Gene Expression Of Carnitine Palmitoyltransferase Research Articles

Effects of three beta adrenergic receptor agonists on growth performance, blood biochemical parameters, fatty acids composition and carnitine palmitoyltransferase I gene expression of rainbow trout, Oncorhynchus mykiss

Different beta 1 and 2 adrenergic receptor agonists may be various biological and physiological effects on fish species. An experiment was designed to study the effects of feeding ractopamine, terbutaline and metaproterenol; as beta1, beta2 and less selective beta 2 adrenergic receptor agonists, respectively; on body weight gain, feed conversion ratio, concentration of biochemical parameters in the serum, gene expression of carnitine palmitoyltransferase I in liver and meat and also, fatty acids profile of filet meat of the rainbow trout. One hundred ninety two of juvenile rainbow trout were randomly assigned to 16 fiber glass tanks and fed one of four dietary treatments containing control (0), ractopamine, terbutaline and metaproterenol at levels of 10 ppm in diet for 8-week feeding trials. It was found that metaproterenol and ractopamine improved final body weight, body weight gain and feed conversion ratio of fish. The serum concentrations of phosphorus and albumin were also significantly increased by all beta adrenergic agonists and ractopamine reduced triglyceride level (p<0.05). The fatty acids level of fish filet was significantly increased by the dietary supplement of various beta adrenergic agonists (p<0.05) but ractopamine had a greater effect. The gene expression of carnitine palmitoyltransferase I in liver was significantly (P<0.05) increased by all beta adrenergic agonists. The present study showed that various beta1 and beta 2 adrenergic receptor agonists had same physiological effects on rainbow trout but it seems ractopamine, as a beta 1 adrenergic receptor, had more potential on fatty acid metabolism and growth response of rainbow trout.

Molecular characterization and tissue distribution of carnitine palmitoyltransferases in Chinese mitten crab Eriocheir sinensis and the effect of dietary fish oil replacement on their expression in the hepatopancreas.

The carnitine palmitoyltransferase (CPT) family includes CPT 1 and CPT 2 that transport long-chain fatty acids into the mitochondrial compartment for β-oxidation. In this study, three isoforms (CPT 1α, CPT 1β and CPT 2) of the CPT family were cloned from Chinese mitten crab (Eriocheir sinensis) and their complete coding sequences (CDS) were obtained. Sequence analysis revealed deduced amino acid sequences of 915, 775 and 683 amino acids, respectively. Gene expression analysis revealed a broad tissue distribution for all three isoforms, with high CPT 1α and CPT 2 mRNA levels in the hepatopancreas of males and females. In males, CPT 1β was highly expressed in gill, heart, brain ganglia and muscle, while in females, CPT 1β-mRNA levels were relatively high in muscle, hepatopancreas and ovary tissue. The effects of dietary fish oil replacement on the expression of the three CPT isoforms in the hepatopancreas during gonadal development were investigated using five experimental diets formulated with replacement of 0, 25, 50, 75 and 100% fish oil by 1:1 rapeseed oil: soybean oil. The results showed that Diets 2# and 5# yielded higher CPT 1α and CPT 2 mRNA expression in males (P < 0.05), while in females, expression of all three CPT isoforms increased then declined in the hepatopancreas with increasing dietary fish oil replacement. The observed changes in CPT gene expression varied in different isoforms and gender, suggesting the three CPT genes might play different roles in fatty acid β-oxidation in E. sinensis.

Antihyperlipidemic effect of a Rhamnus alaternus leaf extract in Triton-induced hyperlipidemic rats and human HepG2 cells

