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LPA (lysophosphatidic acid) is a lipid mediator that stimulates cell proliferation and growth, and is involved in physiological and pathological processes such as wound healing, platelet activation, angiogenesis and the growth of tumours. Therefore defining the mechanisms of LPA production and degradation are of interest in understanding the regulation of these processes. Extracellular LPA synthesis is relatively well understood, whereas the mechanisms of its degradation are not. One route of LPA degradation is dephosphorylation. A candidate enzyme is the integral membrane exophosphatase LPP1 (lipid phosphate phosphohydrolase type 1). In the present paper, we report the development of a mouse wherein the LPP1 gene (Ppap2a) was disrupted. The homozygous mice, which are phenotypically unremarkable, generally lack Ppap2a mRNA, and multiple tissues exhibit a substantial (35-95%) reduction in LPA phosphatase activity. Compared with wild-type littermates, Ppap2a(tr/tr) animals have increased levels of plasma LPA, and LPA injected intravenously is metabolized at a 4-fold lower rate. Our results demonstrate that LPA is rapidly metabolized in the bloodstream and that LPP1 is an important determinant of this turnover. These results indicate that LPP1 is a catabolic enzyme for LPA in vivo.

LPA (lysophosphatidic acid), a potent bioactive phospholipid, elicits diverse cellular responses through activation of the G-protein-coupled receptors LPA1-LPA4. LPA-mediated signalling is partially regulated by LPPs (lipid phosphate phosphatases; LPP-1, -2 and -3) that belong to the phosphatase superfamily. This study addresses the role of LPPs in regulating LPA-mediated cell signalling and IL-8 (interleukin-8) secretion in HBEpCs (human bronchial epithelial cells). Reverse transcription-PCR and Western blotting revealed the presence and expression of LPP-1-3 in HBEpCs. Exogenous [3H]oleoyl LPA was hydrolysed to [3H]-mono-oleoylglycerol. Infection of HBEpCs with an adenoviral construct of human LPP-1 for 48 h enhanced the dephosphorylation of exogenous LPA by 2-3-fold compared with vector controls. Furthermore, overexpression of LPP-1 partially attenuated LPA-induced increases in the intracellular Ca2+ concentration, phosphorylation of IkappaB (inhibitory kappaB) and translocation of NF-kappaB (nuclear factor-kappaB) to the nucleus, and almost completely prevented IL-8 secretion. Infection of cells with an adenoviral construct of the mouse LPP-1 (R217K) mutant partially attenuated LPA-induced IL-8 secretion without altering LPA-induced changes in intracellular Ca2+ concentration, phosphorylation of IkappaB, NF-kappaB activation or IL-8 gene expression. Our results identify LPP-1 as a key regulator of LPA signalling and IL-8 secretion in HBEpCs. Thus LPPs could represent potential targets in regulating leucocyte infiltration and airway inflammation.

The regulation of protein kinase C (PKC)zeta in relation to its turnover, cell growth and transformation was investigated in Rat2 fibroblasts by over-expressing wild-type or mutant forms of PKCzeta. Deletion of the pseudosubstrate site (PSS) produced the most active mutant (PKCzeta Delta PSS), but mutants designed to mimic phosphorylated PKCzeta had lower specific activities in an in vitro assay. The mutant lacking phosphorylation at the Thr-560 site (T560A) had similar specific activity to wild-type PKCzeta. The T560A mutant also protected PKCzeta against proteolysis, whereas phosphorylation at Thr-410 targeted it towards proteosomal degradation. Blocking proteosomal degradation with lactacystin caused the accumulation of full-length PKCzeta Delta PSS, T410E, PKCzeta Delta PSS T410/560E, PKCzeta and T560A. Expressed PKCzeta activity was paralleled by extracellular-regulated protein kinase activation, increased cell division, serum-independent growth and focus formation. These foci were seen for cells expressing higher PKCzeta activity (PKCzeta Delta PSS, PKCzeta Delta PSS T410/560E and T560A mutants), but these fibroblasts did not show significant anchorage-independent growth. This work provides novel information concerning the role of the PSS and phosphorylation sites in regulating the activity and turnover of an atypical PKC and shows how this activity can induce cell transformation with respect to focus formation.

Differential effects of acetyl(C2-) ceramide (N-acetylsphingosine) were studied on coated vesicle formation from Golgi-enriched membranes of Chinese hamster ovary (CHO) and Madin-Darby canine kidney (MDCK) cells. C2-ceramide blocked the translocation of ADP-ribosylation factor-1 (ARF-1) and protein kinase C-alpha (PKC-alpha) to the membranes from CHO cells, but not those of MDCK cells. Consequently, C2-ceramide blocked the stimulation of phospholipase D1 (PLD1) by the cytosol and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in membranes from CHO cells. Basal specific activity of PLD1 and the concentration of ARF-1 were 3-4 times higher in Golgi-enriched membranes from MDCK cells compared with CHO cells. Moreover, PLD1 activity in MDCK cells was stimulated less by cytosol and GTP[S]. PLD2 was not detectable in the Golgi-enriched membranes. Incubation of intact CHO cells or their Golgi-enriched membranes with C2-ceramide also inhibited COP1 vesicle formation by membranes from CHO, but not MDCK, cells. Specificity was demonstrated, since dihydro-C2-ceramide had no significant effect on ARF-1 translocation, PLD1 activation or vesicle formation in membranes from both cell types. C2-ceramide also decreased the secretion of virus-like particles to a greater extent in CHO compared with MDCK cells, whereas dihydro-C2-ceramide had no significant effect. The results demonstrate a biological effect of C2-ceramide in CHO cells by decreasing ARF-1 and PKC-alpha binding to Golgi-enriched membranes, thereby preventing COP1 vesicle formation.

Lipid phosphate phosphatase-1 (LPP-1) dephosphorylates exogenous lysophosphatidate and thereby regulates the activation of lysophosphatidate receptors and cell division. Mutation of seven amino acids in three conserved domains of mouse LPP-1 abolished its activity. A glycosylation site was demonstrated between conserved Domains 1 and 2. LPP-1 is expressed in the plasma membrane, and the present results demonstrate the active site to be located on the outer surface.

Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling.

Cultured rat hepatocytes were preincubated with glucagon or a cyclic AMP analogue for up to 24 h and lipid synthesis and secretion were determined during the next 2 h. Glucagon or cyclic AMP did not change the incorporation of choline or glycerol into phosphatidylcholine, or choline into sphingomyelin, in the cells after 0-12 h of preincubation. After 12 h these incorporations were increased. Incorporations into hepatic lysophosphatidylcholine were decreased after preincubation with glucagon or cyclic AMP for 0-12 h, but by 24 h they increased. There was no change in the lysophosphatidylcholine in the medium after preincubation with glucagon or cyclic AMP for up to 6 h, but increases occurred after preincubation from 12 to 24 h. The secretion of triacylglycerol was decreased after preincubation for 0-1 h, but it returned to control values after 4 h. After preincubation for 18-24 h the incorporation of glycerol into secreted triacylglycerol was increased. The results are discussed in relation to the control of lipid metabolism in starvation and diabetes.

