Dietary Clofibrate Research Articles

Intestinal Carnitine Status and Fatty Acid Oxidation in Response to Clofibrate and Medium-Chain Triglyceride Supplementation in Newborn Pigs

To investigate the role of peroxisome proliferator-activated receptor alpha (PPARα) in carnitine status and intestinal fatty acid oxidation in neonates, a total of 72 suckled newborn piglets were assigned into 8 dietary treatments following a 2 (±0.35% clofibrate) × 4 (diets with: succinate+glycerol (Succ), tri-valerate (TC5), tri-hexanoate (TC6), or tri-2-methylpentanoate (TMPA)) factorial design. All pigs received experimental milk diets with isocaloric energy for 5 days. Carnitine statuses were evaluated, and fatty acid oxidation was measured in vitro using [1-14C]-palmitic acid (1 mM) as a substrate in absence or presence of L659699 (1.6 µM), iodoacetamide (50 µM), and carnitine (1 mM). Clofibrate increased concentrations of free (41%) and/or acyl-carnitine (44% and 15%) in liver and plasma but had no effects in the intestine. The effects on carnitine status were associated with the expression of genes involved in carnitine biosynthesis, absorption, and transportation. TC5 and TMPA stimulated the increased fatty acid oxidation rate induced by clofibrate, while TC6 had no effect on the increased fatty acid oxidation induced by clofibrate (p > 0.05). These results suggest that dietary clofibrate improved carnitine status and increased fatty acid oxidation. Propionyl-CoA, generated from TC5 and TMPA, could stimulate the increased fatty acid oxidation rate induced by clofibrate as anaplerotic carbon sources.

Transplacental induction of fatty acid oxidation in term fetal pigs by the peroxisome proliferator-activated receptor alpha agonist clofibrate.

BackgroundTo induce peroxisomal proliferator-activated receptor α (PPARα) expression and increase milk fat utilization in pigs at birth, the effect of maternal feeding of the PPARα agonist, clofibrate (2-(4-chlorophenoxy)-2-methyl-propanoic acid, ethyl ester), on fatty acid oxidation was examined at full-term delivery (0 h) and 24 h after delivery in this study. Each group of pigs (n = 10) was delivered from pregnant sows fed a commercial diet with or without 0.8% clofibrate for the last 7 d of gestation. Blood samples were collected from the utero-ovarian artery of the sows and the umbilical cords of the pigs as they were removed from the sows by C-section on day 113 of gestation.ResultsHPLC analysis identified that clofibric acid was present in the plasma of the clofibrate-fed sow (~4.2 μg/mL) and its offspring (~1.5 μg/mL). Furthermore, the maternal-fed clofibrate had no impact on the liver weight of the pigs at 0 h and 24 h, but hepatic fatty acid oxidation examined in fresh homogenates showed that clofibrate increased (P < 0.01) 14C-accumulation in CO2 and acid soluble products 2.9-fold from [1-14C]-oleic acid and 1.6-fold from [1-14C]-lignoceric acid respectively. Correspondingly, clofibrate increased fetal hepatic carnitine palmitoyltransferase (CPT) and acyl-CoA oxidase (ACO) activities by 36% and 42% over controls (P < 0.036). The mRNA abundance of CPT I was 20-fold higher in pigs exposed to clofibrate (P < 0.0001) but no differences were detected for ACO and PPARα mRNA between the two groups.ConclusionThese data demonstrate that dietary clofibrate is absorbed by the sow, crosses the placental membrane, and enters fetal circulation to induce hepatic fatty acid oxidation by increasing the CPT and ACO activities of the newborn.

Dietary clofibrate stimulates the formation and size of estradiol-induced breast tumors in female August-Copenhagen Irish (ACI) rats

