Glucose uptake is required for efficient lipid deposition in adipose tissue. Glucose uptake is controlled by the number of Glut4 transport proteins at the plasma membrane (PM). Under fasting/basal conditions, almost no glucose is taken up by adipocytes. After feeding, insulin increases PM Glut4 20-50 fold. Under both conditions, Glut4 at the PM continuously exchanges with intracellular Glut4. Glut4 traffics through both the general endocytic pathway and through highly regulated storage compartments (GSVs). By analyzing the effects of protein disruption on trafficking kinetics, and using mathematical modeling to test hypotheses, eight rate-limiting steps that control PM Glut4 were identified. Three of these: GSV priming, GSV fusion, and recycling from endosomes into GSVs (sequestration) are highly regulated, increasing 20-100-fold with insulin. This complex, multi-step trafficking and regulatory pathway allows for precise control of PM Glut4 while simultaneously allowing efficient constitutive recycling of endosomal proteins from the same compartments. This pathway utilizes both cytosolic (e.g. Glut4 sorting sequence mutants) and intra-vesicular protein sorting machineries (e.g. sortilin, LRP1).
Using a forward Chemical Genetics screen, we discovered novel activators and inhibitors of Glut4 and insulin action. These include drugs that acutely affect glucose and lipid metabolism in humans through unknown mechanisms. Interestingly, these studies showed that the intra-vesicular sorting machinery is pH dependent- with high affinity in mildly acidic “early” endosomes (pH 6 - 7), and lower affinity as endosomal acidification increases (pH ~5). This is analogous to the mannose-6-phosphate receptors that deliver enzymes from the Golgi to endosomes. The enzymes remain bound to the receptors in the early endosomes, allowing efficient recycling of constitutive proteins, but retention of the enzymes. After maturation to more acidic pH, the enzymes are released and the receptors are recycled back to the Golgi. The remaining proteins are delivered to lysosomes (pH ~4). Sequential requirements for Rabs 14 and 10 and their effector protein TBC-1D4/AS160 suggest that Glut4 may follow a similar pathway.