1. Induction of hypothyroidism in rats by feeding propylthiouracil (PTU) significantly increased serum cholesterol concentrations, and the effect was more pronounced for cholesterol in low-density lipoproteins (LDL) rather than high-density lipoproteins (HDL). The concentrations of serum triacylglycerol were decreased in hypothyroidism. These effects on serum lipids were also seen when the normal rats were pair-fed with the PTU-treated group. 2. Feeding a diet rich in saturated fat and cholesterol further increased cholesterol concentrations in LDL and also elevated that in very-low-density lipoprotein (VLDL) of hypothyroid rats. In euthyroid rats such a diet resulted in a relatively small increase in VLDL cholesterol, whereas LDL cholesterol was decreased. 3. Steady-state concentrations of mRNA for the hepatic LDL receptor were significantly decreased in the livers of hypothyroid rats, but were not significantly changed by high-fat feeding in euthyroid or hypothyroid rats. 4. The expression of the LDL receptor in hepatocytes cultured from hypothyroid rats was decreased relative to the euthyroid controls. 5. Whereas the esterification of cholesterol with oleate in hepatocytes cultured from hypothyroid rats was decreased, the activity of acyl-CoA:cholesterol acyltransferase (ACAT) in the livers of these animals was not changed. 6. High-fat feeding increased the hepatic ACAT activity in normal and hypothyroid rats. 7. Incubation of rat hepatocytes with 10 nM-tri-iodothyronine for 4 h increased the relative concentration of the mRNA for the LDL receptor by 25%. 8. It is therefore concluded that thyroid hormones stimulate the synthesis and expression of the hepatic LDL receptor. Elevated cholesterol concentrations in LDL in hypothyroidism probably result from a primary defect in the expression of the hepatic receptor, rather than indirectly via changes in ACAT activity.

Oleate, linoleate, linolenate, arachidonate and eicosapentaenoate, but not myristate, palmitate and stearate, stimulated glycogen phosphorylase activity by 2-8-fold when added to cultured rat hepatocytes. Addition of BSA or Ca2- to the incubation medium decreased the stimulating effects of the unsaturated fatty acids. The combination of oleate or linolenate, with corticosterone, testosterone or estradiol produced synergistic stimulations of phosphorylase activity. The stimulation of glycogen phosphorylase activity by linolenate was inhibited by staurosporine or sphingosine. Staurosporine (80 nM) alone also decreased basal phosphorylase activities by about 60%. The results show that unsaturated fatty acids can be used as model agonists to stimulate phosphorylase activity by a mechanism that probably involves protein kinase C. On the basis of the fatty acid: BSA ratios used, this stimulation should only occur in vivo at high fatty acid concentrations when accompanied by hypoalbuminaemia.

The effects of dexamethasone (a synthetic glucocorticoid) and insulin on the secretion of very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) were investigated. Rat hepatocytes in monolayer culture were preincubated for 15 h in the presence or absence of combinations of 100 nM-dexamethasone and 2 nM-, 10 nM- or 50 nM-insulin. Dexamethasone increased [3H]oleate incorporation into secreted triacylglycerol by 2.7-fold and the mass of triacylglycerol secreted by 1.5-fold. Insulin alone decreased these parameters and antagonized the effect of dexamethasone. Dexamethasone increased the secretion of [3H]leucine in apolipoprotein (apo) E, and in the large (BH) and small (BI) forms of apo B in VLDL by about 7.1-, 3.6- and 4.0-fold respectively. Insulin alone decreased the secretion of these 3H-labelled apolipoproteins in VLDL. However, 2 nM-insulin with dexamethasone increased the secretion of 3H-labelled apo BH and apo BL by a further 0.8- and 3.2-fold respectively; 50 nM-insulin decreased the secretions of apo E, apo BH and apo BL in VLDL. Similar effects for dexamethasone or insulin alone were also obtained for the masses of apo E and apo BL + H secreted in VLDL. Albumin secretion was not significantly altered by either dexamethasone or insulin alone, but in combination they stimulated by 2.1-2.6-fold. Insulin or dexamethasone alone had little effect on the secretion of apolipoproteins in the HDL fraction. However, dexamethasone plus 2 nM-insulin increased the incorporation of [3H]leucine into apo AI, apo AH plus apo C, apo AIV and apo E of HDL by about 1.8-, 1.6-, 1.7- and 2.0-fold respectively. The apo E in the bottom fraction represented about 69% of the total 3H-labelled apo E secreted. The responses in the total secretion of apo E from the hepatocytes resembled those seen in HDL. The interactions of insulin and dexamethasone are discussed in relation to the general regulation of lipoprotein metabolism, the development of hyperlipidaemias and the predisposition to premature atherosclerosis.

The effects of vasopressin on the short-term control of fatty acid metabolism were studied in isolated rat hepatocytes. Vasopressin increased the oxidation of oleate to CO2 and decreased the formation of ketones in hepatocytes from Wistar rats, but not from Brattleboro rats. Incubation with vasopressin for 30 min increased the conversion of oleate into triacylglycerol by 17% and 32% in hepatocytes from Wistar and Brattleboro rats respectively. The corresponding increases for the phospholipid fraction were 19% and 42%. When Wistar-rat hepatocytes were incubated with corticosterone for 6 h there was a 19% increase in triacylglycerol synthesis, and a 52% increase if vasopressin was added 30 min before the end of the incubation. Glycerol phosphate acyltransferase activity was not significantly increased by vasopressin. Incubation for 5-60 min with vasopressin increased the Vmax. of phosphatidate phosphohydrolase by 48% and 32% respectively in hepatocytes from Wistar and Brattleboro rats. These increases were antagonized if EGTA was added to the medium used for incubating the hepatocytes. The replacement of vasopressin by 5 microM-ionophore A23187 produced a significant increase of 13% in the phosphohydrolase activity. It is therefore likely that the effects of vasopressin on the phosphohydrolase are mediated by Ca2+. These results are discussed in relation to the possible function of phosphatidate phosphohydrolase in controlling the turnover of phosphoinositides, the synthesis of phosphatidylethanolamine, phosphatidylcholine and triacylglycerol, and the secretion of very-low-density lipoproteins.

