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A bstract The ability to infer properties of primordial inflation relies on the conservation of the superhorizon perturbations between their exit during inflation, and their re-entry during radiation era. Any considerable departure from this property would require reinterpreting the data. This is why it is important to understand how superhorizon perturbations interact with the thermal plasma driving the radiation dominated Universe. We model the plasma by free photons in a thermal state and compute the one-loop correction to the power spectrum of primordial tensor perturbations. This correction grows in time and is not suppressed by any small parameter. While one-loop result is not reliable because it invalidates perturbation theory, it signals potentially interesting effects that should be investigated further.

Neutrophils migrate into tissues as innate immune responders. Nourshargh and colleagues demonstrate that neutrophils can migrate in the reverse direction in vivo under inflammatory conditions and may contribute to the dissemination of systemic inflammation. The migration of neutrophils into inflamed tissues is a fundamental component of innate immunity. A decisive step in this process is the polarized migration of blood neutrophils through endothelial cells (ECs) lining the venular lumen (transendothelial migration (TEM)) in a luminal-to-abluminal direction. By real-time confocal imaging, we found that neutrophils had disrupted polarized TEM ('hesitant' and 'reverse') in vivo . We noted these events in inflammation after ischemia-reperfusion injury, characterized by lower expression of junctional adhesion molecule C (JAM-C) at EC junctions, and they were enhanced by blockade or genetic deletion of JAM-C in ECs. Our results identify JAM-C as a key regulator of polarized neutrophil TEM in vivo and suggest that reverse TEM of neutrophils can contribute to the dissemination of systemic inflammation.

Imbalance of the excitatory neurotransmitter glutamate and of the inhibitory neurotransmitter GABA is one of several causes of seizures. ATP has also been implicated in epilepsy. However, little is known about the mechanisms involved in the release of ATP from cells and the consequences of the altered ATP signaling during seizures. Pannexin1 (Panx1) is found in astrocytes and in neurons at high levels in the embryonic and young postnatal brain, declining in adulthood. Panx1 forms large-conductance voltage sensitive plasma membrane channels permeable to ATP that are also activated by elevated extracellular K(+) and following P2 receptor stimulation. Based on these properties, we hypothesized that Panx1 channels may contribute to seizures by increasing the levels of extracellular ATP. Using pharmacological tools and two transgenic mice deficient for Panx1 we show here that interference with Panx1 ameliorates the outcome and shortens the duration of kainic acid-induced status epilepticus. These data thus indicate that the activation of Panx1 in juvenile mouse hippocampi contributes to neuronal hyperactivity in seizures.

Cell volume homeostasis is vital for the maintenance of optimal protein density and cellular function. Numerous mammalian cell types are routinely exposed to acute hypertonic challenge and shrink. Molecular crowding modifies biochemical reaction rates and decreases macromolecule diffusion. Cell volume is restored rapidly by ion influx but at the expense of elevated intracellular sodium and chloride levels that persist long after challenge. Although recent studies have highlighted the role of molecular crowding on the effects of hypertonicity, the effects of ionic imbalance on cellular trafficking dynamics in living cells are largely unexplored. By tracking distinct fluorescently labeled endosome/vesicle populations by live-cell imaging, we show that vesicle motility is reduced dramatically in a variety of cell types at the onset of hypertonic challenge. Live-cell imaging of actin and tubulin revealed similar arrested microfilament motility upon challenge. Vesicle motility recovered long after cell volume, a process that required functional regulatory volume increase and was accelerated by a return of extracellular osmolality to isosmotic levels. This delay suggests that, although volume-induced molecular crowding contributes to trafficking defects, it alone cannot explain the observed effects. Using fluorescent indicators and FRET-based probes, we found that intracellular ATP abundance and mitochondrial potential were reduced by hypertonicity and recovered after longer periods of time. Similar to the effects of osmotic challenge, isovolumetric elevation of intracellular chloride concentration by ionophores transiently decreased ATP production by mitochondria and abated microfilament and vesicle motility. These data illustrate how perturbed ionic balance, in addition to molecular crowding, affects membrane trafficking. Significance Maintenance of cell volume and ionic homeostasis are fundamental to cell function. Cells shrink when extracellular osmolality is increased, and although mechanisms controlling cell volume recovery are well documented, the mechanisms underlying changes in membrane trafficking that occur during adaptation to high osmolality are not well understood. Molecular crowding arising from cell shrinkage can alter protein structure and has been proposed to provoke changes in trafficking events. However, cell volume recovery involves a rapid influx of ions, and the effects of ionic imbalance on trafficking dynamics in living cells remain largely unexplored. We found that high levels of chloride and loss of ATP, in addition to molecular crowding, contribute to altered cytoskeletal and vesicular dynamics during hypertonic stress.

