Distribution and metabolism of arachidonic and docosahexaenoic acids in rat pineal cells. Effect of norepinephrine

The time-course incorporation of 10 μM [¹⁴C]arachidonic (AA) and docosahexaenoic (DHA) acids into glycerolipids was studied in rat pineal cells. The incorporation of both labeled fatty acids into total lipids was approximately equal, but their distribution profiles among the various cell lipids showed marked differences. The esterification of [¹⁴C]DHA in the neutral lipids, triacylglycerols (TAG) and cholesterol esters (CE), was 2-fold higher than that of [¹⁴C]AA whereas the opposite could be observed in total phospholipids (PL). The order of incorporation into PL was phosphatidylcholine (PC) > phosphatidylinositol (PI) = phosphatidylethanolamine (PE) for [¹⁴C]AA and PC = PE for [¹⁴C]DHA, the incorporation of both fatty acids being not detected in phosphatidylserine (PS) and that of DHA not in PI. When using 0.5 μM [³H] fatty acids, the respective distribution patterns resembled that of fatty acids at 10 μM, except for a lower proportion in TAG. The stimulation of ³H-labeled cells by 100 μM norepinephrine induced a 170% increase of basal release of [³H]AA into the medium, while [³H]DHA was virtually not released. However, the analysis of cell labeling revealed that both [³H] fatty acid levels were decreased in PL and increased in TAG. These findings suggest different involvement for AA and DHA in the pineal function. The preferential incorporation of DHA in TAG suggests that TAG might play an important role in the pineal enrichment with DHA. The absence of DHA release after NE stimulation, which however cannot be ascertained, may raise the question of the role of DHA in NE transduction.     

Protein Kinase D1 (PKD1) Phosphorylation Promotes Dopaminergic Neuronal Survival during 6-OHDA-Induced Oxidative Stress

Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson’s disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1^WT) or constitutively active PKD1 (PKD1^S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Neurotransmitter-induced changes in arachidonate incorporation into rat brain phospholipids: pharmacological manipulations of receptors,presynaptic uptake,mao activity and deacylation-reacylation processes

The incorporation of [³H]arachidonic acid ([³H]AA) into phospholipids (PL) of rat brain, was studied in cerebral cortex slices in the presence and absence of norepinephrine (NE), serotonin (5-HT) and carbamylcholine (CCH). Both NE and 5-HT produced a concentration-dependent effect of stimulating [³H]AA incorporation into phosphatidylinositol (PI) while attenuating incorporation into other PL. Addition of CCH had no apparent effect. The β-adrenergic agonist, isoproterenol, had an effect similar to that seen with equimolar concentrations of NE, whereas the α₁ agonist, phenylephrine, or the α₂ agonist, clonidine, did not produce significant changes. However, application of the NE-receptor blockers, propranolol or prazosin, in the presence of NE, did not modify the NE-induced effects. Similarly, the 5-HT-receptor blockers, methysergide or ketanserine, failed to modify the 5-HT-induced effects, indicating that the neurotransmitter-produced changes may not be receptor mediated. Manipulations of the NE or 5-HT reuptake systems by imipramine (IMI) or desipramine (DMI) had a small additive effect on the neurotransmitter-produced changes in [³H]AA incorporation, suggesting that a functional presynaptic reuptake system is not required for the NE or 5-HT-produced effects. The possibility that the NE or 5-HT effects involve the oxidative metabolism of the monoamines by MAO was also investigated. The MAO inhibitors tranylcypromine and pargyline had no appreciable effect on the neurotransmitter-induced changes in [³H]AA incorporation whereas clorgyline clearly reduced the increase in [³H]AA incorporation into PI seen in the presence of NE or 5-HT, but this clorgyline effect may not be related to its activity as MAO inhibitor. The phospholipase A₂ inhibitor mepacrine had no significant effect on the NE-produced increase in [³H]AA incorporation into PI, but it antagonized the NE-produced decrease in [³H]AA incorporation into PC. Delta-9-Tetrahydrocannabinol, which acts as acyltransferase inhibitor, antagonized the NE-produced increase in [³H]AA incorporation into PI without appreciably influencing the NE-produced decrease in [³H]AA incorporation into PC. These findings suggest that the neurotransmitter-produced increase in [³H]AA incorporation into PI is mediated by stimulation of a specific lyso-PI arachidonyl transferase. The neurotransmitter effects on arachidonate incorporation may have physiological significance in view of the importance of processes of deacylation and reacylation of membrane PL in regulating the function of neuronal membranes.     

