Inhibition of serine palmitoyltransferase in vitro and long-chain base biosynthesis in intact Chinese hamster ovary cells by beta-chloroalanine

The effects of beta-chloroalanine (beta-Cl-alanine) on serine palmitoyltransferase activity and the de novo biosynthesis of sphinganine and sphingenine were investigated in vitro with rat liver microsomes and in vivo with intact Chinese hamster ovary (CHO) cells. The inhibition in vitro was rapid (5 mM beta-Cl-alanine caused complete inactivation in 10 min), irreversible, and concentration and time dependent and apparently involved the active site because inactivation only occurred with beta-Cl-L-alanine (not beta-Cl-D-alanine) and was blocked by L-serine. These are characteristics of mechanism-based ("suicide") inhibition. Serine palmitoyltransferase (SPT) was also inhibited when intact CHO cells were incubated with beta-Cl-alanine (complete inhibition occurred in 15 min with 5 mM), and this treatment inhibited [14C]serine incorporation into long-chain bases by intact cells. The concentration dependence of the loss of SPT activity and of long-chain base synthesis was identical. The effects of beta-Cl-L-alanine appeared to occur with little perturbation of other cell functions: the cells exhibited no loss in cell viability, [14C]serine uptake was not blocked, total lipid biosynthesis from [14C]acetic acid was not decreased (nor was the appearance of radiolabel in cholesterol and phosphatidylcholine), and [3H]thymidine incorporation into DNA was not affected. There appeared to be little effect on protein synthesis based on the incorporation of [3H]leucine, which was only decreased by 14%. Although beta-Cl-L-alanine is known to inhibit other pyridoxal 5'-phosphate dependent enzymes, alanine and aspartate transaminases were not inhibited under these conditions. These results establish the close association between the activity of serine palmitoyltransferase and the cellular rate of long-chain base formation and indicate that beta-Cl-alanine and other mechanism-based inhibitors might be useful to study alterations in cellular long-chain base synthesis.     

Enzymology of long-chain base synthesis by aorta: induction of serine palmitoyltransferase activity in rabbit aorta during atherogenesis

Serine palmitoyltransferase [EC 2.3.1.50] initiates the biosynthesis of sphingolipids by catalyzing the condensation of a fatty acyl-CoA with serine to yield the committed intermediate 3-ketosphinganine or one of its homologues. The presence of serine palmitoyltransferase in aorta was established under optimal assay conditions using microsomes from New Zealand White rabbits. Its activity was dependent on microsomal protein, L-serine, pyridoxal 5'-phosphate, and palmitoyl-CoA. Although several different saturated and unsaturated fatty acyl-CoA thioesters were utilized as substrates, maximal activity was with palmitoyl-CoA, suggesting that this enzyme contributes to the predominance of 18-carbon long-chain bases in sphingolipids from aorta. Rabbits, fed a Purina lab chow supplemented with 2% cholesterol, were used to study serine palmitoyltransferase activity in aorta during experimental atherogenesis. An increase in activity from intimal-medial preparations was detectable prior to prominent lipid accumulation or cellular proliferation. Activity continued to elevate over the 12-week duration of feeding concurrent with the increase in serum cholesterol and in proportion to the development of plaques resulting in a 3.7-fold increase in activity (20.7 +/- 2.6 pmol per min per mg microsomal protein +/- SE in the cholesterol-fed group versus 5.6 +/- 1.9 in the pair-fed controls also matched for age and sex; P less than 0.005). Thus, the accumulation of sphingomyelin that occurs in aorta during experimental atherogenesis may be related to increased long-chain base synthesis by serine palmitoyltransferase.     

Activities of serine palmitoyltransferase (3-ketosphinganine synthase) in microsomes from different rat tissues

