Effects of an acetyl-coenzyme A carboxylase inhibitor and a sodium-sparing diuretic on aldosterone-stimulated sodium transport, lipid synthesis, and phospholipid fatty acid composition in the toad urinary bladder.

A correlation study of the effects of two agents, 2-methyl-2-[p-(1,2,3,4-tetrahydro-1-naphthyl)phenoxy]propionic acid (TPIA) and amiloride, on aldosterone-induced alterations in Na+ transport, lipid synthesis, and phospholipid fatty acid composition has been carried out in the toad urinary bladder. TPIA, an inhibitor of acetyl-CoA carboxylase, inhibits aldosterone-stimulated Na+ transport as well as hormone-induced lipid synthesis and the increase in weight percentage of phospholipid long-chain polyunsaturated fatty acids. Amiloride, a diuretic which blocks sodium entry into the transporting epithelium, does not alter aldosterone's effects on lipid and fatty acid metabolism but prevents the hormone-induced increase in Na+ transport. These results support the conclusion that aldosterone increases Na+ transport in the toad urinary bladder by altering membrane fatty acid metabolism and that the lipid biosynthetic events following aldosterone treatment are a primary response to the hormone and not secondary to increased Na+ transport.     

Inhibition of T lymphocyte activation by cyclosporin A: interference with the early activation of plasma membrane phospholipid metabolism

Rabbit lymph node and thymus lymphocytes were stimulated with concanavalin A (Con A). Cyclosporin A (CSA) inhibited in a dose-dependent way the induction of RNA and DNA synthesis; nearly complete inhibition was observed at a concentration of 200 ng/ml. Results of kinetic studies suggested that the immunosuppressive drug interfered with an early event occurring in activated lymphocytes. Among the earliest changes detectable in activated lymphocytes, the turnover of plasma membrane phospholipids is increased, predominantly of their fatty acid moieties, catalyzed by the membrane-bound lysophosphatide acyltransferase. CSA, at concentrations identical with those inhibiting macromolecular synthesis, also inhibited the Con A-stimulated specific increase in the incorporation of labeled fatty acids into plasma membrane phospholipids. When lymphocytes were stimulated with Con A for 1 hr, incorporation of labeled oleic acid and arachidonic acid approximately doubled in plasma membrane phospholipids. CSA at a concentration of 200 ng/ml prevented the elevated incorporation of labeled fatty acids into plasma membrane phospholipids of Con A-stimulated thymocytes. Concomitantly, the activation of lysolecithin acyltransferase, the key enzyme for the incorporation of long-chain fatty acids into phospholipids, was strongly inhibited. Up to high concentrations, CSA had no effect on the phospholipid metabolism of unstimulated lymphocytes. The results suggest that CSA inhibits the activation of T lymphocytes by interfering with the early activation of plasma membrane phospholipid metabolism.     

Lipid metabolism during bacterial growth, sporulation, and germination: kinetics of fatty acid and macromolecular synthesis during spore germination and outgrowth of Bacillus thuringiensis.

The timing and kinetics of fatty acid synthesis are delineated for Bacillus thuringiensis spore germination and outgrowth by analyzing [U-14C]acetate and [2-3H]glycerol incorporation into chloroform-methanol-extractable and trichloroacetic acid-precipitable lipids. In addition to measurement of pulsed and continuous labeling of fatty acids, monitoring the incorporation of radioactive phenylalanine, thymidine, and uridine from the onset of germination through first cell division provides a profile of biochemical activities related to membrane differentiation and cellular development. Upon germination, ribonucleic acid synthesis is initiated, immediately followed by rapid and extensive fatty acid synthesis that in turn precedes protein, deoxyribonucleic acid and triglyceride synthesis. Significantly, formation of fatty acids from acetate exhibits further developmental periodicity in which a large transient increase in fatty acid synthetic activity coincides with the approach of cell division. Radiorespirometric analyses indicates only slight oxidative decarboxylation of acetate and corroborates the extreme involvement of acetate in specific fatty acid biosynthetic reactions throughout cellular modification. These findings graphically demonstrate an intimate association of fatty acid metabolism with commitment to spore outgrowth and subsequent cell division.     

