Glyceride metabolism in cultured cells dissociated from rat cerebral cortex

Abstract— [1-¹⁴C]stearic acid and [2-³H]glycerol were rapidly taken up and esterified into triacylglycerol and phospholipids by rat brain cells cultivated in monolayers. Expressed in terms of pool size, the incorporation of glycerol and stearate into triacylglycerol was 6- and 8-fold, respectively, higher than the incorporation into the choline phosphoglycerides. Tritium-labelled glycerol in both triacylglycerol and glycerophosphatides was diluted more rapidly than the [¹⁴C] labelled fatty acids. Chase experiments indicated a transfer of fatty acid from one lipid class to another, mainly from triacylglycerol to phospholipids, with no apparent loss of radioactivity. The accumulation of triacylglycerol in the brain cells was a function of both the presence of exogenous fatty acids in the culture medium and the metabolic needs of the cells; as long as the cells were involved in active formation of membranes the proportion of triacylglycerol was relatively small; its concentration increased while cell division slowed down in older, fully monolayered cultures.     

TURNOVER OF PHOSPHATIDYLCHOLINE IN MICROSOMES AND MYELIN IN BRAINS OF YOUNG AND ADULT RATS

We have tested the hypothesis that the turnover of phosphatidylcholine in subcellular fractions of rat brain is a function of the age at which this lipid is deposited. Rats, 60 days of age, were injected intracranially with [2-³H]glycerol and either [methyl-¹⁴C]choline (to label the base moiety) or [U-¹⁴C]glucose (to label acyl moieties). Littermates were killed up to 90 days after injection and brain microsomes and myelin isolated. Lipids were extracted and the phosphatidylcholine was isolated by 2-dimensional TLC and hydrolyzed to its constituent moieties. The ³H in the glycerol backbone and ¹⁴C in the choline or acyl residues was quantitated. The microsomal and myelin ³H/¹⁴C ratios decreased with time with either set of precursors, indicating that labeled choline and acyl moieties were reutilized more efficiently than the glycerol backbone. The various precursors exhibited first order decay curves with half-lives for the glycerol backbone of 6 and 11 days for the microsomal and myelin fractions respectively. These results contrast with those previously obtained with identical experimental procedures when 17-day-old animals were injected. In that study, although much of the phosphatidylcholine turned over rapidly as for the older animals, by 2 weeks after injection most of the remaining phosphatidylcholine was turning over more slowly with a half-life of 13 and 25 days for microsomes and myelin respectively (Miller et al., 1977). The base and acyl moieties also had a corresponding shorter half-life in older animals relative to the slow turnover phase in younger rats.     

Axonal transport of phospholipids in rat visual system.

