Isotope Effects in the Predissociation of Excited States of N2+ Produced by Photoionization of 14N2 and 15N2 at Energies Between 24.2 and 25.6 eV

Photoelectron/photoion imaging spectrometry employing dispersed VUV radiation from the SOLEIL synchrotron has been used to study the predissociation of N2+ states located up to 1.3 eV above the ion's first dissociation limit. Branching ratios for unimolecular decay into either N2+ or N+ were obtained by measuring coincidences between threshold electrons and mass-selected product ions, using a supersonic beam of either 14N2 or 15N2 as photoionization target. The results confirm that predissociation of the C2Σu+ state of 14N2+ is faster than emission to the electronic ground-state by a factor 10 or more for all vibrational levels v′ ≥ 3, while for 15N2+ the two decay modes have comparable probabilities for the levels v′ = 3, 4, and 5. In contrast, no significant isotope effect could be observed for the other states of N2+ identified in the photoelectron spectrum. For both 14N2+ and 15N2+ isotopologues all vibrational levels of these other states decay to an extent of at least 95% by predissociation

A Serum Factor Induces Insulin-Independent Translocation of GLUT4 to the Cell Surface which Is Maintained in Insulin Resistance

In response to insulin, glucose transporter GLUT4 translocates from intracellular compartments towards the plasma membrane where it enhances cellular glucose uptake. Here, we show that sera from various species contain a factor that dose-dependently induces GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes, human adipocytes, myoblasts and myotubes. Notably, the effect of this factor on GLUT4 is fully maintained in insulin-resistant cells. Our studies demonstrate that the serum-induced increase in cell surface GLUT4 levels is not due to inhibition of its internalization and is not mediated by insulin, PDGF, IGF-1, or HGF. Similarly to insulin, serum also augments cell surface levels of GLUT1 and TfR. Remarkably, the acute effect of serum on GLUT4 is largely additive to that of insulin, while it also sensitizes the cells to insulin. In accordance with these findings, serum does not appear to activate the same repertoire of downstream signaling molecules that are implicated in insulin-induced GLUT4 translocation. We conclude that in addition to insulin, at least one other biological proteinaceous factor exists that contributes to GLUT4 regulation and still functions in insulin resistance. The challenge now is to identify this factor

Is ionic choline and geranate (CAGE) liquid caging diet-derived fat, limiting its absorption?

International audienc

Regulated transport of the glucose transporter GLUT4.

International audienceIn muscle and fat cells, insulin stimulates the delivery of the glucose transporter GLUT4 from an intracellular location to the cell surface, where it facilitates the reduction of plasma glucose levels. Understanding the molecular mechanisms that mediate this translocation event involves integrating our knowledge of two fundamental processes--the signal transduction pathways that are triggered when insulin binds to its receptor and the membrane transport events that need to be modified to divert GLUT4 from intracellular storage to an active plasma membrane shuttle service

Involvement of the proteasome in activation of endothelial nitric oxide synthase.

International audienceNitric oxide originating from the endothelial cells of the vessel wall is essential for the vascular system. It is produced by the enzyme endothelial nitric oxide synthase (eNOS). Cellular eNOS activity is affected by changes in eNOS synthesis. To address whether degradation also contributes to eNOS activity, the effect of proteasome inhibitors on eNOS-mediated NO synthesis was studied in the microvascular endothelial cell line bEnd.3 and in cultured primary aortic endothelial cells. Surprisingly, agonist-induced increases in eNOS activity were reduced to 42 and 50% in the presence of the proteasome inhibiting drugs MG132 and clasto-lactacystin-beta-lactone, respectively (P < 0.01). The decrease in activity occurred within 1 hour of drug treatment and was not accompanied by a change in intracellular levels of either eNOS or its inhibitor caveolin-1. Taken together, these data may indicate that eNOS is regulated by an interacting protein, different from caveolin-1, that inhibits its activity and is rapidly degraded by the proteasome in the presence of eNOS agonists

Akt activation is required at a late stage of insulin-induced GLUT4 translocation to the plasma membrane.

International audienceInsulin stimulates the translocation of glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (PM). This involves multiple steps as well as multiple intracellular compartments. The Ser/Thr kinase Akt has been implicated in this process, but its precise role is ill defined. To begin to dissect the role of Akt in these different steps, we employed a low-temperature block. Upon incubation of 3T3-L1 adipocytes at 19 C, GLUT4 accumulated in small peripheral vesicles with a slight increase in PM labeling concomitant with reduced trans-Golgi network labeling. Although insulin-dependent translocation of GLUT4 to the PM was impaired at 19 C, we still observed movement of vesicles toward the surface. Strikingly, insulin-stimulated Akt activity, but not phosphatidylinositol 3 kinase activity, was blocked at 19 C. Consistent with a multistep process in GLUT4 trafficking, insulin-stimulated GLUT4 translocation could be primed by treating cells with insulin at 19 C, whereas this was not the case for Akt activation. These data implicate two insulin-regulated steps in GLUT4 translocation: 1) redistribution of GLUT4 vesicles toward the cell cortex-this process is Akt-independent and is not blocked at 19 C; and 2) docking and/or fusion of GLUT4 vesicles with the PM-this process may be the major Akt-dependent step in the insulin regulation of glucose transport

