Endogenous Lipid Metabolism Research Articles

Partially defatted rather than native poppy seeds beneficially alter lipid metabolism in rats fed a high-fat diet

Partially defatted poppy seeds, a by-product of poppy oil cold pressing, could be an interesting dietary supplement for obesity management. The aim of this study was to compare the effects of dietary supplementation with a small amount of native or partially defatted poppy seeds on gastrointestinal function and lipid metabolism in rats fed a high-fat diet. The defatted poppy seeds had, among others, lower fat content and higher fibre and protein content than native poppy seeds. The rats fed with a high-fat diet were characterised by severe metabolic disorders, especially in the liver, and poppy seeds were unable to prevent them. However, depending on the seed form, dietary supplementation with poppy seeds differentially affected the microbial and endogenous lipid metabolism in rats. In the distal intestine, both dietary seed forms stimulated microbial acetate production, and the supplementation with partially defatted poppy seeds additionally inhibited isobutyrate and isovalerate formation, which indicates a reduction in putrefaction. Both dietary seed forms increased cholesterol accumulation in the liver. Only dietary supplementation with partially defatted poppy seeds attenuated visceral fat and hepatic triglyceride accumulations and lowered blood triglyceride concentrations, and at the transcriptional level, the inhibition of SREBP-1c, which upregulates genes responsible for de novo lipogenesis, was additionally observed in this organ. In conclusion, a low and regular consumption of partially defatted poppy seeds may be beneficial in managing obesity-related disorders.

Bisphenol S exposure accelerates the progression of atherosclerosis in zebrafish embryo-larvae

Bisphenol S (BPS), widely utilized in manufacturing of daily necessities, is a toxicant with potential to induce atherosclerotic cardiovascular disease (ASCVD). However, the mode of action by which BPS exposure induces ASCVD remains unknown. Here, macrophages that were exposed to BPS in combination with oxidized low-density lipoprotein (oxLDL) exhibited enhanced formation of foam cells, a hallmark of ASCVD. Furthermore, zebrafish embryo-larvae were exposed to BPS (0, 1, 10 and 100 μg/L) for 15 days (d) and the characteristic symptoms of ASCVD including an inflammatory response, macrophage recruitment around blood vessels, and accumulation of oxLDL on vascular endothelium, were induced in 15-d larvae. After zebrafish were exposed to BPS for 45 d, BPS mobilized fatty acid metabolism and activated peroxisome proliferator-activated receptor signaling in larval liver, the hub of endogenous lipid metabolism, causing an increase in plasma LDL. Driven by high plasma LDL levels, the caudal artery of zebrafish larvae exhibited lipid accumulation and a thickened area with a large number of collagen fibers, accompanied by characteristic lesions, as well as hyperlipidemia, erythrocyte aggregation, thinner blood vessel walls and increased levels of leukocytes and thromboocytes in plasma. Our data demonstrate that BPS accelerates the progression of ASCVD using zebrafish embryo-larvae as a model.

Tanshinol A Ameliorates Triton-1339W-Induced Hyperlipidemia and Liver Injury in C57BL/6J Mice by Regulating mRNA Expression of Lipemic-Oxidative Injury Genes.

Tanshinol A, which is derived from a traditional Chinese herbal Radix Salviae Miltiorrhizae is indicative of a hypolipidemic candidate. Therefore, we aim to validate its hypolipidemic activity of tanshinol A and explore its mechanism in triton-1339W-induced hyperlipidemic mice model, which possess multiply pathogenesis for endogenous lipid metabolism disorder. Experimental hyperlipidemia mice are treated with or without tanshinol A (i.g. 40, 20, 10 mg/kg), and blood and liver tissue were collected for validating its hypolipidemic and hepatic protective effect, and hepatic mRNA expression profile, which was associated with lipid metabolism dysfunction and liver injury, was detected by RT-qPCR. As results show, triton-1339W-induced abnormal of serum TC, TAG, HDL-C, LDL-C, SOD, MDA, GOT, and GPT is remarkably attenuated by tanshinol A. In pathological experiment, triton-1339W-induced hepatocellular ballooning degeneration, irregular central vein congestion, and inflammation infiltration are alleviated by tanshinol A. Correspondingly, hepatic mRNA expression of Atf4, Fgf21, Vldlr, Nqo1, Pdk4, and Angptl4, which are genes regulating lipemic-oxidative injury, are significantly increased by tanshinol A by 2~6 fold. Abcg5, Cd36, and Apob, which are responsible for cholesterol metabolism, are mildly upregulated. Noticeably, triton-1339W-suppressed expressions of Ptgs2/Il10, which are genes responsible for acute inflammation resolution in liver injury, are remarkably increased by tanshinol A. Conclusively, tanshinol A exerted hypolipidemic effect and hepatoprotective effect through restoring triton-1339W-suppressed mRNA expression, which may be involved in Atf4/Fgf21/Vldlr and Ptgs2/Il-10 signaling pathways.

