Induction and origin of hepatocytes in rat pancreas.

2-[4(2,2- Dichlorocyclopropyl )phenoxy]2-methyl propionic acid (ciprofibrate), a peroxisome proliferator , induced hepatocytes in the pancreas of adult male F-344 rats when added to their diet at a dosage of 10 mg/kg body weight for 60-72 wk. These cells are morphologically indistinguishable from hepatic hepatocytes and were usually localized adjacent to islets of Langerhans with extensions into surrounding acinar tissue. A significant increase in the volume density of peroxisomes, together with immunochemically detectable amounts of two peroxisome-associated enzymes, was observed in pancreas with hepatocytes of rats maintained on ciprofibrate. Uricase-containing crystalloid nucleoids, specific for rat hepatocyte peroxisomes, were present in pancreatic hepatocytes. These structures facilitated the identification of cells with hybrid cytoplasmic features characteristic of pancreatic acinar and endocrine cells and hepatocytes. Such cells are presumed to represent a transitional state in which pancreas specific genes are being repressed while liver specific ones are simultaneously expressed. The presence of exocrine and/or endocrine secretory granules in transitional cells indicates that acinar/intermediate cells represent the precursor cell from which pancreatic hepatocytes are derived.

Comparison of the Effects of Clofibrate and Silafibrate on Sperm Parameters Quality and Sex Hormones in Male Rats.

PURPOSE: Fibrates are drugs widely used for the treatment of hyperlipidemic disorders. Previous studies on a novel analogue of clofibrate, called silafibrate, have shown good lipid lowering effects. This study was designed to assess the role of silafibrate as a peroxisome proliferator-activated receptors (PPARs) agonist on sperm health and spermatogenesis in adult male rats. MATERIAL AND METHODS: Seventy male Wistar rats were randomly allocated into 7 groups: Cl-10, Cl-20, and Cl-40 mg/kg/day (clofibrate); Si-10, Si-20, and Si-40 mg/kg/day (silafibrate); and C, control. After a 28-day treatment, all rats were euthanized. Blood samples were taken for determination of testosterone, total antioxidant capacity, levels of malondialdehyde, and oxidized low-density lipoprotein. Reproductive organs were dissected and spermatozoa collected from the epididymis for analysis. RESULT: Sperm parameters (count, motility, viability, and morphology) and total serum testosterone decreased significantly in clofibrate-treated (20 and 40 mg/kg) rats (P < 0.05) as compared with normal rats. CONCLUSION: We conclude that PPARs agonists have significant adverse effect on sperm viability, motility, and total serum testosterone, and could be harmful for sperm parameters and male reproductive function in rats.

Branched chain amino acid oxidation in cultured rat skeletal muscle cells. Selective inhibition by clofibric acid.

Leucine metabolism in skeletal muscle is linked to protein turnover. Since clofibrate is known both to cause myopathy and to decrease muscle protein content, the present investigations were designed to examine the effects of acute clofibrate treatment on leucine oxidation. Rat skeletal muscle cells in tissue culture were used in these studies because cultivated skeletal muscle cells, like muscle in vivo, have been shown to actively utilize branched chain amino acids and to produce alanine. The conversion of [1-(14)C]leucine to (14)CO(2) or to the [1-(14)C]keto-acid of leucine (alpha-keto-isocaproate) was linear for at least 2 h of incubation; the production of (14)CO(2) from [1-(14)C]leucine was saturable with a K(m) = 6.3 mM and a maximum oxidation rate (V(max)) = 31 nmol/mg protein per 120 min. Clofibric acid selectively inhibited the oxidation of [1-(14)C]leucine (K(i) = 0.85 mM) and [U-(14)C]isoleucine, but had no effect on the oxidation of [U-(14)C]glutamate, -alanine, -lactate, or -palmitate. The inhibition of [1-(14)C]leucine oxidation by clofibrate was also observed in the rat quarter-diaphragm preparation. Clofibrate primarily inhibited the production of (14)CO(2) and had relatively little effect on the production of [1-(14)C]keto-acid of leucine. A physiological concentration-3.0 g/100 ml-of albumin, which actively binds clofibric acid, inhibited but did not abolish the effects of a 2-mM concentration of clofibric acid on leucine oxidation. Clofibrate treatment stimulated the net consumption of pyruvate, and inhibited the net production of alanine. The drug also increased the cytosolic NADH/NAD(+) ratio as reflected by an increase in the lactate/pyruvate ratio, in association with a decrease in cell aspartate levels. The changes in pyruvate metabolism and cell redox state induced by the drug were delayed compared with the nearly immediate inhibition of leucine oxidation. These studies suggest that clofibric acid, in concentrations that approximate high therapeutic levels of the drug, selectively inhibits branched chain amino acid oxidation, possibly at the level of the branched chain keto-acid dehydrogenase.

