Hypolipidemic Compounds Research Articles

Induction of the peroxisome proliferator activated receptor by fenofibrate in rat liver

The process of peroxisome proliferation in rodent liver by hypolipidemic compounds and related substances has recently been shown to be receptor-madiated. In the present study, we have examined the effect of oral administration of the strong peroxisome proliferator fenofibrate on the hepatic expression level of the peroxisome proliferator activated receptor (PPAR) in rats. Immunoblots of rat liver cytosols and nuclear extracs using antibodies raised against recombinant PPAR/β-galactosidase fusion proteins revealed a pronounced increase in the amount of PPAR protein in response to fenofibrate treatment. This induction could also be confirmed at the level or RNA by Northern blotting. A time-course investigation showed a delayed accumulation of mRNA in response to the treatment, starting on day 2 after a latency period of at least one day. Thus, induction of the PPAR as a response to peroxisome proliferators represents one important dimension of the pleiotropic effects of peroxisome proliferators.

Induction of peroxisomal fatty acyl-coenzyme A oxidase and total carnitine acetyl-coenzyme A transferase in primary cultures of rat hepatocytes by garlic extracts

Garlic has been proposed as a natural hypolipidemic substance. Most hypolipidemic compounds induce peroxisomal proliferation and increase enzyme activities associated with peroxisomal β-oxidation in rat liver. Here we report that garlic methanol-extracts behave as hypolipidemic drugs, increasing the activity of peroxisomal fatty acyl-coenzyme A oxidase and of total carnitine acetyl-coenzyme A transferase in primary cultures of rat hepatocytes. Both enzymes are considered markers associated with increased peroxisomal β-oxidation. As in the case of hypolipidemic peroxisome proliferators, garlic extracts partially prevented the decrease in fatty acyl-coenzyme A oxidase as the culture aged. No changes were observed in the activity of microsomal NADPH cytochrome c reductase or of mitochondrial glutamate dehydrogenase.

Urinary glucuronide excretion of fenofibric and clofibric acid glucuronides in man. Is it polymorphic?

The possible polymorphism of the glucuronidation reaction in man has been investigated using two hypolipidaemic compounds, fenofibrate and clofibrate, as the test probes. The formation of fenofibryl and clofibryl glucuronides was identified by their susceptibility to hydrolyses by beta-glucuronidase. The urinary excretion of the glucuronides was measured in 72 healthy volunteers after a single dose of fenofibrate, and in 104 subjects given a single dose of clofibric acid. Fenofibrate was excreted at a lower rate than clofibrate, since 13.94% and 26.55% of the doses of fenofibrate and clofibrate respectively, were recovered in urine in 8 h. Correlation analysis indicated that sex and body mass index significantly influenced the formation of fenofibryl glucuronide, whereas age and oral contraceptives affected the excretion of clofibryl acid glucuronide. The 8-hour urinary excretion patterns of clofibryl glucuronide and of clofibric acid presented a Gaussian distribution, whereas those of fenofibryl glucuronide and fenofibric acid showed 2 populations. When the metabolic ratio free fenofibric acid/glucuronide was considered, 84.7% of subjects presented the ratio 0.147, and 15.3% had the 3-fold higher ratio of 0.421. The study has shown, in the human population studied, that the glucuronidation of fenofibric acid but not that of clofibric acid may present a polymorphism.

The effect of beclobric acid and clofibric acid on peroxisomal β-oxidation and peroxisome proliferation in primary cultures of rat, monkey and human hepatocytes

