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.

Treatment of lactating sows with clofibrate as a synthetic agonist of PPARα does not influence milk fat content and gains of litters.

BACKGROUND: In rats, it has been observed that treatment with activators of peroxisome proliferator-activated receptor α (PPARα) disturbs metabolic adaptations during lactation, which in turn lead to a reduction of milk fat content and gains of litters during the suckling period. It has not yet been investigated whether agonists of PPARα are impairing milk production of lactating sows in a similar manner as in rats. Therefore, the present study aimed to investigate the effect of treatment with clofibrate, a strong synthetic agonist of PPARα, on milk composition and litter gains in lactating sows. RESULTS: Twenty lactating sows received either a basal diet (control group) or the same diet with supplementation of 2 g of clofibrate per kg of diet (clofibrate group). In the clofibrate group, mRNA concentrations of various PPARα target genes involved in fatty acid utilization in liver and skeletal muscle were moderately up-regulated. Fat and energy content of the milk and gains of litters during the suckling period were not different between the control group and the clofibrate group. CONCLUSION: It is shown that treatment with clofibrate induces only a moderate up-regulation of PPARα target genes in liver and muscle of lactating sows and in turn might have limited effect on whole body fatty acid utilization. This may be the reason why clofibrate treatment did not influence milk fat content and gains of litters during the suckling period. Thus, the present study indicates that activation of PPARα induced either by native agonists such as dietary polyunsaturated fatty acids or a by negative energy balance might be largely uncritical in lactating sows with respect to milk production and litter gains in lactating sows.

Clofibrate increases long-chain fatty acid oxidation by neonatal pigs.

BACKGROUND: Utilization of energy-dense lipid fuels is critical to the rapid development and growth of neonates. OBJECTIVE: To increase efficiency of milk fat utilization by newborn pigs, the effect of clofibrate on in vivo and in vitro long-chain fatty acid (LCFA) oxidation was evaluated. METHODS: Newborn male pigs were administered 5 mL of vehicle (2% Tween 80) with or without clofibrate (75 mg/kg body weight) once daily via i.g. gavage for 4 d. Total LCFA oxidative capacity was measured in respiration chambers after gastric infusion (n = 5 per treatment) with isoenergetic amounts of [1-(14)C]triglycerides (TGs), either oleic acid (18:1n-9) TG [3.02 mmol/kg body weight (BW)(0.75)] or erucic acid (22:1n-9) TG (2.46 mmol/kg BW(0.75)). Total expired (14)CO2 was collected and quantified at 20-min intervals over 24 h. Hepatic in vitro LCFA oxidation was determined simultaneously using [1-(14)C]oleic acid and erucic acid substrates. RESULTS: The in vivo 24-h accumulative [1-(14)C]TG oxidation (percentage of energy intake/kg BW(0.75)) tended to increase with clofibrate supplementation (P = 0.10), although there was no difference in the peak or mean utilization rate. The maximal extent of oleic acid TG oxidation was 1.6-fold that of erucic acid TG (P < 0.006). Hepatic in vitro LCFA oxidation increased 61% with clofibrate (P < 0.0008). The increase in mitochondria was 4-fold greater than in peroxisomes. The relative abundance of mRNA increased 2- to 3-fold for hepatic peroxisome proliferator-activated receptor α and its target genes (fatty acyl-coenzyme A oxidase and carnitine palmitoyltransferase) in the pigs that were administered clofibrate (P < 0.04). CONCLUSION: Clofibrate may improve in vivo LCFA oxidative utilization in neonatal pigs.

Novel use of a lipid-lowering fibrate medication to prevent nicotine reward and relapse: preclinical findings.

