The overall goal of this project is to investigate the synthesis of the zinc binding protein, metallothionein, as it relates to mammalian zinc metabolism. Emphasis is placed on integrating the induction of the protein to zinc absorption by the small intestine, regulation of the p lasma zinc concentration and intracellular zinc metabolism in liver, kidney, intestine and prostate. Research supported by this project has demonstrated that metallothionein is induced by dietary and parenteral zinc as well as glucocorticoids. This is effected by a change in the translatable metallothionein mRNA pool, most likely as the result of altered metallothionein gene experssion. A partially purified rat liver metallothionein mRNA was used to prepare double stranded cDNA for insertion into a pBR325 plasmid and transfection of an E. coli host. A cDNA containing metallothionein sequencee was isolated. This cloned cDNA, when nick translated to incorporate 32P-dCTP, is used as a probe to quantitate metallothionein mRNA levels in liver kidney, intestine and prostate. The induction of metallothionein mRNA by altered dietary zinc supply, endotoxin and leukocytic endogenous mediator administration, fasting, acute infection, parenteral alimentation and streptozotocin-induced diabetes as well as glucagon and glucocorticoids administration are variables that will be examined. The metallothionein mRNA levels during gestation and lactation will be examined in both the fetus, neonate and dam. The effect of maternal zinc deficiency, alcohol consumption and endotoxin exposure on fetal metallothionein mRNA will be evaluated. For all experiments metallothionein-bound zinc and copper and total metallothionein will be measured as variables and related to mRNA levels. The induction of renal metallothionein synthesis on the renal handling of zinc and copper by the isolated, perfused kidney will be examined. The absorption of zinc by the isolated vascularly perfused intestine will be related to metallothionein synthesis under a variety of conditions that will alter absorption. The relationship of liver zinc metabolism and metallothionein induction to fructose 1,6-bisphosphatase activity will be examined.