The Mediterranean buckthorn, Rhamnus alaternus L., is a plant used in traditional medicine in Mediterranean countries. We aimed at characterizing its phenolic compounds and explore potential antihyperlipidemic activity of this plant. The profile of phenolic compounds in R. alaternus leaf crude methanolic extract (CME) and its liquid-liquid extraction-derived fractions were analyzed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS2). Effects of CME on: circulating lipids in rats with Triton WR-1339-induced hyperlipidemia, intracellular lipid accumulation and expression of genes of fatty acid metabolism in human hepatoma HepG2 cells, and adipogenesis in the 3T3-L1 murine adipocyte cell model were assessed. The HPLC/ESI-MS2 analytical profile revealed a total of fifteen compounds, of which eleven were identified. Oral CME administration decreased blood levels of cholesterol and triacylglycerols in hyperlipidemic rats (by 60% and 70%, respectively, at 200 mg CME/kg). In HepG2 cells, CME exposure dose-dependently decreased intracellular lipids and up-regulated gene expression of carnitine palmitoyltransferase 1 involved in fatty acid oxidation. In the 3T3-L1 model, CME favored preadipocyte proliferation and adipogenesis, pointing to positive effects on adipose tissue expandability. These results suggest novel uses of R. alaternus by showing that its leaves are rich in flavonoids and flavonoid derivatives with an antihyperlipidemic effect in vivo and in hepatic cells.

Effect of dietary copper addition on lipid metabolism in rabbits

ABSTRACTThe present study was conducted to investigate the effect of copper supplementation on lipid metabolism in rabbits. Our study showed dietary copper addition (5-45 mg/kg) increased body mass gain, but decreased fat and liver weights compared with those in the control group (P < 0.05). Copper (45 mg/kg) addition significantly increased the skeletal muscle weight, but inhibited cytoplasmic lipid accumulation in liver, skeletal muscle and adipose tissue (P < 0.05). Compared with the control group, dietary copper addition (45 mg/kg) significantly increased plasma triglyceride levels but decreased very low density lipoprotein levels (P < 0.05). Copper treatment significantly increased gene expression of carnitine palmitoyltransferase (CPT) 1, CPT2 and peroxisome proliferator-activated receptor (PPAR) a in liver (P < 0.05). In skeletal muscle, CPT1, CPT2, fatty acid transport protein, fatty acid-binding protein, and PPARa mRNA as well as phosphorylated AMP-activated protein kinase (AMPK) levels were significantly up-regulated by copper treatment (P < 0.05). Rabbits receiving copper supplementation had higher CPT1, CPT2, PPARa and hormone-sensitive lipase mRNA levels in adipose tissue (P < 0.05). In conclusion, copper promoted skeletal muscle growth and reduced fat accretion. PPARa signaling in liver, skeletal muscle and adipose tissues and AMPK signaling in skeletal muscle tissue were involved in the regulation of lipid metabolism by copper.

Inhibition of Gene Expression of Carnitine Palmitoyltransferase I and Heart Fatty Acid Binding Protein in Cyclophosphamide and Ifosfamide-Induced Acute Cardiotoxic Rat Models

This study investigated whether cyclophosphamide (CP) and ifosfamide (IFO) therapy alters the expression of the key genes engaged in long-chain fatty acid (LCFA) oxidation outside rat heart mitochondria, and if so, whether these alterations should be viewed as a mechanism during CP- and IFO-induced cardiotoxicity. Adult male Wistar albino rats were assigned to one of the six treatment groups: Rats in group 1 (control) and group 2 (L-carnitine) were injected intraperitoneal (i.p.) with normal saline and L-carnitine (200mg/kg/day), respectively, for 10 successive days. Animals in group 3 (CP group) were injected i.p. with normal saline for 5days before and 5days after a single dose of CP (200mg/kg, i.p.). Rats in group 4 (IFO group) received normal saline for 5 successive days followed by IFO (50mg/kg/day, i.p.) for 5 successive days. Rats in group 5 (CP-carnitine supplemented) were given the same doses of L-carnitine as group 2 for 5days before and 5days after a single dose of CP as group 3. Rats in group 6 (IFO-carnitine supplemented) were given the same doses of L-carnitine as group 2 for 5days before and 5days concomitant with IFO as group 4. Immediately, after the last dose of the treatment protocol, blood samples were withdrawn and animals were killed for biochemical, histopathological and gene expression studies. Treatment with CP and IFO significantly decreased expression of heart fatty acid binding protein (H-FABP) and carnitine palmitoyltransferase I (CPT I) genes in cardiac tissues. Moreover, CP but not IFO significantly increased acetyl-CoA carboxylase2 mRNA expression. Conversely, IFO but not CP significantly decreased mRNA expression of malonyl-CoA decarboxylase. Both CP and IFO significantly increased serum lactate dehydrogenase, creatine kinase isoenzyme MB and malonyl-CoA content and histopathological lesions in cardiac tissues. Interestingly, carnitine supplementation completely reversed all the biochemical, histopathological and gene expression changes induced by CP and IFO to the control values, except CPT I mRNA, and protein expression remained inhibited by IFO. Data from the current study suggest, for the first time, that (1) CP and IFO therapy is associated with the inhibition of the expression of H-FABP and CPT I genes in cardiac tissues with the consequent inhibition of mitochondrial transport and oxidation of LCFA. (2) The progressive increase in cardiotoxicity enzymatic indices and the decrease in H-FABP and CPT I expression may point to the possible contribution of these genes to CP- and IFO-induced cardiotoxicity.

