Glut4 trafficking: Roles of LRP1, endosomal pH and Rab GTPases
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Insulin regulates Glut4 trafficking between the intracellular storage compartments and the PM of fat and muscle cells, which is a key process in glucose homeostasis. Insulin is released in response to post-prandial excess blood sugar and facilitates glucose clearance by stimulating Glut4 translocation. The detailed Glut4 trafficking itinerary and how insulin regulates this process is not known. Our research was focused in understanding the effect of insulin in Glut4 trafficking and the pathways Glut4 take during its trafficking between the Glut4 storage compartments (GSVs) and the PM. Using a flow cytometric based assays, we show that insulin increased Glut4 exocytic constant (kex) and the amount of Glut4 cycling consistent with previous studies, but also showed that insulin does not affect the Glut4 endocytic constant (ken). Previous studies have estimated ken values but in this study we measured it directly using multiple experimental approaches and showed it is not inhibited by insulin. We also showed all the AS160 substrate Rab GTPases found on Glut4 vesicles are involved in Glut4 trafficking. Even though Rab10 was already previously shown to be involved in Glut4 trafficking, we show here that Rabs 8A, 8B and 14 are also involved in Glut4 trafficking in adipocytes. These knockdowns also affected LRP1 trafficking. Using the Rab knockdown's effect on Glut4 and LRP1 trafficking, we modeled their effect in membrane trafficking in the constitutive and regulated pathways. We also show evidence that one of the major cargo proteins of GSVs, LRP1, might be involved in Glut4 trafficking using its luminal domain. Endosomal pH is required for receptor mediated endocytosis but in this study we showed for the first time that it is also required for Glut4 trafficking. We show evidence that there are two Glut4 trafficking pathways: pH-dependent and pH-independent trafficking.