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Peroxisome Proliferator-activated Receptor α Controls the Hepatic CYP4A Induction Adaptive Response to Starvation and Diabetes

1998; Elsevier BV; Volume: 273; Issue: 47 Linguagem: Inglês

10.1074/jbc.273.47.31581

1083-351X

Deanna L. Kroetz, Philip Yook, Phillipe Costet, Pascale Bianchi, Thierry Pineau,

Pharmacogenetics and Drug Metabolism

The hepatic CYP4A enzymes are important fatty acid and prostaglandin ω-hydroxylases that are highly inducible by fibric acid hypolipidemic agents and other peroxisome proliferators. Induction of the CYP4A enzymes by peroxisome proliferators is mediated through the nuclear peroxisome proliferator-activated receptor α (PPARα). Fatty acids have recently been identified as endogenous ligands of PPARα, and this receptor has been implicated in the regulation of lipid homeostasis. In the present report we characterized the induction of the hepatic CYP4A genes in rats during the altered lipid metabolism associated with starvation and diabetes. The mRNA levels of CYP4A1, CYP4A2, and CYP4A3 were induced 7–17-fold in the livers of fasted animals and 3–8-fold in the livers of diabetic animals. This was accompanied by corresponding changes in CYP4A protein levels and arachidonic and lauric acid ω-hydroxylase activity. Interestingly, feeding animals after the fasting period caused as much as an 80% suppression of CYP4A mRNA levels, whereas CYP4A protein levels and functional activity returned to control values. A second PPARα-responsive gene, acyl-CoA oxidase, was also induced in rat liver by diabetes and fasting. By using PPARα-deficient mice, we unambiguously demonstrated that PPARα is strictly required for hepatic CYP4A induction by starvation and diabetes. Similarly, induction of hepatic thiolase and bifunctional enzyme also required expression of PPARα. This represents the first evidence for the pathophysiologically induced activation of a nuclear receptor. The hepatic CYP4A enzymes are important fatty acid and prostaglandin ω-hydroxylases that are highly inducible by fibric acid hypolipidemic agents and other peroxisome proliferators. Induction of the CYP4A enzymes by peroxisome proliferators is mediated through the nuclear peroxisome proliferator-activated receptor α (PPARα). Fatty acids have recently been identified as endogenous ligands of PPARα, and this receptor has been implicated in the regulation of lipid homeostasis. In the present report we characterized the induction of the hepatic CYP4A genes in rats during the altered lipid metabolism associated with starvation and diabetes. The mRNA levels of CYP4A1, CYP4A2, and CYP4A3 were induced 7–17-fold in the livers of fasted animals and 3–8-fold in the livers of diabetic animals. This was accompanied by corresponding changes in CYP4A protein levels and arachidonic and lauric acid ω-hydroxylase activity. Interestingly, feeding animals after the fasting period caused as much as an 80% suppression of CYP4A mRNA levels, whereas CYP4A protein levels and functional activity returned to control values. A second PPARα-responsive gene, acyl-CoA oxidase, was also induced in rat liver by diabetes and fasting. By using PPARα-deficient mice, we unambiguously demonstrated that PPARα is strictly required for hepatic CYP4A induction by starvation and diabetes. Similarly, induction of hepatic thiolase and bifunctional enzyme also required expression of PPARα. This represents the first evidence for the pathophysiologically induced activation of a nuclear receptor. cytochrome P450 hydroxyeicosatetraenoic acid peroxisome proliferator-activated receptor acyl-CoA oxidase enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (bifunctional enzyme) 3-ketoacyl-CoA thiolase nucleotide kilobase pair glyceraldehyde-3-phosphate dehydrogenase. The cytochrome P450 4A (CYP4A)1 enzymes are fatty acid and prostaglandin hydroxylases that are abundantly expressed in the liver and kidney (1Simpson A.E. Gen. Pharmacol. 1997; 28: 351-359Crossref PubMed Scopus (248) Google Scholar). Of recent interest has been the physiological significance of CYP4A-catalyzed arachidonic acid metabolites. The ω-hydroxylated metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE), has been implicated in diverse biological functions, including the regulation of vascular tone, renal tubular ion transport, and bronchoconstriction (2Rahman M. Wright Jr., J.T. Douglas J.G. Am. J. Hypertens. 