Obesity Resistance Promotes Mild Contractile Dysfunction Associated with Intracellular Ca2+ Handling

Abstract Background: Diet-induced obesity is frequently used to demonstrate cardiac dysfunction. However, some rats, like humans, are susceptible to developing an obesity phenotype, whereas others are resistant to that. Objective: To evaluate the association between obesity resistance and cardiac function, and the impact of obesity resistance on calcium handling. Methods: Thirty-day-old male Wistar rats were distributed into two groups, each with 54 animals: control (C; standard diet) and obese (four palatable high-fat diets) for 15 weeks. After the experimental protocol, rats consuming the high-fat diets were classified according to the adiposity index and subdivided into obesity-prone (OP) and obesity-resistant (OR). Nutritional profile, comorbidities, and cardiac remodeling were evaluated. Cardiac function was assessed by papillary muscle evaluation at baseline and after inotropic maneuvers. Results: The high-fat diets promoted increase in body fat and adiposity index in OP rats compared with C and OR rats. Glucose, lipid, and blood pressure profiles remained unchanged in OR rats. In addition, the total heart weight and the weight of the left and right ventricles in OR rats were lower than those in OP rats, but similar to those in C rats. Baseline cardiac muscle data were similar in all rats, but myocardial responsiveness to a post-rest contraction stimulus was compromised in OP and OR rats compared with C rats. Conclusion: Obesity resistance promoted specific changes in the contraction phase without changes in the relaxation phase. This mild abnormality may be related to intracellular Ca2+ handling.

Roles of Bassoon in assembling the presynaptic active zone for neurotransmitter release

Magdeburg, Univ., Fak. f��r Naturwiss., Diss., 2010

Influence de deux milieux s��quentiels pour la culture d'embryons sur la production d'hormone gonadotrophine chorionique et de progest��rone par des cellules JEG-3 et BeWo [Effects of different embryo development media on hCG and progesterone release by JEG-3 and BeWo cells]

Current in vitro fertilisation (IVF) practice requires synchronisation between the��environment of cultured oocytes and embryos and the surroundings to what they would have��been exposed to in vivo. Commercial, sequential media follow this requirement but their exact��composition is not available. We have compared two widely used IVF culture media systems using��the two choriocarcinoma cell lines JEG-3 and BeWo. The two hormones hCG and progesterone��were determined in the culture supernatants as endpoints. In both cell lines, but in a more��pronounced way in JEG-3, progesterone rather than hCG production was stimulated, and a��higher hormone release was observed in the fertilisation than in the cleavage media. Differences��between manufacturers were small and did not favour one system over the other. We conclude��that both sequential media systems can be equally well used in current IVF laboratory practice.���� 2012 Elsevier Masson SAS. All rights reserved.

D-amphetamine fails to increase extracellular dopamine levels in mice lacking alpha 1b-adrenergic receptors: relationship between functional and nonfunctional dopamine release.

It was found recently that locomotor and rewarding effects of psychostimulants and opiates were dramatically decreased or suppressed in mice lacking alpha1b-adrenergic receptors [alpha1b-adrenergic receptor knock-outs (alpha1bAR-KOs)] (Drouin et al., 2002). Here we show that blunted locomotor responses induced by 3 and 6 mg/kg d-amphetamine in alpha1bAR-KO mice [-84 and -74%, respectively, when compared with wild-type (WT) mice] are correlated with an absence of d-amphetamine-induced increase in extracellular dopamine (DA) levels in the nucleus accumbens of alpha1bAR-KO mice. Moreover, basal extracellular DA levels in the nucleus accumbens are lower in alpha1bAR-KO than in WT littermates (-28%; p < 0.001). In rats however, prazosin, an alpha1-adrenergic antagonist, decreases d-amphetamine-induced locomotor hyperactivity without affecting extracellular DA levels in the nucleus accumbens, a finding related to the presence of an important nonfunctional release of DA (Darracq et al., 1998). We show here that local d-amphetamine releases nonfunctional DA with the same affinity but a more than threefold lower amplitude in C57BL6/J mice than in Sprague Dawley rats. Altogether, this suggests that a trans-synaptic mechanism amplifies functional DA into nonfunctional DA release. Our data confirm the presence of a powerful coupling between noradrenergic and dopaminergic neurons through the stimulation of alpha1b-adrenergic receptors and indicate that nonfunctional DA release is critical in the interpretation of changes in extracellular DA levels. These results suggest that alpha1b-adrenergic receptors may be important therapeutic pharmacological targets not only in addiction but also in psychosis because most neuroleptics possess anti-alpha1-adrenergic properties.