The effects of dietary quercetin supplementation and sex on the fatty acid profile of rabbit meat, dissectible fat and caecotrophes

This study examined the effects of dietary quercetin on the fatty acid (FA) profile of rabbit caecotrophes, dissectible fat, Longissimus thoracis et lumborum (LTL) muscle and hindleg (HL) meat. Sixteen male and sixteen female New Zealand White rabbits were fed a control or quercetin-supplemented (2 g quercetin dihydrate/kg feed) diet from 5 to 12 weeks old, then slaughtered. Caecotrophes were collected from the gut, and the dissectible fat, LTL and deboned HL were sampled. Lipids in the samples were transmethylated, then identified and quantified using GC-FID. Quercetin-supplementation increased C18:0 in the fat, and C20:4n-6 in the LTL — suggesting an interaction with endogenous lipid metabolism — but had no effect on the HL and caecotrophes. Sex affected the caecotrophe FAs, but had little effect on the meat's nutritional value. The FA profiles of the LTL and HL differed, but both aligned to nutritional recommendations. The caecotrophe FA profile was indicative of microbial biohydrogenation, but this had minimal effect on the carcass FA.

Endocannabinoid system and the expression of endogenous ceramides in human hepatocellular carcinoma.

The endogenous lipid metabolism network is associated with the occurrence and progression of malignancies. Endocannabinoids and ceramides have demonstrated their anti-proliferative and pro-apoptotic properties in a series of cancer studies. The aim of the present study was to evaluate the expression patterns of endocannabinoids and endogenous ceramides in 67 pairs of human hepatocellular carcinoma (HCC) tissues and non-cancerous counterpart controls. Anandamide (AEA), the major endocannabinoid, was reduced in tumor tissues, probably due to the high expression and activity of fatty acid amide hydrolase. Another important endocannabinoid, 2-arachidonylglycerol (2-AG), was elevated in tumor tissues compared with non-tumor controls, indicating that the biosynthesis of 2-AG is faster than the degradation of 2-AG in tumor cells. Furthermore, western blot analysis demonstrated that cannabinoid receptor 1 was downregulated, while cannabinoid receptor 2 was elevated in HCC tissues, in accordance with the alterations in the levels of AEA and 2-AG, respectively. For HCC tissues, the expression levels of C18:0, 20:0 and 24:0-ceramides decreased significantly, whereas C12:0, 16:0, 18:1 and 24:1-ceramides were upregulated, which may be associated with cannabinoid receptor activation and stearoyl-CoA desaturase protein downregulation. The exact role of endocannabinoids and ceramides in regulating the fate of HCC cells requires further investigation.

APOE hypermethylation is significantly associated with coronary heart disease in males

