Oleic Ester Research Articles

Conformationally constrained analogues of diacylglycerol. 10. Ultrapotent protein kinase C ligands based on a racemic 5-disubstituted tetrahydro-2-furanone template.

5,5-Bis(hydroxymethyl)tetrahydro-2-furanone and its isomer 4,4-bis(hydroxymethyl)tetrahydro-2-furanone were investigated as possible templates for the construction of conformationally constrained analogues of the biologically important second messenger, diacylglycerol (DAG). The former lactone contains embedded within its structure an exact glycerol moiety, while in the latter the ring oxygen has been transposed to the other side of the carbonyl group. All target compounds were synthesized as racemates from 1,3-dihydroxy-2-propanone. The 5,5-bis(hydroxymethyl)tetrahydro-2-furanone proved to be the better template for the construction of DAG surrogates that were demonstrated to have high binding affinities for the biological target, protein kinase C (PK-C). The simplest target compounds derived from this template (3e and 3f) have one of the hydroxyl moieties functionalized either as a myristate or as an oleate ester. The simplest target compound (9c) derived from the ineffective 4,4-bis-(hydroxymethyl)tetrahydro-2-furanone template was investigated only with a myristoyl acyl chain. Reducing the long acyl chain to an acetyl moiety and attaching a compensating lipophilic chain to the lactone ring as an alpha-alkylidene moiety produced compounds 10e and 10f (Z-isomers) and 11e and 11f (E-isomers), which were constructed on the more effective 5,5-bis(hydroxymethyl)tetrahydro-2-furanone template. Targets 14c (Z-isomer) and 15c (E-isomer) were derived, in turn, from 4,4-bis(hydroxymethyl)tetrahydro-2-furanone. The affinities of these ligands for PK-C were assessed in terms of their ability to displace bound [3H-20]phorbol 12,13-dibutyrate (PDBU) from the single isozyme PK-C alpha. The biological data support the hypothesis that the increase in binding affinity for PK-C shown by some of these constrained DAG mimetics appears to be entropic in nature. Two of the designed ligands (10e and 10f) showed the highest affinities (34 and 24 nM, respectively) reported so far for a DAG analogue. Assuming that the interaction between these racemic compounds and PK-C is stereospecific, the potency of the active enantiomer is anticipated to double.

Characterization of enzymatically prepared biosurfactants

AbstractVarious fatty monoesters of sugars and sugar alcohols were prepared enzymatically in organic solvent. Water produced during esterification was removed by refluxing through a dessicant under reduced pressure. Surface properties of these esters such as surface and interfacial tensions and their ability to stabilize emulsions at 30°C were evaluated: oleate esters of glucose, fructose, and sorbitol show similar behavior in reduction of surface and interfacial tensions, and values for the critical micelle concentration are about 8·10−5 M. It was also observed with sorbitol esters that the shorter the alkyl chain, the higher the critical micelle concentration. Generally, emulsions prepared with the emulsifier dissolved in the water or in the oil phase lead to oil‐in‐water or water‐in‐oil emulsions, respectively. Sorbitol monolaurate significantly increased the stability of oil‐in‐water emulsions, with only 5% separation of the phases after 48h at 30°C, compared to 10% for chemically prepared sorbitan monolaurate under the same conditions. Sorbitol monoerucate was very efficient in stabilizing water‐in‐oil emulsions, with only 1% separation of the phases.

On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety.

Combined treatment with trypsin, cholesterol esterase, and neuraminidase transforms LDL, but not HDL or VLDL, to particles with properties akin to those of lipid extracted from atherosclerotic lesions. Single or double enzyme modifications, or treatment with phospholipase C, or simple vortexing are ineffective. Triple enzyme treatment disrupts the ordered and uniform structure of LDL particles, and gives rise to the formation of inhomogeneous lipid droplets 10-200 nm in diameter with a pronounced net negative charge, but lacking significant amounts of oxidized lipid. Enzymatically modified LDL (E-LDL), but not oxidatively modified LDL (ox-LDL), is endowed with potent complement-activating capacity. As previously found for lipid isolated from atherosclerotic lesions, complement activation occurs to completion via the alternative pathway and is independent of antibody. E-LDL is rapidly taken up by human macrophages to an extent exceeding the uptake of acetylated LDL (ac-LDL) or oxidatively modified LDL. After 16 h, cholesteryl oleate ester formation induced by E-LDL (50 micrograms/ml cholesterol) was in the range of 6-10 nmol/mg protein compared with 3-6 nmol/mg induced by an equivalent amount of acetylated LDL. At this concentration, E-LDL was essentially devoid of direct cytotoxic effects. Competition experiments indicated that uptake of E-LDL was mediated in part by ox-LDL receptor(s). Thus, approximately 90% of 125I-ox-LDL degradation was inhibited by a 2-fold excess of unlabeled E-LDL. Uptake of 125I-LDL was not inhibited by E-LDL. We hypothesize that extracellular enzymatic modification may represent an important step linking subendothelial deposition of LDL to the initiation of atherosclerosis.