The myocardial infarct size-limiting and antiarrhythmic effects of acyl-CoA:cholesterol acyltransferase inhibitor VULM 1457 protect the hearts of diabetic-hypercholesterolaemic rats against ischaemia/reperfusion injury both in vitro and in vivo.

The study was designed to characterise the influence of a novel acyl-CoA:cholesterol acyltransferase inhibitor, VULM 1457, on the severity of myocardial ischaemia-reperfusion injury in a model of diabetes mellitus and hypercholesterolaemia induced by co-administration of streptozotocin and a high fat-cholesterol diet. We used Langendorff-perfused rat hearts to measure the size of myocardial infarction after 30 min of regional ischaemia, followed by a 2-h reperfusion period, and open-chest rats were exposed to 6 min of ischaemia and 10 min of reperfusion to analyse ventricular arrhythmias. In addition to the high fat-cholesterol diet, VULM 1457 was administered to the diabetic-hypercholesterolaemic rats for 5 days. Decreased plasma and liver cholesterol levels and a significantly reduced occurrence of ventricular fibrillation (29% vs. 100%, P<0.01), determined via the mean number and duration of episodes (0.6+/-0.4 and 2.1+/-1.4 s vs. 2.8+/-0.8 and 53.5+/-14.4 s in diabetic-hypercholesterolaemic rats, both P<0.01), were observed in these animals. Lethal ventricular fibrillation was suppressed, and arrhythmia severity was also significantly decreased in these animals as compared to the non-treated animals (2.9+/-0.6 vs. 4.9+/-0.2; P<0.05). A smaller infarct size, normalised to the size of area at risk, was observed in the treated diabetic-hypercholesterolaemic group as compared to the non-treated group (16.3+/-1.9% vs. 37.3+/-3.1%; P<0.01). Aside from remarkable hypolipidaemic activity, VULM 1457 improved the overall myocardial ischaemia-reperfusion injury outcomes in the diabetic-hypercholesterolaemic rats by suppressing arrhythmogenesis as well as by reducing myocardial necrosis.

Effect of VULM 1457, an ACAT inhibitor, on serum lipid levels and on real time red blood cell flow in diabetic and non-diabetic hamsters fed high cholesterol-lipid diet.

Acyl-coenzyme A: cholesterol O-acyltransferase (ACAT) catalyzes the formation of cholesterol/fatty acyl-coenzyme A esters. Accumulation of cholesterol esters leads to pathological changes connected with atherosclerosis. We have evaluated effects of a newly synthesized ACAT inhibitor, 1-(2,6-diisopropyl-phenyl)-3-[4-(4'-nitrophenylthio)phenyl] urea (VULM 1457), on serum lipid (cholesterol and triglycerides) levels and velocity of red blood cells (RBC) in non-diabetic and diabetic hamsters fed on high cholesterol-lipid (HCHL) diet during 3 months. The VULM 1457 effects on the paw microcirculation were assessed using capillary microscopy by measuring (RBC) velocity in vivo. Hamsters fed on HCHL diet became hypercholesterolemic with a dramatic increase in serum lipids accompanied with significantly decreased RBC velocity. Diabetic hamsters fed on HCHL diet had further increased serum lipids with reduction of RBC velocity. The VULM 1457 inhibitor lowered cholesterol levels in both non-diabetic and diabetic hamsters fed on HCHL diet. The greater VULM 1457 effect was shown in diabetic hamsters fed on HCHL diet where VULM 1457 expressed hypotriglycerides effects, too. An improved RBC velocity-pronounced effect was observed in diabetic hamsters fed on HCHL diet treated with VULM 1457. These results suggest that the ACAT inhibitor, VULM 1457, is a prospective hypolipidemic and anti-atherogenic drug which treats diabetes.

Peroxisome proliferator-induced hepatocarcinogenesis: histochemical analysis of ciprofibrate-induced preneoplastic and neoplastic lesions for gamma-glutamyl transpeptidase activity.