APOE is involved in exogenous and endogenous lipid metabolism and is a candidate gene for coronary heart disease (CHD). Using the SYBR-Green quantitative methylation-specific PCR method, we measured APOE methylation levels in 640 cases of CHD and 436 controls. Meanwhile, we used the Sequenom MassARRAY platform to genotype APOE rs7412 and rs7259620. In men, we found that CHD patients had significantly lower levels of APOE methylation than non-CHD controls (P = 0.044). In addition, rs7412-T and rs7259620-A were protective factors for CHD in males (rs7412-T: OR = 0.527, allele P = 0.004; rs7259620-A: OR = 0.743, allele P = 0.029). In the CHD group and the non-CHD control group, APOE methylation levels were inversely correlated with age (total patients: r = −0.015, P = 0.009, total controls: r = −0.185, P = 1E−04). In the female control group, the APOE methylation levels were inversely correlated with plasma APOA1 protein levels (female controls: r = −0.188, P = 0.008). In the total controls and female controls, APOE methylation levels were inversely correlated with plasma APOE protein levels (total controls: r = −0.110, P = 0.031, female controls: r = −0.188, P = 0.008). Our GDMR interaction analysis showed that there was an interaction between APOE methylation and rs7412 (P = 0.011). In addition, there was a significant interaction between APOE methylation, rs7259620, gender, smoking, LDL, TC, and APOE protein levels in the risk of CHD (P = 0.011). In summary, our research showed that the methylation of APOE was important for the risk of CHD in men.

Beyond detoxification: a role for mouse mEH in the hepatic metabolism of endogenous lipids

Microsomal and soluble epoxide hydrolase (mEH and sEH) fulfill apparently distinct roles: Whereas mEH detoxifies xenobiotics, sEH hydrolyzes fatty acid (FA) signaling molecules and is thus implicated in a variety of physiological functions. These epoxy FAs comprise epoxyeicosatrienoic acids (EETs) and epoxy-octadecenoic acids (EpOMEs), which are formed by CYP epoxygenases from arachidonic acid (AA) and linoleic acid, respectively, and then are hydrolyzed to their respective diols, the so-called DHETs and DiHOMEs. Although EETs and EpOMEs are also substrates for mEH, its role in lipid signaling is considered minor due to lower abundance and activity relative to sEH. Surprisingly, we found that in plasma from mEH KO mice, hydrolysis rates for 8,9-EET and 9,10-EpOME were reduced by 50% compared to WT plasma. This strongly suggests that mEH contributes substantially to the turnover of these FA epoxides—despite kinetic parameters being in favor of sEH. Given the crucial role of liver in controlling plasma diol levels, we next studied the capacity of sEH and mEH KO liver microsomes to synthesize DHETs with varying concentrations of AA (1–30 μM) and NADPH. mEH-generated DHET levels were similar to the ones generated by sEH, when AA concentrations were low (1 μM) or epoxygenase activity was curbed by modulating NADPH. With increasing AA concentrations sEH became more dominant and with 30 μM AA produced twice the level of DHETs compared to mEH. Immunohistochemistry of C57BL/6 liver slices further revealed that mEH expression was more widespread than sEH expression. mEH immunoreactivity was detected in hepatocytes, Kupffer cells, endothelial cells, and bile duct epithelial cells, while sEH immunoreactivity was confined to hepatocytes and bile duct epithelial cells. Finally, transcriptome analysis of WT, mEH KO, and sEH KO liver was carried out to discern transcriptional changes associated with the loss of EH genes along the CYP-epoxygenase–EH axis. We found several prominent dysregulations occurring in a parallel manner in both KO livers: (a) gene expression of Ephx1 (encoding for mEH protein) was increased 1.35-fold in sEH KO, while expression of Ephx2 (encoding for sEH protein) was increased 1.4-fold in mEH KO liver; (b) Cyp2c genes, encoding for the predominant epoxygenases in mouse liver, were mostly dysregulated in the same manner in both sEH and mEH KO mice, showing that loss of either EH has a similar impact. Taken together, mEH appears to play a leading role in the hydrolysis of 8,9-EET and 9,10-EpOME and also contributes to the hydrolysis of other FA epoxides. It probably profits from its high affinity for FA epoxides under non-saturating conditions and its close physical proximity to CYP epoxygenases, and compensates its lower abundance by a more widespread expression, being the only EH present in several sEH-lacking cell types.

Decreased activity of butyrylcholinesterase in blood plasma of patients with chronic obstructive pulmonary disease.

