Induction of the lactose transport system in a lipid-synthesis-defective mutant of Escherichia coli

In order to relate the biogenesis of the lactose transport system to lipid synthesis, a glycerol-requiring mutant of Escherichia coli K-12 with a specific defect in l-glycerol-3-phosphate synthesis was isolated and characterized. The defective enzyme is the biosynthetic l-glycerol-3-phosphate dehydrogenase [l-glycerol-3-phosphate: NAD (P) oxidoreductase, EC 1.1.1.8] which functions as a dihydroxyacetone phosphate reductase to provide l-glycerol-3-phosphate for lipid synthesis. In this mutant, removal of glycerol from the growth medium results in inhibition of the synthesis of protein, deoxyribonucleic acid, and phospholipid. Inhibition of phospholipid synthesis immediately follows glycerol removal, whereas the inhibition of deoxyribonucleic acid and protein synthesis is preceded by a short lag period. Glycerol starvation does not change the turnover pattern of previously synthesized phospholipids. The blocking of lipid synthesis by glycerol starvation causes a drastic decrease in inducibility of beta-galactoside transport activity relative to beta-galactosidase, indicating that induction of lactose transport requires de novo lipid synthesis.     

Lipid composition and metabolism in megakaryocytes at different stages of maturation

The lipid composition and metabolism of isolated guinea pig megakaryocyte subgroups at various stages of maturation were investigated. Three groups were studied: 1) 67% of megakaryocytes in Group A were immature; 2) Group B was heterogeneous and contained both immature and mature subgroups of megakaryocytes; 3) 92% of megakaryocytes in Group C were mature. Lipid composition was determined by thin-layer chromatography, lipid-phosphorus, and gas-liquid chromatography. Cholesterol, ceramide, and de novo fatty acid synthesis were evaluated with [14C]acetate. [14C]Glycerol was used to assess de novo phospholipid synthesis. 14C-Labeled fatty acids were used to evaluate fatty acid uptake. The phospholipid and cholesterol content was found to be four times greater in mature megakaryocytes than that in immature megakaryocytes, which paralleled the protein content and volume of mature and immature cells. The cholesterol-phospholipid ratio was similar and there were no differences in the phospholipid species in the three groups. Phospholipid and cholesterol synthesis were established in immature megakaryocytes and persisted at about the same level in mature megakaryocytes. The uptake of arachidonic and palmitic acids also occurred primarily in immature cells, while the de novo synthesis of palmitic acid occurs predominantly in mature megakaryocytes. There was an inverse relationship between the uptake of exogenous palmitic acid and fatty acid synthesis, but the uptake of palmitic acid primarily inhibited fatty acid synthesis in mature megakaryocytes. There were differences in the acylation of phospholipid species with arachidonic acid in megakaryocytes at different stages of maturation since the acylation of phosphatidylcholine occurred primarily in immature megakaryocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

An update on the role of phospholipid metabolism in the action of steroidogenic agents

Most steroidogenic agents which bind to cell surface receptors activate adenylate cyclase and/or phospholipase C. Activation of either signaling system may also be associated with rapid increases in de novo phospholipid synthesis, but it is at present uncertain whether this is a secondary or parallel event. Activation of phospholipase C leads to hydrolysis of phosphatidylinositol-4',5'-PO4 (PIP2) and generation of two second messengers, inositol-triphosphate and diacylglycerol (DAG), which mobilize Ca2+ and activate protein kinase C, respectively. Increases in de novo phospholipid synthesis lead to rapid increases in phosphatidic acid, DAG and C-kinase activity. The PIP2-phospholipase C system appears to initiate the steroidogenic response to certain agents, such as angiotensin-II, and this may be amplified by concomitant increases in phospholipid synthesis. With other agonists, the role of phospholipase C activation and de novo phospholipid (and DAG) synthesis is less certain. In some tissues, activation of protein kinase C by exogeneously added DAG analogues provokes an increase in steroidogenesis. However, this is not observed in other tissues, and it is uncertain whether this rules out involvement of the C-kinase system for steroidogenesis in these tissues, or whether endogenously produced DAG is a more effective activator of the relevant C-kinase system then exogenously added DAG analogues. The role of other potential intracellular signaling substances that may be derived from phospholipase C activation and de novo phospholipid synthesis is also at present uncertain, as are the interrelationships between these two phospholipid responses, cyclic nucleotides, and other steroidogenic factors.     

