Growth and metabolism of inositol-starved Saccharomyces cerevisiae.

Upon starvation for inositol, a phospholipid precursor, an inositol-requiring mutant of Saccharomyces cerevisiae has been shown to die if all other conditions are growth supporting. The growth and metabolism of inositol-starved cells has been investigated in order to determine the physiological state leading to "inositolless death". The synthesis of the major inositol-containing phospholipid ceases within 30 min after the removal of inositol from the growth medium. The cells, however, continue in an apparently normal fashion for one generation (2 h under the growth conditions used in this study). The cessation of cell division is not preceded or accompanied by any detectable change in the rate of macromolecular synthesis. When cell division ceases, the cells remain constant in volume, whereas macromolecular synthesis continues at first at an unchanged rate and eventually at a decreasing rate. Macromolecular synthesis terminates after about 4 h of inositol starvation, at approximately the time when the cells begin to die. Cell death is also accompanied by a decline in cellular potassium and adenosine triphosphate levels. The cells can be protected from inositolless death by several treatments that block cellular metabolism. It is concluded that inositol starvation results in a imbalance between the expansion of cell volume and the accumulation of cytoplasmic constituents. This imbalance is very likely the cause of inositolless death.     

The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+.

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated 'second messenger' which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular 'messenger' for controlling metabolic processes during insulin action.     

Lipid synthesis during morphogenesis of Mucor racemosus.

Lipid synthesis increases coordinately with protein and RNA synthesis during morphogenesis of Mucor racemosus. The lipid synthesis inhibitor cerulenin can completely block morphogenesis under conditions in which cell growth continues. An increase in phospholipid turnover may be an important correlate to morphogenesis of Mucor spp., especially the turnover of phosphotidyl inositol and phosphatidyl ethanolamine. The increase in ornithine decarboxylase, which occurs during morphogenesis, is inhibited by the addition of cerulenin.     

Effects of insulin and protein synthesis inhibitors on phospholipid metabolism, diacylglycerol levels, and pyruvate dehydrogenase activity in BC3H-1 cultured myocytes

BC3H-1 myocytes were cultured with 32PO4 for 3 days to label phospholipids to constant specific activity. Subsequent treatment with physiological concentrations of insulin provoked 40-70% increases in 32PO4 levels (reflecting increases in mass) in phosphatidic acid, phosphatidylinositol, and polyphosphoinositides, and, lesser, 20-25% increases in phosphatidylserine and the combined chromatographic area containing phosphatidylethanolamine plus phosphatidylcholine plus phosphatidylcholine. Insulin-induced increases in phospholipids were significant within 5 min and near-maximal at 15-30 min. Comparable rapid insulin-induced increases in [3H]phosphatidylinositol were observed in myocytes prelabeled with [3H]inositol. These insulin effects (as per prolonged pulse-chase experiments) were due to increase phospholipid synthesis rather than decreased phospholipid degradation. Cycloheximide (and puromycin) pretreatment prevented insulin-induced increases in phospholipids and rapidly reversed ongoing insulin effects on phospholipids and pyruvate dehydrogenase activity. Insulin also rapidly increased diacylglycerol levels. These findings suggest that: (a) insulin provokes rapid increases in de novo synthesis of phosphatidic acid and its derivatives, e.g. phosphoinositides and diacylglycerol; (b) protein synthesis inhibitors diminish phospholipid levels in insulin-treated (but not control) tissues by increasing phospholipid degradation (?phospholipase(s) activation); and (c) changes in phospholipids and diacylglycerol may be important for changes in pyruvate dehydrogenase and other enzymatic activities during treatment with insulin and/or protein synthesis inhibitors.     