The binding of human low-density lipoprotein labelled with 125I to rat hepatocytes in monolayer culture was measured at 4 degrees C. Evidence for two different specific binding sites was obtained. Binding to Site 1 was characterized by: being displaced by dextran sulphate or heparin; being dependent on Ca2+; having a Kd value of about 15 micrograms of protein/ml; not being significantly displaced by a 20-fold excess unlabelled low-density lipoprotein that had been reductively methylated; being displaced by approx. 40% by a 20-fold protein excess of unlabelled human high-density lipoprotein, HDL3, and increasing with time in culture when newborn-calf serum was present in the medium. The binding to Site 2 had the following properties: it was not displaced by sulphated polysaccharides; it was only partially Ca2+-dependent, and the presence of EDTA increased the Kd value; the apparent Kd value in the presence of Ca2+ was approx. 30 micrograms of protein/ml, which was significantly higher than for Site 1; it was displaced by approx. 30% with a 20-fold excess of low-density lipoprotein that had been methylated; it was displaced by unlabelled HDL3 to a similar extent to Site 1; it did not increase significantly with time in culture. The characteristics of binding to Sites 1 and 2 are discussed in relation to the receptors for low-density lipoproteins that have previously been described in various cell types. It is proposed that the experimental system described in this paper is suitable for studying the regulation of the binding of low-density lipoproteins to hepatocytes.

1. The effects of dietary modification, including starvation, and of corticotropin injection on the activities of acyl-CoA synthetase, glycerol phosphate acyltransferase, dihydroxyacetone phosphate acyltransferase, phosphatidate phosphohydrolase, diacylglycerol acyltransferase and lipoprotein lipase were measured in adipose tissue. 2. Lipoprotein lipase activities in heart were increased and those in adipose tissue were decreased when rats were fed on diets enriched with corn oil or beef tallow rather than with sucrose or starch. The lipoprotein lipase activity was lower in the adipose tissue of rats fed on the sucrose rather than on the starch diet. 3. Rats fed on the beef tallow diet had slightly higher activities of the total glycerol phosphate acyltransferase in adipose tissue than did rats fed on the sucrose or starch diet. The diacylglycerol acyltransferase and the mitochondrial glycerol phosphate acyltransferase activities were higher for the rats fed on the tallow diet than for those fed on the corn-oil diet. 4. Starvation significantly decreased the activities of lipoprotein lipase (after 24 and 48 h), acyl-CoA synthetase (after 24 h) and of the mitochondrial glycerol phosphate acyltransferase and the N-ethylmaleimide-insensitive dihydroxyacetone phosphate acyltransferase (after 48 h) in adipose tissue. The activities of the microsomal glycerol phosphate acyltransferase, diacylglycerol acyltransferase and the soluble phosphatidate phosphohydrolase were not significantly changed after 24 or 48 h of starvation. 5. The activities of lipoprotein lipase and phosphatidate phosphohydrolase in adipose tissue were decreased 15 min after corticotropin was injected into rats during November to December. No statistically significant differences were found when these experiments were performed during March to September. These differences may be related to the seasonal variation in acute lipolytic responses. 6. These results are discussed in relation to the control of triacylglycerol synthesis and lipoprotein metabolism.

1. Microsomal membranes from rat liver were incubated with ATP, CoA, Mg2+, [14C]palmitate, F- and sn-glycerol 3-phosphate in order to label them with [14C]phosphatidate. These membranes were isolated and used in a second incubation in which [3H]CTP was present, and the simultaneous synthesis of [14C]diacylglycerol and [3H]CDP-diacylglycerol was measured. 2. The addition of phosphatidate phosphohydrolase, which had been partially purified from the particle-free supernatant, supplemented the activity of the endogenous phosphohydrolase, but it did not alter the rate of CDP-diacylglycerol formation. 3. Adding EDTA inhibited phosphatidate cytidylyl-transferase activity and stimulated the activity of the phosphohydrolases by removing excess of Mg2+. 4. Increasing the concentration of Mg2+, norfenfluramine or chlorpromazine in the assay system stimulated cytidylyltransferase activity, but decreased the activities of both phosphohydrolases. 5. The mechanism for the stimulation of cytidylyl=transferase activity by the cationic drugs and Mg2+ was investigated with emulsions of phosphatidate and the microsomal fraction of rat liver. 6. There was a threshold concentration of about 5mM-MgCl2 below which no cytidylyltransferase activity was detected in the presence or absence of norfenfluramine. Just above this threshold concentration norfenfluramine stimulated cytidylyltransferase activity, but this stimulation disappeared as the Mg2+ concentration was raised to its optimum of 20mM. Norfenfluramine therefore partially replaced the bivalent-cation requirement. 7. At 30 mM-MgCl2 amphiphilic cationic drugs inhibited cytidylyltransferase activity at relatively high concentrations in a non-competitive manner with respect to phosphatidate. 8. The implications of these results are discussed with respect to the regulation of the synthesis of the acidic phospholipids compared with the synthesis of phosphatidylcholine, phosphatidylethanolamine and triacylglycerol.

Testosterone (40-300 microM), oestradiol (20-500 microM), progesterone (20-500 microM), dexamethasone (10 nM-1 microM) and corticosterone (1-10 microM) activate glycogen phosphorylase rapidly when added directly to hepatocytes. The activation of phosphorylase was concentration-dependent and occurred after 10 min for dexamethasone, 30 min for testosterone and 60 min for oestradiol and progesterone. This rapid effect does not appear to be dependent on a stimulation of protein synthesis, it is independent of an increase in cyclic AMP, and it is not diminished by the presence of ornithine decarboxylase inhibitors. The stimulation of phosphorylase activity is diminished by depleting the incubation medium of Ca2+ in the presence of 0.5 mM-EGTA, and therefore it may involve changes in the distribution of Ca2+ in the hepatocytes. These results may explain some of the pharmacological effects of sex steroids, and also might contribute to the physiological actions of glucocorticoids.

The incubation of hepatocytes with 1-4mM-oleate increased the total activity of phosphatidate phosphohydrolase that was measured in the presence of Mg2+ to about 2-fold. This was accompanied by an increase in the proportion of the enzyme that was isolated with the particulate fractions. Conversely, the addition of up to 4mM-oleate decreased the recovery of phosphatidate phosphohydrolase in the cytosolic fraction from about 70% to 3% when hepatocytes were lysed with digitonin. Most of the increase in the membrane-associated phosphohydrolase activity was isolated after cell fractionation in the microsomal fraction that was enriched with the endoplasmic-reticulum marker arylesterase. It is proposed that the translocation of phosphatidate phosphohydrolase facilitates the increased synthesis of triacylglycerols in the liver when it is presented with an increased supply of fatty acids.