Serine palmitoyltransferase [EC 2.3.1.50] catalyzes the first unique reaction of sphingolipid biosynthesis. To determine whether or not different rat tissues are capable of initiating this pathway, its activity was determined for microsomes from rat liver, lung, brain, kidney, intestine, spleen, muscle, heart, pancreas, testes, ovary, and stomach. Serine palmitoyltransferase was found in every tissue, and, when compared to the microsomal glycerol 3-phosphate acyltransferase, the activities correlated directly with their sphingomyelin levels as a percentage of total phospholipids. This suggests that the activities were comparable to expected cellular needs for long-chain bases, if the initial enzymes of glycerolipid and sphingolipid biosynthesis influence the phospholipid composition of cells by determining the relative partitioning of fatty acyl-CoA's toward these two lipid classes. Serine palmitoyltransferase activities were also determined using different fatty acyl-CoA's and were consistently greatest with CoA thioesters of saturated fatty acids with 16 +/- 1 carbon atoms. This suggests that the predominance of 18-carbon long-chain bases in vivo is due to the higher activity of this enzyme with palmitoyl-CoA. Together, these findings indicate a role for serine palmitoyltransferase in regulating both the type and amount of long-chain bases found in tissues.     

Induction of Ganglioside Biosynthesis and Neurite Outgrowth of Primary Cultured Neurons by l-threo-1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol

Abstract: We reported previously that stereoisomers of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the d - threo and l - threo forms, exerted inhibitory and stimulatory effects on glycosphingolipid (GSL) biosynthesis in B16 melanoma cells, respectively. In the present study, the primary cultured rat neocortical explants were treated with l - or d - threo -PDMP. These isomers exhibited opposite effects on neurite outgrowth: d -PDMP was inhibitory at concentrations ranging from 5 to 20 µ M , whereas l -PDMP was stimulatory over the same concentration range, and the maximal effect was observed at 10–15 µ M . Rat neocortical explants were doubly labeled with [¹⁴C]serine and [³H]galactose at 15 µ M l - or d -PDMP. l -PDMP increased the incorporations of both labels into sphinganine, sphingosine, ceramide, sphingomyelin, neutral GSLs, and gangliosides, whereas d -PDMP inhibited the glucosylation of ceramide resulting in a reduction of ganglioside biosynthesis and accumulation of precursors of glucosylceramide, ceramide, and sphingomyelin. To clarify the stimulatory effect of l -PDMP on GSL biosynthesis, serine palmitoyltransferase, sphingosine N -acyltransferase, glucosylceramide synthase, lactosylceramide synthase, GM3 synthase, and GD3 synthase were quantified in cell lysates of explants pretreated with this agent. Serine palmitoyltransferase was fully activated up to 150% of the control. Furthermore, marked increases in the activities of lactosylceramide synthase (200%), GM3 synthase (240%), and GD3 synthase (300%) were observed. These results suggest that the neurotrophic action of l -PDMP may be ascribable to its stimulatory effect on the biosynthesis of GSLs, especially that of gangliosides.     

Enzymology of long-chain base synthesis by liver: characterization of serine palmitoyltransferase in rat liver microsomes

Serine palmitoyltransferase [palmitoyl-CoA:L-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50] catalyzes the initial and committed step in the biosynthesis of the long-chain bases of sphingolipids. A simple assay, based upon the incorporation of [3H]serine into the chloroform-soluble product 3-ketosphinganine, has been developed and demonstrated to be valid for analyzing this enzyme in rat liver microsomes. More than 75% of the serine palmitoyltransferase of rat liver was associated with the microsomal subfraction. The dependencies of activity on the incubation time, pH, temperature, other assay components (e.g., dithiothreitol, EDTA, and pyridoxal 5'-phosphate), and the concentrations of microsomal protein, L-serine, and palmitoyl-CoA were investigated. The requirement of pyridoxal 5'-phosphate for activity was established by formation of the apoenzyme by dialysis against cysteine, and recovery of full activity upon reconstitution with the coenzyme. Activities with fatty acyl-CoA's of varying alkyl chain length were distributed nearly symmetrically around a maximum at 16 carbons (palmitoyl-CoA) for the fully saturated substrates. Less activity was obtained with the CoA thioesters of cis-unsaturated fatty acids, but trans-9-hexadecenoyl-CoA yielded essentially the same activity as palmitoyl-CoA. Hence, this enzyme is capable of initiating the synthesis of the major long-chain bases, as well as compounds that may constitute the unidentified bases reported in analyses of mammalian sphingolipids.     