The peroxisome-proliferating effects of clofibric acid and beclobric acid were studied in primary cultures of hepatocytes derived from rat, monkey ( Macaco fascicularis) and human liver. Determination of peroxisomal fatty acid β-oxidation and morphometrical analysis of the peroxisomal compartment were performed after incubation of 1-day-old hepatocyte cultures for 3 days with either compound. In rat liver cell cultures both compounds gave a 10-fold increase in peroxisomal β-oxidation, a 3-fold increase in the relative number of peroxisomes and a 1.5-fold increase in the mean size of peroxisomes. Beclobric acid gave its maximal effect at a concentration of 10 μM, which is at least one order of magnitude lower than the maximum-effect concentration of clofibric acid. At concentrations > 300 μM beclobric acid was cytotoxic. No stimulation of peroxisomal fatty acid β-oxidation was found in either monkey or human hepatocyte cultures. Morphometrical analysis also showed no increase in the peroxisomal compartment in cultures derived from these species, as indicated by the lack of increase in both relative number and size of peroxisomes. In all three species tested beclobric acid was equally cytotoxic for hepatocytes in vitro. These results are of relevance for the interpretation of the peroxisomeproliferating effects of clofibrate and similar compounds in rats. Since peroxisome proliferation may be correlated to increased hepatic tumour incidences in the rat, the absence of peroxisome proliferation in primates suggests the absence of tumourogenic activity by hypolipidemic compounds in these species.

Effect of diabetes and starvation on the activity of rat liver epoxide hydrolases, glutathione S-transferases and peroxisomal β-oxidation

The activities of peroxisomal β-oxidation, cytosolic and microsomal epoxide hydrolase as well as soluble glutathione S-transferases have been determined in the livers of alloxan- and streptozotocin-diabetic male Fischer-344 rats. Five, seven and ten days after initiation of diabetes serum glucose levels were elevated 3.6-, 5.7- to 6.2- and 6-fold, while the activities of peroxisomal β-oxidation and cytosolic epoxide hydrolase were elevated 1.5- and 2.5-fold, 1.4- and 2.7-fold and 1.3- and 2.0-fold, respectively. The activities of microsomal epoxide hydrolase and glutathione S-transferases were reduced to about 71% and 80% of controls. Application of 10 I.U./kg depot insulin twice a day for 10 consecutive days to alloxan-diabetic individuals approximately restored the initial glucose levels and enzyme activities except for peroxisomal β-oxidation. Starvation of Fischer-344 rats for 48 hours and 5 days similarly resulted in a 1.3-fold to 2.1-fold and 1.2- to 1.6-fold increase in peroxisomal β-oxidation and cytosolic epoxide hydrolase activity, respectively. Microsomal epoxide hydrolase was significantly decreased to 57% and 61% of control activity whereas glutathione S-transferase was only marginally reduced to 91% and 92%. Except for glutathione S-transferases initial enzyme activities were restored upon refeeding within 10 days. These results are similar to those obtained upon feeding of hypolipidemic compounds with peroxisome proliferating activity, and may indicate that high levels of free fatty acids or their metabolites which are known to accumulate in liver in both metabolic states may act as endogenous peroxisome proliferators.

Effect of hypolipidemic compounds on lauric acid hydroxylation and phase II enzymes

Treatment of male Fischer 344 rats with various hypolipidemic drugs of different peroxisome proliferating potency (1-benzylimidazole, acetylsalicylic acid, clofibrate, tiadenol) led to an induction of liver lauric acid hydroxylase, whereas probucol, which is not a peroxisome proliferator, did not induce this enzyme. Activity of bilirubin UDP-glucuronosyltransferase was increased by all the compounds tested. The highest increase was observed after treatment with acetylsalicylic acid (2.3-fold). High correlation ( r = 0.953) was observed between the activities of lauric acid hydroxylase and the corresponding activities of cytosolic epoxide hydrolase reported previously. The amount of microsomal expoxide hydrolase was not changed by any of the compounds. Whereas clofibrate and tiadenol decreased glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene as substrate, 1-benzylimidazole and probucol increased this activity. With 4-hydroxynonenal as a substrate qualitatively the same results were obtained with the exception that probucol did not affect the enzyme activity. When glutathione S-transferase activity was measured with cis-stilbene oxide as substrate only the more than five-fold increase after treatment with 1-benzylimidazole was significantly different from control values. Activity of dihydrodiol dehydrogenase was increased after treatment of rats with 1-benzylimidazole (1.5-fold), whereas application of tiadenol led to a decrease of enzyme activity. Feeding of male guinea pigs with clofibrate did not change the activity of peroxisomal β-oxidation, cytosolic epoxide hydrolase or lauric acid hydroxylase. However, treatment with tiadenol caused an increase of these activities.

