Molecular cloning of a cDNA for rat diabetes-inducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene.
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 lambda gt 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.
Richardson
T H
TH
School of Biological Sciences, University of Surrey, Guildford, UK.
Schenkman
J B
JB
Turcan
R
R
Goldfarb
P S
PS
Gibson
G G
GG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D004790:Enzyme Induction; D005215:Fasting / Q000378:metabolism; D007328:Insulin / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D013739:Testosterone / Q000502:physiology
D007328:Insulin; D012333:RNA, Messenger; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating