Protein kinase C activity in neonatal cultured rat cardiomyocytes supplemented with docosahexaenoic acid.

In vitro studies have indicated that the 1-stearoyl, 2-arachidonyl diacylglycerol (DAG) is the most effective one for the activation of protein kinase C, although many other DAGs having a different fatty acid composition are active, but to a different extent. Using cultures of neonatal rat ventricular cells, grown in a medium enriched in docosahexaenoic acid (DHA), we previously obtained a cell population that, after alpha 1-adrenoceptor stimulation, produced a DHA enriched DAG. In this study, we have tested the "in vivo" ability of this modified DAG as protein kinase C activator, demonstrating a lower but more persistent translocation of the enzyme from cytosol to particulate fraction in the DHA treated cells. The differences in the PKC activation pattern could be explained by a different metabolism of the DHA enriched DAG by DAG kinase.     

Alteration of glycerolipid and sphingolipid-derived second messenger kinetics in ras transformed 3T3 cells

The effect of ras transformation (rasB fibroblasts) on basal and serum-stimulated diacylglcerol (DAG) composition and mass was examined over time with respect to changes in membrane phospholipid composition and ceramide mass. RasB cells vs. Nontransformed control cells (rasD and NR6) had chronically elevated DAG levels (up to 240 min) following serum stimulation, indicating a defect in the recovery phase of the intracellular DAG pulse. Ras transformation also had a dramatic effect on DAG composition. Molecular species analysis revealed that DAG from unstimulated rasB cells was enriched inthe Δ9 desaturase fatty acyl species (monoenoate 18:1 (n − 7) and 18:1 (n−9)), and depleted in arachidonic acid (20:4(n−6)). With the exception of glycerophosphoinositol (GPI), DAG remodeling paralleled the compositional alterations in individual phospholipid classes. Importantly, ras transformation altered the fatty acyl composition of sphingomyelin, a precursor to the ceramide second messenger. With the addition of serum, control cells (rasD) had a progressive increase in ceramide mass with levels approximately 5-fold higher by 240 min. In contrast, ceramide levels did not increase in rasB cells at either 4 or 240 min. These results demonstrate that ras-oncogene, in addition to its effects on DAG metabolism, can also abolish the cellular increase in ceramide mass in response to serum stimulation. Since DAG and ceramide may have opposing biological functions, the prolonged elevation of DAG and the suppression of ceramide levels would be consistent with an enhanced proliferative capacity.     

Stimulus-response coupling in insulin-secreting HIT cells. Effects of secretagogues on cytosolic Ca2+, diacylglycerol, and protein kinase C activity

The hamster islet B cell line HIT retains the ability to secret insulin in response to glucose and several receptor agonists. We used HIT cells to study the initial signaling events in glucose or receptor agonist-stimulated insulin secretion. Glucose stimulated insulin release from HIT cells in a dose-dependent manner with a half-maximal effect seen already at 1 mM. Insulin release was also stimulated by carbachol in a glucose-dependent manner. Glucose depolarized the HIT cell membrane potential as assessed with the fluorescent probe bisoxonol and raised intracellular Ca2+ as revealed by fura-2 measurements. Using a Mn2+ fura-2 quenching technique, we could show that the rise in intracellular Ca2+ was due to Ca2+ influx following opening of voltage-gated Ca2+ channels. Glucose is thought to increase the diacylglycerol (DAG) content of insulin-secreting cells. However, although HIT cells respond to glucose in terms of insulin secretion, membrane depolarization, and Ca2+ rise, the hexose was unable to increase the proportion of protein kinase C activity associated with membranes. In contrast, the membrane-associated protein kinase C activity increased in HIT cells exposed to the two receptor agonists carbachol and bombesin. Bombesin was shown to generate DAG with the expected fatty acid composition of activators of phospholipase C. Glucose, in contrast, only caused minor increases in DAG containing myristic and palmitic acid without affecting total DAG mass. The failure to detect stimulation of protein kinase C by glucose could be due to both the limited amount and to the different fatty acid composition of the metabolically generated DAG. The latter was in part supported by experiments performed on protein kinase C partially purified from HIT cells. Indeed, 1,2-dipalmitoylglycerol, presumed to be the main DAG species generated by glucose, was only one-third as active as 1,2-dioleoylglycerol and 1-stearoyl-2-arachidonylglycerol in stimulating the isolated enzyme at physiological Ca2+ concentration. It is therefore unlikely that DAG and protein kinase C play a major role in glucose-stimulated insulin secretion.     

