Effects of octylglucoside and triton X-100 on the kinetics and specificity of carnitine palmitoyltransferase

The effects of octylglucoside on the substrate specificity, kinetics and aggregation state of purified carnitine palmitoyltrasferase (CPT) from beef heart mitochondria were investigated and compared to the effects of Triton X-100. Conditions in which CPT can be assayed in the absence of micelles and albumin, thereby eliminating miceller effects on the kinetic parameters, are described. When octylglucoside is substituted for Triton X-100, the specificity of CPT in the forward direction shifts towards the long-chain acyl-CoAs, and large changes in the kinetic constants are observed. The K_0.5 for L-carnitine varied as much as 50-fold, depending on the acyl-CoA and detergent used. At pH 8.0 and 200 μM palmitoyl-CoA, the K_0.5 for L-carnitine is 4.9 mM in 12 mM octylglucoside and 0.2 mM in 0.1% Triton X-100. Octylglucoside enhances the activity of CPT with long-chain acyl-CoA and lowers the K_0.5 for these substrates. At pH 6.0, the K_0.5 for palmitoyl-CoA is 24.2 μM in 0.1% Triton X-100, in contrast to 3.1 μM in 12 mM octylglucoside. Octylglucoside is a competitive inhibitor of CPT with octanoyl-CoA as substrate with a K_i of 15 mM. Nonlinear kinetics for both acyl-CoAs and L-carnitine are observed when the concentration of octylglucoside is reduced to less than half of its critical micellar concentration (cmc). Gel filtration of CPT in octylglucoside below its cmc gives a single protein peak with a molecular mass of ca. 660,000 daltons. These data indicate that the catalytically active form of purified CPT is an aggregate that has quaternary structure and must have a very flexible catalytic site whose affinity for substrate and catalytic efficiency can be altered greatly by changes in environment and experimental conditions.     

Cardiolipin Stabilizes and Increases Catalytic Efficiency of Carnitine Palmitoyltransferase II and Its Variants S113L,P50H,and Y479F

Muscle carnitine palmitoyltransferase II (CPT II) deficiency is associated with various mutations in CPT2 gene. In the present study, the impact of the two CPT II variants P50H and Y479F were characterized in terms of stability and activity in vitro in comparison to wildtype (WT) and the well investigated variant S113L. While the initial enzyme activity of all variants showed wild-type-like behavior, the activity half-lives of the variants at different temperatures were severely reduced. This finding was validated by the investigation of thermostability of the enzymes using nano differential scanning fluorimetry (nanoDSF). Further, it was studied whether the protein stabilizing diphosphatidylglycerol cardiolipin (CL) has an effect on the variants. CL indeed had a positive effect on the stability. This effect was strongest for WT and least pronounced for variant P50H. Additionally, CL improved the catalytic efficiency for CPT II WT and the investigated variants by twofold when carnitine was the varied substrate due to a decrease in K_M. However, there was no influence detected for the variation of substrate palmitoyl-CoA. The functional consequences of the stabilization by CL in vivo remain open.     

Effects of dietary conjugated linoleic acids on hepatic and muscle lipids in hybrid striped bass

Conjugated linoleic acids (CLA) are the focus of numerous studies, yet the effects of these isomers of octadecadienoic acids have not been evaluated in many species of fish. In this study, graded amounts of CLA-0,0.5, 0.75, or 1.0% of the diet—were fed to juvenile hybrid striped bass for 8 wk. Dietary treatments were fed to apparent satiation twice daily to triplicate groups of fish initially weighing 13.4 g/fish. Feed intake and weight gain of fish fed 1.0% CLA were significantly reduced compared to fish fed no CLA. Fish fed 0.5 and 0.75% CLA exhibited reduced feed intake similar to fish fed 1.0% CLA, but had growth rates that were not significantly different from those of fish fed no CLA. Feed efficiency improved significantly in fish as dietary CLA concentrations increased. Total liver lipid concentrations were significantly reduced in fish fed the diets containing CLA compared to those of fish fed the control diet, and intraperitoneal fat ratio was significantly lower in fish fed 1.0% CLA compared to fish fed no CLA. Fish fed dietary CLA exhibited significant increases in hepatosomatic index and moisture content of muscle and carcass. The CLA isomers were detected in liver and muscle of fish fed the diets containing CLA, while a low concentration of one isomer was detected in liver and muscle of fish fed the control diet. Dietary CLA resulted in a significant increase in 18∶2(c-9,c-12) concentration in liver and muscle, but a significant reduction in 18∶1n−7 in these tissues. Furthermore, feeding CLA resulted in a significant increase in the concentration of 20∶5n−3 and 22∶6n−3 in liver, but a reduction of these fatty acids in muscle. This study showed that feeding CLA elevated tissue concentrations of these fatty acid isomers, reduced tissue lipid contents, improved feed efficiency, and altered fatty acid concentrations in liver and muscle of fish.     

