Akihiko Hirose

Modification by clofibric acid of acyl composition of glycerolipids in rat liver. Possible involvement of fatty acid chain elongation and desaturation.

Administration of p-chlorophenoxyisobutyric acid (clofibric acid) to rats induced a marked change in acyl composition of hepatic glycerolipids; a considerable increase in the proportion of octadecenoic acid (18:1) was accompanied by a marked decrease in the proportion of octadecadienoic acid (18:2). Among the glycerolipids, the changes in the proportions of 18:1 and 18:2 were the most marked in phosphatidylcholine. The change in the acyl composition of phosphatidylcholine paralleled the change in free fatty acid composition in microsomes. The treatment of rats with clofibric acid resulted in a 2.3-fold increase in activity of microsomal palmitoyl-CoA chain elongation and a 4.8-fold increase in activity of stearoyl-CoA desaturation. The activities of acyl-CoA synthetase, 1-acylglycerophosphate acyltransferase and 1-acylglycerophosphorylcholine acyltransferase in hepatic microsomes were increased approx. 3-, 1.7- and 3.6-times, respectively, by the treatment of rats with clofibric acid. These findings are discussed with respect to the role of fatty acid modification systems in the regulation of acyl composition of phosphatidylcholine.

cDNA cloning of rat liver 2,4-dienoyl-CoA reductase.

cDNA clones of 2,4-dienoyl-CoA reductase were isolated from rat liver cDNA libraries constructed in phages lambda gt11 and lambda gt10. Hybrid selected translation analysis revealed that 2,4-dienoyl-CoA reductase was translated as a polypeptide with a molecular weight of about 36,000, which was about 3,000 molecular weight units larger than mature reductase. Sequencing analysis revealed that the open reading frame encoded a polypeptide consisting of 335 amino acid residues (predicted molecular weight = 36,132), which contained an N-terminal extension peptide of 34 amino acid residues (presequence) in addition to the mature enzyme. Thus, 2,4-dienoyl-CoA reductase is synthesized as a larger precursor polypeptide, and the N-terminal extension peptide may be acting as the mitochondrial import signal.

Distribution of carbon-11 labeled methamphetamine and the effect of its chronic administration in mice

[11C]Methamphetamine, a psychotropic agent, was synthesized by N-methylation of amphetamine with [11C]CH3 I in hopes that it could be applied in the near future to assist positron emission tomography (PET) in the imaging of its distribution in the human brain. The regional distribution of [11C]methamphetamine was investigated in the mice brain at various intervals after an intravenous (i.v.) injection. Radioactivity was higher in the hypothalamus, cortex, striatum and hippocampus. Furthermore, in chronically administered mice, the uptake of [11C]methamphetamine was higher in the striatum than those in other regions. The regional differences in the distribution of methamphetamine in the mice brain may enable the imaging of its distribution by PET using [11C]methamphetamine.

Effect of clofibric acid on the molecular species composition of diacyl glycerophosphocholine of rat liver microsomes

The effect of administeringp-chlorophenoxyisobutyric (clofibric) acid to rats on the molecular species composition of diacyl-glycerophosphocholine (GPC) of rat liver microsomes was studied. Microsomal choline glycerophospholipids were converted to 1,2-diradyl-3-acetylglycerol and were separated into molecular species by reverse-phase high performance liquid chromatography. Diacyl-GPC consisted of 17 different molecular species. The predominant species were arachidonoyl derivatives, such as 18∶0–20∶4 (22.2% of the total) and 16∶0–20∶4 (22.0%). Administration of clofibric acid to rats caused a marked increase in 16∶0–18∶1 species of diacyl-GPC from 8% to 30%, making these the predominant species of diacyl-GPC in clofibric acid-fed rats. Also, a significant decrease (50% of controls) in 18∶0–18∶2 and 18∶0–20∶4 species was observed, whereas the decrease in molecular species containing 16∶0 at the 1-position such as 16∶0–18∶2 and 16∶0–20∶4 was small (approximately 85% of control). The results show that clofibric acid caused marked changes in the molecular species composition of diacyl-GPC. The participation of 1-acyl-GPC acyltransferase and stearoyl-CoA desaturase in the regulation of the molecular species composition of diacyl-GPC is discussed.