Capsaicin attenuates palmitate-induced expression of macrophage inflammatory protein 1 and interleukin 8 by increasing palmitate oxidation and reducing c-Jun activation in THP-1 (human acute monocytic leukemia cell) cells

Capsaicin, a spicy component of hot peppers, has been shown to improve inflammatory disease and obesity. In this study, we tested the hypothesis that the anti-inflammatory activity of capsaicin can be used to improve free fatty acid (FFA)-induced inflammation by reducing gene expression of macrophage inflammatory protein 1 (MIP-1) and interleukin 8 (IL-8) in THP-1 (human acute monocytic leukemia cell) macrophages. To investigate whether capsaicin ameliorates palmitate-induced MIP-1 and IL-8 gene expressions, we treated THP-1 cells with palmitate in the presence or absence of capsaicin and measured MIP-1 and IL-8 by real-time polymerase chain reaction. To elucidate the mechanism by which capsaicin effects on palmitate-induced MIP-1 and IL-8 gene expressions, we performed immunoblotting with stress kinase-related antibodies and measured palmitate oxidation and palmitate oxidation-related gene expression. Palmitate and stearate but not the unsaturated FFA oleate significantly increased MIP-1 and IL-8 expressions in THP-1 macrophages. Treatment with capsaicin or FFA oxidation stimulators inhibited palmitate-induced MIP-1 and IL-8 expressions in THP-1 macrophages. Capsaicin increased the gene expression of carnitine palmitoyltransferase 1 and the β-oxidation of palmitate. Furthermore, capsaicin significantly reduced palmitate-stimulated activation of c-Jun N-terminal kinase, c-Jun, and p38. Our data suggest that the attenuation of palmitate-induced MIP-1 and IL-8 gene expressions by capsaicin is associated with reduced activation of c-Jun N-terminal kinase, c-Jun, and p38 and preserved β-oxidation activity.

Dissimilar Properties of Vaccenic Versus Elaidic Acid in β‐Oxidation Activities and Gene Regulation in Rat Liver Cells

Vaccenic acid (trans-11-C(18:1)) chemically resembles elaidic acid (trans-9-C(18:1)) which is assumed to increase the risk of cardiovascular diseases, and thus could exert similar effects. Possible different oxidation rates of vaccenic versus elaidic acid were checked in muscles and liver, and through related gene expression in normal rat liver cells. In hepatic mitochondria, carnitine palmitoyltransferase (CPT) I exhibited comparable activity rates with both trans-isomers. CPT II activity was 30% greater (P < 0.05) with vaccenic than with elaidic acid as nonesterified fatty acids (NEFAs) or acyl-CoAs. Activity of the first beta-oxidation step was similar between the isomers in all the tissue slices and liver extracts assayed. Respiration rates were comparable with both trans-isomers as NEFAs in various liver extracts, but were 30% greater (P < 0.05) with vaccenoyl-CoA than with elaidoyl-CoA in liver mitochondria. Vaccenic acid was oxidised 25% more (P < 0.05) by liver peroxisomes than elaidic acid. In hepatocytes cultured with trans- and corresponding cis-C(18:1) isomers, gene expression of CPT I, hydroxyacyl-CoA dehydrogenase and hydroxymethylglutaryl-CoA synthase was at least 100% increased (P < 0.05), but was unchanged with vaccenic acid, relative to controls. In conclusion, the position and geometry of the double bonds in acyl chains are suggested to confer on vaccenic and elaidic acid specific biochemical properties that might differently affect their fates in tissues.