Effect of hormones on phospholipid metabolism in human cultured fibroblasts

The effect of hormones on phospholipid metabolism, pool size, 32P labeling and changes in fatty acid of human adult fibroblasts was determined. Simultaneously the change in membrane fluidity of single cells was recorded via fluorescence recovery after photobleaching under the influence of hormones. From all substances tested (isoproterenol, phenylephrine, adrenalin, histamine, angiotensin II, dansylcadaverine, propranolol) only isoproterenol and adrenalin slightly decreased total amount of phosphatidylcholine (PC). The amount of the other phospholipids analyzed remained unchanged. The 32P incorporation rate into phospholipids (PC, phosphatidylinositol (PI), phosphatidylethanolamine (PE)) was affected basicly different analyzing either PC, PI or PE. Histamine and propranolol provoked the highest incorporation of 32P (240% increase in PI labeling). Isoproterenol and adrenalin decreased PC labeling (45% and 18%) whereas isoproterenol decreased 32P incorporation into PI (18%), and adrenalin led to an increase (37%). PE labeling showed no or a slight increase in 32P incorporation applying the other agonists or antagonists. The fatty acid pattern of the respective phospholipids changed only to a minor extend. A decrease in hexadecanoic acid content of PI was found after administration of either isoproterenol, adrenalin or histamine. Parallel determination of membrane fluidity of single cells by fluorescence recovery after photobleaching showed an increase in the diffusion coefficient of a fluorescent lipid probe sticking in the membrane, following administration of isoproterenol and adrenalin, other substances tested exerted no effect. A relationship to changes in phospholipid metabolism became obvious. These results are discussed considering known mechanisms of receptor coupling and change in phospholipid metabolism and fluidity.     

Effects of inhibitors of protein and RNA synthesis on aldosterone-stimulated changes in phospholipid fatty acid metabolism in the toad urinary bladder.

The effects of an inhibitor of RNA synthesis, cordycepin, and an inhibitor of protein synthesis, cycloheximide, on aldosterone-induced changes in lipid metabolism and phospholipid fatty acid composition have been studied in the toad urinary bladder. At the concentrations employed, the inhibitors abolish the hormone-induced increases in total lipid synthesis, phospholipid fatty acid specific activities, and weight percentage of phospholipid long-chain polyunsaturated fatty acids as well as blocking the aldosterone-mediated increase in sodium transport.     

Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores

LéJohn, Herbert B. (Purdue University, Lafayette, Ind.), and James S. Lovett. Ribonucleic acid and protein synthesis in Rhizophlyctis rosea zoospores. J. Bacteriol. 91:709-717. 1966.-The uniflagellate zoospores of Rhizophlyctis rosea display active motility and a high endogenous respiratory metabolism, but neither growth nor net ribonucleic acid (RNA) or protein synthesis can be measured by ordinary procedures. Nevertheless, synthesis can be detected with isotopic precursors. Uracil-C(14) is incorporated slowly into both the soluble and ribosomal RNA. Analysis of zoospore extracts (on diethylaminoethyl cellulose columns or sucrose gradients) after various periods of labeling suggested that most of the uracil incorporation represents slow synthesis of ribosomal precursor RNA and, ultimately, ribosomes. Actinomycin D caused an 80% inhibition of uracil incorporation. The most rapidly labeled RNA was susceptible to extensive degradation in cells treated with actinomycin, but the percentage of stable RNA increased with the time of incorporation before addition of the antibiotic. Neither the effects of actinomycin nor the results of chase experiments have established unequivocally the existence of turnover or the presence of a short-lived "messenger" fraction in motile spores. Both leucine and methionine were slowly incorporated into a spectrum of cellular proteins. The methyl group of C(14)-methylmethionine also served as a methyl donor for the methylation of soluble RNA but not of ribosomal RNA. The observations that some of the newly synthesized RNA and protein occur in the intact 82S ribosomes and that actinomycin inhibits the low level of protein synthesis provide some indirect evidence for a very low rate of "messenger" synthesis and turnover in zoospores.     

