Étude de la fonction de GLUT8 sur un modèle de souris "knock-out"

Résumé GLUT8 est la première des nouvelles isoformes des GLUT récemment identifiés. Il est fortement exprimé dans les testicules et plus faiblement dans les blastocystes, le cerveau, particulièrement au niveau de l'hippocampe, et le coeur. En conditions basales, il est retenu dans un compartiment intracellulaire. Si on l'exprime en surface cellulaire, par la mutation du motif d'internalisation dileucine, il transporte le glucose avec une bonne affinité. Dans le but d'étudier sa fonction au niveau de l'organisme, nous avons créé un modèle de knock out conditionnel, en entourant le dernier exon du gène de GLUT8 par deux sites loxP. En croisant nos souris avec une souche de souris transgénique exprimant la cre-recombinase dans les cellules de la lignée germinale, nous avons généré un modèle de souris portant la délétion totale de GLUT8 de manière constitutionnelle. Les statistiques effectuées sur les premières naissances indiquent qu'une partie des souris knock out ne survit pas, suggérant un rôle de GLUT8 au niveau du développement embryonnaire. Les souris qui ont survécu ne présentent toutefois pas d'anomalies durant la croissance et sont fertiles. Elles ont des taux de glucose et d'insuline sanguins normaux. Au niveau cérébral, la structure de l'hippocampe n'est pas modifiée par la suppression de GLUT8, cependant, les souris GLUT8-/- présentent une prolifération cellulaire augmentée dans le gyrus denté. Cette augmentation de division cellulaire pourrait être la réponse adaptée à une éventuelle augmentation de la mort cellulaire au niveau de l'hippocampe. Elles ne semblent toutefois pas présenter de défauts cognitifs majeurs dans le bassin de Morris en conditions normales. Toutefois, en conditions de jeûne, elles tendent à une meilleure mémorisation à court terme. Les études morphologiques et histologiques au niveau cardiaque n'ont pas révélé de d'hypertrophie au niveau ventriculaire. La stimulation de la contraction à l'isoprotérénol n'a pas mis en évidence de défaut d'adaptation des coeurs GLUT8-/-. Cependant l'analyse fonctionnelle par électrocardiogramme, en conditions basales, a montré une augmentation de la durée de l'onde P, suggérant un défaut dans la dépolarisation des oreillettes. Nos résultats indiquent que GLUT8 ne joue pas un rôle prédominant dans la survie et la fonction basale des souris. Il pourrait jouer un rôle plus important dans des situations stressantes pour l'organisme, comme l'hypoglycémie ou les conditions d'ischémie qui induiraient son expression à la membrane plasmique et stimuleraient le captage du glucose. Abstract GLUT8 was the first of the recently identified isoform of the GLUT family proteins. It is strongly expressed in the testis. It is also found at a lower level in the blastocyst, in heart and in the brain. Under basal conditions, it is retained in the intracellular compartment, but when the internalization motif dileucine is mutated, GLUT8 translocates to the plasma membrane and transports glucose with a relatively high affinity. To study its function in vivo, we created a conditional knock out mouse model. To do so, we targeted the last exon of the GLUT8 gene with two loxP sites. We then crossed these mice with a transgenic model expressing the cre-recombinase in the gem' line to generate a constitutional total knock out mouse. The statistics made on the first breedings showed that some of the knock out mice do not survive, suggesting a role of GLUT8 in the embryonic development. Conversely mice who survive do not show developmental defects and they are fertile with normal glucose and insulin blood levels. In the brain, the general structure of the hippocampus is not modified by the deletion of GLUT8. However, GLUT8-/- mice show an increase in the cell proliferation in the dentate gyms. This cell proliferation could be due to an increase in the cell death in the hippocampus. When tested in the morris water maze, these mice do not show any cognitive defects in the basal conditions, but they have a tendency to learn better in fasted conditions. The morphological and histological studies made at the heart level did not show any cardiac hypertrophy in the ventricles. The stimulation with isoproterenol did not show any adaptation defects in the GLUT8-/- hearts. However, the functional analysis made in basal conditions with the electrocardiogram showed an increase in the P wave length, suggesting a defect in the atrial depolarization in the knock out mice. Overall, our results show that GLUT8 does not play an important role in the basal general functions in the mice, but might play a more important role during whole organism stress. Hypoglycaemia or ischemia, for example could stimulate the GLUT8 translocation to the plasma membrane to increase specifically glucose uptake. Résumé tout public Les différentes cellules de l'organisme possèdent des propriétés particulières, qui leur permettent de maintenir les fonctions de l'organe auquel elles appartiennent. La membrane plasmique qui les délimite sélectionne les substances qui vont pénétrer à l'intérieur de la cellule et permet ainsi de maintenir un environnement interne constant. Le glucose est une source d'énergie importante pour la cellule et doit pouvoir pénétrer à l'intérieur de la cellule. Il utilise pour cela des protéines de transport qui le feront passer de part et d'autre de la membrane. Les protéines de la famille des GLUT (pour GLUcose Transporter) possèdent cette capacité. GLUT8 est un membre de la famille des GLUT identifié récemment. Il possède la capacité de transporter le glucose quand il se présente à la surface de la cellule. Il est principalement exprimé dans les testicules, dans le coeur et le cerveau et durant le développement embryonnaire. Son rôle n'est toutefois pas encore défini. Ce travail consiste à étudier la fonction de GLUT8 au niveau de l'organisme entier. Nous avons créé un modèle de souris dans lesquelles l'expression de GLUT8 a été supprimée pour mettre en évidence son importance dans le maintien de l'intégrité des fonctions du corps. Les observations effectuées sur les souris qui n'expriment plus GLUT8 nous indiquent que leurs cellules prolifèrent plus vite au niveau de l'hippocampe. L'hippocampe est une structure située dans le cerveau qui est impliquée dans les phénomènes d'apprentissage. Les souris qui ont été testées dans des tâches d'apprentissage n'ont malgré cela pas montré une amélioration de la mémorisation. Dans le coeur, la suppression de GLUT8 semble présenter un défaut quand on mesure l'activité électrique du coeur par électrocardiogramme. Toutefois, ils fonctionnent normalement et ne présentent pas de défauts morphologiques en conditions normales. Les expériences effectuées sur les modèles de souris indiquent que GLUT8 ne jouerait pas un rôle prédominant dans le fonctionnement normal du corps. Il pourrait exercer sa fonction dans des situations plus particulières comme l'hypoglycémie, où il permettrait une meilleure capacité à transporter le glucose dans les cellules.

