Implication of the endocannabinoid system in the locomotor hyperactivity associated with congenital hypothyroidism

Alterations in motor functions are well-characterized features observed in humans and experimental animals subjected to thyroid hormone dysfunctions during development. Here we show that congenitally hypothyroid rats display hyperactivity in the adult life. This phenotype was associated with a decreased content of cannabinoid receptor type 1 (CB(1)) mRNA in the striatum and a reduction in the number of binding sites in both striatum and projection areas. These findings suggest that hyperactivity may be the consequence of a thyroid hormone deficiency-induced removal of the endocannabinoid tone, normally acting as a brake for hyperactivity at the basal ganglia. In agreement with the decrease in CB(1) receptor gene expression, a lower cannabinoid response, measured by biochemical, genetic and behavioral parameters, was observed in the hypothyroid animals. Finally, both CB(1) receptor gene expression and the biochemical and behavioral dysfunctions found in the hypothyroid animals were improved after a thyroid hormone replacement treatment. Thus, the present study suggests that impairment in the endocannabinoid system can underlay the hyperactive phenotype associated with hypothyroidism.

The alpha 1a and alpha 1b-adrenergic receptor subtypes: molecular mechanisms of receptor activation and of drug action.

In this chapter we summarize some aspects of the structure-functional relationship of the alpha 1a and alpha 1b-adrenergic receptor subtypes related to the receptor activation process as well as the effect of different alpha-blockers on the constitutive activity of the receptor. Molecular modeling of the alpha 1a and alpha 1b-adrenergic receptor subtypes and computational simulation of receptor dynamics were useful to interpret the experimental findings derived from site directed mutagenesis studies.

A combined approach for the assessment of cell viability and cell functionality of human fibrochondrocytes for use in tissue engineering.

Temporo-mandibular joint disc disorders are highly prevalent in adult populations. Autologous chondrocyte implantation is a well-established method for the treatment of several chondral defects. However, very few studies have been carried out using human fibrous chondrocytes from the temporo-mandibular joint (TMJ). One of the main drawbacks associated to chondrocyte cell culture is the possibility that chondrocyte cells kept in culture tend to de-differentiate and to lose cell viability under in in-vitro conditions. In this work, we have isolated human temporo-mandibular joint fibrochondrocytes (TMJF) from human disc and we have used a highly-sensitive technique to determine cell viability, cell proliferation and gene expression of nine consecutive cell passages to determine the most appropriate cell passage for use in tissue engineering and future clinical use. Our results revealed that the most potentially viable and functional cell passages were P5-P6, in which an adequate equilibrium between cell viability and the capability to synthesize all major extracellular matrix components exists. The combined action of pro-apoptotic (TRAF5, PHLDA1) and anti-apoptotic genes (SON, HTT, FAIM2) may explain the differential cell viability levels that we found in this study. These results suggest that TMJF should be used at P5-P6 for cell therapy protocols.

Impact of dietary betaine and conjugated linoleic acid on insulin sensitivity, protein and fat metabolism of obese pigs.

To determine possible mechanisms of action that might explain the nutrient partitioning effect of betaine and conjugated linoleic acid (CLA) in Iberian pigs and to address potential adverse effects, twenty gilts were restrictively fed from 20 to 50 kg BW Control, 0.5% betaine, 1% CLA or 0.5% betaine + 1% CLA diets. Serum hormones and metabolites profile were determined at 30 kg BW and an oral glucose test was performed before slaughter. Pigs were slaughtered at 50 kg BW and livers were obtained for chemical and histological analysis. Decreased serum urea in pigs fed betaine and betaine + CLA diets (11%; P = 0.0001) indicated a more efficient N utilization. The increase in serum triacylglycerol (58% and 28%, respectively; P = 0.0098) indicated that CLA and betaine + CLA could have reduced adipose tissue triacylglycerol synthesis from preformed fatty acids. Serum glucose, low-density lipoprotein (LDL) cholesterol and non-esterified fatty acids were unaffected. CLA and betaine + CLA altered serum lipids profile, although liver of pigs fed CLA diet presented no histopathological changes and triglyceride content was not different from Control pigs. Compared with controls, serum growth hormone decreased (20% to 23%; P = 0.0209) for all treatments. Although serum insulin increased in CLA, and especially in betaine + CLA pigs (28% and 83%; P = 0.0001), indices of insulin resistance were unaffected. In conclusion, CLA, and especially betaine + CLA, induced changes in biochemical parameters and hormones that may partially explain a nutrient partitioning effect in young pigs. Nevertheless, they exhibited weak, although detrimental, effects on blood lipids. Moreover, although livers were chemically and histologically normal, pigs fed CLA diet challenged with a glucose load had higher serum glucose than controls.

