Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Molecular cloning of a cDNA for rat diabetesinducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene T. H. Richardson, John B. Schenkman, Rob Turcan, Peter S. Goldfarb & G. Gordon Gibson To cite this article: T. H. Richardson, John B. Schenkman, Rob Turcan, Peter S. Goldfarb & G. Gordon Gibson (1992) Molecular cloning of a cDNA for rat diabetes-inducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene, Xenobiotica, 22:6, 621-631, DOI: 10.3109/00498259209053125 To link to this article: http://dx.doi.org/10.3109/00498259209053125
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XENOBIOTICA,
1992, VOL. 22,
NO.
6, 621-631
Molecular cloning of a cDNA for rat diabetes-inducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene
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T. H. RICHARDSON?, JOHN B. SCHENKMANS, ROB TURCANg, PETER S. GOLDFARB? and G. GORDON GIBSONTI f School of Biological Sciences, Molecular Toxicology Group, University of Surrey, Guildford, Surrey GU2 SXH, U K $ Pharmacology Department, University of Connecticut Health Center, Farmington, C T 06032, USA 5 Drug Metabolism Department, Hoechst Pharmaceutical Research Laboratories, Walton Manor, Milton Keynes, Bucks, U K Received 20 September 1991; accepted 6 February 1992 1. A polyclonal, monospecific antibody to a constitutive, diabetes-inducible and insulin-reversible cytochrome P-450 isozyme (RLM,) was used to screen a male rat liver cDNA library in Apt 11. Six clones harbouring the RLM, 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 RLM, cDNA to that of ethanol-inducible P4502E1 rat cDNA showed the two cDNAs to be identical, the RLM, cDNA corresponding to nucleotides 31G1402 of the P4502E1 sequence. 2. RLM, 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 RLM, mRNA in male rat liver. Increased RLM, mRNA level in the diabetic rat resulted in increased RLM, apoprotein synthesis when polysomal RNA was used in a cell-free, protein-synthesizing system, indicating that the elevated RLM, level observed in diabetic rats was correlated directly with the increased RLM, mRNA concentration. 3. Daily insulin treatment of diabetic rats reversed the diabetes-dependent increase in RLM, mRNA in a time-dependent manner, returning to control values after approximately 2 weeks of continuous insulin treatment. This insulin-dependent decrease of the RLM, 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 RLM, mRNA and in witro translated apoprotein. 5. Modulation of RLM, 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.
Introduction Cytochrome P-450 comprises a large gene family of closely related enzymes which play a central role in the oxidative metabolism of xenobiotics and endogenous substrates. I t has also been established that certain chemicals can selectively induce individual microsomal cytochrome P-450 isozymes. For example, cytochromes
7 T o whom correspondence
should be addressed.
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P4501A1 and P4502B1 are elevated in the rat following treatment with 3methylcholanthrene or phenobarbital respectively (Botelho et al. 1979). Cytochrome P-450 levels are also modulated by hormonal stimuli and in certain pathophysiological states. In diabetes, this can result in the altered metabolism of compounds such as hexobarbital, aminopyrine and aniline (Dixon et al. 1961, Past and Cook 1980). Favreau and Schenkman have isolated and characterized a cytochrome P-450 isozyme (RLM,) that is elevated 5 - to %fold in streptozotocin-pretreated, diabetic male rats (Favreau et al. 1987)as well as in starvation; RLM, catalyses hydroxylation of aniline. On subsequent insulin treatment, the diabetes-dependent induction of RLILI, protein, and related catalytic activity, is reversed. When the N-terminal amino acid sequence (residues 1-10) of RLM, was determined (Favreau et al. 1987) and compared with the corresponding sequences of other constitutive cytochromes P-450, it was found to be identical to cytochrome P4502E1 (P-450j, CYP2El), a cytochrome P-450 induced by ethanol, acetone and the antituberculosis drug isoniazid (Ryan et al. 1985). It was therefore considered desirable to determine whether P4502E1 and P450RLM6 are identical or closely-related, yet different, members of a cytochrome P-450 sub-family. In this study we have used cDNA cloning and sequencing and an analysis of mRNA modulation to discriminate between these two possibilities.
Materials and methods Animals. Male rats, 6-8 weeks old (Wistar and Sprague-Dawley) were housed in suspended metal cages and maintained on a 12 h light/dark cycle. Animals were allowed unlimited access to laboratory chow (Spratt's Laboratory Diet 1, Barking, Essex, UK) and water. Fasted rats received no food for 48 h but water ad libitum animals were made diabetic with a single tail vein injection of streptozotocin (65 mg/kg) and control rats were given an equal volume of the dosing vehicle (50 mM sodium citrate, p H 4.5). Animals were killed 10-12 weeks later. Development of diabetes was confirmed by positive urinary glucose dip stick tests with values of 1000 or higher (Bili Lab Stix, Ames, Inc). T o study the time course of insulin reversal, diabetic animals (10 weeks after a single dose of streptozotocin as above) were dosed daily with human insulin (NPH Isophane, Squibb) 2 Units subcutaneously at 07.00 hours and 8-12 units at 17.00 hours. Animals were killed by decapitation at 6, 12, 24, 48, 72 h and 14 days after commencement of insulin treatment. Daily testosterone treatments were performed for 14 days as previously described (Thummel and Schenkman 1990) at a dose level of 250pg/male rat; this dosing protocol resulted in testosterone plasma levels within the normal physiological range (Favreau et al. 1987). After slaughter, livers were removed, immediately frozen in liquid nitrogen and stored at -70°C until the preparation of RNA. Enzymes and chemicals L-(35S)methionine (approximately 1200Ci/mmol)and the nuclease-treated rabbit reticulocyte lysate in vitro translation kit was purchased from Dupont NEN Research Products (Boston, MA, USA). Restriction endonucleases, DNA multiprime labelling kit and [ a d * P ] d C T P (3000Ci/mmol) were obtained from Amersham. Unless otherwise stated all chemicals were purchased through BDH, Sigma or Boehringer Mannheim and were of the purest grades available. Isolation and sequencing of c D N A clones A male rat liver cDNA library in l g t l l (Earnshaw et al. 1988) was kindly provided by D r D. Earnshaw (Biochemistry Department, University of Surrey). Specific phage clones containing the diabetesinducible RLM, insert were isolated by screening the cDNA expression library with a mono-specific rabbit polyclonal antibody to RLM, (Favreau et al. 1987), as previously described (Huynh et al. 1985). Positive RLM,-containing clones were identified using a biotinylated sheep anti-rabbit I g C second antibody followed by staining with a streptavidin-horseradish peroxidase complex (Sera Lab Ltd). Between 100000 and 150000 plaques from the cDNA library were screened and six positive clones isolated. Three of these stained very strongly with the RLM, antisera, and were further purified by two additional rounds of screening. Phage DNA was isojated (Maniatis et al. 1982) and cDNA inserts sized following digestion with Eco RI on a 1%agarose gel. The largest insert (approximately 1.1 K b and termed RLM,-l) was subcloned into pUC19 and characterized by restriction endonuclease mapping. Plasmid DNA was prepared by the alkaline lysis method as previously described (Birnboin and Doly 1979).