Oleic Acid Alleviates Cadmium-Induced Oxidative Damage in Rat by Its Radicals Scavenging Activity

Toxic heavy metal cadmium wildly pollutes the environment and threats the human health. Effective treatment of cadmium-induced toxicity and organ damage is an important issue. Cadmium causes organ damage through inducing oxidative stress. Our previous study also found oleic acid (OA) synthesis-related gene can confer resistance to cadmium and alleviate cadmium-induced stress in yeast. However, its alleviation mechanism on cadmium stress especially in animals is still unclear. In this study, the alleviative effects of OA on cadmium and cadmium-induced oxidative stress in rats were investigated. Oral administration of 10, 20, and 30 mg/kg/day OA can significantly increase the survival rate of rats intraperitoneally injected with 30 mg/kg/day cadmium continuously for 7 days. Similar to ascorbic acid (AA), OA can significantly reduce the cadmium-induced lipid peroxidation in multiple organs of rats. The investigation of OA on superoxide dismutase (SOD) activity showed that OA increased the SOD activity of cadmium-treated rat organs. More important, OA reduced the level of superoxide radical O²⁻ of cadmium-treated rat organs. And OA exhibited a strong DPPH radicals scavenging activity at dose of 10, 20 and 30 mg/mL, which may contributed to alleviating cadmium-induced oxidative stress. This study revealed that OA could significantly alleviate cadmium stress via reducing cadmium-induced lipid peroxidation and SOD activity inhibition through its radicals scavenging activity.

     

Effect of estradiol and progesterone on the rate of incorporation of radiolabeled arachidonate into phospholipids and triglycerides of the rat uterus

To obtain information on the effect of estradiol (E) and progesterone (P) on the overall rate of the acylation and deacylation reactions with [3H]arachidonate ([3H]AA) in the rat uterus, we investigated the effect of chronic treatment of ovariectomized rats with 2-200 micrograms/day E-dipropionate (EPP) and with 2 mg P alone or with the combination of these steroids on the extent and the time course of the in vitro incorporation of [3H]AA into various phospholipids (PLs) and the triacylglycerol fraction (TG). The results demonstrate that physiologic doses of E leads to a rapid equilibrium of the deacylation-acylation cycle only in the case of phosphatidylinositol (PI) whereas at high (200 micrograms/day) dose level it exerts the same effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) as well as on TG. P alone has no remarkable effects in the ovariectomized rat, but it decreases markedly the incorporation of [3H]AA into PI and TG in intact animals. Furthermore, P decreases the incorporation of [3H]AA into PI in the ovariectomized rat treated with 2 micrograms/day EPP as well as attenuating the enhanced labeling of PC, PE and PI caused by higher doses. Time-course studies provide evidence that all of these effects of progesterone can be accounted for by its ability to decrease the rate of deacylation and, therefore, to prolong the time needed to attain equilibrium in the rates of acylation and deacylation reactions of the various lipids with arachidonate. These data offer a novel outlook on the regulatory role of progesterone and estrogens in uterine function.     

Neutral Lipid Metabolism Influences Phospholipid Synthesis and Deacylation in Saccharomyces cerevisiae

Establishment and maintenance of equilibrium in the fatty acid (FA) composition of phospholipids (PL) requires both regulation of the substrate available for PL synthesis (the acyl-CoA pool) and extensive PL turnover and acyl editing. In the present study, we utilize acyl-CoA synthetase (ACS) deficient cells, unable to recycle FA derived from lipid deacylation, to evaluate the role of several enzymatic activities in FA trafficking and PL homeostasis in Saccharomyces cerevisiae. The data presented show that phospholipases B are not contributing to constitutive PL deacylation and are therefore unlikely to be involved in PL remodeling. In contrast, the enzymes of neutral lipid (NL) synthesis and mobilization are central mediators of FA trafficking. The phospholipid:DAG acyltransferase (PDAT) Lro1p has a substantial effect on FA release and on PL equilibrium, emerging as an important mediator in PL remodeling. The acyl-CoA dependent biosynthetic activities of NL metabolism are also involved in PL homeostasis through active modulation of the substrate available for PL synthesis. In addition TAG mobilization makes an important contribution, especially in cells from stationary phase, to FA availability. Beyond its well-established role in the formation of a storage pool, NL metabolism could play a crucial role as a mechanism to uncouple the pools of PL and acyl-CoAs from each other and thereby to allow independent regulation of each one.     