Induction of microsomal 1-acylglycerophosphocholine acyltransferase by peroxisome proliferators in rat kidney; co-induction with peroxisomal β-oxidation

Induction of microsomal 1-acyl-glycerophosphocholine (GPC) acyltransferase in rat tissues by four peroxisome proliferators, clofibric acid, tiadenol, DEHP and PFOA, was examined. Among the nine tissues examined, kidney, liver and intestinal mucosa responded to the challenges by the peroxisome proliferators to induce the enzyme. The treatment of rats with various dose of clofibric acid, tiadenol, DEHP or PFOA resulted in an induction of kidney microsomal 1-acyl-GPC acyltransferase in a dose-dependent manner. Despite the structural dissimilarity of peroxisome proliferators, the induction of microsomal 1-acyl-GPC acyltransferase was highly correlated with the induction of peroxisomal beta-oxidation. The activity of microsomal 1-acyl-GPC acyltransferase was not affected by changes in hormonal (adrenalectomy, diabetes, hyperthyroidism and hypothyroidism) and nutritional (starvation, starvation-refeeding, fat-free-diet feeding and high-fat-diet feeding) states. The induction of renal microsomal 1-acyl-GPC acyltransferase was seen in mice subsequent to the administration of clofibric acid and tiadenol and in guinea pigs subsequent to the administration of tiadenol. These results may indicate that kidney microsomal 1-acyl-GPC acyltransferase is a highly specific parameter responsive to the challenges by peroxisome proliferators and may suggest that the possibility that the inductions by peroxisome proliferators of microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation in kidney are co-regulated.

cDNA cloning of mitochondrial Δ3, Δ2-enoyl-CoA isomerase of rat liver

A 1.08 kbp cDNA encoding rat liver mitochondrial Δ 3 , Δ 2 -enoyl-CoA isomerase (ECI) of 298 amino acid residues ( M r 32 895) was isolated from rat liver λgt11, λgt10 cDNA libraries by the combination of an immunochemical method with a rabbit-antibody against rat liver ECI and a plaque hybridization method. The deduced amino acid sequence from the cDNA indicates that ECI is synthesized with an amino-terminal extrasequence of 35 amino acid residues and processed to the mature enzyme ( M r 29 256).

Immunocytochemical localization of 2,4-dienoyl-CoA reductase in the liver of normal and di-(2-ethylhexyl) phthalate-fed rats

SummaryLocalization of 2,4-dienoyl-CoA reductase (DCR) in rat liver was studied using immunoenzyme and immunogold techniques. The animals were fed on a laboratory diet with or without 2% di-(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, for two weeks. For light microscopy (LM), semithin Epon sections were stained by immunoenzyme technique after removal of the epoxy resin. For electron microscopy (EM), ultrathin Lowicryl K4M sections were stained by the protein A-gold technique. By LM, in untreated rats reaction deposits showing the antigenic sites for DCR were present in the cytoplasmic granules. Hepatocytes, epithelial cells of interlobular bile duct, and sinus-lining cells contained these granules. After administration of DEHP, the cytoplasmic granules stained similarly. The staining intensity of the heaptocytes increased markedly, but that of the other cells decreased. The sinus-lining cells became mostly negative. By EM, gold particles indicating the antigenic sites for DCR were present in both the mitochondria and peroxisomes of hepatocytes of untreated rats. In the other cells, the gold label was confined to the mitochondria. After administration of DEHP, labelling intensity of the hepatocyte mitochondria increased markedly, but that of the peroxisomes conversely decreased. Quantitative analysis of labelling density showed that the mitochondrial DCR increased to about three times that in the untreated rat, but the peroxisomal DCR decreased to 1/6. The results show that in the rat liver, DCR exists in both, mitochondria and peroxisomes. DEHP can induce mitochondrial DCR, but not peroxisomal DCR.

Immunocytochemical localization of Δ3, Δ2-enoyl-CoA isomerase in rat liver. The effects of di-(2-ethylhexyl)phthalate, a peroxisome proliferator

Immunocytochemical localization of Δ3, Δ2‐enoyl‐CoA isomerase (isomerase) was investigated in rat liver. Livers of di‐(2‐ethylhexyl)phthalate) (DEHP)‐treated or untreated rats were perfusion‐fixed and embedded in Epon or Lowicryl K4M. By light microscopy, reaction deposits for the enzyme were present in the cytoplasmic granules of hepatocytes and interlobular bile duct epithelium. Weak staining was noted in sinus‐lining cells. After administration of DEHP, the granular staining of the hepatocytes was markedly enhanced, whereas the staining reaction of the sinus‐lining cells decreased. The isomerase staining pattern was quite similar to that of long‐chain acyl‐CoA dehydrogenase (a mitochondrial marker), but different from that of catalase (a peroxisomal marker). Under electron microscopy, gold particles for isomerase were seen to be confined mainly to mitochondria of the hepatocytes, the bile duct epithelial cells and sinus‐lining cells. Peroxisomes were weakly labeled. After DEHP administration, the peroxisomes were markedly induced, but the mitochondria were not. Quantitative analysis showed that the induction of the peroxisomal isomerase was only 2‐fold whereas the mitochondrial isomerase was enhanced about 5‐fold, 40 times as high as the peroxisomal enzyme. The results show that the mitochondria are the main intracellular site for isomerase and the peroxisomes a minor site. The mitochondrial isomerase of the rat liver is markedly induced by peroxisome proliferators, DEHP and clofibrate.