University of Surrey

Test tubes in the lab Research in the ATI Dance Research

The Isolation and Characterization of a Diabetes-Inducible P450 Gene.

Richardson, Toby Howard. (1990) The Isolation and Characterization of a Diabetes-Inducible P450 Gene. Doctoral thesis, University of Surrey (United Kingdom)..

[img]
Preview
Text
27726971.pdf
Available under License Creative Commons Attribution Non-commercial Share Alike.

Download (49MB) | Preview

Abstract

The objective of this study was to isolate a cDNA probe corresponding to a constitutive diabetes-inducible and insulin-reversible cytochrome P450 (RLM6). This cDNA would then be used in conjunction with a polyclonal monospecific antibody to RLM6 to study the regulation of this enzyme at the mRNA and protein level. The polyclonal, monospecific antibody to RLM6 was used to screen a male rat liver cDNA library in the phage vector gt11. Six recombinant phage harbouring RLM6 cDNA inserts were isolated from the expression library. Three of these were plaque purified and their cDNA inserts examined. The largest insert (approximately 1.1Kb, and termed RLM6-1) was subcloned into the plasmid vector pUC 19 and characterised. Restriction endonuclease mapping and nucleotide sequencing revealed that when the RLM6 cDNA was compared to that of the ethanol-inducible P450IIE1, the two cDNAs were identical, the RLM6-1 cDNA corresponding to nucleotides 310 to 1402 of the P450 IIE1 sequence. The RLM6-1 cDNA was subsequently used as a molecular probe to study the regulation of this gene in pathophysiological conditions. Northern blot and dot blot hybridisation analyses demonstrated that both streptozotocin-induced diabetes and fasting significantly elevated the steady state levels of RLM6 mRNA in male rat liver. The increased RLM6 mRNA level in the diabetic rat resulted in increased RLM6 apoprotein synthesis when the polysomal RNA was used in a cell-free, protein synthesising system, indicating the elevated RLM6 level observed in diabetic rats correlated with the increased RLM6 mRNA concentration. Daily insulin treatment of diabetic rats reversed the diabetes-dependent increase in RLM6 mRNA in a time-dependent manner, returning to control values after approximately 2 weeks of continuous insulin treatment. This insulin-dependent reduction of the RLM6 mRNA level was paralleled by a similar time-dependent decrease in serum acetone concentration. Testosterone treatment of the diabetic rat also resulted in a decrease in both RLM6 mRNA and in vitro translated apoprotein. Preliminary studies investigating the effects of endotoxaemia on P450 expression would indicate that in obstructive jaundice there is a marked suppression of RLM6 mRNA. and P450 specific content. In vitro studies indicated that Kupffer cells may be indirectly involved in the suppression of P450s. Finally arachidonic acid was found not to be a substrate for RLM6. Taken collectively, the data presented reveal that the diabetes-inducible cytochrome P450 RLM6 is probably the same gene as P450 IIEI and appears to be under a complex mode of regulation, both by dietary factors and by the hormonal status of the animal.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Richardson, Toby Howard.
Date : 1990
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1990.
Depositing User : EPrints Services
Date Deposited : 06 May 2020 14:37
Last Modified : 06 May 2020 14:42
URI: http://epubs.surrey.ac.uk/id/eprint/856282

Actions (login required)

View Item View Item

Downloads

Downloads per month over past year


Information about this web site

© The University of Surrey, Guildford, Surrey, GU2 7XH, United Kingdom.
+44 (0)1483 300800