Abstract— Seventeen day old rats were injected intraocularly with a phospholipid precursor, [³²P]phosphate, and a glycoprotein precursor, [³H]fucose. Animals were killed between 1 h and 21 days later, and structures of the visual pathway (retina, optic nerve, optic tract, lateral geniculate body, and superior colliculus) were dissected. Radioactivity in phospholipids ([³²P] in solvent-extracted material) and in glycoproteins ([³H] in solvent-extracted residue) was determined. Incorporation of [³H]fucose into retinal glycoproteins peaked at 6–8 h. Labelled glycoproteins were present in superior colliculus by 2h after injection, indicating a rapid rate of transport; maximal labelling was at 8–10 h after injection. Incorporation of [³²P]phosphate into retinal phospholipids peaked at 1 day after injection. Phospholipids were also rapidly transported since label was present in the superior colliculus by 3 h after injection: however, maximal labelling did not occur until 5–6 days. These results indicate that newly synthesized phospholipids enter a preexisting pool, part of which is later committed to transport at a rapid rate. Transported phospholipids were catabolized at the nerve endings with a maximum half-life of several days; there was minimal recycling of precursor label. Lipids were fractionated by thin-layer chromatography, and radioactivity in individual phospholipid classes determined. Choline and ethanolamine phosphoglycerides were the major transported phospholipids, together accounting for approx 85% of the total transported lipid radioactivity. At early time points, the ratio of radioactivity in choline phosphoglycerides to that in ethanolamine phosphoglycerides increased in structures progressively removed from the site of synthesis (retina) but by 2 days approached a constant value. In each structure, choline phosphoglyceride-ethanolamine phosphoglyceride radioactivity ratios decreased with time, rapidly at first, but plateaued by 2 days. These results indicate that choline phosphoglycerides are committed to transport sooner than ethanolamine phosphoglycerides. Some experiments were also conducted using [2-³H]glycerol as a phospholipid precursor. Results concerning incorporation of this precursor into individual phospholipid classes and their subsequent axonal transport were comparable to those obtained using [³²P]phosphate, with the following exceptions: (a) incorporation of [2-³H]glycerol into retinal phospholipids was relatively rapid (near-maximal levels at 1 h after injection) although transport to the superior colliculus showed an extended time course very similar to [³²P]-labelled lipids; (b) [2-³H]glycerol was somewhat less efficient than [³²P]phosphate in labelling lipids committed to transport relative to labelling those which remained in the retina; and (c) [2-³H]glycerol did not label plasmalogens.     

Changes in phospholipid composition during the development of Dictyostelium discoideum

The phospholipid composition of Dictyostelium discoideum cells was determined at various stages of development by two-dimensional, thin-layer chromatography and reaction thin-layer chromatography. Major phospholipids of D. discoideum which were detectable throughout all stages of development were ethanolamine phosphoglyceride and choline phosphoglyceride. Ethanolamine phosphoglyceride and choline phosphoglyceride were found as their plasmalogen forms at 45–58 and 10–24%, respectively. There were no qualitative changes in phospholipid composition during the development, but quantitative changes did occur. The relative content of ethanolamine phosphoglyceride in the total phospholipids gradually decreased from 60% at the vegetative stage to 44% at the 1-day-sorocarp stage. In contrast, choline phosphoglyceride gradually increased from 27% at the vegetative stage to 48% at the preculmination stage, and then gradually decreased to 43% during the culmination. The decrease in ethanolamine phosphoglyceride content during the middle and late development was due mainly to the decreased amount of its plasmalogen form but the increase of choline phosphoglyceride was independent of quantitative changes of its plasmalogen form. Other minor components of phospholipid did not show significant changes in their levels. The causes of these changes in contents of ethanolamine phosphoglyceride and choline phosphoglyceride were examined by label and chase experiments with [³H]ethanolamine and [¹⁴C]choline. It seems that one-third to one-half of the increased amount of choline phosphoglyceride was due to stepwise methylation of ethanolamine phosphoglyceride, and the remaining two-thirds to one-half was caused by de novo synthesis of choline phosphoglyceride from CDP-choline and diglyceride.     

Analysis of Acyl Fluxes through Multiple Pathways of Triacylglycerol Synthesis in Developing Soybean Embryos

The reactions leading to triacylglycerol (TAG) synthesis in oilseeds have been well characterized. However, quantitative analyses of acyl group and glycerol backbone fluxes that comprise extraplastidic phospholipid and TAG synthesis, including acyl editing and phosphatidylcholine-diacylglycerol interconversion, are lacking. To investigate these fluxes, we rapidly labeled developing soybean (Glycine max) embryos with [¹⁴C]acetate and [¹⁴C]glycerol. Cultured intact embryos that mimic in planta growth were used. The initial kinetics of newly synthesized acyl chain and glycerol backbone incorporation into phosphatidylcholine (PC), 1,2-sn-diacylglycerol (DAG), and TAG were analyzed along with their initial labeled molecular species and positional distributions. Almost 60% of the newly synthesized fatty acids first enter glycerolipids through PC acyl editing, largely at the sn-2 position. This flux, mostly of oleate, was over three times the flux of nascent [¹⁴C]fatty acids incorporated into the sn-1 and sn-2 positions of DAG through glycerol-3-phosphate acylation. Furthermore, the total flux for PC acyl editing, which includes both nascent and preexisting fatty acids, was estimated to be 1.5 to 5 times the flux of fatty acid synthesis. Thus, recycled acyl groups (16:0, 18:1, 18:2, and 18:3) in the acyl-coenzyme A pool provide most of the acyl chains for de novo glycerol-3-phosphate acylation. Our results also show kinetically distinct DAG pools. DAG used for TAG synthesis is mostly derived from PC, whereas de novo synthesized DAG is mostly used for PC synthesis. In addition, two kinetically distinct sn-3 acylations of DAG were observed, providing TAG molecular species enriched in saturated or polyunsaturated fatty acids.     