IntroductionButyrylcholinesterase (BChE) is involved in the metabolism of endogenous lipids and xenobiotics, such as esters of carboxylic or phosphoric acids. Butyrylcholinesterase activity is associated with both inflammation and oxidative stress. Changes in the activity of this enzyme have been observed in various diseases such as liver cirrhosis, diabetes, neurodegenerative disease and others.Material and methodsThe study involved 30 patients with chronic obstructive pulmonary disease (COPD) and 18 healthy subjects. The COPD patients were divided according to the severity of the disease by applying the classification of COPD based on GOLD standards for forced expiratory volume in 1 s (FEV1) and the FEV1/forced expiratory volume (FVC) ratio. The control group comprised blood samples collected from healthy subjects without concomitant diseases related to the respiratory system. Butyrylcholinesterase activity, lipid peroxidation and total antioxidant capacity (TAC) were determined in the blood plasma.ResultsA significant (p < 0.05) decrease in the activity of BChE, associated with an increase in lipid peroxidation and a decrease in the total antioxidant capacity, was observed in blood plasma of patients with chronic obstructive pulmonary disease.ConclusionsThe study shows for the first time that activity of BChE in the blood plasma of patients diagnosed with chronic obstructive pulmonary disease is considerably reduced compared with healthy subjects. These changes were accompanied by a decrease of TAC and an increase of lipid peroxidation, which suggests that they may be related to the oxidative stress induced by COPD disease.

Berberine Reduces Neurotoxicity Related to Nonalcoholic Steatohepatitis in Rats.

Berberine is a plant alkaloid that has several pharmacological effects such as antioxidant, antilipidemic, and anti-inflammatory effects. Nonalcoholic steatohepatitis (NASH) triggers different aspects of disorders such as impaired endogenous lipid metabolism, hypercholesterolemia, oxidative stress, and neurotoxicity. In this study, we examined the mechanism by which NASH induces neurotoxicity and the protective effect of berberine against both NASH and its associated neurotoxicity. NASH induced rats showed significant impairments in lipid metabolism with increased serum triglycerides, cholesterol, and low-density lipoprotein (LDL). The NASH induced group also demonstrated a significant oxidative stress which is characterized by increased TBARs level and decreased antioxidant capacity such as GSH and SOD levels. Moreover, the NASH induction was associated with inflammation which was demonstrated by increased TNFα and nitric oxide levels. Hyperglycemia and hyperinsulinemia were observed in the NASH induced group. Also, our results showed a significant increase in the expression of the acetylcholine esterase (AChE) and amyloid beta precursor protein (AβPP). These changes were significantly correlated with decreased insulin degrading enzyme (IDE) and beta-amyloid40 (Aβ 40) and increased beta-amyloid42 (Aβ 42) in the hippocampal region. Daily administration of berberine (50 mg/kg) for three weeks ameliorated oxidative stress, inflammation, hyperlipidemia, hyperglycemia, hyperinsulinemia, and the observed neurotoxicity.

Metabolism of Xenobiotic Carboxylic Acids: Focus on Coenzyme A Conjugation, Reactivity, and Interference with Lipid Metabolism

While xenobiotic carboxylic acids (XCAs) have been studied extensively with respect to their enzymatic conversion to potentially reactive acyl glucuronides with implications to drug induced hepatotoxicity, the formation of xenobiotic-S-acyl-CoA thioesters (xenobiotic-CoAs) have been much less studied in spite of data indicating that such conjugates may be equally or more reactive than the corresponding acyl glucuronides. This review addresses enzymes and cell organelles involved in the formation of xenobiotic-CoAs, the reactivity of such conjugates toward biological macromolecules, and in vitro and in vivo methodology to assess consequences of such reactivity. Further, the propensity of xenobiotic-CoAs to interfere with endogenous lipid metabolism, e.g., inhibition of β-oxidation or depletion of the CoA or carnitine pools, adds to the complexity of the potential contribution of XCAs to hepatotoxicity by a number of mechanisms in addition to those in common with the corresponding acyl glucuronides. On the basis of our review of the literature on xenobiotic-CoA conjugates, there appear to be a number of gaps in our understanding of the bioactivation of XCA both with respect to the mechanisms involved and the experimental approaches to distinguish between the role of acyl glucuronides and xenobiotic-CoA conjugates. These aspects are focused upon and described in detail in this review.