Comparative induction of drug-metabolizing enzymes by hypolipidaemic compounds

1. 1. The inducing effect of hypolipidaemic compounds containing arylcarboxylic structure (fenofibrate, bezafibrate, ciprofibrate) on drug-metabolizing enzymes was compared with that of other chemically different molecules (probucol, 1-benzylimidazole) in rat liver. 2. 2. Hepatomegaly was closely associated to the decrease in triglycerides and cholesterol contents in plasma. 3. 3. Lauric acid hydroxylation and hydratation of trans-stilbene oxide by the cytosolic epoxide hydrolase were markedly increased by the arylcarboxylic acids (fenofibrate, bezafibrate and ciprofibrate). The determination of the concentration in microsomal epoxide hydrolase by immunoassay revealed no change in the protein amount within the membrane; only the specific activity was enhanced by bezafibrate and 1-benzylimidazole, suggesting an activation process. 4. 4. Bilirubin glucuronidation was increased by the arylcarboxylic structures and by 1-benzylimidazole; by contrast probucol decreased this activity.

Gastric Morphological Changes Including Carcinoid Tumors in Animals Treated with a Potent Hypolipidemic Agent, Ciprofibrate

Oral administration of ciprofibrate, a potent hypolipidemic compound, to rats for 2 or more weeks at doses of 20 mg/kg/day or more resulted in hypertrophy and increased eosinophilia of the oxyntic cells in the gastric mucosa. Ultrastructural evaluation revealed small secretory canaliculi with small microvilli in these cells, changes consistent with the inhibition of acid secretion. After longer administration (e.g., greater than 2 months at 20 mg/kg/day), hyperplasia of the neuroendocrine cells (in particular, the enterochromaffin-like cells) was present in the fundic mucosa of the stomach. After life-time (2-year) administration at 10 mg/kg/day, neuroendocrine cell hyperplasia was accompanied by formation of malignant carcinoid tumors in the fundus of 5 of 59 male and 1 of 60 female rats. In contrast, administration of ciprofibrate to mice at 20 mg/kg/day for 2 months was not associated with oxyntic or neuroendocrine cell changes, a finding consistent with the lack of gastric carcinoid tumors in a 2-year mouse study. Similarly, no significant changes were induced in the marmoset stomach by doses as high as 100 mg/kg/day for 6 months. These findings are consistent with the hypothesis that the formation of gastric carcinoid tumors following ciprofibrate administration is a phenomenon that occurs specifically in those species such as the rat where this compound has significant gastric antisecretory activity.

Induction of peroxisome proliferation and hepatic tumours in C57BL/6N mice by ciprofibrate, a hypolipidaemic compound.

The hepatic effects of ciprofibrate, a potent peroxisome proliferator, were evaluated in male C57BL/6N mice, a mouse strain with very low incidence of spontaneous liver tumour development. Dietary feeding of ciprofibrate (0.0125% or 0.025% w/w) for 2 weeks resulted in a marked proliferation of peroxisomes (9-fold increase) and several-fold increase (8- to 10-fold) in the activity of peroxisomal beta-oxidation enzymes. Feeding ciprofibrate at 0.025% concentration for 15 months followed by a 0.0125% for 6 months led to the development of hepatic adenomas in 8/14 (57%) and hepatocellular carcinomas (HCC) in 3/14 (21%) mice. In mice given 0.0125% ciprofibrate for 18 months 5 of 8 (62%) and 3 of 8 (37%) developed adenomas and HCC respectively. Similar to the findings observed in rats, both the adenomas and HCC were negative for gamma-glutamyltranspeptidase. These results in C57BL/6N mice of hepatocarcinogenic effect of ciprofibrate, a non-genotoxic chemical, indicate that peroxisome proliferation can be used as a reliable parameter to evaluate the carcinogenicity of hypolipidaemic compounds.

Lymphatic absorption of hypolipidemic compound, 1-O-[p-(myristyloxy)-alpha-methylcinnamoyl] glycerol (LK-903).