Regulation of VLDL secretion in primary culture of rat hepatocytes: involvement of cAMP and cAMP‐dependent protein kinases

When hepatocytes were cultured for 24 h in the presence of forskolin (10(-4) mol l-1) or isobutylmethylxanthine (IBMX, 10(-3) mol l-1), the intracellular cAMP concentration peaked (320-380 pmol mg-1 protein) after 10-20 min of culture. This increase was accompanied by a decrease in the secretion of triacylglycerol, cholesterol and apoprotein B associated with VLDL. After 4 h cAMP levels had returned almost to basal values but the inhibition of VLDL secretion persisted. There was a small intracellular accumulation of triacylglycerol but not of apoprotein B. Addition of forskolin and IBMX together led to a further increase in intracellular cAMP and a further suppression of VLDL output. Similar effects on the secretion of VLDL were also observed after addition of Bt2cAMP. Exposure of cell cultures to glucagon (10(-7) mol l-1) for only 10 min raised cellular cAMP levels to > 200 pmol mg-1 protein, and suppressed VLDL secretion during the next 24 h to < 40% of control. All of the substances tested inhibited de novo synthesis of fatty acids but had little or no effect on cholesterol synthesis and did not inhibit oleate esterification to triacylglycerol. The cAMP-dependent protein kinase antagonist Rp-cAMPS prevented suppression of VLDL triacylglycerol secretion induced by glucagon (10(-7) mol l-1) and abolished glucagon-induced ketogenesis. Rp-cAMPS also inhibited Bt2cAMP (7.5 x 10(-6) mol l-1)-induced suppression of VLDL secretion and enhancement of ketogenesis. It is concluded that rat hepatic VLDL metabolism can be regulated by cAMP and cAMP-dependent protein kinases, and that the initial transient rise in cellular cAMP levels induced by glucagon is sufficient to maintain a long-term inhibitory effect on assembly and secretion of VLDL.

Alveolar permeability enhancement by oleic acid and related fatty acids: evidence for a calcium-dependent mechanism.

Pulmonary exposure to oleic acid (OA) is associated with permeability alterations and cellular damage; however, the exact relationship between these two effects has not been clearly established. Using cultured alveolar epithelial monolayers, we demonstrated that OA and some other fatty acids (< or = 50 microM) can induce permeability changes without detectable cellular damage. At higher concentrations, however, OA caused severe membrane damage and leakage to solute flux. The permeability enhancing effect of OA was observed with both the paracellular marker 3H-mannitol and the lipophilic transcellular indicator 14C-progesterone. While the effect of OA on transcellular permeability may be attributed to its known effect on membrane fluidity, the paracellular promoting effect of OA and its mechanism are not well established. We postulated that OA may increase paracellular permeability through a Ca(2+)-dependent tight junction mechanism. Using dual-excitation fluorescence microscopy, we demonstrated that OA can increase intracellular calcium, [Ca2+]i, in a dose-dependent manner. This effect was transient at low OA concentrations (< or = 50 microM) but became more pronounced and sustained at higher concentrations. Free hydroxyl and unsaturated groups were required for this activation since esterified OA (oleic methyl ester) and stearic acid (a saturated fatty acid with equal chain length) had much reduced effects on both the [Ca2+]i and the permeability alterations. Degree of unsaturation was unimportant since linolenic acid (18:3), linoleic acid (18:2), and OA (18:1) had similar and comparable effects on the two parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of nonionic surfactant on enzymatic hydrolysis of used newspaper

Effects of five types of nonionic surfactant having a polyoxyethylene glycol (POG) group on enzymatic hydrolysis of used newspaper were studied. The surfactants examined in this work always enhanced the saccharification. The optimum surfactant concentration was 0.05% (wt/substrate wt) in the case of POG(21) sorbitane oleic ester. Among the surfactants, POG phenyl ether types showed the highest enhancement effect, for example, with two times higher conversion at 80 h than that without surfactant. Using POG nonylphenyl ether series, the effects of surfactant were considered from the point of the HLB (hydrophile-lypophile balance) value. The GFC (gel filtration chromatography) analysis of free enzyme quantity were also done to study the effect of surfactant on enzyme adsorption onto substrate. As the HLB value increased, the free enzyme quantity and the conversion both increased. It appears that surfactants help the enzyme to desorb from the binding site on the substrate surface after the completion of saccharification at that site.