The effects on glycerolipid synthesis of a series of compounds including many drugs were investigated in cell-free preparations and slices of rat liver. p-Chlorobenzoate, p-chlorophenoxyisobutyrate, halofenate, D-amphetamine, adrenaline, procaine and N-[2-(4-chloro-3-sulphamoylbenzoyloxy)ethyl]norfenfluramine had little inhibitory effect on any of the systems investigated. Two amphiphilic anions, clofenapate and 2-(p-chlorophenyl)-2-(m-trifluoromethylphenoxy)acetate, both inhibited glycerol phosphate acyltransferase and diacylglycerol acyltransferase at approx. 1.6 and 0.7 mm respectively. Clofenapate (1 mm) also inhibited the incorporation of glycerol into lipids by rat liver slices without altering the relative proportions of the different lipids synthesized. The amphilic amines, mepyramine, fenfluramine, norfenfluramine, hydroxyethylnorfenfluramine, N-(2-benzoyloxyethyl)norfenfluramine, cinchocaine, chlorpromazine and demethylimipramine inhibited phosphatidate phosphohydrolase by 50% at concentrations between 0.2 and 0.9 mm. The last four compounds inhibited glycerol phosphate acyltransferase by 50% at concentrations between 1 and 2.6 mm. None of the amines examined appeared to be an effective inhibitor of diacylglycerol acyltransferase. Norfenfluramine, hydroxyethylnorfenfluramine and N-(2-benzoyloxyethyl)norfenfluramine produced less inhibition of glycerol incorporation into total lipids than was observed with equimolar clofenapate. The major effect of these amines in liver slices was to inhibit triacylglycerol and phosphatidylcholine synthesis and to produce a marked accumulation of phosphatidate. The results are discussed in terms of the control of glycerolipid synthesis. They partly explain the observed effects of the various drugs on lipid metabolism. The possible use of these compounds as biochemical tools with which to investigate the reactions of glycerolipid synthesis is considered.

Feeding rats with sorbitol, fructose, glycerol and ethanol increases the concentration of serum corticosterone without significantly altering the concentration of insulin. This increase appears to be partly responsible for the increases in the hepatic activity of phosphatidate phosphohydrolase (compared with rats fed glucose or 0.9% NaCl) that has been reported [Sturton, Pritchard, Han & Brindley (1978) Biochem. J. 174, 667--670] and the enhanced capacity of the liver to synthesize triacylglycerols. The ethanol-induced increase in phosphohydrolase activity was largely, but not completely, prevented by adrenalectomy.

The measurement of phosphate release from phosphatidate overestimates the microsomal activity of phosphatidate phosphohydrolase from rat liver, since phosphate is also produced via the glycerol phosphate that results from the deacylation of phosphatidate. The determination of phosphate production can be a reliable assay for the soluble phosphatidate phosphohydrolase in rat liver, because the glycerol phosphate formed is not hydrolysed under the conditions used.

1. Male rats were injected daily for 5 days with 0.15m-NaCl, corticotropin, cortisol or l-thyroxine and the rates of glycerolipid synthesis were measured in the livers after intraportal injection of [(14)C]palmitate and [(3)H]glycerol. 2. Injection of all three hormones decreased the rates of body-weight gain. 3. Cortisol treatment increased the weight of the liver relative to body weight. 4. Thyroxine treatment increased the relative rate of triacylglycerol synthesis from [(3)H]glycerol and decreased the relative accumulation of (3)H and (14)C in diacylglycerol. It did not significantly alter the accumulation of these isotopes in phosphatidate nor the activity of the soluble phosphatidate phosphohydrolase in the total liver. However, this activity increased by 1.5-fold when expressed relative to the soluble protein of the liver. The increased triacylglycerol synthesis appears to be related to a general increase in the turnover of fatty acids in the liver. 5. Treatment with cortisol and corticotropin increased the relative rate of triacylglycerol synthesis from [(3)H]glycerol, decreased the accumulation of (3)H in phosphatidate and increased the flux of both isotopes from phosphatidate to diacylglycerol. This appeared to be caused by the increased activity of the soluble phosphatidate phosphohydrolase that was observed in the livers of the cortisol-treated rats. 6. It is proposed that cortisol could be directly or indirectly involved in increasing the activity of hepatic phosphatidate phosphohydrolase in starvation, diabetes, laparotomy, subtotal hepatectomy, liver damage, ethanol feeding and in obesity. This enzyme adaptation could contribute to the potential of the liver to increase its synthesis and accumulation of triacylglycerols or to secrete very-low-density lipoproteins.

1. The synthesis and secretion of glycerolipids by monolayer cultures of rat hepatocytes was measured by using radioactive choline, glycerol and fatty acids and by measuring the concentration of triacylglycerols in the cells. 2. The incorporation of glycerol into triacylglycerol and the accumulation of this lipid in hepatocytes showed little specificity for fatty acids, except for eicosapentaenoate, which stimulated least. Oleate was more effective at stimulating triacylglycerol secretion than were palmitate, stearate, arachidonate and eicosapentaenoate. 3. Linoleate, linolenate, arachidonate and eicosapentaenoate stimulated the incorporation of glycerol and choline into phosphatidylcholine that was secreted into the medium. By contrast, palmitate and stearate produced relatively high incorporations into the phosphatidylcholine that remained in the cells. 4. The incorporation of glycerol and choline into lysophosphatidylcholine in the medium was stimulated 2-3-fold by all of the unsaturated fatty acids tested, whereas palmitate and stearate failed to stimulate if the acids were added separately. When 1 mM-stearate was added with 1 mM-linoleate, the incorporation of linoleate into lysophosphatidylcholine was about 4 times higher than that of stearate. 5. It is proposed that the secretion of lysophosphatidylcholine by the liver could provide a transport system for choline and essential unsaturated fatty acids to other organs.

1. Fat-free homogenates from the epididymal fat-pads of rats were used to measure the rate of palmitate esterification with different substrates. The effectiveness of the acyl acceptors decreased in the order glycerol phosphate, dihydroxyacetone phosphate, 2-octadecenyl-glycerol and 2-hexadecylglycerol. 2. Glycerol phosphate and dihydroxyacetone phosphate inhibited their rates of esterification in a mutually competitive manner. 3. The esterification of glycerol phosphate was also inhibited in a partially competitive manner by 2-octadecenylglycerol and to a lesser extent by 2-hexadecylglycerol. However, glycerol phosphate did not inhibit the esterification of 2-octadecenylglycerol. 4. The esterification of dihydroxyacetone phosphate and 2-hexadecylglycerol was more sensitive to inhibition by clofenapate than was that of glycerol phosphate. Norfenfluramine was more effective in inhibiting the esterification of 2-hexadecylglycerol than that of glycerol phosphate or dihydroxyacetone phosphate. 5 It is concluded that rat adipose tissue can synthesize glycerolipids by three independent routes.

