Addition Of palmitoyl-CoA Research Articles

TYPE 2 DIABETIC HEARTS ARE ENERGETICALLY DEFECTIVE BOTH GLOBALLY AND AT THE LEVEL OF THE MITOCHONDRIA

Introduction Cardiac metabolism in type 2 diabetes is abnormal, with increased fatty acid oxidation, and decreased glucose oxidation. The ratio of phosphocreatine/adenosine triphosphate (PCr/ATP) is decreased in the hearts of diabetic patients, indicating an energetic defect. However, an underlying mechanism is yet to be determined. Methods Diabetic and control hearts were perfused in an 11.7T magnet and high energy phosphates measured in contracting hearts via magnetic resonance spectroscopy. Cardiac subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated from different diabetic and control rats, and respired using an oxygen-sensitive electrode with glutamate, pyruvate, malate (GPM), and fatty acids. Results Diabetic hearts had a 20% reduction in ATP levels with no change in phosphocreatine. Despite this, cardiac function was unaltered between groups. Citrate synthase assay data, and mtDNA copy number showed that diabetic hearts have 30% more mitochondria than control heart. However, both SSM and IFM from diabetic rats had decreased respiratory rates when respired solely on GPM (normalised to mitochondrial protein), which was rescued by the addition of fats. Addition of palmitoyl-CoA (P-CoA) to GPM resulted in a 50% decrease in respiration in both diabetic and control mitochondria. Suppression was prevented if P-CoA was co-infused with carnitine, or if palmitoyl-carnitine was used. Inhibition from P-CoA was abolished in the presence of an uncoupling agent (FCCP), demonstrating inhibition was mediated via the phosphorylation apparatus. Conclusion Diabetic hearts are energetically impaired when compared to control hearts. This energetic defect pairs with a mitochondrial defect, with diabetic hearts potentially compensating by increasing mitochondrial number.

The selective COX-2 inhibitor celecoxib modulates sphingolipid synthesis

Sphingolipids such as ceramides (Cers) play important roles in cell proliferation, apoptosis, and cell cycle regulation. An increased Cer level is linked to the cytotoxic effects of several chemotherapeutics. Various selective cyclooxygenase-2 (COX-2) inhibitors induce anti-proliferative effects in tumor cells. We addressed the possible interaction of the selective COX-2 inhibitors, coxibs, with the sphingolipid pathway as an explanation of their anti-proliferative effects. Sphingolipids were measured using liquid chromatography tandem mass spectrometry. Treatment of various cancer cell lines with celecoxib significantly increased sphinganine, C(16:0)-, C(24:0)-, C(24:1)-dihydroceramide (dhCer) and led to a depletion of C(24:0)-, C(24:1)-Cer in a time- and concentration-dependent manner, whereas other coxibs had no effect. Using (13)C,(15)N-labeled l-serine, we demonstrated that the augmented dhCers after celecoxib treatment originate from de novo synthesis. Celecoxib inhibited the dihydroceramide desaturase (DEGS) in vivo with an IC(50) of 78.9 +/- 1.5 muM and increased total Cer level about 2-fold, indicating an activation of sphingolipid biosynthesis. Interestingly, inhibition of the sphingolipid biosynthesis by specific inhibitors of l-serine palmitoyltransferase diminished the anti-proliferative potency of celecoxib. In conclusion, induction of de novo synthesis of sphingolipids and inhibition of DEGS contribute to the anti-proliferative effects of celecoxib.

Is there a single malonyl‐CoA pool in the heart for regulation of carnitine palmitoyltransferase‐I and fatty acid chain elongation

In non-lipogenic tissues malonyl-CoA serves two functions: 1) regulation of mitochondrial fatty acid oxidation via inhibition of carnitine palmitoyltransferase-I (CPT-I), and 2) providing two-carbon units for fatty acid chain elongation. In the presence of acetyl-CoA carboxylase (ACC-2) substrates acetyl-CoA, ATP-Mg2+, and bicarbonate isolated rat heart mitochondria synthesize malonyl-CoA that results in inhibition of CPT-I. Furthermore, addition of palmitoyl-CoA and NADPH/NADH to isolated intact rat heart mitochondria catalyze the incorporation of radioactive malonyl-CoA into lipid soluble products suggesting an outer mitochondrial membrane localization of the long-chain fatty acid elongation pathway. These data suggest that malonyl-CoA formed by mitochondrial ACC-2 is available for both, regulation of fatty acid oxidation and for fatty acid chain elongation. The finding with perfused heart that stable isotope labeled palmitate is chain-elongated to stearate without prior chain shortening by β-oxidation and isotope dilution as judged from the near identical isotope enrichment of palmitoyl- and stearoylcarnitine is in support of this. These data and the exclusive outer mitochondrial membrane localization of CPT-I suggests that mitochondria catalyze long-chain fatty acid elongation also in vivo. Our data raise the question about metabolic channeling of malonyl-CoA between ACC-2, CPT-I, and fatty acid chain elongation and how these processes are regulated. Supported by: DK-066107, POI AG15885, and VA Med. Res. Serv.