In vivo and in vitro effects of somatostatin and insulin on glucagon release in a human glucagonoma.

Inhibition of pancreatic glucagon secretion has been reported to be mediated by glucose, insulin and somatostatin. As no human pancreatic alpha-cell lines are available to study in vitro the relative importance of insulin and glucose in the control of pancreatic glucagon release, we investigated a patient presenting with a malignant glucagonoma who underwent surgical resection of the tumour. Functional somatostatin receptors were present as octreotide administration decreased basal glucagon and insulin secretion by 52 and 74%, respectively. The removed tumour was immunohistochemically positive for glucagon, chromogranin A and pancreatic polypeptide but negative for insulin, gastrin and somatostatin. The glucagonoma cells were also isolated and cultured in vitro. Incubation experiments revealed that change from high (10 mM) to low (1 mM) glucose concentration was unable to stimulate glucagon secretion. A dose-dependent inhibition of glucagon release by insulin was however, observed at low glucose concentration. These findings demonstrate that insulin could inhibit glucagon secretion in vitro in the absence of elevated glucose concentrations. These data suggest, as observed in vivo and in vitro in several animal studies, that glucopenia-induced glucagon secretion in humans is not mediated by a direct effect of low glucose on alpha-cells but possibly by a reduction of insulin-mediated alpha-cell suppression and/or an indirect neuronal stimulation of glucagon release.

Etude des propri��t��s dynamiques de la s��cr��tion r��gul��e des v��sicules glutamatergiques des astrocytes