1997; 10: 356-365Crossref PubMed Scopus (77) Google Scholar). Recent studies with recombinant proteins have confirmed the narrow substrate specificity of the CYP4A enzymes. These enzymes can hydroxylate saturated and unsaturated fatty acids at the ω and ω-1 positions, with a clear preference for the ω position (3Aoyama T. Hardwick J.P. Imaoka S. Funae Y. Gelboin H.V. Gonzalez F.J. J. Lipid Res. 1990; 31: 1477-1482Abstract Full Text PDF PubMed Google Scholar, 4Roman L.J. Palmer C.N. Clark J.E. Muerhoff A.S. Griffin K.J. Johnson E.F. Masters B.S. Arch. Biochem. Biophys. 1993; 307: 57-65Crossref PubMed Scopus (79) Google Scholar). The selectivity of the CYP4A enzymes for prostaglandins E1, A1, and F2α is limited to specific isoforms, and hydroxylation of these substrates is measurable only at the ω position (3Aoyama T. Hardwick J.P. Imaoka S. Funae Y. Gelboin H.V. Gonzalez F.J. J. Lipid Res. 1990; 31: 1477-1482Abstract Full Text PDF PubMed Google Scholar, 4Roman L.J. Palmer C.N. Clark J.E. Muerhoff A.S. Griffin K.J. Johnson E.F. Masters B.S. Arch. Biochem. Biophys. 1993; 307: 57-65Crossref PubMed Scopus (79) Google Scholar). Detailed kinetic studies have not yet been carried out to clearly identify the relative importance of the multiple CYP4A isoforms in the in vivo metabolism of fatty acids and prostaglandins. Fatty acid ω-hydroxylation is generally a minor pathway of hepatic fatty acid metabolism relative to mitochondrial β-oxidation. However, it becomes increasingly important during periods of increased delivery of fatty acids to the liver such as in uncontrolled diabetes mellitus or starvation and in clinical conditions such as Reye's syndrome and alcoholic liver disease where mitochondrial β-oxidation is severely impaired (5Ma X. Baraona E. Lieber C.S. Hepatology. 1993; 18: 1247-1253Crossref PubMed Scopus (61) Google Scholar, 6Mortensen P.B. Gregersen N. Biochim. Biophys. Acta. 1982; 710: 477-484Crossref PubMed Scopus (54) Google Scholar, 7Heubi J.E. Partin J.C. Partin J.S. Schubert W.K. Hepatology. 1987; 7: 155-164Crossref PubMed Scopus (74) Google Scholar). The hepatic and renal expression of the CYP4A genes is highly inducible by a diverse group of compounds referred to as peroxisome proliferators. Peroxisome proliferators include the widely prescribed lipid-lowering drugs of the fibrate class (including clofibrate, fenofibrate, and nafenopin), phthalate ester plasticizers such as di-(2-ethylhexyl)phthalate, the endogenous steroid dehydroepiandrosterone, and chlorinated phenoxy and benzoic acid herbicides (8Reddy J.K. Azarnoff D.L. Hignite C.E. Nature. 1980; 283: 397-398Crossref PubMed Scopus (774) Google Scholar, 9Bacher M.A. Gibson G.G. Chem. Biol. Interact. 1988; 65: 145-156Crossref PubMed Scopus (32) Google Scholar, 10Wu H.Q. Masset-Brown J. Tweedie D.J. Milewich L. Frenkel R.A. Martin-Wixtrom C. Estabrook R.W. Prough R.A. Cancer Res. 1989; 49: 2337-2343PubMed Google Scholar, 11Okita J.R. Castle P.J. Okita R.T. J. Biochem. Toxicol. 1993; 8: 135-144Crossref PubMed Scopus (53) Google Scholar, 12Espandiari P. Thomas V.A. Glauert H.P. O'Brien M. Noonan D. Robertson L.W. Fundam. Appl. Toxicol. 1995; 26: 85-90Crossref PubMed Google Scholar). Administration of peroxisome proliferators to rats results in hepatomegaly, a dramatic increase in the number of peroxisomes in the liver, and a decreased level of serum triglycerides (13Reddy J.K. Lalwai N.D. Crit. Rev. Toxicol. 