Hydroxylamine Treatment Differentially Inactivates Purified Rat Hepatic Asialoglycoprotein Receptors and Distinguishes Two Receptor Populations

We previously showed that two subpopulations of asialoglycoprotein receptors (ASGP-Rs), designated State 1 and State 2 ASGP-Rs, are present in intact cells and that State 2 ASGP-Rs can be inactivated in permeable rat hepatocytes in a temperature- and ATP-dependent manner. These inactivated ASGP-Rs can be quantitatively reactivated by the addition of palmitoyl-CoA (Weigel, P. H., and Oka, J. A. (1993) J. Biol. Chem. 268, 27186-27190). Here we show that approximately 50% of purified rat ASGP-Rs are inactivated by treatment with hydroxylamine under mild conditions. The activity of affinity-purified ASGP-Rs was assessed by measuring the specific binding of 125I-asialo-orosomucoid (ASOR) in a dot-blot assay after immobilization onto nitrocellulose. Treatment of ASGP-Rs in solution with 0.0125-1.0 M NH2OH, pH 7.4, at 4 degrees C for 4 h resulted in a progressive loss of ASOR binding activity. ASGP-R inactivation with NH2OH occurred more readily at basic pH or at room temperature. Similar treatment with Tris had no effect on ASGP-R activity. The kinetics of ASGP-R activity loss and the dose-response for this inactivation were both biphasic, indicating the presence of two equal populations of ASGP-Rs with different sensitivities to NH2OH. The more sensitive population of ASGP-Rs (approximately 50%) was inactivated by treatment with 0.2 M NH2OH (4 degrees C, 4 h) or with 1.0 M NH2OH (4 degrees C, 1 h) without detectable peptide cleavage as assessed by SDS-polyacrylamide gel electrophoresis. State 1 ASGP-Rs, purified from chloroquine- or monensin-treated hepatocytes, showed significantly less sensitivity to NH2OH treatment (both in kinetics and dose dependence). Furthermore, under mild conditions NH2OH caused dissociation and inactivation of approximately 50% of the total ASGP-Rs (State 1 and State 2) that were prebound to ASOR-Sepharose, whereas the same treatment caused dissociation of only < 20% of State 1 ASGP-Rs from such preformed complexes. As shown in the accompanying paper (Zeng, F. Y., Kaphalia, B. S., Ansari, G. A. S., and Weigel, P. H. (1995) J. Biol. Chem. 270, 21382-21387) all three RHL subunits of active ASGP-Rs, in fact, contain covalently attached palmitate and stearate. In cultured cells, [3H]palmitic acid is metabolically incorporated into all three subunits. These radiolabeled fatty acids are completely released from purified ASGP-Rs by mild NH2OH treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel cycle involving fatty acyl-coenzyme A regulates asialoglycoprotein receptor activity in permeable hepatocytes.

Asialoglycoprotein receptors (ASGP-Rs) in permeable rat hepatocytes can be inactivated in the absence of ligand. This cytosol-independent effect is relatively slow (t1/2 approximately 12 min) and is temperature and ATP dependent. Here we show that in the absence of cytosol, the addition of palmitoyl-CoA (Pal-CoA) rapidly (t1/2 < 0.4 min) and quantitatively reactivates the inactivated receptors. Receptor reactivation was half-maximal at approximately 10-12 microM free Pal-CoA at 37 degrees C. Although substantially higher total concentrations were used, much of the added Pal-CoA was cell associated and not free. The effects of Pal-CoA were eliminated by bovine serum albumin at concentrations sufficient to bind all free monomeric fatty acyl-CoA, suggesting that micellar effects are not responsible for the ability to reactivate ASGP-Rs. Also, palmitoyl-carnitine did not substitute for Pal-CoA. The initial ASGP-R inactivation is not affected by treating cells with N-ethylmaleimide or by a KCl wash but is inhibited by sodium orthovanadate or high Ca2+ levels. Myristoyl-CoA (C14) was also able to reactivate inactive ASGP-Rs about as well as Pal-CoA. Fatty acyl-CoAs with chain lengths of C12 (lauroyl) or C18 (steroyl) were < 50% as active. The ligand binding activity of these receptors can subsequently be modulated within minutes by the further addition of ATP or Pal-CoA to achieve additional rounds of ASGP-R inactivation or reactivation, respectively. These in vitro data demonstrate the occurrence of a novel asialoglycoprotein receptor inactivation-reactivation cycle that could regulate receptor activity during endocytosis and receptor recycling.