Triacylglycerol Bioassembly in Microspore-Derived Embryos of Brassica napus L. cv Reston

Erucic acid (22:1) was chosen as a marker to study triacylglycerol (TAG) biosynthesis in a Brassica napus L. cv Reston microspore-derived (MD) embryo culture system. TAGs accumulating during embryo development exhibited changes in acyl composition similar to those observed in developing zygotic embryos of the same cv, particularly with respect to erucic and eicosenoic acids. However, MD embryos showed a much higher rate of incorporation of ¹⁴C-erucoyl moieties into TAGs in vitro than zygotic embryos. Homogenates of early-late cotyledonary stage MD embryos (14-29 days in culture) were assessed for the ability to incorporate 22:1 and 18:1 (oleoyl) moieties into glycerolipids. In the presence of [1-¹⁴C]22:1-coenzyme A (CoA) and various acyl acceptors, including glycerol-3-phosphate (G-3-P), radiolabeled erucoyl moieties were rapidly incorporated into the TAG fraction, but virtually excluded from other Kennedy Pathway intermediates as well as complex polar lipids. This pattern of erucoyl incorporation was unchanged during time course experiments or upon incubation of homogenates with chemicals known to inhibit Kennedy Pathway enzymes. In marked contrast, parallel experiments conducted using [1-¹⁴C]18:1-CoA and G-3-P indicated that ¹⁴C oleoyl moieties were incorporated into lyso-phosphatidic acids, phosphatidic acids, diacylglycerols, and TAGs of the Kennedy Pathway, as well as other complex polar lipids, such as phosphatidylcholines and phosphatidylethanolamines. When supplied with l-[2-³H(N)]G-3-P and [1-¹⁴C]22:1-CoA, the radiolabeled TAG pool contained both isotopes, indicating G-3-P to be a true acceptor of erucoyl moieties. Radio-high-performance liquid chromatography, argentation thin-layer chromatography/gas chromatography-mass spectrometry, and stereospecific analyses of radiolabeled TAGs indicated that 22:1 was selectively incorporated into the sn-3 position by a highly active diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), while oleoyl moieties were inserted into the sn-1 and sn-2 positions. In the presence of sn-1,2-dierucin and [1-¹⁴C]22:1-CoA, homogenates and microsomal preparations were able to produce radiolabeled trierucin, a TAG not found endogenously in this species. A 105,000g pellet fraction contained 22:1-CoA:DGAT exhibiting the highest specific activity. The rate of 22:1-CoA:DGAT activity in vitro could more than account for the maximal rate of TAG biosynthesis observed in vivo during embryo development. In double label experiments, G-3-P was shown to stimulate the conversion of [³H]phosphatidylcholines to [³H]diacylglycerols, which subsequently acted as acceptors for ¹⁴C erucoyl moieties. In vitro, 22:1 moieties did not enter the sn-1 position of TAGs by a postsynthetic modification or transacylation of preformed TAGs.     

