Specific 125I-iodination of cell surface lipids: plasma membrane alterations induced during humoral immune attack.

A method whereby lactoperoxidase-catalyzed 125I-iodination of plasma membrane lipids can be achieved is described. The reaction results in a uniform and stable labeling of neutral lipids, phospholipids, lysophosphatides, free fatty acids, and triacylglycerides. By the use of this method, the action of antibody plus complement (C) on the specific release of lipid from the plasma membrane of line-10 tumor cells was studied. Within 15 min after the addition of C to antibody-sensitized cells, the enhanced release of specific lipid classes from the cell surface was observed; these lipids included sphingomyelin, phosphatidylserine, and phosphatidylcholine. The release of phosphatidylethanolamine and, in some instances, triglycerides, was reduced after antibody-C attack. Neither the specificity of the antibody used to sensitize the cells nor the ability of the antibody plus C to be cytotoxic to the cells appeared to affect the identity or amounts of lipids released from the cells.     

Identification of lipids associated with the ability of tumor cells to resist humoral immune attack.

Certain metabolic inhibitors or chemotherapeutic agents that increase the susceptibility of line-1 or line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of the cells to synthesize lipids. A direct correlation was found between the drug-induced increase in sensitivity to antibody-C mediated killing and the inhibition of the ability of the cells to incorporate acetate, glycerol, and fatty acids into complex cellular lipids. Drug-treated cells recultured in drug-free medium regained their resistance to antibody-C mediated killing; these cells recovered their ability for complex lipid synthesis at this time. Thin layer chromatography of CHCl3:CH3OH lipid extracts from these cells indicated that the drug-induced increase in susceptibility to humoral immune attack correlated with the inhibition of acetate, glycerol, and fatty acid incorporation into cardiolipin and triglyceride in line-10 cells and the inhibition of incorporation of these compounds into cardiolipin alone in line-1 cells. No direct correlation was found between the sensitivity of the cells to humoral immune attack and the ability of the cells to incorporate precursors of lipid synthesis into other lipid moieties (sphyngomyelin, phosphatidyl serine, phosphatidyl choline, phosphatidyl glycerol, or cholesterol esters). The synthesis of cardiolipin and triglycerides, therefore, appears to be associated with the mechanism whereby these tumor cells resist antibody-C mediated killing.     

Effect of concanavalin A on the killing of tumor cells by antibody and complement.

Concanavalin A (Con A) was found to inhibit the killing of antibody-sensitized line-1 tumor cells (TA) by guinea pig complement (GPC) but not by human complement (HuC). Other plant lectins (wheat germ, leucoagglutinin, and pokeweed mitogen) were also tested but Con A was the only lectin found to inhibit antibody-GPC-mediated killing. The inhibitory effect of Con A was observed when the GPC was mixed with Con A or when the antibody-sensitized cells were pretreated with Con A (TA-Con A) before the addition of GPC. The effect could be reversed by treatment of such cells with alpha-D-methylglucopyranoside or by incubation at 37 degrees C for approximately 2 hr. Con A appeared to act by preventing the binding of the first component of GPC (GPC1) to antibody-sensitized tumor cells. Differences in the binding of the first component of HuC (HuC1) and GPC1 to TA-Con A suggested that a difference in the binding site for HuC1 and GPC1 might exist. There was no difference in the number of GPC1 molecules fixed to antibody-sensitized sheep erythrocytes (EA) or EA treated with Con A in experiments using the same antibody as used with the tumor cells and the same Con A preparation. It would consequently appear that the inhibitory effect of Con A on the binding of GPC1 to TA is not due solely to an interaction of Con A with the antibody.     

Lysis of tumor cells by antibody and complement. VII. Complement-dependent 86Rb release--a nonlethal event?

The guinea pig hepatoma (line-1) treated with anti-Forssman antibody (TA) and GPC sequentially released 86Rb, 14C from 14C aminoidobutyric acid and failed to exclude trypan blue. Incubation of TA with fluid phase GPC for 1 min caused maximal 86Rb release; however, if the GPC was removed at this time, the cells were not subsequently killed. Using a number of naturally occurring human sera deficient in a complement component we have shown 86Rb release requires the binding of the complement components 1 through 8, but there was no absolute requirement for C9. Irreversible damage to the cell as measured by 14C AIB release or uptake of trypan blue required the complete sequence of complete sequence of complement components. These observations indicate that 86Rb release is not a relible indicator cytotoxicity.     