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DNA sequencing was performed by the dideoxy chain termination method (Chen and Seeburg 1985), using the Sequenase system (version 2.0) from the United States Biochemical Corporation according to the suppliers instructions. Template, double-stranded plasmid DNA was prepared according to the method of Zhang et al. 1988. In addition to the universal and reverse primers, appropriate internal start oligonucleotides (20 mers) were synthesized on an Applied Biosystems 381A D N A synthesizer. RLM,-1 cDNA was sequenced between three and five times to ensure total confidence in the derived cDNA for each nucleotide.
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Total R N A and polysomal R N A isolation Total hepatic RNA was prepared from a 5~ guanidinium isothiocyanate homogenate of frozen tissue by direct precipitation using 4M LiCl (Cathala et al. 1983). Polysomes were isolated from a Tris-sucrose homogenate by magnesium precipitation (Palmiter 1974). Southern, Northern and dot blot analysis Southern analysis was carried out essentially as described by Southern (1975). T h e filter was washed in 2 x SSC/O.l% (w/v) S D S at room temperature for 2 x 10min and 0.1 x SSC/O.l% (w/v) S D S at 55°C for 1 x 40 min. Total RNA for Northern blots was electrophoretically separated in 1% (w/v) agarose gels, containing 2.2 M formaldehyde and transferred to nitrocellulose (Maniatis et al. 1982). For dot blots, RNA was applied directly to nitrocellulose paper after denaturation using a BRL Hybridot-Manifold. Nitrocellulose filters were baked at 80°C for 2 h. Prehybridization was carried out overnight at 42°C in 50% deionized formamide, 5 x SSC (1 x SSC is 1 5 0 m ~NaCl in 1 5 m ~sodium citrate, pH7.0), 5 x Denhardt's solution (bovine serum albumin, 0.1%, ficoll, 0.1%, and polyvinylpyrrolidone, 01%), 10mg/rnl sheared and denatured salmon sperm DNA and 2.5mM sodium phosphate buffer, pH6.6. Filters were hybridized overnight (42°C) in the same solution with the "P labelled probe. Following hybridization, filters were washed at room temperature in 3 x SSC, 0.1% sodium dodecyl sulphate (SDS), 2 x lOmin, followed by 1 x SSC, 0.1% SDS (2 x 15 min) and at finally 42°C in 0.5 x SSC, 0.1% S D S (2 x 10min). Filters were exposed at -70°C to Kodak XAR-5 film with a Lightening Plus intensifying screen. RNA dot blots were reprobed with an actin cDNA probe to confirm that equivalent amounts of RNA had been loaded (data not shown). Preparation of c D N A probe T h e 1.1 K b RLM,-1 cDNA fragment was isolated by Eco RI digestion and recovered following electrophoresis in 1% (w/v) low melting point agarose by phenol extraction and ethanol precipitation. DNA was labelled by the random priming method of Feinberg and Vogelstein (1983) using the Amersham multiprime DNA-labelling system and [ u - ~ ~ dCTP. P] Cell free translation of polysomes Total polysomes (1Opg) were translated in vitro using a rabbit reticulocyte lysate kit (BRL) and L[35S]methionine as recommended by the kit manufacturers. Translation was performed at 26°C for 60min at which point an aliquot (1 pl) was used to determine translational activity by measuring TCAprecipitable incorporated radioactivity. T h e remaining incubation mixture was boiled for 4 min with 4% (w/v) SDS and then diluted with immunoprecipitation buffer (1% nonidet P-40, 0 . 1 5 ~NaCl, 2mM EDTA, 0.5%Aprotonin and lOmM Tris-HC1, pH7.4) such that the final S D S concentration was 0.3% (w/v). Translation products were immunoprecipitated by the method of Anderson and Blobel (1983) using 5Opg RLM, antibody (IgG fraction), and immunoprecipitates recovered with protein-A-sepharose CL-4B. SDS-PAGE was performed as described previously (Favreau et al. 1987) and the dried gel autoradiographed using Kodak XAR-5 film. Serum acetone determination Blood was removed before liver isolation, allowed to clot and centrifuged to separate the serum. Acetone levels were measured as described previously (Thummel and Schenkman 1990).
Results Characterization of RL.M6-l cDNA. Clone RLM6-1 was characterized initially by restriction mapping using single enzyme digests. Results (figure 1) indicated that the restriction map of clone RLM6-1 was similar to that of an internal portion of the known P4502E1 cDNA coding sequence (Song et al. 1986). Subsequent nucleotide sequence analysis (strategy shown in figure 1) revealed that clone RLM6-1 contained an open reading frame of 1092 nucleotides (figure 2), which was identical to that published previously for P4502E1 (Song et al. 1986). In the light of this identity of
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Figure 1. Restriction map and sequencing strategy for the RLM,-l clone. T o p line shows restriction enzyme recognition sites for the previously published P4502El(P450j) cDNA (Song et al. 1986). Shaded lower box shows the corresponding map for the RLM,-1 cDNA clone. Arrows indicate the direction and extent of sequence determined for the RLM,-I cDNA subcloned into pUC19. Arrowheads indicate the position and direction of synthetic oligonucleotides used as primers for sequencing.
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Figure 2. Nucleotide and deduced amino acid sequence for RLM,-I cDNA. RLM, sequence was determined by the dideoxy chain termination method as described in the Materials and methods section, and deduced amino acid sequence given in the single letter nomenclature.
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nucleotide sequence and the Southern blot data indicating the presence of only one gene corresponding to P4502E1 (Song et al. 1986) or to RLM6-1 (figure 3), it is probable that diabetes-inducible P450RLM6 and ethanol-inducible P4502E1 are identical proteins coded for by the same gene.
Elevation of P450RLM6 mRNA level by diabetes and fasting T o substantiate further the above relationship between P450RLM6 and P4502E1, Northern blot analysis of liver RNA using the RLM6-1 cDNA probe revealed a single mRNA species of approximately 1700 nucleotides in length, in both control and diabetic animals (figure 4(a)). Level of RLM6 mRNA was increased noticeably by approximately five-fold in the diabetic animal. This increase of RLM, mRNA level was also seen when total hepatic mRNA from fasted rats was analysed by dot blots. In this case, the RLM, level appeared to be induced to a similar extent (figure 4(b)). These results show that RLM, mRNA levels can be modulated not only by pathophysiological states such as diabetes, but also in the normal rat by fasting which alters the hepatic metabolic flux.