Experimental drugs that activate α-type peroxisome proliferator-activated receptors (PPARα) have recently been shown to reduce the rewarding effects of nicotine in animals, but these drugs have not been approved for human use. The fibrates are a class of PPARα-activating medications that are widely prescribed to improve lipid profiles and prevent cardiovascular disease, but these drugs have not been tested in animal models of nicotine reward. Here, we examine the effects of clofibrate, a representative of the fibrate class, on reward-related behavioral, electrophysiological, and neurochemical effects of nicotine in rats and squirrel monkeys. Clofibrate prevented the acquisition of nicotine-taking behavior in naive animals, substantially decreased nicotine taking in experienced animals, and counteracted the relapse-inducing effects of re-exposure to nicotine or nicotine-associated cues after a period of abstinence. In the central nervous system, clofibrate blocked nicotine's effects on neuronal firing in the ventral tegmental area and on dopamine release in the nucleus accumbens shell. All of these results suggest that fibrate medications might promote smoking cessation. The fact that fibrates are already approved for human use could expedite clinical trials and subsequent implementation of fibrates as a treatment for tobacco dependence, especially in smokers with abnormal lipid profiles.

Clofibrate relaxes the longitudinal smooth muscle of the mouse distal colon through calcium-mediated desensitisation of contractile machinery.

Peroxisome proliferator-activated receptor α (PPAR-α) is a ligand-activated transcription factor that exerts strong effects on metabolic pathways. Our aim was to elucidate the effect of clofibrate, a PPAR-α agonist, on the longitudinal muscle of the mouse distal colon. We initially found that clofibrate induced a relaxation response in this muscle. Notably, the PPAR-α antagonists GW9662 and T0070907 did not attenuate this clofibrate-induced relaxation. The structurally related PPAR-α agonists fenofibrate and bezafibrate induced relaxation in the distal colon as effectively as clofibrate. In contrast, wy-14643, which activates PPAR-α more selectively than clofibrate, had no effect. Furthermore, clofibrate-induced relaxation was not affected by N-nitro-L-arginine, an NO synthase inhibitor, 1H-[1,2,4]-oxadiazolo-[4,3- a]quinoxaline-1-one, a soluble guanylate cyclase inhibitor, or H89, a protein kinase A inhibitor. Tetrodotoxin, an Na⁺ channel blocker, and glibenclamide, apamin, charybdotoxin and XE991, various K⁺ channel blockers, had no effect on clofibrate-induced relaxation. Importantly, clofibrate induced a relaxation response that was not accompanied by any alteration in the cytoplasmic Ca²âº concentration in the longitudinal muscle of the mouse distal colon. Moreover, calyculin A, a myosin light-chain phosphatase (MLCP) inhibitor, attenuated clofibrate-induced relaxation. Our findings indicate that clofibrate relaxes the longitudinal smooth muscle of the mouse distal colon by regulating MLCP activity.

Wild bitter gourd extract up-regulates mRNA expression of PPARα, PPARγ and their target genes in C57BL/6J mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Wild bitter gourd (Momordica charantia Linn. var. abbreviata ser.) was commonly used as a medicinal herb in Asia, Africa, and South America because of its anti-diabetic, antibacterial, anti-viral, and chemopreventive functions. MATERIALS AND METHODS: C57BL/6J mice were orally administered with 250, 500 or 1000mg/kg BW of WBGE in 0.2mL/mouse of olive oil daily for 2 weeks. RESULTS: Compared to control (vehicle treated) mice, mice receiving WBGE showed significantly higher PPARα, ACO (acyl-CoA oxidase) and L-FABP (liver-fatty acid binding protein) mRNA expression, ACO activity and protein in the liver (P<0.05), as clofibrate-treated mice. WBGE treatment also resulted in significantly higher PPARγ and LPL (lipoprotein lipase) mRNA (P<0.05) in the epididymal adipose tissue. Liver triglyceride and non-esterified fatty acid concentration in WBGE treated mice were significantly lower than those of control mice (P<0.05). Plasma adiponectin level was significantly higher in mice receiving WBGE than in control mice (P<0.05), as the rosiglitazone treated mice. CONCLUSION: Results of this study demonstrated that WBGE also activates PPARα and PPARγ signaling pathway in vivo.

Anti-atherogenic property of ferulic acid in apolipoprotein E-deficient mice fed Western diet: comparison with clofibrate.