Digestion of phosphatidylcholines,absorption, and esterification of lipolytic products by Aeshna cyanea larvae as studied in vivo and in vitro

Digestion and absorption of phosphatidylcholine by Aeshna cyanea larvae were studied in vivo and in vitro with the isolated digestive juice and isolated midgut. The experiments were performed with stable ether analogues (1-alkyl-2-acyl-,1,2-dialkyl phosphatidylcholine, and 1-monoalkyl-lysophosphati-dylcholine), with radioactive 1,2-diacylphosphatidylcholine alternatively labelled in the acyl- and choline moieties, and with several phosphatidylcholine derivatives (1-[1-¹⁴C]acyl- and 1-[³H] alkyl-lysophosphatidylcholine, [1-¹⁴C]oleic acid, [2-¹⁴C]glycerol, phosphoryl[methyl-¹⁴C]choline, and [methyl-¹⁴C]choline). Chromatographic analyses of the digestion products revealed that phosphatidylcholine was degraded via two interconnected hydrolytic pathways involving phospholipase C, phospholipase A₂, lipase, and alkaline phosphatase. Complete hydrolysis by these pathways yielded the same four end products: free fatty acid, glycerol, choline, and P_i, which were absorbed by the midgut enterocytes. Of the intermediate hydrolysates, lysophosphatidylcholine, monoacylglycerol, and possibly phosphorylcholine were also absorbed. Radiolabelled oleic acid, glycerol, lysophosphatidylcholine and monoacylglycerol (as judged from monoalkylglycerol absorption) were incorporated into phospholipids and acylglycerols of the midgut enterocytes and were released into the haemolymph primarily in the form of diacylglycerols. In the case of glycerol ingestion, a small fraction of haemolymph radioactivity was associated with free glycerol and glycerolphosphate. After absorption by the enterocytes, radiolabelled choline was partly oxidized to betaine, partly phosphorylated, and partly incorporated into lyso- and phosphatidylcholine. It was recovered from the haemolymph predominantly as free choline, phosphorylcholine, and betaine. Arch. Insect Biochem. Physiol. 36:273–293, 1997. © 1997 Wiley-Liss, Inc.     

Acyltransferase capacity of membranes from cotyledons of germinating peas

The incorporation of 1-[¹⁴C]-palmitate into the lipids of microsomal and mitochondrial membranes from peas (Pisum sativum L., var. Massey Gem) and the relative effects of ATP and coenzyme A(CoA) on the process have been examined. Both mitochondrial and microsomal pellets possessed acyltransferase capacity, which responded similarly to additions of ATP and CoA. Incorporation of 1-[¹⁴C]-palmitate into phospholipid was promoted by ATP alone, but incorporation into triacylglycerols was not. The addition of CoA alone did not promote incorporation. The addition of CoA and ATP further promoted incorporation into phospholipids and also stimulated incorporation into triacylglycerol. It was concluded that some CoA must be membrane-bound and available for phospholipid but not for triacylglycerol synthesis. Phospholipase A, treatment of microsomal and mitochondrial phospholipids, previously labelled with 1-[¹⁴C]-palmitate in the presence of ATP and coenzyme A, showed that incorporation occurred only into the 2-position of phosphatidyl choline and phosphatidyl ethanolamine. There was enough lyso-phosphatidyl choline in the phospholipids of microcomal membranes (obtained from a 100 000 g pellet) to account for the observed incorporations of palmitate. Using microsomal membranes whose fatty acyl groups were pre-labelled by incubation of tissue with 1-[¹⁴C]-acetate, no evidence of acyl exchange was found during subsequent incubations with unlabelled palmitate. Similar observations were made using oleate instead of palmitate. It was concluded that acyl-CoA: 1-acylglycerophosphocholine o-acyltransferase (E.C. 2.3.1.23) was responsible for the observed acyl transfer to phosphatidyl choline. Sucrose gradient analysis of whole homogenates and of the 10 000 g pellet showed that both mitochondrial and rough endoplasmic reticulum possessed acyltransferase capacity, with the bulk of this residing in the mitochondria. The possible significance of this widely distributed membrane activity is briefly discussed.     