The intestinal absorption process of 1-O-[p-(myristyloxy)-alpha-methylcinnamoyl] glycerol (LK-903), a new hypolipidemic compound, was studied in rats. When 3H-LK-903 or 3H-LKA [3H-p- (myristyloxy)-alpha-methyl cinnamic acid], labeled at the cinnamic acid moiety, or 14C-LK-903, labeled at the glycerol moiety, were administered orally to thoracic duct-cannulated rats at a dose of 0.233 mmol/kg, 31.1, 6.7 and 18.1% of the dose, respectively, appeared in the lymph within 24 h. In this case, radioactive compounds in the lymph lipids consisted of LKA (radioactivity was not detected in the fraction of LKA with 14C-LK-903), LK-903, diglyceride analogues and triglyceride analogues. The percentages of the triglyceride analogues were the highest, followed by the diglyceride analogues. On the other hand, when doubly labeled LK-903 (3H/14C = 1, corrected ratio) was administered orally, the values of 3H/14C for the monoglyceride, diglyceride and triglyceride analogues in the lymph were 1.2-1.5, 1.7-1.9 and 1.9-2.7, respectively. The lymphatic absorption of LK-903 was stimulated by the presence of lecithin but inhibited by a high dose of triolein. The results indicated that (1) LK-903 formed micelles in the intestine, (2) a large part of LK-903 was absorbed as such, (3) a part of LK-903 was hydrolyzed in the intestinal mucosa, and (4) a part of LKA formed by hydrolysis was again utilized to synthesize the higher glycerides and absorbed via the lymphatic absorption route for lipids.

MODIFICATION OF N-NITROSOBIS (2-HYDROXYPROPYL) AMINE-INDUCED CARCINOGENESIS BY ORALLY ADMINISTERED CLOFIBRATE IN HAMSTERS

Clofibrate, one of hypolipidemic compounds, has been shown as a hepatocarcinogen in rats. In the present experiment, effects of clofibrate given in the promotion stage on liver, gallbladder, pancreas, lung, and kidney carcinogenesis were investigated in male Syrian golden hamsters. After subcutaneous administration of BHP at a dose of 500 mg/kg body weight once a week for 5 weeks, hamsters were maintained on diet containing 0.25% or 0.5% clofibrate for 30 weeks. The hepato-promoting activity of clofibrate was indicated by multiplicity of liver lesions including hyperplastic nodules and hepatocellular carcinomas, and it was greater in hamsters given 0.25% clofibrate than in those receiving 0.5% clofibrate. Peroxisome proliferation was observed in the hepatocytes of hamster given 0.5% clofibrate for 13 weeks under the electron microscope. Inhibitry effect of clofibrate was demonstrated in the development of pancreatic duct adenocarcinoma and lung neoplasms, including adenoma and adenocarcinoma. Hypolipidemic status shown in the serum of hamsters given clofibrate might be involved in the inhibitory effect on pancreas and lung carcinogenesis. Clofibrate did not affect gall- bladder and kidney carcinogenesis. These results indicated that clofibrate modulated liver, pancreas, and lung carcinogenesis in hamsters.

Comparison of the short-term hepatic effects of orally administered citral in long evans hooded and wistar albino rats

The short-term effects of citral on the liver have been studied in two strains of rat. Hepatomegaly was accompanied in citral-treated rats by an altered distribution of lipid and glycogen in the liver and peroxisome proliferation occurred in a manner reminiscent of that associated with some hypolipidaemic compounds. Specific biochemical markers supported the morphological changes in the peroxisomes. Cyanide-insensitive palmitoyl CoA oxidation showed, at the maximum, fourfold and threefold inductions in Wistar albino and Long Evans hooded rats, respectively. In addition, induction of cytochrome P-450 levels was greater in the Long Evans than in the Wistar rats, the maximal increases recorded being 81 and 27% respectively. A peroxisome-associated polypeptide of molecular weight 80,000 daltons (PPA-80) was induced, especially in Long Evans rats. No alterations in plasma triglycerides or total cholesterol were detected. The differential induction of the mixed-function oxidase system and the differential proliferation of peroxisomes in these two strains of rat suggest that citral may be metabolized differently in the two strains. The study indicates that peroxisomal and possibly also mitochondrial changes are involved in the action of citral on lipid metabolism.