Uncoupling of ganglioside biosynthesis by Brefeldin A

We have studied the effect of Brefeldin A (BFA), an antiviral antibiotic, on glycosphingolipid metabolism in primary cultured cerebellar cells. Cells were labeled metabolically with [14C]galactose, or pulse-labeled with precursors of glycosphingolipid biosynthesis; i.e., [14]serine, [3H]palmitic acid or [3H]sphingosine. In all cases BFA (1 microgram/ml) strongly inhibited (75-95%) ganglioside biosynthesis beyond the stage of GM3 and GD3, that is the formation of GM1, GD1a, GT1b and GQ1b. Simultaneously an accumulation of GlcCer, LacCer, GM3 and GD3 was observed (up to 2000%). These effects could be reversed fully by removal of the BFA from the culture medium. These results indicate that the LacCer-, GM3- and GD3-synthases of murine cerebellar cells are localized together on the proximal site of the Golgi apparatus, probably in the cis-Golgi compartment. It is probable that sphingomyelin synthase and some of the other glycosyltransferases involved in ganglioside biosynthesis are localized in distinct compartments beyond the cis Golgi.     

Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes)

The activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC 2.3.1.50), the enzyme catalyzing the first step in the synthesis of the long-chain base required for sphingolipid assembly, has been characterized in a plant system. Enzyme activity in a microsomal membrane fraction from summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of L-[3H]Ser into the chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme exhibited a strong preference for palmitoyl-CoA, with optimal activity at a substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was required as a coenzyme. The pH optimum was 7.6, and the temperature optimum was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. Two known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and [beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. The results from this study suggest that the properties and catalytic mechanism of Ser palmitoyltransferase from squash are similar to those of the animal, fungal, and bacterial enzyme in most respects. The specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher than those previously reported for preparations from animal tissues.     

D-Serine inhibits serine palmitoyltransferase, the enzyme catalyzing the initial step of sphingolipid biosynthesis

Serine palmitoyltransferase (SPT), responsible for the initial step of sphingolipid biosynthesis, catalyzes condensation of palmitoyl coenzyme A and L-serine to produce 3-ketodihydrosphingosine (KDS). For determination of the stereochemical specificity of the amino acid substrate, a competition analysis of the production of [(3)H]KDS from L-[(3)H]serine was performed using purified SPT. D-Serine inhibited [(3)H]KDS production as effectively as non-radioactive L-serine, whereas neither D-alanine nor D-threonine showed any significant effect. Incubation of purified SPT with [palmitoyl 1-(14)C]palmitoyl coenzyme A and D-serine did not produce [(14)C]KDS, while the control incubation with L-serine did. These results suggest that D-serine competes with L-serine for the amino acid recognition site of SPT, but that D-serine is not utilized by this enzyme to produce KDS.     

Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s)

The de novo biosynthesis of sphinganine and sphingosine was studied using LM cells incubated with [14C] serine in serum-free media. Most of the radiolabeled long-chain bases were initially found in dihydroceramides (as sphinganine) and the proportion appearing in complex sphingolipids (as sphingosine) increased over time. Since free long-chain bases were not detected (although formation of 3-ketosphinganine, the first condensation product of serine and palmitoyl-CoA, could be demonstrated in vitro), it appears that the first step is rate-limiting for dihydroceramide biosynthesis. The kinetics suggested that after N-acyl-sphinganines were formed they were dehydrogenated to N-acylsphingosines. No evidence was found for the formation in vivo or in vitro of the putative intermediates of the direct biosynthesis of sphingosine from sphinganine (i.e. 3-ketosphingosine and free sphingosine). The conversion of N-acylsphinganines to N-acyl-sphingosines was confirmed by incubating cells with [14C] serine followed by unlabeled serine, which resulted in a rapid increase in the sphingosine-to-sphinganine ratio in amide-linked sphingolipids during the chase. These findings are most consistent with a pathway for long-chain base biosynthesis in which N-acyl-sphinganines are first synthesized by LM cells and the 4-trans-double bond is added to this or subsequent products, as opposed to the most cited pathway wherein sphingosine is made directly from sphinganine.     