Coordination of storage lipid synthesis and membrane biogenesis: evidence for cross-talk between triacylglycerol metabolism and phosphatidylinositol synthesis

Despite the importance of triacylglycerols (TAG) and steryl esters (SE) in phospholipid synthesis in cells transitioning from stationary-phase into active growth, there is no direct evidence for their requirement in synthesis of phosphatidylinositol (PI) or other membrane phospholipids in logarithmically growing yeast cells. We report that the dga1Δlro1Δare1Δare2Δ strain, which lacks the ability to synthesize both TAG and SE, is not able to sustain normal growth in the absence of inositol (Ino(-) phenotype) at 37 °C especially when choline is present. Unlike many other strains exhibiting an Ino(-) phenotype, the dga1Δlro1Δare1Δare2Δ strain does not display a defect in INO1 expression. However, the mutant exhibits slow recovery of PI content compared with wild type cells upon reintroduction of inositol into logarithmically growing cultures. The tgl3Δtgl4Δtgl5Δ strain, which is able to synthesize TAG but unable to mobilize it, also exhibits attenuated PI formation under these conditions. However, unlike dga1Δlro1Δare1Δare2Δ, the tgl3Δtgl4Δtgl5Δ strain does not display an Ino(-) phenotype, indicating that failure to mobilize TAG is not fully responsible for the growth defect of the dga1Δlro1Δare1Δare2Δ strain in the absence of inositol. Moreover, synthesis of phospholipids, especially PI, is dramatically reduced in the dga1Δlro1Δare1Δare2Δ strain even when it is grown continuously in the presence of inositol. The mutant also utilizes a greater proportion of newly synthesized PI than wild type for the synthesis of inositol-containing sphingolipids, especially in the absence of inositol. Thus, we conclude that storage lipid synthesis actively influences membrane phospholipid metabolism in logarithmically growing cells.     

Proteolipids in developing rat brain

In this report data are summarized on changes in the quantity of proteolipid protein (PLP), its amino acid composition, and the lipid moiety of these lipid-protein complexes in rat brain during postnatal development. In all three parts of the central nervous system (CNS) studied (cerebral hemispheres, medulla oblongata and spinal cord) the main pattern of PLP accumulation is on the whole similar. PLP content is very low in the newborn, and it increased 12 to 20-fold during development. The highest rate of PLP accumulation is observed in the periodfrom 10 to 30 days after birth. Against the background of general similarity the concentration of some amino acids such as lysine, proline, tyrosine in PLP somewhat increased during development, while that of aspartic acid, glutamic acid, glycine, and leucine decreased. Soluble proteolipid complexes, purified to various degree from lipids were isolated from brain of rats of different ages. As compared with the original lipid extracts from which they were obtained, the crude and especially purified proteolipids in all the animals studied were enriched in acidic phospholipids (PhL). This prevalence of acidic PhL increased with age. During the development in phospholipid moiety of proteolipids (PL) the content of phosphatidyl serine, sphingomyelin and mainly diphosphatidyl glycerol increases and that of phosphatidyl inositol and especially phosphatidyl choline decreases. The concentration of acidic PhL more tightly bound with PLP appreciably increases with age. Most of these changes occur mainly during the second decade after birth.Special Issue dedicated to Dr. Eugene Kreps.     

Metabolism of the phospholipid precursor inositol and its relationship to growth and viability in the natural auxotroph Schizosaccharomyces pombe.

Phospholipid metabolism in the fission yeast Schizosaccharomyces pombe was examined. Three enzymes of phospholipid biosynthesis, cytidine diphosphate diacylglycerol synthase (CDP-DG), phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase, were characterized in extracts of S. pombe cells. Contrary to an earlier report, we were able to demonstrate that CDP-DG served as a precursor for PI and PS biosynthesis in S. pombe. S. pombe is naturally auxotrophic for the phospholipid precursor inositol. We found that S. pombe was much more resistant to loss of viability during inositol starvation than artificially generated inositol auxotrophs of Saccharomyces cerevisiae. The phospholipid composition of S. pombe cells grown in inositol-rich medium (50 microM) was similar to that of S. cerevisiae cells grown under similar conditions. However, growth of S. pombe at low inositol concentrations (below 30 microM) affected the ratio of the anionic phospholipids PI and PS, while the relative proportions of other glycerophospholipids remained unchanged. During inositol starvation, the rate of PI synthesis decreased rapidly, and there was a concomitant increase in the rate of PS synthesis. Phosphatidic acid and CDP-DG, which are precursors to these phospholipids, also increased when PI synthesis was blocked by lack of exogenous inositol. The major product of turnover of inositol-containing phospholipids in S. pombe was found to be free inositol, which accumulated in the medium and could be reused by the cell.     