Figure 3.
Southern blot analysis (RLM,-1 cDNA probe) of rat genomic DNA.
Genomic DNA from a male Wistar rat was digested separately overnight with endonuclease restriction enzymes shown (Bam HI, Eco RI, Hind 111 or Pst I), fractionated by electrophoresis in ‘1% agarose gels and blotted on to nitrocellulose. T h e filter was probed with 32P-labelled RLM,-l cDNA and washed as indicated in the Materials and methods section. Lambda Hind I1 I molecular size markers (Kbp) are shown on the right hand side of the gel.
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Figure 4. Northern and dot blot analysis of RNA from control, diabetic and fasted rats. ( a ) Total RNA (20pg) isolated from control (lane 1) and 2-week diabetic male rats (lane 2) was denatured and electrophoresed on a 2.2 M formaldehyde/l.2% (w/v) agarose gel, blotted on to nitrocellulose and hybridized with 32P-labelled RLM,-l cDNA. (b) One and 10pg of total RNA from control, diabetic and fasted male rats were applied directly on to nitrocellulose with a hybridot filtration device, hybridized with the 32P-labelled probe and visualized by autoradiography.
Effectof insulin on P450RLM6 mRNA levels Treatment of diabetic rats with insulin has been shown to reverse the induction of RLM, levels and reduce the specific content of this isozyme to that seen in control rats (Favreau and Schenkman 1988). In order to investigate the mechanism of regulation by insulin, RLM, mRNA levels were determined in total RNA after 6, 12,24,72 h and 2 weeks of insulin treatment. RLM, mRNA levels decreased rapidly in the initial 12 h of insulin treatment followed by a gradual decrease over the remainder of the 14-day period to below the control mRNA level (figure 5), indicating that insulin reversed the diabetes-dependent accumulation of RLM, mRNA. In this context, it has been proposed that P4502E1 mRNA levels in the diabetic rat are elevated due to mRNA stabilization (Song et al. 1986). In parallel with the analysis of changes in RLM, mRNA levels, serum acetone concentrations were determined in the same animals. As shown in table 1, streptozotocin-induced diabetes increases the serum acetone concentrations approximately 56-fold over the control value. These elevated serum acetone levels dropped rapidly after 6-12 h of insulin treatment, returning to just above control levels following 14 days of continuous insulin treatment. Accordingly, the rise in RLMs mRNA levels in the diabetic state, and its reversal by insulin, are
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Figure 5.
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Effect of insulin on total RLM, RNA in the diabetic rat.
Male rats were made diabetic with streptozotocin and daily treatment with insulin continued for up to 2 weeks. RNA was prepared from two animals at each time point, pooled, applied directly on to nitrocellulose and subsequently probed with 32P-labelled RLM,-1 cDNA.
Table 1. Serum acetone concentrations during insulin reversal of diabetic rats. Serum acetone (mM)
Treatment Control Diabetic Diabetic plus Diabetic plus Diabetic plus Diabetic plus Diabetic plus Diabetic plus
insulin 6 h) insulin (1 2 h) insulin (24 h) insulin (48 h) insulin (72 h) insulin ( 2 weeks)
0.07 k0.03 4.10+ 2.26 5.94 (n = 2) 0.49k0.35 0 5 9 0.10 0.75 If: 0.36 0.87k0.49 0 2 8 k 0.1 9
+
Rats were made diabetic with a single streptozotocin injection (65 mg/kg i.p.) at the start of week 1 and diabetes allowed to develop over a 10-week period. At the end of week 10, twice-daily insulin treatment wascommenced (see Materialsand methods for full description of insulin treatment) and continued over a period of 2 weeks with interim kills as indicated in the table. Times refer to the period after insulin treatment had commenced. Results are expressed as meankS.D. from three rats per group unless otherwise indicated.
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accompanied by similar changes in serum acetone concentrations. These results parallel the relationship between cytochrome P4502E1 induction and serum acetone levels (Yo0 et al. 1990).
EfSect of testosterone on RLM6 m R N A level in the diabetic male rat Streptozotocin-induced diabetes is accompanied by a substantial fall in serum testosterone levels (Murray et al. 1981). It was of interest therefore to examine the influence of testosterone treatment on the regulation of RLM, expression in the male diabetic rat liver. Accordingly, total RNA derived from control, diabetic and diabetic plus testosterone treated rats was analysed by RNA dot blots using the RLM, cDNA probe. As shown in figure 6 ( a ) , testosterone treatment of diabetic animals resulted in a partial reversal of the diabetes-induced elevation of RLM, mRNA. This result was confirmed when the polysomal RNAs were translated in vitro (figure 6 ( b ) ) . However it should be noted that in insulin-treated diabetic animals, there appears also to be a decrease in immunoprecipitable RLM, apoprotein.
Figure 6.
Effect of testosterone on RLM, mRNA-directed protein synthesis and corresponding mRNA levels in the male diabetic rat. (a)Total RNA (5 pg) from control (C), diabetic (D) or diabetic plus testosterone ( D + T ) treated male rats were pooled from four animals and dot blot analysis carried out with the RLM,-1 cDNA probe (left hand panel) or actin cDNA probe (right hand panel). ( b ) Liver polysomal RNA (10pg) was used to programme a rabbit reticulocyte protein-synthesizing system, and 2 x lo6dpm from each translated mix was immunoprecipitated with RLM, antibody (SOpg) and protein Asepharose CL-4B. Immunoprecipates were run on SDS-PAGE and visualized by autoradiography. Polysomal RNA was derived from either control (track l ) , diabetic (track 2), diabetic plus insulin (track 3) or diabetic plus testosterone (track 4) treated male rats.
