The rate of glucose stimulated in vitro (pro)insulin synthesis in islets of Langerhans isolated from fed male rats was determined. Increases in the glucose concentration of the incubation medium, over the physiological range (2 to 20 mM), stimulated the rates of both (pro)insulin and total protein synthesis during a two hour incubation. This stimulation was preferential for (pro)insulin synthesis and had a sigmoidal dose response curve with a concentration threshold of between 2.5 mM and 5.0 mM and a maximal rate at 20 mM glucose. Inhibition of islet RNA synthesis by.;ctinomycin D depressedthe rate of total protein synthesis within 30 minutes of the application of a 20 mM glucose stimulus. A specific inhibition of (pro)insulin synthesis by actinomycin D, that occured 60 minutes after the application of a 20 mM glucose stimulus, was thought to reflect the inhibition of glucose stimulated preproinsulin mRNA synthesis. The effect of glucose on islet preproinsulin mRNA content during in vitro incubations was also determined. To achieve this a dot blot hybridization assay for preproinsulin mRNA was 3 2 developed which used a P-labeled cloned human insulin gene as the hybridization probe. The assay proved to be sensitive enough to detect preproinsulin mRNA in as few as fifty islets. Incubation of islets, for 2 hours, at 2.5 mM glucose had no effect on islet preproinsulin mRNA content but incubation at 20 mM glucose increased islet preproinsulin mRNA content. Actinomycin D had no effect on this pattern of glucose stimulation and it is suggested that the degradation of preproinsulin mRNA is in some way dependent on RNA synthesis and that its inhibition by glucose plays an important role in the regulation of islet preproinsulin mRNA content.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:376755 |
| Date | January 1986 |
| Creators | Marshall, Jonathon J. A. |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/35103 |
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