The phosphatidylcholine diacylglycerol cholinephosphotransferase is required for efficient hydroxy fatty acid accumulation in transgenic Arabidopsis

We previously identified an enzyme, phosphatidylcholine diacylglycerol cholinephosphotransferase (PDCT), that plays an important role in directing fatty acyl fluxes during triacylglycerol (TAG) biosynthesis. The PDCT mediates a symmetrical interconversion between phosphatidylcholine (PC) and diacylglycerol (DAG), thus enriching PC-modified fatty acids in the DAG pool prior to forming TAG. We show here that PDCT is required for the efficient metabolism of engineered hydroxy fatty acids in Arabidopsis (Arabidopsis thaliana) seeds. When a fatty acid hydroxylase (FAH12) from castor (Ricinus communis) was expressed in Arabidopsis seeds, the PDCT-deficient mutant accumulated only about half the amount of hydroxy fatty acids compared with that in the wild-type seeds. We also isolated a PDCT from castor encoded by the RcROD1 (Reduced Oleate Desaturation1) gene. Seed-specific coexpression of this enzyme significantly increased hydroxy fatty acid accumulation in wild type-FAH12 and in a previously produced transgenic Arabidopsis line coexpressing a castor diacylglycerol acyltransferase 2. Analyzing the TAG molecular species and regiochemistry, along with analysis of fatty acid composition in TAG and PC during seed development, indicate that PDCT acts in planta to enhance the fluxes of fatty acids through PC and enrich the hydroxy fatty acids in DAG, and thus in TAG. In addition, PDCT partially restores the oil content that is decreased in FAH12-expressing seeds. Our results add a new gene in the genetic toolbox for efficiently engineering unusual fatty acids in transgenic oilseeds.     

Changes in fatty acid compositions of myocardial lipids in rats with heart failure following myocardial infarction

Changes in fatty acid composition of myocardial lipids were examined in rats with heart failure following myocardial infarction. Left ventricular systolic pressure (LVSP) was decreased and left ventricular end-diastolic pressure (LVEDP) was elevated 24 h, 1 and 12 weeks after left coronary artery ligation (CAL), suggesting the development of heart failure at these periods in this model. Hearts were isolated 24 h, 1 week and 12 weeks after the operation. Myocardial lipids in the infarcted scar tissue, non-infarcted remaining left ventricle including interseptum and right ventricle were separated into phospholipid (PL), triacylglycerol (TG), diacylglycerol (DAG) and free fatty acid (FFA) fractions. In the scar tissue PL content markedly decreased whereas TG, DAG and FFA contents increased 24 h after CAL. Despite a marked decrease in constituted fatty acids of PL fraction in the scar tissue the percentage of arachidonic acid in PL was elevated 12 weeks after CAL, suggesting that release of arachidonic acid during PL degradation was suppressed. In the non-infarcted viable left ventricle PL content remained unchanged throughout the experiment whereas TG, DAG and FFA contents were elevated 24 h after CAL. Despite no changes in PL and other lipid contents in the non-infarcted tissue the percentage of linoleic acid in PL was reduced and that of docosahexaenoic acid in PL was elevated 12 weeks after CAL. Our findings showed that myocardial lipid composition of the non-infarcted left ventricle was altered only in an early stage of the development of heart failure and fatty acid compositions of PL was exchanged in a late stage of the development of heart failure. The exchange may be related to cardiac dysfunction or myocardial remodelling in the rat with heart failure.     