Effects of antibacterial agents and drugs monitored by atomic force microscopy.

Originally invented for topographic imaging, atomic force microscopy (AFM) has evolved into a multifunctional biological toolkit, enabling to measure structural and functional details of cells and molecules. Its versatility and the large scope of information it can yield make it an invaluable tool in any biologically oriented laboratory, where researchers need to perform characterizations of living samples as well as single molecules in quasi-physiological conditions and with nanoscale resolution. In the last 20 years, AFM has revolutionized the characterization of microbial cells by allowing a better understanding of their cell wall and of the mechanism of action of drugs and by becoming itself a powerful diagnostic tool to study bacteria. Indeed, AFM is much more than a high-resolution microscopy technique. It can reconstruct force maps that can be used to explore the nanomechanical properties of microorganisms and probe at the same time the morphological and mechanical modifications induced by external stimuli. Furthermore it can be used to map chemical species or specific receptors with nanometric resolution directly on the membranes of living organisms. In summary, AFM offers new capabilities and a more in-depth insight in the structure and mechanics of biological specimens with an unrivaled spatial and force resolution. Its application to the study of bacteria is extremely significant since it has already delivered important information on the metabolism of these small microorganisms and, through new and exciting technical developments, will shed more light on the real-time interaction of antimicrobial agents and bacteria.

Rab18 dynamics in adipocytes in relation to lipogenesis, lipolysis and obesity.

Lipid droplets (LDs) are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K), the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the β-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER) is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in β-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed.

Circulating antioxidant defences are decreased in healthy people after a high-fat meal.

The aim of this study was to examine the responses of uric acid, antioxidant defences and pro-oxidant variables after a high-fat meal. Twenty-five healthy persons without criteria for the metabolic syndrome, underwent a high-fat meal with Supracal (60 g fat). Measurements were made at baseline and 3 h after the meal of TAG, uric acid, HDL-cholesterol, total proteins and oxidative stress. Following the high-fat meal, we detected a significant increase in pro-oxidative variables and a decrease in antioxidative variables. The uric acid concentrations were significantly lower after the high-fat meal and the reduction correlated significantly with the oxidative stress variables. The inverse relation between reduced uric acid and increased carbonylated proteins remained in multiple regression analysis. We conclude that uric acid is a powerful antioxidant and its reduction following a high-fat meal may be related with its acute antioxidative action.

Amniotic fluid iodine concentrations do not vary in pregnant women with varying iodine intake.

Iodine deficiency is an important clinical and public health problem. Its prevention begins with an adequate intake of iodine during pregnancy. International agencies recommend at least 200 microg iodine per d for pregnant women. We assessed whether iodine concentrations in the amniotic fluid of healthy pregnant women are independent of iodine intake. This cross-sectional, non-interventional study included 365 consecutive women who underwent amniocentesis to determine the fetal karyotype. The amniocentesis was performed with abdominal antisepsis using chlorhexidine. The iodine concentration was measured in urine and amniotic fluid. The study variables were the intake of iodized salt and multivitamin supplements or the prescription of a KI supplement. The mean level of urinary iodine was 139.0 (SD 94.5) microg/l and of amniotic fluid 15.81 (SD 7.09) microg/l. The women who consumed iodized salt and those who took a KI supplement had significantly higher levels of urinary iodine than those who did not (P = 0.01 and P = 0.004, respectively). The urinary iodine levels were not significantly different in the women who took a multivitamin supplement compared with those who did not take this supplement, independently of iodine concentration or multivitamin supplement. The concentrations of iodine in the amniotic fluid were similar, independent of the dietary iodine intake. Urine and amniotic fluid iodine concentrations were weakly correlated, although the amniotic fluid values were no higher in those women taking a KI supplement. KI prescription at recommended doses increases the iodine levels in the mother without influencing the iodine levels in the amniotic fluid.

Analyse fonctionnelle et moléculaire d'un nouveau gène (VIT32) impliqué dans le mécanisme d'action de la vasopressine

Résumé Les mécanismes de régulation de la réabsorption fine du sodium dans la partie distale (tube distal et tube collecteur) du néphron ont un rôle essentiel dans le maintien de l'homéostasie de la composition ionique et du volume des fluides extracellulaires. Ces mécanismes permettent le maintien de la pression sanguine. Dans la cellule principale du tube collecteur cortical (CCD), le taux de réabsorption de sodium dépend essentiellement de l'activité du canal épithélial à sodium (ENaC) à la membrane apicale et de la pompe sodium-potassium-adénosine-triphosphatase (Na+-K±ATPase) à la membrane basolatérale. L'activité de ces deux molécules de transport est en partie régulée par des hormones dont l'aldostérone, la vasopressine et l'insuline. Dans les cellules principales de CCD, la vasopressine régule le transport de sodium en deux étapes : une étape précoce dite « non-génomique » et une étape tardive dite « génotnique ». Durant l'étape précoce, la vasopressine régule l'expression de gènes, dont certains peuvent être impliqués dans le transport de sodium, comme ENaC et la Na+ -K+ATP ase. Le but de mon travail a été d'étudier l'implication d'une protéine appelée VIP32 (vasopressin induced protein : VIP) dans le transport de sodium. L'expression de VIP32 est augmentée par la vasopressine dans les cellules principales de CCD. Dans l'ovocyte de Xenopus laevis utilisé comme système d'expression hétérologue, nous avons montré que l'expression de VIP32 provoque la maturation méiotique de l'ovocyte par l'activation de la voie des MAPK (mitogen-activated protein kinase : MAPK) et du facteur de promotion méiotique (MPF). La co-expression d'ENaC et de VIP32 diminue l'activité d'ENaC de façon sélective, par l'activation de la voie des MAPK, sans affecter l'expression du canal à la surface membranaire. Nous avons également montré que la régulation de l'activité d'ENaC par la voie des MAPK est dépendante du mécanisme de régulation d'ENaC lié à un motif du canal appelé PY. Ce motif est impliqué dans le contrôle de la probabilité d'ouverture ainsi que l'expression à la surface membranaire d'ENaC. Dans les cellules principales, VIP32 par l'activation de la voie des MAPK peut être impliqué dans la régulation négative du transport transépithélial qui a lieu après plusieurs heures de traitement à la vasopressine. Le tube collecteur de reins normaux présente un taux basal significatif d'activité de la voie MAPK MEK1/2-ERK1/2. Dans la lignée mpkCCDc14 de cellules principales de CCD de souris, que nous avons utilisé pour cette partie du travail, nous avons montré la présence d'un taux basal d'activité d'ERK1/2 (pERK1/2). L'aldostérone et la vasopressine, connus pour stimuler le courant sodique transépithélial dans le CCD, ne changeaient pas le taux basal de pERK1/2. Le transport de sodium transépithélial basal, ou stimulé par l'aldostérone ou la vasopressine est diminué par l'effet de PD98059, un inhibiteur de MEK1/2 qui diminue parallèlement le taux de pERK1/2. Nous avons également montré dans des cellules non stimulées, ou stimulées par de l'aldostérone ou de la vasopressine, que l'activité de la Na+-K+ ATPase, mais pas celle d'ENaC est inhibée par des traitements avec différents inhibiteurs de MEK1/2. Par un marquage de la Na±-K+ATPase à la surface membranaire nous avons montré que la voie d'ERK1/2 contrôle l'activité intrinsèque de la Na+-K+ ATPase, plutôt que son expression à la surface membranaire. Ces données ont montré que l'activité de la Na+-K+ATPase et le transport transépithélial de sodium sont contrôlés par l'activité basal et constitutive de la voie d'ERK1/2. Summary The regulation of sodium reabsorption in the distal nephron (distal tubule and cortical collecting duct) in the kidney plays an essential role in the control of extracellular fluids composition and volume, and thereby blood pressure. In the principal cell of the collecting duct (CCD), the level of sodium reabsorption mainlly depends on the activity of both epithlial sodium channel (ENaC) and sodium-potassium-adenosine-triphosphatase (Na+-K+ATPase). The activity of these