Differential changes in lipid metabolism of myeloid and lymphoid cell lines induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA)

Treatment of the myeloid cell lines, U-937 or HL-60, with 10 nM of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), for 24 h increased the rate of incorporation of [3H]glycerol into total chloroform extracts. A proportionally greater labeling of the non-polar lipid (NL) fraction compared to the polar, phospholipid (PL), fraction was observed. Chromatographic analysis showed a 6-fold increase in the labeling of triacylglycerols (TAG), a 2-fold increase in diacylglycerols, and no changes in monoacylglycerols. PL labeling showed a 3-fold increase in phosphatidylcholine (PC). The effect of TPA on TAG labeling was selectively observed in myeloid cell lines. No such a change was found in the lymphoid cell line. MOLT-3, which did respond to TPA with increased PC labeling. Incorporation of [3H]arachidonic acid (AA) into TAG by U-937 cells was selectively increased (2.5-fold) after treatment with TPA for 24 h. Treatment of U-937 cells with TPA in serum-free medium resulted in no increased labeling of TAG. These studies suggest that changes in TAG metabolism may be characteristic of myeloid differentiation and depend on the presence of serum factor(s).     

多巴胺能神经元线粒体的氧化损伤与帕金森病

帕金森病(Parkinson’s disease,PD)的一个主要病理特征就是中脑黑质多巴胺能神经元的丧失,目前研究认为该病理变化与多种因素有关,包括蛋白质异常积聚、泛素蛋白酶体系统功能异常、神经炎症、线粒体损伤和氧化应激。在帕金森病人和动物模型中,中脑黑质有着明显的氧化改变。帕金森病的遗传和环境因素均会作用于线粒体,尤其对线粒体呼吸链复合体I有着抑制作用,造成线粒体损伤,产生活性氧(ROS)。活性氧的大量产生造成脂类、蛋白质和DNA的氧化,从而加剧多巴胺能神经元的线粒体和细胞损伤。多巴胺代谢过程中会产生活性氧,该自身代谢特点决定了多巴胺能神经元存在有较高的氧化应激,易受环境因素的影响。因而,线粒体的氧化损伤在帕金森病病理发生中起着重要作用。     

Triacylglycerol accumulation is not primarily affected in myotubes established from type 2 diabetic subjects

In the present study, we investigated triacylglycerol (TAG) accumulation, glucose and fatty acid (FA) uptake, and glycogen synthesis (GS) in human myotubes from healthy, lean, and obese subjects with and without type 2 diabetes (T2D), exposed to increasing palmitate (PA) and oleate (OA) concentrations with/without high glucose and/or high insulin concentrations for 4 days. We showed that these myotubes expressed an increased TAG accumulation (P<0.001) without differences between groups. Chronically high insulin, but not high glucose concentrations, increases TAG accumulation by 25% (P<0.001). Inhibition of oxidative phosphorylation by antimycin A and oligomyin was followed by a reduced lipid oxidation (P<0.05) and increased TAG accumulation (P<0.05), but only in the presence of FAs. Both chronic PA and OA exposure reduced the insulin-mediated PA and OA uptake (fold change) (P<0.001), but could not induce insulin resistance at the level of glucose uptake, whereas high insulin concentrations induced insulin resistance (P<0.001). Chronic, high PA, but not OA, induced insulin resistance at the GS level in control subjects (P<0.05). The TAG content correlated negatively with insulin-stimulated FA uptake (P<0.001), but did not correlate with insulin-stimulated glucose uptake for PA or OA (P>0.05). These results indicate that (1) TAG accumulation is not primarily affected in skeletal muscle tissue of obese and T2D; (2) induced inhibition of oxidative phosphorylation is followed by TAG accumulation; (3) increasing FA and insulin availability, and reduced oxidative phosphorylation, and to a lesser extent glucose, are determinants for differences in intramyocellular TAG accumulation; (4) quantitative TAG content may not be the best marker for insulin resistance. Thus, increased TAG content in skeletal muscle of obese and T2D subjects is adaptive.     