Molecular species analysis of 1,2-diacylglycerol released in response to progesterone binding to the amphibian oocyte plasma membrane

Progesterone, the physiological inducer of amphibian meiosis, acts within minutes at plasma membrane receptors of the Rana pipiens oocyte to release 1,2-diacylglycerol (DAG) from plasma and intracellular membranes. High-performance liquid chromatography (HPLC) analysis of lipid extracts of uninduced oocytes indicates the presence of at least three classes of DAG with a total DAG content of about 150 micromol/kg wet weight. Within 3-5 min after exposure to progesterone, there was a differential increase in all three DAG classes with a twofold increase in total DAG by 10 min. The fatty acid composition of the DAGs in uninduced and progesterone-stimulated oocytes was compared using thin layer chromatographic analysis of lipid extracts from oocytes double-labeled with [14C] or [3H]glycerol and [14C] or [3H]fatty acids. The ratio of labeled fatty acid/labeled glycerol was measured in phosphatidylcholine (PC), phosphatidylinositol (PI) and DAG. The linoleic (18:2) or arachidonic (20:4) acid/glycerol ratios in basal DAG were low compared to that in PC or PI. In contrast, the myristic (14:0), palmitic (16:0) or oleic (18:1) acid/glycerol ratios in basal DAG were relatively high compared to the ratio in PC and PI. A transient increase in both linoleic and palmitic acid labeling of DAG occurred within the first 1-2 min in progesterone-treated oocytes, followed by a return to or below the basal level. Arachidonic and myristic acid labeling of DAG fall within the first minute after progesterone treatment, followed by a sustained rise over the next 10 min. The [3H]oleic acid/[14C]glycerol ratio of DAG does not change significantly following exposure to progesterone. Pretreatment with a phospholipid N-methylation inhibitor (2-methylaminoethane) precluded the rise in linoleic and palmitic acid-rich DAG, whereas pretreatment with a diglyceride kinase inhibitor (D102) produced a sustained elevation of linoleic and palmitic acid-rich DAG. These results indicate that the DAG released in response to progesterone is composed of multiple new molecular species of DAG and that both the palmitate and linolate-rich forms are rapidly phosphorylated to form phosphatidic acid (PA). The newly formed DAG species differ from the basal DAG species and reflect sequential activation of sphingomyelin (SM) synthase, PC-specific phospholipase D (PLD) and PI-specific phospholipase C in response to progesterone, which we have described previously.     

Lipid Mixtures Containing a Very High Proportion of Saturated Fatty Acids Only Modestly Impair Insulin Signaling in Cultured Muscle Cells

In vitro examinations of the effect of saturated fatty acids on skeletal muscle insulin action often use only one or two different fatty acid species, which does not resemble the human plasma fatty acid profile. We compared graded concentrations (0.1-0.8mM) of 3 different lipid mixtures: 1) a physiologic fatty acid mixture (NORM; 40% saturated fatty acids), 2) a physiologic mixture high in saturated fatty acids (HSFA; 60% saturated fatty acids), and 3) 100% palmitate (PALM) on insulin signaling and fatty acid partitioning into triacylglycerol (TAG) and diacylglycerol (DAG) in cultured muscle cells. As expected, PALM readily impaired insulin-stimulated pAkt^Thr308/Akt and markedly increased intracellular DAG content. In contrast, the fatty acid mixtures only modestly impaired insulin-stimulated pAkt^Thr308M/Akt, and we found no differences between NORM and HSFA. Importantly, NORM and HSFA did not increase DAG content, but instead dose-dependently increased TAG accumulation. Therefore, the robust impairment in insulin signaling found with palmitate exposure was attenuated with physiologic mixtures of fatty acids, even with a very high proportion of saturated fatty acids. This may be explained in part by selective partitioning of fatty acids into neutral lipid (i.e., TAG) when muscle cells were exposed to physiologic lipid mixtures.     

Dietary 1-monoolein decreases postprandial GIP release by reducing jejunal transport of glucose and fatty acid in rodents