1983; 12: 1-58Crossref PubMed Scopus (859) Google Scholar). An induction of a number of genes that encode proteins involved in fatty acid metabolism and transport accompanies these changes in liver structure and lipid metabolism. Included in this group are the CYP4A enzymes, acyl-coenzyme A oxidase (AOX), the bifunctional enzyme (BIEN), 3-ketoacyl-CoA thiolase (thiolase), the cytosolic liver fatty acid-binding protein, HMG-CoA synthase, and apolipoproteins A-I and A-II (14Schoonjans K. Staels B. Auwerx J. J. Lipid Res. 1996; 37: 907-925Abstract Full Text PDF PubMed Google Scholar). Significant progress has been made in understanding the mechanisms involved in the induction of CYP4A and other responsive genes by peroxisome proliferators. Early studies indicated a transcriptional regulation of induction characterized by a 5–7-fold increase in CYP4A mRNA levels following treatment with clofibrate (15Hardwick J.P. Song B.J. Huberman E. Gonzalez F.J. J. Biol. Chem. 1987; 262: 801-810Abstract Full Text PDF PubMed Google Scholar). Induction is regulated through the peroxisome proliferator-activated receptor (PPAR), a member of the nuclear receptor superfamily of ligand-activated transcription factors. Three different PPARs have been identified (α, β/δ, and γ), and each displays a distinct pattern of expression (14Schoonjans K. Staels B. Auwerx J. J. Lipid Res. 1996; 37: 907-925Abstract Full Text PDF PubMed Google Scholar). The α isoform is predominantly expressed in hepatocytes, cardiomyocytes, renal proximal tubule cells, and enterocytes and is associated with the peroxisome proliferator effects. Targeted disruption of PPARα in mice clearly demonstrated that this was the major isoform mediating the pleiotropic effects of peroxisome proliferators (16Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1487) Google Scholar). Following treatment with the peroxisome proliferators clofibrate and Wy-14,643, mice lacking the PPARα gene showed no hepatomegaly, peroxisome proliferation, or induction of the normally responsive genes. PPARs contain a DNA-binding domain that recognizes a peroxisome proliferator response element in the promoters of target genes. Peroxisome proliferator response elements consist of a directly repeating core sequence separated by one nucleotide and have been identified in a number of genes involved in lipid metabolism and transport, including rabbit CYP4A6 and rat CYP4A1 (17Johnson E.F. Palmer C.N. Griffin K.J. Hsu M.H. FASEB J. 1996; 10: 1-8Crossref Scopus (169) Google Scholar). The PPAR binds to the response element as a heterodimeric complex with the 9-cis-retinoic acid (rexinoid) receptor (18Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Nature. 1992; 358: 771-774Crossref PubMed Scopus (1510) Google Scholar). PPARα was originally considered an orphan receptor with no identified endogenous ligand. In addition to peroxisome proliferators, a number of fatty acids have been shown to activate PPARα, but until recently, attempts to demonstrate binding of these compounds to the receptor failed (19Gottlicher M. Widmark E. Li Q. Gustafsson J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4653-4657Crossref PubMed Scopus (795) Google Scholar, 20Keller H. Dreyer C. Medin J. Mahfoudi A. Ozato K. Wahli W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2160-2164Crossref PubMed Scopus (846) Google Scholar, 21Issemann I. Prince R.A. Tugwood J.D. Green S. J. Mol. Endocrinol. 1993; 11: 37-47Crossref PubMed Scopus (282) Google Scholar). In the past year several groups have identified unsaturated fatty acids, the lipoxygenase metabolite (8S)-HETE, and fibrates as ligands for PPARα (22Kliewer S.A. Sundseth S.S. Jones S.A. Brown P.J. Wisely G.B. Koble C.S. Devchand P. Wahli W. Willson T.M. Lenhard J.M. Lehmann J.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4318-4323Crossref PubMed Scopus (1859) Google Scholar, 23Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1844) Google Scholar). Based on the results of competition binding and conformation-based assays, oleic, linoleic, linolenic, and arachidonic acids were identified as ligands at concentrations similar to those found in human plasma, providing strong evidence that these compounds act as endogenous ligands of PPARα. Other eicosanoids activate but do not bind the receptor, raising the possibility that further metabolism may be required for the formation of the actual ligand. Recent data show that in rodents dietary polyunsaturated fatty acids induce microsomal and peroxisomal fatty acid oxidation through activation of PPARα and suppress lipogenic gene expression through a PPARα-independent mechanism (24Ren B. Thelen A.P. Peters J.M. Gonzalez F.J. Jump D.B. J. Biol. Chem. 1997; 272: 26827-26832Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). The identification of fatty acids and eicosanoids as PPAR ligands suggests that