Differential Incorporation of Precursor Moieties into Cerebral Cortex and Cerebellum Glycerophospholipids During Aging

The incorporation of polar and non-polar moieties into cerebral cortex (CC) and cerebellum (CRBL) phospholipids of adult (3.5-month-old) and aged (21.5-month-old) rats was studied in a minced tissue suspension. The biosynthesis of acidic phospholipids through [³H]glycerol appears to be slightly increased with respect to that of zwitterionic or neutral lipids in CC of aged rats with respect to adult rats. On the contrary, the synthesis of phosphatidylcholine (PC) from [³H]choline was inhibited. However, the incorporation of [¹⁴C]serine into phosphatidylserine (PS) was higher in CC and CRBL in aged rats with respect to adult rats. The synthesis of phosphatidylethanolamine (PE) from PS was not modified during aging. Saturated ([³H]palmitic) and polyunsaturated ([³H]arachidonic) acids were incorporated successfully by adult and aged brain lipids. In addition [³H]palmitic, [³H]oleic and [³H]arachidonic acid were employed as glycerolipid precursors in brain homogenate from aged (28.5 month old) and adult (3.5 month old) rats. [³H]oleic acid incorporation into neutral lipids (NL) and [³H]arachidonic acid incorporation into PC, PE and phosphatidylinositol (PI) were increased in aged rats with respect to adult rats. Present results show the ability and avidity of aged brain tissue in vitro to incorporate unsaturated fatty acids when they are supplied exogenously. They also suggest a different handling of choline and serine by base exchange enzyme activities to synthesize PC and PS during aging.     

Remodelling of triacylglycerols in microsomal preparations from developing castor bean (Ricinus communis L.) endosperm

Microsomal preparations from developing castor bean (Ricinus communis L.) endosperm catalyzed remodelling of in-situ-formed triacylglycerol (TAG) species. Castor bean microsomal membranes synthesized [¹⁴C]TAGs from either glycerol 3-phosphate and [¹⁴C]ricinoleoyl-CoA or [¹⁴C]glycerol 3-phosphate and ricinoleoyl-CoA. Upon repelleting and subsequent incubation of the microsomes a redistribution occurred of both the [¹⁴C]glycerol and [¹⁴C]ricinoleoyl moieties of the in-situ-synthesized [¹⁴C]TAGs. Radioactivity was transferred from TAG species with three (3HO-TAG) or two (2HO-TAG)ricinoleoyl groups into species with two or one (HO-TAG) ricinoleoyl groups. Mass analysis of the lipid and fatty acid movements in the membranes showed that a net synthesis of TAGs with no, one and two ricinoleoyl groups occurred at the expense of 3HO-TAG and polar lipids. Thus, the non-hydroxylated acyl groups from polar lipids were used in the remodelling of TAGs. In-vivo feeding of [¹⁴C]ricinoleic acid to slices of castor bean endosperm demonstrated the presence of two radioactive pools of TAGs one in the oil bodies, which was rich in [¹⁴C]3HO-TAG, and one associated with the microsomal membranes, which was dominated by radioactive 1HO-TAG and 2HO-TAG. The microsomal TAG pool was remodelled in vivo in a similar way as in the in-vitro experiments with microsomal membranes. Received: 8 November 1996 / Accepted: 5 February 1997     

Inactivation of microsomal triglyceride transfer protein impairs the normal redistribution but not the turnover of newly synthesized glycerolipid in the cytosol,endoplasmic reticulum and Golgi of primary rat hepatocytes