Factors to consider in using [U-C]palmitate for analysis of sphingolipid biosynthesis by tandem mass spectrometry

This study describes the use of a stable-isotope labeled precursor ([U-13C]palmitate) to analyze de novo sphingolipid biosynthesis by tandem mass spectrometry. It also describes factors to consider in interpreting the data, including the isotope's location (13C appears in three isotopomers and isotopologues: [M + 16] for the sphingoid base or N-acyl fatty acid, and [M + 32] for both); the isotopic enrichment of palmitoyl-CoA; and its elongation, desaturation, and incorporation into N-acyl-sphingolipids. For HEK293 cells incubated with 0.1 mM [U-13C]palmitic acid, ～60% of the total palmitoyl-CoA was 13C-labeled by 3 h (which was near isotopic equilibrium); with this correction, the rates of de novo biosynthesis of C16:0-ceramide, C16:0-monohexosylceramide, and C16:0-sphingomyelins were 62 ± 3, 13 ± 2, and 60 ± 11 pmol/h per mg protein, respectively, which are consistent with an estimated rate of appearance of C16:0-ceramide using exponential growth modeling (119 ± 11 pmol/h per mg protein). Including estimates for the very long-chain fatty acyl-CoAs, the overall rate of sphingolipid biosynthesis can be estimated to be at least ～1.6-fold higher. Thus, consideration of these factors gives a more accurate picture of de novo sphingolipid biosynthesis than has been possible to-date, while acknowledging that there are inherent limitations to such approximations.     

Biosynthesis of willardiine and isowillardiine in germinating pea seeds and seedlings

The synthesis of the pyrimidinyl amino acids willardiine and isowillardiine was studied in vivo and in vitro. Uracil derivatives stimulate the biosynthesis of both compounds; the free base is the most effective. Significant incorporation of [2-(14)C]uracil and [6-(14)C]orotate into willardiine and isowillardiine was found. Incorporation of [6-(14)C]orotate was substantially decreased in the presence of uracil, and to a lesser extent by uridine and UMP. [3-(14)C]Serine was incorporated into the alanine side chain of the two uracilylalanines but not into the ring. The effect of a number of uracil analogues and inhibitors of pyrimidine metabolism was examined. Some were shown to stimulate the biosynthesis; the most noticeable effects were obtained with 6-azauracil and 2-thiouracil. Attempts to obtain extracts capable of synthesizing the uracilylalanines from uracil and serine were unsuccessful, but weak activity was observed when serine was replaced by O-acetylserine.     

Etomoxir mediates differential metabolic channeling of fatty acid and glycerol precursors into cardiolipin in H9c2 cells

We examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL.     

Sphingolipid biosynthesis in cultured neurons. Down-regulation of serine palmitoyltransferase by sphingoid bases.

Addition of exogenous sphingosine homologues (D-erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of serine palmitoyltransferase activity in a time- and concentration-dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half-maximal reduction of enzyme activity occurred after a 4-h treatment with 25 microM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4-8 h with 50 microM long-chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10-50 microM), was fed to the cells, decrease of 3-ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D-erythro-sphingosine, L-threo-C18-sphingosine did not yield any decrease of serine palmitoyltransferase activity when added to the cells under identical experimental conditions. Decrease of serine palmitoyltransferase activity was fully reversible after removal of the long-chain bases from the culture medium. Activities of other enzymes of lipid metabolism, ceramide synthase, long-chain acyl-CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of serine palmitoyltransferase is quite specific.     

Purine biosynthesis de novo in rat skeletal muscle.

Purine biosynthesis by the 'de novo' pathway was demonstrated in isolated rat extensor digitorum longus muscle with [1-14C]glycine, [3-14C]serine and sodium [14C]formate as nucleotide precursors. Evidence is presented which suggests that the source of glycine and serine for purine biosynthesis is extracellular rather than intracellular. The relative incorporation rates of the three precursors were formate greater than glycine greater than serine. Over 85% of the label from formate and glycine was recovered in the adenine nucleotides, principally ATP. Azaserine markedly inhibited purine biosynthesis from both formate and glycine. Cycloserine inhibited synthesis from serine, but not from formate. Adenine, hypoxanthine and adenosine markedly inhibited purine synthesis from sodium [14C]formate.     