Modification of CaCo-2 cell membrane fatty acid composition by eicosapentaenoic acid and palmitic acid: effect on cholesterol metabolism

Membrane fatty acid composition of CaCo-2 cells was modified by incubating the cells for 8 days in medium containing 100 microM eicosapentaenoic acid or palmitic acid. The effect of membrane fatty acid changes on cholesterol metabolism was then studied. Cells incubated with eicosapentaenoic acid had significant changes in membrane fatty acid composition with an accumulation of 20:5 and 22:5 and a reduction in monoenoic fatty acids compared to cells grown in palmitic acid. Intracellular cholesteryl esters could not be detected in CaCo-2 cells grown in the presence of the n-3 polyunsaturated fatty acid. In contrast, cells incubated with the saturated fatty acid contained 2 micrograms/mg protein of cholesteryl esters. Cells grown in eicosapentaenoic acid, however, accumulated significantly more triglycerides compared to cells modified with palmitic acid. The rate of oleic acid incorporation into triglycerides was significantly increased in cells incubated with eicosapentaenoic acid. CaCo-2 cells modified by eicosapentaenoic acid had lower rates of HMG-CoA reductase and ACAT activities compared to cells modified with palmitic acid. The incorporation of the two fatty acids into cellular lipids also differed. Palmitic acid was predominantly incorporated into cellular triglycerides, whereas eicosapentaenoic acid was preferentially incorporated into phospholipids with 60% of it in the phosphatidylethanolamine fraction. The data indicate that membrane fatty acid composition is significantly altered by growing CaCo-2 cells in eicosapentaenoic acid. These modifications in membrane fatty acid saturation are accompanied by a decrease in the rates of cholesterol synthesis and cholesterol esterification.     

Sterol carrier protein-2 localization in endoplasmic reticulum and role in phospholipid formation

Although sterol carrier protein-2 (SCP-2; also called nonspecific lipid transfer protein) binds fatty acids and fatty acyl-CoAs, its role in fatty acid metabolism is not fully understood. L-cell fibroblasts stably expressing SCP-2 were used to resolve the relationship between SCP-2 intracellular location and fatty acid transacylation in the endoplasmic reticulum. Indirect immunofluorescence double labeling and laser scanning confocal microscopy detected SCP-2 in peroxisomes > endoplasmic reticulum > mitochondria > lysosomes. SCP-2 enhanced incorporation of exogenous [(3)H]oleic acid into phospholipids and triacylglycerols of overexpressing cells 1.6- and 2.5-fold, respectively, stimulated microsomal incorporation of [1-(14)C]oleoyl-CoA into phosphatidic acid in vitro 13-fold, and exhibited higher specificity for unsaturated versus saturated fatty acyl-CoA. SCP-2 enhanced the rate-limiting step in microsomal phosphatidic acid biosynthesis mediated by glycerol-3-phosphate acyltransferase. SCP-2 also enhanced microsomal acyl-chain remodeling of phosphatidylethanolamine up to fivefold and phosphatidylserine twofold, depending on the specific fatty acyl-CoA, but had no effect on other phospholipid classes. In summary, these results were consistent with a role for SCP-2 in phospholipid synthesis in the endoplasmic reticulum.     

Oxytocin regulates Ca2+ level in myometrium by influencing phosphoinositide metabolism

The effect of oxytocin on phosphoinositide metabolism as well as on membrane protein phosphorylation in myometrial tissue was studied. Oxytocin enhanced the 32P incorporation into phospholipids in myometrial tissue. The effect of oxytocin on phosphoinositide metabolism was also detected in plasma membrane of 20 days pregnant rats. Phosphorylated membrane lipids have been analysed and phosphatidylinositol 4, 5-bisphosphate proved to be the main reaction product. Oxytocin enhanced the 32P incorporation into phospholipids measured in the first 30 sec then the labeling decreased more rapidly then in case of the control. The effect of oxytocin proved to be concentration dependent. The protein phosphorylation was also influenced by oxytocin. However the amount of alkylphosphate formed depended on the presence or absence of Ca2+, Ca2+-calmodulin and cyclic AMP, oxytocin influenced the protein phosphorylation in the presence of Ca2+-calmodulin only.     