Correlation between the ability of tumor cells to resist humoral immune attack and their ability to synthesize lipid.

Agents that increase (certain metabolic inhibitors, chemotherapeutic agents, and x-irradiation), decrease (hormones), or have no effect (hyperthermia) on the susceptibility of line-1 and line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of these tumor cells to synthesize macromolecules. A correlation was found between the drug-induced increase in sensitivity of these cells to antibody-C mediated killing and the loss of their ability to incorporate fatty acids into complex cellular lipids. Similarly, the hormone-induced increase in resistance of the cells to killing was accompanied by an enhancement in complex lipid synthesis by these cells was also observed after the cells were exposed to physical means of insult (x-irradiation or hyperthermia). No correlation was found between the sensitivity of the cells to antibody-C mediated killing and their ability to synthesize DNA, RNA, protein, or complex carbohydrate, or their capacity for de novo lipid synthesis as measured by incorporation of acetate and glycerol into cellular macromolecules. The assembly of free fatty acids into complex lipid moieties is therefore proposed to be of fundamental importance for the ability of the tumor cells to resist humoral immune killing.     

Lysis of tumor cells by antibody and complement. II. Lack of correlation between amount of C4 and C3 fixed and cell lysis.

Two antigenically distinct diethylnitrosamine-induced guinea pig hepatoma cell lines, line-1 and line-10, sensitized with rabbit anti-Forssman or with tumor-specific antibody, were more susceptible to killing by human complement (HuC) than by guinea pig complement (GPC). This difference could not be ascribed to differences in the amount of C1, C4, and C3 fixed: millions of C4 and hundreds of thousands of C3 were detected on cells whether they were killed or not killed by the C sources. Tumor cells sensitized with anti-Forssman IgM antibody generally had more GP C4 and C3 than Hu C4 and C3 bound to their surfaces. Cells sensitized with anti-tumor antibody generally had more Hu C4 and C3 than GP C4 and C3 bound to their surfaces. The resistance to killing of nucleated cells by antibody and C may be due in part to intrinisic properties of the cell.     

Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis

Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-¹⁴C]-fatty acids and in cells treated with [1-¹⁴C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-¹⁴C]acetate or Tween-[1-¹⁴C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-¹⁴C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-¹⁴C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism.     

Influence of chain length of pyrene fatty acids on their uptake and metabolism by Epstein-Barr-virus-transformed lymphoid cell lines from a patient with multisystemic lipid storage myopathy and from control subjects.

The uptake and intracellular metabolism of 4-(1-pyrene)butanoic acid (P4), 10-(1-pyrene)decanoic acid (P10) and 12-(1-pyrene)dodecanoic acid (P12) were investigated in cultured lymphoid cell lines from normal individuals and from a patient with multisystemic lipid storage myopathy (MLSM). The cellular uptake was shown to be dependent on the fatty-acid chain length, but no significant difference in the uptake of pyrene fatty acids was observed between MLSM and control lymphoid cells. After incubation for 1 h the distribution of fluorescent fatty acids taken up by the lymphoid cell lines also differed with the chain length, most of the fluorescence being associated with phospholipid and triacylglycerols. In contrast with P10 and P12, P4 was not incorporated into neutral lipids. When the cells were incubated for 24 h with the pyrene fatty acids, the amount of fluorescent lipids synthesized by the cells was proportional to the fatty acid concentration in the culture medium. After a 24 h incubation in the presence of P10 or P12, at any concentration, the fluorescent triacylglycerol content of MLSM cells was 2-5-fold higher than that of control cells. Concentrations of pyrene fatty acids higher than 40 microM seemed to be more toxic for mutant cells than for control cells. This cytotoxicity was dependent on the fluorescent-fatty-acid chain length (P12 greater than P10 greater than P4). Pulse-chase experiments permitted one to demonstrate the defect in the degradation of endogenously biosynthesized triacylglycerols in MLSM cells (residual activity was around 10-25% of controls on the basis of half-lives and initial rates of P10- or P12-labelled-triacylglycerol catabolism); MLSM lymphoid cells exhibited a mild phenotypic expression of the lipid storage (less severe than that observed in fibroblasts). P4 was not utilized in the synthesis of triacylglycerols, and thus did not accumulate in MLSM cells: this suggests that natural short-chain fatty acids might induce a lesser lipid storage in this disease.     