De novo fatty acid synthesis at the mitotic exit is required to complete cellular division

Although the regulation of the cell cycle has been extensively studied, much less is known about its coordination with the cellular metabolism. Using mass spectrometry we found that lysophospholipid levels decreased drastically from G₂/M to G₁ phase, while de novo phosphatidylcholine synthesis, the main phospholipid in mammalian cells, increased, suggesting that enhanced membrane production was concomitant to a decrease in its turnover. In addition, fatty acid synthesis and incorporation into membranes was increased upon cell division. The rate-limiting reaction for de novo fatty acid synthesis is catalyzed by acetyl-CoA carboxylase. As expected, its inhibiting phosphorylation decreased prior to cytokinesis initiation. Importantly, the inhibition of fatty acid synthesis arrested the cells at G₂/M despite the presence of abundant fatty acids in the media. Our results suggest that de novo lipogenesis is essential for cell cycle completion. This “lipogenic checkpoint” at G₂/M may be therapeutically exploited for hyperproliferative diseases such as cancer.     

Kinetic analyses of liver phosphatidylcholine and phosphatidylethanolamine biosynthesis using 13C NMR spectroscopy

Choline and ethanolamine are substrates for de novo synthesis of phosphatidylcholine (PtdC) and phosphatidylethanolamine (PtdE) through the CDP-choline and CDP-ethanolamine pathways. In liver, PtdE can also be converted to PtdC by PtdE N-methyltransferase (PEMT). We investigated these kinetics in rat liver during a 60 min infusion with ¹³C-labeled choline and ethanolamine. NMR analyses of liver extracts provided concentrations and ¹³C enrichments of phosphocholine (Pcho), phosphoethanolamine (Peth), PtdC, and PtdE. Kinetic models showed that the de novo and PEMT pathways are ‘channeled’ processes. The intermediary metabolites directly derived from exogenous choline and ethanolamine do not completely mix with the intracellular pools, but are preferentially used for phospholipid synthesis. Of the newly synthesized PtdC, about 70% was derived de novo and 30% was by PEMT. PtdC and PtdE de novo syntheses displayed different kinetics. A simple model assuming constant fluxes yielded a modest fit to the data; allowing upregulated fluxes significantly improved the fit. The ethanolamine-to-Peth flux exceeded choline-to-Pcho, and the rate of PtdE synthesis (1.04 μmol/h/g liver) was 2–3 times greater than that of PtdC de novo synthesis. The metabolic pathway information provided by these studies makes the NMR method superior to earlier radioisotope studies.     

Fatty acid synthesis in Escherichia coli is indirectly inhibited by phenethyl alcohol.

Experiments were performed to determine how phenethyl alcohol inhibits phospholipid synthesis in E. coli. At a nonbacteriostatic concentration, the drug reduces the rate of de novo fatty acid and phospholipid synthesis by 60 to 70%. The inhibition of fatty acid synthesis was found to be a secondary consequence of the inhibition of phospholipid synthesis. Phenethyl alcohol reduces the rate of incorporation of exogenous fatty acids into the phospholipids of a fatty acid auxotroph by 60%. These results indicate that this drug controls phospholipid synthesis beyond the level of fatty acid synthesis. Phenethyl alcohol inhibits the synthesis of phospholipids containing saturated fatty acids to a greater extent than it does the synthesis of phospholipids containing unsaturated fatty acids. It controls the synthesis of phospholipids containing saturated fatty acids at both the level of fatty acid synthesis and the level of incorporation of the saturated fatty acids into phospholipids. The synthesis of phospholipids containing unsaturated fatty acids is inhibited at the level of incorporation of the fatty acids into phospholipids.     

The origin of long-chain fatty acids required for de novo ether lipid/plasmalogen synthesis

Peroxisomes are single-membrane bounded organelles that in humans play a dual role in lipid metabolism, including the degradation of very long-chain fatty acids and the synthesis of ether lipids/plasmalogens. The first step in de novo ether lipid synthesis is mediated by the peroxisomal enzyme glyceronephosphate O-acyltransferase, which has a strict substrate specificity reacting only with the long-chain acyl-CoAs. The aim of this study was to determine the origin of these long-chain acyl-CoAs. To this end, we developed a sensitive method for the measurement of de novo ether phospholipid synthesis in cells and, by CRISPR-Cas9 genome editing, generated a series of HeLa cell lines with deficiencies of proteins involved in peroxisomal biogenesis, beta-oxidation, ether lipid synthesis, or metabolite transport. Our results show that the long-chain acyl-CoAs required for the first step of ether lipid synthesis can be imported from the cytosol by the peroxisomal ABCD proteins, in particular ABCD3. Furthermore, we show that these acyl-CoAs can be produced intraperoxisomally by chain shortening of CoA esters of very long-chain fatty acids via beta-oxidation. Our results demonstrate that peroxisomal beta-oxidation and ether lipid synthesis are intimately connected and that the peroxisomal ABC transporters play a crucial role in de novo ether lipid synthesis.     