Determination of protein replacement rates by deuterated water: validation of underlying assumptions

H(2)O administration has recently been proposed as a simple and convenient method to measure protein synthesis rates. (2)H(2)O administration results in deuterium labeling of free amino acids such as alanine, and incorporation into proteins of labeled alanine can then be used to measure protein synthesis rates. We examined first whether during (2)H(2)O administration plasma free alanine enrichment is a correct estimate of the enrichment in the tissue amino acid pools used for protein synthesis. We found that, after (2)H(2)O administration, deuterium labeling in plasma free alanine equilibrated rapidly with body water, and stable enrichment values were obtained within 20 min. Importantly, oral administration of (2)H(2)O induced no difference of labeling between portal and peripheral circulation except for the initial 10 min after a loading dose. The kinetics of free alanine labeling were comparable in various tissues (liver, skeletal muscle, heart) and in plasma with identical plateau values. We show next that increased glycolytic rate or absorption of unlabeled amino acids from ingested meals do not modify alanine labeling. Calculated synthesis rates of mixed proteins were much higher (20- to 70-fold) in plasma and liver than in muscle and heart. Last, comparable replacement rates of apoB100-VLDL were obtained in humans by using the kinetics of incorporation into apoB100 of infused labeled leucine or of alanine labeled by (2)H(2)O administration. All of these results support (2)H(2)O as a safe, reliable, useful, and convenient tracer for studies of protein synthesis, including proteins with slow turnover rate.     

Labeling of adipocyte membranes by sulfo-N-succinimidyl derivatives of long-chain fatty acids: Inhibition of fatty acid transport

Summary Sulfo-N-succinimidyl derivatives of the long-chain fatty acids, oleic and myristic, were synthesized and covalently reacted with isolated rat adipocytes. The plasma membrane proteins labeled by these compounds and the effect of labeling on the transport of long-chain fatty acids were investigated. Sulfo-N-succinimidyl oleate (SSO) and myristate (SSM) inhibited the transport of fatty acids (by about 70%). Inhibition of fatty acid transport was not a result of alterations in cell integrity, as intracellular water volume was not changed. It did not reflect effects on fatty acid metabolism, since it was observed under conditions where greater than 90% of the fatty acid taken up was recovered in the free form. The inhibitory effect was specific to the fatty acid transport system, as the transport of glucose and the permeation of retinoic acid, a substance with structural similarities to long-chain fatty acids, were unaffected. Sulfosuccinimidyl oleate reacted exclusively with a plasma membrane protein with an apparent size of 85 kDa while sulfosuccinimidyl myristate also labeled a 75-kDa while sulfosuccinimidyl myristate also labeled a 75-kDa protein. These proteins were among the ones labeled by diisothiocyanodisulfonic acid (DIDS) which also inhibits fatty acid transport irreversibly. The data suggest that the 85-kDa protein, which is the only one labeled by all three inhibitors is involved in facilitating membrane permeation of long-chain fatty acids.     

Synthesis and metabolism of arachidonyl- and eicosapentaenoyl-CoA in rat aorta

In studies on the metabolism of polyunsaturated fatty acids, acyl-CoA synthetase for 5,8,11,14-20:4 (arachidonic acid) and 5,8,11,14,17-20:5 (eicosapentaenoic acid) and the incorporation of these fatty acids into complex lipids and their conversion to CO2 were investigated in rat aorta. The activity of acyl-CoA synthetase was 35.9 for arachidonic acid and 63.0 for eicosapentaenoic acid (nmol/mg protein per 10 min) and the apparent Km values were 45 microM for arachidonic acid and 56 microM for eicosapentaenoic acid. Inhibition of eicosapentaenoyl-CoA synthesis by arachidonic acid was stronger than that of arachidonyl-CoA synthesis by eicosapentaenoic acid. Arachidonic acid and eicosapentaenoic acid were mostly incorporated into phospholipids. The incorporation of these fatty acids into cholesterol ester and their conversion to CO2 were less than those of palmitic acid, but their incorporation into triacyglycerol was greater. The incorporation of these fatty acids into phosphatidylserine + phosphatidylinositol and phosphatidylethanolamine was also greater than that of palmitic acid. The patterns of incorporation of arachidonic acid and eicosapentaenoic acid were similar. The physiological roles of these polyunsaturated fatty acids and the interference of eicosapentaenoic acid in arachidonic acid metabolism are discussed on the basis of these results.     

