Molecular cloning of a cDNA for rat diabetes-inducible cytochrome P450RLM₆: hormonal regulation and similarity to the cytochrome P4502E1 gene

Abstract

1. A polyclonal, monospecific antibody to a constitutive, diabetes-inducible and insulin-reversible cytochrome P-450 isozyme (RLM₆) was used to screen a male rat liver cDNA library in λgt 11. Six clones harbouring the RLM₆ cDNA insert were isolated initially from the expression library and three of these were further plaque-purified and sub-cloned. A 1.1 Kb cDNA insert, representing approximately 65% of the expected full length cDNA was characterized by restriction endonuclease mapping and sequenced by the dideoxy chain-termination method. Comparison of the nucleotide sequence of RLM₆ cDNA to that of ethanol-inducible P4502E1 rat cDNA showed the two cDNAs to be identical, the RLM₆ cDNA corresponding to nucleotides 310–1402 of the P4502E1 sequence.

2. RLM₆ cDNA probe was used in Northern blot and RNA dot blot hybridization analysis to demonstrate that both streptozotocin-induced diabetes and fasting significantly elevated the steady-state level of RLM₆ mRNA in male rat liver. Increased RLM₆ mRNA level in the diabetic rat resulted in increased RLM₆ apoprotein synthesis when polysomal RNA was used in a cell-free, protein-synthesizing system, indicating that the elevated RLM₆ level observed in diabetic rats was correlated directly with the increased RLM₆ mRNA concentration.

3. 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 decrease of the RLM₆ mRNA level was paralleled by a similar time-dependent decrease in serum acetone concentration.

4. Treatment of the male diabetic rat with testosterone also resulted in a decrease in both RLM₆ mRNA and in vitro translated apoprotein.

5. Modulation of RLM₆ mRNA level in the diabetic rat by insulin and testosterone, and the nucleotide sequence similarity with that of P4502E1 confirms that diabetes-inducible P450RLM₆ and ethanol-inducible P4502E1 are coded for by the same gene.