RESUME GRAND PUBLICLe cerveau est compos�� de diff��rents types cellulaires, dont les neurones et les astrocytes. Faute de moyens pour les observer, les astrocytes sont tr��s longtemps rest��s dans l'ombre alors que les neurones, b��n��ficiant des outils ad hoc pour ��tre stimul��s et ��tudi��s, ont fait l'objet de toutes les attentions. Le d��veloppement de l'imagerie cellulaire et des outils fluorescents ont permis d'observer ces cellules non ��lectriquement excitables et d'obtenir des informations qui laissent penser que ces cellules sont loin d'��tre passives et participent activement au fonctionnement c��r��bral. Cette participation au fonctionnement c��r��bral se fait en partie par le biais de la lib��ration de substances neuro-actives (appell��es gliotransmetteurs) que les astrocytes lib��rent �� proximit�� des synapses permettant ainsi de moduler le fonctionnement neuronal. Cette lib��ration de gliotransmetteurs est principalement caus��e par l'activit�� neuronale que les astrocytes sont capables de sentir. N��anmoins, nous savons encore peu de chose sur les propri��t��s pr��cises de la lib��ration des gliotransmetteurs. Comprendre les propri��t��s spatio-temporelles de cette lib��ration est essentiel pour comprendre le mode de communication de ces cellules et leur implication dans la transmission de l'information c��r��brale. En utilisant des outils fluorescents r��cemment d��velopp��s et en combinant diff��rentes techniques d'imagerie cellulaire, nous avons pu obtenir des informations tr��s pr��cises sur la lib��ration de ces gliotransmetteurs par les astrocytes. Nous avons ainsi confirm�� que cette lib��ration ��tait un processus tr��s rapide et qu'elle ��tait contr��l��e par des augmentations de calcium locales et rapides. Nous avons ��galement d��crit une organisation complexe de la machinerie supportant la lib��ration des gliotransmetteurs. Cette organisation complexe semble ��tre �� la base de la lib��ration extr��mement rapide des gliotransmetteurs. Cette rapidit�� de lib��ration et cette complexit�� structurelle semblent indiquer que les astrocytes sont des cellules particuli��rement adapt��es �� une communication rapide et qu'elles peuvent, au m��me titre que les neurones dont elles seraient les partenaires l��gitimes, participer �� la transmission et �� l'int��gration de l'information c��r��brale.RESUMEDe petites v��sicules, les �� SLMVs �� ou �� Synaptic Like MicroVesicles ��, exprimant des transporteurs v��siculaires du glutamate (VGluTs) et lib��rant du glutamate par exocytose r��gul��e, ont r��cemment ��t�� d��crites dans les astrocytes en culture et in situ. N��anmoins, nous savons peu de chose sur les propri��t��s pr��cises de la s��cr��tion de ces SLMVs. Contrairement aux neurones, le couplage stimuluss��cr��tion des astrocytes n'est pas bas�� sur l'ouverture des canaux calciques membranaires mais n��cessite l'intervention de seconds messagers et la lib��ration du calcium par le reticulum endoplasmique (RE). Comprendre les propri��t��s spatio-temporelles de la s��cr��tion astrocytaire est essentiel pour comprendre le mode de communication de ces cellules et leur implication dans la transmission de l'information c��r��brale. Nous avons utilis�� des outils fluorescents r��cemment d��velopp��s pour ��tudier le recyclage des v��sicules synaptiques glutamatergiques comme les colorants styryles et la pHluorin afin de pouvoir suivre la s��cr��tion des SLMVs �� l'��chelle de la cellule mais ��galement �� l'��chelle des ��v��nements. L'utilisation combin��e de l'��pifluorescence et de la fluorescence �� onde ��vanescente nous a permis d'obtenir une r��solution temporelle et spatiale sans pr��c��dent. Ainsi avons-nous confirm�� que la s��cr��tion r��gul��e des astrocytes ��tait un processus tr��s rapide (de l'ordre de quelques centaines de millisecondes). Nous avons d��couvert que cette s��cr��tion est contr��l��e par des augmentations de calcium locales et rapides. Nous avons ��galement d��crit des compartiments cytosoliques d��limit��s par le RE �� proximit�� de la membrane plasmique et contenant les SLMVs. Cette organisation semble ��tre �� la base du couplage rapide entre l'activation des GPCRs et la s��cr��tion. L'existence de compartiments subcellulaires ind��pendants permettant de contenir les messagers intracellulaires et de limiter leur diffusion semble compenser de mani��re efficace la nonexcitabilit�� ��lectrique des astrocytes. Par ailleurs, l'existence des diff��rents pools de v��sicules recrut��s s��quentiellement et fusionnant selon des modalit��s distinctes ainsi que l'existence de m��canismes permettant le renouvellement de ces pools lors de la stimulation sugg��rent que les astrocytes peuvent faire face �� une stimulation soutenue de leur s��cr��tion. Ces donn��es sugg��rent que la lib��ration de gliotransmetteurs par exocytose r��gul��e n'est pas seulement une propri��t�� des astrocytes en culture mais bien le r��sultat d'une forte sp��cialisation de ces cellules pour la s��cr��tion. La rapidit�� de cette s��cr��tion donne aux astrocytes toutes les comp��tences pour pouvoir intervenir de mani��re active dans la transmission et l'int��gration de l'information.ABSTRACTRecently, astrocytic synaptic like microvesicles (SLMVs), that express vesicular glutamate transporters (VGluTs) and are able to release glutamate by Ca2+-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Nevertheless, little is known about the specific properties of regulated secretion in astrocytes. Important differences may exist between astrocytic and neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca2+ from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We took advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses like styryl dyes and pHluorin in order to follow exocytosis and endocytosis of SLMVs at the level of the entire cell or at the level of single event. We combined epifluorescence and total internal reflection fluorescence imaging to investigate, with unprecedented temporal and spatial resolution, the events underlying the stimulus-secretion in astrocytes. We confirmed that exo-endocytosis process in astrocytes proceeds with a time course on the millisecond time scale. We discovered that SLMVs exocytosis is controlled by local and fast Ca2+ elevations; indeed submicrometer cytosolic compartments delimited by endoplasmic reticulum (ER) tubuli reaching beneath the plasma membrane and containing SLMVs. Such complex organization seems to support the fast stimulus-secretion coupling reported here. Independent subcellular compartments formed by ER, SLMVs and plasma membrane containing intracellular messengers and limiting their diffusion seem to compensate efficiently the non-electrical excitability of astrocytes. Moreover, the existence of two pools of SLMVs which are sequentially recruited suggests a compensatory mechanisms allowing the refill of SLMVs and supporting exocytosis process over a wide range of multiple stimuli. These data suggest that regulated secretion is not only a feature of cultured astrocytes but results from a strong specialization of these cells. The rapidity of secretion demonstrates that astrocytes are able to actively participate in brain information transmission and processing.