The endocrine regulation of milk lipid synthesis and secretion in tammar wallaby (Macropus eugenii)

Lipids in tammar milk are predominantly triacylglycerols, and the fatty acid composition varies during the lactation cycle. Little is known about the regulation of their synthesis. This study investigates the endocrine regulation of lipid synthesis in mammary explants from pregnant tammars. Treatment of mammary explants with insulin resulted in a high level of lipid synthesis, but the lipids accumulated in the cytosol. Culture with prolactin resulted in a small increase in lipid synthesis, but electron microscopy showed lipid globules were synthesized in the mammary epithelial cells and secreted into the lumen. Culture with both insulin and prolactin demonstrated elevated levels of synthesis and secretion of lipid. Analysis of the type of fatty acids synthesized in these mammary explants showed that the initiation of synthesis of C(16:0), which also occurs in the first week of lactation, could be reproduced in the pregnant explants cultured with prolactin alone. However, treatment of mammary explants with hydrocortisone did not show a significant effect on lipid synthesis, secretion or the fatty acid synthesized. These results provide new information identifying the role of insulin and prolactin in regulating milk lipid synthesis and secretion in the tammar.     

Cancer cells incorporate and remodel exogenous palmitate into structural and oncogenic signaling lipids

De novo lipogenesis is considered the primary source of fatty acids for lipid synthesis in cancer cells, even in the presence of exogenous fatty acids. Here, we have used an isotopic fatty acid labeling strategy coupled with metabolomic profiling platforms to comprehensively map palmitic acid incorporation into complex lipids in cancer cells. We show that cancer cells and tumors robustly incorporate and remodel exogenous palmitate into structural and oncogenic glycerophospholipids, sphingolipids, and ether lipids. We also find that fatty acid incorporation into oxidative pathways is reduced in aggressive human cancer cells, and instead shunted into pathways for generating structural and signaling lipids. Our results demonstrate that cancer cells do not solely rely on de novo lipogenesis, but also utilize exogenous fatty acids for generating lipids required for proliferation and protumorigenic lipid signaling. This article is part of a special issue entitled Lipid Metabolism in Cancer.     

Lipogenic capacity and relative contribution of the different tissues and organs to lipid synthesis in male rat

The rates of 3H2O incorporation into total lipids and fatty acids were measured in vivo in the different organs and tissues of 7-week old male Wistar rats to compare the lipogenic capacity of those organs and tissues and to determine their relative contributions to body lipid synthesis. Our results were the following; (1) liver was the major site of the synthesis of total lipids and fatty acids (37 and 42%, respectively, of body synthesis); (2) white adipose tissues synthesized about 24% of the total lipids; mesenteric adipose tissue alone synthesizing 40% of the fatty acids produced in dissectable white adipose tissues; (3) skin showed low lipid synthesis but played an appreciable role in that synthesis (8% of the total) due to its large contribution to total body weight; (4) other organs (excluding liver) showed low lipid synthesis; however, that of the small intestine was 1-2% of body synthesis; (5) the rest of the carcass (mainly musculature and skeleton) contributed 25% to body lipid synthesis. The putative roles of the different tissues and organs in adipose tissue development have been discussed.     

Spin labelling studies of cytolysis induced by fatty acids

Rat thymocytes, spleen lymphocytes and isolated nuclei were incubated with fatty acids and then labelled with 5-doxylstearic acid and 12-doxylstearic acid. The ESR spectra only in the case of 5-doxylstearic acid showed changes which were demonstrable only under those conditions which resulted in cytolysis. Thymocytes in medium with 10% serum showed the effect at 10 microM, splenic lymphocytes at 100 microM. The effect was maximal at 2 min and was not enhanced by higher concentrations. The uptake of fatty acid by spleen cells required to cause this change was determined using 14C-oleic acid, to be 0.6 mumol/g tissue. This quantity is less than that required (label:lipid ratio less than 1:10) to produce major perturbations in membranes. Free fatty acids of C-8 to C-18 produced the effect, but not esters or amides. It was concluded that free fatty acids induce changes proximal to the polar region of membrane lipids which, if not progressive and essential to the ultimate process of lysis, are at least indicative of impending cell death at an early time.     