Previous studies revealed that putative preneoplastic and neoplastic lesions induced in the liver by Wy-14,643, a peroxisome proliferator, were gamma-glutamyl transpeptidase (GGT) negative. For ascertainment as to whether phenotypes of foci and carcinomas induced by all peroxisome proliferators are similarly GGT negative, altered areas (AAs), neoplastic nodules (NNs), and hepatocellular carcinomas (HCCs) induced in the livers of male F344 rats by chronic dietary administration of ciprofibrate (0.025% wt/wt in chow; CAS: 52214-84-3) were analyzed histochemically for GGT activity. Eighty-nine percent of AAs, 91% of NNs, and 91% of HCCs were GGT negative. The GGT-negative property of these various hepatic preneoplastic and neoplastic lesions persisted at 8 weeks after the withdrawal of ciprofibrate treatment. The results of this study indicate that the absence of GGT activity is a common feature in hepatic lesions induced by structurally unrelated peroxisome proliferators and is not related to the drug toxicity. The proposal was made that peroxisome proliferators do not derepress the activity of the GGT gene during hepatocarcinogenesis in the rat.

Protective effect of simvastatin and VULM 1457 in ischaemic-reperfused myocardium of the diabetic-hypercholesterolemic rats.

This study examined the effects of simvastatin (10 mg/ kg) and VULM 1457 (50 mg/kg), an ACAT inhibitor, in the heart model of 6 min ischemia followed by 10 min reperfusion injury in the diabetic-hypercholesterolaemic (DM-HCH) rats. In the DM-HCH rats, the incidence of ventricular tachycardia (VT) had a tendency to be increased, while ventricular fibrillation (VF) occurred in all diseased rats (p < 0.01). Simvastatin and VULM 1457 with the shown hypolipidemic effect, significantly (p < 0.01) suppressed a formation of VF (38% and 29%; respectively).

Induction of altered hepatic foci in rats by the administration of hypolipidemic peroxisome proliferators alone or following a single dose of diethylnitrosamine.

The effect of feeding hypolipidemic peroxisome proliferators on the induction of altered hepatic foci (AHF) in Fischer rats was studied in order to determine whether such agents can induce or promote the development of AHF. In the first study, rats were fed ciprofibrate (10 mg/kg/day) for 1 yr. AHF, neoplastic nodules, and hepatocellular carcinomas were induced. The presence of putative gamma-glutamyltranspeptidase (GGT) activity was numerically the most common marker, although it was absent in larger foci and nodules. A deficiency in canalicular ATPase and glucose-6-phosphatase provided the best markers for the larger foci and nodules. In the second study, rats were subjected to partial hepatectomy, and half of the animals were then intubated with diethylnitrosamine (10 mg/kg). One wk later, rats were fed Wy-14,643 at concentrations of 0, 0.05, and 0.1% in the diet for 6 mo. At 6 mo, the number and volume of foci were increased by the feeding of Wy-14,643 after partial hepatectomy alone and were greatly increased when Wy-14,643 was fed after partial hepatectomy/diethylnitrosamine administration. Canalicular adenosine triphosphatase and glucose-6-phosphatase deficiencies were the most common markers of AHF, and AHF of these phenotypes occupied practically all of the focal volume. The larger AHF did not express GGT, and those foci exhibiting GGT were much less common and occupied very little volume. The absence of the GGT protein itself, as opposed to an inhibition of GGT activity, was verified by immunohistochemical staining using an antibody to GGT. These studies show that hypolipidemic peroxisome proliferators can stimulate an increase in AHF following a single dose of diethylnitrosamine and a mitotic stimulus, and they thus can act as promoters in two-stage liver carcinogenesis. GGT is a poor marker for identifying AHF induced by peroxisome proliferators during the early, premalignant phase of hepatocarcinogenesis.

Peroxisome proliferator-induced alterations in the expression and modification of rat hepatocyte plasma membrane proteins.