Research letter: is neuroticism a risk factor for postpartum depression?

Although the relationship between personality and depressive illness is complex (Shea, 2005), there is empirical evidence that some personality features such as neuroticism, harm avoidance, introversion, dependency, self-criticism or perfectionism are related to depressive illness risk (Gunderson et al. 1999).

FGF-2 controls the differentiation of resident cardiac precursors into functional cardiomyocytes.

Recent evidence suggests that the heart possesses a greater regeneration capacity than previously thought. In the present study, we isolated undifferentiated precursors from the cardiac nonmyocyte cell population of neonatal hearts, expanded them in culture, and induced them to differentiate into functional cardiomyocytes. These cardiac precursors appear to express stem cell antigen-1 and demonstrate characteristics of multipotent precursors of mesodermal origin. Following infusion into normal recipients, these cells home to the heart and participate in physiological and pathophysiological cardiac remodeling. Cardiogenic differentiation in vitro and in vivo depends on FGF-2. Interestingly, this factor does not control the number of precursors but regulates the differentiation process. These findings suggest that, besides its angiogenic actions, FGF-2 could be used in vivo to facilitate the mobilization and differentiation of resident cardiac precursors in the treatment of cardiac diseases.

Peroxisome proliferator-activated receptor beta/delta as a therapeutic target for metabolic diseases.