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Discussion This study was prompted by the reported similarity between ethanol-inducible cytochrome P4502E1 and diabetes-inducible RLM,. Both isozymes are induced by diabetes or fasting (Hong et al. 1987) and both proteins are identical in their first 20 amino acid residues (Favreau et al. 1987, Ryan et al. 1985). In addition, they have a similar substrate specificity profile (Favreau et al. 1987, Earnshaw et al. 1988, Song et al. 1986, Murray et al. 1981). Although the P4502E1 gene has been isolated and its regulation studied in the past 5 years (Song et al. 1986, Thomas et al. 1987, Peng et al. 1983, Song et al. 1987, Dong et al. 1988, Yamazoe et al. 1989), no such study has been reported for RLM,. Identity of the RLM, cDNA sequence presented here to that of P4502E1 is strong evidence that diabetes-inducible RLM, and ethanol-inducible P4502E1 are probably the same protein. Although the complete cDNA sequence for RLM, was not determined in our studies, one would expect there to be some nucleotide differences in the cDNAs if P4502E1 and RLM, were distinctly different gene products, as has been reported for the two very closely related phenobarbitalinducible P-450 genes, 2B1 and 2B2 (Yuan et al. 1983). This was not the case, and of the 1092 nucleotides sequenced for the RLM, cDNA (approximating to 65% of the P4502E1 gene) there was complete sequence homology (figure 2) with the reported P4502E1 cDNA sequence. Further evidence of the identity of RLM, and P4502E1 comes from Southern blot analysis of rat genomic DNA. Use of either P4502E1 probe (Song et al. 1986) or our RLM, probe (figure 3) leads to the conclusion that there is only one genomic sequence corresponding to these cDNAs, and confirms the findings of Song et al. (1986). It should be noted that the hybridization band pattern for genomic DNA in our studies using the RLM6-1 cDNA probe is different from that reported by Song et al. (1986) because a different sized cDNA probe and a different rat strain were used in their studies. Confirmation of the existence of one gene also comes from analysis of the Northern blot and RNA dot blot data (figure 4) which indicate that RLM, mRNA is the same size as P4502E1 mRNA and like P4502E1, its level is elevated during diabetes or fasting (Hong et al. 1987). T h e demonstration of identity between P4502E1 and RLM, confirms the suggestion that this gene is regulated by two different post-transcriptional mechanisms, namely mRNA stabilization (Song et al. 1987) and protein stabilization (Eliasson et al. 1988), depending on the inducing stimulus. In streptozotocininduced diabetes and fasting, loss of insulin through the destruction of pancreatic cells is accompanied by decreases in testosterone (Thummel and Schenkman 1990, Murray et al. 1981) and thyroid hormone (Thummel and Schenkman 1990, Chopra et al. 1981) levels and the pulsatile secretion pattern of growth hormone is abolished (Tannenbaum 1981). It is thus difficult to study the phenomenon of mRNA stabilization when a number of hormonal parameters are changing. Results in this paper indicate that insulin may not have a direct effect on RLM, mRNA level, but may indeed function through changes in hormone levels such as testosterone (figure 6). This would further substantiate the proposal that P450RLM6/2E1 is modulated in diabetes both at the mRNA level by hormones and at the protein level by small molecules such as acetone (Eliasson et al. 1988). Our data has also shown that RLM, mRNA levels are elevated in the fasted rat. This may indicate that a mechanism similar to that in diabetes is operative, in that fasting results in an increase in hepatic fatty acid metabolism with a concomitant increase in serum ketones. Whether the fasting-dependent increase in RLM, mRNA level results directly from changes in lipid metabolism or is an indirect effect of hormone changes (as noted above) has yet to be determined.
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In conclusion, it is now clear that constitutive cytochrome P-450 isozymes are regulated both by physiological and nutritional factors and a number of molecular mechanisms may be involved in modulation of enzyme activity. An understanding of these mechanisms may ultimately provide the basis for rationalization of interindividual variations of drug metabolism in man.
Acknowledgements Research was supported in part by an SERC-CASE studentship (T.H.R.) in conjunction with Hoechst Pharmaceuticals, UK, a travel grant from the BurroughsWellcome Fund (J.B.S.), USPH grant G M 261 14 (J.B.S.) and the Medical Research Council (G.G.G.).
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References ANDERSON, D. J., and BLOBEL, G., 1983, Immunoprecipitation of proteins from cell-free translations. Methods in Enzymology, 96, 111-120. BIRNBOIN, H. C., and DOLY,J., 1979, A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research, 7, 1513-1523. BOTELHO, L. H., RYAN,D. E., and LEVIN,W., 1979, Amino acid compositions and partial amino acid sequences of three highly purified forms of liver microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls, phenobarbital, or 3-methylcholanthrene. Journal of Biological Chemistry, 254, 5635-5643. CATHALA, G., SAVOURET, J. F., MENDEZ, B., WEST,B. L., KARIN, M., MARTIAL, J. A,, and BAXTER, J. D., 1983, A method for isolation of intact, translationally active ribonucleic acid. D N A , 2, 329-335. CHEN,E. Y., and SEEBURG, P. H., 1985, Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. D N A , 4, 165-170. CHOPRA, I. J., WIERSINGA, W., and FRANK,H., 1981, Alterations in hepatic monodeiodination of iodothyronines in the diabetic rat. Life Sciences, 28, 1765-1 776. DIXON, R. L., HART,L. G., and FOUTS, J. R., 1961, The metabolism of drugs by liver microsomes from alloxan-diabetic rats. Journal of Pharmacology and Experimental Therapeutics, 133, 7-1 1. DONG,Z., HONG,J., MA, Q., LI, D., BULLOCK, J., GONZALEZ, F. J., PARK,S. S., Gelboin, H. V., and YANG,C. S., 1988, Mechanism of induction of cytochrome P45OAC(P450j) in chemically induced and spontaneously diabetic rats. Archives of Biochemistry and Biophysics, 263, 29-35. EARNSHAW, D., DALE,J. W., GOLDFARB, P. S., and GIBSON,G. G., 1988, Differential splicing in the 3’ non-coding region of rat cytochrome P452 (P450IVAI) mRNA, FEBS Letters, 236, 357-361. ELIASSON, E., JOHANSSON, I., and INGELMAN-SUNDBERG, M., 1988, Ligand dependent maintenance of ethanol-inducible cytochrome P450 in primary rat hepatocyte cell cultures. Biochemical and Biophysical Research Communications, 150, 436-443. FAVREAU, L. V., MALCHOFF, D. M., MOLE,J. E., and SCHENKMAN, J. B., 1987,Responses to insulin by two forms of rat hepatic microsomal cytochrome P-450 that undergo major (RLM,) and minor (RLM,,) elevation in diabetes. JournaE of Biological Chemistry, 262, 14319-14326. FAVREAU, L. V., and SCHENKMAN, J. B., 1988, Composition changes in hepatic microsomal cytochrome P-450 during onset of streptozocin-induced diabetes and during insulin treatment. Diabetes, 37, 577-584. FEINBERG, A. P., and VOGELSTEIN, B., 1983, A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry, 132, 6-1 3. HONG,J., PAN,J., GONZALEZ, F. J., GELBOIN, H. V., and YANG,C. S., 1987, The induction of a specific form of cytochrome P-450 (P-45OJ) by fasting. Biochemical and Biophysical Research Communications, 142, 1077-1083. HUYNH, T. V., YOUNG,R. A., and DAVIFS,R. W., 1985, D N A Cloning-A Practical Approach, Vol. 1, edited by D. M. Glover (IRL Press, Oxford), pp. 49-78. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J., 1982, Molecular Cloning. A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). MURRAY, F. T., ORTH,J., GUNSALUS, G., WEISZ,J., LI, J. B., JEFFERSON, L. S., MUSTO,N. A., and BARDIN, C. W., 1981, The pituitary-testicularaxis in the streptozotocin diabetic male rat: evidence for gonadotroph, sertoli cell and Leydig cell dysfunction. International Journal of Andrology, 4, 265-280. PALMITER, R. D., 1974, Magnesium precipitation of ribonucleoprotein complexes. Expedient techniques for the isolation of undegraded polysomes and messenger ribonucleic acid. Biochemistry, 13, 3606-36 14. PAST,M. R., and COOK,D. E., 1980, Alterations in hepatic microsomal cytochrome P-450 hemeproteins in diabetic rats. Research Communications in Chemical Pathology and Pharmacology, 27, 329-337.