Kinetics of Carnitine Palmitoyltransferase I (CPT I) in Chinese sucker (Myxocyprinus asiaticus) Change with its Development

The present study was conducted to evaluate the ontogeny and kinetic of CPT I in several tissues of the Chinese sucker Myxocyprinus asiaticus. To this end, liver, muscle and intestine tissues were examined at five various developmental stages of Chinese sucker: newly-hatched larvae, 68-day-old, 4-month-old, 1- and 2-year-old Chinese sucker, respectively. The total carnitine (TC) content in the liver increased from birth to 1-year-old Chinese sucker and then declined in the 2-year-old Chinese sucker. From the 68-day-old to the 1-year-old Chinese sucker, both free and total concentrations in muscle increased. Both acyl carnitine (AC) and TC concentrations in intestine were very variable at different stages. The ratio of AC to free carnitine (FC) in liver progressively increased from hatching to 4 months, and declined at the age of 1 year, and then increased by 2 years. In muscle, the highest and lowest ratios of AC/FC were observed at 68-days-old and 4 month-old Chinese sucker. The highest proportion of AC/FC in intestine was also observed at 68-day-old Chinese sucker. All the Chinese sucker larvae at hatching had a high value of the Michaelis constant (K _m) for carnitine. Maximal velocity (V _max) in intestine increased with age. V _max in liver and muscle increased with age except a decrease at 4 months in liver and at 2 years in muscle. The FC concentration in the examined tissues at all developmental stages were less than the respective K _m, indicating that Chinese suckers require supplemental carnitine in their food to ensure that CPT I activity is not constrained by carnitine availability.     

Alterations in CPT‐1 mRNA and fatty acid profile in hepatic cell lines in response to treatment with t10,c12‐ or c9,t11‐conjugated linoleic acid

Trans10,cis12‐conjugated linoleic acid (t10,c12‐CLA) increases liver weights and hepatic lipids in mice. The purpose of this study was to determine the effects of CLA isomers (t10,c12‐CLA or c9,t11‐CLA) and carnitine palmitoyl transferase‐1 (CPT‐1) inhibitors (etomoxir or hemipalmitoylcarnitinium) on CPT‐1 mRNA, fatty acid profile, and cholesterol synthesis in AML‐12 and HepG2 cells. t10,c12‐CLA was incorporated to a greater extent in both cell lines than c9,t11‐CLA. In addition, t10,c12‐CLA increased the free cholesterol content of AML‐12 and HepG2 cells four‐ and fivefold, respectively. Cells incubated with medium containing CPT‐1 inhibitors or t10,c12‐CLA had higher levels of mRNA for CPT‐1 in both cell lines, indicating an increased fatty acid oxidation in hepatic cell lines due to t10,c12‐CLA. Following treatment withdrawal, percentages of c9,t11‐CLA or t10,c12‐CLA remained elevated in cells initially treated with c9,t11‐CLA or t10,c12‐CLA, suggesting a potential for carryover effects of the CLA isomers. The results presented here demonstrate a potential role for t10,c12‐CLA in the modulation of hepatic fatty acid oxidation and cholesterol synthesis.     

Lipid molecular order in liver mitochondrial outer membranes, and sensitivity of carnitine palmitoyltransferase I to malonyl-CoA

Mitochondrial outer membranes were prepared from livers of rats that were in the normal fed state, starved for 48 h, or made diabetic by injection of streptozotocin. Membranes were also prepared from starved late-pregnant rats. The latter three conditions have previously been shown to induce varying degrees of desensitization of mitochondrial overt carnitine palmitoyltransferase (CPT I) to malonyl-CoA inhibition. We measured the fluorescence polarization anisotropy of two probes, 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene-p-toluenesulfonate (TMA-DPH) which, when incorporated into membranes, report on the hydrophobic core and on the peripheral regions of the bilayer, respectively. The corresponding polarization indices (r _DPH and r _TMA-DPH) were calculated. In membranes of all three conditions characterized by CPT I desensitization to malonyl-CoA, r _DPH was decreased, whereas there was no change in r _TMA-DPH, indicating that CPT I is sensitive to changes in membrane core, rather than peripheral, lipid order. The major lipid components of the membranes were analyzed. Although significant changes with physiological state were observed, there was no consistent pattern of changes in gross lipid composition accompanying the changes to membrane fluidity and CPT I sensitivity to malonyl-CoA. We conclude that CPT I kinetic characteristics are sensitive to changes in lipid composition that are localized to specific membrane microdomains.     

<Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12 Conjugated Linoleic Acid Enhances Endurance Capacity by Increasing Fatty Acid Oxidation and Reducing Glycogen Utilization in Mice

The supplementation of conjugated linoleic acid (CLA) has been shown to improve endurance by enhancing fat oxidation during exercise in rodents and humans. This study was designed to investigate the isomer-specific effects of CLA on endurance capacity and energy metabolism in mice during exercise. Male 129Sv/J mice were divided into three dietary groups and fed treatment diet for 6 weeks; control, 0.5 % cis-9,trans-11 (c9,t11) CLA, or 0.5 % trans-10,cis-12 (t10,c12) CLA. Dietary t10,c12 CLA induced a significant increase in maximum running time and distance until exhaustion with a dramatic reduction of total adipose depots compared to a control group, but there were no significant changes in endurance with the c9,t11 CLA treatment. Serum triacylglycerol and non-esterified fatty acid concentrations were significantly lower in the t10,c12 fed mice after exercise compared to control and the c9,t11 CLA fed-animals. Glycogen contents in livers of the t10,c12 fed-mice were higher than those in control mice, concomitant with reduction of serum l-lactate level. There were no differences in non-exercise physical activity among all treatment groups. In addition, the mRNA expression levels of carnitine palmitoyl transferase 1β, uncoupling protein 2 and peroxisome proliferator-activated receptor δ (PPARδ) in skeletal muscle during exercise were significantly up-regulated by the t10,c12 CLA but not the c9,t11 CLA. These results suggest that the t10,c12 CLA is responsible for improving endurance exercise capacity by promoting fat oxidation with a reduction of the consumption of stored liver glycogen, potentially mediated via PPARδ dependent mechanisms.     

Interactive Effects of Dietary Lipid and Phenotypic Feed Efficiency on the Expression of Nuclear and Mitochondrial Genes Involved in the Mitochondrial Electron Transport Chain in Rainbow Trout

A 2 × 3 factorial study was conducted to evaluate the effects of dietary lipid level on the expression of mitochondrial and nuclear genes involved in electron transport chain in all-female rainbow trout Oncorhynchus mykiss. Three practical diets with a fixed crude protein content of 40%, formulated to contain 10% (40/10), 20% (40/20) and 30% (40/30) dietary lipid, were fed to apparent satiety to triplicate groups of either low-feed efficient (F120; 217.66 ± 2.24 g initial average mass) or high-feed efficient (F136; 205.47 ± 1.27 g) full-sib families of fish, twice per day, for 90 days. At the end of the experiment, the results showed that there is an interactive effect of the dietary lipid levels and the phenotypic feed efficiency (growth rate and feed efficiency) on the expression of the mitochondrial genes nd1 (NADH dehydrogenase subunit 1), cytb (Cytochrome b), cox1 (Cytochrome c oxidase subunits 1), cox2 (Cytochrome c oxidase subunits 2) and atp6 (ATP synthase subunit 6) and nuclear genes ucp2α (uncoupling proteins 2 alpha), ucp2β (uncoupling proteins 2 beta), pparα (peroxisome proliferator-activated receptor alpha), pparβ (peroxisome proliferatoractivated receptor beta) and ppargc1α (proliferator-activated receptor gamma coactivator 1 alpha) in fish liver, intestine and muscle, except on ppargc1α in the muscle which was affected by the diet and the family separately. Also, the results revealed that the expression of mitochondrial genes is associated with that of nuclear genes involved in electron transport chain in fish liver, intestine and muscle. Furthermore, this work showed that the expression of mitochondrial genes parallels with the expression of genes encoding uncoupling proteins (UCP) in the liver and the intestine of rainbow trout. This study for the first time presents the molecular basis of the effects of dietary lipid level on mitochondrial and nuclear genes involved in mitochondrial electron transport chain in fish.     

