David N. Brindley

Increased concentrations of phosphatidate, diacylglycerol and ceramide in ras - and tyrosine kinase ( fps )-transformed fibroblasts

Concentrations of the bioactive lipids, phosphatidate and diacylglycerol, increased with time in culture in ras- and tyrosine kinase (fps)-transformed fibroblasts but not in control fibroblasts. On Day 3, diacylglycerol and phosphatidate concentrations were about 3.3- and 5.5-fold higher respectively in the ras-transformed compared to control fibroblasts. These concentrations in fps-transformed fibroblasts were increased about twofold. The changes in phosphatidate and diacylglycerol resulted from enhanced phospholipid turnover rather than from synthesis de novo. The increased ratio of phosphatidate to diacylglycerol is explained by decreased activities of two distinct phosphatidate phosphohydrolases and increased diacylglycerol kinase in ras-transformed fibroblasts. Ceramide concentrations were about 2.5- and threefold higher in the fps- and ras-transformed cells respectively on Day 3 compared to the controls. Incubating control fibroblasts from Days 1 to 3 with phosphatidylcholine-specific phospholipase C increased diacylglycerol, phosphatidate and ceramide concentrations, and decreased Mg2+-independent-phosphatidate phosphohydrolase activity. 8-(4-chlorophenylthio)-cAMP had a cytostatic effect in ras-transformed cells, it decreased the concentrations of phosphatidate and diacylglycerol, but increased that of ceramide. The consequences of increased ceramide and phosphatidate concentrations in ras-transformed cells are discussed in relation to signal transduction, cell division and the transformed phenotype.

Long-chain fatty acids and their acyl-CoA esters cause the translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction of rat liver

A translocation of phosphatidate phosphohydrolase from the cytosolic to the microsomal fraction was promoted in cell‐free extracts of rat liver by oleate and palmitate and their CoA esters. Oleate was more potent in this respect than palmitate and the CoA esters were more effective than the unesterified acids. Octanoate, octanoyl‐CoA and CoA did not cause the translocation. It is proposed that the interaction of phosphatidate phosphohydrolase with the membranes that synthesize glycerolipids causes it to become metabolically active. This enables the liver to increase its capacity for triacylglycerol synthesis in response to an increased supply of fatty acids.

Characterization and assay of phosphatidate phosphatase.

Publisher Summary Phosphatidate phosphatase is involved in the synthesis de novo of triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine. It lies at a branch point in the pathway and can help to regulate the relative rates of flux of substrates passing through CDP-diacylglycerol, and to diacylglycerol. This form of the enzyme is thought to be Mg 2+ -dependent. Phosphatidate phosphatase is involved in signal transduction following the stimulation of a phospholipase D. The latter enzyme degrades phosphatidylcholine in the plasma membrane in response to agonists and produces phosphatidate that is converted to diacylglycerol so as to activate protein kinase C. The technique for assaying phosphatidate phosphatase activity is discussed by taking rat liver as a source of enzyme activities. Rat liver contains two distinct phosphatidate phosphatases: one has an absolute requirement for Mg 2+ and is inhibited by N -ethylmaleimide, whereas the other is insensitive to inhibition by N -ethylmaleimide and does not require Mg 2+ . The N -ethylmaleimide-insensitive phosphatase is inhibited by concentrations of Mg 2+ above about 3.5 mM.

Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes

Glycerolipid synthesis was studied in isolated hepatocytes by using 177 microM [14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 microM) inhibited the synthesis of phosphatidylcholine and triacylglycerol. This was accompanied by an average increase of 12-fold in the accumulation of the labelled precursors in phosphatidate at 200 microM chlorpromazine and a decrease in the conversion of phosphatidate to diacylglycerol of 76%. These results indicate that part of the inhibition of the synthesis of phosphatidylcholine and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conversion of phosphatidate to diacylglycerol. Phosphatidylcholine synthesis increased at higher rates of conversion of phosphatidate to diacylglycerol, but it was relatively independent of the latter rate when this was inhibited by more than about 30% with chlorpromazine. The addition of oleate to the hepatocytes caused a translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Chlorpromazine had the opposite effect and displaced the phosphohydrolase from the membranes in the presence or absence of oleate. There was a highly significant correlation between the activity of phosphatidate phosphohydrolase that was associated with the membranes of the hepatocytes and the calculated conversion of [3H]phosphatidate to diacylglycerol. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spermine. Furthermore, it inhibited the transfer of the soluble phosphohydrolase to microsomal membranes that were labelled with [14C]phosphatidate and thereby decreased diacylglycerol production. It is concluded that part of the action of chlorpromazine in inhibiting the synthesis of triacylglycerol and phosphatidylcholine occurs because it prevents the interaction of the soluble phosphatidate phosphohydrolase with the membranes on which glycerolipid synthesis occurs. This in turn prevents the conversion of phosphatidate to diacylglycerol.