Rats were fed the peroxisome proliferator ciprofibrate (0.025%), and the effects on the expression, modification, and localization of seven domain-specific integral proteins of the rat hepatocyte plasma membrane were assessed using a combination of immunoblotting, -precipitation, and -fluorescence. Ciprofibrate caused the down-regulation of five of the plasma membrane proteins (the epidermal growth factor receptor, the asialoglycoprotein receptor, HA 321, HA 4, and dipeptidylpeptidase IV) and induced the expression of a more basic, lower-Mr isoform of the basolateral plasma membrane protein CE 9. Pulse labeling, chemical deglycosylation, and 125I-wheat germ lectin blotting suggested that the ciprofibrate-induced isoform of CE 9 differed in the posttranslational modification of its oligosaccharides and contained more sialic acid. These changes in hepatocyte surface differentiation were first observed between Days 1 and 5 on the ciprofibrate-containing diet, coincident with other aspects of the pleiotropic response of the hepatocyte to peroxisome proliferators, e.g., the induction of the Mr 78,000 peroxisome proliferation-associated protein. The effects were reversed within 2-3 weeks upon removal of ciprofibrate. The three other peroxisome proliferators tested, di(2-ethylhexyl)phthalate, clofibrate, and Wy-14,643, were found to exert most of these same effects on the expression and modification of the hepatocyte plasma membrane proteins, but the compounds differed in relative potency. The ciprofibrate-induced decreases in the concentrations of the epidermal growth factor receptor, the asialoglycoprotein receptor, HA 321, and HA 4 were similar to the selective down-regulation of these proteins observed transiently during the period of hepatocyte proliferation following two-thirds hepatectomy. Other compounds frequently used in studies of liver enzyme induction and carcinogenesis, the antioxidants ethoxyquin and butylated hydroxyanisole and the liver tumor promoter phenobarbital, were not as effective as ciprofibrate or two-thirds hepatectomy at causing the down-regulation of these proteins. The induction of the lower-Mr isoform of the basolateral plasma membrane protein CE 9 was not observed following two-thirds hepatectomy or upon the feeding of the antioxidants or phenobarbital but was specific to the feeding of the peroxisome proliferators.

Formation of 8-hydroxydeoxyguanosine in liver DNA of rats following long-term exposure to a peroxisome proliferator.

The mechanism by which nongenotoxic peroxisome proliferators induce hepatocellular carcinomas in rats and mice remains intriguing. The available experimental evidence suggests that the proliferation of peroxisomes and induction of peroxisome-associated enzymes results in oxidative stress which then leads to tumorigenesis. However, so far no direct evidence for oxidative DNA damage in livers of peroxisome proliferator-treated animals has been established. In the present study we have examined the DNA obtained from the livers of rats treated with ciprofibrate, a potent peroxisome proliferator, for variable periods of time for 8-hydroxydeoxyguanosine (8-OH-dG), an adduct that results from the damage of DNA caused by hydroxyl radical. Administration of ciprofibrate in diet at a concentration of 0.025% for 16, 28, 36, or 40 weeks resulted in progressive increases in the levels of 8-OH-dG. At 16, 28, and 40 weeks of ciprofibrate treatment, the 8-OH-dG in the liver DNA was significantly increased as compared to controls. This increase in 8-OH-dG levels is attributed to persistent peroxisome proliferation resulting from chronic ciprofibrate treatment as no increase in 8-OH-dG was found in liver DNA of rats that received a single large dose of ciprofibrate. The results of this study clearly demonstrate, for the first time, that persistent proliferation of peroxisomes leads to specific oxidative DNA damage.

Inhibitory effect of antioxidants ethoxyquin and 2(3)-tert-butyl-4-hydroxyanisole on hepatic tumorigenesis in rats fed ciprofibrate, a peroxisome proliferator.

The objective of this study was to test the hypothesis that hepatocarcinogenesis by peroxisome proliferators, a novel class of chemical carcinogens, is mediated either directly by carcinogenic H2O2, generated by peroxisomal oxidase(s) or indirectly by free radicals produced from H2O2, and that antioxidants could retard or inhibit neoplasia by scavenging active oxygen (super-oxide radicals O(2), hydrogen peroxide, hydroxyl radicals HO, and singlet oxygen 1O2). Accordingly, the effect of synthetic antioxidants 2(3)-tert-butyl-14-hydroxyanisole and ethoxyquin on the peroxisome proliferator 2-[4-(2,2-dichlorocyclopropyl)phenoxy]2-methyl-propionic acid (ciprofibrate)-induced hepatic tumorigenesis has been examined in male Fischer 344 rats. Rats were fed either a 2(3)-tert-butyl-4-hydroxyanisole (0.5% w/w)- or ethoxyquin (0.5% w/w)-containing diet with or without ciprofibrate (10 mg/kg of body weight) for 60 weeks. Rats fed ciprofibrate (10 mg/kg of body weight) in the diet or fed a diet with no added chemicals served as controls. Results of this study demonstrated that ethoxyquin markedly inhibited the hepatic tumorigenic effect of ciprofibrate, as evidenced by a decreased incidence of tumors, a decreased number of tumors per liver, and a reduced tumor size. 2(3)-tert-Butyl-4-hydroxyanisole also caused a significant decrease in the incidence and number of hepatocellular carcinomas that were larger than 5 mm. The present data suggest that the inhibitory effect of antioxidants on ciprofibrate-induced hepatic tumorigenesis may be due to H2O2 and free radical-scavenging property of ethoxyquin and 2(3)-tert-butyl-4-hydroxyanisole, since these antioxidants do not prevent peroxisome proliferation and induction of H2O2-generating peroxisomal enzymes in livers of rats fed ciprofibrate. Whether the inhibitory effect of antioxidants is exercised on the presumptive H2O2 initiation process and/or on the postinitiation growth phase of foci and nodules in liver is, at present, unknown.