The requirements for microsomal triglyceride transfer protein (MTP) during the turnover and transfer of glycerolipids from intracellular compartments into secretory very low-density lipoprotein (VLDL) were studied by pre-labelling lipids with [³H]glycerol and [¹⁴C]oleate in primary cultures of rat hepatocytes. The intracellular redistribution of pre-labelled glycerolipids was then compared at the end of subsequent chase periods during which the MTP inhibitor BMS-200150 was either present or absent in the medium. Inhibition of MTP resulted in a decreased output of VLDL triacylglycerol (TAG) and a delayed removal of labelled TAG from the cytosol and from the membranes of the smooth endoplasmic reticulum (SER), the cis- and the trans-Golgi. Inactivation of MTP did not decrease the bulk lipolytic turnover of cellular TAG as reflected by changes in its [³H]glycerol:[¹⁴C]oleate ratios. However, a larger proportion of the resultant TAG fatty acids was re-esterified and remained with the membranes of the various subcellular fractions rather than emerging as VLDL. The effects of BMS-200150 on the pattern of phospholipid (PL) mechanism and redistribution suggested that inhibition of MTP prevented the normal lipolytic transfer of PL-derived fatty acids out of the SER, cis- and trans-Golgi membrane pools. Finally, changes in the ¹⁴C specific radioactivities of the cytosolic and membrane pools of TAG suggested that inhibition of MTP prevented a normal influx of relatively unlabelled fatty acids into these pools during the chase period.     

Comparison in genetically hyperlipoproteinemic and normal rats of the secretion of lipids synthesized by isolated livers perfused with isotopic oleic acid and glycerol at high concentrations

After perfusing isolated livers of Zucker fa/fa ad Fa/- rats with loads of [9,10-3H2] oleic acid (346 mumol) and [1-14C] glycerol (115 mumol), glycerol inhibited the hepatic secretion of triacylglycerol and phospholipids in the two groups of rats. However, the amount of acylglycerols synthetized from these exogenous substrates is slightly higher in the obese rats than in the normal rats. These results suggest that glycerol, present in high amounts in blood of fa/fa rats, failed to regulate triacylglycerols and phospholipids secretion.     

Elevation of cerebrospinal fluid choline levels by nicotinamide involves the enzymatic formation of N1-methylnicotinamide in brain tissue

Nicotinamide administration can elevate plasma and brain choline levels and produce a marginal increase in striatal acetylcholine levels in the rat. We now report that subcutaneous nicotinamide produces a substantial and long-lasting rise in asternal cerebrospinal fluid (CSF) levels of choline in free-moving rats, possibly through the enzymatic formation of N¹-methylnicotinamide (NMN) in brain. CSF choline levels peaked 2 hours after nicotinamide administration and were accompanied by increases in striatal, cortical, hippocampal and plasma choline levels. The enzymatic formation of [³H]NMN in rat brain was evaluated by incubating aliquots of rat brain cytosol with unlabelled nicotinamide and the methyl donor [³H]S-adenosylmethionine. High performance liquid chromatography and radiochemical detection demonstrated that [³H]NMN was specifically formed by a brain cytosolic enzyme. The production of [³H]NMN was dependent on exogenous nicotinamide and could be prevented by denaturing the cytosol. The metabolism of nicotinamide to NMN in rat brain may explain the rise in CSF choline levels since NMN, a quaternary amine, can inhibit choline transport at the choroid villus and reduce choline clearance.     

Turnover of ethanolamine phosphoglycerides in different brain areas of adult and aged rats

The turnover of ethanolamine glycerophospholipids (EGP) has been determined in six different cerebral areas of 4-month and 22-month-old rats, by injecting [³H]glycerol together with [¹⁴C]ethanolamine into the lateral ventricle of the brain. The areas examined behave quite differently in respect to their utilization of the most simple precursors of phosphoglyceride biosynthesis. The incorporation of both glycerol and ethanolamine is already complete as early as 2–4 hours and then reutilization begins, at least for the so called fast pools of phosphoglycerides. The different slopes of the specific activity of the two precursors in EGP suggest a high degree of variance among catabolic rates in the different brain regions. In aged rats the utilization of the water-soluble precursors of EGP synthesis decreases in all brain areas and these data suggest that aging may have a different effect on the catabolic activities as well as phospholipid biosynthesis.     

