The long-range objective of the work proposed in this application is to provide new morphological information related to subcellular mechanisms of hepatic glycogen metabolism through the use of light and electron microscopy autoradiographs and immunocytochemistry using colloidal gold and cDNA probes. Four model systems will be studied: (1) the fasted adrenalectomized rat in which depleted hepatic glycogen stores are restored following glucocorticoid administration; (2) the action of insulin on hepatic glycogen synthesis in diabetic rats; (3) the developing mouse embryo in which hepatic glycogen is absent in early fetal stages and abundant in later stages; and (4) primary cultures of embryonic chick and adult rat hepatocytes in which the medium can be supplemented with various quantities of glucose, insulin, glucocorticoids, and glucagon in order to follow their direct effects on glycogen metabolism. Using these model systems we will continue to study the cellular location of sites of glycogen synthesis and the relationship of newly synthesized glycogen to smooth endoplasmic reticulum (SER) and other organelles by identifying the location of silver grains in light and electron microscopy autoradiographs after a pulse label of 3H-galactose. In order to correlate the biochemistry of hepatic glycogen synthesis with results from the autoradiographic experiments we will isolate cellular organelles and glycogen by cell fractionation procedures, quantitate the amount of label in these cellular components after pulse labels of either 3H-glucose or 3H-galactose, and analyze membrane fractions with respect to changes in structure, function, and composition related to experimental manipulations. We will study the morphology of hepatic glycogen deposition in hepatocytes of mouse embryos from developmental days hepatic glycogen deposition in hepatocytes of mouse embryos from developmental days 15-19 giving special attention to the relationship of SER to the newly deposited glycogen granules. Using electron microscopy we propose to identify the subcellular location of glycogen synthase phosphatase, synthase, and phosphorylase by coupling colloidal gold particles to antibodies to these enzymes and studying the distribution of gold particles in ultrathin sections of hepatocytes. Finally, using the in situ hybridization technique, we will identify those cells which are synthesizing mRNAs encoding glycogen synthase phosphatase, synthase and phosphorylase. In these studies, we will use 3H- labeled anti-sense RNAs complementary to appropriate mRNAs as probes.