Insulin Increases Cell Surface GLUT4 Levels by Dose Dependently Discharging GLUT4 into a Cell Surface Recycling Pathway

The insulin-responsive glucose transporter GLUT4 plays an essential role in glucose homeostasis. A novel assay was used to study GLUT4 trafficking in 3T3-L1 fibroblasts/preadipocytes and adipocytes. Whereas insulin stimulated GLUT4 translocation to the plasma membrane in both cell types, in nonstimulated fibroblasts GLUT4 readily cycled between endosomes and the plasma membrane, while this was not the case in adipocytes. This efficient retention in basal adipocytes was mediated in part by a C-terminal targeting motif in GLUT4. Insulin caused a sevenfold increase in the amount of GLUT4 molecules present in a trafficking cycle that included the plasma membrane. Strikingly, the magnitude of this increase correlated with the insulin dose, indicating that the insulin-induced appearance of GLUT4 at the plasma membrane cannot be explained solely by a kinetic change in the recycling of a fixed intracellular GLUT4 pool. These data are consistent with a model in which GLUT4 is present in a storage compartment, from where it is released in a graded or quantal manner upon insulin stimulation and in which released GLUT4 continuously cycles between intracellular compartments and the cell surface independently of the nonreleased pool

High-throughput analysis of the dynamics of recycling cell surface proteins.

International audienceRecycling via the plasma membrane is a key feature that is shared by many membrane proteins. Using a combination of indirect immunofluorescence labeling and fluorescence detection using a fluorescence multiwell plate reader, we exploited the possibilities of quantitatively measuring the trafficking kinetics of transmembrane proteins. Parameters that can be studied include dynamic appearance/presence at the cell surface, recycling via the cell surface, and internalization. For the insulin-responsive glucose transporter GLUT4 (glucose transporter number 4), details are presented on how to quantitatively measure insulin-induced GLUT4 translocation toward the plasma membrane (transition state) and to analyze cell surface recycling of GLUT4 in basal and insulin-stimulated cells (steady state)

Akt activation is required at a late stage of insulin-induced GLUT4 translocation to the plasma membrane.

International audienceInsulin stimulates the translocation of glucose transporter GLUT4 from intracellular vesicles to the plasma membrane (PM). This involves multiple steps as well as multiple intracellular compartments. The Ser/Thr kinase Akt has been implicated in this process, but its precise role is ill defined. To begin to dissect the role of Akt in these different steps, we employed a low-temperature block. Upon incubation of 3T3-L1 adipocytes at 19 C, GLUT4 accumulated in small peripheral vesicles with a slight increase in PM labeling concomitant with reduced trans-Golgi network labeling. Although insulin-dependent translocation of GLUT4 to the PM was impaired at 19 C, we still observed movement of vesicles toward the surface. Strikingly, insulin-stimulated Akt activity, but not phosphatidylinositol 3 kinase activity, was blocked at 19 C. Consistent with a multistep process in GLUT4 trafficking, insulin-stimulated GLUT4 translocation could be primed by treating cells with insulin at 19 C, whereas this was not the case for Akt activation. These data implicate two insulin-regulated steps in GLUT4 translocation: 1) redistribution of GLUT4 vesicles toward the cell cortex-this process is Akt-independent and is not blocked at 19 C; and 2) docking and/or fusion of GLUT4 vesicles with the PM-this process may be the major Akt-dependent step in the insulin regulation of glucose transport

Insulin stimulates the entry of GLUT4 into the endosomal recycling pathway by a quantal mechanism.

International audienceThe insulin-sensitive glucose transporter GLUT4 mediates the uptake of glucose into adipocytes and muscle cells. In this study we have used a novel 96-well plate fluorescence assay to study the kinetics of GLUT4 trafficking in 3T3-L1 adipocytes. We have found evidence for a graded release mechanism whereby GLUT4 is released into the plasma membrane recycling system in a nonkinetic manner as follows: the kinetics of appearance of GLUT4 at the plasma membrane is independent of the insulin concentration; a large proportion of GLUT4 molecules do not participate in plasma membrane recycling in the absence of insulin; and with increasing insulin there is an incremental increase in the total number of GLUT4 molecules participating in the recycling pathway rather than simply an increased rate of recycling. We propose a model whereby GLUT4 is stored in a compartment that is disengaged from the plasma membrane recycling system in the basal state. In response to insulin, GLUT4 is quantally released from this compartment in a pulsatile manner, leaving some sequestered from the recycling pathway even in conditions of excess insulin. Once disengaged from this location we suggest that in the continuous presence of insulin this quanta of GLUT4 continuously recycles to the plasma membrane, possibly via non-endosomal carriers that are formed at the perinuclear region