Postprandial secretion of insulin and glucose-dependent insulinotropic polypeptide (GIP) is differentially regulated by not only dietary carbohydrate but also fat. Recent studies have shown that the ingestion of diacylglycerol (DAG) results in lower postprandial insulin and GIP release than that of triacylglycerol (TAG), suggesting a possible mechanism for the antiobesity effect of DAG. The structural and metabolic characteristics of DAG are believed to be responsible for its beneficial effects. This study was designed to clarify the effect of 1-monoacylglycerol [oleic acid-rich (1-MO)], the characteristic metabolite of DAG, on postprandial insulin and GIP secretion, and the underlying mechanism. Dietary 1-MO dose dependently stimulated whole body fat utilization, and reduced high-fat diet-induced body weight gain and visceral fat accumulation in mice, both of which are consistent with the physiological effect of dietary DAG. Although glucose-stimulated insulin and GIP release was augmented by the addition of fat, coingestion of 1-MO reduced the postprandial hormone release in a dose-dependent manner. Either glucose or fatty acid transport into the everted intestinal sacs and enteroendocrine HuTu-80 cells was also reduced by the addition of 1-MO. Reduction of either glucose or fatty acid transport or the nutrient-stimulated GIP release by 1-MO was nullified when the intestine was pretreated with sodium-glucose cotransporter-1 (SGLT-1) or fatty acid translocase (FAT)/CD36 inhibitor. We conclude that dietary 1-MO attenuates postprandial GIP and insulin secretion by reducing the intestinal transport of the GIP secretagogues, which may be mediated via SGLT-1 and FAT/CD36. Reduced secretion of these anabolic hormones by 1-MO may be related to the antiobesity effect of DAG.     

Channeling of eukaryotic diacylglycerol into the biosynthesis of plastidial phosphatidylglycerol

Plastidial glycolipids contain diacylglycerol (DAG) moieties, which are either synthesized in the plastids (prokaryotic lipids) or originate in the extraplastidial compartment (eukaryotic lipids) necessitating their transfer into plastids. In contrast, the only phospholipid in plastids, phosphatidylglycerol (PG), contains exclusively prokaryotic DAG backbones. PG contributes in several ways to the functions of chloroplasts, but it is not known to what extent its prokaryotic nature is required to fulfill these tasks. As a first step toward answering this question, we produced transgenic tobacco plants that contain eukaryotic PG in thylakoids. This was achieved by targeting a bacterial DAG kinase into chloroplasts in which the heterologous enzyme was also incorporated into the envelope fraction. From lipid analysis we conclude that the DAG kinase phosphorylated eukaryotic DAG forming phosphatidic acid, which was converted into PG. This resulted in PG with 2-3 times more eukaryotic than prokaryotic DAG backbones. In the newly formed PG the unique Delta3-trans-double bond, normally confined to 3-trans-hexadecenoic acid, was also found in sn-2-bound cis-unsaturated C18 fatty acids. In addition, a lipidomics technique allowed the characterization of phosphatidic acid, which is assumed to be derived from eukaryotic DAG precursors in the chloroplasts of the transgenic plants. The differences in lipid composition had only minor effects on measured functions of the photosynthetic apparatus, whereas the most obvious phenotype was a significant reduction in growth.     

The essential fatty acid status in phenylketonuria patients under treatment

Phenylketonuric patients are on a special diet that lacks certain essential fatty acids. This study evaluates the essential fatty acid status of a group of phenylketonuric patients in the Netherlands undergoing dietary treatment. To this end, the essential fatty acid status of nine phenylketonuria patients was studied. On the basis of age and gender, two control subjects were selected for each patient. The essential fatty acid composition of duplicate food portions and the essential fatty acid status of plasma and erythrocytes were analyzed. Phenylketonuria subjects had a different essential fatty acid profile from their peers, especially concerning the n-3 fatty acids. N-6 and n-3 fatty long-chain polyenes were hardly consumed by phenylketonuria subjects, in contrast to the control subjects. Linoleic acid, on the other hand, was consumed in significantly higher amounts by phenylketonuria subjects and made up about 40% of their daily fat consumption. The essential fatty acid consumption pattern of the phenylketonuria subjects is mirrored by the essential fatty acid concentrations in blood. The essential fatty acid status of the phenylketonuric diet should be improved in order to prevent deficiency in n-3 fatty acids.     

Biphasic changes in the level and composition of Dunaliella salina plasma membrane diacylglycerols following hypoosmotic shock.