Lipase-Catalyzed Synthesis and Hydrolysis of Cholesterol Oleate in Aot/Isooctane Microemulsions

We have examined a lipase-catalyzed bidirectional ester synthesis/hydrolysis reaction in a water-in-oil microemulsion system. The reactants were cholesterol (alcohol), oleic acid (acid) and cholesterol oleate (ester), and the solvent system consisted of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/water. The reactions were assayed by using [3H]oleic acid, [3H]cholesterol, or [3H]cholesterol oleate for the synthesis and hydrolysis reactions, respectively (separate incubations). The lipase that we used derived from Candida cylindracea, and was used at a concentration of 0.1mg/ml microemulsion. The reactions were performed at 22°C as the reactions proceeded more slowly at higher temperatures. With the initial reactant concentrations set to 10 mM cholesterol, 1 min oleic acid, and 1 mM cholesterol oleate, it was observed that the optimal [H2O]/[AOT] ratio was at about 9 both for the esterification reaction and for the hydrolysis reaction (after 24 h). The hydrolysis reaction was slower than the synt...

Regulation of fatty-acid metabolism by pancreatic hormones in cultured human hepatocytes

The effects of pancreatic hormones and cyclic AMP on long-chain fatty-acid metabolism were investigated in human hepatocytes isolated from 12 liver biopsy specimens and cultured for 4 days in an insulin-free medium. Glucagon (10(-6) mol/L) increased endogenous ketone body production by 150%. This resulted from alterations in the partition of long-chain fatty acids from esterification toward oxidation. Glucagon or cyclic AMP enhanced (14C) oleate oxidation (basal = 45.8% +/- 5.0%; glucagon = 66.8% +/- 5.3%; cyclic AMP = 67.6% +/- 5.0% of metabolized oleate) at the expense of oleate esterification. Insulin (10(-7) mol/L) antagonized the glucagon-induced oleate oxidation. After 24 hr in basal culture conditions, the rate of lipogenesis decreased to the same low rate as in glucagon-treated cells. The presence of insulin did not restore a high rate of lipogenesis. These results are the first direct evidence of a control of ketone body production by glucagon in the human liver.

α1-Adrenergic regulation of ketogenesis in isolated rat hepatocytes

Studies were conducted to clarify the effects of noradrenaline on oleate metabolism in isolated hepatocytes from fed rats. Noradrenaline caused an inhibition of ketogenesis from oleate along with a stimulation of glucose release through α 1-adrenergic receptors. Anti-ketogenic action of noradrenaline was confirmed by the suppression of the formation of radioactive acid-soluble products from [1- 14C]oleate in response to this agent. Noradrenaline increased the conversion of [1- 14C]oleate into 14CO 2 but failed to affect [1- 14C]oleate esterification. When hepatocytes were incubated in a medium containing 1 mM EGTA but no Ca 2+, the effects of noradrenaline on oleate oxidation were negated. On the other hand, noradrenaline-induced increase in glucose release remained unchanged even in the absence of Ca 2+ in the incubation medium. Decrease in ketogenesis and increase in glucose release produced by vasopressin was completely abolished by calcium depletion. Noradrenaline caused a significant increase in cAMP levels in both the presence and absence of Ca 2+, although the effect was more marked in the latter. Vasopressin did not affect it. The noradrenaline-induced increase in cAMP and glucose release in the absence of Ca 2+ was also mediated by α 1-adrenergic receptors. These data are discussed and it is suggested that α 1-adrenergic agonists may control hepatic ketogenesis and glycogenolysis through two separate signal transduction mechanisms, i.e., a calcium-mobilizing system which is common with vasopressin, and a cAMP generation system which vasopressin lacks.