Action of clofibrate and its analogs in rats. Dissociation of hypolipidemic effects and the induction of peroxisomal beta-oxidation.

Experiments were conducted to compare directly the effects of clofibrate (ethyl 2-(p-chlorophenoxy)isobutyrate) and its analogs on serum lipids and on the rate-limiting enzyme of the hepatic peroxisomal beta-oxidation pathway, fatty acyl-coenzyme A oxidase. Clofibrate feeding (5 g/kg diet) led to 8-10-fold increases in enzyme activity in 7 days. The enzyme remained elevated during the 28-day course of the experiment. The treatment did not lead to a lowering of serum cholesterol or triacylglycerol at any time during the experiment. In separate experiments rats were given clofibrate or one of five analogs by gastric intubation for 4 consecutive days. Four of the five analogs studied caused a significant lowering of serum triacylglycerol to about 50% of pretreatment level; another analog and clofibrate itself did not significantly affect serum triacylglycerol levels. None of the five analogs caused an induction of hepatic fatty acyl-CoA oxidase, while clofibrate treatment led to a 3-4-fold increase in enzyme activity. The results demonstrate a complete dissociation between the hypolipidemic action of these compounds and the induction of peroxisomal beta-oxidation. Thus it appears that the two phenomena are mechanistically unrelated.

Structure-dependent induction of bilirubin glucuronidation and lauric acid 12-hydroxylation by arylcarboxylic acids chemically related to clofibrate.

The inductive potency of carboxylic acids, structurally related to clofibrate, on bilirubin UDPglucuronosyltransferase was investigated in the rat. For this purpose, structure-induction relationships were established using ten different arylcarboxylic or chlorophenoxycarboxylic acids. 4'-Chlorophenoxyacetic, -propionic and -isobutyric (clofibric) acids progressively increased hepatic glucuronidation of bilirubin (17%, 43%, 60% greater than controls, respectively) after a 5-day treatment in rat (100 mg/kg per day). 2-Phenylpropionic acid also enhanced bilirubin UDPglucuronosyltransferase activity (50%) in contrast to phenylacetic acid. The other compounds did not, or only slightly, affect this parameter. These results indicate that specific structural features are required for the induction property. Moreover, a good correlation (r = 0.962) was found between the extent of induction and the physiochemical descriptors which characterize the electronic state of the molecules, when analysed by multidimensional regression. Fluorescence polarization revealed that the compounds tested, especially clofibric acid, did not affect, in vivo or in vitro, the anisotropy of two different probes embedded in the microsomal membranes. Finally, since the interaction of the carboxylic acids with the membranes did not modify the latency state of bilirubin UDPglucuronosyltransferase, it was concluded that the increase in enzyme activity was due more to a real induction than to activation of bilirubin UDPglucuronosyltransferase. A close linkage was established between bilirubin UDPglucuronosyltransferase induction and that of cytochrome P-452, as shown by enhanced omega-oxidation of lauric acid. This led to the hypothesis that both processes could be under coordinate regulation and mediated by a molecular interaction depending on the physicochemical properties of the carboxylic acids.

Sex-related difference in the effect of clofibric acid on induction of two novel long-chain acyl-CoA hydrolases in rat liver.