Hypoosmotic shock has been shown to trigger an immediate and selective increase of plasma membrane diacylglycerols (DAG) in the green alga Dunaliella salina, coinciding with an approximately equivalent loss of phosphatidylinositol 4,5-bisphosphate from this membrane [Ha, K.S., & Thompson, G.A., Jr. (1991) Plant Physiol. 97, 921-927]. Following a slight decline in amount, DAG levels of the plasma membrane resumed their rise by 2 min after the shock and by 40 min had achieved a maximum concentration equivalent to 230% of DAG levels in unstressed cells. This second, more sustained increase of plasma membrane DAG was matched by a DAG increase in the microsome-enriched cytoplasmic membrane fraction, commencing at 2 min and peaking at 140% of control values. The changing pattern of DAG molecular species produced in the plasma membrane during the early phases of hypoosmotic stress was compatible with their derivation from phospholipase C hydrolysis of inositol phospholipids and phosphatidylcholine. From 8 min following hypoosmotic shock, as relatively larger scale DAG accumulations developed in the cytoplasmic membranes, the molecular species composition changed to reflect a marked increase in de novo synthesis of sn-1-oleoyl, sn-2-palmitoylglycerol, and dioleoylglycerol. The former molecular species appears to be synthesized in the chloroplast while the latter is produced in the endoplasmic reticulum. The radioisotope labeling data with Na2(14)CO3 confirmed that the biphasic formation of DAG triggered by hypoosmotic shock culminates in a large-scale de novo synthesis of DAG. This is the first clear evidence for de novo synthesis as a source of DAG following PIP2-mediated signaling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acid:Coenzyme A Ligase in Brain: Fatty Acid Specificity in Cellular and Subcellular Fractions

Abstract: We measured long-chain fatty acid:coenzyme A (CoA) ligase (EC 6.2.1.3) activity with four fatty acids in brain homogenates, and cellular and subcellular fractions to determine whether there are differences in activity that could be correlated with differences in fatty acid composition and metabolism. In rat brain homogenates, ligase activity varied appreciably with the four acids, with 18:2 > 18:1 > 16:0 > 22:1 (nmol acyl-CoA formed/min/mg protein; 1.46, 1.20, 0.96, and 0.57, respectively). This order was similar under all incubation conditions tested, including variable pH and fatty acid concentrations. The relative specific activities (RSA, 16:0 = 1.0) with the four substrates were similar in rat brain homogenate, mitochondria, and microsomes, with the highest specific activities in the latter fraction. The RSA were also similar in ox brain homogenates, in rabbit brain microsomes prepared from gray and white matter, in neurons isolated from rat brain, and in cultured neuroblastoma cells. Rat liver homogenates had a significantly different pattern of RSA. These results indicate that the ligase(s) has a preference for certain fatty acids, but suggest that the major control of fatty acid composition and metabolism is a function of subsequent metabolic steps.     

Phosphatidylcholine is a quantitatively more important source of increased 1,2-diacylglycerol than is phosphatidylinositol in mast cells

The widely accepted hypothesis that the increased 1,2-diacylglycerol (DAG) in stimulated cells is derived from phosphoinositides was tested by comparing the pattern of molecular species of phosphatidylinositol (PI) to that of DAG in mast cells. For any glycerol-based lipid, molecular species are defined by unique combinations of the two fatty acids esterified to glycerol. The quantitative frequency distribution of these molecular species represent a "fingerprint" that provides a sensitive approach to assessing precursor/product relationships. Based on mass, the molecular species fingerprints PI, phosphatidylcholine (PC), phosphatidylethanolamine, and phosphatidylserine were determined in unstimulated mast cells and compared to that of the DAG found after stimulation by IgE R bridging, compound 48/80 and the Ca+2 ionophore A23187. The molecular species fingerprint of DAG before stimulation was quite different from that of PI.IgE R cross-linking caused a 1.5 to 2-fold increase in DAG mass 1 to 3 min after stimulation with a concomitant shift in the pattern of DAG molecular species to one that bore only a partial resemblance to that of PI suggesting that considerably less than half of the incremental DAG is likely derived from PI. Ten to 20 min after Ag challenge, DAG levels became maximal (3.2- and 2.9-fold, respectively), but its molecular species pattern returned toward that seen in unstimulated cells suggesting that only perhaps 25% of the incremental DAG might be derived from PI. The molecular species fingerprint of DAG much more closely resembled that PC suggesting that as much as 75% of the incremental DAG might be derived from PC. Similar observations were made when 48/80 and A23187 were used as secretory agonists. These experiments indicate that the DAG participating in the "phosphoinositide cycle" represents a quantitatively modest fraction of the DAG accumulating in stimulated mast cells and suggest that mechanisms other than PI hydrolysis, including perhaps a "PC cycle," are more important than previously assumed in causing the rise in DAG during activation.     