Role of oxidative stress in paraquat-induced dopaminergic cell degeneration

Systemic treatment of mice with the herbicide paraquat causes the selective loss of nigrostriatal dopaminergic neurons, reproducing the primary neurodegenerative feature of Parkinson's disease. To elucidate the role of oxidative damage in paraquat neurotoxicity, the time-course of neurodegeneration was correlated to changes in 4-hydroxy-2-nonenal (4-HNE), a lipid peroxidation marker. When mice were exposed to three weekly injections of paraquat, no nigral dopaminergic cell loss was observed after the first administration, whereas a significant reduction of neurons followed the second exposure. Changes in the number of nigral 4-HNE-positive neurons suggest a relationship between lipid peroxidation and neuronal death, since a dramatic increase in this number coincided with the onset and development of neurodegeneration after the second toxicant injection. Interestingly, the third paraquat administration did not cause any increase in 4-HNE-immunoreactive cells, nor did it produce any additional dopaminergic cell loss. Further evidence of paraquat-induced oxidative injury derives from the observation of nitrotyrosine immunoreactivity in the substantia nigra of paraquat-treated animals and from experiments with ferritin transgenic mice. These mice, which are characterized by a decreased susceptibility to oxidative stress, were completely resistant to the increase in 4-HNE-positive neurons and the cell death caused by paraquat. Thus, paraquat exposure yields a model that emphasizes the susceptibility of dopaminergic neurons to oxidative damage.     

Superoxide dismutase/catalase mimetics are neuroprotective against selective paraquat-mediated dopaminergic neuron death in the substantial nigra: implications for Parkinson disease

Exposure of mice to the herbicide paraquat has been demonstrated to result in the selective loss of dopaminergic neurons of the substantia nigra, pars compacta (SNpc) akin to what is observed in Parkinson disease (PD). In this study, we investigate the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK-134 and EUK-189) in protecting against paraquat-induced dopaminergic cell death in both the rat dopaminergic cell line 1RB3AN27 (N27) and primary mesencephalic cultures in vitro and in adult mice in vivo. Our data demonstrate that pretreatment with either EUK-134 or EUK-189 significantly attenuates paraquat-induced neurotoxicity in vitro in a concentration-dependent manner. Furthermore, systemic administration of EUK-189 decreases paraquat-mediated SNpc dopaminergic neuronal cell death in vivo. These findings support a role for oxidative stress in paraquat-induced neurotoxicity and suggest novel therapeutic approaches for neurodegenerative disorders associated with oxidative stress such as PD.     

Berberine treatment attenuates the palmitate-mediated inhibition of glucose uptake and consumption through increased 1,2,3-triacyl-sn-glycerol synthesis and accumulation in H9c2 cardiomyocytes

Dysfunction of lipid metabolism and accumulation of 1,2-diacyl-sn-glycerol (DAG) may be a key factor in the development of insulin resistance in type 2 diabetes. Berberine (BBR) is an isoquinoline alkaloid extract that has shown promise as a hypoglycemic agent in the management of diabetes in animal and human studies. However, its mechanism of action is not well understood. To determine the effect of BBR on lipid synthesis and its relationship to insulin resistance in H9c2 cardiomyocytes, we measured neutral lipid and phospholipid synthesis and their relationship to glucose uptake. Compared with controls, BBR treatment stimulated 2-[1,2-³H(N)]deoxy-D-glucose uptake and consumption in palmitate-mediated insulin resistant H9c2 cells. The mechanism was though an increase in protein kinase B (AKT) activity and GLUT-4 glucose transporter expression. DAG accumulated in palmitate-mediated insulin resistant H9c2 cells and treatment with BBR reduced this DAG accumulation and increased accumulation of 1,2,3-triacyl-sn-glycerol (TAG) compared to controls. Treatment of palmitate-mediated insulin resistant H9c2 cells with BBR increased [1,3-³H]glycerol and [1-¹⁴C]glucose incorporation into TAG and reduced their incorporation into DAG compared to control. In addition, BBR treatment of these cells increased [1-¹⁴C]palmitic acid incorporation into TAG and decreased its incorporation into DAG compared to controls. BBR treatment did not alter phosphatidylcholine or phosphatidylethanolamine synthesis. The mechanism for the BBR-mediated decreased precursor incorporation into DAG and increased incorporation into TAG in palmitate-incubated cells was an increase in DAG acyltransferase-2 activity and its expression and a decrease in TAG hydrolysis. Thus, BBR treatment attenuates palmitate-induced reduction in glucose uptake and consumption, in part, through reduction in cellular DAG levels and accumulation of TAG in H9c2 cells.     

