The overall aim of the work described in this thesis was to examine the effects of diabetes and insulin on the metabolism of mRNA in rat skeletal muscle. The primary objective was to determine whether insulin stimulates protein synthesis in the skeletal muscle of diabetic rats by promoting the translation of pre-existing mRNA. Initially the most suitable conditions for the isolation of skeletal muscle ribosomes, and the sedimentation of these on sucrose density gradients were determined. Ionic conditions of 50 mM Tris-HCl, 200 mM KC1, 5 mM MgC12 (pH 7.6) in the sucrose density gradients were found to be best for decreasing the dimerization of 80S ribosomes which hindered discrimination between monosomes and polysomes. Ribosomes extracted at high ionic strength (which gave superior yields) had similar sedimentation profiles to those isolated at low ionic strength, with about 60% of the total ribosomes in polysomes. Skeletal muscle polysomes were found to be small in size compared to those from some other cells and tissues, with the most predominant species being those containing three, four and five ribosomes. This small size did not appear to be due to nicking of the mRNA by ribo- nuclease, for ribosomes prepared under sterile conditions, in the presence of heparin (an inhibitor of ribonuclease) did not give larger polysomes. Neither did addition of cycloheximide, suggesting that the small size of the polysomes was not due to the ribosomes running off the mRNA during isolation. When skeletal muscle ribosomes were translated in a cell-free system from wheat germ, the major products were of molecular weight 15,500 - 17,500, although smaller amounts of species with molecular weights as high as 100,000 could be detected. Initially diabetes was induced with alloxan or streptozotocin and found to decrease the number of polysomes in rat skeletal muscle ribosomes, as judged by sucrose density gradient analysis. When insulin was administered to either alloxan- or streptozotocin- diabetic rats, and the ribosomes isolated at low ionic strength, the polysomes rapidly reassembled. However, when the ribosomes were extracted at high ionic strength, reassembly of polysomes was only observed with the ribosomes from the rats that had been made diabetic with streptozotocin. These observations imply that alloxan induces a ribonuclease activity that is extracted from another sub- cellular fraction of skeletal muscle by the medium of high ionic strength during isolation, breaking down the polysomes which had reassembled in vivo after injection of insulin. Attempts to detect this putative nuclease activity directly using yeast RNA or radioactive HeLa cell ribosomal RNA as a substrate were unsuccessful. The concentration of RNA per gram of DNA in rat skeletal muscle was found to decrease by about 25% from that in normal rats, 3 days after the induction of either alloxan- or streptozotocin-diabetes. Thus the putative ribonuclease, referred to above, cannot be responsible for the decreased skeletal muscle RNA in diabetes. (Abstract shortened by ProQuest.)