1. Rats were fed on diets enriched with starch, sucrose, corn oil or beef tallow for 3 weeks and the activities of various enzymes in the liver were measured. 2. The mitochondrial glycerol phosphate acyltransferase activity was lower in rats fed on the starch diet than on the two high-fat diets. 3. The non-microsomal (presumably peroxisomal) dihydroxyacetone phosphate acyltransferase activity was higher in rats fed on the starch diet and corn-oil diets than in those fed on the sucrose and beef-tallow diets. Urate oxidase activity was higher in rats fed on the starch diet than in the three other groups. There were no significant differences in the activity of acyl-CoA oxidase among the groups. 4. The activity of soluble phosphatidate phosphohydrolase was not significantly different among the dietary groups. There were increases of 3.3--4.3-fold in this activity in the dietary groups 6h after injection of corticotropin. The equivalent increases for the mitochondrial glycerol phosphate acyltransferase were 1.4--1.6 fold. 5. The corticosterone responses to the corticotropin injection were not significantly different between dietary groups. However, the corticosterone response of the rats fed on the two high-fat diets was prolonged when the rats were given an acute load of fructose [Brindley, Cooling, Glenny, Burditt & McKechnie (1981) Biochem. J. 200. 275--283]. 6. Rats fed on the high-fat diets had higher concentrations of circulating cholesterol than those fed on the starch and sucrose diets. Serum triacylglycerol concentrations were lower in the rats fed on the starch diet than in the three other groups. 7. The results are discussed in terms of the relationship between diet, hormonal balance and hepatic glycerolipid metabolism.

1. We have previously shown that the capacity for specific binding of human 125I-labelled low-density lipoprotein (LDL) to rat hepatocytes increases with time in culture [Salter, Bugaut, Saxton, Fisher & Brindley (1987) Biochem. J. 247, 79-84]. 2. In the present study we show that this up-regulation is accompanied by a rise in the cholesterol ester content of the cells. 3. Inhibition of cholesterol esterification with the drug 58-035 (Sandoz) significantly decreases the time-dependent 'up-regulation' of LDL receptors. 4. Incubation of hepatocytes with LDL itself has little effect on subsequent LDL binding. However, when cholesterol esterification is inhibited, incubation with LDL decreases binding below that attained with the drug alone. 5. Inhibition of cholesterol synthesis with Lovastatin significantly increases LDL binding and antagonizes the effect of 58-035. 6. We conclude that in hepatocytes the rate of cellular cholesterol esterification can become the major determinant of LDL-receptor activity.

1. Phosphatidate phosphohydrolase from the particle-free supernatant of rat liver was assayed by using emulsions of phosphatidate as substrate. 2. The inhibition of the phosphohydrolase by chlorpromazine was of a competitive type with respect to phosphatidate. The potency of various amphiphilic cationic drugs as inhibitors of this reaction was related to their partition coefficients into a phosphatidate emulsion. 3. The effect of chlorpromazine on the phosphohydrolase activity was complementary rather than antagonistic towards Mg2+. Chlorpromazine stimulated the phosphohydrolase activity in the absence of added Mg2+ and was able to replace the requirement for Mg2+. However, at optimum concentrations of Mg2+, chlorpromazine inhibited the reaction, as did Ca2+. The phosphohydrolase activity was also stimulated by Co2+ and to a lesser extent by Mn2+, Fe2+, Fe3+, Ca2+, spermine and spermidine when Mg2+ was not added to the assays. 4. It is concluded that the inhibition of phosphatidate phosphohydrolase by amphiphilic cations can largely be explained by the interaction of these compounds with phosphatidate, which changes the physical properties of the lipid, making it less available for conversion into diacylglycerol. 5. The implications of these results to the effects of amphiphilic cations in redirecting glycerolipid synthesis at the level of phosphatidate are discussed.

1. The activities of fatty acid synthetase, acyl-CoA synthetase, glycerol phosphate acyltransferase and phosphatidate phosphatase were measured in the mammary glands of rabbits from day 16 of pregnancy to day 15 of post partum. 2. There were significant correlations between the increases in activities of these enzymes during this period. This was the case whether the activities were expressed per mg of homogenate protein, per g wet wt. of tissue or per total wet weight of the whole glands. The only exception was the lack of correlation between the activities of fatty acid synthetase and of phosphatidate phosphatase per g wet wt. of tissue. 3. These co-ordinate increases are discussed in relation to the changes which occur in fatty acid metabolism in the mammary gland during pregnancy and lactation.

1. Male rats were fed for 14 days on diets containing (by wt.) 53% of starch, or on diets in which 20% of the starch was replaced by sucrose, corn oil or lard. 2. The hepatic activities of the microsomal glycerol phosphate acyltransferase, dihydroxyacetone phosphate acyltransferase, phosphatidate cytidylyltransferase, diacylglycerol acyltransferase and choline phosphotransferase, and of the soluble phosphatidate phosphohydrolase, were measured. 3. The soluble phosphatidate phosphohydrolase activity was higher in those rats fed on lard than in those fed on the starch diet. Choline phosphotransferase activity was higher in the rats fed on corn oil than in those fed on the starch diet. 4. The rate of hepatic glycerolipid synthesis was measured in vivo 1 min after injection of [1,3-3H]glycerol and [1-14C]palmitate into the portal veins. 5. The relative rate of phosphatidylcholine synthesis in vivo was increased after feeding with corn oil and the higher specific activity of choline phosphotransferase may contribute to this result. The equivalent rate of triacylglycerol synthesis was increased by feeding with lard rather than corn oil, and the increased activity of phosphatidate phosphohydrolase may partly explain this. The latter changes probably contribute to the increased concentration of triacylglycerol which other authors have observed in the livers and sera of animals fed on saturated and monounsaturated fats.

A method is described by which the Mg2+-stimulated phosphatidate phosphohydrolase can be purified from the soluble fraction of liver from ethanol-treated rats. The increase in specific activity was about 416-fold. This involved purification by adsorption on calcium phosphate, chromatography on DE-52 DEAE-cellulose, separation on Ultrogel AcA-34 and chromatography on CM-Sepharose 6B. The effects of phosphatidylcholine, phosphatidate and Mg2+, Mn2+ and Zn2+ on the activity are described. Inhibitor studies indicate that the phosphohydrolase contains functional thiol groups and arginine residues.

Male rats were kept for 14 days with alternating 12h periods of light and darkness. The hepatic activity of soluble phosphatidate phosphohydrolase and the concentration of serum insulin were maximum at about 2h after dark. The peak concentration of serum corticosterone occurred 2h before the dark period. It is proposed that corticosterone is partly responsible for the increased phosphohydrolase activity, and that this enables the liver to increase its capacity to synthesize triacylglycerols during the period of maximum feeding.

Hepatocytes were preincubated with 10mM-glucagon and 100 microM-corticosterone to increase phosphatidate phosphohydrolase activity. Addition of 10 nM-glucagon or 100 microM-8-bromo cyclic GMP to a second incubation mixture that contained cycloheximide increased the half-life of the phosphohydrolase activity. Dexamethasone (100 nM) had no significant effect, but insulin (500 pM) or spermine (1 mM) decreased the half-life. None of these compounds altered the general rate of degradation of proteins labelled with [3H]leucine. There appears to be a specific control of the half-life of phosphatidate phosphohydrolase activity, which could contribute to its long-term regulation in the liver.