Manoalide,a Phospholipase A2 Inhibitor,Inhibits Arachidonate Incorporation and Turnover in Brain Phospholipids of the Awake Rat

The Fatty Acid method was used to determine whether incorporation of plasma radiolabeled arachidonic acid into brain phospholipids is controlled by phospholipase A₂. Awake rats received an i.v. injection of a phospholipase A₂ inhibitor, manoalide (10 mg/kg), and then were infused i.v. with [1-¹⁴C]arachidonate or [³H]arachidonate. Animals were killed after infusion by microwave irradiation, and tracer distribution was analyzed in brain phospholipid, neutral lipid and acyl-CoA pools. Calcium-independent phospholipase A₂ activity in brain homogenate was reduced by manoalide, whereas phospholipase C activity was unaffected. At 60 min but not at 20 or 40 min after its injection, manoalide had significantly decreased by 50% incorporation of unesterified arachidonate into and turnover within brain phospholipids, taking into account dilution of the brain arachidonoyl-CoA pool by recycled arachidonate. Manoalide also increased by 100% the net rate of unesterified arachidonate incorporation into brain triacylglycerol. This study indicates that manoalide can be used to inhibit brain phospholipase A₂ in vivo, and that phospholipase A₂ plays a critical role in arachidonate turnover in brain phospholipids and neutral lipids.     

Separation and Characterization of Inositol Phospholipids from the Pulvini of Samanea saman

To supplement current thin-layer chromatographic methods for separation and quantitation of plant phospholipids, an alternative method, high-performance liquid chromatography was developed. The major inositol-containing lipids from the pulvini of Samanea saman Merr. were identified as phosphatidylinositol, phosphatidylinositol phosphate, and phosphatidylinositol bisphosphate based on comigration with authentic standards on high-performance liquid chromatography and on thin-layer chromatography. The patterns of incorporation of radioactivity into the putative phosphatidylinositol and phosphatidylinositol phosphate were consistent with these identifications when pulvini were labeled with [³H]glycerol, [³H]inositol, or [³²P]orthophosphate. Analysis of the products of enzymic hydrolysis, of chemical deacylation, and of `fingerprint' methanolysis of these phospholipids confirmed the identifications.     

The effects of inhibitors of protein synthesis on incorporation of lipids into myelin

Abstract— Following intracranial injections of puromycin, the incorporation of [³H]leucine into brain protein was inhibited by 80 per cent. Conversely, incorporation of [³⁵S]sulphate into sulphatide or [2-³H]glycerol into phosphatidyl choline was not inhibited. Under these conditions, appearance of labelled protein in myelin was inhibited by 90 per cent, while the appearance of newly labelled sulphatide and phosphatidyl choline in myelin membrane was not greatly affected. Experiments with cycloheximide gave similar results with phosphatidyl choline, but incorporation of [³⁵S]sulphate into total sulphatide was decreased by about 30 per cent in animals given cycloheximide. Neither puromycin nor cycloheximide had any inhibitory effect on galactocerebroside sulphotransferase.     

