Hypothalamic glucose sensing is involved in the control of feeding behavior and peripheral glucose homeostasis, and glial cells are suggested to play an important role in this process. Diazepam-binding inhibitor (DBI) and its processing product the octadecaneuropeptide (ODN), collectively named endozepines, are secreted by astroglia, and ODN is a potent anorexigenic factor. Therefore, we investigated the involvement of endozepines in brain glucose sensing. First, we showed that intracerebroventricular administration of glucose in rats increases DBI expression in hypothalamic glial-like tanycytes. We then demonstrated that glucose stimulates endozepine secretion from hypothalamic explants. Feeding experiments indicate that the anorexigenic effect of central administration of glucose was blunted by coinjection of an ODN antagonist. Conversely, the hyperphagic response elicited by central glucoprivation was suppressed by an ODN agonist. The anorexigenic effects of centrally injected glucose or ODN agonist were suppressed by blockade of the melanocortin-3/4 receptors, suggesting that glucose sensing involves endozepinergic control of the melanocortin pathway. Finally, we found that brain endozepines modulate blood glucose levels, suggesting their involvement in a feedback loop controlling whole-body glucose homeostasis. Collectively, these data indicate that endozepines are a critical relay in brain glucose sensing and potentially new targets in treatment of metabolic disorders.

Chemotactic migration is a fundamental behavior of cells and its regulation is particularly relevant in physiological processes such as organogenesis and angiogenesis, as well as in pathological processes such as tumor metastasis. The majority of chemotactic stimuli activate cell surface receptors that belong to the G protein-coupled receptor (GPCR) superfamily. Although the autophagy machinery has been shown to play a role in cell migration, its mode of regulation by chemotactic GPCRs remains largely unexplored. We found that ligand-induced activation of 2 chemotactic GPCRs, the chemokine receptor CXCR4 and the urotensin 2 receptor UTS2R, triggers a marked reduction in the biogenesis of autophagosomes, in both HEK-293 and U87 glioblastoma cells. Chemotactic GPCRs exert their anti-autophagic effects through the activation of CAPNs, which prevent the formation of pre-autophagosomal vesicles from the plasma membrane. We further demonstrated that CXCR4- or UTS2R-induced inhibition of autophagy favors the formation of adhesion complexes to the extracellular matrix and is required for chemotactic migration. Altogether, our data reveal a new link between GPCR signaling and the autophagy machinery, and may help to envisage therapeutic strategies in pathological processes such as cancer cell invasion.

Chemotactic migration is a fundamental cellular behavior relying on the coordinated flux of lipids and cargo proteins toward the leading edge. We found here that the core autophagy protein ATG9A plays a critical role in the chemotactic migration of several human cell lines, including highly invasive glioma cells. Depletion of ATG9A protein altered the formation of large and persistent filamentous actin (F-actin)–rich lamellipodia that normally drive directional migration. Using live-cell TIRF microscopy, we demonstrated that ATG9A-positive vesicles are targeted toward the migration front of polarized cells, where their exocytosis correlates with protrusive activity. Finally, we found that ATG9A was critical for efficient delivery of β1 integrin to the leading edge and normal adhesion dynamics. Collectively, our data uncover a new function for ATG9A protein and indicate that ATG9A-positive vesicles are mobilized during chemotactic stimulation to facilitate expansion of the lamellipodium and its anchorage to the extracellular matrix.

Autophagy is a highly conserved self-degradative process that plays a key role in diverse cellular processes such as stress response or differentiation. A growing body of work highlights the direct involvement of autophagy in cell migration and cancer metastasis. Specifically, autophagy has been shown to be involved in modulating cell adhesion dynamics as well as epithelial-to-mesenchymal transition. After providing a general overview of the mechanisms controlling autophagosome biogenesis and cell migration, we discuss how chemotactic G protein-coupled receptors, through the repression of autophagy, may orchestrate membrane trafficking and compartmentation of specific proteins at the cell front in order to support the critical steps of directional migration.

The cloning and production of a human anti-rhesus (Rh) D monoclonal antibody (mAb) using the baculovirus-insect cell expression system is described. This monoclonal recombinant antibody R.D7C2 derived from a human parental IgM lambda immunoglobulin was obtained after immortalization of lymphocytes by Epstein-Barr virus (EBV). The human heavy (VH) and light (VL) variable regions were cloned from the parental cell line and genetically fused to the human constant IgG1 heavy (H) and light (L) chain genes (gamma 1 and lambda, respectively). A recombinant baculovirus was constructed that directs the co-expression of genes encoding both genetically fused heavy and light chains under the control of two late and strong baculovirus promoters. After infecting the Spodoptera frugiperda (Sf9) insect cell line with this baculovector, a complete IgG1 mAb was secreted in the culture medium indicating that each immunoglobulin chain was correctly processed and assembled with a functional glycosylation into a tetrameric form. In vitro analysis showed that the functional properties of R.D7C2 using agglutination tests were efficient for the specific recognition of Rh-D-positive red blood cells (RBC). In addition, R.D7C2 showed effector functions of the gamma 1 heavy chain resulting in the lysis of Rh+ papain RBC by an antibody-directed cellular cytotoxicity mechanism. These results demonstrate that R.D7C2 can be produced in the baculovirus-insect cell expression system as a source for potential therapeutic application in the treatment of the haemolytic disease of the newborn.

Introduction: Recent work has shown that benzodiazepines interact with the immune system and exhibit anti-inflammatory effects. By usingin vitromodels, researchers in several studies have shown that the peptidergic endogenous ligands of benzodiazepine receptors, namedendozepines, are involved in the immune response. All endozepines identified so far derive from diazepam-binding inhibitor (DBI), which generates several biologically active fragments. The aim of the present study was to measure plasma levels of DBI-like immunoreactivity (DBI-LI) in a rat model of sepsis and in patients with systemic inflammation from septic or non-septic origin.Methods: Cecal ligation and puncture (CLP) or sham surgery was performed in rats. Blood samples were taken from animals, patients hospitalized for digestive surgery with inflammatory diseases, and healthy volunteers. Measurements of plasma DBI-related peptides were carried out by radioimmunoassay in animal and human samples.Results: In the rats, CLP provoked an increase of plasma DBI-LI (+37%) 6 hours postsurgery. In humans, DBI-LI levels were significantly higher in the systemic inflammation group than in the healthy volunteer group (48.6 (32.7 to 77.7) pg/mlversus11.1 (5.9 to 35.3) pg/ml,P < 0.001). We found a positive correlation between endozepine levels and Acute Physiology and Chronic Health Evaluation II score (r_s = 0.33 (0.026 to 0.58),P < 0.05) and tumor necrosis factor α levels (r_s = 0.43 (0.14 to 0.65),P < 0.01). The area under the receiver operating characteristic curve for endozepines was 0.842 (95% CI (0.717 to 0.966),P < 0.0001) for discriminating patients with inflammation from healthy volunteers.Conclusions: Endozepines might be involved in the inflammatory response in patients with systemic inflammation.