Hydrogen in Drinking Water Reduces Dopaminergic Neuronal Loss in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Mouse Model of Parkinson's Disease

It has been shown that molecular hydrogen (H₂) acts as a therapeutic antioxidant and suppresses brain injury by buffering the effects of oxidative stress. Chronic oxidative stress causes neurodegenerative diseases such as Parkinson''s disease (PD). Here, we show that drinking H₂-containing water significantly reduced the loss of dopaminergic neurons in PD model mice using both acute and chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The concentration-dependency of H₂ showed that H₂ as low as 0.08 ppm had almost the same effect as saturated H₂ water (1.5 ppm). MPTP-induced accumulation of cellular 8-oxoguanine (8-oxoG), a marker of DNA damage, and 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation were significantly decreased in the nigro-striatal dopaminergic pathway in mice drinking H₂-containing water, whereas production of superoxide (O₂•⁻) detected by intravascular injection of dihydroethidium (DHE) was not reduced significantly. Our results indicated that low concentration of H₂ in drinking water can reduce oxidative stress in the brain. Thus, drinking H₂-containing water may be useful in daily life to prevent or minimize the risk of life style-related oxidative stress and neurodegeneration.     

Preferential distribution of long chain polyunsaturated fatty acids in phospatidyl ethanolamine fraction of guinea pig alveolar apical membranes

We investigated the fatty acid distribution in guinea pig alveolar apical membranes at different developmental stages. Fatty acid composition of the purified membranes isolated from guinea pig fetuses (at 65 day, term=68 day), neonates (day 1) and adult males was determined. The levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) were higher in the adult guinea pig alveolar apical membrane phosphatidylethanolamine (PE) fraction (9. 3+/-2.2 and 2.9+/-1.0%, respectively) while in other phospholipids (PL) fractions their levels were low or absent (P<0.01). Furthermore, levels of AA and DHA in the PE fraction of apical membrane increased significantly from fetal (6.6+/-3.0 and 0.8+/-0.4%, respectively) to neonatal life (10.3+/-1.5 and 3.0+/-0.8%, respectively). Increase in the level of DHA (almost four-fold) was much more pronounced than that of AA (P<0.05). As for guinea pig alveolar membranes, EPA and AA were mostly present in the PE fraction in pulmonary adenocarcinoma derived cells (A549 cells), a parallel model of type II pneumocytes, with the levels of AA around three-fold greater than that of EPA, Binding of radiolabelled fatty acids to A549 cells showed no significant differences between the maximum uptake achieved for different fatty acids (AA, 1.7+/-0.2, EPA, 2.3+/-0.3, LA, 1.7+/-0.2, OA, 2.0+/-0.2nmol/mg protein, P>0.5). Once the fatty acids were taken up by these cells AA was mostly identifiable in the monoacylglycerol (MAG) fraction, whereas EPA was equally distributed between the MAG and PL fractions. Oleic acid was mainly present in the triglyceride (TAG) fraction whereas LA was evenly distributed between the TAG, MAG, and PL fractions. Our data demonstrate a preferential distribution of AA and DHA in PE fractions of alveolar apical membranes during development.     