Type 1 diabetes mellitus (T1DM) is due to the selective destruction of islet beta cells by immune cells. Current therapies focused on repressing the immune attack or stimulating beta cell regeneration still have limited clinical efficacy. Therefore, it is timely to identify innovative targets to dampen the immune process, while promoting beta cell survival and function. Liver receptor homologue-1 (LRH-1) is a nuclear receptor that represses inflammation in digestive organs, and protects pancreatic islets against apoptosis. Here, we show that BL001, a small LRH-1 agonist, impedes hyperglycemia progression and the immune-dependent inflammation of pancreas in murine models of T1DM, and beta cell apoptosis in islets of type 2 diabetic patients, while increasing beta cell mass and insulin secretion. Thus, we suggest that LRH-1 agonism favors a dialogue between immune and islet cells, which could be druggable to protect against diabetes mellitus. Type 1 diabetes mellitus (T1DM) is characterized by beta cell loss because of an autoimmune attack. Here the authors show that an agonist for LRH-1/NR5A2, a nuclear receptor known to be protective against beta cell apoptosis, inhibits immune-mediated inflammation and hyperglycemia in T1DM mouse models.

Previous work has revealed that Cx36, the sole connexin expressed in the insulin-producing beta cells, enhances the secretion of insulin, and promotes the resistance of beta cells against pro-inflammatory cytokines. In parallel, the anti-diabetic sulphonylurea glibenclamide was shown to promote the assembly and function of Cx36 channels. Here, we assessed whether glibenclamide could protect the insulin-producing cells against conditions mimicking those expected at the onset of type 1 diabetes. We found that the drug 1) protected in vitro the mouse MIN6 cells from the apoptosis and loss of Cx36, which are induced by Th1 cytokines; 2) prevented the development of hyperglycemia as well as the loss of beta cells and Cx36, which rapidly develop with aging in untreated NOD mice; 3) modified the proportion of effector CD4+ and CD8+ T cells in pancreatic draining lymph nodes. The data imply that an early glibenclamide treatment may help protecting beta cells against the autoimmune attack, which triggers the development of type 1 diabetes.

Cx36-Mediated Coupling Reduces β-Cell Heterogeneity, Confines the Stimulating Glucose Concentration Range, and Affects Insulin Release Kinetics Stephan Speier 1 , Asllan Gjinovci 2 , Anne Charollais 2 , Paolo Meda 2 and Marjan Rupnik 1 1 Neuroendocrinology, European Neuroscience Institute Göttingen, Göttingen, Germany 2 Department of Cell Physiology and Metabolism, University of Geneva, Genève, Switzerland Address correspondence and reprint requests to Stephan Speier, The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital L1, Stockholm, Sweden. E-mail: stephan.speier{at}ki.se Abstract We studied the effect of gap junctional coupling on the excitability of β-cells in slices of pancreas, which provide a normal environment for islet cells. The electrophysiological properties of β-cells from mice (C57Bl/6 background) lacking the gap junction protein connexin36 (Cx36 −/− ) were compared with heterozygous (Cx36 +/− ) and wild-type littermates (Cx36 +/+ ) and with frequently used wild-type NMRI mice. Most electrophysiological characteristics of β-cells were found to be unchanged after the knockout of Cx36, except the density of Ca 2+ channels, which was increased in uncoupled cells. With closed ATP-sensitive K + (K ATP ) channels, the electrically coupled β-cells of Cx36 +/+ and Cx36 +/− mice were hyperpolarized by the membrane potential of adjacent, inactive cells. Additionally, the hyperpolarization of one β-cell could attenuate or even stop the electrical activity of nearby coupled cells. In contrast, β-cells of Cx36 −/− littermates with blocked K ATP channels rapidly depolarized and exhibited a continuous electrical activity. Absence of electrical coupling modified the electrophysiological properties of β-cells consistent with the reported increase in basal insulin release and altered the switch on/off response of β-cells during an acute drop of the glucose concentration. Our data indicate an important role for Cx36-gap junctions in modulating stimulation threshold and kinetics of insulin release. Cx36, connexin36 KATP channel, ATP-sensitive K+ channel Footnotes The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted December 15, 2006. Received February 19, 2006. DIABETES