Conjugated Linoleic Acid (t-10, c-12) Reduces Fatty Acid Synthesis de Novo, but not Expression of Genes for Lipid Metabolism in Bovine Adipose Tissue ex Vivo

We hypothesized that exogenous fatty acids, and especially or 18:2 trans-10, cis-12 conjugated linoleic acid (CLA), would decrease adipogenic and lipogenic gene expression and de novo fatty acid biosynthesis in intramuscular (i.m.) and subcutaneous (s.c.) adipose tissues. Fresh i.m. and s.c. adipose tissues were collected from the longissimus thoracis muscle of Angus steers at 12, 14, and 16 months of age (n = 4 per time point). Adipose tissue explants were incubated in duplicate for 48 h with 40 μM α-linolenic (ALA), oleic, stearic, trans-vaccenic, or CLA. Adipocyte size, acetate and glucose incorporation into fatty acids in vitro and mRNA levels for C/EBPβ, CPT1β, GPR43, PPARγ, PRKAA1 (AMPKα) and SCD1 were measured following the incubations. PRKAA1 and SCD1gene expression were greater (P < 0.001) in s.c. adipose tissue than in i.m. adipose tissue and acetate incorporation into lipids and C/EBPβ, PPARγ, and SCD1gene expression were greater at 16 months of age than at 12 months of age in i.m. adipose (P < 0.01). C/EBPβ gene expression increased by 16 months of age and PRKAA1 gene expression decreased by 16 months of age in s.c. adipose tissue. All fatty acids increased s.c. adipocyte volumes whereas CLA decreased acetate incorporation into lipids in s.c. adipose tissue (P < 0.05), but none of the fatty acids affected gene expression in i.m. or s.c. adipose tissue (P > 0.10). Thus, CLA depressed de novo fatty acid biosynthesis from acetate but neither CLA nor other fatty acids significantly affected adipogenic or lipogenic gene expression.     

Rosiglitazone,a PPAR-γ Agonist,Fails to Attenuate CLA-Induced Milk Fat Depression and Hepatic Lipid Accumulation in Lactating Mice

Our objective was to investigate the combination of rosiglitazone (ROSI) and conjugated linoleic acid (CLA) on mammary and hepatic lipogenesis in lactating C57Bl/6 J mice. Twenty-four lactating mice were randomly assigned to one of four treatments applied from postpartum day 6 to day 10. Treatments included: (1) control diet, (2) control plus 1.5 % dietary CLA (CLA) substituted for soybean oil, (3) control plus daily intra-peritoneal (IP) rosiglitazone injections (10 mg/kg body weight) (ROSI), and (4) CLA plus ROSI (CLA-ROSI). Dam food intake and milk fat concentration were depressed with CLA. However, no effects were observed with ROSI. The CLA-induced milk fat depression was due to reduced expression for mammary lipogenic genes involved in de-novo fatty acid (FA) synthesis, FA uptake and desaturation, and triacyglycerol synthesis. Liver weight (g/100 g body weight) was increased by CLA due to an increase in lipid accumulation triggering a compensatory reduction in mRNA abundance of hepatic lipogenic enzymes, including acetyl-CoA carboxylase I and stearoyl-CoA desaturase I. On the contrary, no effects were observed with ROSI on hepatic and mammary lipogenic gene and enzyme expression. Overall, feeding CLA to lactating mice induced milk fat depression and increased hepatic lipid accumulation, probably due to the presence of trans-10, cis-12 CLA isomer, while ROSI failed to significantly attenuate both hepatic steatosis and reduction in milk fat content.     

Conjugated linoleic acids (CLA) as precursors of a distinct family of PUFA

One of the possibilities for distinct actions of c9,t11- and the t10,c12-conjugated linoleic acid (CLA) isomers may be at the level of metabolism since the conjugated diene structure gives to CLA isomers and their metabolites a distinct pattern of incorporation into the lipid fraction and metabolism. In fact, CLA appears to undergo similar transformations as linoleic acid but with subtle isomer differences, which may account for their activity in lowering linoleic acid metabolites in those tissues rich in neutral lipids where CLA is preferentially incorporated. Furthermore, c9,t11 and t10,c12 isomers are metabolized at a different rate in the peroxisomes, where the shortened metabolite from t10,c12 is formed at a much higher proportion than the metabolite from c9,t11. This may account for the lower accumulation of t10,c12 isomer into cell lipids. CLA isomers may therefore be viewed as a “new” family of polyunsaturated fatty acids (PUFA) producing a distinct range of metabolites using the same enzymatic system as the other (i.e., n−3, n−6 and n−9) PUFA families. It is likely that perturbation of PUFA metabolism by CLA will have an impact on eicosanoid formation and metabolism, closely linked to the biological activities attributed to CLA.     