The peroxisome proliferator-activated receptor (PPAR) family comprises three distinct isotypes: PPARalpha, PPARbeta/delta and PPARgamma. PPARs are nuclear hormone receptors that mediate the effects of fatty acids and their derivatives at the transcriptional level. Until recently, the characterisation of the important role of PPARalpha in fatty acid oxidation and of PPARgamma in lipid storage contrasted with the sparse information concerning PPARbeta/delta. However, evidence is now emerging for a role of PPARbeta/delta in tissue repair and energy homeostasis. Experiments with tissue-specific overexpression of PPARbeta/delta or treatment of mice with selective PPARbeta/delta agonists demonstrated that activation of PPARbeta/delta in vivo increases lipid catabolism in skeletal muscle, heart and adipose tissue and improves the serum lipid profile and insulin sensitivity in several animal models. PPARbeta/delta activation also prevents the development of obesity and improves cholesterol homeostasis in obesity-prone mouse models. These new insights into PPARbeta/delta functions suggest that targeting PPARbeta/delta may be helpful for treating disorders associated with the metabolic syndrome. Although these perspectives are promising, several independent and contradictory reports raise concerns about the safety of PPARbeta/delta ligands with respect to tumourigenic activity in the gut. Thus, it appears that further exploration of PPARbeta/delta functions is necessary to better define its potential as a therapeutic target.

Population pharmacokinetics of ganciclovir in solid-organ transplant recipients receiving oral valganciclovir.

Valganciclovir (VGC) is an oral prodrug of ganciclovir (GCV) recently introduced for prophylaxis and treatment of cytomegalovirus infection. Optimal concentration exposure for effective and safe VGC therapy would require either reproducible VGC absorption and GCV disposition or dosage adjustment based on therapeutic drug monitoring (TDM). We examined GCV population pharmacokinetics in solid organ transplant recipients receiving oral VGC, including the influence of clinical factors, the magnitude of variability, and its impact on efficacy and tolerability. Nonlinear mixed effect model (NONMEM) analysis was performed on plasma samples from 65 transplant recipients under VGC prophylaxis or treatment. A two-compartment model with first-order absorption appropriately described the data. Systemic clearance was markedly influenced by the glomerular filtration rate (GFR), patient gender, and graft type (clearance/GFR = 1.7 in kidney, 0.9 in heart, and 1.2 in lung and liver recipients) with interpatient and interoccasion variabilities of 26 and 12%, respectively. Body weight and sex influenced central volume of distribution (V(1) = 0.34 liter/kg in males and 0.27 liter/kg in females [20% interpatient variability]). No significant drug interaction was detected. The good prophylactic efficacy and tolerability of VGC precluded the demonstration of any relationship with GCV concentrations. In conclusion, this analysis highlights the importance of thorough adjustment of VGC dosage to renal function and body weight. Considering the good predictability and reproducibility of the GCV profile after treatment with oral VGC, routine TDM does not appear to be clinically indicated in solid-organ transplant recipients. However, GCV plasma measurement may still be helpful in specific clinical situations.

Perioperative thirst: an analysis from the perspective of the Symptom Management Theory

A theoretical study aimed to analyze the existing knowledge in the literature on the perioperative thirst symptom from the perspective of Symptom Management Theory, and supplemented with the experience of the study group and thirst research. Thirst is described as a very intense symptom occurring in the perioperative period, and for this reason it cannot be ignored. The Symptom Management Theory is adequate for understanding the thirst symptom and is a deductive theory, focused on the domains of the Person, Environment and Health / Illness Status, as well as on the dimensions of Experience, Management Strategies and Symptom Outcomes. Using the theory leads us to consider perioperative thirst in its multifactorial aspects, analyzing the interrelation of its domains and dimensions in order to draw attention to this symptom that has been insufficiently valued, recorded and treated in clinical practice.