1. Male rats were fed for 5 weeks after weaning on a diet containing (by weight) 59% of starch or on diets that contained 39% of starch and 20% of either sucrose, beef tallow or corn oil. 2. The rats fed on the beef tallow consumed more energy than did the rats fed on the starch and sucrose diets. The rats fed on the corn oil drank less water than did the other groups of rats. 3. There were no significant differences between the four groups in terms of body-weight gain, epididymal-fat-pad weight and in the size, number and triacylglycerol content of the adipocytes in the fat-pads. 4. There was a significant correlation (P less than 0.001) between the activities of glycerol phosphate acyltransferase and monoacylglycerol acyltransferase in individual rats. Both of these activities were highest in the group fed on the high-starch diet and both correlated with the consumption of glucose by individual rats in the four groups. 5. The percentage of glycerol phosphate converted into diacylglycerol and triacylglycerol was positively correlated with the mean diameters, surface area and triacylglycerol content of the adipocytes for individual rats and was greates in the sucrose-fed rats. 6. The specific activity of dihydroxyacetone phosphate acyltransferase was highest in the rats fed on beef tallow. This activity was positively correlated with the energy intake for all dietary groups over the 5-week feeding period. 7. The results are discussed in terms of the functions of the three routes of glycerolipid synthesis in adipose tissue.

Rat hepatocytes were preincubated for 16 h with hormones or drugs and then for a further 8 h with 125I-human low-density lipoprotein (LDL). Glucagon (via cyclic AMP) and adrenaline (via cyclic AMP and alpha-effects) increased the binding of 125I-LDL to the LDL receptor, and the degradation of LDL to [125I]iodotyrosine. The effects on degradation were antagonized by dexamethasone, and the action of cyclic AMP on binding and degradation was inhibited by actinomycin D. The results are discussed in relation to the control of lipoprotein metabolism in diabetes.

Rat hepatocytes were incubated in monolayer culture for 8 h. Glucagon (10nM) increased the total phosphatidate phosphohydrolase activity by 1.7-fold. This effect was abolished by adding cycloheximide, actinomycin D or 500 pM-insulin to the incubations. The glucagon-induced increase was synergistic with that produced by an optimum concentration of 100 nM-dexamethasone. Theophylline (1mM) potentiated the effect of glucagon, but it did not affect the dexamethasone-induced increase in the phosphohydrolase activity. The relative proportion of the phosphohydrolase activity associated with membranes was decreased by glucagon when 0.15 mM-oleate was added 15 min before the end of the incubations to translocate the phosphohydrolase from the cytosol. This glucagon effect was not seen at 0.5 mM-oleate. Since glucagon also increased the total phosphohydrolase activity, the membrane-associated activity was maintained at 0.15 mM-oleate and was increased at 0.5 mM-oleate. This activity at both oleate concentrations was also increased in incubations that contained dexamethasone, particularly in the presence of glucagon. Insulin increased the relative proportion of phosphatidate phosphohydrolase that was associated with membranes at 0.15 mM-oleate, but not at 0.5 mM-oleate. It also decreased the absolute phosphohydrolase activity on the membranes at both oleate concentrations in incubations that also contained glucagon and dexamethasone. None of the hormonal combinations significantly altered the total glycerol phosphate acyltransferase activity. However, glucagon significantly increased the microsomal activities, and insulin had the opposite effect. Glucagon also decreased the mitochondrial acyltransferase activity. There was a highly significant correlation between the total phosphatidate phosphohydrolase activity and the synthesis of neutral lipids from glycerol phosphate and 0.5 mM-oleate in homogenates of cells from all of the hormonal combinations. Phosphatidate phosphohydrolase activity is increased in the long term by glucocorticoids and also by glucagon through cyclic AMP. In the short term, glucagon increases the concentration of fatty acid required to translocate the cytosolic reservoir of activity to the membranes on which phosphatidate is synthesized. Insulin opposes the combined actions of glucagon and glucocorticoids. The long-term events explain the large increases in the phosphohydrolase activity that occur in vivo in a variety of stress conditions. The expression of this activity depends on increases in the net availability of fatty acids and their CoA esters in the liver.

Rat hepatocytes were incubated in monolayer culture in modified Leibovitz L-15 medium containing either 10% (v/v) newborn-calf serum or 0.2% (w/v) fatty-acid-poor bovine serum albumin. The addition of 100 nM-dexamethasone increased the activities of both phosphatidate phosphohydrolase and tyrosine aminotransferase by about 3.5-fold after 8h, and these activities continued to rise until at least 24h. Incubating the hepatocytes in the albumin-containing medium with 10 microM- or 100 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate increased the activities of the phosphohydrolase and aminotransferase by 2.6- and 3.4-fold respectively after 8h. These increases were blocked by actinomycin D. The increases in the activities that were produced by the cyclic AMP analogue and dexamethasone were independent and approximately additive. Insulin when added alone did not alter the phosphohydrolase activity, but it increased the aminotransferase activity by 34%. The dexamethasone-induced increase in the phosphohydrolase activity was completely blocked by 7-144 microM-insulin, whereas that of the aminotransferase was only partly suppressed. Insulin had no significant Effects on the increases in the activities of phosphatidate phosphohydrolase and tyrosine aminotransferase that were produced by the cyclic AMP analogue, but this may be because the analogue is fairly resistant to degradation by the phosphodiesterase. The activity of glycerol kinase was not significantly changed by incubating the hepatocytes with insulin, dexamethasone and the cyclic AMP analogue alone or in combinations. It is proposed that high concentrations of cyclic AMP and glucocorticoids increase the total activity of phosphatidate phosphohydrolase in the liver and provide it with an increased capacity for synthesizing triacylglycerols and very-low-density lipoproteins, which is expressed when the availability of fatty acids is high. There appears to be a co-ordinated hormonal control of triacyglycerol synthesis and gluconeogenesis in diabetes and in metabolic stress to enable the liver to supply other organs with energy.

1. There are two distinct binding sites (Site 1 and Site 2) for human low-density lipoprotein (LDL) on rat hepatocytes in monolayer culture [Salter, Saxton & Brindley (1986) Biochem. J. 240, 549-557]. 2. Binding of 125I-LDL to Site 1, but not to Site 2, is up-regulated between 20 and 44 h in culture by preincubation of the cells with human high-density lipoprotein 3 (HDL3). 3. A similar preincubation with HDL2 had no significant effect on binding to either site. 4. Preincubation with human LDL led to a partial down-regulation of subsequent binding of 125I-LDL to Site 1. Since binding after incubation with LDL was measured at 37 degrees C, binding to Site 2 could not be distinguished from LDL that had been internalized by the cells. 5. Hepatocytes were shown to degrade 125I-LDL, resulting in the accumulation of [125I]iodotyrosine in the medium. Evidence was found that iodotyrosine may be further degraded by deiodinase produced by the cells. 6. Regulation of binding to Site 1 by preincubation with LDL or HDL3 was found to lead to a parallel regulation of LDL degradation. 7. It is concluded that rat hepatocytes not only bind but also metabolize human LDL and that these processes are under metabolic regulation.