Utilization of endogenous fatty acid stores for energy production in bovine preimplantation embryos

Although current embryo culture media are based on carbohydrate metabolism of embryos, little is known about metabolism of endogenous lipids. L-carnitine is a β-oxidation cofactor absent in most culture media. The objective was to investigate the influence of L-carnitine supplementation on bovine embryo development. Abattoir-derived bovine cumulus oocyte complexes were cultured and fertilized. Post-fertilization, presumptive zygotes were transferred into a basic cleavage medium ± carbohydrates (glucose, lactate and pyruvate) ± 5 mm L-carnitine and cultured for 4 days in vitro. In the absence of carbohydrates during culture, embryos arrested at the 2- and 4-cell stages. Remarkably, +L-carnitine increased development to the morula stage compared to +carbohydrates alone (P < 0.001). The beneficial effects of L-carnitine were further demonstrated by inclusion of carbohydrates, with 14-fold more embryos reaching the morula stage after culture in the +carbohydrates +L-carnitine group compared to the +carbohydrates group (P < 0.05). Whereas there was a trend for +L-carnitine to increase ATP (P = 0.09), ADP levels were higher and ATP: ADP ratio were 1.9-fold lower (main effect, P < 0.05) compared to embryos cultured in –L-carnitine. Therefore, we inferred that +L-carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. In conclusion, L-carnitine supplementation supported precompaction embryo development and there was an additive effect of +L-carnitine +carbohydrates on early embryo development, most likely through increased β-oxidation within embryos.

Lipids in Alzheimer’s disease and their potential for therapy

Alzheimer’s disease pathology is a multifactorial, long-lasting, irreversible process within the CNS resulting in neuronal death. This process starts years prior to the appearance of the first clinical symptoms, such as cognitive decline and loss of short-term memory. Pathological processes mainly include accumulation of neurotoxic amyloid-b in plaques and neurofibrillary tangles containing hyperphosphorylated tau protein. Disorders in endogenous lipid metabolism have been associated with the development and progress of Alzheimer’s disease pathology. Here, we present the recent advances in our understanding of the coherency between the proteinaceous initiators, amyloid-b and tau, and phospholipids, sphingolipids, cholesterol and oxysterols as representatives of membrane lipids within the CNS. Furthermore, new strategies for the overall interruption of these harmful interactions are discussed.

92 UTILISATION OF ENDOGENOUS FATTY ACID STORES FOR ENERGY PRODUCTION IN BOVINE PRE-IMPLANTATION EMBRYOS

Current embryo culture media are based on the carbohydrate metabolism of embryos. However, little is known about the metabolism of endogenous lipids. This is surprising given the high intracellular lipid densities of embryos of some species and the potential for ATP production via β-oxidation. L-carnitine is a β-oxidation co-factor that is absent in most culture media. The aim of this study is to investigate the influence of carnitine supplementation ± carbohydrates on bovine embryo development. Abattoir-derived cattle cumulus–oocyte complexes were cultured and fertilised (Sutton-McDowall et al. 2006 Biol. Reprod. 74, 881–888). Post-fertilisation (24 h), presumptive zygotes were transferred into an amino acid-free cleavage media ± carbohydrates (glucose, lactate and pyruvate) ±5 mM carnitine and cultured for 4 days. The absence of carbohydrates during culture resulted in embryos arresting at the 2- and 4-cell stages. Remarkably, +carnitine significantly increased development to the morula stage compared with +carbohydrates alone (20.4 ± 3% vs 4.7 ± 2.5% morula development; P &lt; 0.001). The combination of carbohydrates and carnitine supplementation further improved embryo development, with 14-fold more embryos reaching the morula stage after culture in the +carbohydrates +carnitine group compared with the +carbohydrates group (+carbohydrates = 3.1 ± 1.9 vs +carbohydrates +carnitine = 43.8 ± 9.1% morula development; P &lt; 0.05). The beneficial effects of carnitine supplementation on embryo development were reversed when embryos were cultured in presence of etomoxir, a non-reversible inhibitor of the rate-limiting enzyme of β-oxidation (development to 8-cell stage; +carnitine = 33.9 ± 8% vs +carnitine +etomoxir = 19.2 ± 4.9%; P &lt; 0.05). Intracellular lipid content of embryos +carnitine was determined by culturing presumptive zygotes in media -carbohydrates ± carnitine for 24 h. Lipid content of embryos was determined by measuring BODIPY 493/503 dye fluorescence. Carnitine supplementation reduced fluorescence intensity 1.8-fold (P &lt; 0.001). Adenosine triphosphate and ATP:ADP levels were measured in embryos after 24 h of culture ± carbohydrates ± carnitine. While there was a trend for +carnitine to increase ATP levels (P = 0.09), ADP levels were higher and ATP:ADP ratio were 1.9-fold lower (main effect, P &lt; 0.05) compared with embryos cultured in –carnitine. This indicates +carnitine embryos were more metabolically active, with higher rates of ATP-ADP conversion. We have shown carnitine supplementation supports pre-compaction embryo development and there is an additive effect of +carnitine +carbohydrate on early embryo development. This is most likely through increased β-oxidation levels within embryos. Current disparities between in vivo and in vitro embryo production, in particular increased lipid content (Romek et al. 2010 Theriogenology 74, 265–276) and decreased developmental potential of in vitro-produced embryos, may be an artefact resulting from limited lipid oxidation in vitro.