Composition and synthesis of fatty acids in atherosclerotic aortas of the pigeon

The composition, synthesis, and esterification of fatty acids were studied in aortas of White Carneau and Show Racer pigeons after perfusion of the aortas with a medium containing acetate-1-(14)C. For both breeds of pigeons the principal change in aortic fatty acids, in response to an atherogenic diet, was a marked increase in the percentage of oleic acid in the cholesteryl ester fraction. In atherosclerotic aortas incorporation of acetate-1-(14)C into the phospholipid and glyceride fractions increased 2-fold, while a much greater increase (up to 10-fold) was seen in incorporation into cholesteryl esters. In those birds receiving the atherogenic diet, palmitic acid accounted for approximately 50% of the fatty acid radioactivity, compared with approximately 25% from control aortas. Calculation of fatty acid synthesis showed the major newly synthesized fatty acids to be stearic acid in the phospholipid fraction; stearic, palmitic, and oleic acids in the glycerides; and oleic acid in the cholesteryl esters. The pattern of fatty acid synthesis was closely similar to the actual fatty acid composition of the aorta. In atherosclerotic aortas an increased synthesis of all fatty acids was seen, but the greatest increase was seen in the synthesis of oleic acid and its esterification to cholesterol.     

The lipid profile of brown adipose tissue is sex-specific in mice

Brown adipose tissue (BAT) is a thermogenic organ with a vital function in small mammals and potential as metabolic drug target in humans. By using high-resolution LC-tandem-mass spectrometry, we quantified 329 lipid species from 17 (sub)classes and identified the fatty acid composition of all phospholipids from BAT and subcutaneous and gonadal white adipose tissue (WAT) from female and male mice. Phospholipids and free fatty acids were higher in BAT, while DAG and TAG levels were higher in WAT. A set of phospholipids dominated by the residue docosahexaenoic acid, which influences membrane fluidity, showed the highest specificity for BAT. We additionally detected major sex-specific differences between the BAT lipid profiles, while samples from the different WAT depots were comparatively similar. Female BAT contained less triacylglycerol and more phospholipids rich in arachidonic and stearic acid whereas another set of fatty acid residues that included linoleic and palmitic acid prevailed in males. These differences in phospholipid fatty acid composition could greatly affect mitochondrial membranes and other cellular organelles and thereby regulate the function of BAT in a sex-specific manner.     

Cerebral Phosphoinositide and Energy Metabolism During and After Insulin-Induced Hypoglycemia

During and after insulin-induced hypoglycemia, changes in levels of cerebral phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidic acid (PA), triacylglycerol (TAG), diacylglycerol (DAG), and free fatty acids (FFAs) as well as the cerebral energy state were studied in relation to the EEG. In hypoglycemic rats with an EEG pattern of quasiperiodic sharp or slow sharp waves, which preceded the development of an isoelectric EEG, PIP2 levels increased significantly, together with a slight decrease in PI content. Levels of the other lipids did not change during this period. The cerebral energy state was affected only slightly in spite of profound decreases in plasma and tissue glucose levels. With 30 min of an isoelectric EEG, levels of all phosphoinositides and PA decreased significantly; total FFA and DAG contents increased seven- and twofold, respectively; the TAG-palmitate level decreased, and that of TAG-arachidonate increased. Plasma and tissue glucose were nearly depleted, and the cerebral energy state deteriorated severely. The increment in fatty acids in the DAG and FFA pools was less than their loss from phosphoinositides and PA, an observation suggesting vascular washout or oxidation of a portion of the FFAs produced. Following 90 min of glucose infusion, PIP and PA levels recovered to control values; however, the PIP2 content exceeded control levels, and that of PI remained below control levels. DAG and FFA contents returned to normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circadian rhythms in Neurospora crassa: membrane composition of a mutant defective in temperature compensation