Circadian variation of the hypocholesterolemic effect of K13.004 in rats

The hypocholesterolemic effect in rats of the new lipid-lowering agent K13.004 was dependent on the time of day of its application. This dependence was shifted together with the time of peak activity of hepatic cholesterol synthesis (CS) when the feeding time of the animals was changed. This compound considerably reduced serum cholesterol only if given before the peak of hepatic CS, whereas application afterwards was ineffective. Our finding suggests that this hypolipidemic compound lowers serum cholesterol by inhibition of hepatic CS. Drugs acting in such a way should be administered prior to the maximum of hepatic sterol synthesis.

Concomitant induction of cytosolic but not microsomal epoxide hydrolase with peroxisomal β-oxidation by various hypolipidemic compounds

The effects of two cholesterol-lowering (probucol and 1-benzyl-imidazole), three triglycerideand cholesterol-lowering (clofibrate, tiadenol and fenofibrate) and one triglyceride-lowering (acetylsalicylic acid) compounds on the specific activities of two lipid-metabolizing enzymes (cyanide-insensitive peroxisomal β-oxidation and palmitoyl-CoA hydrolase) and two xenobiotic metabolizing enzymes (cytosolic (cEH) and microsomal epoxide hydrolase (mEH b)) from the livers of male Fischer F-344 rats were investigated. With the exception of probucol and acetylsalicylic add, all compounds tested caused a dose-dependent hepatomegaly. Taken on a weight basis fenofibrate was the most effective inducer, causing a 20-fold induction of peroxisomal β-oxidation, a 13-fold induction of cEH activity and a 16-fold induction of palmitoyl-CoA hydrolase activity. The other compounds with triglyceride-lowering activity also induced cEH as well as peroxisomal β-oxidation and palmitoyl-CoA hydrolase activity. The potency of each individual drug was similar for induction of cEH activity as compared with that of peroxisomal β-oxidation and palmitoyl-CoA hydrolase activity, but very dissimilar for mEH b, which upon treatment with any of the triglyceride-lowering compounds was either not or only minimally (<1.5-fold) induced. 1-Benzylimidazole possessing exclusively cholesterol-lowering activity increased mEH b, much more than either cEH or peroxisomal β-oxidation. The absence of an enhancement of cEH activity in in vitro studies confirmed that the increase in enzyme activity by the test compounds is not caused by activation. cEH activity was also induced in the kidney but only about 2-fold by fenofibrate, tiadenol and acetylsalicylic acid. With hypolipidemic drugs varying in their peroxisome-proliferating potency from inactive to very potent, the effects on peroxisomal β-oxidation and cEH were similar in all instances, whilst the effects on mEH b, could be clearly dissociated. Thus, in the rat a concomitant regulation of cEH with peroxisomal β-oxidation and peroxisome proliferation by hypolipidemic drugs becomes apparent.

Coordinate induction of peroxisomal β-oxidation activity and cytosolic epoxide hydrolase activity

The effect of four hypolipidemic compounds (tiadenol, clofibrate, acetylsalicylic acid, 1-benzylimidazole) on the specific activities of peroxisomal β-oxidation and cytosolic and microsomal epoxide hydrolase of rat liver was investigated. Since specific activity of cytosolic epoxide hydrolase from outbred Sprague-Dawley rats showed large interindividual variations ( ∼ 38-fold), induction studies were performed with inbred Fischer F-344 rats, which showed only low interindividual variations ( ∼ 2-fold). Clofibrate, tiadenol and acetylsalicylic acid caused a 8-, 13- and 4.5-fold induction of cEH and a 13-, 19- and 5-fold induction of peroxisomal β-oxidation activity, respectively. Microsomal epoxide hydrolase activity was only slightly increased (< 1.5-fold). 1-Benzylimidazole induced both cytosolic epoxide hydrolase and peroxisomal β-oxidation activity about 2-fold, whereas microsomal epoxide hydrolase activity was increased about 4-fold. Increase in cytosolic epoxide hydrolase activity was not due to enzyme activation as demonstrated by in vitro studies. On the other hand, these in vitro studies showed that the increase in microsomal epoxide hydrolase activity by 1-benzylimidazole may partially be due to activation of the enzyme.