Ca 2+-mediated action of long-chain acyl-CoA on liver mitochondria energy-linked processes

The decrease of steady-state transmembrane potential (delta psi) and loss of accumulated Ca2+ are magnified if palmitoyl-CoA is added to rat liver mitochondria exposed to Ca2+ and phosphate. The extent of this damage increases with increasing concentration of long-chain acyl-CoA. Addition of L-carnitine with or without the addition of palmitoyl-CoA considerably delays the deenergization. In the latter case, there is a substantial decrease in the assayed endogenous long-chain acyl-CoA content. This protective action of L-carnitine is abolished by L-aminocarnitine, a powerful inhibitor of carnitine palmitoyl transferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21.). The removal of Ca2+ by EGTA, or the inhibition of its uptake by Ruthenium red or Mg2+ further enhances the degree of protection.

Retinol Esterification by Mouse Epidermal Microsomes: Evidence for Acyl-CoA:Retinol Acyltransferase Activity

In an attempt to characterize the enzyme(s) responsible for retinol esterification in hairless mouse epidermis, various subcellular fractions were incubated with [3H]retinol and the reaction products (retinyl esters) isolated by high-performance liquid chromatography. The microsomal fraction exhibited the highest esterifying activity and was stimulated by the addition of palmitoyl-CoA and dithiothreitol, but not by palmitic acid. Saturation kinetics with an apparent Km of about 6 microM for retinol were noted. Experiments with competitive and noncompetitive inhibitors of [3H]retinol esterification established that the epidermal enzyme was an acyl-CoA:retinol acyltransferase (ARAT; EC 2.3.1.76). The specificity for retinol was not absolute; a few closely related vitamin A alcohols were equally good substrates. The ARAT activity was not significantly altered by physiologic variations in the epidermal vitamin A content. In conclusion, mouse epidermis expresses ARAT activity which may be of importance for the regulation of vitamin A metabolism at the cellular level.

Retinol esterification by rat liver microsomes. Evidence for a fatty acyl coenzyme A: retinol acyltransferase.

To explore the nature of retinyl ester synthesis by liver microsomes, membranes prepared from rat or cat liver were incubated under various conditions with [3H] retinol dispersed in dimethyl sulfoxide. When [3H]retinol, buffer, and microsomes were incubated together (basal conditions), some [3H]retinol esterification was consistently observed. However, the rate of esterification could be increased 6- to 11-fold by addition of either palmitoyl-CoA (100 microM) or a fatty acyl CoA-generating system. To determine whether the fatty acid used to esterify [3H]retinol under basal conditions might be derived from an endogenous pool of fatty acyl-CoA associated with the microsomal preparation, microsomes were pretreated at pH 7.4 with 0.5 M hydroxylamine, a reagent that reacts with coenzyme A thioesters to form hydroxamates. This pretreatment reduced the basal reaction by 69%. However, hydroxylamine-treated microsomes still retained acyltransferase activity, as shown by a 24- to 40-fold increase in retinyl ester synthesis after addition of palmitoyl-CoA. When microsomes were incubated with both [3H]retinol and [14C]palmitoyl-CoA of known specific radioactivities, the ratio of 14C to 3H in newly synthesized retinyl palmitate was essentially equal to that of its putative substrates, indicating that [14C]palmitate did not undergo significant isotope dilution prior to acylation of [3H]retinol. These experiments provide direct evidence for retinol esterification catalyzed by a microsomal acyl-CoA:retinol acyltransferase and indirect evidence for a pool of fatty acyl-CoA in isolated liver microsomes that is available to react with [3H]retinol to form esterified retinol.

The enzymatic synthesis of wax esters by a microsomal preparation from the honeybee Apis mellifera L.

A microsomal preparation obtained from the abdomens of the honeybee Apis mellifera L. incorporated long chain primary alcohols, fatty acids, and the acyl group from acyl-CoA into cuticular wax monoesters. The incorporation of [1 - 14C]-palmitate into monoester was stimulated by ATP, CoA, and MgCl 2, and the addition of palmitoyl-CoA caused a five fold increase in monoester synthesis using [R- 3H]-tetracosanol as the labelled substrate. This indicated that the mechanism of monoester synthesis involved the transfer of an acyl group from acyl-CoA to a primary alcohol. The addition of palmitoyl-CoA at concentrations up to 100 μM caused an increase in the rate of monoester synthesis, whereas at concentrations above 100 μM, palmitoyl-CoA markedly inhibited monoester formation. An apparent K m for tetracosanol was calculated from a double reciprocal plot to be 9 × 10 −4 M. Although the naturally occurring primary alcohols in the monoesters of the cuticular wax from the honeybee range in chain length from C 24 to C 32, labelled primary alcohols of 16, 18, 24 and 28 carbons were readily incorporated into monoester, with the C 16 and C 18 components incorporated at higher rates then the C 24 and C 28 homologues. A C 27 secondary alcohol was not incorporated into wax esters.