Administration of 0.4% clofibrate in the diet stimulated estradiol (E 2)-induced mammary carcinogenesis in the August-Copenhagen Irish (ACI) rat without having an effect on serum levels of E 2. This treatment stimulated by several-fold the NAD(P)H-dependent oxidative metabolism of E 2 and oleyl-CoA-dependent esterification of E 2 to 17β-oleyl-estradiol by liver microsomes. Glucuronidation of E 2 by microsomal glucuronosyltransferase was increased moderately. In contrast, the activity of NAD(P)H quinone reductase 1 (NQO1), a representative monofunctional phase 2 enzyme, was significantly decreased in liver cytosol of rats fed clofibrate. Decreases in hepatic NQO1 in livers of animals fed clofibrate were noted before the appearance of mammary tumors. E 2 was delivered in cholesterol pellets implanted in 7–8-week-old female ACI rats. The animals received AIN-76A diet containing 0.4% clofibrate for 6, 12 or 28 weeks. Control animals received AIN-76A diet. Dietary clofibrate increased the number and size of palpable mammary tumors but did not alter the histopathology of the E 2-induced mammary adenocarcinomas. Collectively, these results suggest that the stimulatory effect of clofibrate on hepatic esterification of E 2 with fatty acids coupled with the inhibition of protective phase 2 enzymes, may in part, enhance E 2-dependent mammary carcinogenesis in the ACI rat model.

Dietary clofibrate inhibits induction of hepatic antioxidant enzymes by chronic estradiol in female ACI rats.

Excess production of H2O2 has been implicated in oncogenesis. The object of the present study was twofold: first, to determine the influence of chronic estradiol (E2) on the activities of selected hepatic antioxidant enzymes in female ACI rats, a strain that is highly sensitive to the induction of estrogen dependent mammary tumors; secondly, to evaluate the actions of dietary clofibrate, a peroxisome proliferator, on activities of these enzymes in control and E2-treated ACI rats. Enzymes selected for study were: NAD(P)H quinone oxidoreductase (NQO1), glutathione S-transferase (GST) and glutathione peroxidase (GPx). Cytosolic catalase (CAT) was also measured as an index of peroxisome proferation in control and E2- treated animals. E2 was administered chronically over 6 and 12 week periods from cholesterol pellet implants containing either 1 or 3 mg E2. Animals were fed AIN-76A diets with or without 0.4% clofibrate over the experimental period. NQO1 and GST but not GPx were induced to varying degrees (NQO1 about 300%, and GST about 45-97%) by chronic E2-treatment. E2-induced increases in these activities were completely prevented in rats exposed to dietary clofibrate. Dietary clofibrate also caused slight but significant reductions in baseline activities of NQO1, GST and GPx in control animals. Serum E2 levels, increased approximately 540% in a dose-dependent manner, and were not altered by dietary clofibrate. It is concluded that chronic E2 treatment markedly induces several important hepatic antioxidant enzymes in female ACI rats, and induction of these activities by E2 is inhibited completely by dietary clofibrate. Both of these actions have the potential to markedly influence the profile of E2 metabolites exported from the liver to E2 sensitive extrahepatic tissues and influence the initiation and progression of hormone-dependent tumors.

NAD + biosynthesis and metabolic fluxes of tryptophan in hepatocytes isolated from rats fed a clofibrate-containing diet

Hepatocytes were isolated from rats fed a diet with or without 0.25% clofibrate, and NAD + synthesis by the hepatocytes was determined using either [ carboxyl- 14C]nicotinic acid or [5- 3H]tryptophan. NAD + and total pyridine nucleotides synthesized from [ 14C]nicotinic acid by the clofibrate-treated cells were not significantly different from those synthesized by the control cells when expressed on the basis of nanomoles per hour per milligram of DNA. On the contrary, NAD + synthesized from [ 3H]tryptophan was significantly higher in the clofibrate-treated cells (158% of the control cells) on the basis of nanomoles per hour per milligram of DNA. Clofibrate was inhibitory to tryptophan metabolism as a whole, affecting the glutarate pathway more (decreased to 37% of control) than the kynureninase flux (decreased to 64% of control). As a result, the quinolinate-NAD flux, estimated as the difference in the amounts of tryptophan metabolized by the two metabolic pathways, increased in the clofibrate-treated hepatocytes. The increase in quinolinate during the incubation was 8 times more in the clofibrate-treated cells than in the control cells, which confirmed alteration in the metabolic fluxes of tryptophan in the clofibrate-treated cells. Hepatic quinolinate phosphoribosyltransferase (EC 2.4.2.19) activity increased with dietary clofibrate and returned to the control level 1 week after removing clofibrate from the diet. Nicotinate phosphoribosyltransferase (EC 2.4.2.11) and NAD + glycohydrolase (EC 3.2.2.5) activities remained unchanged with dietary clofibrate.