The cel mutant of Neurospora, partially blocked in fatty acid synthesis and lacking temperature compensation of its circadian rhythm below 22 degrees C, had a phospholipid fatty acid composition in liquid shaker culture distinctly different from that of a cel+ control strain. During growth, cel+ exhibited a reproducible increase in its linoleic acid level from about 32 to a plateau at 63 mol%, and a corresponding decrease in its linolenic acid level from about 40 to a plateau at 10 mol%. The level of palmitic acid was constant at 19 mol%. In the cel strain, the linoleic acid level was constant at 54 mol% while the palmitic acid level increased from about 12 to about 23 mol%. Supplementation with palmitic or linoleic acids altered the patterns of fatty acid composition of cel, but did not affect the pattern of cel+. Altered fatty acid composition cosegregated with the cel marker. The mitochondrial phospholipids of cel in liquid culture also had abnormal fatty acid composition, as did the whole mycelial phospholipids on solid medium. These results are consistent with the involvement of membrane homeostasis in the temperature compensation of circadian rhythms.     

Dramatic differences in the roles in lipid metabolism of two isoforms of diacylglycerol kinase

Lipid species changes for SV40-transformed fibroblasts from wild-type or from diacylglycerol kinase-epsilon (DGKepsilon) or diacylglycerol kinase-alpha (DGKalpha) knockout mice were determined for glycerophospholipids, polyphosphatidylinositides (GPInsP n ) and diacylglycerol (DAG) using direct infusion mass spectrometry. Dramatic differences in arachidonate (20:4 fatty acid)-containing lipids were observed for multiple classes of glycerophospholipids and polyphosphatidylinositides between wild-type and DGKepsilon knockout cells. However, no difference was observed in either the amount or the acyl chain composition of DAG between DGKepsilon knockout and wild-type cells, suggesting that DGKepsilon catalyzed the phosphorylation of a minor fraction of the DAG in these cells. The differences in arachidonate content between the two cell lines were greatest for the GPInsP n lipids and lowest for DAG. These findings indicate that DGKepsilon plays a significant role in determining the enrichment of GPInsP n with 20:4 and that there is a pathway for the selective translocation of arachidonoyl phosphatidic acid from the plasma membrane to the endoplasmic reticulum. In contrast, no substantial difference was observed in the acyl chain composition of any class of glycerophospholipid or diacylglycerol between lipid extracts from fibroblasts from wild-type mice or from DGKalpha knockout mice. However, the cells from the DGKalpha knockout mice had a higher concentration of DAG, consistent with the lack of downregulation of the major fraction of DAG by DGKalpha, in contrast with DGKepsilon that is primarily responsible for enrichment of GPInsP n with arachidonoyl acyl chains.     

Lipolytic effect of serotonin in vitro

The lipolytic action of serotonin on isolated adipocytes from the adipose tissue of rats has been studied. The adipocytes were incubated in serotonin 10(-6) M. Changes both in concentration and composition of the free intra and extracellular fatty acids as well as diacylglycerides through liquid gas chromatography were evaluated at different intervals. A lower concentration of free fatty acids and diacylglycerides is produced during the first minutes of incubation as well as a subsequent increase in the concentration of both, which becomes greatest after 20-30 minutes. The composition of both lipidic fractions (FFA and DAG) into fatty acids at 5, 10, 20 and 30 minutes, is related to the composition of the triacylglycerides (TAG), since during the esterification process a decline in the DAG of linoleic and palmitoleic acid is observed, both acids arranging themselves preferably in the TAG 2 position. Whereas the inverse process occurs during lipolysis; i.e. an increase in the proportion of the acids in the 2 position. In the FFA fraction, a higher proportion of fatty acids, preferential by arranged in positions 1 + 3 of the TAG's is observed. Similarly a decrease is observed in the extracellular concentration of FFA in the presence of serotonin with respect to the controls, a fact which has been described by other authors. An analysis of the present data leads us to revise the possible role of "Cahill's cycle" (simultaneous activation of the DAG-acyl-transferase and the HSL-TAG-lipase) in the action of serotonin and other hormones.