Effect of Glycerol Deprivation on the Phospholipid Metabolism of a Glycerol Auxotroph of Staphylococcus aureus

A study of the effects of glycerol deprivation on the content and metabolism of the phospholipids of a glycerol auxotroph of Staphylococcus aureus showed that (i) there was an increase in the proportions of lysylphosphatidylglycerol (LPB) and a concomitant decrease in the proportion of phosphatidylglycerol. The total phospholipid content per sample and the proportion of cardiolipin did not change, but the phosphatidic acid increased transiently and then fell to pretreatment levels. (ii) The loss of (32)P from the lipids during the chase in a pulse-chase experiment was essentially the same in phosphatidylglycerol, cardiolipin, and phosphatidic acid during glycerol deprivation or growth in the presence of glycerol. LPG lost half the radioactivity in slightly more than two doubling times when grown with glycerol. In the absence of glycerol, (32)P accumulated in LPG for about 20 min and then stopped, after which time there was no apparent turnover. (iii) During glycerol deprivation, the initial (32)P incorporation decreased sixfold compared to that of the control with glycerol. The initial incorporation into LPG decreased only 2.5-fold, whereas that of PG decreased 45-fold. (iv) During glycerol deprivation, the free fatty acid content increased from 1.2 to 12.5% of the total extractable fatty acids and then slowly decreased. The increase was largely iso- and anti-iso-branched 21-carbon-atom fatty acids. In glycerol-supplemented cultures, the major fatty acids were branched 14- to 18-carbon fatty acids. The decrease in longer chain free fatty acids after 60 min represented their esterification into lipids. (v) During glycerol deprivation ribonucleic acid synthesis and cell growth continued for 40 min and protein synthesis continued for 90 min. Then synthesis and growth stopped. (vi) After the addition of glycerol to glycerol-deprived cells, (32)P and (14)C-glycerol were incorporated into the phospholipids without lag; ribonucleic acid, protein synthesis, and cell growth began after a 5- to 10-min lag at the pretreatment rate. The initial rate of lipid synthesis after the addition of glycerol was three times greater than the growth rate. This rapid rate continued for about 25 min until the lipid content and proportions of LPG and phosphatidylglycerol were restored.     

The endocrine regulation of milk lipid synthesis and secretion in tammar wallaby (Macropus eugenii)

Lipids in tammar milk are predominantly triacylglycerols, and the fatty acid composition varies during the lactation cycle. Little is known about the regulation of their synthesis. This study investigates the endocrine regulation of lipid synthesis in mammary explants from pregnant tammars. Treatment of mammary explants with insulin resulted in a high level of lipid synthesis, but the lipids accumulated in the cytosol. Culture with prolactin resulted in a small increase in lipid synthesis, but electron microscopy showed lipid globules were synthesized in the mammary epithelial cells and secreted into the lumen. Culture with both insulin and prolactin demonstrated elevated levels of synthesis and secretion of lipid. Analysis of the type of fatty acids synthesized in these mammary explants showed that the initiation of synthesis of C_16:0, which also occurs in the first week of lactation, could be reproduced in the pregnant explants cultured with prolactin alone. However, treatment of mammary explants with hydrocortisone did not show a significant effect on lipid synthesis, secretion or the fatty acid synthesized. These results provide new information identifying the role of insulin and prolactin in regulating milk lipid synthesis and secretion in the tammar.     

Interrelation of aerobic glycolysis and lipogenesis in isolated perfused liver of well-fed rats

The rates of glycolysis and lipogenesis in isolated perfused liver of well-fed rats were studied. When liver was allowed to synthesize [14C]glycogen prior to perfusion, no more than 9% of the degraded [14C]glycogen was recovered in lactate and 6% in lipid. Addition of glucose, fructose and sorbitol enhanced concomitantly the formation of lactate and pyruvate and the rate of release of triglyceride and free fatty acid. Glucose was less efficient than fructose or sorbitol. The incorporation of 14C from these 14C-labelled substrates into lactate, pyruvate and lipids confirmed their role as carbon sources. Incorporation of 14C into the glycerol moiety of neutral lipid exceeded that found in the fatty acids, suggesting that these substrates contributed largely to the esterification of fatty acids. The total rate of de novo fatty acid synthesis was correlated with the formation of lactate and pyruvate. It is concluded that increased rates of aerobic glycolysis are related to increased rates of lipogenesis.     