Expression of Cytochrome P450 Genes of the CYP4 Family in Midgut and Fat Body of the Tobacco Hornworm, Manduca sexta

Two conserved regions in the alignment of cytochrome P450 family 4 (CYP4) proteins served as guide to the synthesis of degenerate oligonucleotide primers. The primers were used in PCR from a midgut cDNA library and RT-PCR from fat body mRNA, both from last instar larvae of the tobacco hornworm, Manduca sexta. The PCR products of 443-449 bp were cloned and sequenced. Nine P450 clones representing four new genes were obtained from the midgut. Fifteen P450 clones representing three new genes were obtained from the fat body. Two genes were expressed in both tissues. A number of putative allelic variants were also observed for three of the P450 genes. The resulting sequences of 130-132 amino acids were aligned to generate a parsimony analysis of CYP4 P450 proteins. Two new subfamilies of CYP4 were designated from M. sexta by these procedures, CYP4L and CYP4M. The sequence of a full-length cDNA clone for CYP4M2 (41.2% identity to CYP4C1) confirmed that the PCR products obtained by this method were P450s belonging to the CYP4 family. The developmental expression of the CYP4 genes appeared to be coordinately regulated in both fat body and midgut. In the fat body, CYP4 mRNA levels declined after the first day of the final larval instar, peaked during the wandering stage, and fell again until the prepupal molt. Midgut CYP4 mRNA levels were higher during the active feeding, midwandering, prepupal, and pupal stages. Addition of 2-tridecanone or 2-undecanone to the diet induced several P450s in the midgut and in the fat body. Phenobarbital induced CYP4M1 in the fat body and dietary clofibrate induced the mRNA levels of CYP4M1 and CYP4M3 in the midgut. The results indicate that at least four CYP4 genes are expressed in single tissues of a Lepidopteran insect. Several of these P450 may be involved in tissue responses to xenobiotics.

Fat Intake and Clofibrate Administration Have Interrelated Effects on Liver Cholesterol Concentration and Serum Butyryl Cholinesterase Activity in Rats

Rats were fed for 15 d purified diets with different amounts of coconut fat, and with or without clofibrate. Fat was added at the expense of an isoenergetic amount of glucose. The hypolipidemic action of clofibrate was not influenced by the amount of fat in the diet. Clofibrate did not affect liver cholesterol concentration in rats fed the low fat diet, but it counteracted the rise in liver cholesterol seen in rats fed the high fat diet. This could relate to the observed raised intestinal clofibrate-hydrolyzing activity of rats fed the high fat diet, because hydrolysis of clofibrate gives rise to its biologically active form. In rats fed the low fat diet, but not in those fed the high fat diet, clofibrate raised the activity of serum esterase-1, which (unlike esterase-2) does not hydrolyze clofibrate. Possibly, the dramatic stimulatory effect of fat feeding on serum esterase-1 activity had overruled any influence of clofibrate. Clofibrate elevated serum butyryl cholinesterase activity, with this effect being amplified by fat feeding. High levels of dietary fat in the absence of dietary clofibrate did not alter serum butyryl cholinesterase activity. Clofibrate did not change butyryl cholinesterase and esterase-1 activities in small intestine. The high fat diet caused slightly higher levels of butyryl cholinesterase activity in small intestine, but markedly raised intestinal esterase-1 activity. This study shows that certain effects of clofibrate and a high fat diet are interrelated.

Induction of the major integral membrane protein of mouse liver peroxisomes by peroxisome proliferators.