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

What do we know about gliotransmitter release from astrocytes?

Astrocytes participate in information processing by actively modulating synaptic properties via gliotransmitter release. Various mechanisms of astrocytic release have been reported, including release from storage organelles via exocytosis and release from the cytosol via plasma membrane ion channels and pumps. It is still not fully clear which mechanisms operate under which conditions, but some of them, being Ca(2+)-regulated, may be physiologically relevant. The properties of Ca(2+)-dependent transmitter release via exocytosis or via ion channels are different and expected to produce different extracellular transmitter concentrations over time and to have distinct functional consequences. The molecular aspects of these two release pathways are still under active investigation. Here, we discuss the existing morphological and functional evidence in support of either of them. Transgenic mouse models, specific antagonists and localization studies have provided insight into regulated exocytosis, albeit not in a systematic fashion. Even more remains to be uncovered about the details of channel-mediated release. Better functional tools and improved ultrastructural approaches are needed in order fully to define specific modalities and effects of astrocytic gliotransmitter release pathways.

Contribution of TIR domain-containing adapter inducing IFN-beta-mediated IL-18 release to LPS-induced liver injury in mice.

BACKGROUND/AIMS: After treatment with heat-killed Propionibacterium acnes mice show dense hepatic granuloma formation. Such mice develop liver injury in an interleukin (IL)-18-dependent manner after challenge with a sublethal dose LPS. As previously shown, LPS-stimulated Kupffer cells secrete IL-18 depending on caspase-1 and Toll-like receptor (TLR)-4 but independently of its signal adaptor myeloid differentiation factor 88 (MyD88), suggesting importance of another signal adaptor TIR domain-containing adapter inducing IFN-beta (TRIF). Nalp3 inflammasome reportedly controls caspase-1 activation. Here we investigated the roles of MyD88 and TRIF in P. acnes-induced hepatic granuloma formation and LPS-induced caspase-1 activation for IL-18 release. METHODS: Mice were sequentially treated with P. acnes and LPS, and their serum IL-18 levels and liver injuries were determined by ELISA and ALT/AST measurement, respectively. Active caspase-1 in LPS-stimulated Kupffer cells was determined by Western blotting. RESULTS: Macrophage-ablated mice lacked P. acnes-induced hepatic granuloma formation and LPS-induced serum IL-18 elevation and liver injury. Myd88(-/-) Kupffer cells, but not Trif(-/-) cells, exhibited normal caspase-1 activation upon TLR4 engagement in vitro. Myd88(-/-) mice failed to develop hepatic granulomas after P. acnes treatment and liver injury induced by LPS challenge. In contrast, Trif(-/-) mice normally formed the hepatic granulomas, but could not release IL-18 or develop the liver injury. Nalp3(-/-) mice showed the same phenotypes of Trif(-/-) mice. CONCLUSIONS: Propionibacterium acnes treatment MyD88-dependently induced hepatic granuloma formation. Subsequent LPS TRIF-dependently activated caspase-1 via Nalp3 inflammasome and induced IL-18 release, eventually leading to the liver injury.