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PENG,R., TENNANT, P., LORR, N. A,, and YANG,C. S., 1983, Alterations of microsomal monooxygenase system and carcinogen metabolism by streptozotocin-induced diabetes in rats. Carcinogen&, 4, 703-708. RYAN,D. E., RAMANATHAN, L., IIDA, S., THOMAS, P. E., HANIU, M., SHIVELY, J. E., LEIBER, C. S., and LEVIN, W., 1985, Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid. Journal of Biological Chemistry, 260, 6385-6393. SONG,B. J., GELBOIN, H. V., PARK, S. S., YANG,C. S., and GONZALEZ, F. J., 1986, Complementary DNA and protein sequences of ethanol-inducible rat and human cytochrome P-450s. Journal of Biological Chemistry, 261, 16689-16697. SONG,B. J . , MATSUNAGA, T., HARDWICK, J. P., PARK,S. S., VEECH, R. L., YANG,C. S., and GELBOIN, H. V., 1987, Stabilisation of cytochrome P450j messenger ribonucleic acid in the diabetic rat. Molecular Endocrinology, 1, 542-547. SOUTHERN, E. M., 1975, Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology, 98, 503-51 7. TANNENBAUM, G. S., 1981, Growth hormone secretory dynamics in streptozotocin diabetes: evidence of a role for endogenous circulating somatostatin. Endocrinology, 108, 76-82. THOMAS, P. E., BANDIERA, S., MAINES,R. L., RYAN,D. E., and LEVIN,W., 1987, Regulation of cytochrome P-450j, high-affinity N-nitrosodimethylamine demethylase, in rat hepatic microsomes. Biochemistry, 26, 228G2289. THUMMEL, K. E., and SCHENKMAN, J . B., 1990, Effects of testosterone and growth hormone treatment in hepatic microsomal P450 expression in the diabetic rat. Molecular Pharmacology, 37, 119-129. YAMAZOE, Y., MURAYAMA, N., SHIMADA, M., YAMAGUCHI, K., and KATO, R., 1989, Cytochrome P450 in livers of diabetic rats: regulation by growth hormone and insulin. Archives of Biochemistry and Biophysics, 268, 567-5 75. YUAN,P. M., RYAN,D. E., LEVIN, W., and SHIVELY, J. E., 1983, Identification and localization of amino acid substitutes between the phenobarbital-inducible rat hepatic microsomal cytochromes P-450 by micro sequence analyses. Proceedings of the National Academy of Sciences (USA), 80, 1169-1180. YOO,J. S. H., HONG,J. Y ., NING,S. M., and YANG,C. S., 1990, Roles of dietary corn oil in the regulation of cytochromes P450 and glutathione S-transferases in rat liver. Journal of Nutrition, 120, 1718-1726. ZHANG,H., SCHOLL, R., BROWSE, J., and SOMERVILLE, C., 1988, Double stranded DNA sequencing as a choice for DNA sequencing. Nucleic Acids Research, 16, 1221.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Molecular cloning of a cDNA for rat diabetesinducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene T. H. Richardson, John B. Schenkman, Rob Turcan, Peter S. Goldfarb & G. Gordon Gibson To cite this article: T. H. Richardson, John B. Schenkman, Rob Turcan, Peter S. Goldfarb & G. Gordon Gibson (1992) Molecular cloning of a cDNA for rat diabetes-inducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene, Xenobiotica, 22:6, 621-631, DOI: 10.3109/00498259209053125 To link to this article: http://dx.doi.org/10.3109/00498259209053125
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Date: 15 March 2016, At: 12:22
XENOBIOTICA,
1992, VOL. 22,
NO.
6, 621-631
Molecular cloning of a cDNA for rat diabetes-inducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene
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T. H. RICHARDSON?, JOHN B. SCHENKMANS, ROB TURCANg, PETER S. GOLDFARB? and G. GORDON GIBSONTI f School of Biological Sciences, Molecular Toxicology Group, University of Surrey, Guildford, Surrey GU2 SXH, U K $ Pharmacology Department, University of Connecticut Health Center, Farmington, C T 06032, USA 5 Drug Metabolism Department, Hoechst Pharmaceutical Research Laboratories, Walton Manor, Milton Keynes, Bucks, U K Received 20 September 1991; accepted 6 February 1992 1. A polyclonal, monospecific antibody to a constitutive, diabetes-inducible and insulin-reversible cytochrome P-450 isozyme (RLM,) was used to screen a male rat liver cDNA library in Apt 11. Six clones harbouring the RLM, 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 RLM, cDNA to that of ethanol-inducible P4502E1 rat cDNA showed the two cDNAs to be identical, the RLM, cDNA corresponding to nucleotides 31G1402 of the P4502E1 sequence. 2. RLM, 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 RLM, mRNA in male rat liver. Increased RLM, mRNA level in the diabetic rat resulted in increased RLM, apoprotein synthesis when polysomal RNA was used in a cell-free, protein-synthesizing system, indicating that the elevated RLM, level observed in diabetic rats was correlated directly with the increased RLM, mRNA concentration. 3. Daily insulin treatment of diabetic rats reversed the diabetes-dependent increase in RLM, mRNA in a time-dependent manner, returning to control values after approximately 2 weeks of continuous insulin treatment. This insulin-dependent decrease of the RLM, 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 RLM, mRNA and in witro translated apoprotein. 5. Modulation of RLM, 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.