Loss of Connexin36 Channels Alters β-Cell Coupling, Islet Synchronization of Glucose-Induced Ca 2+ and Insulin Oscillations, and Basal Insulin Release Magalie A. Ravier 1 , Martin Güldenagel 2 , Anne Charollais 3 , Asllan Gjinovci 3 , Dorothée Caille 3 , Goran Söhl 2 , Claes B. Wollheim 3 , Klaus Willecke 2 , Jean-Claude Henquin 1 and Paolo Meda 3 1 Unit of Endocrinology and Metabolism, University of Louvain Faculty of Medicine, Brussels, Belgium 2 Institute of Genetics, University of Bonn, Bonn, Germany 3 Department of Cell Physiology and Metabolism, Geneva University Medical Center, Geneva, Switzerland Address correspondence and reprint requests to Paolo Meda, MD, Department of Cell Physiology and Metabolism, University of Geneva, C.M.U., 1 rue Michel Servet, 1211 Geneva 4, Switzerland. E-mail: paolo.meda{at}medecine.unige.ch . Jean-Claude Henquin, Unit of Endocrinology and Metabolism, University of Louvain Faculty of Medicine, UCL 55.30, Avenue Hippocrate 55, B-1200 Brussels, Belgium. E-mail: henquin{at}endo.ucl.ac.be Abstract Normal insulin secretion requires the coordinated functioning of β-cells within pancreatic islets. This coordination depends on a communications network that involves the interaction of β-cells with extracellular signals and neighboring cells. In particular, adjacent β-cells are coupled via channels made of connexin36 (Cx36). To assess the function of this protein, we investigated islets of transgenic mice in which the Cx36 gene was disrupted by homologous recombination. We observed that compared with wild-type and heterozygous littermates that expressed Cx36 and behaved as nontransgenic controls, mice homozygous for the Cx36 deletion (Cx36 −/− ) featured β-cells devoid of gap junctions and failing to exchange microinjected Lucifer yellow. During glucose stimulation, islets of Cx36 −/− mice did not display the regular oscillations of intracellular calcium concentrations ([Ca 2+ ] i ) seen in controls due to the loss of cell-to-cell synchronization of [Ca 2+ ] i changes. The same islets did not release insulin in a pulsatile fashion, even though the overall output of the hormone in response to glucose stimulation was normal. However, under nonstimulatory conditions, islets lacking Cx36 showed increased basal release of insulin. These data show that Cx36-dependent signaling is essential for the proper functioning of β-cells, particularly for the pulsatility of [Ca 2+ ] i and insulin secretion during glucose stimulation. [Ca2+]i, intracellular calcium concentration Cx36, connexin36 LY, Lucifer yellow Footnotes M.A.R., M.G., and A.C. contributed equally to this work. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted March 11, 2005. Received December 8, 2004. DIABETES