Conjugated Linoleic Acid Isomers Trans‐10, Cis‐12 and Cis‐9, Trans‐11 Prevent Collagen‐Induced Arthritis in a Direct Comparison

Mixed‐isomer conjugated linoleic acid (CLA) and the individual isomers, trans‐10, cis‐12 (CLAt10c12) and cis‐9, trans‐11 (CLAc9t11), decrease severity of collagen‐induced arthritis (CA) when consumed after disease onset. Few studies have been conducted exploring the role of CLA in the prevention of autoimmune diseases. These studies suggest that isomer‐specific effects may be occurring; however, a direct comparison of CLAt10c12 and CLAc9t11 has yet to be conducted. A study to compare the ability of CLAt10c12 and CLAc9t11 to prevent CA and assess their effects on early inflammation was performed. DBA/1 mice were fed a semipurified diet containing 6% corn oil (CO), 5.5% CO and 0.5% CLAt10c12, or 5.5% CO and 0.5% CLAc9t11 (n = 27 per diet) starting three weeks before CA primary immunization. Effects on disease incidence and severity, anticollagen antibodies, plasma and paw cytokines, and hepatic fatty acids were measured. Arthritis incidence was reduced by a minimum of 34% in mice fed either CLA isomer compared to those fed CO diet (p = 0.06). In mice that did develop arthritis (n = 9–12 mice per treatment), CLAt10c12 reduced arthritic severity to a greater extent than CLAc9t11 and CO (p = 0.03). CLA isomer treatment attenuated the increased hepatic arachidonic acid (ARA; 20:4n‐6) observed with arthritis at one‐week postonset (p = 0.03), while no differences in anticollagen antibodies or cytokines were observed between dietary treatments. These results suggest that CLA isomers may be effective at preventing specific immune‐mediated inflammatory diseases, in part, through modulation of the ARA cascade.     

Physical Properties of Two Isomers of Conjugated Linoleic Acid

Thermal properties, powder X-ray diffraction patterns and FT-IR absorption spectra of crystals of two isomers of conjugated linoleic acid (CLA), 9-cis, 11-trans-CLA (c9t11), 10-trans, 12-cis-CLA (t10c12) were examined. To search for polymorphic modifications, we carefully performed crystallization from melt and solution phases, and isolated one type of crystalline form in c9t11 and t10c12. The melting temperature (T _m) was 14.9 °C, enthalpy of fusion (ΔH) was 38.7 kJ/mol, and entropy of fusion (ΔS) was 134 J/mol K for c9t11, and T _m = 19.8 °C, ΔH = 35.6 kJ/mol and ΔS = 122 J/mol K for t10c12. The X-ray diffraction and FT-IR measurements indicated O_⊥ subcell packing in the crystals of c9t11 and t10c12, and long spacing values of 4.22 nm for c9t11 and 3.88 nm for t10c12. The unique molecular structures of the two isomers of CLA are discussed in comparison to the polymorphism of oleic acid, petroselinic acid, elaidic acid and linoleic acid, all of which are unsaturated fatty acids having the same carbon number of 18 as that of the two CLA isomers.     

Dietary Conjugated Linoleic Acid-c9t11 Prevents Collagen-Induced Arthritis,Whereas Conjugated Linoleic Acid-t10c12 Increases Arthritic Severity

Two conjugated linoleic acid (CLA) isomers, cis‐9, trans‐11 (CLAc9t11) and trans‐10, cis‐12 (CLAt10c12), reduce inflammation in a number of animal models, including collagen‐induced arthritis (CA). However, little is known about the ability of individual CLA isomers to prevent autoimmune disease onset. Evidence that mixed isomer CLA drives T helper cell (Th) 1 responses suggests that CLA, or a specific isomer, exacerbates onset of Th1 autoimmune diseases. In two experiments, we examined if prior dietary exposure to CLAt10c12 (experiment 1) or CLAc9t11 (experiment 2) affected the incidence or severity of CA. DBA/1 mice were fed a semi purified diet with either 6% corn oil (CO, w/w), 5.75% CO plus 0.25% CLAt10c12, or 5.5% CO plus 0.5% CLAc9t11 prior to arthritis development. Arthritis incidence and severity, anti‐collagen antibodies, paw cytokines, and hepatic fatty acids were measured. CLAt10c12 had no effect on arthritis incidence but increased arthritic severity (42%, P = 0.02); however, CLAc9t11 decreased arthritis incidence 39% compared to CO fed mice (P = 0.01), but had no effect on disease severity. CLAt10c12‐induced increase in anti‐collagen type II IgG antibodies may be a mechanism by which this isomer increased arthritic severity, and CLAc9t11‐induced increase in Th2 paw cytokines (IL‐4 and IL‐10, P ≤ 0.04) may explain how CLAc9t11 reduced the arthritis incidence. While both isomers are well known to reduce inflammation in arthritic mice, these new data suggest isomer differences when fed prior to autoimmune disease.