Increased uptake of fatty acids by the isolated rat liver after raising the fatty acid binding protein concentration with clofibrate

Following the administration of clofibrate to rats, the concentration of Z protein or fatty acid binding protein in liver cytosol increases by 98 %. Ligandin concentration remains unchanged. Isolated perfused livers of clofibrate-treated rats take up free fatty acids from the perfusate at a significantly higher rate (+ 76 %) than controls. Lipid synthesis from radioactive fatty acids is not modified by clofibrate administration. The yield of plasma membranes obtained from liver homogenates as well as their lipid composition are similar in control and clofibrate treated livers. These results seem to exclude the possibility that the enhancement of FFA uptake could result from an indirect effect of the drug on FFA metabolism and/or plasma membrane surface and thus support the view that Z protein plays a role in intracellular fatty acid transport in the liver.     

Control of phosphatidylcholine synthesis in Hep G2 cells. Effect of fatty acids on the activity and immunoreactive content of choline phosphate cytidylyltransferase.

We examined the effect of fatty acids on phosphatidylcholine synthesis and cytidylyltransferase activity in Hep G2 cells. Treatment of Hep G2 cells with oleic acid caused an increase in the incorporation of [methyl-14C]choline into phosphatidylcholine and a corresponding decrease in radioactivity in choline phosphate using a pulse-chase procedure. This result is consistent with a fatty acid-induced increase in the cytidylyl-transferase step in the choline pathway. We measured cytidylyltransferase activity in membrane fractions and in cytosol (100,000 x g supernatant or soluble enzyme released by digitonin). The activity increased in both membrane and cytosol. Thus, an increase in total activity occurred. Cytidylyltransferase protein determined by Western blot immunoassay increased after oleic acid treatment. Immunotitration of cytidylyltransferase protein also indicated that an increase in enzyme protein resulted from oleic acid treatment. Cycloheximide did not prevent the oleic acid-induced increase in cytidylyltransferase activity. The increase in enzyme activity was apparent when we measured the activity in the presence or absence of lipid activators. Separation of cytosolic cytidylyltransferase into H- and L-forms showed that the increase in cytosolic activity was due to an increase in H-form. The amount of L-form did not change. We interpret these results to suggest that fatty acid treatment of Hep G2 cells promoted the formation of active cytidylyltransferase (H-form) from a preexisting inactive form. The increased activity was distributed between membranes and the lipoprotein form in cytosol (H-form).     

Rapid flip-flop of oleic acid across the plasma membrane of adipocytes

Nonesterified long-chain fatty acids may enter cells by free diffusion or by membrane protein transporters. A requirement for proteins to transport fatty acids across the plasma membrane would imply low partitioning of fatty acids into the membrane lipids, and/or a slower rate of diffusion (flip-flop) through the lipid domains compared to the rates of intracellular metabolism of fatty acids. We used both vesicles of the plasma membrane of adipocytes and intact adipocytes to study transmembrane fluxes of externally added oleic acid at concentrations below its solubility limit at pH 7.4. Binding of oleic acid to the plasma membrane was determined by measuring the fluorescent fatty acid-binding protein ADIFAB added to the external medium. Changes in internal pH caused by flip-flop and metabolism were measured by trapping a fluorescent pH indicator in the cells. The metabolic end products of oleic acid were evaluated over the time interval required for the return of intracellular pH to its initial value. The primary findings were that (i) oleic acid rapidly binds with high avidity in the lipid domains of the plasma membrane with an apparent partition coefficient similar to that of protein-free phospholipid bilayers; (ii) oleic acid rapidly crosses the plasma membrane by the flip-flop mechanism (both events occur within 5 s); and (iii) the kinetics of esterification of oleic acid closely follow the time dependence of the recovery of intracellular pH. Any postulated transport mechanism for facilitating translocation of fatty acid across the plasma membrane of adipocytes, including a protein transporter, would have to compete with the highly effective flip-flop mechanism.     