Nicotine-induced release of vasopressin in the conscious rat: role of opioid peptides and hemodynamic effects.

Nicotine has been shown to stimulate the release of vasopressin and to cause significant hemodynamic changes. The mechanisms leading to enhanced vasopressin secretion and the vascular consequences of the high plasma vasopressin levels during nicotine infusion have not yet been determined. Therefore, the purposes of the present study were 1) to examine in normal conscious rats the role of opioid peptides in the nicotine-induced increase in plasma vasopressin levels and 2) to assess the role of vasopressin in the hemodynamic effects of nicotine (20 micrograms/min for 15 min) using a specific V1 antagonist of the vascular actions of vasopressin. Plasma vasopressin levels were significantly increased in the nicotine-treated animals (39.5 +/- 10 vs. 3.7 +/- 0.6 pg/ml in the controls, P less than .01). Pretreatment with naloxone, an antagonist of opioids at their receptors, did not reduce the vasopressin levels (47.7 +/- 9 pg/ml). Nicotine also increased mean blood pressure (122.5 +/- 2.5 to 145.2 +/- 3.3 mm Hg, P less than .01) and decreased heart rate (461 +/- 6 to 386 +/- 14.5 beats/min, P less than .05). Administration of the vasopressin V1 antagonist before the nicotine infusion did not affect the systemic hemodynamics or the regional blood flow distribution, as assessed by radiolabeled microspheres. Thus, these results suggest that the nicotine-induced secretion of vasopressin is not mediated by opioid receptors and that the high plasma vasopressin levels do not exert any significant hemodynamic effect on cardiac output or blood flow distribution.

Pharmacokinetic profile of two different pharmaceutical forms of theophylline (a slow release tablet and a syrup) after multiple dose administration to healthy human volunteers

Due to the narrow therapeutic range of theophyline, plasma concentrations of this drug are monitored in patients undergoing chronic therapy. Slow-release preparations avoid the fluctuations in plasma levels and improve patient compliance. In this study, we have compared the pharmacokinetic profiles of a theophylline slow-release tablet and a syrup form, when administered in multiple doses to healthy adult volunteers. The classification based upon releasing patterns is confirmed.

Liver hyperplasia and paradoxical regulation of glycogen metabolism and glucose-sensitive gene expression in GLUT2-null hepatocytes. Further evidence for the existence of a membrane-based glucose release pathway.

We investigated the impact of GLUT2 gene inactivation on the regulation of hepatic glucose metabolism during the fed to fast transition. In control and GLUT2-null mice, fasting was accompanied by a approximately 10-fold increase in plasma glucagon to insulin ratio, a similar activation of liver glycogen phosphorylase and inhibition of glycogen synthase and the same elevation in phosphoenolpyruvate carboxykinase and glucose-6-phosphatase mRNAs. In GLUT2-null mice, mobilization of glycogen stores was, however, strongly impaired. This was correlated with glucose-6-phosphate (G6P) levels, which remained at the fed values, indicating an important allosteric stimulation of glycogen synthase by G6P. These G6P levels were also accompanied by a paradoxical elevation of the mRNAs for L-pyruvate kinase. Re-expression of GLUT2 in liver corrected the abnormal regulation of glycogen and L-pyruvate kinase gene expression. Interestingly, GLUT2-null livers were hyperplasic, as revealed by a 40% increase in liver mass and 30% increase in liver DNA content. Together, these data indicate that in the absence of GLUT2, the G6P levels cannot decrease during a fasting period. This may be due to neosynthesized glucose entering the cytosol, being unable to diffuse into the extracellular space, and being phosphorylated back to G6P. Because hepatic glucose production is nevertheless quantitatively normal, glucose produced in the endoplasmic reticulum may also be exported out of the cell through an alternative, membrane traffic-based pathway, as previously reported (Guillam, M.-T., Burcelin, R., and Thorens, B. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 12317-12321). Therefore, in fasting, GLUT2 is not required for quantitative normal glucose output but is necessary to equilibrate cytosolic glucose with the extracellular space. In the absence of this equilibration, the control of hepatic glucose metabolism by G6P is dominant over that by plasma hormone concentrations.

