation of the number of samples required to determine the variance with predetermined precision, and to determine which parameter had the most impact on the variance of the AAFs.Results: The analysis of the five Asian regions showed that 150 000 samples gave a sufficiently accurate estimation of the 95% confidence intervals for each disease. The relative risk functions accounted for most of the variance in the majority of cases.Conclusions: Within reasonable computation time, the method yielded very accurate values for variances of AAFs.

Protection of pancreatic beta-cells by high density lipoproteins

SUMMARYThe incidence of type 2 diabetes (T2D) is increasing worldwide and is linked to the enhancement of obesity. The principal cause of T2D development is insulin resistance, which lead to the increase of insulin production by the pancreatic beta-cells. In a pathological environment, namely dyslipidaemia, hyperglycaemia and inflammation, beta-cell compensation will fail in more vulnerable cells and diabetes will occur. High Density Lipoproteins (HDLs), commonly named "good cholesterol" are known to be atheroprotective. Low levels of HDLs are associated with increased prevalence of cardiovascular disease but are also an independent risk factor for the development of T2D. HDLs were demonstrated to protect pancreatic beta-cells against several stresses. However the molecular mechanisms of the protection are unknown and the objectives of this work were to try to elucidate the way how HDLs protect. The first approach was a broad screening of genes regulated by the stress and HDLs. A microarray analysis was performed on beta-cells stressed by serum deprivation and rescued by HDLs. Among the genes regulated, we focused on 4E-BP1, a cap-dependent translational inhibitor. In addition, HDLs were also found to protect against several other stresses.Endoplasmic reticulum (ER) stress is a mechanism that may play a role in the onset of T2D. The unfolded protein response (UPR) is a physiological process that aims at maintaining ER homeostasis in conditions where the protein folding and secretion is perturbed. Specific signalling pathways are involved in the increase of folding, export and degradation capacity of the ER. However, in case where the stress is prolonged, this mechanism turns to be pathological, by inducing cell death effector pathways, leading to beta-cell apoptosis. In our study, we discovered that HDLs were protective against ER stress induced by drugs and physiological stresses such as saturated free fatty acids. HDLs protected beta-cells by promoting ER homeostasis via the improvement of the folding and trafficking od proteins from the ER to the Golgi apparatus.Altogether our results suggest that HDLs are important for beta-cell function and survival, by protecting them from several stresses and acting on ER homeostasis. This suggests that attempt in keeping normal HDLs levels or function in patients is crucial to lessen the development of T2D.RÉSUMÉL'incidence du diabète de type 2 est en constante augmentation et est fortement liée à l'accroissement du taux d'obésité. La cause principale du diabète de type 2 est la résistance à l'insuline, qui entraîne une surproduction d'insuline par les cellules bêta pancréatiques. Dans un environnement pathologique associé à l'obésité (dyslipidémie, hyperglycémie et inflammation), les cellules bêta les plus vulnérables ne sont plus capables de compenser en augmentant leur production d'insuline, dysfonctionnent, ce qui conduit à leur mort par apoptose. Les lipoprotéines de hautes densités (HDLs), communément appelées (( bon cholestérol », sont connues pour leurs propriétés protectrices contre l'athérosclérose. Des niveaux bas de HDLs sanguins sont associés au risque de développer un diabète de type 2. Les HDLs ont également montré des propriétés protectrices contre divers stresses dans la cellule bêta. Cependant, les mécanismes de protection restent encore inconnus et l'objectif de ce travail a été d'investiguer les mécanismes moléculaires de protection des HDLs. La première approche choisie a été une étude du profil d'expression génique par puce à ADN afin d'identifier les gènes régulés par le stress et les HDLs. Parmi les gènes régulés, notre intérêt s'est porté sur 4E-BP1, un inhibiteur de la traduction coiffe- dépendante, dont l'induction par le stress était corrélée avec une augmentation de l'apoptose. Suite à cette étude, les HDLs ont également montrés un rôle protecteur contre d'autres stresses. Il s'agit particulièrement du stress du réticulum endoplasmique (RE), qui est un mécanisme qui semble jouer un rôle clé dans le développement du diabète. L'UPR (« Unfolded Protein Response ») est un processus physiologique tendant à maintenir l'homéostasie du réticulum endoplasmique, organelle prépondérante pour la fonction des cellules sécrétrices, notamment lorsqu'elle est soumise à des conditions extrêmes telles que des perturbations de la conformation tertiaire des protéines ou de la sécrétion. Dans ces cas, des voies de signalisation moléculaires sont activées, ce qui mène à l'exportation des protéines mal repliées, à leur dégradation et à l'augmentation de l'expression de chaperonnes capables d'améliorer le repliement des protéines mal formées. Toutefois, en cas de stress persistant, ce mécanisme de protection s'avère être pathologique. En induisant des voies de signalisation effectrices de l'apoptose, il conduit finalement au développement du diabète. Dans cette étude, nous avons démontré que les HDLs étaient capables de protéger la cellule bêta contre le stress du RE induits par des inhibiteurs (thapsigargine, tunicamycine) ou des stresses physiologiques tels que les acides gras libres. Les HDLs ont la capacité d'améliorer l'homéostasie du RE, notamment en favorisant le repliement et le transfert des protéines du RE à l'appareil de Golgi.En résumé, ces données suggèrent que les HDLs sont bénéfiques pour la survie des cellules bêta soumises à des stresses impliqués dans le développement du diabète, notamment en restaurant l'homéostasie du RE. Ces résultats conduisent à soutenir que le maintien des taux de cholestérol joue un rôle important dans la limitation de l'incidence du diabète.