this receptor plays an important role in the regulation of lipid homeostasis and in the pathogenesis of a number of metabolic disorders, including diabetes, obesity, and atherosclerosis. Limited data suggest that the hepatic CYP4A genes are also induced by at least two pathophysiological conditions, diabetes and fasting. An increase in CYP4A protein levels and its specific enzymatic activity, lauric acid (ω/ω-1)-hydroxylase activity, has been detected in the livers of streptozotocin-treated rats (25Barnett C.R. Gibson G.G. Wolf C.R. Flatt P.R. Ioannides C. Biochem. J. 1990; 268: 765-769Crossref PubMed Scopus (84) Google Scholar, 26Barnett C.R. Rudd S. Flatt P.R. Ioannides C. Biochem. Pharmacol. 1993; 45: 313-319Crossref PubMed Scopus (40) Google Scholar, 27Shimojo N. Ishizaki T. Imaoka S. Funae Y. Fujii S. Okuda K. Biochem. Pharmacol. 1993; 46: 621-627Crossref PubMed Scopus (148) Google Scholar, 28Ferguson N.L. Donahue B.S. Tenney K.A. Morgan E.T. Drug Metab. Dispos. 1993; 21: 745-746PubMed Google Scholar). Limited data suggest that CYP4A mRNA levels are also increased following induction of diabetes (28Ferguson N.L. Donahue B.S. Tenney K.A. Morgan E.T. Drug Metab. Dispos. 1993; 21: 745-746PubMed Google Scholar, 29Kimura S. Hardwick J.P. Kozak C.A. Gonzalez F.J. DNA (N. Y.). 1989; 8: 517-525Crossref PubMed Scopus (141) Google Scholar). Similarly, CYP4A protein levels and associated lauric acid hydroxylation were increased in hepatic microsomes from animals that had been fasted for 48–72 h immediately prior to sacrifice (30Imaoka S. Terano Y. Funae Y. Arch. Biochem. Biophys. 1990; 278: 168-178Crossref PubMed Scopus (128) Google Scholar). There is no information from these studies as to whether CYP4A mRNA levels were also increased. The current study was designed to test the hypothesis of involvement of the nuclear receptor PPARα in the pathophysiological adaptive response of mammals to diabetes and fasting. This is of particular importance since induction of the formation of the CYP4A metabolites of arachidonic acid may alter their biological effects. We found that both diabetes and fasting induce CYP4A mRNA and protein expression and alter the profile of arachidonic acid metabolism. The diabetes-induced effects were insulin-dependent, and the effects due to fasting were highly dependent on the duration of food deprivation. By using PPARα-deficient mice (PPARα−/−), we unambiguously demonstrated that these effects are mediated through PPARα, providing the first evidence of a pathophysiologically induced activation of this nuclear receptor. Furthermore, similar induction of AOX, BIEN, and thiolase suggests that this pathophysiological activation of PPARα will have widespread effects on hepatic lipid metabolism. Streptozotocin was purchased from Sigma and insulin from the University of California San Francisco Pharmacy Services. Blood glucose was measured using the One Touch®monitoring kit from Lifescan, Milpitas, CA. Radiolabeled nucleotides were purchased from NEN Life Science Products, and radiolabeled arachidonic acid and lauric acid were from Amersham Pharmacia Biotech. Restriction enzymes were obtained from New England Biolabs, Beverly, MA; modifying enzymes were from Life Technologies, Inc., and RNase was from Ambion, Austin, TX. All molecular biology grade chemicals, high pressure liquid chromatography solvents, and ScintiVerse LC were from Fisher. Arachidonic acid and lauric acid were purchased from Nu Chek Prep, Elysian, MN. Nitrocellulose and nylon membranes were from Micron Separations, Inc., Westborough, MA, and the anti-rat CYP4A1 antisera was purchased from Gentest Corp., Woburn, MA. Protein was measured with the Pierce BCA Protein Assay from Pierce. All other reagents were of the highest grade available and were purchased from Fisher or Sigma. Male Sprague-Dawley rats (200–220 g) were purchased from Simonsen Laboratories, Gilroy, CA, and were housed in a controlled environment with a 12-h light/dark cycle and free access to water and standard laboratory chow. All animal use was approved by the University of California San Francisco Committee on Animal Research and followed the National Institutes of Health guidelines for the care and use of experimental animals. Diabetes