Massage with aromatherapy: effectiveness on anxiety of users with personality disorders in psychiatric hospitalization

OBJECTIVE To investigate the effectiveness of aromatherapy massage using the essential oils (0.5%) of Lavandula angustifolia and Pelargonium graveolens for anxiety reduction in patients with personality disorders during psychiatric hospitalization. METHOD Uncontrolled clinical trial with 50 subjects submitted to six massages with aromatherapy, performed on alternate days, on the cervical and the posterior thoracic regions. Vital data (heart and respiratory rate) were collected before and after each session and an anxiety scale (Trait Anxiety Inventory-State) was applied at the beginning and end of the intervention. The results were statistically analyzed with the chi square test and paired t test. RESULTS There was a statistically significant decrease (p < 0.001) of the heart and respiratory mean rates after each intervention session, as well as in the inventory score. CONCLUSION Aromatherapy has demonstrated effectiveness in anxiety relief, considering the decrease of heart and respiratory rates in patients diagnosed with personality disorders during psychiatric hospitalization.

Clinical Proton MR Spectroscopy in Central Nervous System Disorders.

A large body of published work shows that proton (hydrogen 1 [(1)H]) magnetic resonance (MR) spectroscopy has evolved from a research tool into a clinical neuroimaging modality. Herein, the authors present a summary of brain disorders in which MR spectroscopy has an impact on patient management, together with a critical consideration of common data acquisition and processing procedures. The article documents the impact of (1)H MR spectroscopy in the clinical evaluation of disorders of the central nervous system. The clinical usefulness of (1)H MR spectroscopy has been established for brain neoplasms, neonatal and pediatric disorders (hypoxia-ischemia, inherited metabolic diseases, and traumatic brain injury), demyelinating disorders, and infectious brain lesions. The growing list of disorders for which (1)H MR spectroscopy may contribute to patient management extends to neurodegenerative diseases, epilepsy, and stroke. To facilitate expanded clinical acceptance and standardization of MR spectroscopy methodology, guidelines are provided for data acquisition and analysis, quality assessment, and interpretation. Finally, the authors offer recommendations to expedite the use of robust MR spectroscopy methodology in the clinical setting, including incorporation of technical advances on clinical units. © RSNA, 2014 Online supplemental material is available for this article.

How to manage the left subclavian artery during endovascular stenting of the thoracic aorta.

We performed a systematic review of the literature to establish whether revascularisation of the left subclavian territory is necessary when this artery is covered by a stent. We retrieved data from 99 studies incorporating 4906 patients. Incidences of left-arm ischaemia (0.0% vs 9.2%, p=0.002) and stroke (4.7% vs 7.2%, p<0.001) were significantly less following revascularisation, although mortality (10.5% vs 3.4%, p=0.032) and endoleak incidence (25.8% vs 12.6%, p=0.008) were increased. No significant differences in spinal-cord ischaemia were seen. Revascularisation may reduce downstream ischaemic complications but can cause significant risk. Indications must be carefully considered on an individual patient basis.

Postmortem imaging of sudden cardiac death

Postmortem imaging is increasingly used in forensic practice in cases of natural deaths related to cardiovascular diseases, which represent the most common causes of death in developed countries. While radiological examination is generally considered to be a good complement for conventional autopsy, it was thought to have limited application in cardiovascular pathology. At present, multidetector computed tomography (MDCT), CT angiography, and cardiac magnetic resonance imaging (MRI) are used in postmortem radiological investigation of cardiovascular pathologies. This review presents the actual state of postmortem imaging for cardiovascular pathologies in cases of sudden cardiac death (SCD), taking into consideration both the advantages and limitations. The radiological evaluation of ischemic heart disease (IHD), the most frequent cause of SCD in the general population of industrialized countries, includes the examination of the coronary arteries and myocardium. Postmortem CT angiography (PMCTA) is very useful for the detection of stenoses and occlusions of coronary arteries but less so for the identification of ischemic myocardium. MRI is the method of choice for the radiological investigation of the myocardium in clinical practice, but its accessibility and application are still limited in postmortem practice. There are very few reports implicating postmortem radiology in the investigation of other causes of SCD, such as cardiomyopathies, coronary artery abnormalities, and valvular pathologies. Cardiomyopathies representing the most frequent cause of SCD in young athletes cannot be diagnosed by echocardiography, the most widely available technique in clinical practice for the functional evaluation of the heart and the detection of cardiomyopathies. PMCTA and MRI have the potential to detect advanced stages of diseases when morphological substrate is present, but these methods have yet to be sufficiently validated for postmortem cases. Genetically determined channelopathies cannot be detected radiologically. This review underlines the need to establish the role of postmortem radiology in the diagnosis of SCD.

