Roland Govers

Regulated transport of the glucose transporter GLUT4

In 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.

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

ABSTRACT 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.

Subcellular targeting and trafficking of nitric oxide synthases

Unlike most other endogenous messengers that are deposited in vesicles, processed on demand and/or secreted in a regulated fashion, NO (nitric oxide) is a highly active molecule that readily diffuses through cell membranes and thus cannot be stored inside the producing cell. Rather, its signalling capacity must be controlled at the levels of biosynthesis and local availability. The importance of temporal and spatial control of NO production is highlighted by the finding that differential localization of NO synthases in cardiomyocytes translates into distinct effects of NO in the heart. Thus NO synthases belong to the most tightly controlled enzymes, being regulated at transcriptional and translational levels, through co- and post-translational modifications, by substrate availability and not least via specific sorting to subcellular compartments, where they are in close proximity to their target proteins. Considerable efforts have been made to elucidate the molecular mechanisms that underlie the intracellular targeting and trafficking of NO synthases, to ultimately understand the cellular pathways controlling the formation and function of this powerful signalling molecule. In the present review, we discuss the mechanisms and triggers for subcellular routing and dynamic redistribution of NO synthases and the ensuing consequences for NO production and action.

Insulin Stimulates the Entry of GLUT4 into the Endosomal Recycling Pathway by a Quantal Mechanism

The 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.

High-Throughput Analysis of the Dynamics of Recycling Cell Surface Proteins

Recycling 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).