Insulin provokes a transient activation of phospholipase C in the rat epididymal fat pad

Insulin is known to increase the de novo synthesis of inositol phospholipids in rat epididymal fat pads. We presently examined the effects of insulin on the hydrolysis of inositol phospholipids in this tissue. Relatively small (30-40%) but significant increases in inositol phosphates (mono-, di-, and tri-) were apparent within 30-60 s of insulin treatment in fat pads (and adipocytes); thereafter, inositol phosphates returned to control levels. These rapid insulin-induced increases in inositol phosphates appeared to be due to phospholipase C-mediated hydrolysis of inositol phospholipids, since there were associated transient decreases in these lipids during 32P pulse-chase experiments. Increases in the synthesis of inositol phospholipids were also apparent within a few minutes of insulin treatment and persisted for at least 2 h. We conclude that, in the rat epididymal fat pad, insulin has two phospholipid effects, viz. a transient activation of phospholipase C, and a persistent increase in de novo phospholipid synthesis.     

Valproate disrupts regulation of inositol responsive genes and alters regulation of phospholipid biosynthesis

Valproate (VPA) is one of the two drugs approved by the Food and Drug Administration (FDA) for the treatment of bipolar disorder. The therapeutic mechanism of VPA has not been established. We have shown previously that growth of the yeast Saccharomyces cerevisiae in the presence of VPA causes a decrease in intracellular inositol and inositol-1-P, and a dramatic increase in expression of INO1, which encodes the rate limiting enzyme for de novo inositol biosynthesis. To understand the underlying mechanism of action of VPA, INO1, CHO1 and INO2 expression, intracellular inositol and phospholipid biosynthesis were studied as a function of acute and chronic exposure of growing cells to the drug. A decrease in intracellular inositol was apparent immediately after addition of VPA. Surprisingly, expression of genes that are usually derepressed during inositol depletion, including INO1, CHO1 and INO2 (that contain inositol-responsive UASINO sequences) decreased several fold during the first hour, after which expression began to increase. Incorporation of 32Pi into total phospholipids was significantly decreased. Pulse labelling of CDP-DG and PG, shown previously to increase during inositol depletion, increased within 30 min. However, pulse labelling of PS, which normally increases during inositol depletion, was decreased within 30 min. PS synthase activity in cell extracts decreased with time, although VPA did not directly inhibit PS synthase enzyme activity. Thus, in contrast to the effect of chronic VPA treatment, short-term exposure to VPA abrogated the normal response to inositol depletion of inositol responsive genes and led to aberrant synthesis of phospholipids.     

Composition and synthesis of cellular lipids in Neurospora crassa during cellular differentiation.

The synthesis of cellular lipids of Neurospora crassa was measured during growth on low (2% sucrose)- and high (15% glucose)-carbohydrate supplementation. The amount of lipid per dry weight of cells does not change during the germination and early logarithmic growth periods, but the percentage of phospholipid in the lipid does increase, reaching a maximal value of 90% at 4 to 5 h after inoculation, at which time the phospholipid content of the cells is approximately 60 mumol/g (dry weight). The content of the anionic phospholipids, as a percentage of the lipid fraction, is relatively constant during the growth period, but the contents of the zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine change in a reciprocal fashion. During the first 8 h of growth, phosphatidylcholine falls from 53% of the phospholipid to 43%, whereas phosphatidylethanolamine rises from 29 to 38%. The total of these two phospholipids is approximately 83% during the growth period studied. The synthesis of cellular phospholipids, measured either by [32P]H3PO4 or [14C]glucose incorporation, reached maximal levels between 3 and 5 h of growth. The effect of the high-carbohydrate supplement on cellular lipids was minimal. Inclusion of 15% glucose decreased the labeling of phospholipid by [32P]H3PO4, but did not affect lipid composition. This observation is in contrast to the effects of high glucose on mitochondrial phospholipid synthesis.     