Effects of dietary fatty acid composition on the regulation of carnitine palmitoyltransferase (CPT) I in rainbow trout (Oncorhynchus mykiss)

Dietary fatty acid composition, particularly polyunsaturated fatty acids, can affect both genetic and non-genetic regulatory mechanisms of carnitine palmitoyltransferase (CPT) I, the main regulatory enzyme of mitochondrial fatty acid oxidation. We aimed to determine how these regulatory mechanisms were affected by changes in the fatty acid composition of the diet in fish. Specifically, we fed rainbow trout (Oncorhynchus mykiss) either a high polyunsaturated fatty acid (PUFA) diet, a high saturated fatty acid (SFA) diet or a mixed fatty acid control (CTL) diet for 8 weeks to determine if modifications of the dietary fatty acids would affect 1) the genetic expression of CPT I and its transcription factor peroxisome proliferator activated receptor (PPAR), 2) the mitochondrial membrane composition and if these modifications would affect CPT I sensitivity to malonyl-CoA, and 3) levels of malonyl-CoA in the tissues. We found that fish fed the high PUFA diet significantly increased CPT I mRNA expression in red muscle, liver and adipose tissue, while PPAR alpha and beta expressions were variable across tissues. Few significant changes were observed in the mitochondrial membrane composition with the exception of DHA in the red muscle. There were no significant differences in CPT I sensitivity to malonyl-CoA or the malonyl-CoA content of the tissues with either experimental diet. Our present data suggest that changes in gene expression of CPT I and PPARs is the main regulatory mechanism controlling CPT I function in fish using our experimental diet.

Sesamin Increases Alpha‐Linolenic Acid Conversion to Docosahexaenoic Acid in Atlantic Salmon (Salmo salar L.) Hepatocytes: Role of Altered Gene Expression

In vitro cultivated Atlantic salmon (Salmo salar L.), hepatocytes were incubated without or with a mixture of sesamin and episesamin in order to test for possible effects on lipid metabolism. Sesamin/episesamin exposure (0.05 mM, final concentration) led to increased elongation and desaturation of (14)C 18:3n-3 to docosahexaenoic acid ((14)C 22:6n-3, DHA, P < 0.01) and down regulated gene expression of Delta6 and Delta5 desaturases compared to control treatment. Sesamin/episesamin further increased the hepatocytes capacity for fatty acid beta-oxidation of (14)C 18:3n-3 (P < 0.01) to the (14)C acid soluble products, acetate, malate and oxaloacetate, in agreement with an increased gene expression of carnitine palmitoyltransferase I. Also the gene expression of cluster of differentiation 36 was upregulated and the expression of scavenger receptor type B, peroxisome proliferator-activated receptors alpha and gamma were downregulated. The amount of triacylglycerols secreted by the cells tended to be lower in the sesamin/episesamin incubated hepatocytes than the control cells. This study shows that sesamin has favourable effects on lipid metabolism leading to increased level of DHA, which may be of interest for aquaculture use.

Effects of fenofibrate on gene expression of carnitine palmitoyltransferase 1 in liver and skeletal muscle and its influence on insulin sensitivity

To study the effects of fenofibrate (FF), a peroxisome proliferator activated receptor (PPAR) alpha activator, on the expression of carnitine palmitoyltransferase 1 (CPT-1) mRNA in liver and muscle and its influence on insulin sensitivity. Thirty-two normal 8 week-old male SD rats were randomly divided into 3 groups: normal control group, fed with normal food for 3 weeks (NC group, n = 10), high fat diet group, fed with high fat food (HF group, n = 10), and high fat diet supplemented with FF group, fed with high fat food and given with gastric perfusion of FF (50 mg x kg(-1) x d(-1)) (FF group, n = 12). Fast serum triglyceride (TG) level was tested by automatic biochemical analyzer after 8-10 h fasting. Euglycemic-hyperinsulinemic clamp method was used to calculate the glucose infusion rate (GIR) so as to evaluate the insulin sensitivity. By the end of experiment the rats were killed with their internal organs taken out. The triglyceride (TG) contents of liver and skeletal muscle were measured using Folch method. Real-time PCR was used to detect the mRNA expression. of CPT-1 in the liver and skeletal muscles. As compared with the NC group, the TG levels of serum, liver, and skeletal muscle of the HF group were higher than those of the NC group by 0.45-fold, 2.14-fold, and 10.64-fold respectively. The GIR of the HF group was (6.2 +/- 0.8) mg x kg(-1) x min(-1), significantly lower than that of the NC group (15.8 +/- 2.1) mg x kg(-1) x min(-1), (P < 0.01), and that of the P < 0.01. The CPT-1 mRNA expression in liver of the HF group was not significantly different from that of the NC group (P > 0.05); the expression of CPT-1 mRNA in skeletal muscle of the HF group was lower than that of the NC group by 71% (P < 0.01). The CPT-1 mRNA expression in liver and skeletal muscle of the FF group were significantly higher than those of the HF group by 1.00 and 1.05 times respectively (both P < 05). The GIR was negatively correlated with the levels in the liver (r = -0.87, P < 0.01) and in the skeletal muscle (r = -0.78, P < 0.01). Fenofibrate promotes the oxygenation of fatty acids by up-regulating the CPT-1 mRNA expression in the liver and skeletal muscles, thus improving the insulin sensitivity.