Regulation of selectivity of CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn -glycerols

The suitability of monoenoic, dienoic, tetraenoic, and hexaenoic molecular species of 1,2-diacyl-sn-glycerols as substrates for the CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied in rat liver microsomes. No statistically significant difference in the rates of phosphatidylcholine synthesis with the various diacylglycerols was found at 0.40 mM, although a moderate discrimination against hexaenoic species relative to monoenoic and dienoic species was observed at 0.25 mM. The addition of palmitoyl-CoA (7.5 micron) significantly enhanced cholinephosphotransferase activity when tetraenoic diacylglycerols were added at 0.25 or 0.40 mM. CDPethanolamine at 24.4 micron was found to inhibit the rates of phophatidylcholine biosynthesis by 54 and 39% with hexaenoic and monoenoic 1,2-diacyl-sn-glycerols, respectively, whereas no significant effects were observed in the case of dienoic and tetraenoic species. These latter findings may partially explain why 1-saturated 2-docosahexaenoyl diacylglycerols are used to a greater extent for phosphatidylethanolamine than for phosphatidylcholine synthesis in rat liver in vivo. The present results also suggest that the selectivity of the cholinephosphotransferase for certain molecular species of 1,2-diacyl-sn-glycerols is a function of diacylglycerol concentration and may be mediated under physiological conditions by substrates for enzymes which compete for common diacylglycerol precursors.

Fatty acid desaturase system of yeast microsomes: Involvement of cytochrome b5-containing electron-transport chain

The oxidative desaturation of palmitoyl CoA by microsomes from anaerobically grown Saccharomyces cerevisiae has been studied by using NADH as electron donor. The desaturation product was identified as palmitoleic acid by periodate oxidation. The desaturase activity was sensitive to relatively high concentrations of cyanide; the concentration of cyanide causing half-maximal inhibition was determined to be 7.1 m m. The rate of reoxidation of cytochrome b 5 in NADH-reduced microsomes was stimulated by the addition of palmitoyl CoA, and the amount of cytochrome b 5 reoxidized by the palmitoyl CoA added could be closely correlated to the amount of palmitoleate formed. No stimulation of the reoxidation of cytochrome b 5 was induced by palmitoyl CoA in microsomes prepared from the desaturase-repressed cells and from a desaturase-deficient mutant, strain KD-20. It is concluded that the fatty acyl CoA desaturase system of yeast microsomes involves cytochrome b 5 as an electron carrier and that the terminal desaturase is sensitive to relatively high concentrations of cyanide.

Acyltransferase activities in rat lung microsomes

Some properties of acyl-CoA:1-acyl- sn-glycero-3-phosphorylcholine acyltransferase in rat lung microsomes were studied using various acyl-CoAs as the substrate. Depending on the fatty acid moiety of acyl-CoAs, quite different values were obtained on the Michaelis constant, the maximal velocity, and the activation energy. Moreover, the incorporation of fatty acid from an acyl-CoA was affected in a different manner by the addition of other acyl-CoAs. These results suggested that there are at least two different acyltransferases which are tentatively termed as follows: (1) palmitoyl-CoA:1-acylglycerophosphorylcholine acyltransferase; and (2) arachidonoyl-CoA:1-acylglycerophosphorylcholine acyltransferase. A low K m value, a low maximal velocity, and a low value of the activation energy were obtained for the former activity. The activity is readily inhibited by the addition of other acyl-CoAs and also at the higher concentration of palmitoyl-CoA itself. While a high K m value, a high maximal velocity, and a high value of the activation energy were obtained for the latter activity. The activity is not affected by the addition of palmitoyl-CoA or oleoyl-CoA and only slightly inhibited by linoleoyl-CoA, which indicates a high substrate specificity for polyenoyl-CoA especially for arachidono-yl-CoA. It seems that the present result, together with the previous findings obtained in slice experiments and in in vivo studies, do not support the idea that palmitoyl-CoA:1-acylglycerophosphorylcholine acyltransferase participates in the main pathway for the formation of dipalmitoyllecithin in lung.