Circulating soluble CR1 (CD35). Serum levels in diseases and evidence for its release by human leukocytes.

C receptor type 1 (CR1, CD35) is present in a soluble form in plasma (sCR1). Soluble CR1 was measured with a specific ELISA assay in normal individuals and in patients with different diseases. The mean serum concentration of sCR1 in 31 normal donors was 31.4 +/- 7.8 ng/ml, and was identical in plasma. An increase in sCR1 was observed in 36 patients with end-stage renal failure on dialysis (54.8 +/- 11.7 ng/ml, p < 0.0001), and in 22 patients with liver cirrhosis (158.3 +/- 49.9 ng/ml, p < 0.0001). The mean sCR1 levels dropped from 181 +/- 62.7 to 52.1 +/- 24.0 ng/ml (p < 0.001) in nine patients who underwent liver transplantation, and was 33.5 +/- 7.3 in 10 patients with functioning renal grafts, indicating that the increase in sCR1 was reversible. Soluble CR1 was elevated in some hematologic malignancies (> 47 ng/ml), which included B cell lymphoma (12/19 patients), Hodgkin's lymphoma (4/4), and chronic myeloproliferative syndromes (4/5). By contrast, no increase was observed in acute myeloid or lymphoblastic leukemia (10) or myeloma (5). In two patients with chronic myeloproliferative syndromes, sCR1 decreased rapidly after chemotherapy. The mean concentration of sCR1 was not significantly modified in 181 HIV-infected patients at various stages of the disease (34.8 +/- 14.4 ng/ml), and in 13 patients with active SLE (38.3 +/- 19.6 ng/ml), although in both groups the number of CR1 was diminished on E. There was a weak but significant correlation between sCR1 and CR1 per E in HIV infection and SLE (r = 0.39, p < 0.0001, and r = 0.60, p < 0.03 respectively). In vitro, monocytes, lymphocytes, and neutrophils were found to release sCR1 into culture supernatants. In vivo, sCR1 was detected in the serum of SCID mice populated with human peripheral blood leukocytes. The sCR1 levels correlated with those of human IgG (r = 0.97, p < 0.0001), suggesting synthesis of sCR1 by the transferred lymphocytes. The mechanisms underlining the increased levels of sCR1 and its biologic consequences remain to be defined.

Eokines: synthesis, storage and release from human eosinophils

Eosinophils are prominent inflammatory cells in asthma and other allergic disorders, as well as in helminthic parasite infections. Recently, eosinophils have been reported to synthesize and store a range of regulatory proteins within their secretory granules (eokines). Eokines comprise a group of cytokines, chemokines, and growth factors which are elaborated by eosinophils. These proteins, and the messages which encode them, appear to be identical to those produced by lymphocytes and other tissues. Interestingly, immunoreactivity to many of these eokines has been found to co-localize to the eosinophil��s secretory granules. In this review, we have discussed the repertoire of 18 eokines so far identified in eosinophils, and focused on four of these, namely, interleukin-2 (IL-2), IL-4, granulocyte/macrophage colony-stimulating factor (GM-CSF), and RANTES. These four eokines co-localize to the crystalloid granules in eosinophils, as shown in studies using subcellular fractionation and immunogold labeling in electron microscopy. During stimulation by physiological triggers, for example, with serum-coated particles, eosinophils release these mediators into the surrounding supernatant. In addition, eokines are likely to be synthesized within eosinophils rather than taken up by endocytosis, as show in detection of mRNA for each of these proteins using in situ hybridization, RT-PCR, and in the case of RANTES, in situ RT-PCR. Eokines synthesis and release from eosinophils challenges the commonly held notion that these cells act downstream of key elements in immune system, and indicate that they may instead belong to the afferent arm of immunity.