Harold W. Cook

In vitro formation of polyunsaturated fatty acids by desaturation in rat brain: some properties of the enzymes in developing brain and comparisons with liver.

Abstract— In vitro desaturation of [1‐C14]linolenic, linoleic, oleic, and icosatrienoic acids was determined using homogenates and subcellular fractions of developing rat brain and liver. Linolenic, linoleic, and oleic acids were desaturated in the δ6‐position and activity was optimal in the presence of CoA, ATP, MgCl2, and NADH in a citrate‐phosphate buffer at pH 6.0. Icosatrienoic acid was desaturated in the δ5‐position with a much broader pH optimum. The unstable desaturation systems of brain were protected by reduced glutathione and niacinamide and markedly inhibited by dithiothreitol, p‐chloromercuribenzoate, sodium cyanide or bathophenanthroline sulfonate. With brain homogenate of neonatal rats, the relative rates of desaturation of these substrates were 18:3(n ‐ 3) > 18:2(n ‐ 6) > 20:3(n ‐ 6) > 18:l (n ‐ 9). Specific activity of brain enzymes was greatest in neonatal rats with fluctuations in activity between 3 and 6 days of age. During this period, liver enzyme appeared to alter in a reciprocal manner. Total desaturation capacity of brain was maximal and fairly constant between 4 and 20 days of age, whereas liver activity increased dramatically after weaning. The activity of crude microsomal preparations from neonatal brain, like that of liver microsomes, was stimulated by a heat‐labile component of the cytosolic fraction. These results demonstrate that brain has a high capacity for desaturation of the essential fatty acids during crucial stages of brain development when liver activity is relatively low.

Differential effects of sphingomyelin hydrolysis and cholesterol transport on oxysterol-binding protein phosphorylation and Golgi localization

The deposition of de novo synthesized and lipoprotein-derived cholesterol at the plasma membrane and transport to the endoplasmic reticulum is dependent on sphingomyelin (SM) content. Here we show that hydrolysis of plasma membrane SM in Chinese hamster ovary cells by exogenous bacterial sphingomyelinase resulted in enhanced cholesterol esterification at the endoplasmic reticulum and rapid dephosphorylation of the oxysterol-binding protein (OSBP), a cytosolic/Golgi receptor for oxysterols such as 25-hydroxycholesterol. After sphingomyelinase treatment, restoration of OSBP phosphorylation closely paralleled resynthesis of SM and down-regulation of cholesterol ester synthesis. SM hydrolysis activated an okadaic acid-sensitive phosphatase that was not stimulated in Chinese hamster ovary cells by short chain ceramides. Agents that specifically blocked sphingomyelinase-mediated delivery of cholesterol to acyl-CoA:cholesterol acyltransferase (U18666A) or promoted cholesterol efflux to the medium (cyclodextrin) did not inhibit OSBP dephosphorylation. SM hydrolysis also promoted OSBP translocation from a vesicular compartment to the Golgi apparatus. Cyclodextrin and U18666A also caused OSBP translocation to the Golgi apparatus, suggesting that OSBP movement is coupled to changes in the cholesterol content of the plasma membrane or Golgi apparatus. These results identify OSBP as a potential target of SM turnover and cholesterol mobilization at the plasma membrane and/or Golgi apparatus.

Evidence that neutral spingomyelinase of cultured murine neuroblastoma cells is oriented externally on the plasma membrane

The activity of the neutral, Mg2+-stimulated sphingomyelinase of cultured neuroblastoma cells (N1E-115) is enriched in the plasma membrane fraction and is reduced following treatment of intact or broken cells with trypsin, alpha-chymotrypsin, papain, and protease. Two protease-sensitive enzymes of the cell interior (lactate dehydrogenase and NADPH-cytochrome c reductase) are not affected by protease treatment of intact cells. These results indicate that the neutral, Mg2+-stimulated sphingomyelinase is oriented externally on the plasma membrane of the cultured neuroblastoma cell.

Interaction of (n−3) and (n−6) fatty acids in desaturation and chain elongation of essential fatty acids in cultured glioma cells

Recent research in various biological systems has revived interest in interactions between the (n−6) and (n−3) essential fatty acids. We have utilized cultured glioma cells to show that linolenic acid, 18∶3(n−3), is rapidly desaturated and chain elongated; 20∶5(n−3) is the major product and accumulates almost exclusively in phospholipids. We examined effects of various (n−6), (n−3), (n−9) and (n−7) fatty acids at 40 μM concentration on desaturation and chain elongation processes using [1-14C]18∶3(n−3) as substrate. In general, monoenoic fatty acids were without effect. The (n−6) fatty acids (18∶2, 18∶3, 20∶3, 20∶4 and 22∶4) had little effect on total product formed. There was a shift of labeled product to triacylglycerol, and in phospholipids, slightly enhanced conversion of 20∶5 to 22∶5 was evident. In contrast, 22∶6(n−3) was inhibitory, whereas 20∶3(n−3) and 20∶5(n−3) had much less effect. At concentrations <75 μM, all acids were inhibitory. Most products were esterified to phosphatidylcholine, but phosphatidylethanolamine also contained a major portion of 20∶5 and 22∶5. We provide a condensed overview of how the (n−6) and (n−3) fatty acids interact to modify relative rates of desaturation and chain elongation, depending on the essential fatty acid precursor. Thus, the balance between these dietary acids can markedly influence enzymes providing crucial membrane components and substrates for biologically active oxygenated derivatives.