S-adenosyl-L-homocysteine hydrolase, key enzyme of methylation metabolism, regulates phosphatidylcholine synthesis and triacylglycerol homeostasis in yeast: implications for homocysteine as a risk factor of atherosclerosis

In eukaryotes, S-adenosyl-L-homocysteine hydrolase (Sah1) offers a single way for degradation of S-adenosyl-L-homocysteine, a product and potent competitive inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases. De novo phosphatidylcholine (PC) synthesis requires three AdoMet-dependent methylation steps. Here we show that down-regulation of SAH1 expression in yeast leads to accumulation of S-adenosyl-L-homocysteine and decreased de novo PC synthesis in vivo. This decrease is accompanied by an increase in triacylglycerol (TG) levels, demonstrating that Sah1-regulated methylation has a major impact on cellular lipid homeostasis. TG accumulation is also observed in cho2 and opi3 mutants defective in methylation of phosphatidylethanolamine to PC, confirming that PC de novo synthesis and TG synthesis are metabolically coupled through the efficiency of the phospholipid methylation reaction. Indeed, because both types of lipids share phosphatidic acid as a precursor, we find in cells with down-regulated Sah1 activity major alterations in the expression of the INO1 gene as well as in the localization of Opi1, a negative regulatory factor of phospholipid synthesis, which binds and is retained in the endoplasmic reticulum membrane by phosphatidic acid in conjunction with VAMP/synaptobrevin-associated protein, Scs2. The addition of homocysteine, by the reversal of the Sah1-catalyzed reaction, also leads to TG accumulation in yeast, providing an attractive model for the role of homocysteine as a risk factor of atherosclerosis in humans.     

The elongation of exogenous fatty acids and the control of phospholipid acyl chain length in Micrococcus cryophilus.

The synthesis of fatty acids de novo from acetate and the elongation of exogenous satuated fatty acids (C12-C18) by the psychrophilic bacterium Micrococcus cryophilus (A.T.C.C. 15174) grown at 1 or 20 degrees C was investigated. M. cryophilus normally contains only C16 and C18 acyl chains in its phospholipids, and the C18/C16 ratio is altered by changes in growth temperature. The bacterium was shown to regulate strictly its phospholipid acyl chain length and to be capable of directly elongating myristate and palmitate, and possibly laurate, to a mixture of C16 and C18 acyl chains. Retroconversion of stearate into palmitate also occurred. Fatty acid elongation could be distinguished from fatty acid synthesis de novo by the greater sensitivity of fatty acid elongation to inhibition by NaAsO2 under conditions when the supply of ATP and reduced nicotinamide nucleotides was not limiting. It is suggested that phospholipid acyl chain length may be controlled by a membrane-bound elongase enzyme, which interconverts C16 and C18 fatty acids via a C14 intermediate; the activity of the enzyme could be regulated by membrane lipid fluidity.     

Fatty acid synthesis and metabolism of phospholipid acyl groups in strain L mouse fibroblasts

Strain L mouse fibroblasts grown in medium supplemented with 2.5% delipidized horse serum were found capable of desaturating oleic and linoleic acid to dienoic and trienoic acid(s), respectively. Although 40-60% of de novo fatty acid synthesis from [2-3H]acetate was inhibited by the administration of exogenous oleic or linoleic acid, sterole synthesis was only slightly affected. Within 24-48 h after incorporation, phospholipid fatty acyl groups could undergo active exchange between phospholipids. After this dynamic transition period was over, not only were the phospholipid acyls retained but some vicinal fatty acyl pairs of phospholipid also appeared to be stable and remained together throughout the depletion period. At any time in the experiment, however, introduction of exogenous fatty acid perturbed this phospholipid acyl retention, delayed the time at which the phospholipid acyl groups no longer moved between phospholipids and also decreased the ultimate number of phospholipid acyl groups retained by strain L mouse fibroblasts.     