Incorporation of myristate and palmitate into the sheep reticulocyte transferrin receptor: evidence for identical sites of labeling

The ability of sheep reticulocytes and plasma membranes isolated from them to incorporate fatty acids into the transferrin receptor has been examined using both [3H]palmitate and [3H]myristate. Both fatty acids, when incorporated into the transferrin receptor, can be released by treating the protein with 1 M hydroxylamine at pH 7.0. After treatment of the 3H-acylated receptor with borohydride, an 3H-labeled alcohol is released, suggesting that the receptor-bound fatty acid is in thioester linkage. With both [3H]myristate and [3H]palmitate, Cleveland maps from immunoprecipitates of the transferrin receptor labeled in intact cells and isolated membranes show that identical peptides are labeled. No evidence was obtained for qualitatively different labeling with the two fatty acids. In intact reticulocytes, incorporation of [3H]palmitate into the transferrin receptor is approximately 3.5 times greater than the incorporation of [3H]myristate from equivalent concentrations of the labeled fatty acids. However, in isolated reticulocyte plasma membranes, there is much less difference between palmitate and myristate incorporation (with ATP) or between their acyl-CoA derivatives. The reason for the discrepancy between cells and membranes is unknown but may be due to the presence in intact cells of more than one enzyme for activating the fatty acids. Acylation of the receptor in isolated plasma membranes is fourfold greater with the CoA derivatives than with the free fatty acids. The fatty acid activating enzyme(s) as well as the acyltransferase(s) appear to be membrane bound in reticulocytes.     

Pathway of carbon flow during fatty acid synthesis from lactate and pyruvate in rat adipose tissue

The metabolism of pyruvate and lactate by rat adipose tissue was studied. Pyruvate and lactate conversion to fatty acids is strongly concentration-dependent. Lactate can be used to an appreciable extent only by adipose tissue from fasted-refed rats. A number of compounds, including glucose, pyruvate, aspartate, propionate, and butyrate, stimulated lactate conversion to fatty acids. Based on studies of incorporation of lactate-2-(3)H and lactate-2-(14)C into fatty acids it was suggested that the transhydrogenation sequence of the "citrate-malate cycle"(1) was not providing all of the NADPH required for fatty acid synthesis from lactate. An alternative pathway for NADPH formation involving the conversion of isocitrate to alpha-ketoglutarate via cytosolic isocitrate dehydrogenase was proposed. Indirect support for this proposal was provided by the rapid labeling of glutamate from lactate-2-(14)C by adipose tissue incubated in vitro, as well as the demonstration that glutamate can be readily metabolized by adipose tissue via reactions localized largely in the cytosol. Furthermore, isolated adipose tissue mitochondria convert alpha-ketoglutarate to malate, or in the presence of added pyruvate, to citrate. Glutamate itself can not be metabolized by these mitochondria, a finding in keeping with the demonstration of negligible levels of NAD-glutamate dehydrogenase activity in adipose tissue mitochondria. Pyruvate stimulated alpha-ketoglutarate and malate conversion to citrate and reduced their oxidation to CO(2). It is proposed that under conditions of excess generation of NADH malate may act as a shuttle carrying reducing equivalents across the mitochondrial membrane. Malate at low concentrations increased pyruvate conversion $$Word$$ citrate and markedly decreased the formation of CO(2) by isolated adipose tissue mitochondria. Malate also stimulated citrate and isocitrate metabolism by these mitochondria, an effect that could be blocked by 2-n-butylmalonate. This potentially important role of malate in the regulation of carbon flow during lipogenesis is underlined by the observation that 2-n-butylmalonate inhibited fatty acid synthesis from pyruvate, but not from glucose and acetate, and decreased the stimulatory effect of pyruvate on acetate conversion to fatty acids.     

Plasma membrane phospholipid translocation in the mouse peritoneal macrophage: differential response to stimulation of eicosanoid production.