Effects of Energy Balance on Hepatic Capacity for Oleate and Propionate Metabolism and Triglyceride Secretion

The purpose of this study was to identify conditions that could decrease accumulation of triglyceride in liver, preferably by increasing hepatic secretion of triglyceride-rich lipoproteins. Hepatocytes isolated from lactating goats were incubated in vitro, and the fate of [1-14C]oleate was measured to determine hepatic capacity for various routes of long-chain fatty acid metabolism. The effect of in vivo energy balance and modifications of the nutrients present in the culture media were tested. Addition of linoleic acid, isovalerate, niacin, propionate, or propylene glycol did not affect triglyceride accumulation or secretion. Pyruvate decreased intracellular triglyceride accumulation. Changes in oxidation of oleate through manipulation of carnitine acyl transferase activity did not influence oleate esterification rate.Livers and hepatocytes isolated from goats in negative energy balance contained more lipid and triglyceride. Liver cells from goats in negative energy balance had decreased capacity for converting propionate to glucose with no change in ketogenic capacity as judged by acid soluble product formation from oleate. Hepatocytes from goats in negative energy balance retained less oleate as cell triglyceride with no change in triglyceride export, indicating a decreased net rate of esterification. Lactating goats, either in negative or positive energy balance, demonstrated the same low capacity for export of newly synthesized triglyceride as previously repored for fed wethers.

Esterification of Glycosides with Glycerol and Trimethylolpropane Moieties by Candida cylidracea Lipase.

Glycosides, the reducing ends of which were glycerol and 2-ethyl-2-(hydroxymethyl)-1,3-propanediol (trimethylolpropane, TMP), were esterified with oleic acid (OA) by a lipase from Candida cylindracea. Two glycosides, 1-O-β-d-galactosyl glycerol (Gal-1-Gl) and O-β-d-glucosyl TMP (Glc-TMP), were enzymatically synthesized by transglycosylation. GIc-TMP was esterified at a higher water content than Gal-1-Gl. When an equal amount of the glycosides and OA were used as substrates, almost 20% and 50% of OA were consumed for the esterification of Gal-1-Gl and Glc-TMP, respectively. Laurie, capric, and caprylic acids also gave esters. Oleic ester synthesis was compared among several related glycosides. The enzyme esterified the glycosides with a TMP moiety better than those with a glycerol moiety. The reaction was scarcely affected not only by the kind of monosaccharide moieties but also by anomeric linkage types between the moieties.

Induction of ketogenesis and fatty acid oxidation by glucagon and cyclic AMP in cultured hepatocytes from rabbit fetuses. Evidence for a decreased sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition after glucagon or cyclic AMP treatment

The effects of pancreatic hormones and cyclic AMP on the induction of ketogenesis and long-chain fatty acid oxidation were studied in primary cultures of hepatocytes from fetal and newborn rabbits. Hepatocytes were cultivated during 4 days in the presence of glucagon (10(-6) M), forskolin (2 x 10(-5) M), dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-4) M) or insulin (10(-7) M). Ketogenesis and fatty acid metabolism were measured using [1-14C]oleate (0.5 mM). In hepatocytes from fetuses at term, the rate of ketogenesis remained very low during the 4 days of culture. In hepatocytes from 24-h-old newborn, the rate of ketogenesis was high during the first 48 h of culture and then rapidly decreased to reach a low value similar to that measured in cultured hepatocytes from term fetuses. A 48 h exposure to glucagon, forskolin or cyclic AMP derivatives is necessary to induce ketone body production in cultured fetal hepatocytes at a rate similar to that found in cultured hepatocytes from newborn rabbits. In fetal liver cells, the induction of ketogenesis by glucagon or cyclic AMP results from changes in the partitioning of long-chain fatty acid from esterification towards oxidation. Indeed, glucagon, forskolin and cyclic AMP enhance oleate oxidation (basal, 12.7 +/- 1.6; glucagon, 50.0 +/- 5.5; forskolin, 70.6 +/- 5.4; cyclic AMP, 77.5 +/- 3.4% of oleate metabolized) at the expense of oleate esterification. In cultured fetal hepatocytes, the rate of fatty acid oxidation in the presence of cyclic AMP is similar to the rate of oleate oxidation present at the time of plating (85.1 +/- 2.6% of oleate metabolized) in newborn rabbit hepatocytes. In hepatocytes from term fetuses, the presence of insulin antagonizes in a dose-dependent fashion the glucagon-induced oleate oxidation. Neither glucagon nor cyclic AMP affect the activity of carnitine palmitoyltransferase I (CPT I). The malonyl-CoA concentration inducing 50% inhibition of CPT I (IC50) is 14-fold higher in mitochondria isolated from cultured newborn hepatocytes (0.95 microM) compared with fetal hepatocytes (0.07 microM), indicating that the sensitivity of CPT I decreases markedly in the first 24 h after birth. The addition of glucagon or cyclic AMP into cultured fetal hepatocytes decreased by 80% and 90% respectively the sensitivity of CPT I to malonyl-CoA inhibition. In the presence of cyclic AMP, the sensitivity of CPT I to malonyl-CoA inhibition in cultured fetal hepatocytes is very similar to that measured in cultured hepatocytes from 24-h-old newborns.