The inhibition of phospholipid synthesis in escherichia coli by phenethyl alcohol.

The kinetics of lipid metabolism during phenethyl alcohol treatment of Escherichia coli were examined. Phenethyl alcohol at a non-bacteriostatic concentration reduces the accumulation of [32-P] phosphate into phospholipids and alters the phospholipid composition of the cell membrane. The changes in phospholipid composition are a result of the inhibitory effect of phenethyl alcohol on the rates of synthesis of the individual phospholipids. The inhibition in the rate of phosphatidylethanolamine synthesis by phenethyl alcohol was twice the inhibition in the rate of phosphatidyglycerol synthesis. The de novo rate of cardiolipin synthesis was only slightly inhibited. However, net cardiolipin accumulation increased during phenethyl alcohol treatment due to a more rapid turnover of phosphatidylglycerol to cardiolipin. Phenethyl alcohol also altered the fatty acid composition of the cell as a result of its inhibitory effect on the rate of individual fatty acid synthesis. However, the inhibition of phospholipid synthesis was not reversed by fatty acid supplementation of phenethyl alcohol treated cells. This result indicates that phenethyl alcohol does not inhibit phospholipid synthesis solely at the level of fatty acid synthesis.     

Acylation of glycerol 3-phosphate is the sole pathway of de novo phospholipid synthesis in Escherichia coli.

The inhibition of phospholipid synthesis engendered by starving glycerol 3-phosphate (G3P) auxotrophs of Escherichia coli (plsB or gpsA) for G3P is incomplete; 5 to 10% of the normal rate of phospholipid synthesis remains, even after prolonged starvation. We report that G3P starvation of a strain having lesions in both the gpsA and plsB genes resulted in essentially complete (greater than 98.5%) inhibition of phospholipid synthesis, indicating that all de novo glycerolipid synthesis in E. coli proceeds by acylation of G3P.     

A yeast acetyl coenzyme A carboxylase mutant links very-long-chain fatty acid synthesis to the structure and function of the nuclear membrane-pore complex.

The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope. Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space. MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis. Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids. We provide evidence that synthesis of very-long-chain fatty acids (C26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope.     

Differential effects of pertussis toxin on insulin-stimulated phosphatidylcholine hydrolysis and glycerolipid synthesis de novo. Studies in BC3H-1 myocytes and rat adipocytes

Insulin-induced increases in diacylglycerol (DAG) have been suggested to result from stimulation of de novo phosphatidic acid (PA) synthesis and phosphatidylcholine (PC) hydrolysis. Presently, we found that insulin decreased PC levels of BC3H-1 myocytes and rat adipocytes by approximately 10-25% within 30 s. These decreases were rapidly reversed in both cell types, apparently because of increased PC synthesis de novo. In BC3H-1 myocytes, pertussis toxin inhibited PC resynthesis and insulin effects on the pathway of de novo PA-DAG-PC synthesis, as evidenced by changes in [3H]glycerol incorporation, but did not inhibit insulin-stimulated PC hydrolysis. Pertussis toxin also blocked the later, but not the initial, increase in DAG production in the myocytes. Phorbol esters activated PC hydrolysis in both myocytes and adipocytes, but insulin-induced stimulation of PC hydrolysis was not dependent upon activation of PKC, since this hydrolysis was not inhibited by 500 microM sangivamycin, an effective PKC inhibitor. Our results indicate that insulin increases DAG by pertussis toxin sensitive (PA synthesis de novo) and insensitive (PC hydrolysis) mechanisms, which are mechanistically separate, but functionally interdependent and integrated. PC hydrolysis may contribute importantly to initial increases in DAG, but later sustained increases are apparently largely dependent on insulin-induced stimulation of the pathway of de novo phospholipid synthesis.     

Regulation of fetal lung disaturated phosphatidylcholine synthesis by de novo palmitate supply

Lung surfactant disaturated phosphatidylcholine (PC) is highly dependent on the supply of palmitate as a source of fatty acid. The purpose of this study was to investigate the importance of de novo fatty acid synthesis in the regulation of disaturated PC production during late prenatal lung development. Choline incorporation into disaturated PC and the rate of de novo fatty acid synthesis was determined by the relative incorporation of [14C]choline and 3H2O, respectively, in 20-day-old fetal rat lung explants and in 18-day-old explants which were cultured 2 days. Addition of exogenous palmitate (0.15 mM) increased (26%) choline incorporation into disaturated PC but did not inhibit de novo fatty acid synthesis, as classically seen in other lipogenic tissue. Even in the presence of exogenous palmitate, de novo synthesis accounted for 87% of the acyl groups for disaturated PC. Inhibition of fatty acid synthesis by agaric acid or levo-hydroxycitrate decreased the rate of choline incorporation into disaturated PC. When explants were subjected to both exogenous palmitate and 60% inhibition of de novo synthesis, disaturated PC synthesis was below control values and 75% of disaturated PC acyl moieties were still provided by de novo synthesis. These data show that surfactant disaturated PC synthesis is highly dependent on the supply of palmitate from de novo fatty acid synthesis.     