Introduction Cytochrome P-450 comprises a large gene family of closely related enzymes which play a central role in the oxidative metabolism of xenobiotics and endogenous substrates. I t has also been established that certain chemicals can selectively induce individual microsomal cytochrome P-450 isozymes. For example, cytochromes
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P4501A1 and P4502B1 are elevated in the rat following treatment with 3methylcholanthrene or phenobarbital respectively (Botelho et al. 1979). Cytochrome P-450 levels are also modulated by hormonal stimuli and in certain pathophysiological states. In diabetes, this can result in the altered metabolism of compounds such as hexobarbital, aminopyrine and aniline (Dixon et al. 1961, Past and Cook 1980). Favreau and Schenkman have isolated and characterized a cytochrome P-450 isozyme (RLM,) that is elevated 5 - to %fold in streptozotocin-pretreated, diabetic male rats (Favreau et al. 1987)as well as in starvation; RLM, catalyses hydroxylation of aniline. On subsequent insulin treatment, the diabetes-dependent induction of RLILI, protein, and related catalytic activity, is reversed. When the N-terminal amino acid sequence (residues 1-10) of RLM, was determined (Favreau et al. 1987) and compared with the corresponding sequences of other constitutive cytochromes P-450, it was found to be identical to cytochrome P4502E1 (P-450j, CYP2El), a cytochrome P-450 induced by ethanol, acetone and the antituberculosis drug isoniazid (Ryan et al. 1985). It was therefore considered desirable to determine whether P4502E1 and P450RLM6 are identical or closely-related, yet different, members of a cytochrome P-450 sub-family. In this study we have used cDNA cloning and sequencing and an analysis of mRNA modulation to discriminate between these two possibilities.
Materials and methods Animals. Male rats, 6-8 weeks old (Wistar and Sprague-Dawley) were housed in suspended metal cages and maintained on a 12 h light/dark cycle. Animals were allowed unlimited access to laboratory chow (Spratt's Laboratory Diet 1, Barking, Essex, UK) and water. Fasted rats received no food for 48 h but water ad libitum animals were made diabetic with a single tail vein injection of streptozotocin (65 mg/kg) and control rats were given an equal volume of the dosing vehicle (50 mM sodium citrate, p H 4.5). Animals were killed 10-12 weeks later. Development of diabetes was confirmed by positive urinary glucose dip stick tests with values of 1000 or higher (Bili Lab Stix, Ames, Inc). T o study the time course of insulin reversal, diabetic animals (10 weeks after a single dose of streptozotocin as above) were dosed daily with human insulin (NPH Isophane, Squibb) 2 Units subcutaneously at 07.00 hours and 8-12 units at 17.00 hours. Animals were killed by decapitation at 6, 12, 24, 48, 72 h and 14 days after commencement of insulin treatment. Daily testosterone treatments were performed for 14 days as previously described (Thummel and Schenkman 1990) at a dose level of 250pg/male rat; this dosing protocol resulted in testosterone plasma levels within the normal physiological range (Favreau et al. 1987). After slaughter, livers were removed, immediately frozen in liquid nitrogen and stored at -70°C until the preparation of RNA. Enzymes and chemicals L-(35S)methionine (approximately 1200Ci/mmol)and the nuclease-treated rabbit reticulocyte lysate in vitro translation kit was purchased from Dupont NEN Research Products (Boston, MA, USA). Restriction endonucleases, DNA multiprime labelling kit and [ a d * P ] d C T P (3000Ci/mmol) were obtained from Amersham. Unless otherwise stated all chemicals were purchased through BDH, Sigma or Boehringer Mannheim and were of the purest grades available. Isolation and sequencing of c D N A clones A male rat liver cDNA library in l g t l l (Earnshaw et al. 1988) was kindly provided by D r D. Earnshaw (Biochemistry Department, University of Surrey). Specific phage clones containing the diabetesinducible RLM, insert were isolated by screening the cDNA expression library with a mono-specific rabbit polyclonal antibody to RLM, (Favreau et al. 1987), as previously described (Huynh et al. 1985). Positive RLM,-containing clones were identified using a biotinylated sheep anti-rabbit I g C second antibody followed by staining with a streptavidin-horseradish peroxidase complex (Sera Lab Ltd). Between 100000 and 150000 plaques from the cDNA library were screened and six positive clones isolated. Three of these stained very strongly with the RLM, antisera, and were further purified by two additional rounds of screening. Phage DNA was isojated (Maniatis et al. 1982) and cDNA inserts sized following digestion with Eco RI on a 1%agarose gel. The largest insert (approximately 1.1 K b and termed RLM,-l) was subcloned into pUC19 and characterized by restriction endonuclease mapping. Plasmid DNA was prepared by the alkaline lysis method as previously described (Birnboin and Doly 1979).
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DNA sequencing was performed by the dideoxy chain termination method (Chen and Seeburg 1985), using the Sequenase system (version 2.0) from the United States Biochemical Corporation according to the suppliers instructions. Template, double-stranded plasmid DNA was prepared according to the method of Zhang et al. 1988. In addition to the universal and reverse primers, appropriate internal start oligonucleotides (20 mers) were synthesized on an Applied Biosystems 381A D N A synthesizer. RLM,-1 cDNA was sequenced between three and five times to ensure total confidence in the derived cDNA for each nucleotide.
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Total R N A and polysomal R N A isolation Total hepatic RNA was prepared from a 5~ guanidinium isothiocyanate homogenate of frozen tissue by direct precipitation using 4M LiCl (Cathala et al. 1983). Polysomes were isolated from a Tris-sucrose homogenate by magnesium precipitation (Palmiter 1974). Southern, Northern and dot blot analysis Southern analysis was carried out essentially as described by Southern (1975). T h e filter was washed in 2 x SSC/O.l% (w/v) S D S at room temperature for 2 x 10min and 0.1 x SSC/O.l% (w/v) S D S at 55°C for 1 x 40 min. Total RNA for Northern blots was electrophoretically separated in 1% (w/v) agarose gels, containing 2.2 M formaldehyde and transferred to nitrocellulose (Maniatis et al. 1982). For dot blots, RNA was applied directly to nitrocellulose paper after denaturation using a BRL Hybridot-Manifold. Nitrocellulose filters were baked at 80°C for 2 h. Prehybridization was carried out overnight at 42°C in 50% deionized formamide, 5 x SSC (1 x SSC is 1 5 0 m ~NaCl in 1 5 m ~sodium citrate, pH7.0), 5 x Denhardt's solution (bovine serum albumin, 0.1%, ficoll, 0.1%, and polyvinylpyrrolidone, 01%), 10mg/rnl sheared and denatured salmon sperm DNA and 2.5mM sodium phosphate buffer, pH6.6. Filters were hybridized overnight (42°C) in the same solution with the "P labelled probe. Following hybridization, filters were washed at room temperature in 3 x SSC, 0.1% sodium dodecyl sulphate (SDS), 2 x lOmin, followed by 1 x SSC, 0.1% SDS (2 x 15 min) and at finally 42°C in 0.5 x SSC, 0.1% S D S (2 x 10min). Filters were exposed at -70°C to Kodak XAR-5 film with a Lightening Plus intensifying screen. RNA dot blots were reprobed with an actin cDNA probe to confirm that equivalent amounts of RNA had been loaded (data not shown). Preparation of c D N A probe T h e 1.1 K b RLM,-1 cDNA fragment was isolated by Eco RI digestion and recovered following electrophoresis in 1% (w/v) low melting point agarose by phenol extraction and ethanol precipitation. DNA was labelled by the random priming method of Feinberg and Vogelstein (1983) using the Amersham multiprime DNA-labelling system and [ u - ~ ~ dCTP. P] Cell free translation of polysomes Total polysomes (1Opg) were translated in vitro using a rabbit reticulocyte lysate kit (BRL) and L[35S]methionine as recommended by the kit manufacturers. Translation was performed at 26°C for 60min at which point an aliquot (1 pl) was used to determine translational activity by measuring TCAprecipitable incorporated radioactivity. T h e remaining incubation mixture was boiled for 4 min with 4% (w/v) SDS and then diluted with immunoprecipitation buffer (1% nonidet P-40, 0 . 1 5 ~NaCl, 2mM EDTA, 0.5%Aprotonin and lOmM Tris-HC1, pH7.4) such that the final S D S concentration was 0.3% (w/v). Translation products were immunoprecipitated by the method of Anderson and Blobel (1983) using 5Opg RLM, antibody (IgG fraction), and immunoprecipitates recovered with protein-A-sepharose CL-4B. SDS-PAGE was performed as described previously (Favreau et al. 1987) and the dried gel autoradiographed using Kodak XAR-5 film. Serum acetone determination Blood was removed before liver isolation, allowed to clot and centrifuged to separate the serum. Acetone levels were measured as described previously (Thummel and Schenkman 1990).