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.     

Effects of light on phospholipid metabolism in DunaUella salina

Dunalliella salina (Teodoresco) is a unicellular, wall-less, halotolerant green alga. Previous work has shown that levels of inositol phospholipiils in whole cells of D. salina fluctuate in response to hyper- and hypo-osmotic shock. In this paper, we report the effects of changes in the light environment on levels of phospholipids, including inositol phospholipids, in D. scilina. Utilizing both short-term and long-term labeling of phospholipids with ³²PO₄, we were able to compare both immediate and long-term changes in lipid metabolism during changes in the light environment. Relative to the other phospholipids. phosphotidic acid and the inositol phospholipids phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were rapidly labeled, even in the dark, suggesting that the metabolism of these compounds is more active than that of the bulk cellular phospholipids. There was little change in inositol phospholipid metabolism when cells were illuminated following a 1 h dark adaptation period, Furthermore, the inositol phospholipid signal transduction pathway did not respond to severe photoinhibition treatment. Apparently this plasma-membrane-based signal transduction pathway, which responds to changes in the external environment, is relatively insensitive to major changes in chloroplast metabolism.     

Differential changes in galactolipid and phospholipid species in soybean leaves and roots under nitrogen deficiency and after nodulation

The availability of nitrogen (N) to plants has a profound impact on carbohydrate and protein metabolism, but little is known about its effect on membrane lipid species. This study examines the changes in galactolipid and phospholipid species in soybean as affected by the availability of N, either supplied to soil or obtained through Bradyrhizobium japonicum nodulation. When N was limited in soil, the content of galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacyglycerol (DGDG), decreased drastically in leaves, while a smaller decrease of DGDG was observed in roots. In both leaves and roots, the overall content of different phospholipid classes was largely unchanged by N limitation, although some individual phospholipid molecular species did display significant changes. Nodulation with Bradyrhizobium of soybean grown in N-deficient soil resulted in a large increase in levels of plastidic lipid classes, MGDG, DGDG, and phosphatidylglycerol, along with smaller increases in non-plastidic phospholipids in leaves. Nodulation also led to higher levels of phospholipids in roots without changes in root levels of MGDG and DGDG. Overall, N availability alters lipid content more in leaves than roots and more in galactolipids than phospholipids. Increased N availability leads to increased galactolipid accumulation in leaves, regardless of whether N is supplied from the soil or symbiotic fixation.     

Inositol Depletion Restores Vesicle Transport in Yeast Phospholipid Flippase Mutants

In eukaryotic cells, type 4 P-type ATPases function as phospholipid flippases, which translocate phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of the lipid bilayer. Flippases function in the formation of transport vesicles, but the mechanism remains unknown. Here, we isolate an arrestin-related trafficking adaptor, ART5, as a multicopy suppressor of the growth and endocytic recycling defects of flippase mutants in budding yeast. Consistent with a previous report that Art5p downregulates the inositol transporter Itr1p by endocytosis, we found that flippase mutations were also suppressed by the disruption of ITR1, as well as by depletion of inositol from the culture medium. Interestingly, inositol depletion suppressed the defects in all five flippase mutants. Inositol depletion also partially restored the formation of secretory vesicles in a flippase mutant. Inositol depletion caused changes in lipid composition, including a decrease in phosphatidylinositol and an increase in phosphatidylserine. A reduction in phosphatidylinositol levels caused by partially depleting the phosphatidylinositol synthase Pis1p also suppressed a flippase mutation. These results suggest that inositol depletion changes the lipid composition of the endosomal/TGN membranes, which results in vesicle formation from these membranes in the absence of flippases.     