Dissociation of phosphorylation and translocation of a myristoylated protein kinase C substrate (MARCKS protein) in C6 glioma and N1E-115 neuroblastoma cells.

Abstract: An 80‐kDa protein labeled with [3H]myristic acid in C6 glioma and N1E‐115 neuroblastoma cells has been identified as the myristoylated alanine‐rich C kinase substrate (MARCKS protein) on the basis of its calmodulin‐binding, acidic nature, heat stability, and immunochemical properties. When C6 cells preincubated with [3H]myristate were treated with 200 nM 4β‐12‐O‐tetradecanoylphorbol 13‐acetate (β‐TPA), labeled MARCKS was rapidly increased in the soluble digitonin fraction (maximal, fivefold at 10 min) with a concomitant decrease in the Triton X‐100–soluble membrane fraction. However, phosphorylation of this protein was increased in the presence of β‐TPA to a similar extent in both fractions (maximal, fourfold at 30 min). In contrast, β‐TPA–stimulated phosphorylation of MARCKS in N1E‐115 cells was confined to the membrane fraction only and no change in the distribution of the myristoylated protein was noted relative to α‐TPA controls. These results indicate that although phosphorylation of MARCKS by protein kinase C occurs in both cell lines, it is not directly associated with translocation from membrane to cytosol, which occurs in C6 cells only. The cell‐specific translocation of MARCKS appears to correlate with previously demonstrated differential effects of phorbol esters on stimulation of phosphatidylcholine turnover in these two cell lines.

Turnover of phospholipid fatty acyl chains in cultured neuroblastoma cells: involvement of deacylation-reacylation and de novo synthesis in plasma membranes

Cultured neuroblastoma cells (NIE-115) rapidly incorporated the essential fatty acid, linoleic acid (18:2 (n = 6), into membrane phospholipids. Fatty acid label appeared rapidly (2-10 min) in plasma membrane phospholipids without evidence of an initial lag. Specific activity (nmol fatty acid/mumol phospholipid) was 1.5-2-fold higher in microsomes than in plasma membrane. In these membrane fractions phosphatidylcholine had at least 2-fold higher specific activity than other phospholipids. With 32P as radioactive precursor, the specific activity of phosphatidylinositol was 2-fold higher compared to other phospholipids in both plasma membrane and microsomes. Thus a differential turnover of fatty acyl and head group moieties of both phospholipids was suggested. This was confirmed in dual-label (3H fatty acid and 32P), pulse-chase studies that showed a relatively rapid loss of fatty acyl chains compared to the head group of phosphatidylcholine; the opposite occurred with phosphatidylinositol. A high loss of fatty acyl chain relative to phosphorus indicated involvement of deacylation-reacylation in fatty acyl chain turnover. The patterns of label loss in pulse-chase experiments at 37 and 10 degrees C indicated some independent synthesis and modification of plasma membrane phospholipids at the plasma membrane. Lysophosphatidylcholine acyltransferase and choline phosphotransferase activities were demonstrated in isolated plasma membrane in vitro. Thus, studies with intact cells and with isolated membrane fractions suggested that neuroblastoma plasma membranes possess enzyme activities capable of altering phospholipid fatty acyl chain composition by deacylation-reacylation and de novo synthesis at the plasma membrane itself.

The influence oftrans-acids on desaturation and elongation of fatty acids in developing brain

Abstracttrans-Monounsaturated acids account for up to 3% of the total octadecenoic acyl chains of human brain lipids. To investigate the influence oftrans-acids on desaturation and chain elongation of fatty acids, in vitro and in vivo experiments with rat brain were performed. In the in vitro assays of Δ9 desaturation, Δ6 desaturation and chain elongation,trans,trans-dienoic acid was inhibitory, particularly to chain elongation. Slight differences between the inhibitory effects oftrans-monoenoic acids and theircis-isomers were observed. In an in vivo model, unlabeled fatty acid (stearate, oleate, elaidate, linoleate, linoelaidate, arachidonate, ortrans-monoene from margarine) was injected simultaneously with [1-14C] linoleic acid into the brains of suckling rats. Linoelaidate and oleate inhibited desaturation and elongation of linoleate, whereas elaidate, stearate andtrans-monoene from margarine were stimulatory. While the demonstration of differences betweencis andtrans monoenic isomers required relatively high levels of the test acids, it appears thattrans-acids can influence desaturation and elongation enzymes that lead to acyl chain modification in the central nervous system.