Results Characterization of RL.M6-l cDNA. Clone RLM6-1 was characterized initially by restriction mapping using single enzyme digests. Results (figure 1) indicated that the restriction map of clone RLM6-1 was similar to that of an internal portion of the known P4502E1 cDNA coding sequence (Song et al. 1986). Subsequent nucleotide sequence analysis (strategy shown in figure 1) revealed that clone RLM6-1 contained an open reading frame of 1092 nucleotides (figure 2), which was identical to that published previously for P4502E1 (Song et al. 1986). In the light of this identity of
T . H . Richardson et al.
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4
Figure 1. Restriction map and sequencing strategy for the RLM,-l clone. T o p line shows restriction enzyme recognition sites for the previously published P4502El(P450j) cDNA (Song et al. 1986). Shaded lower box shows the corresponding map for the RLM,-1 cDNA clone. Arrows indicate the direction and extent of sequence determined for the RLM,-I cDNA subcloned into pUC19. Arrowheads indicate the position and direction of synthetic oligonucleotides used as primers for sequencing.
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nucleotide sequence and the Southern blot data indicating the presence of only one gene corresponding to P4502E1 (Song et al. 1986) or to RLM6-1 (figure 3), it is probable that diabetes-inducible P450RLM6 and ethanol-inducible P4502E1 are identical proteins coded for by the same gene.
Elevation of P450RLM6 mRNA level by diabetes and fasting T o substantiate further the above relationship between P450RLM6 and P4502E1, Northern blot analysis of liver RNA using the RLM6-1 cDNA probe revealed a single mRNA species of approximately 1700 nucleotides in length, in both control and diabetic animals (figure 4(a)). Level of RLM6 mRNA was increased noticeably by approximately five-fold in the diabetic animal. This increase of RLM, mRNA level was also seen when total hepatic mRNA from fasted rats was analysed by dot blots. In this case, the RLM, level appeared to be induced to a similar extent (figure 4(b)). These results show that RLM, mRNA levels can be modulated not only by pathophysiological states such as diabetes, but also in the normal rat by fasting which alters the hepatic metabolic flux.
Figure 3.
Southern blot analysis (RLM,-1 cDNA probe) of rat genomic DNA.
Genomic DNA from a male Wistar rat was digested separately overnight with endonuclease restriction enzymes shown (Bam HI, Eco RI, Hind 111 or Pst I), fractionated by electrophoresis in ‘1% agarose gels and blotted on to nitrocellulose. T h e filter was probed with 32P-labelled RLM,-l cDNA and washed as indicated in the Materials and methods section. Lambda Hind I1 I molecular size markers (Kbp) are shown on the right hand side of the gel.
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Figure 4. Northern and dot blot analysis of RNA from control, diabetic and fasted rats. ( a ) Total RNA (20pg) isolated from control (lane 1) and 2-week diabetic male rats (lane 2) was denatured and electrophoresed on a 2.2 M formaldehyde/l.2% (w/v) agarose gel, blotted on to nitrocellulose and hybridized with 32P-labelled RLM,-l cDNA. (b) One and 10pg of total RNA from control, diabetic and fasted male rats were applied directly on to nitrocellulose with a hybridot filtration device, hybridized with the 32P-labelled probe and visualized by autoradiography.
Effectof insulin on P450RLM6 mRNA levels Treatment of diabetic rats with insulin has been shown to reverse the induction of RLM, levels and reduce the specific content of this isozyme to that seen in control rats (Favreau and Schenkman 1988). In order to investigate the mechanism of regulation by insulin, RLM, mRNA levels were determined in total RNA after 6, 12,24,72 h and 2 weeks of insulin treatment. RLM, mRNA levels decreased rapidly in the initial 12 h of insulin treatment followed by a gradual decrease over the remainder of the 14-day period to below the control mRNA level (figure 5), indicating that insulin reversed the diabetes-dependent accumulation of RLM, mRNA. In this context, it has been proposed that P4502E1 mRNA levels in the diabetic rat are elevated due to mRNA stabilization (Song et al. 1986). In parallel with the analysis of changes in RLM, mRNA levels, serum acetone concentrations were determined in the same animals. As shown in table 1, streptozotocin-induced diabetes increases the serum acetone concentrations approximately 56-fold over the control value. These elevated serum acetone levels dropped rapidly after 6-12 h of insulin treatment, returning to just above control levels following 14 days of continuous insulin treatment. Accordingly, the rise in RLMs mRNA levels in the diabetic state, and its reversal by insulin, are
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Figure 5.
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Effect of insulin on total RLM, RNA in the diabetic rat.
Male rats were made diabetic with streptozotocin and daily treatment with insulin continued for up to 2 weeks. RNA was prepared from two animals at each time point, pooled, applied directly on to nitrocellulose and subsequently probed with 32P-labelled RLM,-1 cDNA.