LIPID COMPOSITION AND METABOLISM OF CULTURED HAMSTER BRAIN ASTROCYTES

Abstract— The lipid composition and metabolism of confluent cultures of cells derived from newborn hamster brain and having morphology characteristic of immature astrocytes or spongioblasts was investigated and compared to that of newborn hamster brain dispersions and cloned glioma cells (C6). The cells displayed stable morphology for at least 30 subcultures; thereafter spontaneous transformation occurred. No appreciable changes were observed in either composition or metabolic characteristics of any major neutral lipid or phospholipid class in successive subcultures or following transformation. The overall lipid composition of the hamster astrocyte cultures closely resembled that of newborn hamster brain, but the phospholipid composition showed substantial differences. The cells contained as a percent of lipid P relatively more ethanolamine plasmalogen, choline plasmalogen and sphingomyelin and somewhat less phosphatidylcholine and phosphatidylethanolamine. The phospholipids of the hamster astrocyte and C6 cells were similar. Of the lipid precursors examined, [U-¹⁴C]glucose was incorporated best into all preparations. C6 glioma cells incorporated both [U-¹⁴C]glucose and [1-¹⁴C]acetate most actively. From 69–88% of ³²P incorporated into hamster astrocyte phospholipids was present in choline phosphoglycerides, whereas the corresonding figure for hamster brain dispersions was 53%. The ratio of specific activities of phosphatidylcholine to phosphatidylinositol was substantially higher in the cultured cells than in the brain preparations. The small pool of choline plasmalogen in the hamster astrocytes usually achieved the highest specific activity of any phospholipid. When [U-¹⁴C]glucose and [1-¹⁴C]acetate were precursors, the bulk of label in the astrocytes appeared in choline phosphoglycerides and triacyglycerol. Our results indicate that the hamster astrocyte cell line as grown expresses distinctive features of lipid composition and metabolism which are nearly constant through many generations.     

Regulation of triacylglycerol biosynthesis in embryos and microsomal preparations from the developing seeds of Cuphea lanceolata.

Embryos of Cuphea lanceolata have more than 80 mol% of decanoic acid ('capric acid') in their triacylglycerols, while this fatty acid is virtually absent in phosphatidylcholine (PtdCho). Seed development was complete 25-27 days after pollination, with rapid triacylglycerol deposition occurring between 9 and 24 days. PtdCho amounts increased until day 15 after pollination. Analysis of embryo lipids showed that the diacylglycerol (DAG) pool consisted of mainly long-chain molecular species, with a very small amount of mixed medium-chain/long-chain glycerols. Almost 100% of the fatty acid at position sn-2 in triacylglycerols (TAG) was decanoic acid. When equimolar mixtures of [14C]decanoic and [14C]oleic acid were fed to whole detached embryos, over half of the radioactivity in the DAG resided in [14C]oleate, whereas [14C]decanoic acid accounted for 93% of the label in the TAG. Microsomal preparations from developing embryos at the mid-stage of TAG accumulation catalysed the acylation of [14C]glycerol 3-phosphate with either decanoyl-CoA or oleoyl-CoA, resulting in the formation of phosphatidic acid (PtdOH), DAG and TAG. Very little [14C]glycerol entered PtdCho. In combined incubations, with an equimolar supply of [14C]oleoyl-CoA and [14C]decanoyl-CoA in the presence of glycerol 3-phosphate, the synthesized PtdCho species consisted to 95% of didecanoic and dioleic species. The didecanoyl-glycerols were very selectively utilized over the dioleoylglycerols in the production of TAG. Substantial amounts of [14C]oleate, but not [14C]decanoate, entered PtdCho. The microsomal preparations of developing embryos were used to assess the acyl specificities of the acyl-CoA:sn-glycerol-3-phosphate acyltransferase (GPAT, EC 2.3.1.15) and the acyl-CoA:sn-1-acyl-glycerol-3-phosphate acyltransferase (LPAAT, EC 2.3.1.51) in Cuphea lanceolata embryos. The efficiency of acyl-CoA utilization by the GPAT was in the order decanoyl = dodecanoyl greater than linoleoyl greater than myristoyl = oleoyl greater than palmitoyl. Decanoyl-CoA was the only acyl donor to be utilized to any extent by the LPAAT when sn-decanoylglycerol 3-phosphate was the acyl acceptor. sn-1-Acylglycerol 3-phosphates with acyl groups shorter than 16 carbon atoms did not serve as acyl acceptors for long-chain (greater than or equal to 16 carbon atoms) acyl-CoA species. On the basis of the results obtained, we propose a schematic model for triacylglycerol assembly and PtdCho synthesis in a tissue specialized in the synthesis of high amounts of medium-chain fatty acids.     

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.