Anti-atherogenic effect of ferulic acid (0.02%, w/w) was investigated in comparison with the clofibrate (0.02%, w/w) in apolipoprotein E-deficient (apo E(-/-)) mice fed Western diet. Concentrations of total cholesterol (total-C), apolipoprotein B (apo B) in the plasma and epididymal adipose tissue weight were significantly lower in the ferulic acid and clofibrate supplemented groups compared to the control group. The ratio of apo B to apo A-I was also significantly lower in those groups than in the control group. Activities of hepatic ACAT and HMG-CoA reductase were only significantly lower in the ferulic acid and clofibrate groups, respectively than in the control group. The numbers of mice that exhibited aortic fatty plaque were 8/10 in control groups vs. 0/10 in the ferulic acid or clofibrate group. The activities of anti-oxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and paraoxonase) in the hepatocyte and erythrocyte were significantly higher in the ferulic acid group than in the control group. In contrast, hepatic TBARS level was only markedly lower in the ferulic acid group. These results provide a new insight into the anti-atherogenic property of ferulic acid in the apo E(-/-) mice fed a Western diet.

Mitochondrial HMG-CoA synthase partially contributes to antioxidant protection in the kidney of stroke-prone spontaneously hypertensive rats.

OBJECTIVE: Increased oxidative stress plays an important role in cardiovascular diseases including hypertension and stroke. Evidence has indicated that ketone bodies could exert antioxidative effects. We explored the role of renal mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS2) expression, a key control site of ketogenesis, in stroke-prone spontaneously hypertensive rats (SHRSPs) and their ancestral hypertensive but stroke-resistant spontaneously hypertensive rats (SHRs). METHODS: Two groups of SHRSPs were fed a standard chow or standard chow supplemented with clofibrate (an agonist of HMGCS2 promoter), respectively, and SHRs fed with a standard chow were used as controls. The renal levels of HMGCS2, Akt, and phosphorylated protein kinase B (Akt) were measured by western blotting. Malondialdehyde, catalase, superoxide dismutase, and glutathione peroxidase were detected by assay kits. RESULTS: Compared with SHRs, lower HMGCS2 protein expression, enhanced phosphorylated Akt signal, higher malondialdehyde levels, and higher catalase activity were observed in kidney tissues in SHRSPs (P < 0.05). No differences in superoxide dismutase and glutathione peroxidase activities were observed between SHRSPs and SHRs. Clofibrate treatment significantly upregulated renal HMGCS2 expressions, inhibited phosphorylation of Akt, and decreased malondialdehyde levels and catalase activities in SHRSP kidney tissues (P < 0.05). CONCLUSION: These results demonstrated the difference in HMGCS2 expression and oxidative stress in kidney tissues between SHRSPs and their SHR controls. The enhanced oxidative stress was partly due to the lower HMGCS2 expression regulated possibly by the Akt signaling pathway.

Temporal evaluation of CYP mRNA in mice administered with prototypical P450 inducers: comparison with conventional protein/enzyme methods.

Assessment of cytochrome P450 (CYP) induction at the mRNA level in preclinical rodent studies has gained interest in recent years, but there are still concerns regarding correlations between the mRNA and the enzyme activity levels, especially in mice. The purpose of the present study was to systematically evaluate patterns of temporal changes of CYPs 1a1, 1a2, 2b10, 3a11, and 4a10 at mRNA, protein, and activity levels in order to determine to what extent mRNA levels could be used either qualitatively or quantitatively for the assessment of CYP enzyme induction. In this study, livers from male CD-1 mice treated daily with beta-naphthoflavone, phenobarbital, dexamethasone, clofibrate, and control vehicles were collected for RNA and microsomal analysis after 0.5, 1, 2, 4, and 8 days of daily dose. The results revealed a good correlation among mRNA, protein, and enzyme activity levels, with the best correlation at the time points between Days 2 and 8, suggesting that the appropriate time to monitor CYP mRNA may be beyond Day 2 of chemical treatments. Based on these results, we concluded that the mRNA approach is a useful tool to monitor CYP induction in mice, particularly when treatment duration is beyond 2 days.

Treatment with pharmacological peroxisome proliferator-activated receptor alpha agonist clofibrate increases intestinal carnitine absorption in rats.