Peroxisome proliferators are known to increase the volume of the peroxisomal compartment in rodent liver. We have examined the induction of the major integral membrane protein of mouse liver peroxisomes (PMP68) by a number of these agents, and compared this with their effect on the peroxisomal bifunctional protein (PBP), an enzyme of the beta-oxidation pathway which is located in the peroxisome matrix. Dietary clofibrate, di-2-(ethylhexyl)phthalate and Wy-14,643, three structurally unrelated proliferators, all increased the mRNA and protein content of PMP68 approx. 2-fold, whereas PBP was induced 8-13-fold. The kinetics and sequence of induction of PMP68 and PBP following a single dose of Wy-14,643 were compared and shown to be similar, and the effects were reversible. Another proliferator, BM 15766, caused maximal induction of PMP68 but only a low induction of PBP; further PBP induction was achieved by the administration of BM 15766 in combination with Wy-14,643. Similarly, BM 15766 and Wy-14,643 increased transcription of the PMP68 gene in vitro, whereas PBP gene transcription was increased by Wy-14,643 but not by BM 15766. Thus peroxisome proliferators enhance the expression of the genes for both the membrane protein PMP68 and the matrix protein PBP, but the regulation of this expression appears to be mediated by different mechanisms.

Elevated concentrations of α-tocopherol, ascorbic acid, and serum lipids in rats fed polychlorinated biphenyls, chlorobutanol, or phenobarbital

Experiments were conducted with growing rats to investigate the effects of dietary 0.05% polychlorinated biphenyls (PCB), 0.3% chlorobutanol (Chloretone), and 0.2% phenobarbital sodium on intestinal absorption of α-tocopherol and serum and tissue lipids. and the influence of dietary 5% pectin and 0.2% ethyl p-chlorophenoxyisobutyrate (clofibrate) on the changes in these lipids due to dietary 0.4% Chloretone. Dietary addition of PCB increased serum concentrations of α-tocopherol, cholesterol, and phospholipids. These were mainly attributed to the increments in the fraction of high-density lipoproteins. PCB intake increased tissue α-tocopherol and apparent absorption of α-tocopherol from intestine. Similar changes in serum and tissue lipids and in intestinal absorption of α-tocopherol were observed with dietary Chloretone and phenobarbital. Dietary clofibrate and pectin depressed the increase in serum concentrations of these lipids by Chloretone. Changes in serum levels of α-tocopherol, cholesterol, and phospholipids correlated with those in apparent absorption of α-tocopherol. These results suggest that the increase in serum cholesterol and phospholipids due to xenobiotics relates to the increase in serum and tissue α-tocopherol.

Apparent induction of microsomal carboxylesterase activities in tissues of clofibrate-fed mice and rats

Treatment with 0.5% ( w w ) dietary clofibrate, a peroxisome proliferator, for 14 days induced microsomal carboxylesterase activities for five substrates including malathion, clofibrate, diethylsuccinate, diethylphthalate, and p-nitrophenylacetate in liver and kidney of male Swiss-Webster mice and Sprague-Dawley rats. The induction was substrate, tissue, and species dependent. The carboxylesterase activity was induced in mouse from 1.2- to 2.2-fold (liver) and from 1.1- to 1.7-fold (kidney) depending upon substrate used. Analogous values from rat ranged from 1.0- to 1.4-fold (liver) and from 1.1- to 1.8-fold (kidney). Enzyme activities were either decreased or not affected in testes of treated mice and rats. Substituted trifluoroketones (“transition-state” inhibitors of carboxylesterase) were found to be very potent inhibitors of clofibrate-metabolizing carboxylesterase(s) and to be potentially useful in distinguishing among isozymes. The inhibition data suggested that changes in carboxylesterase activity following clofibrate treatment were both qualitative and quantitative.

Epoxide hydrolysis in the cytosol of rat liver, kidney, and testis: Measurement in the presence of glutathione and the effect of dietary clofibrate