Cytoprotective properties of α-tocopherol are related to gene regulation in cultured d-galactosamine-treated human hepatocytes

Vitamin E (α-tocopherol) has demonstrated antioxidant activity and gene-regulatory properties. d-Galactosamine (D-GalN)-induced cell death is mediated by nitric oxide in hepatocytes, and it is associated with hepatic steatosis. The beneficial properties of α-tocopherol and their relation to oxidative stress and gene regulation were assessed in D-GalN-induced cell death. Hepatocytes were isolated from human liver resections by a collagenase perfusion technique. α-Tocopherol (50 μM) was administered at the advanced stages (10 h) of D-GalN-induced cell death in cultured hepatocytes. Cell death, oxidative stress, α-tocopherol metabolism, and NF-κB-, pregnane X receptor (PXR)-, and peroxisome proliferator-activated receptor (PPAR-α)-associated gene regulation were estimated in the hepatocytes. D-GalN increased cell death and α-tocopherol metabolism. α-Tocopherol exerted a moderate beneficial effect against apoptosis and necrosis induced by D-GalN. Induction (rifampicin) or inhibition (ketoconazole) of α-tocopherol metabolism and overexpression of PXR showed that the increase in PXR-related CYP3A4 expression caused by α-tocopherol enhanced cell death in hepatocytes. Nevertheless, the reduction in NF-κB activation and inducible nitric oxide synthase expression and the enhancement of PPAR-α and carnitine palmitoyl transferase gene expression by α-tocopherol may be relevant for cell survival. In conclusion, the cytoprotective properties of α-tocopherol are mostly related to gene regulation rather than to antioxidant activity in toxin-induced cell death in hepatocytes.

Sesamin, a sesame lignan, is a potent inducer of hepatic fatty acid oxidation in the rat.

The effects of sesamin, one of the most abundant lignans in sesame seed, on hepatic fatty acid oxidation were examined in rats that were fed experimental diets containing various amounts (0%, 0.1%, 0.2%, and 0.5%) of sesamin (a 1:1 mixture of sesamin and episesamin) for 15 days. Dietary sesamin dose-dependently increased both mitochondrial and peroxisomal palmitoyl-coenzyme A (CoA) oxidation rates. Mitochondrial activity almost doubled in rats on the 0.5% sesamin diet. Peroxisomal activity increased more than 10-fold in rats fed a 0.5% sesamin diet in relation to rats on the sesamin-free diet. Dietary sesamin greatly increased the hepatic activity of fatty acid oxidation enzymes, including carnitine palmitoyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase, 3-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and 3-ketoacyl-CoA thiolase. Dietary sesamin also increased the activity of 2,4-dienoyl-CoA reductase and delta3,delta2-enoyl-CoA isomerase, enzymes involved in the auxiliary pathway for beta-oxidation of unsaturated fatty acids dose-dependently. Examination of hepatic mRNA levels using specific cDNA probes showed a sesamin-induced increase in the gene expression of mitochondrial and peroxisomal fatty acid oxidation enzymes. Among these various enzymes, peroxisomal acyl-CoA oxidase and bifunctional enzyme gene expression were affected most by dietary sesamin (15- and 50-fold increase by the 0.5% dietary level). Sesamin-induced alterations in the activity and gene expression of carnitine palmitoyltransferase I and acyl-CoA oxidase were in parallel with changes in the mitochondrial and peroxisomal palmitoyl-CoA oxidation rate, respectively. In contrast, dietary sesamin decreased the hepatic activity and mRNA abundance of fatty acid synthase and pyruvate kinase, the lipogenic enzymes. However, this lignan increased the activity and gene expression of malic enzyme, another lipogenic enzyme. An alteration in hepatic fatty acid metabolism may therefore account for the serum lipid-lowering effect of sesamin in the rat.