The metabolism and translocation of exogenously introduced plasma membrane phosphatidylcholine (PC) having the fluorescent fatty acid analog aminocaproyl NBD (N-nitrobenzo-2-oxa-1,3 diazole) (NBD-PC), in the sn2 position was studied in cultured murine peritoneal macrophages using biochemical and morphological techniques. Following labeling of the cell plasma membrane at 2 degrees C by vesicle lipid exchange, macrophages were warmed in the presence or absence of pharmacological stimuli of eicosanoid production and release. Fluorescence microscopy indicated that the phospholipid was translocated to an internal cellular pool upon stimulation with zymosan. In contrast, the membrane PC analog was primarily metabolized and released after being found diffusely associated with the cytoplasm in macrophages stimulated with the calcium ionophore A23187. Evidence obtained by double labeling zymosan-treated macrophages with NBD-PC and a monoclonal antibody directed against a lysosomal membrane protein demonstrated that the fluorescent lipid is internalized in association with the zymosan particles and both are found in lysosomes. The results suggest that multiple pathways exist in peritoneal macrophages which target plasma membrane PC into different cellular compartments for hydrolysis and conversion to eicosanoid products and release from cells.     

The effect of hypolipidemic drugs on plant lipid metabolism

The effect of hypolipidemic drugs, WY14643 and DH990, on plant lipid metabolism has been studied. The total incorporation of [14C]acetate into lipids was inhibited by addition of both drugs to aged potato (Solanum tuberosum) tuber discs, spinach (Spinacia oleracea) leaves, and spinach chloroplasts, while the incorporation in Chlorella vulgaris cells was affected only by DH990. Moreover, DH990 inhibited the incorporation of 14C-labeled fatty acids into phosphatidylcholine and phosphatidylethanolamine of potato discs, and decreased the incorporation into phosphatidylglycerol of Chlorella cells. DH990 inhibited the formation of polyunsaturated fatty acids in potato discs, Chlorella cells, and spinach leaves, whereas WY14643 had no effect on the formation of these fatty acids. Stearoyl-ACP desaturase from safflower (Carthamus tinctorius) seeds was very sensitive to both drugs, especially DH990, which completely blocked the activity at 2 mM levels. When safflower lysophospholipid acyltransferases were solubilized by detergent treatment, only DH990 inhibited the incorporation of [14C]oleoyl-CoA into lysophosphatidylcholine or lysophosphatidylethanolamine. Both drugs inhibited fatty acid synthesis from [14C]malonyl-CoA in the microsomal fraction from safflower seeds, but only DH990 inhibited FAS activity in the soluble fraction; both drugs inhibited severely the formation of stearic acid. Both acetyl-CoA carboxylase and acetyl-CoA synthetase were sensitive to both drugs.     

Acute regulation of cardiac metabolism by the hexosamine biosynthesis pathway and protein O-GlcNAcylation

Objective

The hexosamine biosynthesis pathway (HBP) flux and protein O-linked N-acetyl-glucosamine (O-GlcNAc) levels have been implicated in mediating the adverse effects of diabetes in the cardiovascular system. Activation of these pathways with glucosamine has been shown to mimic some of the diabetes-induced functional and structural changes in the heart; however, the effect on cardiac metabolism is not known. Therefore, the primary goal of this study was to determine the effects of glucosamine on cardiac substrate utilization.

Methods

Isolated rat hearts were perfused with glucosamine (0–10 mM) to increase HBP flux under normoxic conditions. Metabolic fluxes were determined by ¹³C-NMR isotopomer analysis; UDP-GlcNAc a precursor of O-GlcNAc synthesis was assessed by HPLC and immunoblot analysis was used to determine O-GlcNAc levels, phospho- and total levels of AMPK and ACC, and membrane levels of FAT/CD36.

Results

Glucosamine caused a dose dependent increase in both UDP-GlcNAc and O-GlcNAc levels, which was associated with a significant increase in palmitate oxidation with a concomitant decrease in lactate and pyruvate oxidation. There was no effect of glucosamine on AMPK or ACC phosphorylation; however, membrane levels of the fatty acid transport protein FAT/CD36 were increased and preliminary studies suggest that FAT/CD36 is a potential target for O-GlcNAcylation.

Conclusion/Interpretation

These data demonstrate that acute modulation of HBP and protein O-GlcNAcylation in the heart stimulates fatty acid oxidation, possibly by increasing plasma membrane levels of FAT/CD36, raising the intriguing possibility that the HBP and O-GlcNAc turnover represent a novel, glucose dependent mechanism for regulating cardiac metabolism.