Propionate inhibits hepatocyte lipid synthesis

Oat bran lowers serum cholesterol in animals and humans. Propionate, a short-chain fatty acid produced by colonic bacterial fermentation of soluble fiber, is a potential mediator of this action. We tested the effect of propionate on hepatocyte lipid synthesis in rats using [1-14C]acetate, 3H2O, and [2-14C]mevalonate as precursors. Propionate produced a statistically significant inhibition of cholesterol biosynthesis from [1-14C]acetate at a concentration of 1.0 mM and from 3H2O and [2-14C]mevalonate at concentrations of 2.5 mM. Propionate also produced a significant inhibition of fatty acid biosynthesis at concentrations of 2.5 mM using [1-14C]acetate as a precursor. The demonstration of propionate-mediated inhibition of cholesterol and fatty acid biosynthesis at these concentrations suggests that propionate may inhibit cholesterol and fatty acid biosynthesis in vivo and may mediate in part the hypolipidemic effects of soluble dietary fiber. Further studies are needed to clarify this action of propionate and to establish the exact mechanisms by which the inhibition occurs.     

Sphingolipid metabolism in Bacteroideaceae.

The lipid composition of the anaerobic Bacteroides thetaiotaomikron has been analyzed. Sphingomyelin, ceramide phosphinicoethanolamine, free even-numbered and branched chain sphingosine bases and ceramide represented about 50% of the total lipid extract. The main ester phospholipid was phosphatidylethanolamine. The alkali-stable sphingophospholipids were predominantly N-acylated with 3-hydroxypalmitic acid, whereas the ester phospholipids are preferentially substituted with normal even and odd-numbered and branched-chain fatty acids. When Bacteroides was grown in a medium supplemented with labelled palmitic acid, this fatty acid was utilized for acylation reactions and to a large extent for the de novo synthesis of sphinganine. This long-chain base was incorporated into the sphingolipids and was also present in free form. The 3-hydroxypalmitic acid present in sphingolipids is not derived from palmitic acid, since labelled palmitate did not serve as a precursor. Free sphinganine added to the culture medium was also utilized efficiently for the biosynthesis of the sphingolipids by growing Bacteroides cultures. The 3H/14C ratio in sphingomyelin and ceramide phosphinicoethanolamine is the same, when [1-14C]palmitic acid and [3-3H]sphinganine serve as precursors. Sphingomyelin, which is usually only present in higher animals, is synthesized de novo in this Bacteroides strain.     

The biosynthesis of phosphatidylserine and phosphatidylethanolamine from L-[3-14C]serine in isolated rat hepatocytes

Incorporation of L-[3-14C]serine into phosphatidylserine (PS) and phosphatidylethanolamine (PE) has been studied in isolated rat hepatocytes. Ethanolamine inhibited the incorporation, indicating competition with serine in the base-exchange reaction. Choline, monomethylethanolamine, dimethylethanolamine and dimethyl-3-aminopropan-1-ol had no such effect. The observed rate of PS biosynthesis corresponded to 7-17 nmol/min per liver at 0.55 mM L-serine. The results indicate that only a small fraction (1/25 to 1/70) of the PS pool equilibrates with the base-exchange enzyme, and that decarboxylation to PE occurs preferentially from this pool. The rate of PS synthesis and decarboxylation can therefore not be calculated by methods which assume random, homogeneous labelling of the total PS pool. The apparent rate of PS decarboxylation increased approx. 4-fold when L-serine increased from 0.5 to 2.25 mM, suggesting that decarboxylation of PS to PE might be regulated by the concentration of L-serine or by the amount of PS present in the hepatocyte cell membranes. Lauric, palmitic, stearic, oleic and linoleic acid decreased the rate of PS synthesis. At 0.5 mM, lauric and palmitic acid were most inhibitory. At 1.0 mM, linoleic acid was the least inhibitory fatty acid. The saturated hexaenoic and saturated tetraenoic species of PS contained 51 and 29%, respectively, of the incorporated L-[3-14C]serine. The combined monoene dienoic/diene dienoic fraction had the highest rate of synthesis judged by its relative specific activity. At 0.9 mM concentration, linoleic acid doubled the relative specific activity of the combined monoene dienoic/diene dienoic fraction of PS. Incorporation of L-[3-14C]serine into molecular species of PE resembled that into PS, both in the absence and presence of linoleic acid, suggesting that the phosphatidylserine decarboxylase (EC 4.1.1.65) has a low specificity towards the fatty acid composition of PS. The results indicate that biosynthesis of PS from L-serine occurs mainly by the base-exchange with only negligible contribution from direct incorporation of phosphatidic acid or diacylglycerol. Furthermore, the deacylation-reacylation pathway seem to contribute only little to the determination of the fatty acid composition of hepatocyte PS. Active PS turnover seems to be confined to a small fraction of the PS pool.     

