The liver is a key target for insulin action and also appears to regulate the plasma insulin level by insulin receptor mediated internalization and degradation. During this process the insulin receptor is internalized and then recycled to the plasma membrane. It is the goal of this proposal to understand the cellular compartments involved in this complex process and the molecular mechanisms which underlie it. There is now evidence, including our own, that coated vesicles are transient intermediates in insulin internalization. It will be determined whether a single coated vesicle can transport insulin as well as other physiologically significant ligands including asialoglycoproteins into the cell. This goal will be accomplished using galactosylated acetylcholinesterase (gal AChE), which is a high affinity ligand for asialoglycoprotein receptors on hepatocytes. Any organelle containing this ligand can be separated from those not containing it by density gradient centrifugation following AChE mediated iron-copper deposition. This technology should also allow the separation and purification of acidic endosomes from 125I-insulin perfused livers. This organelle appears to be a key compartment for the separation and sorting of many ligands and receptors and it will be determined if insulin-insulin/receptor separation occurs here as well. It has recently been found by us that highly purified liver coated vesicles contain a very active protein kinase. Using biochemical and immunocytochemical techniques it is proposed to purify this enzyme, raise antibodies against it and investigate its possible role(s) in receptor mediated endocytosis of insulin and other ligands by liver. It is hoped that these studies will add to our understanding of insulin homeostasis in normal tissues. They may also aid in determining the mechanisms involved in the pathogenesis of diabetes.
