Protein Kinase C Isoforms: Insulin Signalling, Cyclic AMP Metabolism and Diabetes

Sweeney, Gary (1994) Protein Kinase C Isoforms: Insulin Signalling, Cyclic AMP Metabolism and Diabetes. PhD thesis, University of Glasgow.

Abstract

Protein kinase C has been implicated in the regulation of insulin signalling by many previous studies. This phenomenon was further investigated here using both established and novel experimental systems. Using the streptozotocin-induced diabetic rat model, protein kinase C activity was assessed after partial purification of the enzyme from the liver of both normal and diabetic rats. It was found that inducing diabetes in rats using streptozotocin resulted in a decrease in total protein kinase C activity. Further analysis of protein kinase C activity showed that this difference was accounted for both by a reduction in activity of Ca2+-dependent and Ca2+-independent isoforms. To enable correlation of protein kinase C isoform expression with activity changes in these samples, antibodies were raised to specifically detect individual protein kinase C isoforms. Results from immunoblotting studies using isoform specific antisera suggested that, in rat liver, the reduced Ca2+-dependent protein kinase C activity might be associated with alterations in the alpha and betaII isoforms, whilst modified activity of the PKC-epsilon species may have been responsible for the reduced Ca2+-independent activity observed. The availability of a set of Chinese hamster ovary cell clones overexpressing the human insulin receptor, both alone and combined with a specific protein kinase C isoform, allowed direct in vitro analysis of the interaction of insulin and protein kinase C. In this case their control of intracellular cyclic AMP metabolism was studied. Results obtained by directly measuring intracellular cyclic AMP concentration in cells suggest that insulin can attenuate agonist stimulated increases in intracellular cyclic AMP concentration. However, this effect of insulin was only evident in cells overexpressing both the human insulin receptor and PKC-epsilon (CHO-epsilon cells). Upon directly measuring cyclic AMP phosphodiesterase activity in all CHO cell clones available, it was observed that CHO-epsilon cells exhibited a higher basal phosphodiesterase activity than any other cell clone and that this activity was substantially increased by treating cells with insulin. Both of these observations were no longer apparent after cells had undergone long-term phorbol ester treatment to down-regulate protein kinase C. These results confirmed that PKC-epsilon plays a crucial role in regulating both basal phosphodiesterase activity and insulin induced changes in phosphodiesterase activity within these cells. They also suggested that insulin's ability to attenuate agonist induced increases in intracellular cyclic AMP concentrations in these cells was due, at least partly, to stimulation of cyclic AMP phosphodiesterase activity.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Miles Houslay
• Keywords: Biochemistry, Molecular biology
• Date of Award: 1994
• Depositing User: Enlighten Team
• Unique ID: glathesis:1994-76338
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 15:24
• Last Modified: 19 Nov 2019 15:24
• URI: https://theses.gla.ac.uk/id/eprint/76338