Activation of PPARalpha by clofibrate has recently been shown to cause upregulation of the high-affinity carnitine transporter novel organic cation transporter (OCTN) 2 in small intestine. This strongly suggests that PPARalpha activation in response to clofibrate treatment improves the absorption of carnitine from the diet. To test this hypothesis, we performed an experiment with rats which were fed diets with or without 5 g clofibrate/kg diet and with or without 5 g L-carnitine/kg diet. PPARalpha was significantly activated by clofibrate in small intestine as evidenced by increased relative mRNA concentrations of the PPARalpha target gene acyl-CoA oxidase (P < 0.05). Relative mRNA concentration of OCTN2 in small intestine was significantly increased by clofibrate (P < 0.05) but not the carnitine supplementation, whereas relative mRNA concentrations of other carnitine transporters (OCTN1, ATB(0+)) in small intestine were not influenced by either clofibrate or carnitine. The absorption rate of carnitine in small intestine was markedly higher in rats treated with clofibrate than in those treated without clofibrate (P < 0.05). In conclusion, the present study shows that administration of clofibrate to rats increases carnitine absorption in small intestine which is probably due to the observed upregulation of OCTN2 mediated by activation of PPARalpha.

Clofibrate treatment up-regulates novel organic cation transporter (OCTN)-2 in tissues of pigs as a model of non-proliferating species.

Recent studies have shown that treatment of rodents with agonists of peroxisome proliferator-activated receptor (PPAR)-alpha causes an up-regulation of novel organic cation transporter (OCTN)-2, a carnitine transporter, and increases carnitine concentration in the liver. This study was performed to investigate whether such effects occur also in pigs which like humans have a lower expression of PPAR alpha and are less responsive to treatment with PPAR alpha agonists than rodents. An experiment with 18 pigs was performed which were fed a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had higher relative mRNA concentrations of OCTN2 in liver (3.1-fold), skeletal muscle (1.5-fold) and epithelial cells from small intestine (1.8-fold) than control pigs (P<0.05). Pigs treated with clofibrate had also higher concentrations of free and total carnitine in the liver and a higher concentration of free carnitine in skeletal muscle than control pigs (P<0.05). Concentrations of gamma-butyrobetaine, the precursor of endogenous formation of carnitine, in liver, muscle and plasma did not differ between both groups; the activity of gamma-butyrobetaine dioxygenase, the rate limiting enzyme of carnitine synthesis, in the liver was lower in pigs treated with clofibrate than in control pigs (P<0.05). This study shows for the first time that treatment with a PPAR alpha agonist causes an up-regulation of OCTN2 in liver, muscle and enterocytes from small intestine of pigs. This in turn increases carnitine concentrations in liver and muscle probably by enhancing carnitine uptake into cells.

Does the capacity for energy utilization affect the survival of post-smolt Atlantic salmon, Salmo salar L., during natural outbreaks of infectious pancreatic necrosis?

If osmotic stress and reduced seawater tolerance are predisposing factors for infectious pancreatic necrosis (IPN) outbreaks in farmed Atlantic salmon, increased survival by enhancing access to energy would be expected. The aim of the present study was, therefore, to increase energy access in 1-year old Atlantic salmon after sea transfer by increasing the level of dietary fat, by exchanging some of the dietary oil with more easily oxidized medium chain triacylglycerols, or by dietary supplementation of potentially energy enhancing additives such as clofibrate and tetradecylthioacetic acid (TTA). A natural outbreak of IPN occurred 8 weeks after sea transfer, and a significant dietary effect explaining 76% of the variation in mortality was observed. Relative percentage survival for the fish fed TTA in sea water was 70% when compared with the unsupplemented control, reducing mortality from 7.8 to 2.3%. Muscle fat content and plasma chloride were related to IPN mortality, suggesting that reduced hypoosmoregulatory capacity might be a predisposing factor to the onset of an IPN outbreak. Based on the observation of a threefold increase in white muscle mitochondrial fatty acid oxidizing activity by TTA, it is suggested that TTA has resulted in a re-allocation of dietary fatty acids from storage to energy producing oxidation.

Clofibrate causes an upregulation of PPAR-{alpha} target genes but does not alter expression of SREBP target genes in liver and adipose tissue of pigs.