Phosphatidylcholine metabolism in cultured cells: catabolism via glycerophosphocholine

The catabolism of phosphatidylcholine (PtdCho) has been studied in cultured murine neuroblastoma (N1E-115), C6 glioma, rat brain primary glia, and human fibroblast cells. Cells were pulse labelled for 96 h with [methyl-3H]choline followed by a chase for up to 24 h in medium containing 4 mM choline. Measurement of the radioactivity and mass of choline-containing compounds in these cells indicated that the major degradative pathway is PtdCho----lysophosphatidylcholine (lysoPtdCho)----glycerophosphocholine (GroPCho)----choline. At all times during the chase, PtdCho, sphingomyelin and lysoPtdCho comprised 72-92% of the cell-associated radioactivity; the remaining 10-30% was water-soluble and was chiefly GroPCho (30-80%) in all cell lines. In fibroblasts, however, phosphocholine (PCho) was also a major labelled water-soluble component (33-54%). The specific activity of GroPCho closely parallelled that of PtdCho in fibroblasts, but decreased faster than PtdCho in C6 and N1E-115 cells. We postulate that this may be due to distinct pools of PtdCho in the cell with differing rates of turnover. The changes in specific activity of PCho suggest that the major portion is formed by synthesis rather than as a degradative product. However, the inability to reduce the specific activity of this fraction to that of the intracellular choline suggests that a portion may be derived from either PtdCho or GroPCho.

Incorporation, metabolism and positional distribution of trans-unsaturated fatty acids in developing and mature brain. Comparison of elaidate and oleate administered intracerebrally.

Mixtures of [1-14C]elaidic acid and [9,10-3H]oleic acid, as well as [1-14C]elaidic or [1-14C]oleic acid alone, were administered by intracerebral injection to 10-day old and adult rats to examine the relative incorporation and metabolism of trans- and cis-monounsaturated fatty acids in developing and mature brain. Rates and extents of total incorporation of label from trans- and cis-acid into complex lipids were similar. Maximum labeling of the neutral lipid, mainly triacylglycerol and cholesterol, occurred prior to 4 h after injection whereas phospholipid, mainly choline phosphoglyceride, was maximally labeled at 4--8 h after injection. The decrease in labeled phospholipid from 24 to 90 h was greater with elaidate than with oleate. At 8 h labeled elaidic acid was preferentially esterified in the 1-position of all phosphoglycerides whereas labeled oleic acid, in contrast to the positional distribution of endogenous oleate, was randomly distributed. Label from elaidate found in palmitate increased with time to 26% of the total recovered label by 48 h. Thus, although some of the trans-fatty acid is oxidized and its labeled carbon is redistributed, most is incorporated unaltered into complex lipid of brain at rates similar to those for its cis-isomer. The developing central nervous system, therefore, does not metabolically exclude the trans-acid, elaidic acid, from membrane lipids.

Biosynthesis of fatty acids in vitro by homogenate of developing rat brain: desaturation and chain-elongation

Abstract 1. 1. Chain-elongation of [1-14C]16:1(n−7) to 18:1(n−7) in rat brain homogenates was dependent on exogenous NADPH and malonyl-CoA; slight additive stimulation occurred when NADH and acetyl CoA were added. Activity was greatest in developing brain and lower in the adult. 2. 2. Chain-elongation of [1-14C]16:0, 18:0, and 18:1 showed a similar trend. 16:0 exceeded 18:0 elongation 5–20-fold at all ages examined; 18:1 elongation was even less. 3. 3. The formation of 18:1 from [1-14C]16:0 by desaturation and chain-elongation was higher in developing than in adult brain, and was stimulated 2-fold (adult) to 20–30-fold (fetal to 20 days) by NADPH and malonyl CoA. 18:1(n−7) accounted for 12–19% of total 18:1 formed at all ages. Thus, in rat brain the major pathway of 18:1 formation appears to be 16:0 → 18:0 → 18:1(n−9), and the minor pathway 16:0 → 16:1 → 18:l(n−7). 4. 4. Δ6 desaturation of exogenous [1-14C]16:0 and 18:0, and of 18:0 formed endogenously from exogenous 16:0, occurred between fetal stage and 20 days of age only. At birth, this activity accounted for 62% of the 16:1 formed from 16:0, and 28% of the 18:1 formed from 18:0. 5. 5. The previous demonstration of Δ9 desaturating activity, and the present findings of (a) chain-elongation of 16- and 18-carbon saturates and monoenes and (b) Δ6 desaturation of 16:0 and 18:0, show that developing rat brain has the necessary enzymes for forming all the major 16:1 and 18:1 monoenoic fatty-acid isomers.