Effects of exogenous phospholipase enzymes, arachidonic acid and 1-oleoyl-2-acetyl- sn-glycerol on ketogenesis in isolated rat hepatocytes

Studies were conducted to see whether exogenous phospholipase C from Clostridium perfringens, phospholipase A2 from Crotalus adamanteus venom, arachidonic acid and 1-oleoyl-2-acetyl-sn-glycerol (OAG) mimic the anti-ketogenic action of vasopressin in isolated rat hepatocytes. Exogenous phospholipase C inhibited ketogenesis in the presence of 0.5 mM oleate. Experiments employing [1-14C]oleate, however, indicated that the mechanism involved in the anti-ketogenic action of exogenous phospholipase C is distinct from that of vasopressin. The decreased rate of the production of acid-soluble products from [1-14C]oleate in response to vasopressin could be explained by the sum of the increased rates of 14CO2 formation and [1-14C]oleate esterification. By contrast, exogenous phospholipase C suppressed not only the formation of acid-soluble products but also 14CO2 production and [1-14C]oleate esterification. Indeed, phospholipase C greatly inhibited [1-14C]oleate uptake into hepatocytes. It is suggested that the alteration of the architecture of plasma membrane by exogenous phospholipase C may lead to the disturbance of oleate uptake and consequent general suppression of oleate metabolism. Exogenous phospholipase A2, arachidonic acid and OAG increased ketogenesis regardless of the presence of oleate. The ketogenic effects may be attributed to the supply of fatty acids by these agents to hepatocytes.

Inhibitors of sterol synthesis. 15-Oxygenated steryl ester hydrolase activity of rat liver at neutral and acid pH

5α-Cholest-8(14)-en-3β-ol-15-one (15 ketosterol) is a potent inhibitor of cholesterol biosynthesis with significant hypocholesterolemic activity. The results of a recent study (Schroepfer, G.J., Jr., Christophe, A., Chu, A.J., Izumi, A., Kisic, A. and Sherrill, B.C. (1988) Chem. Phys. Lipids 48, 29–58) have indicated that, after intragastric administration of the 15-ketosterol in triolein to rats, most of the compound in intestinal lymph occurs in the form of the oleate ester, which is associated with chylomicrons. Moreover, after intravenous administration of chylomicrons containing the oleate ester of 15-[2,4- 3H]ketosterol, rapid and selective uptake of 3H by liver was observed, which was associated with the rapid and substantial appearance of labeled free 15-ketosterol in liver. The present study concerns the capabilities of rat liver fractions to catalyze the hydrolysis of 15-ketosteryl oleate. Efficient hydrolysis was observed at acid pH with a digitonin-solubilized extract of rat liver, with a rate similar to that for the hydrolysis of cholesteryl oleate. The distribution of acid 15-ketosteryl oleate hydrolase of whole liver homogenate on a metrizamide isopycnic density gradient was similar to that of acid cholesteryl oleate hydrolase and acid phosphatase, suggesting that the lysosomal acid lipase is the enzyme responsible for the hydrolysis of the 15-ketosteryl oleate at acid pH. At neutral pH, 15-ketosteryl oleate and cholesteryl oleate were hydrolyzed at similar rates by the microsomal fraction of liver homogenate, whereas the 15-ketosteryl oleate was hydrolyzed at a much lower rate than cholesteryl oleate by the cytosolic fraction. The distribution of neutral 15-ketosteryl oleate hydrolase activity of whole liver homogenate on a metrizamide isopycnic density gradient was most correlated to a microsomal esterase, whereas cholesteryl oleate hydrolase activity was most correlated to a cytosolic enzyme. Both 15-ketosteryl oleate and cholesteryl oleate hydrolase activities were correlated to a mitochondrial marker enzyme.