The Neutral Lipids of Paramecium tetraurelia: Changes with Culture Age and the Detection of Steryl Esters in Ciliary Membranes1

Neutral lipids, particularly triglycerides, accounted for the major decrease in the total lipid content in Paramecium cells that occurs with culture age. Sterols, triglycerides, and steryl esters were the major classes of neutral lipids in cells and isolated cilia. Free as well as high concentrations of esterified sterols were detected in purified ciliary membrane preparations. Stigmasterol and 7-dehydrostigmasterol were the major components of both free and esterified sterols of cells and cilia; however, when cholesterol was present in the growth medium, it was desaturated to 7-dehydrocholesterol and incorporated into cellular and ciliary lipids. Free fatty acids from cells and triglycerides from cells and cilia were low in polyunsaturated fatty acids and reflected the composition of fatty acids in the culture medium. An exception was the reduced concentration of stearate in triglycerides from whole cells. Greater than 50% of triglyceride fatty acids from cilia were saturated. The fatty acid compositions of cellular triglycerides and ciliary steryl esters did not change with culture age, but those of cellular steryl esters and ciliary triglycerides did change. In comparison with phospholipids, these neutral lipid fatty acid compositional changes were smaller. The sensitivity of these stigmasterol-containing cells to polyene antibiotics indicated that they were killed by nystatin > filipin > amphotericin B. The unexpected finding of high concentrations of steryl esters in ciliary membrane preparations is discussed.     

Endothelial cell fatty acid unsaturation mediates cold-induced oxidative stress

Ultraprofound hypothermia (< 5 degrees C) induces changes to cell membranes such as liquid-to-gel lipid transitions and oxidative stress that have a negative effect on membrane function and cell survival. We hypothesized that fatty acid substitution of endothelial cell lipids and alterations in their unsaturation would modify cell survival at 0 degrees C, a temperature commonly used during storage and transportation of isolated cells or tissues and organs used in transplantation. Confluent bovine aortic endothelial cells were treated with 18-carbon fatty acids (C18:0, C18:1n-9, C18:2n-6, or C18:3n-3), C20:5n-3 or C22:6n-3 (DHA), and then stored at 0 degrees C without fatty acid supplements. Storage of control cells caused the release of lactate dehydrogenase (LDH) and a threefold increase in lipid peroxidation (LPO) when compared to control cells not exposed to cold. Pre-treating cells with C18:0 decreased the unsaturation of cell lipids and reduced LDH release at 0 degrees C by 50%, but all mono- or poly-unsaturated fatty acids increased injury in a concentration-dependent manner and as the extent of fatty acid unsaturation increased. DHA-treatment increased cell fatty acid unsaturation and caused maximal injury at 0 degrees C, which was prevented by lipophilic antioxidants BHT or vitamin E, the iron chelator deferoxamine, and to a lesser extent by vitamin C. Furthermore, the cold-induced increase in LPO was reduced by C18:0, vitamin E, or DFO but enhanced by DHA. In conclusion, the findings implicate iron catalyzed free radicals and LPO as a predominant mechanism of endothelial cell injury at 0 degrees C, which may be reduced by increasing lipid saturation or treating cells with antioxidants.     

A rapid method for determining the fatty acid composition of lipid A

Lipid A is the most conservative part of LPS. Its fatty acids composition can serve as an important taxonomic marker of bacteria. The isolation of LPS and studying their chemical composition are difficult and protracted procedure. We propose the rapid method of determining the prevailed fatty acids of lipid A without isolation of LPS from the cell. The essence of the method is in the release of cell from the lipids which are not components of LPS. These lipids, in contrast to the lipid A, are more easily extractable from the cell structures. The fatty acids, which prevailed in the lipid-free cells, are the structural components of lipid A.     

Neonatal hypoxic hyperlipidemia in the rat: effects on aldosterone and corticosterone synthesis in vitro

Neonatal hypoxia increases aldosterone production and plasma lipids. Because fatty acids can inhibit aldosterone synthesis, we hypothesized that increases in plasma lipids restrain aldosteronogenesis in the hypoxic neonate. We exposed rats to 7 days of hypoxia from birth to 7 days of age (suckling) or from 28 to 35 days of age (weaned at day 21). Plasma was analyzed for lipid content, and steroidogenesis was studied in dispersed whole adrenal glands untreated and treated to wash away lipids. Hypoxia increased plasma cholesterol, triglycerides, and nonesterified fatty acids in the suckling neonatal rat only. Washing away lipids increased aldosterone production in cells from 7-day-old rats exposed to hypoxia, but not in cells from normoxic 7-day-old rats or from normoxic or hypoxic 35-day-old rats. Addition of oleic or linolenic acid to washed cells inhibited both aldosterone and corticosterone production, although cells from hypoxic 7-day-old rats were less sensitive. We conclude that hypoxia induces hyperlipidemia in the suckling neonate and that elevated nonesterified fatty acids inhibit aldosteronogenesis.