The translocation of phosphatidate phosphohydrolase between the cytosol and the microsomal membranes was investigated by using a cell-free system from rat liver. Linoleate, alpha-linolenate, arachidonate and eicosapentenoate promoted the translocation to membranes with a similar potency to that of oleate. The phosphohydrolase that associated with the membranes in the presence of [14C]oleate or 1mM-spermine coincided on Percoll gradients with the peak of rotenone-insensitive NADH-cytochrome c reductase, and in the former case with a peak of 14C. Microsomal membranes were enriched with the phosphohydrolase activity by incubation with [14C]oleate or spermine and then incubated with albumin. The phosphohydrolase activity was displaced from the membranes by albumin, and this paralleled the removal of [14C]oleate from the membranes when this acid was present. Chlorpromazine also displaced phosphatidate phosphohydrolase from the membranes, but it did not displace [14C]oleate. The effects of spermine in promoting the association of the phosphohydrolase with the membranes was inhibited by ATP, GTP, CTP, AMP and phosphate. ATP at the same concentration did not antagonize the translocating effect of oleate. From these results and previous work, it was concluded that the binding of long-chain fatty acids and their CoA esters to the endoplasmic reticulum acts as a signal for more phosphatidate phosphohydrolase to associate with these membranes and thereby to enhance the synthesis of glycerolipids, especially triacylglycerol. The translocation of the phosphohydrolase probably depends on the increased negative charge on the membranes, which could also be donated by the accumulation of phosphatidate. Chlorpromazine could oppose the translocation by donating a positive charge to the membranes.

Rat hepatocytes in monolayer culture were preincubated for 19 h with 1 microM-dexamethasone, and the incubation was continued for a further 23 h with [14C]oleate, [3H]glycerol and 1 microM-dexamethasone. Dexamethasone increased the secretion of triacylglycerol into the medium in particles that had the properties of very-low-density lipoproteins. The increased secretion was matched by a decrease in the triacylglycerol and phosphatidylcholine that remained in the hepatocytes. Preincubating the hepatocytes for the total 42 h period with 36 nM-insulin decreased the amount of triacylglycerol in the medium and in the cells after the final incubation for 23 h with radioactive substrates. However, insulin had no significant effect on the triacylglycerol content of the cell and medium when it was present only in the final 23 h incubation. Insulin antagonized the effects of dexamethasone in stimulating the secretion of triacylglycerol from the hepatocytes, especially when it was present throughout the total 42 h period. The labelling of lysophosphatidylcholine in the medium when hepatocytes were incubated with [14C]oleate and [3H]glycerol was greater than that of phosphatidylcholine. The appearance of this lipid in the medium, unlike that of triacylglycerol and phosphatidylcholine, was not stimulated by dexamethasone, or inhibited by colchicine. However, the presence of lysophosphatidylcholine in the medium was decreased when the hepatocytes were incubated with both dexamethasone and insulin. These findings are discussed in relation to the control of the synthesis of glycerolipids and the secretion of very-low-density lipoproteins and lysophosphatidylcholine by the liver, particularly in relation to the interactions of glucocorticoids and insulin.

Rats were fed by stomach tube with a single dose of glucose, sorbitol, fructose, glycerol or ethanol of equivalent energy contents or with 0.15 M-NaCl. They were killed 6 h later and the relative rates of phosphatidate deacylation and dephosphorylation measured in the microsomal and supernatant fractions of the livers. Treatment with sorbitol, fructose, glycerol and ethanol increased phosphohydrolase activities in the microsomal and supernatant fractions. The only significant change in deacylase activity was an increase in the microsomal fraction produced by ethanol. It is proposed that hepatic triacylglycerol synthesis is partly controlled by the balance between phosphatidate phosphohydrolase and phospholipase A-type activities.

1. The activities of some enzymes of glycerolipid synthesis were measured in homogenates obtained from the intestinal scrapings of 62-66-day foetuses and 2- and 8-day-old guinea pigs. 2. The ratio of protein concentration/DNA concentration was significantly higher (P greater than 0.001) in homogenized tissue from the neonatal compared with the foetal guinea pigs. Enzyme activities were therefore expressed relative to both protein and to DNA. 3. The specific activities (relative to DNA) of palmitoyl-CoA synthetase, glycerol phosphate acyltransferase and phosphatidate phosphatase were higher in homogenized tissues from neonatal than in those from the foetal guinea pigs. These activities are probably involved more in cell proliferation than in the absorption and transport of triacylglycerol. Its activity was not significantly different in the foetal and neonatal guinea pigs when expressed relative to DNA but it was lower in the neonatal guinea pigs when expressed relative to protein. The entry of food into the intestine after birth is therefore not necessary for its activity.

1. A new assay system is described for monoglyceride acyltransferase (acylglycerol palmitoyltransferase, EC 2.3.1.22) in which palmitoyl-CoA is generated from palmitoyl-(-)-carnitine. 2. With the microsomal fraction from homogenates of guinea-pig intestinal mucosa, the V(max.) of this enzyme decreased with different acyl acceptors in the order 2-monopalmitoylglycerol>2-hexadecylglycerol>rac-1-monopalmitoylglycerol. 3. There were highly significant correlations between the monoglyceride acyltransferase activity as measured with these three substrates. This demonstrates that each of these substrates can be used to measure the same enzyme activity. 4. The advantages of using generated palmitoyl-CoA with 2-hexadecylglycerol and rac-1-monopalmitoylglycerol as model substrates for this enzyme are discussed.

1. Male rats were fed for 14 days on powdered diets containing (by weight) 53% of starch, or on diets in which 20g of starch per 100g of diet was replaced by lard or corn oil. They were then fed acutely by stomach tube with a single dose of glucose, fructose or ethanol of equivalent energy contents, or with 0.15m-NaCl. The serum concentrations of corticosterone, insulin, glucose, glycerol, triacylglycerol and cholesterol were measured up to 6h after this treatment. 2. Feeding saline (0.9% NaCl) acutely to the rats maintained on the three powdered diets produced a small transient increase in circulating corticosterone that was similar to that in rats maintained on the normal 41B pelleted diet. 3. Feeding glucose acutely to the rats on the powdered diets produced peak concentrations of corticosterone that were 2-3-fold higher than those seen in rats maintained on the 41B diet. The duration of this response increased in the order starch diet

Incubating the particle-free supernatant of rat liver with alkaline phosphatase decreased the activity of phosphatidate phosphohydrolase by 21-29%. When the particle-free supernatant was incubated with various combinations of Mg2+, ATP, cyclic AMP and cyclic AMP-dependent protein kinase this failed to alter significantly phosphatidate phosphohydrolase activity under the conditions employed. The incubation of hepatocytes in monolayer culture with 0.5 mM-8-(4-chlorophenylthio)adenosine 3',5'-monophosphate increased the total activity of phosphatidate phosphohydrolase as measured in vitro. This also decreased the proportion of the phosphohydrolase that was associated with the membrane fraction of the cells and increased that in the cytosolic fraction. Adding 1 mM-oleate to the hepatocytes promoted the translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Oleate overcame the effect of the cyclic AMP analogue in favouring the cytosolic distribution of the phosphohydrolase. These results are discussed in relation to the interaction of hormonal balance and substrate supply in controlling the synthesis of phosphatidylcholine and triacylglycerol in the liver in stress and in diabetes. It is proposed that the cytosolic phosphatidate phosphohydrolase activity represents a reservoir of potential activity that becomes expressed when the enzyme translocates to the membranes on which the synthesis of glycerolipids occurs.