Dietary Phytoestrogens Activate AMP-Activated Protein Kinase With Improvement in Lipid and Glucose Metabolism Christopher R. Cederroth 1 , Manlio Vinciguerra 2 , Asllan Gjinovci 2 , Françoise Kühne 1 , Marcella Klein 3 , Manon Cederroth 1 , Dorothée Caille 2 , Mariane Suter 4 , Dietbert Neumann 4 , Richard W. James 5 , Daniel R. Doerge 6 , Theo Wallimann 4 , Paolo Meda 2 , Michelangelo Foti 2 , Françoise Rohner-Jeanrenaud 3 , Jean-Dominique Vassalli 1 and Serge Nef 1 1 Department of Genetic Medicine and Development and National Centre of Competence in Research–Frontiers in Genetics, University of Geneva, Geneva, Switzerland 2 Department of Cellular Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland 3 Laboratory of Metabolism, University of Geneva, Geneva, Switzerland 4 Institute of Cell Biology, ETH Zürich, Zürich, Switzerland 5 Clinical Diabetes Unit, Division of Endocrinology, Diabetology, and Nutrition, Faculty of Medicine, Department of Internal Medicine, University of Geneva, Geneva, Switzerland 6 National Center for Toxicological Research, Jefferson, Arkansas Corresponding author: Serge Nef, Department of Genetic Medicine and Development and National Centre of Competence in Research–Frontiers in Genetics, University of Geneva, 1211 Geneva 4, Switzerland. E-mail: serge.nef{at}medecine.unige.ch Abstract OBJECTIVE— Emerging evidence suggests that dietary phytoestrogens can have beneficial effects on obesity and diabetes, although their mode of action is not known. Here, we investigate the mechanisms mediating the action of dietary phytoestrogens on lipid and glucose metabolism in rodents. RESEARCH DESIGN AND METHODS— Male CD-1 mice were fed from conception to adulthood with either a high soy–containing diet or a soy-free diet. Serum levels of circulating isoflavones, ghrelin, leptin, free fatty acids, triglycerides, and cholesterol were quantified. Tissue samples were analyzed by quantitative RT-PCR and Western blotting to investigate changes of gene expression and phosphorylation state of key metabolic proteins. Glucose and insulin tolerance tests and euglycemic-hyperinsulinemic clamp were used to assess changes in insulin sensitivity and glucose uptake. In addition, insulin secretion was determined by in situ pancreas perfusion. RESULTS— In peripheral tissues of soy-fed mice, especially in white adipose tissue, phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase was increased, and expression of genes implicated in peroxisomal fatty acid oxidation and mitochondrial biogenesis was upregulated. Soy-fed mice also showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity due to increased glucose uptake into skeletal muscle. Thus, mice fed with a soy-rich diet have improved adipose and glucose metabolism. CONCLUSIONS— Dietary soy could prove useful to prevent obesity and associated disorders. Activation of the AMPK pathway by dietary soy is likely involved and may mediate the beneficial effects of dietary soy in peripheral tissues. ACC, acetyl-CoA carboxylase AMPK, AMP-activated protein kinase AUC, area under curve DEXA, dual-energy X-ray absorptiometry ER, estrogen receptor ERRα, estrogen receptor–related receptor α FFA, free fatty acid GLP-1, glucagon-like peptide 1 GTT, glucose tolerance test HPLC, high-performance liquid chromatography IRβ, insulin receptor β IRS, insulin receptor substrate ITT, insulin tolerance test mAb, monoclonal antibody mTOR, mammalian target of rapamycin PGC, peroxisome proliferator–activated receptor γ co-activator PPAR, peroxisome proliferator–activated receptor ROS, reactive oxygen species TG, triglyceride WAT, white adipose tissue Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on day month year. DOI: 10.2337/db07-0630. Additional information for this article can be found in an online appendix at http://dx.doi.org/10.2337/db07-0630 . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted January 31, 2008. Received May 10, 2007. DIABETES

Aims/hypothesis Non-invasive imaging of beta cells is a much-needed development but is one that faces significant biological and technological hurdles. A relevant imaging method should at least allow for an evaluation over time of the mass of beta cells under physiological and pathological conditions, and for an assessment of novel therapies. We, therefore, investigated the ability of a new MRI probe to repeatedly measure the loss of beta cells in a rodent model. Methods We developed an innovative nanoparticle probe that targets the glucagon-like peptide 1 receptor, and can be used for both fluorescence imaging and MRI. Using fluorescence, we characterised the specificity and biodistribution of the probe. Using 1.5T MRI, we longitudinally imaged the changes in insulin content in male and female mice of the RIP-DTr strain, which mimic the changes expected in type 1 and type 2 diabetes, respectively. Results We showed that this probe selectively labelled beta cells in situ, imaged in vivo native pancreatic islets and evaluated their loss after diphtheria toxin administration, in a model of graded beta cell deletion. Thus, using clinical MRI, the probe quantitatively differentiates, in the same mouse strain, between female animals featuring a 50% loss of beta cells and the males featuring an almost complete loss of beta cells. Conclusions/interpretation The approach addresses several of the hurdles that have so far limited the non-invasive imaging of beta cells, including the potential to repeatedly monitor the very same animals using clinically available equipment, and to differentiate graded losses of beta cells.