This study investigated the effect of clofibrate treatment on expression of target genes of peroxisome proliferator-activated receptor (PPAR)-alpha and various genes of the lipid metabolism in liver and adipose tissue of pigs. An experiment with 18 pigs was performed in which pigs were fed either a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had heavier livers, moderately increased mRNA concentrations of various PPAR-alpha target genes in liver and adipose tissue, a higher concentration of 3-hydroxybutyrate, and markedly lower concentrations of triglycerides and cholesterol in plasma and lipoproteins than control pigs (P < 0.05). mRNA concentrations of sterol regulatory element-binding proteins (SREBP)-1 and -2, insulin-induced genes (Insig)-1 and Insig-2, and the SREBP target genes acetyl-CoA carboxylase, 3-methyl-3-hydroxyglutaryl-CoA reductase, and low-density lipoprotein receptor in liver and adipose tissue and mRNA concentrations of apolipoproteins A-I, A-II, and C-III in the liver were not different between both groups of pigs. In conclusion, this study shows that clofibrate treatment activates PPAR-alpha in liver and adipose tissue and has a strong hypotriglyceridemic and hypocholesterolemic effect in pigs. The finding that mRNA concentrations of some proteins responsible for the hypolipidemic action of fibrates in humans were not altered suggests that there were certain differences in the mode of action compared with humans. It is also shown that PPAR-alpha activation by clofibrate does not affect hepatic expression of SREBP target genes involved in synthesis of triglycerides and cholesterol homeostasis in liver and adipose tissue of pigs.

Clofibrate increases hepatic triiodothyronine (T3)- and thyroxine (T4)-glucuronosyltransferase activities and lowers plasma T3 and T4 concentrations in pigs.

In rats, clofibrate acts as a microsomal enzyme inducer and disrupts the metabolism of thyroid hormones by increasing hepatic glucuronidation of thyroxine. Whether similar effects occur in the pig has not yet been investigated. This study was performed to investigate the effect of clofibrate treatment on metabolism of thyroid hormones in pigs. To this end, an experiment with 18 pigs, which were assigned to two groups, was performed. One group received a control diet, and the other group was fed the same diet supplemented with 5 g of clofibrate/kg for 28 days. Pigs treated with clofibrate had higher hepatic activities of T(3)- and T(4)-UDP glucuronosyltransferases (UGT) and lower concentrations of total and free T(4) and total T(3) in plasma than control pigs (P < 0.05). Weights and histology of the thyroid gland (epithelial height, follicle lumen diameter) did not differ between the two groups, but pigs treated with clofibrate had higher mRNA concentrations of various genes in the thyroid responsive to thyroid-stimulating hormone (TSH) such as TSH receptor, sodium iodine symporter, thyroid peroxidase, and cathepsin B than control pigs (P < 0.05). Pigs treated with clofibrate also had lower hepatic mRNA concentrations of proteins involved in plasma thyroid hormone transport [thyroxine-binding globulin (P < 0.10), transthyretin (P < 0.05), and albumin (P < 0.05)] and thyroid hormone receptor alpha(1) (P < 0.05) than control pigs. In conclusion, this study shows that clofibrate treatment induces a strong activation of T(3)- and T(4)-UGT in pigs, leading to increased glucuronidation and markedly reduced plasma concentrations of these hormones, accompanied by a moderate stimulation of thyroid function.

Anticholesterolemic effect of 3,4-di(OH)-phenylpropionic amides in high-cholesterol fed rats.

Two amide synthetic derivatives of 3,4-di(OH)-hydrocinnamate (HC), 3,4-dihydroxyphenylpropionic (l-serine methyl ester) amide (E030) and 3,4-dihydroxyphenylpropionic (l-aspartic acid) amide (E076), were investigated to compare their lipid-lowering efficacy with HC. Male rats were fed a 1 g/100 g high-cholesterol diet for 6 weeks with supplements of either clofibrate (0.02%, w/w), HC (0.025%, w/w), E030 (0.039%, w/w) or E076 (0.041%, w/w). The clofibrate supplement was used as a positive control for the lipid-lowering efficacy. The food intakes and body weight gains were not significantly different among the groups. The plasma and hepatic cholesterol and triglyceride levels were lower in clofibrate, HC, E030, and E076-supplemented groups compared to the control group. The supplementation of HC and its amide derivatives was as effective as clofibrate in increasing the ratio of HDL-cholesterol to total plasma cholesterol and reducing the atherogenic index (AI). The hepatic cholesterol level in the HC and E076 groups was significantly lower than that in the clofibrate group. The hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA reductase) and acyl-CoA:cholesterol acyltransferase (ACAT) activities were significantly lower in the all test groups than in the control group. The excretion of neutral sterol was significantly higher in the HC, E030, and E076-supplemented groups compared to the control group. The plasma AST and ALT activities, indirect indexes of hepatic toxicity, were significantly lower in the HC, E030, and E076-supplemented groups than in the control group. Accordingly, the current results suggest that E030 and E076, two amide synthetic derivatives of HC, are effective in lowering lipid activity.