Table 1. Serum acetone concentrations during insulin reversal of diabetic rats. Serum acetone (mM)
Treatment Control Diabetic Diabetic plus Diabetic plus Diabetic plus Diabetic plus Diabetic plus Diabetic plus
insulin 6 h) insulin (1 2 h) insulin (24 h) insulin (48 h) insulin (72 h) insulin ( 2 weeks)
0.07 k0.03 4.10+ 2.26 5.94 (n = 2) 0.49k0.35 0 5 9 0.10 0.75 If: 0.36 0.87k0.49 0 2 8 k 0.1 9
+
Rats were made diabetic with a single streptozotocin injection (65 mg/kg i.p.) at the start of week 1 and diabetes allowed to develop over a 10-week period. At the end of week 10, twice-daily insulin treatment wascommenced (see Materialsand methods for full description of insulin treatment) and continued over a period of 2 weeks with interim kills as indicated in the table. Times refer to the period after insulin treatment had commenced. Results are expressed as meankS.D. from three rats per group unless otherwise indicated.
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accompanied by similar changes in serum acetone concentrations. These results parallel the relationship between cytochrome P4502E1 induction and serum acetone levels (Yo0 et al. 1990).
EfSect of testosterone on RLM6 m R N A level in the diabetic male rat Streptozotocin-induced diabetes is accompanied by a substantial fall in serum testosterone levels (Murray et al. 1981). It was of interest therefore to examine the influence of testosterone treatment on the regulation of RLM, expression in the male diabetic rat liver. Accordingly, total RNA derived from control, diabetic and diabetic plus testosterone treated rats was analysed by RNA dot blots using the RLM, cDNA probe. As shown in figure 6 ( a ) , testosterone treatment of diabetic animals resulted in a partial reversal of the diabetes-induced elevation of RLM, mRNA. This result was confirmed when the polysomal RNAs were translated in vitro (figure 6 ( b ) ) . However it should be noted that in insulin-treated diabetic animals, there appears also to be a decrease in immunoprecipitable RLM, apoprotein.
Figure 6.
Effect of testosterone on RLM, mRNA-directed protein synthesis and corresponding mRNA levels in the male diabetic rat. (a)Total RNA (5 pg) from control (C), diabetic (D) or diabetic plus testosterone ( D + T ) treated male rats were pooled from four animals and dot blot analysis carried out with the RLM,-1 cDNA probe (left hand panel) or actin cDNA probe (right hand panel). ( b ) Liver polysomal RNA (10pg) was used to programme a rabbit reticulocyte protein-synthesizing system, and 2 x lo6dpm from each translated mix was immunoprecipitated with RLM, antibody (SOpg) and protein Asepharose CL-4B. Immunoprecipates were run on SDS-PAGE and visualized by autoradiography. Polysomal RNA was derived from either control (track l ) , diabetic (track 2), diabetic plus insulin (track 3) or diabetic plus testosterone (track 4) treated male rats.
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Discussion This study was prompted by the reported similarity between ethanol-inducible cytochrome P4502E1 and diabetes-inducible RLM,. Both isozymes are induced by diabetes or fasting (Hong et al. 1987) and both proteins are identical in their first 20 amino acid residues (Favreau et al. 1987, Ryan et al. 1985). In addition, they have a similar substrate specificity profile (Favreau et al. 1987, Earnshaw et al. 1988, Song et al. 1986, Murray et al. 1981). Although the P4502E1 gene has been isolated and its regulation studied in the past 5 years (Song et al. 1986, Thomas et al. 1987, Peng et al. 1983, Song et al. 1987, Dong et al. 1988, Yamazoe et al. 1989), no such study has been reported for RLM,. Identity of the RLM, cDNA sequence presented here to that of P4502E1 is strong evidence that diabetes-inducible RLM, and ethanol-inducible P4502E1 are probably the same protein. Although the complete cDNA sequence for RLM, was not determined in our studies, one would expect there to be some nucleotide differences in the cDNAs if P4502E1 and RLM, were distinctly different gene products, as has been reported for the two very closely related phenobarbitalinducible P-450 genes, 2B1 and 2B2 (Yuan et al. 1983). This was not the case, and of the 1092 nucleotides sequenced for the RLM, cDNA (approximating to 65% of the P4502E1 gene) there was complete sequence homology (figure 2) with the reported P4502E1 cDNA sequence. Further evidence of the identity of RLM, and P4502E1 comes from Southern blot analysis of rat genomic DNA. Use of either P4502E1 probe (Song et al. 1986) or our RLM, probe (figure 3) leads to the conclusion that there is only one genomic sequence corresponding to these cDNAs, and confirms the findings of Song et al. (1986). It should be noted that the hybridization band pattern for genomic DNA in our studies using the RLM6-1 cDNA probe is different from that reported by Song et al. (1986) because a different sized cDNA probe and a different rat strain were used in their studies. Confirmation of the existence of one gene also comes from analysis of the Northern blot and RNA dot blot data (figure 4) which indicate that RLM, mRNA is the same size as P4502E1 mRNA and like P4502E1, its level is elevated during diabetes or fasting (Hong et al. 1987). T h e demonstration of identity between P4502E1 and RLM, confirms the suggestion that this gene is regulated by two different post-transcriptional mechanisms, namely mRNA stabilization (Song et al. 1987) and protein stabilization (Eliasson et al. 1988), depending on the inducing stimulus. In streptozotocininduced diabetes and fasting, loss of insulin through the destruction of pancreatic cells is accompanied by decreases in testosterone (Thummel and Schenkman 1990, Murray et al. 1981) and thyroid hormone (Thummel and Schenkman 1990, Chopra et al. 1981) levels and the pulsatile secretion pattern of growth hormone is abolished (Tannenbaum 1981). It is thus difficult to study the phenomenon of mRNA stabilization when a number of hormonal parameters are changing. Results in this paper indicate that insulin may not have a direct effect on RLM, mRNA level, but may indeed function through changes in hormone levels such as testosterone (figure 6). This would further substantiate the proposal that P450RLM6/2E1 is modulated in diabetes both at the mRNA level by hormones and at the protein level by small molecules such as acetone (Eliasson et al. 1988). Our data has also shown that RLM, mRNA levels are elevated in the fasted rat. This may indicate that a mechanism similar to that in diabetes is operative, in that fasting results in an increase in hepatic fatty acid metabolism with a concomitant increase in serum ketones. Whether the fasting-dependent increase in RLM, mRNA level results directly from changes in lipid metabolism or is an indirect effect of hormone changes (as noted above) has yet to be determined.
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In conclusion, it is now clear that constitutive cytochrome P-450 isozymes are regulated both by physiological and nutritional factors and a number of molecular mechanisms may be involved in modulation of enzyme activity. An understanding of these mechanisms may ultimately provide the basis for rationalization of interindividual variations of drug metabolism in man.
Acknowledgements Research was supported in part by an SERC-CASE studentship (T.H.R.) in conjunction with Hoechst Pharmaceuticals, UK, a travel grant from the BurroughsWellcome Fund (J.B.S.), USPH grant G M 261 14 (J.B.S.) and the Medical Research Council (G.G.G.).
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