The sex differences in the induction of two novel long-chain acyl-CoA hydrolases in hepatic cytosol of rats by clofibric acid (p-chlorophenoxyisobutyric acid)-feeding and the properties of the induced acyl-CoA hydrolases were investigated. Marked sex-related difference was observed in the induction of acyl-Coa hydrolase activity. The sex difference was mainly due to the difference in the induction of acyl-CoA hydrolase with higher molecular weight (hydrolase I), but not to the difference in the induction of acyl-CoA hydrolase with lower molecular weight (hydrolase II). The extent of the induction of the hydrolase I in hepatic cytosol of male rats was 3.5 times over that of female rats. Castration of male rats resulted in the marked depression of the ability to induce hydrolase I. The administration of testosterone to the castrated male rats recovered completely the ability to induce hydrolase I. Unlike hydrolase I, the ability to induce hydrolase II did not respond to the changes in state of androgen. The administration of di-(2-ethylhexyl)phthalate also induced both hydrolase I and II, although the extent of the induction of hydrolase I was less compared to that by clofibric acid treatment. Likewise, marked sex difference was observed in the induction of the hydrolase I on di-(2-ethylhexyl)phthalate administration. These two hydrolases showed different kinetic properties and different substrate specificities to each other. Hydrolase I was inhibited by bovine serum albumin in vitro, but was not affected by Mg2+. Hydrolase II was activated slightly in the presence of lower concentrations of bovine serum albumin, Mg2+ or Ca2+.

Induction of hepatic long-chain acyl-CoA hydrolase by clofibric acid administration.

The induction of hepatic long-chain acyl-CoA hydrolase in the cytosolic fraction by administration of clofibric acid (p-chlorophenoxyisobutyric acid) was compared in rats, mice and guinea-pigs. In rats, two long-chain acyl-CoA hydrolases were induced by the administration of clofibric acid. In mice, only one long-chain acyl-CoA hydrolase was induced, and this hydrolase had properties similar to those of the lower-molecular-weight hydrolase induced in the hepatic cytosol of rats. In hepatic cytosol of guinea-pig, no hydrolase was induced by the administration of clofibric acid.

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.

Increased activity of stearoyl-CoA desaturation in liver from rat fed clofibric acid.

Male rats were fed a diet containing 0.5% (w/w) p-chlorophenoxyisobutyric acid (clofibric acid), a hypolipidemic drug. Activities of stearoyl-CoA desaturation in hepatic microsomes were increased approx. 4 times following the administration of clofibric acid for 7 days. An increase in the activity of desaturation of stearic acid was also observed in the liver of clofibric acid-fed rats in vivo. The increase in the activity of microsomal stearoyl-CoA desaturation by clofibric acid-feeding was due to the increase in the activity of terminal desaturase as measured by the rate constant for cytochrome b5 reoxidation, but not due to the changes in cytochrome b5 content and NADH-cytochrome b5 reductase activity. Increases in the activity of stearoyl-CoA desaturation by clofibric acid-feeding were also observed in rats of hormonally altered state, such as diabetic rats, hyperthyroid rats and hypothyroid rats. Percentages of octadecenoic acid in total fatty acid of hepatic lipid were increased with the increase in the activity of stearoyl-CoA desaturation.

Differential effects of altered hormonal state on the induction of acyl-CoA hydrolases and peroxisomal beta-oxidation by clofibric acid.

Induction of hydrolase I, hydrolase II, peroxisomal beta-oxidation and hepatomegaly caused by clofibric acid (rho-chlorophenoxyisobutyric acid) administration was investigated in relation to alterations in hormonal state of glucocorticoid, thyroid hormone and insulin. (1) In adrenalectomized rats, the ability to induce hydrolase I was depressed effectively and little hepatomegaly was produced. Hydrolase II and peroxisomal beta-oxidation were induced to a similar extent, compared to those of intact rats. (2) In hypothyroid rats, induction of hydrolase I, hydrolase II, peroxisomal beta-oxidation and hepatomegaly was reduced. In hyperthyroid rats, the ability to induce hydrolase I, hydrolase II and peroxisomal beta-oxidation was depressed, although hepatomegaly was produced by the same or a greater extent as in intact rats. (3) In diabetic rats, marked reduction of ability to induce both hydrolase I and II was observed and induction of hepatomegaly was depressed slightly, although peroxisomal beta-oxidation was induced normally. Differences in the response of four parameters (hydrolase I, hydrolase II, peroxisomal beta-oxidation and liver size) to alterations in hormonal state suggest that the four biological responses to clofibric acid may each be mediated through distinct mechanism(s) other and regulated by distinct hormone(s).