A Comparison of the Fatty Acid Esters of Estradiol and Corticosterone Synthesized by tIssues of the Rat

The biosynthesis of the fatty acid esters of the corticoid (corticosterone) and estrogen (estradiol) was compared in parallel incubations of corticosterone and estradiol with several tissues of the rat. The fatty acid composition of the esters of the two steroids was characterized in mammary and uterine tissue. In both of these tissues, the esters of estradiol were extremely heterogeneous. To the contrary, in the same tissues only one predominant ester of corticosterone, corticosterone-21-oleate, was formed. It comprised 70-80% of the total. The oleate ester of estradiol accounted for only 20% of the esters of this estrogen. In addition, fatty acid esters of an A-ring reduced metabolite of corticosterone, 5 beta-dihydrocorticosterone, was also identified. Its fatty acid composition is identical to that of corticosterone. In other experiments the fatty acid esters of both steroids were isolated from several tissues and quantified. When the amount of steroidal ester formed was compared, there was over a 100-fold difference among the various tissues in the ratio of estradiol to corticosterone ester synthesized. Thus, the rate of synthesis of the fatty acid esters of each class of steroid varies dramatically from tissue to tissue, and their fatty acid composition differs markedly as well. If the same enzyme synthesized both the estrogen and corticoid esters, then it would be expected that the relative amount of both esters synthesized in various tissues should be constant and likewise that their composition should be the same. Since neither occurred, these results suggest that the enzyme which produces the C-17 fatty acid esters of the estrogens may be different from the one which synthesizes the C-21 esters of the corticoids. The existence of separate enzyme systems for the synthesis of the fatty acid esters of these steroid hormones opens the possibility of specific physiological controls of each of these unusual steroidal metabolites.

Inhibitors of sterol synthesis. Oleate ester of 5α-cholest-8(14)-en-3β-ol-15-one as a substrate for pancreatic cholesterol esterase

5α-Cholest-8(14)-en-3β-yl-15-one oleate (15-ketosteryl oleate), the oleate ester of a compound with the capacity to lower serum cholesterol, was effectively hydrolyzed by partially purified porcine pancreatic cholesterol esterase with an apparent K m of 0.28 ± 0.01 mM and a V max of 0.62 ± 0.01 μmol/min per mg protein compared to an apparent K m of 0.19 ± 0.02 mM and a V max of 0.37 ± 0.02 μmol/min per mg protein for cholesteryl oleate. The 15-ketosteryl oleate was also hydrolyzed by highly purified rat pancreatic cholesterol esterase with an apparent K m of 0.20 ± 0.01 mM and a V max of 86.7 ± 3.0 μmol/min per mg protein compared to an apparent K m of 0.43 ± 0.01 mM and a V max of 119.8 ± 2.6 μmol/min per mg protein for cholesteryl oleate. 15-Ketosteryl oleate is, therefore, a good substrate for pancreatic cholesterol esterase from either source. The 15-ketosterol is a weak competitive inhibitor of partially purified porcine pancreatic cholesterol esterase when cholesteryl oleate is the substrate.

Inhibitors of sterol synthesis. A major role of chylomicrons in the metabolism of 5α-cholest-8(14)-en-3β-ol-15-one in the rat

The metabolism of 5α-cholest-8(14)-en-3β-ol-15-one ( I), a potent regulator of cholesterol (Chol) metabolism which has significant hypocholesterolemic activity upon oral administration to animals, has been investigated in male rats. After intragastric administration of [2,4- 3H] I and [4- 14C]Chol in triolein to intestinal lymph duct-cannulated rats, most of the 3H of the lymph was associated with chylomicrons. Most of the 3H in the chylomicrons was associated with fatty acid esters of I and the oleate ester represented the major species of the esters of I. After intravenous injection of the isolated doubly-labeled chylomicrons to intact rats, rapid clearance of 3H and 14C from blood was observed which was associated with a rapid and selective uptake of 3H and 14C by liver. The rate of disappearance of 3H from blood and the rate of uptake of 3H by liver were similar, if not identical, to those for 14C. In contrast, the disappearance of 3H from the liver was much more rapid than that of 14C. Studies of the distribution of 3H in liver demonstrated rapid formation of free I and the formation of [ 3H]Chol. In addition, significant amounts of the 3H in liver were associated with polar materials, a finding which was not observed in the case of 14C. After intravenous administration of the doubly-labeled chylomicrons to bile duct-cannulated rats, very rapid and substantial metabolism of the administered 3H to polar biliary metabolites was observed. The bulk of the 3H not recovered in bile at 49 h after the injection of the labeled chylomicrons was recovered in blood and tissues and almost all (∼94%) of this material was associated with Chol and Chol esters. The combined results indicate an important role for chylomicrons in the overall metabolism of I. The selective delivery of I to liver as its oleate ester in chylomicrons (or, more probably, as chylomicron remnants) and the subsequent metabolism of the oleate ester of I in liver has important consequences with respect to the actions of I which are discussed herein.