Inhibition of serine palmitoyltransferase activity in rabbit aorta by L-cycloserine

Serine palmitoyltransferase (EC 2.3.1.50) initiates the biosynthesis of sphingolipids. Its activity is induced in the aortas of rabbits fed a Purina lab chow supplemented with 2% cholesterol (Williams, R. D., D. S. Sgoutas, and G. S. Zaatari. 1986. J. Lipid Res. 27: 763-770). Induction occurs during atherogenesis in parallel with increased arterial sphingomyelin concentrations. In this study, L-cycloserine was shown to be a potent inhibitor of serine palmitoyltransferase in aortas from New Zealand White rabbits. Activity was reduced in vitro by 50% using 5 microM L-cycloserine with 50 micrograms of microsomal protein. To assess in vivo inhibition, L-cycloserine was administered by intraperitoneal injection to rabbits maintained on either a standard Purina laboratory chow or one supplemented with 2% cholesterol. Serine palmitoyltransferase activity was inhibited by 76% in the aortas of rabbits on the standard chow 4 hr after a single 25 mg/kg body weight dose and 52% after a 10 mg/kg dose. Activity was reduced by 36% in animals on the standard chow and by 37% in the cholesterol-fed group after 1 week of daily doses. These experiments demonstrate that L-cycloserine inhibits serine palmitoyltransferase in aorta, and thus may be used to reduce sphingomyelin concentrations during experimental atherogenesis.     

OBSERVATIONS ON THE BIOSYNTHESIS OF RAT BRAIN CERAMIDE AND CEREBROSIDE IN VIVO

Abstract— The incorporation in vivo of l -[¹⁴C]serine into ceramide and cerebroside of young rat brain has been studied. Acid hydrolysis of labelled ceramide and galactosyl-ceramide followed by selective partitioning of the resulting components indicated that 88 per cent of the radioactivity was present in the long-chain base portion. At early time points (10 min, 20 min) the precursor was incorporated into ceramide and to a lesser degree into glucosyl-ceramide. During time intervals of 5 and 10 h, the specific activity values (d.p.m./μmol) for ceramide and glucosyl-ceramide decreased, while values for galactosyl-ceramide, containing either unsubstituted fatty acids (NFA) or α-hydroxy fatty acids (HFA), increased 50 and 30 per cent, respectively. Analysis of labelled ceramide at all time points studied (10 min-10 h) indicated that l -[¹⁴C]serine was incorporated onto the NFA type. This observation suggests that HFA-ceramide may not be the physiological precursor of HFA-galactosyl-ceramide. In this context, the postulated precursor roles of both ceramide and psychosine in the biosynthesis of brain cerebrosides are discussed.     

Serine regulates phosphatidylethanolamine biosynthesis in the hamster heart.

The role of serine as a precursor and metabolic regulator for phosphatidylethanolamine biosynthesis in the hamster heart was investigated. Hearts were perfused with 50 microM [1-3H]ethanolamine in the presence or absence of serine for up to 60 min. Ethanolamine uptake was attenuated by 0.05-10 mM serine in a noncompetitive manner, and the incorporation of labeled ethanolamine into phosphatidylethanolamine was also inhibited by serine. Analysis of the ethanolamine-containing metabolites in the CDP-ethanolamine pathway revealed that the conversion of ethanolamine to phosphoethanolamine was reduced. The reduction was a result of an inhibition of ethanolamine kinase activity by an elevated pool of intracellular serine. Perfusion of the heart with 1 mM serine caused a 5-fold increase in intracellular serine pool. In order to examine the action of serine on other phosphatidylethanolamine metabolic pathways, hearts were perfused with [1-3H]glycerol in the presence and absence of serine. Serine did not cause any enhancement of phosphatidylethanolamine hydrolysis. The base-exchange reaction for phosphatidylserine formation or the decarboxylation of phosphatidylserine was not affected by serine perfusion. We conclude that circulating serine plays an important role in the modulation of phosphatidylethanolamine biosynthesis via the CDP-ethanolamine pathway in the hamster heart but does not affect the contribution of the decarboxylase pathway for phosphatidylethanolamine formation.