Autoreactive natural killer T cells: promoting immune protection and immune tolerance through varied interactions with myeloid antigen‐presenting cells

Natural killer T (NKT) cells are innate T lymphocytes that are restricted by CD1d antigen-presenting molecules and recognize lipids and glycolipids as antigens. NKT cells have attracted attention for their potent immunoregulatory effects. Like other types of regulatory lymphocytes, a high proportion of NKT cells appear to be autoreactive to self antigens. Thus, as myeloid antigen-presenting cells (APCs) such as monocytes, dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) constitutively express CD1d, NKT cells are able to interact with these APCs not only during times of immune activation but also in immunologically quiescent periods. The interactions of NKT cells with myeloid APCs can have either pro-inflammatory or tolerizing outcomes, and a central question is how the ensuing response is determined. Here we bring together published results from a variety of model systems to highlight three critical factors that influence the outcome of the NKT-APC interaction: (i) the strength of the antigenic signal delivered to the NKT cell, as determined by antigen abundance and/or T-cell receptor (TCR) affinity; (ii) the presence or absence of cytokines that costimulate NKT cells [e.g. interleukin (IL)-12, IL-18 and interferon (IFN)-alpha]; (iii) APC intrinsic factors such as differentiation state (e.g. monocyte versus DC) and Toll-like receptor (TLR) stimulation. Together with recent findings that demonstrate new links between NKT cell activation and endogenous lipid metabolism, these results outline a picture in which the functions of NKT cells are closely attuned to the existing biological context. Thus, NKT cells may actively promote tolerance until a critical level of danger signals arises, at which point they switch to activating pro-inflammatory immune responses.

On Mechanism of Quorum Sensing in Candida albicans by 3(R)-Hydroxy-Tetradecaenoic Acid

Quorum sensing (QS) enables microorganisms to monitor their own density of population, and also their pathogenicity by intracellular signals, and synchronizing their specialized gene system in a particular cell density. QS system has been shown in Candida sp. as switching mechanism between successive phases in Candida cell morphology. The lag phase that occurs due to QS is commonly attributed to auto-stimulatory compounds, such as farnesol and farnesoic acid, which are released in the medium. The aim of this manuscript is to demonstrate the involvement of 3(R)-HTDE, a metabolite of linoleic acid, in the QS mechanism of Candida albicans. We show that 3(R)-HTDE, a β-oxidation metabolite of endogenously present linoleic acid, accelerates cell morphogenesis in C. albicans, with alteration of gene expressions necessary for hyphal formation at right density of population utilizing aerobic pathway of endogenous lipid metabolism. We also explore the mechanistic underpinnings of the process where we are able to show that alteration of gene expressions are necessary for hyphal formation at the right population density which is achieved by the proper utilization of an aerobic pathway of endogenous lipid metabolism. In addition, we showed how this mediates biofilm formation itself, and the understanding of these mechanisms can be crucial in designing successful interventional strategies to combat Candida related infections.