Effects of Cyanide on Rates and Products of Fatty Acid Synthesis by Chloroplasts Isolated from Spinacia oleracea

Cyanide inhibited unesterified fatty acid synthesis but stimulated glyceride synthesis from [1-¹⁴C]acetate when Spinacia oleracea chloroplasts were incubated in basal media. Both unesterified fatty acid and glyceride accumulation were inhibited when chloroplasts were incubated in a diacylglycerol mode. Stimulation of chloroplast fatty acid synthesis by either exogenous coenzyme A or Triton X-100 was almost completely abolished in the presence of cyanide. Stearoyl-ACP desaturation is considered to be inhibited to a greater extent than is fatty acid synthesis de novo.     

DGK1-encoded diacylglycerol kinase activity is required for phospholipid synthesis during growth resumption from stationary phase in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, triacylglycerol mobilization for phospholipid synthesis occurs during growth resumption from stationary phase, and this metabolism is essential in the absence of de novo fatty acid synthesis. In this work, we provide evidence that DGK1-encoded diacylglycerol kinase activity is required to convert triacylglycerol-derived diacylglycerol to phosphatidate for phospholipid synthesis. Cells lacking diacylglycerol kinase activity (e.g. dgk1Δ mutation) failed to resume growth in the presence of the fatty acid synthesis inhibitor cerulenin. Lipid analysis data showed that dgk1Δ mutant cells did not mobilize triacylglycerol for membrane phospholipid synthesis and accumulated diacylglycerol. The dgk1Δ phenotypes were partially complemented by preventing the formation of diacylglycerol by the PAH1-encoded phosphatidate phosphatase and by channeling diacylglycerol to phosphatidylcholine via the Kennedy pathway. These observations, coupled to an inhibitory effect of dioctanoyl-diacylglycerol on the growth of wild type cells, indicated that diacylglycerol kinase also functions to alleviate diacylglycerol toxicity.     

Inhibition of the Development of Induced Respiration and Cyanide-insensitive Respiration in Potato Tuber Slices by Cerulenin and Dimethylaminoethanol

The interdependence of the development of wound-induced respiration and membrane-related phospholipid biosynthesis in potato tuber (Solanum tuberosum var. Russet) slices was established by the use of agents which selectively affect lipid and phospholipid synthesis. Cerulenin, a specific inhibitor of de novo fatty acid synthesis, inhibited the ultimate development of wound-induced respiration and of cyanide resistance only when given in the critical first 10 to 12 hours of slice aging. Similarly, when slices were exposed to the choline analogue dimethylaminoethanol within the first 10 hours, the phospholipid composition of the membrane lipids was drastically altered, the wound-induced respiration in a 24-hr period was substantially curtailed, and the development of cyanide insensitivity was sharply inhibited. These observations indicate that time-restricted membrane-related phospholipid synthesis is prerequisite to the development of wound-induced respiration and concurrent cyanide insensitivity.     

Intestinal aspects of lipid absorption: in review

The rapidly evolving field of lipid absorption is reviewed with the thrust of new knowledge focused on the interpendency of the luminal and cellular phases of absorption. To date little attention has been paid to factors that regulate the phospholipid biosynthesis in the enterocyte. The availability of 20:4 omega 6 may be the rate-limiting factor for phospholipid synthesis. The source of 20:4 omega 6 is unknown, whether it be synthesized de novo the enterocyte or entirely originating from degradation of bile phospholipid. It has been established that dietary fat can modulate the enterocyte membrane lipid composition and transport properties. Specified fats such as as fish oils rich in 20:5 omega 3 and 22:6 omega 3 have been implicated as protective against hypercholesterolemia. However, the effects of these dietary fats on the transport of nutrients across the enterocyte are not yet known, nor are the mechanisms responsible for the adaptive responses of the brush border identified.