Induction of hepatocytes in the pancreas of copper-depleted rats following copper repletion

Pancreatic hepatocytes are induced in rats maintained on copper-deficient diet containing 0.6% d-penicillamine for 8–10 weeks, followed by copper repletion. These induced hepatocytes are morphologically and functionally very similar to parenchymal cells of the liver. Immunofluorescence stains demonstrated the presence of albumin and catalase in these cells. Strains for pancreatic enzymes and hormones were negative. As expected, the hypolipidemic compounds, ciprofibrate, induced peroxisome proliferation in these cells. These results indicate that a simple depletion and repletion of copper can trigger transdifferentiation in the pancreas of adult rats.

Comparative effect of a series of hypolipidemic compounds oh the release of enzymes from isolated hepatocytes

Comparative effect of a series of hypolipidemic compounds oh the release of enzymes from isolated hepatocytes

Effects of benfluorex and fenofibrate treatment on mitochondrial and peroxisomal marker enzymes in rat liver

Our results demonstrate that benfluorex at doses that are strongly hypotriglyceridemic does not increase hepatic peroxisomal enzyme activities, whereas fenofibrate at doses that are only slightly hypolipidemic induces a dramatic increase in the activity of these enzymes. Thus, the biochemical approach used in this study reveals that the hypolipidemic drug benfluorex does not belong to the class of hypolipidemic compounds known to induce hepatomegaly, hepatic peroxisome proliferation and hepatocarcinoma in rodents. Morphological studies should confirm the absence of peroxisomal induction.

Controlled evaluation of pantethine, a natural hypolipidemic compound, in patients with different forms of hyperlipoproteinemia

Pantethine (P), the stable disulphate form of pantetheine, major component and precursor of coenzyme A, was evaluated within a double-blind protocol (8 weeks for P or for a corresponding placebo) in 29 patients, 11 with type IIB hyperlipoproteinemia, 15 with type IV, and 3 with an isolated reduction of high density lipoprotein cholesterol (HDL-C) levels. In type IIB patients, P (300 mg t.i.d.) determined a highly significant lowering of plasma total and low density lipoprotein (LDL) associated cholesterol (−13.5% for both parameters). In the same patients, HDL-C levels increased about 10% at the end of treatment. Switching from P to placebo was associated with a rapid return to the baseline cholesterolemia. Both in type IIB and type IV patients, plasma triglyceride levels were reduced around 30%, when P was given as the first treatment; when it was preceded by placebo, reductions were less striking (respectively, −17.8% for type IIB and −13.0% for type IV, at the end of P treatment). HDL-C levels were not increased by P, either in type IV, and in the patients with low HDL cholesterolemia. In type IV, LDL cholesterol levels showed a variable response to P: they tended to increase when below 132 mg/dl, prior to treatment, and to be reduced when above this level. This study provides evidence for a significant hypocholesterolemic effect of P, a natural compound free of overt side effects. It also indicates that P may raise HDL-C levels in type IIB patients, while moderately reducing triglyceridemia.

Detection of a nafenopin-binding protein in rat liver cytosol associated with the induction of peroxisome proliferation by hypolipidemic compounds

[ 3H]nafenopin, a known inducer of liver peroxisomal enzymes, was shown to bind to a specific, saturable pool of binding sites in cytosols from rat liver and kidney cortex. Tissue levels of this binding protein (liver > kidney cortex; not detectable in myocardium, skeletal muscle) were seen to correlate with the ability of nafenopin to induce peroxisomal enzymes in these organs. Clofibrate and ciprofibrate, which are structurally similar to nafenopin, competitively blocked the specific binding of [ 3H]nafenopin. Phenobarbital, a non-inducer of peroxisomes, and [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid and 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-β-hydroxyethyl)acetamide, which are structurally unrelated peroxisome proliferators, did not compete for the specific [ 3H]nafenopin binding sites. The [ 3H]nafenopin binding protein is proposed as a mediator of the drug-induced increase in peroxisomes and associated peroxisomal enzymes.