1. Rats were injected with a single dose of 35mg of streptozotocin/kg body wt. They exhibited a diabetes that was characterized by glycosuria, polyuria, polydipsia, hyperphagia, hyperglycaemia, increased concentrations of unesterified fatty acids, glycerol and triacylglycerols in the serum and an increased activity of glucose 6-phosphatase in the liver. 2. After 10 weeks the hepatic activities of the microsomal glycerol phosphate acyltransferase, phosphatidate phosphohydrolase, phosphatidate cytidylyltransferase, diacylglycerol acyltransferase, choline phosphotransferase, CDP-diacylglycerol--inositol phosphatidyltransferase and the soluble phosphatidate phosphohydrolase were measured. 3. The only significant changes were an increase in the activity of the soluble phosphatidate phosphohydrolase and a decrease in that of the CDP-diacylglycerol--inositol phosphatidyltransferase in the diabetic rats. 4. These results are discussed in relation to the control of glycerolipid synthesis.

The influence of cyclic AMP analogues and fatty acids on glycerolipid biosynthesis in monolayer cultures of rat hepatocytes was investigated. Chlorophenylthio-cyclic AMP and adenosine 3':5'-cyclic phosphorothioate inhibited the rate of triacylglycerol synthesis from [1(3)-3H]glycerol, and phosphatidylcholine synthesis from [Me-3H]-choline. Supplementation of the hepatocytes with palmitate (1 mM) reversed chlorophenylthio-cyclic AMP inhibition of triacylglycerol synthesis. Similarly, cyclic AMP analogue-inhibition of phosphatidylcholine synthesis was abolished when the cells were simultaneously incubated with oleate (3 mM). Reactivation of phosphatidylcholine synthesis in chlorophenylthio-cyclic AMP-supplemented cells with oleate was accompanied by conversion of CTP: phosphocholine cytidylyltransferase into the membrane-bound form, since these cells released the enzyme more slowly after treatment with digitonin. The opposing actions of cyclic AMP and fatty acids are discussed in relation to the regulation of glycerolipid biosynthesis during starvation, diabetes and stress.

Rats were treated for 5 days with benfluorex [1-(3-trifluoromethylphenyl)-2-[N-(2-benzoyloxyethyl)amino]propane] or with suspending medium (controls). They were then intubated with an acute intoxicating dose of ethanol or with glucose of equivalent energy content. Treatment of the control rats with ethanol specifically increases the hepatic activity of the soluble phosphatidate phosphohydrolase by about 5-fold in 6 h. The equivalent increase for the benfluorex-treated rats were about 2-fold. The results are discussed in relation to the effects of ethanol and benfluorex on glycerolipid synthesis.

1. The Vmax. activity of pyruvate kinase of sheep adipose tissue increased during tissue culture up to 48 h; the increase was blocked by actinomycin D (an inhibitor of transcription) and was promoted by insulin and antagonized by dexamethasone. 2. In contrast with their effects on pyruvate kinase, insulin and dexamethasone acted synergistically to increase the activity of glucose-6-phosphate dehydrogenase of sheep adipose tissue maintained in culture. 3. Insulin stimulated, whereas dexamethasone inhibited, glucose utilization by sheep adipose tissue maintained in culture; the two agents were mutually antagonistic, and their effects were prevented by actinomycin D. 4. Antimycin A (an inhibitor of the electron-transport chain) stimulated glucose uptake and lactate output by sheep adipose tissue in the presence of dexamethasone and insulin, suggesting that the effects of dexamethasone on glucose utilization by sheep adipose tissue were not due to an inhibition of glucose transport. 5. Comparison of these findings with previous studies on the endocrine control of hepatic pyruvate kinase shows that there are major differences in the control of these Vmax. activities in liver and adipose tissue. 6. Although glucocorticoid hormones inhibit glucose utilization themselves and can antagonize the stimulatory effects of insulin on glucose utilization in adipose tissue from both sheep and rats, there appear to be major differences in the sites of action of these hormones in the two species.

Palmitoyl-CoA synthetase activity in the microsomal fraction of rat liver was measured directly by palmitoyl-CoA production, and indirectly by converting the palmitoyl-CoA into palmitoylcarnitine under optimum conditions. Even in the latter system, palmitoyl-CoA accumulated. The rate of palmitoyl-CoA hydrolysis and the inhibition of palmitoyl-CoA synthetase by palmitoyl-CoA were each estimated to be less than 10% of the maximum rate of palmitoyl-CoA production. The concentration of palmitoyl-CoA present in the assay systems used for measuring palmitate esterification to glycerol phosphate and the activity of palmitoyl-CoA synthetase by using the carnitine-linked determination were measured. These concentrations were not altered by the addition of glycerol phosphate, or of carnitine plus carnitine palmitoyltransferase. The relationship between the activity of palmitoyl-CoA synthetase and the rate of glycerolipid synthesis was investigated. The latter activity was measured by using palmitoyl-CoA generated from palmitate, palmitoyl--AMP or palmitoylcarnitine. It is concluded that, at optimum substrate concentrations, the activity of glycerol phosphate acyltransferase is rate-limiting in the synthesis of phosphatidate by rat liver microsomal fractions. The implications of these results in the measurement of palmitoyl-CoA synthetase and in the control of glycerolipid synthesis are discussed.

1. The synthesis and secretion of glycerolipid by monolayer cultures of rat hepatocytes was measured by determining the incorporations of [3H]glycerol, [3H]oleate and [14C]choline and by the absolute concentration of triacylglycerol. 2. The presence of albumin in the medium stimulated the accumulation of lysophosphatidylcholine in the medium by 11-13-fold. 3. Cycloheximide did not significantly alter the accumulation of lysophosphatidylcholine. 4. This process was particularly sensitive to inhibition by chlorpromazine and verapamil, compared with the secretion of triacylglycerol and phosphatidylcholine. By contrast, it was relatively less sensitive to EGTA. 5. It is suggested that intracellular Ca2+ may be important in the production of lysophosphatidylcholine, which then accumulates in the medium by binding to albumin. In vivo this lysophosphatidycholine may be a means of delivering choline and polyunsaturated fatty acids to other organs.