was induced by a single intraperitoneal injection of streptozotocin (65 mg/kg) in 50 mm citrate buffer, pH 4.5. Control animals were given an injection of buffer only. One group of diabetic animals was given subcutaneous injections of insulin (2 units at 8:00 a.m. and 4 units at 8:00 p.m.) on days 14–20 following streptozotocin treatment. Body weight and blood glucose were determined prior to induction of diabetes and weekly throughout the study period. All animals were sacrificed 21 days after streptozotocin injection. In the fasting studies, food was removed from the animals for 24–72 h, and animals were sacrificed at the end of the treatment. In some cases, the fasting period was followed by a period in which animals were fed for various lengths of time before sacrifice. Control animals had free access to food for an identical length of time, and all groups of animals had free access to water throughout the study. In all cases, rats were anesthetized with ether, and their livers were perfused with ice-cold saline, removed, and immediately frozen in liquid nitrogen. Livers were stored at −80 °C for the preparation of RNA and microsomes. Similar diabetes and fasting protocols were also carried out in C57BL/6 control mice and PPARα−/− mice. All of the mouse studies were carried out at Laboratoire de Pharmacologie et Toxicologie in Toulouse, France, according to institutional guidelines. The development of the PPARα−/− line from homologous recombination of 129Sv-derived cells has been described previously (16Lee S.S. Pineau T. Drago J. Lee E.J. Owens J.W. Kroetz D.L. Fernandez-Salguero P.M. Westphal H. Gonzalez F.J. Mol. Cell. Biol. 1995; 15: 3012-3022Crossref PubMed Scopus (1487) Google Scholar). Chimeric males were initially backcrossed to C57BL/6 females. Several additional rounds of backcrossing were performed in our animal facility to increase the C57BL/6 genetic background and to generate the animals used in these studies. C57BL/6 controls were from IFFA-CREDO, Les Oncins, France. Diabetes was induced in control and PPARα−/− mice (5 mice/strain) with a single intraperitoneal injection of 170 mg/kg streptozotocin. Control animals received vehicle only. Ten days after the streptozotocin treatment the mice were sacrificed, and blood was collected for blood glucose determinations, and livers were removed and immediately frozen in liquid nitrogen. Six groups of five mice were used for the fasting protocol. Control groups of C57BL/6 and PPARα−/− mice were allowed free access to food, and the remaining two groups of each strain were fasted for 30 h. One set of fasted animals was sacrificed at the end of this period, and a second set was fed for 42 h before sacrifice. All animals had free access to water throughout the entire experimental period. At the end of the experimental period, blood was collected for glucose determinations, and livers were removed and immediately frozen in liquid nitrogen. Total RNA was isolated from livers by acid/phenol extraction (31Ausbel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1994: 4.2.4-4.2.5Google Scholar). Construction of specific CYP4A riboprobes and details of the ribonuclease protection assays were described previously (32Kroetz D.L. Huse L.M. Thuresson A. Grillo M.P. Mol. Pharmacol. 1997; 52: 362-372Crossref PubMed Scopus (63) Google Scholar). The CYP4A3 probe is used to detect both the CYP4A3 and CYP4A2 mRNA transcripts. The protected fragments spanned the following regions of the cDNA sequence: 1285–1557 nt of CYP4A1; 343–549 nt of CYP4A2; 214–526 nt of CYP4A3; and 2–250 nt of GAPDH. Autoradiographs were scanned with a Ultro Scan XL laser densitometer from Amersham Pharmacia Biotech, and the level of a given CYP4A mRNA was expressed relative to the level of GAPDH mRNA. Hepatic microsomes were prepared from frozen liver tissue using differential centrifugation and stored at −80 °C (33Imaoka S. Funae Y. Biochim. Biophys. Acta. 1991; 1074: 209-213Crossref PubMed Scopus (36) Google Scholar). Arachidonic acid and lauric acid metabolism were measured in hepatic microsomes at a final substrate concentration of 0.25 and 0.10 mm, respectively. Reaction conditions, metabolite extraction, and separation and quantification of arachidonic acid and lauric acid metabolites by reverse phase high pressure liquid chromatography with radiometric detection were described previously (32Kroetz D.L. Huse L.M. Thuresson A. Grillo M.P. Mol. Pharmacol. 