A possible metabolic explanation for drug‐induced phospholipidosis

A large variety of amphiphilic cationic drugs which are in widespread clinical use produce a generalized phospholipidosis when administered for prolonged periods. These drugs, which vary widely in their potency in causing phospholipidosis, include chlorphentermine, fenfluramine, triparanol, trans-1,4-bis (2-chlorobenzylaminoethy1)-cyclohexane (A79944), azacosterol, 53 ’ diethylaminoethyoxyhexestrol, 1-chloroamitriptyline, iprindole, 2-N-methyl-piperazino-methyl-1,3-diazofluoroanthen 1-oxide (AC 3579), chlorcyclizine, chloroquine, chlorpromazine, thioridazine, imipramine, clomipramine, haloperidol and boxidine (Yamamoto, Adachi & others, 1971a, b; Shikata, Kanetaka &others, 1972; Hruban, Slesers & Ashenbrenner, 1973 ; Lullman, Lullman-Rauch & Wasserman, 1973; Wherrett & Huterer, 1973; De La Iglesia, Feuer & others, 1974; Kasama, Yoshida & others, 1974; Lullman-Rauch, 1974a, b, 1975; Schmien, Seiler & Wasserman, 1974). Although these drugs have a variety of therapeutic effects they are physicochemically rather similar, in that they all possess both a hydrophobic region and a primary or substituted amine group which can bear a net positive charge. This amphiphilic nature enables the drugs to interact with phospholipids, particularly the anionic phospholipids which are quantitatively minor constituents of membranes (e.g. phosphatidate, phosphatidylinositol, phosphatidylserine, cardiolipin). Their capacity both to cause phospholipidosis and to interact with lipids depends largely on the size and hydrophobicity of the apolar portions of the molecule. We recently suggested that interactions of these drugs with anionic phospholipids might cause some of the therapeutic actions or side-effects of these drugs (Brindley, Allan & Michell, 1975). The lipids which accumulate in the lysosomes of a variety of tissues during drug treatment are mainly glycerophospholipids. There are clear indications that compared with normal tissue, these tend to include increased proportions of anionic phospholipids (phosphatidate, phosphatidylinositol, phosphatidylglycerol and lysobisphosphatidate) and decreased proportions of triglyceride and of the major zwitterionic glycerophospholipids (phosphatidylcholine and phosphatidylethanolamine) (Yamamoto & others, 1971a, b; Wherrett & Huterer, 1973; Kasama, Yoshida & others, 1974; Allan & Michell, 1975; Karabelnik & Zbinden, 1975). This pattern of lipid accumulation is in marked contrast to that seen in the classical hereditary lipidoses in which sphingolipids, particularly glycosphingolipids, are the main lipids which accumulate in lysosomes. One proposed explanation of this effect is that phospholipids are normally degraded in lysosomes by phospholipases, but that when amphiphilic cationic drugs form complexes with the phospholipids this prevents phospholipase attack and the phospholipid-drug complexes therefore accumulate and engorge the lysosomes (Liillman & others, 1973 ; Lullman-Rauch, 1974a). Although this mechanism would explain many of the experimental findings it does not provide a complete explanation. For example, it does not explain

Binding of low-density lipoprotein to monolayer cultures of rat hepatocytes is increased by insulin and decreased by dexamethasone

Rat hepatocytes were maintained in monolayer culture for 20 h in the presence of 10% (v/v) new‐born calf serum and then for a further 1–24 h in serum‐free medium containing 2 g bovine serum albumin/l. The specific binding of human 125‐I‐LDL to two distinct sites was then measured at 4°C. Binding to site 1 was displaced by dextran sulphate while that to site 2 was not. The presence of 1–100 nM insulin for 24 h in the second incubation significantly increased binding to site 1. Significant increases were also seen when cells were incubated with 10 nM insulin for 1 h. No significant effects of insulin on binding to site 2 were observed. In contrast, 10 nM—1 μM dexamethasone decreased binding to both sites. The effects of these hormones were mutually antagonistic.