Lipids of Cuscuta reflexa and changes in lipids of its host plants after infection

The lipid level (fresh weight basis) of Cuscuta reflexa Roxb. was related to the lipid content of the host plants Meilicago saliva L., Helianthus annuus L., Pisum sativum L. and Lantana camara L. Parasitizing by the dodder significantly increased the total lipid level of the hosts. The increase was mainly due to enhancement in the neutral lipid fraction.
The level of phospholipid in the parasite was always higher than in its hosts. Phospholidyl choline and phosphatidyl ethanolamine constituted about 65% of the total phospholipid of Cuscuta. This was followed by phosphatidyl inositol (ca 20%) and phosphatidyl glycerol (ca 12%). Phosphatidic acid constituted only ca 3% of the phospholipids of Cuscuta. Although the total phospholipid levels of various host plants were not affected as a result of the infection by Cuscuta, a significant decrease occurred in the levels of phosphatidyl eholine and phosphatidyl ethanolamine as well as marked increases in phosphatidyl inositol and phosphatidic acid. The infected tissue showed an increase in phospholipase D activity as compared with the controls. The results have been discussed in relation to changes in permeability of the infected tissue.     

Expression of C-Type Natriuretic Peptide in the Bovine Pineal Gland

Abstract: The effect of lithium on inositol phospholipid resynthesis in primary cultures of cerebellar granule cells was studied. During activation of phospholipase C by the combined action of a muscarinic agonist and mild depolarization, the levels of inositol phospholipids as well as the inositol phospholipid precursor CMP-phosphatidate appeared highly sensitive to lithium with half-maximal accumulation of CMP-phosphatidate attained at 0.5 m M LiCl, a concentration close to that in the plasma of patients subjected to lithium therapy. Under the same conditions, the effect of lithium on inositol phospholipid metabolism appeared to be mediated by depletion of cytoplasmic free inositol content. This was indicated by the observation that preincubation for 48 h in high extracellular inositol concentrations could decrease or delay the depletion of inositol phospholipids and the accumulation of CMP-phosphatidate induced by 10 m M LiCl. Because even relatively high concentrations of extracellular inositol (500 µ M ) only partially prevented inositol phospholipid depletion, cerebellar granule cells appear to have a comparatively low capacity to accumulate inositol intracellularly, in comparison with other brain cells in culture. The relationship between CMP-phosphatidate accumulation and phospholipase C activity has also been investigated using a range of agonists that have been reported to act on cerebellar granule cells.     

Breakdown of polyphosphoinositides and not phosphatidylinositol accounts for muscarinic agonist-stimulated inositol phospholipid metabolism in rat parotid glands.

The molecular mechanisms underlying the ability of muscarinic agonists to enhance the metabolism of inositol phospholipids were studied using rat parotid gland slices prelabelled with tracer quantities of [3H]inositol and then washed with 10 mM unlabelled inositol. Carbachol treatment caused rapid and marked increases in the levels of radioactive inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and an accumulation of label in the free inositol pool. There were much less marked changes in the levels of [3H]phosphatidylinositol, [3H]phosphatidylinositol 4-phosphate and [3H]phosphatidylinositol 4,5-bisphosphate. At 5 s after stimulation with carbachol there were large increases in [3H]inositol 1,4-bisphosphate and [3H]inositol 1,4,5-trisphosphate, but not in [3H]inositol 1-phosphate. After stimulation with carbachol for 10 min the levels of radioactive inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate greatly exceeded the starting level of radioactivity in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate respectively. When carbachol treatment was followed by addition of sufficient atropine to block all the muscarinic receptors the radioactive inositol phosphates rapidly returned towards control levels. The carbachol-evoked changes in radioactive inositol phosphate and phospholipid levels were blocked in the presence of 2,4-dinitrophenol (an uncoupler of oxidative phosphorylation). The results suggest that muscarinic agonists stimulate a polyphosphoinositide-specific phospholipase C and that these lipids are continuously replenished from the labelled phosphatidylinositol pool. [3H]Inositol 1-phosphate in the stimulated glands probably arises via hydrolysis of inositol 1,4-bisphosphate and not directly from phosphatidylinositol.