1997; 52: 362-372Crossref PubMed Scopus (63) Google Scholar). Hepatic microsomes (5 μg of protein) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblotting using goat polyclonal antibodies against rat liver CYP4A1 (31Ausbel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1994: 4.2.4-4.2.5Google Scholar). The electrophoresis conditions, protein transfer, and immunodetection have been described previously in detail (32Kroetz D.L. Huse L.M. Thuresson A. Grillo M.P. Mol. Pharmacol. 1997; 52: 362-372Crossref PubMed Scopus (63) Google Scholar). Hepatic message levels of mouse CYP4A, BIEN, and thiolase and rat AOX were detected by Northern blotting. Total hepatic RNA was isolated with Trizol reagent from Life Technologies, Inc., Paris, France, and 10-μg samples were fractionated on a 1% agarose gel containing 2.2m formaldehyde and transferred to a nylon membrane. A 1.8-kb fragment of the CYP4A3 cDNA, a 0.5-kb fragment of rat AOX cDNA, a 1.1-kb fragment of rat BIEN cDNA, a 1.0-kb fragment of rat thiolase cDNA, and a 1.2-kb fragment of rat GAPDH cDNA were labeled with [α-32P]dCTP using the random primer technique (31Ausbel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1994: 4.2.4-4.2.5Google Scholar). The membranes were hybridized with the radiolabeled probe at 65 °C overnight in a solution of 6× SSC (1× SSC: 150 mm NaCl, 15 mm sodium citrate, pH 7.0), 1× Denhardt's reagent (10 mg/ml each of BSA fraction V, Ficoll, and polyvinylpyrrolidone), 0.5% sodium dodecyl sulfate, and 20 μg/ml salmon sperm DNA. After hybridization the blots were washed to a final stringency of 0.1× SSC, 0.1% SDS at 65 °C. Bands were visualized with a PhosphorImager and analyzed using ImageQuant Software, Molecular Dynamics, Sunnyvale, CA. All measurements were performed on RNA or protein samples from individual animals, and the results are expressed as mean ± S.D. of five to six animals per treatment group. Statistical significance of differences between treatment groups was evaluated by an analysis of variance with post hoc multiple comparison testing with a modified t test. A value of p < 0.05 was considered to be statistically significant. All rats treated with streptozotocin showed a significant decrease in weight gain and increase in blood glucose levels relative to controls, consistent with the induction of diabetes. One week following streptozotocin treatment blood glucose levels were 88 ± 11 mg/dl in control animals and 379 ± 64 mg/dl in the diabetic animals (p < 0.005). This elevation in blood glucose was maintained throughout the entire study period in the untreated diabetic rats. Both weight gain and blood glucose levels returned to control values upon treatment of diabetic rats with insulin. The effect of diabetes on CYP4A mRNA levels was measured using gene-specific RNase protection assays. These assays quantify the message level of each individual CYP4Agene in a given sample relative to the level of GAPDH mRNA. Diabetic animals showed a significant induction of CYP4A mRNA levels (Fig. 1). The message level for each of the hepatically expressed CYP4A genes (CYP4A1, CYP4A2 and CYP4A3) was increased up to 8-fold in the diabetic rat livers, and this increased expression was completely reversed by treatment of diabetic rats with insulin. The order of induction was CYP4A1 > CYP4A2 > CYP4A3. Lauric acid and arachidonic acid ω- and (ω-1)-hydroxylase activity were measured in liver microsomes as indicators of CYP4A functional activity. Hydroxylation at the terminal position of both of these fatty acids is catalyzed solely by the CYP4A enzymes, whereas metabolism at the ω-1 position is also dependent on CYP2E1 and CYP2C enzymes (3Aoyama T. Hardwick J.P. Imaoka S. Funae Y. Gelboin H.V. Gonzalez F.J. J. Lipid Res. 1990; 31: 1477-1482Abstract Full Text PDF PubMed Google Scholar,34Imaoka S. Tanaka S. Funae Y. Biochem. Int. 1989; 18: 731-740PubMed Google Scholar, 35Laethem R.M. Koop D.R. Mol. Pharmacol. 