ESCMID* guideline for the diagnosis and management of Candida diseases 2012: diagnostic procedures.

As the mortality associated with invasive Candida infections remains high, it is important to make optimal use of available diagnostic tools to initiate antifungal therapy as early as possible and to select the most appropriate antifungal drug. A panel of experts of the European Fungal Infection Study Group (EFISG) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) undertook a data review and compiled guidelines for the clinical utility and accuracy of different diagnostic tests and procedures for detection of Candida infections. Recommendations about the microbiological investigation and detection of candidaemia, invasive candidiasis, chronic disseminated candidiasis, and oropharyngeal, oesophageal, and vaginal candidiasis were included. In addition, remarks about antifungal susceptibility testing and therapeutic drug monitoring were made.

Mécanisme moléculaire de protection des HDLs contre le stress du réticulum endoplasmique dans la cellule beta pancréatique. [Molecular mechanisms of HDLs to protect against the endoplasmic reticulum stress in pancreatic beta cell.]

Introduction: Les particules de HDL (High Density Lipoproteins) ont des fonctions très diverses notamment anti-inflamatoires, anti-apoptotiques ou anti-oxydatives. Chez les patients diabétiques, les niveaux de HDLs sont bas, les prédisposants ainsi à un risque élévé à développer une maladie cardiovasculaire. Sachant que le s HDLs ont également un effet protecteur sur la cellule beta, le but de cette étude est dinvestigué les mécanismes moléculaires de cette protection contre le stress du réticulum, stress qui contriubue au développement du diabéte de type 2. Résultats: La thapsigargine et la tunicamycine induisent lapoptose en induisant un stress dans le réticulum endoplasmique (RE) par un mauvais repliement des protéines dans le RE, ainsi que l'activation de l'UPR (Unfolded Protein Respons) avec trois voies communes de signalisation intracellulaire (IRE1, PREK et ATF6). Ces voix veillent tout d'abord à augmenter la capacité de repliement des protéines et le cas échéant à lapoptose. Nos résultats montrent que les HDLs sont capable d'inhuber lapoptose induite par la thapsigargine et la tunicamycine dans les MIN6. Dans le cas du traitement avec la thapsigargine, plusieurs marqueurs des voix UPR sont bloqués en présence des HDLs, suggérant que l'effet anti-apoptotiques des HDLs s'exerce au niveau ou en amont du RE. Les HDLS par contre ne bloquent par la sortie de calcium du RE induite par la thapsigargine ce qui indique que les HDLs n'interfèrent pas avec l'action de cette drogue sur sa cible (SERCA). Dans le cas de la la tunicamycine, les HDLs ne bloquent pas, ou très légèrement, l'activation des voix de l'UPR. La protection induite par les HDLs contre la mort engendrée par la tunicamycine s'sexerce dont apparement en aval de l'UPR et reste à être déterminer. Conclusions: Nos données suggérent que les HDLs sont capable de protéger la cellule beta contre le stress du réticulum mais apparement de façon différente selon les modalités d'inductions de ce stress.

The zebrafish is an animal model in ophthalmic diseases : Fleck corneal dystrophy and Schorderet-Munier syndrome

RAPPORT DE SYNTHÈSE : Pip5k3 : Pip5k3 is a kinase responsible for fleck corneal dystrophy when mutated. It is a well conserved gene that has only been characterized in human and mouse. Characterization of pip5k3 in zebrafish was necessary before using it as a model. The protein is 70 % similar to the human homologue. The full coding sequence encompasses 6303 by and presented four isoforms. They were differentially expressed during development. All the analyzed organs of the adult zebrafish expressed pip5k3. The adult eye expressed pip5k3 in the cornea, lens, ganglion cell layer (GCL), inner nuclear layer (INL) and outer limiting membrane (OLM). During development, pip5k3 was first uniformly expressed before to be restricted to the head region and to the somites. The expression of pip5k3 in the cornea of the larval eye could make possible the study of fleck corneal dystrophy on this animal. NkxS-3 : NKXS-3 is a transcription factor responsible for a new oculo-auricular syndrome in human when mutated. This recessive disorder is characterized by defects in ear lobule and multiple defects in eye, including microphthalmia and cataract. During development, the zebrafish expressed nkx5-3 in the lens, in the anterior retina and in otic vesicles. Knockdown experiments partially phenocopied the human disease. Microphthalmia and cataract were reproduced, but zebrafish showed also defects in the cartilage of the jaw associated with a microcephaly and fins abnormalities. The retinal cell differentiation was delayed, possibly linked with the delayed expression of at`h5 and crx also observed in morphants. Shh, a regulator of ath5, was normally expressed in morphant. Overexpression of nkx5-3 lead to an anophthalmia, suggesting a role at the early organogenesis of the eye. All the phenotypes observed in morphants and embryos overexpressing nkx5-3 suggest a potential involvement of the FGF and hedgehog signaling pathways.

Perception et communication du risque: du diabète à la maladie cardiovasculaire [Risk perception and communication: from diabetes to cardiovascular diseases].

Evidence-based medicine has enabled to approach disease in a more rational and scientific way. Clinical research has identified behaviours and risk factors that could cause disease often "silent" at the beginning, such as diabetes. Despite the clear impact of these evidences on public health, it seems that the individual risk perception level remains weak. To mention as well, the health professionals very often have a different views, which makes it difficult to communicate the risk with patients. In this article we describe the principles of risk perception, the diabetes related risk perception concerning cardiovascular complications, and suggest some practical strategies and tools which could improve risk communication in the everyday practice.