A mouse model for alpha-methylacyl-CoA racemase deficiency: adjustment of bile acid synthesis and intolerance to dietary methyl-branched lipids.

alpha-Methylacyl-CoA racemase (Amacr) deficiency in humans leads to sensory motor neuronal and liver abnormalities. The disorder is recessively inherited and caused by mutations in the AMACR gene, which encodes Amacr, an enzyme presumed to be essential for bile acid synthesis and to participate in the degradation of methyl-branched fatty acids. To generate a model to study the pathophysiology in Amacr deficiency we inactivated the mouse Amacr gene. As per human Amacr deficiency, the Amacr(-/-) mice showed accumulation (44-fold) of C27 bile acid precursors and decreased (over 50%) primary (C24) bile acids in bile, serum and liver, however the Amacr(-/-) mice were clinically symptomless. Real-time quantitative PCR analysis showed that, among other responses, the level of mRNA for peroxisomal multifunctional enzyme type 1 (pMFE-1) was increased 3-fold in Amacr(-/-) mice. This enzyme can be placed, together with CYP3A11 and CYP46A1, to make an Amacr-independent pathway for the generation of C24 bile acids. Exposure of Amacr(-/-) mice to a diet supplemented with phytol, a source for branched-chain fatty acids, triggered the development of a disease state with liver manifestations, redefining the physiological significance of Amacr. Amacr is indispensable for the detoxification of dietary methyl-branched lipids and, although it contributes normally to bile acid synthesis from cholesterol, the putative pMFE-1-mediated cholesterol degradation can provide for generation of bile acids, allowing survival without Amacr. Based upon our mouse model, we propose elimination of phytol from the diet of patients suffering from Amacr deficiency.

Anti-hyperlipidemic sesquiterpenes and new sesquiterpene glycosides from the leaves of artichoke (Cynara scolymus L.): structure requirement and mode of action.

The methanolic extract from the leaves of artichoke (Cynara scolymus L.) was found to suppress serum triglyceride elevation in olive oil-loaded mice. Through bioassay-guided separation, sesquiterpenes (cynaropicrin, aguerin B, and grosheimin) were isolated as the active components together with new sesquiterpene glycosides (cynarascolosides A, B, and C). The oxygen functional groups at the 3- and 8-positions and exo-methylene moiety in alpha-methylene-gamma-butyrolactone ring were found to be essential for the anti-hyperlipidemic activity of guaiane-type sesquiterpene. In addition, inhibition of gastric emptying was shown to be partly involved in anti-hyperlipidemic activity.

Pharmacological interventions for the treatment of neonatal jaundice.

In the neonate, hyperbilirubinaemia is usually due to a combination of an increased bilirubin load and decreased bilirubin elimination. Despite an understanding of the enzymatic pathways leading to bilirubin production and elimination, very few pharmacological interventions to prevent hyperbilirubinaemia are utilized and the mainstay of treatment remains phototherapy. Previously studied pharmacological agents such as D-penicillamine, phenobarbital and clofibrate may yet prove useful. Recent clinical trials using metalloporphyrins to inhibit heme catabolism and bilirubin production provides a novel pharmacological intervention for the treatment of neonatal jaundice. The safety and efficacy of these therapies will need to be confirmed prior to widespread use.

Topical peroxisome proliferator activated receptor-alpha activators reduce inflammation in irritant and allergic contact dermatitis models.