Regulation of rat carboxylesterase expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): a dose-dependent decrease in mRNA levels but a biphasic change in protein levels and activity.

Carboxylesterases play an important role in the metabolism of endogenous lipids and foreign compounds; therefore, xenobiotic regulation of carboxylesterase gene expression has both physiological and pharmacological significance. We previously reported that beta-naphthoflavone and 3-methylcholanthrene, two potent inducers for cytochrome P4501A enzymes, had opposing effects on the expression of hydrolase S, a secretory carboxylesterase. Beta-naphthoflavone caused suppression, whereas 3-methylcholanthrene caused induction of the expression of this enzyme. The aim of the present study was to determine the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), another prototypical cytochrome P4501A inducer, on the expression of this and several other rat carboxylesterases (hydrolases A, B, and C) in liver and extrahepatic tissues. Rats received TCDD treatment at nonlethal (<or=10 microg/kg), sublethal (30 microg/kg), or lethal doses (90 microg/kg). The overall hydrolytic activity in the liver microsomes toward both 1-naphthyl- and para-nitrophenylacetate was markedly increased in the nonlethal dosage groups, but markedly decreased in the lethal dosage group. Consistent with such a biphasic dose-response relationship, the levels of enzyme proteins exhibited an initial increase, followed by a decrease in response to nonlethal and lethal doses, respectively. In contrast, treatment with TCDD caused a dose-dependent decrease on the levels of mRNA encoding these enzymes. All liver carboxylesterases showed a similar pattern of change on activity, protein, and mRNA levels, suggesting that TCDD coregulates the expression of these genes. In the extrahepatic tissues, a similar biphasic change was observed in activity and in protein and mRNA levels. In both liver and kidney, the expression of cytochrome P4501A1 (CYP1A1) was significantly induced in a dose-dependent manner. TCDD is known to upregulate the expression of CYP1A1 gene through the aryl hydrocarbon receptor (AhR). The differential effects on the expression of liver carboxylesterases and CYP1A1 suggest that TCDD regulates the expression of hydrolytic enzymes via a mechanism(s) other than the AhR-mediated transcription activation, as observed in the CYP1A1 regulation. The different patterns of change on protein and mRNA levels in the nonlethal dosage groups suggest that TCDD regulates the expression of hepatic carboxylesterases by acting on both transcription and translation.

Effects of prior moderate exercise on exogenous and endogenous lipid metabolism and plasma factor VII activity

Effects of prior moderate exercise on exogenous and endogenous lipid metabolism and plasma factor VII activity

Effects of prior moderate exercise on exogenous and endogenous lipid metabolism and plasma factor VII activity

Moderate exercise reduces postprandial triacylglycerol concentrations, which are a risk marker for coronary heart disease. The present study sought to determine the qualitative nature of exercise-induced changes in lipid metabolism and their association (if any) with changes in factor VII activation. Eleven normotriglyceridaemic men, aged 51.7+/-6.1 years (mean+/-S.D.), participated in two oral fat tolerance tests after different pre-conditions: control (no exercise), and exercise (90 min of brisk walking the day before). Venous blood samples were obtained in the fasted state and for 8 h after ingestion of a high-fat meal (1.32 g of fat, 1.36 g of carbohydrate, 0.30 g of protein and 10 mg of [1,1,1-(13)C] tripalmitin x kg(-1) body mass). Prior exercise reduced postprandial plasma triacylglycerol concentrations by 25+/-3% (mean+/-S.E.M.), with lower concentrations in the Svedberg flotation rate (Sf) 20--400 (very-low-density lipoprotein) fraction accounting for 79+/-10% of this reduction. There was no effect on plasma factor VII coagulant activity or on the concentration of the active form of factor VIIa. Prior exercise increased postprandial serum 3-hydroxybutyrate and plasma fatty acid concentrations, decreased serum postprandial insulin concentrations and increased exogenous (8 h (13)C breath excretion of 15.1+/-0.9% of ingested dose compared with 11.9+/-0.8%; P=0.00001) and endogenous postprandial fat oxidation. These data raise the possibility that reduced hepatic secretion of very-low-density lipoprotein plays a role in the attenuation of plasma triacylglycerol concentrations seen after exercise, although it is possible that increased triacylglycerol clearance also contributes to this effect.