two transporters is regulated by hormones especially aldosterone, vasopressin and insuline.In the principal cell of the CCD, vasopressin regulates sodium transport via a short-term effect and a late genomic effect. During the genomic effect vasopressin activates a complex network of vasopressin-dependent genes involved in the regulation of sodium transport as ENaC and Na+-K+ATPase. We were interested in the role of a recently identified vasopressin induced protein (VIP32) and its implication in the regulation of sodium transport in principal cell of the CCD. The Xenopus oocyte expression system revealed two functions : expressed alone VIP32 rapidly induces oocyte meiotic maturation through the activation of the mitogen-activated protein kinase (MAPK) pathway and the meiotic promoting factor and when co-expressed with ENaC, V1P32 selectively dowrn-egulates channel activity, but not channel cell surface expression. We have shown that the ENaC downregulation mediated by the activation of the MAPK pathway is related to the PY motif of ENaC. This motif is implicated in ENaC cell surface expression and open probability regulation. In the kidney principal cell, VIP32 through the activation of MAPK pathway may be involved in the downregulation of transepithelial sodium transport observed within a few hours after vasopressin treatment. The collecting duct of normal kidney exhibits significant activity of the MEK1/2-ERK1/2 MAPK pathway. Using in vitro cultured mpkCCDc14 principal cells we have shown a significant basal level of ERK1/2 activity (pERK1/2). Aldosterone and vasopressin, known to upregulate sodium reabsorption in CCDs, did not change ERK1/2 activity. Basal and aldosterone- or vasopressin-stimulated sodium transport were downregulated by the MEK1/2 inhibitor PD98059 in parallel with a decrease in pERK1/2 in vitro. The activity of Na+-K+ATPase but not that of ENaC was inhibited by MEK1/2 inhibitors in both, unstimulated and aldosterone- or vasopressin-stimulated CCDs in vitro. Cell surface labelling showed that intrinsic activity rather than cell surface expression of Na+-K+ATPase was controlled by pERK1/2. Our data demonstrate that basal constitutive activity of ERK1/2 pathway controls Na+-K+ATPase activity and transepithelial sodium transport in the principal cell. Résumé tout public Les mécanismes de régulation de la réabsorption fine du sodium dans la partie distale du néphron (l'unité fonctionnelle du rein) ont un rôle essentiel dans le maintien de l'homéostasie de la composition et du volume des fluides extracellulaires. Ces mécanismes permettent de maintenir une pression sanguine effective. Dans les cellules principales du tube collecteur, une région spécifique du néphron distal, le transport de sodium dépend essentiellement de l'activité de deux transporteurs de sodium : le canal épithélial à sodium (ENaC) et la pompe sodium-potassium-adénosine-triphosphatase (Na+-K+ATPase). Afin de répondre aux besoins de l'organisme, l'activité de ces deux molécules de transport est en partie régulée par des hormones dont l'aldostérone, la vasopressine et l'insuline. Dans les cellules principales du tube collecteur, la vasopressine régule le transport de sodium en deux étapes : une étape rapide et une étape lente dite « génomique ». Durant l'étape lente, la vasopressine régule l'expression de gènes pouvant être impliqués dans le transport de sodium, dont notamment ceux d'ENaC et de la Na+-K+ATPase. Parmi les gènes dont l'expression est augmentée par la vasopressine, celui de VIP32 (vasopressin induced protein : VIP) fait l'objet de cette étude. Le but de mon travail a été d'étudier, dans un système d'expression hétérologue (l'ovocyte de Xenopus leavis), l'implication de VIP32 dans le transport de sodium. Nous avons montré que VIP32 est capable d'activer un mécanisme moléculaire en cascade appelé MAPK (mitogen-activated protein kinase : MAPK) et est aussi capable de diminuer l'activité d'ENaC. Parallèlement, dans une lignée de cellules principales de tube collecteur les mpkCCDc14, nous avons montré que le taux basal d'activité de la cascade MAPK est capable de réguler l'activité de la Na+-K+ATPase, tandis qu'il n'influence pas l'activité d'ENaC.