1992; 42: 958-963PubMed Google Scholar, 36Laethem R.M. Balazy M. Falck J.R. Laethem C.L. Koop D.R. J. Biol. Chem. 1993; 268: 12912-12918Abstract Full Text PDF PubMed Google Scholar). Lauric acid was metabolized at the ω and ω-1 position in roughly a 1:1 ratio. Lauric acid ω- and (ω-1)-hydroxylation were increased almost 2-fold in hepatic microsomes prepared from diabetic rats, and this activity returned to control values after treatment of diabetic animals with insulin (Table I). Arachidonic acid was metabolized at the ω and ω-1 positions in a 1.8:1 ratio in rat hepatic microsomes. Induction of diabetes resulted in a 1.5-fold increase in the formation of 19- and 20-HETE. As with lauric acid, reversal of the diabetic condition with insulin returned CYP4A functional activity to control levels. Arachidonic acid epoxygenase activity was also induced (1.3-fold) in the livers of diabetic animals. This is consistent with previous reports of CYP2E1 induction by diabetes (26Barnett C.R. Rudd S. Flatt P.R. Ioannides C. Biochem. Pharmacol. 1993; 45: 313-319Crossref PubMed Scopus (40) Google Scholar, 27Shimojo N. Ishizaki T. Imaoka S. Funae Y. Fujii S. Okuda K. Biochem. Pharmacol. 1993; 46: 621-627Crossref PubMed Scopus (148) Google Scholar).Table IEffect of diabetes on hepatic lauric acid and arachidonic acid metabolismMetabolite formation rateControlDiabeticInsulinpmol/min · mg protein11-OH lauric acid755 ± 1121338 ± 3921-ap < 0.0005.608 ± 89.112-OH lauric acid592 ± 1561090 ± 3901-ap < 0.0005.521 ± 84.619-HETE59.8 ± 15.195.5 ± 18.01-bp < 0.05.44.5 ± 19.420-HETE108 ± 27.1154 ± 23.61-bp < 0.05.91.6 ± 20.11-bp < 0.05.Epoxygenase856 ± 2001139 ± 1161-bp < 0.05.536 ± 2421-bp < 0.05.Metabolite formation rates were measured in duplicate from microsomes prepared from six animals per treatment group. Values shown are the mean ± S.D. for each treatment group. Groups of six rats were injected on day 1 with a single dose of streptozotocin (diabetic and insulin groups) or vehicle. The insulin-treated group was given insulin twice daily between days 14 and 21, and all animals were sacrificed on day 21. Hepatic microsomes were prepared, and metabolism of lauric acid (100 μm) and arachidonic acid (250 μm) was measured as described under “Experimental Procedures.” Epoxygenase activity is the sum of epoxyeicosatrienoic acid and dihydroxyeicosatrienoic acid formation. Treatment groups were compared using an analysis of variance and post hoc multiple comparisons with a modified t test.1-a p < 0.0005.1-b p < 0.05. Open table in a new tab Metabolite formation rates were measured in duplicate from microsomes prepared from six animals per treatment group. Values shown are the mean ± S.D. for each treatment group. Groups of six rats were injected on day 1 with a single dose of streptozotocin (diabetic and insulin groups) or vehicle. The insulin-treated group was given insulin twice daily between days 14 and 21, and all animals were sacrificed on day 21. Hepatic microsomes were prepared, and metabolism of lauric acid (100 μm) and arachidonic acid (250 μm) was measured as described under “Experimental Procedures.” Epoxygenase activity is the sum of epoxyeicosatrienoic acid and dihydroxyeicosatrienoic acid formation. Treatment groups were compared using an analysis of variance and post hoc multiple comparisons with a modified t test. An increase in the level of CYP4A-immunoreactive protein in the livers of diabetic rats was consistent with the increase in CYP4A mRNA levels and CYP4A functional activity (Fig. 2). Two immunoreactive proteins were detected with this antibody with the upper band appearing as a doublet in the induced samples. According to Okita and co-workers (37Okita J.R. Johnson S.B. Castle P.J. Dezellem S.C. Okita R.T. Drug Metab. Dispos. 1997; 25: 1008-1012PubMed Google Scholar) the fastest migrating isoform is CYP4A1, the slowest is CYP4A3, and CYP4A2 migrates at an intermediate rate. All of the CYP4A proteins were induced to a similar degree during diabetes, and this induction was reversed by insulin treatment. Fasting of rats for 24–72 h caused the expected loss of body weight, while blood glucose decreased 40–50% after the 24- and 48-h fasts but was normal after the 72-h fasting period. Induction of CYP4A mRNA was apparent after fasting rats for 24 or 48 h (Fig. 3). Maximal induction of each of th