Factors controlling the metabolism of phosphatidate by phosphohydrolase and phospholipase A-type activities: Effects of magnesium, calcium and amphiphilic cationic drugs

1. The simultaneous deacylation and dephosphorylation of 1,2-diacyl-sn-[3H]glycerol 3-phosphate by the microsomal and soluble fraction of rat liver was studied. The substrate was either in the form of an emulsion or bound to microsomal membranes. 2. Mg2+ stimulated the deacylation and dephosphorylation of phosphatidate emulsions by both fractions, although the stimulation of both microsomal activities was less than that in the soluble fraction. The preparations of membrane-bound phosphatidate contained Mg2+. Further addition of Mg2+ inhibited dephosphorylation, whereas low concentrations of EDTA stimulated. Additional Mg2+ had little effect on the deacylation of membrane-bound phosphatidate and EDTA inhibited it. 3. Ca2+ inhibited the phosphohydrolase reactions in both fractions, but had little effect on the deacylation of phosphatidate emulsions or membrane-bound phosphatidate. 4. In the absence of Mg2+, lower concentrations of amphiphilic cations (chlorpromazine and benfluorex) stimulated the deacylation and dephosphorylation of phosphatidate emulsions by the soluble fraction. They also stimulated deacylation by the microsomal fraction, but inhibited dephosphorylation. In the present of 5 mM MgCl2, these drugs inhibited the dephosphorylation and deacylation of phosphatidate emulsions, the deacylation reaction being slightly less sensitive. Chlorpromazine (0.4 and 0.8 mM) also inhibited the dephosphorylation of membrane-bound Mg2+-phosphatidate by microsomal and microsomal plus soluble fractions. The deacylation was stimulated by 0.4 mM chlorpromazine and by 1 and 2 mM norfenfluramine. Chlorpromazine (0.8 mM) inhibited the deacylation by microsomal plus soluble fractions, but not by microsomal fractions alone. 5. The possible importance of the deacylation of phosphatidate in the physiological and pharmacological control of glycerolipid synthesis is discussed.

Spermine promotes the translocation of phosphatidate phosphohydrolase from the cytosol to the microsomal fraction of rat liver and it enhances the effects of oleate in this respect

Spermine (0.5–2 mM) promoted the translocation of phosphatidate phosphohydrolase from the soluble to the microsomal fraction in a cell‐free system derived from rat liver. By contrast, spermidine (1 mM) and putrescine (1 mM) had no significant effect on the translocation when added alone. Spermine, and to a lesser extent, spermidine, enhanced the translocating action of oleate and increased its effectiveness in transferring the phosphohydrolase from the soluble ot the microsomal fraction. It is proposed that the phosphohydrolase becomes metabolically active when it combines with membranes and that polyamines might help to regulate this interaction. This could facilitate the action of fatty acids and enable cells to increase their capacity for triacylglycerol synthesis to match an increased availability of fatty acids.

Possible relationships between changes in body weight set-point and stress metabolism after treating rats chronically with D-fenfluramine: effects of feeding rats acutely with fructose on the metabolism of corticosterone, glucose, fatty acids, glycerol and triacylglycerol

Rats were maintained on a corn oil diet and treated with D-fenfluramine at doses of 2.5 mg/kg twice a day for 11 days or with 10 mg or 25 mg/kg once a day for 12 days. The lower dose of D-fenfluramine produced no marked changes in body weight and after 11 days of treatment the weights of the rats on average were only 2% lower than the controls. The food intake of these rats was only decreased on the first day. The two higher doses of D-fenfluramine decreased the food consumption for about 3 days but thereafter it was similar to that of the control rats. The body weight of these rats fell on the first day, but after about four days the gain in body weight paralleled rather than approached that of the control rats. Increasing the dose of D-fenfluramine progressively decreased the relative size of the epididymal fat pad. At the end of the treatment period the rats were fed acutely with fructose to increase the circulating concentrations of corticosterone and to stimulate triacylglycerol synthesis. All three doses of D-fenfluramine decreased the concentration of circulating triacylglycerol after fructose feeding. The 10 mg/kg dose also decreased the basal concentration of triacylglycerol. The two higher doses of fenfluramine decreased the rises in the circulating concentrations of corticosterone, glycerol and fatty acids that are produced by fructose feeding. The basal concentrations of these compounds in the absence of fructose feeding were not significantly affected by the 10 mg/kg dose of D-fenfluramine. The possible relationship between the effect of chronic treatment with D-fenfluramine in decreasing a metabolic stress response and lipolysis is discussed relative to its hypotriglyceridaemic action and its effect on body weight-set point. The results demonstrate that D-fenfluramine produced persistent changes in metabolism at a time when the treated rats were growing at the same rate as the control rats and when they were eating similar quantities of food.

The intracellular phase of fat absorption.

Most of our present knowledge concerning the transport of fat across the epithelial cells of the small intestine has been gained in little more than ten years. It has resulted from studying the molecular interconversions undergone by lipid in epithelial cells and by determining the subcellular location of the enzymes involved. Thus we have descriptions of the route and time course of fat absorption and are able to relate many of the metabolic activities to the subcellular structures through which the lipid is passing.