Ferrylmyoglobin impairs secretion of VLDL triacylglycerols from stored intracellular pools: involvement of lipid peroxidation

Ferrylmyoglobin (ferrylMb) may play a major role in vivo under certain pathological conditions. Preliminary experiments showed that ferrylmyoglobin induced a mild oxidative stress in rat hepatocytes, mainly reflected by early lipid peroxidation. One of the major functions of hepatocytes is the synthesis, secretion and distribution of lipids to other cells. The aim of this work was to examine whether ferrylMb affected the synthesis and secretion of triacylglycerols (TAG), and the possible involvement of lipid peroxidation on these effects. The heme protein completely impaired VLDL secretion, affecting both the lipid and apoB components of the lipoprotein particle. The incorporation of [(3)H]-oleate into newly synthesized diacylglycerol and TAG was not altered by ferrylMb. The co-treatment of cells with alpha-tocopherol prevented lipid peroxidation and concomitantly reverted VLDL TAG secretion to control values. Importantly, although ferrylMb dramatically blocked prelabeled TAG secretion, newly synthesized TAG secretion was not impaired. These data indicate that lipid peroxidation elicited by ferrylMb modulates the VLDL TAG secretion process, specifically affecting the stored intracellular TAG mobilization, rather than de novo synthesis. Apart from its potential role in vivo, ferrylmyoglobin constitutes a useful model for studying the interactions between lipid peroxidation and the specific TAG pool dependence for VLDL secretion.     

Acyl-CoAs are functionally channeled in liver: potential role of acyl-CoA synthetase

Acyl-CoA synthetase (ACS) catalyzes the activation of long-chain fatty acids to acyl-CoAs, which can be metabolized to form CO(2), triacylglycerol (TAG), phospholipids (PL), and cholesteryl esters (CE). To determine whether inhibiting ACS affects these pathways differently, we incubated rat hepatocytes with [(14)C]oleate and the ACS inhibitor triacsin C. Triacsin inhibited TAG synthesis 70% in hepatocytes from fed rats and 40% in starved rats, but it had little effect on oleate incorporation into CE, PL, or beta-oxidation end products. Triacsin blocked [(3)H]glycerol incorporation into TAG and PL 33 and 25% more than it blocked [(14)C]oleate incorporation, suggesting greater inhibition of de novo TAG synthesis than reacylation. Triacsin did not affect oxidation of prelabeled intracellular lipid. ACS1 protein was abundant in liver microsomes but virtually undetectable in mitochondria. Refeeding increased microsomal ACS1 protein 89% but did not affect specific activity. Triacsin inhibited ACS specific activity in microsomes more from fed than from starved rats. These data suggest that ACS isozymes may be functionally linked to specific metabolic pathways and that ACS1 is not associated with beta-oxidation in liver.     

Arachidonic Acid-Induced Oxidative Injury to Cultured Spinal Cord Neurons

Spinal cord trauma can cause a marked release of free fatty acids, in particular, arachidonic acid (AA), from cell membranes. Free fatty acids, and AA by itself, may lead to secondary damage to spinal cord neurons. To study this hypothesis, cultured spinal cord neurons were exposed to increasing concentrations of AA (0.01-10 microM). AA-induced injury to spinal cord neurons was assessed by measurements of cellular oxidative stress, intracellular calcium levels, activation of nuclear factor-KB (NF-kappaB), and cell viability. AA treatment increased intracellular calcium concentrations and decreased cell viability. Oxidative stress increased significantly in neurons exposed to 1 and 10 microM AA. In addition, AA treatment activated NF-kappaB and decreased levels of the inhibitory subunit, IKB. It is interesting that manganese superoxide dismutase protein levels and levels of intracellular total glutathione increased in neurons exposed to this fatty acid for 24 h, consistent with a compensatory response to increased oxidative stress. These results strongly support the hypothesis that free fatty acids contribute to the tissue injury observed following spinal cord trauma.     

Reversible activation/inactivation of the deacylation/acylation cycle in rat liver microsomes

Rat liver microsomes incorporate [¹⁴C]palmitoyl CoA into membrane phospholipids via the deacylation/acylation cycle. This activity is reversibly inactivated/activated by treatment of the microsomes with ATP, MgCl₂, and 105,000g supernatant or with 105,000g supernatant alone. These observations suggest that the acylation cycle is controlled by a mechanism involving phosphorylation/dephosphorylation. As the pool of lysolecithin in the membranes is not altered by conditions increasing incorporation of palmitoyl CoA into phospholipid, it is probable that the site of regulation of deacylation/acylation is at the acyltransferase rather than the phospholipase.