The Effect of Surgical Treatment Following Peritoneal Sepsis on Hepatic Gene Expression

Peritoneal sepsis results in downregulation of the gene that codes for the hepatic mitochondrial enzyme carnitine palmitoyltransferase (CPT). The inhibition of hepatic CPT transcription by sepsis is thought to be mediated, in part, by increased expression of the leucine-zipper DNA transcription factor c-fos. In a cecal ligation and puncture (CLP) model, we examined the temporal effect of surgical treatment (cecal excision) on sepsis-induced inhibition of CPT gene expression. We investigated the hypothesis that Fos protein level will inversely correlate with the regulation of CPT gene expression. Specifically, we studied hepatic Fos nucleoprotein accumulation and CPT gene expression as measured by total mitochondrial CPT activity, CPT protein, and CPT mRNA. We investigated the following groups: (i) CLP followed by cecal excision 6, 12, or 24 hr following initial insult, (ii) concurrent CLP control group, and (iii) concurrent sham CLP reference group. When measured 48 hr following initial surgical insult, we conclude that: (i) in the absence of surgical treatment, peritoneal contamination results in a decrease in hepatic CPT gene expression and an increase in Fos nucleoprotein accumulation; (ii) surgical treatment at 6 or 12 hr following initial insult prevents the downregulation in hepatic CPT gene expression and does not result in Fos nucleoprotein accumulation; and (iii) surgical treatment at 24 hr following insult did not prevent the downregulation of hepatic CPT gene expression and results in an increase in hepatic Fos nucleoprotein accumulation. These data are consistent with the hypothesis that sepsis-induced regulation of hepatic c-fos gene expression, in part, is responsible for the downregulation of CPT gene expression.

The Regulation of Mitochondrial Fatty Acid Oxidation and Hepatic Gene Expression by Catecholamine

We hypothesized that dopamine or dobutamine may alter hepatic mitochondrial fatty acid oxidation secondary to an effect on hepatic gene expression. We investigated the effect of dopamine or dobutamine on hepatic fat oxidation and gene transcription by studying the enzyme carnitine palmitoyltransferase (CPT), the rate-limiting step in hepatic mitochondrial long-chain fat oxidation. We incubated either H4IIE rat hepatoma cells or rat hepatocytes in primary cell culture with either dopamine (1,0.1, 0.01 μg/ml), dobutamine (1,0.1, 0.01 μg/ml), or vehicle control for 1, 2, 3, or 4 hr. We investigated the effect on (1) CPT mRNA (Northern or dot blotting) and the possible regulatory mechanism by incubating dopamine (0.1 μg/ml) or dobutamine (0.1 μg/ml) with propranolol or phentolamine, (2) CPT translation (CPT [ 35S]methionine incorporation), and (3) hepatic mitochondrial fatty acid oxidation ([1- 14C]palmitate oxidation to acid-soluble products). We conclude that (1) dopamine or dobutamine increases both hepatic CPT mRNA and CPT protein translation, (2) the effect on CPT mRNA is mediated by the β-receptor, (3) the increase in hepatic mitochondrial fat oxidation induced by dopamine or dobutamine may be, in part, secondary to increased CPT transcription and translation, and (4) the significant difference in hepatic fat oxidation induced by dopamine as compared with that by dobutamine is secondary to factors other than transcriptional or translational mechanisms. We speculate that dopamine treatment in the critically ill may increase hepatic mitochondrial fatty acid oxidation and ketogenesis and that this increase in β-oxidation may be, in part, secondary to increased CPT gene expression.