Treatment of cytomegalovirus infection or disease in solid organ transplant recipients with valganciclovir.

Valganciclovir (VGC) has proved efficacious and safe for the prophylaxis against cytomegalovirus (CMV) in high-risk transplant recipients and for the treatment of CMV retinitis in AIDS patients. We used VGC for the treatment of CMV infection (viremia without symptoms) or disease (CMV syndrome or tissue-invasive disease) in kidney, heart, and lung transplant recipients. Fourteen transplant recipients were treated: five for asymptomatic CMV infection and nine for CMV disease. VGC was administered in doses adjusted to renal function for 4 to 12 weeks (induction and maintenance therapy). Clinically, all nine patients with CMV disease responded to treatment. Microbiologically, treatment with VGC turned blood culture negative for CMV within 2 weeks in all patients and was associated with a > or =2 log decrease in blood CMV DNA within 3 weeks in 8 of 8 tested patients. With a follow-up of 6 months (n = 12 patients), asymptomatic recurrent CMV viremia was noted in five cases, and CMV syndrome noted in one case (all cases in the first 2 months after the end of treatment). VGC was clinically well tolerated in all patients; however, laboratory abnormalities occurred in three cases (mild increase in transaminases, thrombocytopenia, and pancytopenia). This preliminary experience strongly suggests that therapy with VGC is effective against CMV in organ transplant recipients; however, the exact duration of therapy remains to be determined: a longer course may be necessary to prevent early recurrence.

Role of the NALP3 Inflammasome in the Development of Two-Kidney, One Clip Hypertension in Mice

Objective: The NALP3 inflammasome functions as a sensor of danger signals and triggers processing and release of IL-1b. Mutations of NALP3 are responsible for the cryopyrin associated periodic syndromes, a group of autoinflammatory disorders that respond to IL1 inhibition. Genetic studies have also linked NALP3 to hypertension in man, but the mechanism is not understood. The aim of this study is to investigate the role of NAPL3 inflammasome in the development of hypertension in an animal model. Design and Method: Six-week old male WT and NALP3 KO mice were used for generating a two-kidney, one clip (2K1C) renovascular hypertension. A U-shaped stainless steel clip (O^ ¼0.12mm) was placed on left renal artery under anaesthesia. The same surgery without clipping was performed in sham mice. At week 6 and 12 after the clipping, intra-arterial blood pressure (BP) was measured in conscious mice. Blood was collected for plasma renin analysis. Heart and kidney were excised and stored for molecular and morphological examinations. n¼5-6 mice per group. Data are mean_SEM. Results: Mean BP was significantly increased at week 6 and 12 in WT-2K1C mice compared to WT-sham group (MBPweek6: 138_2 vs.124_3 mmHg, p<0.01 and MBPweek12: 141_5 vs.122_3 mmHg, p<0.01) followed with an significant increase in heart weight (HW) and a decrease in clipped kidney weight indices in WT-2K1C mice compared to the WT-sham (HW/ BWweek6: 4.65_0.04 vs. 3.99_0.12 mg/g, p<0.001 and HW/BWweek12: 4.94_0.15 vs. 4.22_0.12 mg/g, p<0.001). Interestingly, NALP3 KO-2K1C mice did not develop hypertension. The MBP of KO-2K1C mice was comparable to the KO-sham (MBPweek6: 122_3 vs. 119_3 mmHg, p>0.05 and MBPweek6: 128_5 vs.122_4 mmHg, p>0.05). There was also no significant change in heart and kidney weight indices between KO- 2K1C and KO-sham mice. Conclusion: The preliminary results suggest that absence of NALP3 protects mice from the development of renin-dependent hypertension. Further molecular and morphological examinations are ongoing for the confirmation and mechanism explanation.