Effects of Steric Bulk and Conformational Rigidity on Fatty Acid Omega Hydroxylation by a Cytochrome P450 4A1 Fusion Protein

The action of cytochrome P450 4A1 (CYP4A1) on fatty acid substrates is characterized by a pronounced regioselectivity for ω-hydroxylation. To elucidate the chemical basis of this specificity we probed the active site of a CYP4A1 fusion protein (f4A1) with bulky and/or rigid analogs of lauric acid, the optimum natural substrate for f4A1 and CYP4A1. f4A1 efficiently ω-hydroxylates lauric acid, epoxidizes 11-dodecenoic acid, and oxidizes 11-dodecynoic acid to 1,12-dodecanedioic acid. Medium length fatty acids having ω-terminal groups as large ast-butyl orm-tolyloxy bind tightly to f4A1 as Type I ligands and are efficiently hydroxylated on their methyl termini. ω-Phenylnonanoic acid also induces a Type I binding spectrum (Ks= 0.77 μm) but fails to undergo hydroxylation and strongly inhibits lauric acid hydroxylation by f4A1. Slightly shorter acids such as ω-phenyloctanoic acid, naproxen, and ibuprofen also strongly inhibit lauric acid hydroxylation but do not induce a Type I spectrum and do not undergo hydroxylation. Like 10-methoxydecanoic acid, the rigid and rod-like 4′-methoxy-4-biphenylcarboxylic acid is O-demethylated by f4A1, which also ω-hydroxylatesm- andp-heptyloxybenzoic acids but notm- orp-amyloxyhydrocinnamic acids. The histamine antagonist cimetidine and the peroxisome proliferator perfluorodecanoic acid are both potent inhibitors of f4A1. Thus the active site of f4A1 is quite tolerant of steric bulk and rigidity around the heme region and the polar group recognition site, but perhaps less so in the midchain region. Although CYP4A enzymes are not usually regarded as “drug metabolizing P450s,” the fact that commonly used therapeutic agents strongly inhibit lauric acid ω-hydroxylation by f4A1 as well as liver microsomes from clofibrate-induded rats suggests these and related agents could potentially interfere with the contribution of CYP4A enzymes to the metabolism of endogenous lipids.

Role of hepatic and renal cytochrome p-450 iva1 in the metabolism of lipid substrates

The role of clofibrate-inducible cytochrome P-450 IVA1 in the metabolism of endogenous lipids in both rat liver and kidney microsomal fractions has been investigated. 20(ω)-hydroxyarachidonic acid has been identified as a major metabolite after incubation with both tissue fractions and the structure confirmed by mass spectrometry. The arachidonic acid 20-hydroxylase activity is inducible by clofibrate in both liver and kidney, indicating that cytochrome P-450 IVA1 is probably the enzyme responsible for this activity. In addition, the kidney exhibited higher rates of arachidonate 20-hydroxylase activity than the liver (in both control and induced states). Although leukotriene B 4 was also hydroxylated in the 20-position in both liver and kidney, clofibrate induction resulted in a decrease (approximately 50%) in hydroxylase activity. In addition, the absolute level of leukotriene B 4 20-hydroxylase activity in both tissue homogenates and by purified cytochrome P-450 IVA1 in a reconstituted system, was 2–3 orders of magnitude lower than the corresponding activity for lauric acid and arachidonic acid as substrates, indicating that the leukotriene was not the preferred substrate for this enzyme. Computer modelling of the conformational geometries of the above three potential cytochrome P-450 IVA1 substrates have shown that both lauric and arachidonic acids adopt a compact, ‘hairpin’ structure that are almost superimposed on each other, thereby rationalizing why they are relatively good substrates for this isoenzyme. By contrast, leukotriene B 4 adopts a more bulky geometry than the two fatty acids, thereby providing a coherent structural reason why it is a poorer substrate for the cytochrome P-450 IVA1 isoenzyme.