The hydrolysis of trans- and cis-stilbene oxide and benzo[ a]pyrene-4,5-oxide was measured in cytosol and microsomes of liver, kidney, and testis of control and clofibrate-fed rats. Significant levels of nonprotein sulfhydryls were detected in cytosol from liver (4.6 mM) and testis (1.5 mM). Glutathione was moderately stable in these fractions and interfered with the partition assays as conjugates were retained in the aqueous phase along with diols. When the products were separated by thin-layer chromatography, significant amounts of glutathione-conjugates were found to have been formed in the cytosol of liver and testis. Overnight dialysis or preincubation of cytosol with 0.5 mM diethylmaleate eliminated conjugate formation without affecting diol production. In dialyzed cytosol from clofibrate-fed rats (0.5%, 14 days), the rates of hydrolysis of trans-stilbene oxide were 506, 171, and 96% of controls for liver, kidney, and testis, respectively, and 126% of controls in liver microsomes. Rates of hydrolysis of cis-stilbene oxide were 149, 172, and 96% of controls in microsomes and 154, 124, and 91% of controls in cytosols from livers, kidneys, and testis of clofibrate-fed rats respectively. Hydrolysis of benzo[ a]pyrene-4,5-oxide was similar to that of cis-stilbene oxide. Conjugation of the cis-stilbene oxide with glutathione was detected in cytosols from all three tissues with lesser amounts in the microsomes from liver and kidneys. After clofibrate treatment, the rates of this activity were 200, 173, and 95% of controls in cytosol from liver, kidneys and testis, and 203 and 202% of controls in microsomes from liver and kidneys respectively. These results indicate that epoxide hydrolysis and conjugation in rat liver and kidney are responsive to clofibrate treatment and support other evidence which suggests that hydrolysis of cis- and trans-stilbene oxides in cytosol is catalyzed, in part, by distinct enzymes.

Effect of dietary clofibrate on epoxide hydrolase activity in tissues of mice

The effects of dietary clofibrate on the epoxide-metabolizing enzymes of mouse liver, kidney, lung and testis were evaluated using trans-stilbene oxide as a selective substrate for the cytosolic epoxide hydrolase, cis-stilbene oxide and benzo[ a]pyrene 4,5-oxide as substrates for the microsomal form, and cis-stilbene oxide as a substrate for glutathione S-transferase activity. The hydration of trans-stilbene oxide was greatest in liver followed by kidney > lung > testis. Its hydrolysis was increased significantly in the cytosolic fraction of liver and kidney of clofibrate-treated mice and in the microsomes from the liver. Isoelectric focusing indicates that the same enzyme is responsible for hydrolysis of trans-stilbene oxide in normal and induced liver and kidney. Clofibrate induced glutathione S-transferase activity on cis-stilbene oxide only in the liver. Hydrolysis of both cis-stilbene oxide and benzo[ a]pyrene 4,5-oxide was highest in testis followed by liver>lung>kidney. Hydration of cis-stilbene oxide was induced significantly in both liver and kidney by clofibrate but that of benzo[ a]pyrene 4,5-oxide was induced only in the liver. These and other data based on ratios of hydration of benzo[ a]pyrene 4,5-oxide to cis-stilbene oxide in tissues of normal and induced animals indicate that there are one or more novel epoxide hydrolase activities which cannot be accounted for by either the classical cytosolic or mirosomal hydrolases. These effects are notable in the microsomes of kidney and especially in the cytosol of testis.

Enhancement by dietary clofibrate of peroxisomal palmityl-CoA oxidase in kidney and small intestine of albino mice and liver of genetically lean and obese mice

Using density gradient fractionation palmityl-CoA oxidase was localised in the peroxisomes of kidney and small intestine of albino mice. Dietary clofibrate treatment for 14 days resulted in significant increases in palmityl-CoA oxidase of kidney and small intestine of albino mice and liver of genetically lean and obese mice, and was accompanied in the latter by a proliferation of hepatic peroxisomes.

Rat hepatocyte peroxisomes: ultrastructural alterations following cessation of chronic dietary clofibrate administration.

Es wird festgestellt, dass 3 Wochen nach Beendigung einer Clofibrate-(CPIB)-Diat an Ratten manche «Hepotocyte Peroxisomen» (Mikrokorper) bisher nicht beschriebene, ultra-organische Veranderungen von unbekannter Bedeutung zeigen, die mit der experimentellen Behandlung in Zusammenhang stehen durften.

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Treatment of ovariectomized rats with dietary clofibrate caused a manyfold increase in the liver microsomal esterification of estradiol with fatty acids. The stimulatory effect of clofibrate administration on fatty acid esterification of estradiol by liver microsomes was paralleled in vivo by enhanced estradiol-induced increases in the formation of lobules in the mammary gland and by increased incorporation of bromodeoxyuridine into these lobules. In contrast to the stimulatory effect of clofibrate administration on the action of estradiol in the mammary gland, clofibrate administration had no effect on the uterotropic action of estradiol. These results indicate that clofibrate administration has a selective stimulatory effect on the hormonal action of estradiol in the mammary gland but not in the uterus.