Activators of peroxisome proliferator activated receptor-alpha, a nuclear hormone receptor that heterodimerizes with retinoid X receptor, stimulate epidermal differentiation and inhibit proliferation. Here we determined the anti-inflammatory effects of peroxisome proliferator activated receptor-alpha agonists in models of irritant and allergic contact dermatitis produced in mouse ears by topical treatment with 12-O-tetradecanoylphorbol-13-acetate and oxazalone, respectively. As expected, 12-O-tetradecanoylphorbol-13-acetate treatment resulted in a marked increase in the thickness and weight of the ears and provoked an inflammatory cell infiltrate in the dermis. Topical treatment with three different peroxisome proliferator activated receptor-alpha agonists, clofibrate, WY 14643, or linoleic acid, 45 min and 4 h after 12-O-tetradecanoylphorbol-13-acetate application, resulted in a marked decrease in ear thickness and weight and a reduction in the number of inflammatory cells in the dermis. The reduction in inflammation by these peroxisome proliferator activated receptor-alpha agonists was of similar magnitude to that seen with a potent topical glucocorticoid, clobetasol. In contrast, stearic acid, a free fatty acid that does not activate peroxisome proliferator activated receptor-alpha, had no effect on the 12-O-tetradecanoylphorbol-13-acetate-induced inflammation. Moreover, clofibrate did not significantly alter ear thickness following 12-O-tetradecanoylphorbol-13-acetate treatment in peroxisome proliferator activated receptor-alpha-/- mice, indicating that the anti-inflammatory effect is mediated by peroxisome proliferator activated receptor-alpha. As tumor necrosis factor-alpha and interleukin-1alpha are major mediators of cutaneous inflammation we next used immunohistochemistry to determine whether the peroxisome proliferator activated receptor-alpha agonists reduce the levels of these cytokines in 12-O-tetradecanoylphorbol-13-acetate-treated skin. 12-O-tetradecanoylphorbol-13-acetate treatment resulted in an increase in tumor necrosis factor and interleukin-1alpha staining in the epidermis that was reduced by clofibrate treatment. Finally, clofibrate treatment also reduced ear thickness and weight in oxazalone-induced allergic dermatitis, a change that was accompanied by a reduction in inflammatory cells in the dermis and a decrease in tumor necrosis factor-alpha and interleukin-1alpha levels in the oxazalone-treated epidermis. These studies demonstrate that topically applied peroxisome proliferator activated receptor-alpha agonists possess receptor mediated, anti-inflammatory activity in both irritant and allergic contact dermatitis animal models. The anti-inflammatory properties of peroxisome proliferator activated receptor-alpha agonists, coupled with their anti-proliferative and pro-differentiating effects, suggest that they could be beneficial for the treatment of a variety of cutaneous diseases.

Monitoring expression of genes involved in drug metabolism and toxicology using DNA microarrays.

Oligonucleotide DNA microarrays were investigated for utility in measuring global expression profiles of drug metabolism genes. This study was performed to investigate the feasibility of using microarray technology to minimize the long, expensive process of testing drug candidates for safety in animals. In an evaluation of hybridization specificity, microarray technology from Affymetrix distinguished genes up to a threshold of approximately 90% DNA identity. Oligonucleotides representing human cytochrome P-450 gene CYP3A5 showed heterologous hybridization to CYP3A4 and CYP3A7 RNAs. These genes could be clearly distinguished by selecting a subset of oligonucleotides that hybridized selectively to CYP3A5. Further validation of the technology was performed by measuring gene expression profiles in livers of rats treated with vehicle, 3-methylcholanthrene (3MC), phenobarbital, dexamethasone, or clofibrate and by confirming data for six genes using quantitative RT-PCR. Responses of drug metabolism genes, including CYPs, epoxide hydrolases (EHs), UDP-glucuronosyl transferases (UGTs), glutathione sulfotransferases (GSTs), sulfotransferases (STs), drug transporter genes, and peroxisomal genes, to these well-studied compounds agreed well with, and extended, published observations. Additional gene regulatory responses were noted that characterize metabolic effects or stress responses to these compounds. Thus microarray technology can provide a facile overview of gene expression responses relevant to drug metabolism and toxicology.