Islet transplantation is becoming a tantalizing possibility because of the new methods for harvesting large numbers of healthy islets. The challenge now is to reduce the numbers of islets needed for a successful graft by investigating how to use those islets most effectively. The first part of this proposal focuses on finding ways to optimize the viability of grafted B-cells. We hypothesize that a sizeable number of trans-planted B-cells are lost because of anoxic damage, and that hyperglycemia exacerbates cell wastage. At any point in time, the number of cells within a graft is the result of how many were placed, how many have died, and how much growth has occurred. Islet grafts from hyperglycemic and euglycemic recipients will be studied at multiple time points. We will test the hypothesis using a multistep approach: 1) measure B and non B-cell mass in the grafts of euglycemic and hyperglycemic recipients, 2) assess the rate of cell formation using incorporation of bromodeoxyuridine (BUdR) into DNA, 3) target therapies at the causes of ischemia-induced tissue damage. Ischemic damage is mediated in many tissues by the formation of free radicals. Drugs which block the accumulation and actions of free radicals will be tested. Also, we will decrease the cell's metabolic requirements with exogenous insulin. The second part of this proposal investigates an environmental factor which alters the function of B-cells, chronic hyperglycemia. Studies into the mechanism of this effect have been hampered by the lack of an in vitro model. The major problem has been rapid reversibility of he defects. We propose to fill this need using islet transplants in diabetic rats - the islet tissue is accessible by simply stripping it off the kidney and can be studied in vitro using classical biochemical techniques. We will use a multistep approach: 1) set up a model of subtherapeutic islet transplants in streptozocin diabetic rats; 2) confirm that the graft develops the B- cell secretory defects linked with hyperglycemia; 3) map out how long the defects persist under in vitro conditions; 40 use this system to study B- cell glucose transport and glucose metabolism and oxidation.