Lipid Metabolism in Electroplax

The in vivo labeling of electrocyte lipids is followed after injection of radioactive glycerol and two fatty acids, oleate and arachidonate, into the electric organ of an elasmobranch (Discopyge tschudii). De novo synthesis of lipids and acyl-exchange reactions are operative in the electrocyte. The three precursors are preferentially incorporated into phosphatidylcholine, phosphatidylinositol, and triacylglycerols. The highest specific activities are attained by triacylglycerols and polyphosphoinositides. Electrocyte stacks from electric organ show an efficient and continuous esterification of oleate and arachidonate into lipids after several hours of incubation. Except for an apparently more active labeling of triacylglycerols, which is attributed to the larger availability of free fatty acid precursors under the in vitro experimental conditions, the pattern of lipid labeling is similar to that attained in vivo. 32P-labeled lipids are also steadily produced in electrocyte stacks (24 h of incubation with [32P]phosphate) using glucose as the sole exogenous source of energy. Polyphosphoinositides are the lipids preferentially labeled. The ability to sustain the labeling of lipids under in vitro conditions renders isolated electrocyte stacks an interesting model for future research on lipid involvement in cholinergic function.

Effects of phorbol esters, A23187 and vasopressin on oleate metabolism in isolated rat hepatocytes.

Studies were conducted to compare the metabolic effects of vasopressin, 4 beta-phorbol-12-myristate-13-acetate (PMA) and A23187 on ketogenesis and oleate metabolism in isolated hepatocytes from fed rats. Vasopressin inhibited the formation of acid-soluble products from [1-14C]oleate (0.25 mM, 0.5 mM and 1 mM), the inhibition being most marked at low (0.25 mM) concentration of oleate. Conversion of [1-14C]oleate into 14CO2 and esterified products was stimulated by vasopressin. The stimulatory effect of this hormone on 14CO2 production was most marked at high (1 mM) concentration of oleate, whereas that on [1-14C]oleate esterification was most marked at low (0.25 mM) concentration of oleate. These vasopressin actions were abolished when hepatocytes were incubated in the absence of calcium in the medium. Our results strongly suggest that both increase in esterification and increase in oxidation to CO2 contribute to the anti-ketogenic action of vasopressin when oleate is added as substrate, although the relative extent of their contribution varies according to the oleate concentration. The anti-ketogenic action of vasopressin was mimicked by PMA but not by A23187. PMA also caused a stimulation of [1-14C]oleate esterification although the effect was diminished at 1 mM [1-14C]oleate. A23187 failed to affect [1-14C]oleate esterification. The metabolic effects of PMA were elicited in the absence of extracellular calcium, too. Conversion of [1-14C]oleate into 14CO2 was only slightly increased by both PMA and A23187 when 1 mM [1-14C]oleate was added as substrate. The marked stimulatory effect of vasopressin on 14CO2 production from [1-14C]oleate was not reproduced even by the combination of PMA and A23187.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of sodium 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) on intermediary metabolism in isolated rat-liver cells

In hepatocytes isolated from meal-fed rats, sodium 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) decreased the rate of lipogenesis measured as incorporation of 3H from 3H 2O into glycerolipids and cholesterol. Moreover, POCA inhibited the oxidation of added oleate, whereas oleate esterification was stimulated. In hepatocytes from 24-hr-starved rats, inhibition of gluconeogenesis by POCA was observed only with gluconeogenic precursors which require pyruvate carboxylation. This inhibition was secondary to impaired oxidation of long-chain fatty acids by POCA. It is concluded that, in addition to its inhibition of long-chain fatty acid oxidation, POCA interferes with de novo synthesis of cholesterol and fatty acids. On the other hand, neither fatty acid esterification nor the conversion of oxaloacetate into glucose are affected by POCA.