Limitations of stimulated echo acquisition mode (STEAM) techniques in cardiac applications.

Stimulated echoes are widely used for imaging functional tissue parameters such as diffusion coefficient, perfusion, and flow rates. They are potentially interesting for the assessment of various cardiac functions. However, severe limitations of the stimulated echo acquisition mode occur, which are related to the special dynamic properties of the beating heart and flowing blood. To the well-known signal decay due to longitudinal relaxation and through-plane motion between the preparation and the read-out period of the stimulated echoes, additional signal loss is often observed. As the prepared magnetization is fixed with respect to the tissue, this signal loss is caused by the tissue deformation during the cardiac cycle, which leads to a modification of the modulation frequency of the magnetization. These effects are theoretically derived and corroborated by phantom and in vivo experiments.

Sarcomeric M-band alterations as a marker for human dilated cardiomyopathy

Purpose: The M-band is an important cytoskeletal structure in the centre of the sarcomere, believed to cross-link the thick filament lattice. Its main components are three closely related modular proteins from the myomesin gene family: Myomesin, M-protein and myomesin-3. Each muscle is characterized by its unique M-band protein composition, depending on the contractile parameters of a particular fiber. To investigate the role of the M-band in one of the most relevant and clinically increasing cardiac diseases, we analyzed the expression of myomesin proteins in dilated cardiomyopathy (DCM).Methods: In a previous study we analyzed mouse models suffering from DCM, demonstrating that the embryonic heart specific EH-myomesin splicing isoform was up-regulated directly corresponding to the degree of cardiac dysfunction and ventricular dilation. Based on this study, human ventricular and atrial samples (n=32) were obtained during heart surgery after informed consent and approval by an institutional review board. Patients were aged 30-70 years and suffered from dilated cardiomyopathy (DCM;n=13), Hypertrophic Cardiomyopathy (HCM;n=10) or served as controls (n=9). Patients suffering from DCM or HCM were in endstage heart-failure (NYHA III-IV) and either underwent heart transplantation or Left Ventricular Assist Device (LVAD) implantation. Heart samples from patients who underwent valve surgery or congenital heart surgery served as controls. Heart Samples were analyzed using RT-PCR, Western blot, and immunofluorescence.Results: By investigating the expression pattern of myomesins, we found that DCM is accompanied by specific M-band alterations, which were more pronounced in ventricular samples compared to the atrium. Changes in the amounts of different myomesins during DCM occurred in a cell-specific manner, leading to a higher heterogeneity of the cytoskeleton in cardiomyocytes through the myocardial wall with some cells switching completely to an embryonic phenotype.Conclusions: Here we present that the embryonic heart specific EH-myomesin isoform is up-regulated in human DCM. The alterations of the M-band protein composition might be part of a general adaptation of the sarcomeric cytoskeleton to unfavorable working conditions in the failing heart and may modify the mechanical properties of the cardiomyocytes. We suggest that the upregulation of EH-myomesin might play a pivotal role in DCM and might support classical imagingas a novel sarcomeric marker for this disease.

Existential values transformation in all organ transplantation : a qualitative prospective study

The aim of this qualitative study was to analyze psychological concerns in wait-listed patients T1 and six months after transplantation T2. Semi-structured interviews were conducted and qualitative analysis performed. T1 Kidney patients maintained apparent normality, building emotional protection, and a fatalist attitude. Liver patients set physical limits, reevaluation of life values was reported. Lung patients developed physical and psychological self-protection. Modified life values, fatalism and spirituality were mentioned. Heart patients husbanded ressources and self-protection. Modified life values, fatalist attitude were reported. T2 Kidney patients described new life perspectives and increase of empathy. Liver patients underlined positive identity and life values modifications. Lack of respect of life values generated anger. Heart and lung patients set their existential priorities and underlined increase in spirituality, greater openness and more closeness to significant ones. Lack of respect of human values induced negative feelings. TX comes with physical benefits, but also with positive existential values transformations and a humanistic, altruistic attitude.