Effects of clofibric acid and tiadenol on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation in liver and extrahepatic tissues of rats.

The effects of two peroxisome proliferators, p-chlorophenoxyisobutyric acid (clofibric acid) and 2,2'-(decamethylenedithio)diethanol (tiadenol), on cytosolic long-chain acyl-CoA hydrolase and peroxisomal beta-oxidation were studied in several organs of rat. Among organs of control rats, the brain had the highest activity of long-chain acyl-CoA hydrolase, followed by testis, and a low activity was found in other tissues. Administration of the peroxisome proliferators caused a marked increase in activity of long-chain acyl-CoA hydrolase in both liver and intestinal mucosa and a slight increase in the activity in kidney, but little affected acyl-CoA hydrolase activity in either brain, testis, heart, spleen and skeletal muscle. In accordance with the change in the activity of acyl-CoA hydrolase, the activity of peroxisomal beta-oxidation was markedly increased in liver, intestinal mucosa and kidney, and a slight increase was found in brain and testis, whereas peroxisome proliferators little affected the activity in other organs tested. Gel filtration of cytosol from intestinal mucosa showed that clofibric acid caused an appearance of a new peak in intestinal mucosa. Although cytosol of liver, intestinal mucosa, brain and testis contained two 4-nitrophenyl acetate esterases with different molecular weights (about 105,000 and about 55,000), these esterases are different from cytosolic long-chain acyl-CoA hydrolases of these four organs in respect of molecular weight. The administration of clofibric acid little affected cytosolic 4-nitrophenyl acetate esterases. Comparative studies on cytosolic long-chain acyl-CoA hydrolases from these four organs showed that liver hydrolase I (molecular weight of about 80,000) had properties similar to those of brain and testis enzymes. On the other hand, intestinal mucosa enzyme was different from either hepatic hydrolase I or II (molecular weight of about 40,000). The results from the present study suggest that inductions of peroxisomal beta-oxidation and cytosolic long-chain acyl-CoA hydrolases are essential responses of rats to peroxisome proliferators not only in liver but also in intestinal mucosa and that induced hydrolases are not attributable to non-specific esterases.

Studies with etofibrate in the rat. Part I: Effects on glycerol, free fatty acid and triacylglycerol metabolism.

Etofibrate is the 1,2-ethandiol diester of clofibric acid and nicotinic acid that decreases circulating levels of triacylglycerols and cholesterol. To understand the mechanism by which the drug affects plasma triacylglycerols, normolipemic rats were treated daily with 300 mg of etofibrate/kg body weight or with the medium by a stomach tube. They were decapitated on the 10th day, and showed lower levels of plasma beta-hydroxybutyrate, glycerol, free fatty acids (FFA), total triacylglycerols and cholesterol and VLDL triacylglycerols and cholesterol, whereas glucose and RIA-determined insulin levels were unmodified. Epididymal fat pad pieces from etofibrate-treated rats incubated in vitro released more glycerol but the same amount of FFA to the medium, and had greater uptake of [U-14C]glycerol for [14C]acylglycerol formation. In the presence of heparin, they also showed an enhanced release of lipoprotein lipase activity to the medium. The disappearance from plasma of intravenously administered [1-14C]palmitate was faster in the etofibrate-treated rats, and although they showed a decrease in 14C-esterified fatty acids of neutral lipids in both liver and plasma VLDL, there was an increase in liver 14C-labelled water-soluble components. After intravenous [U-14C]glycerol administration, there was a decrease in plasma VLDL [14C]acylglycerol and [14C]glucose and in liver [14C]acylglycerol, but an increase in plasma [14C]lactate. In the liver, etofibrate treatment heightened the cytosolic glycerol-3-phosphate dehydrogenase activity and the total carnitine concentration, whereas it reduced triacylglycerol and cholesterol concentrations. It is proposed that etofibrate enhances the reesterification of fatty acids and glycerol in adipose tissue, which, together with its augmented lipoprotein lipase activity, may facilitate the clearance of circulating triacylglycerols. These effects may act concomitantly with the decreased synthesis of triacylglycerols, secondary to the increased utilization of their precursors, acyl-CoA and glycerol-3-phosphate, in other pathways, causing the reduction of plasma VLDL triacylglycerols produced by etofibrate treatment.