Acute inhibition of hepatic lipase and increase in plasma lipoproteins after alcohol intake

Chronic alcohol intake is associated with an increase in fasting plasma high density lipoproteins (HDL). To study alcohol's acute effects on plasma lipoproteins, we measured plasma lipoprotein concentrations and activities of postheparin plasma lipases in nine normolipemic males after ingestion of 40 g of ethanol (as whiskey). After alcohol there was no change in lipoprotein lipase activity but hepatic lipase was decreased to 67% of baseline at 6 hr. There were associated increases in HDL phospholipids (12 mg/dl) and cholesterol (10 mg/dl) resulting in prominence of larger, lipid-enriched HDL particles. Changes were most pronounced in the HDL3 and HDL2a subclasses. Very low density lipoprotein (VLDL) phospholipids and cholesterol were also increased by 13 and 9 mg/dl, respectively, with no significant change in triglycerides. Changes in lipoproteins and lipase were largely reversed 10 hr after alcohol intake. The transient increases in VLDL and HDL lipids after alcohol may result in part from acute inhibition of hepatic lipase activity. The results suggest a role of hepatic lipase in the catabolism of phospholipids of VLDL and possibly HDL.     

Lipoprotein lipase-mediated sequestration of long-chain polyunsaturated triacylglycerols in serum LDL from normal and hypothyroid rats

Rat serum VLDL, unlike human, contains significant proportions of triacylglycerols with polyunsaturated C20 and C22 fatty acids. Hypothyroidism in this species is characterized by low levels of serum VLDL, the accumulation of LDL, elevated levels of lipoprotein lipase and depressed hepatic lipase activity. The hypothyroid rat thus represents an interesting model in which to study hepatic VLDL metabolism and the substrate specificity of lipoprotein lipase. This report shows that serum IDL and LDL in both euthyroid and hypothyroid rats contain progressively enhanced proportions of triacylglycerols with polyunsaturated C20 and C22 fatty acids when compared to VLDL. Hypothyroidism resulted in a decrease in the proportion of 22:6 fatty acid within the serum VLDL triacylglycerols when compared to euthyroid VLDL. Lipolysis of VLDL from euthyroid rats in vitro using the perfused rat heart system resulted in increases or sequestration of triacylglycerols containing long-chain polyunsaturated fatty acids within the IDL fraction similar to those seen in vivo. It is concluded that lipoprotein lipase-mediated hydrolysis of VLDL triacylglycerols and the conversion of VLDL to IDL and LDL in the rat results in a progressive sequestration of the longer-chain polyunsaturated triacylglycerol molecular species with the IDL and LDL.     

Lipid class and molecular species interrelationships among plasma lipoproteins of type III and type IV hyperlipemic subjects

As a further appraisal of lipoprotein interconversion and equilibration of lipid components a detailed examination was made of the chemical class and molecular species interrelationships among the major fasting plasma lipoprotein fractions within each of six male Type III and Type IV hyperlipemic subjects subsisting on free choice diets. The lipoprotein fractions were prepared by conventional ultracentrifugation and the lipid class and molecular species composition of the corresponding lipoprotein fractions were determined by gas chromatography of the intact glycerol esters and ceramides. In general, each lipoprotein fraction possessed a well defined lipid class composition, which was characterized by a dramatically decreasing triacylglycerol and increasing phospholipid and cholesteryl ester content, when progressing from the very low (VLDL) to the low (LDL) and high (HDL) density lipoproteins, as already established for normolipemic subjects. Likewise, the LDL, and LDL₂ of the hyperlipemic subjects contained about two times higher proportion of total phospholipid as sphingomyelin than VLDL and HDL. Furthermore, the sphingomyelins of the HDL fraction contained about 30% more of the higher and 30% less of the lower molecular weight species than the sphingomyelins of the VLDL. Smaller differences were seen in the molecular species composition of the phosphatidylcholines, cholesteryl esters and triacylglycerols among the corresponding lipoproteins. In comparison to normolipemic subjects analyzed previously, the hyperlipemic subjects showed greater individual variability. Despite this variability the lipid class and molecular species composition in the hyperlipemic subjects was again incompatible with the hypothesis which postulates direct VLDL conversion into LDL and HDL under the influence of lipoprotein lipase and lecithin: cholesterol acyltransferase. The main differences between normolipemic and hyperlipemic plasma were found to reside in the number of the VLDL and LDL, lipoprotein particles and not in their chemical composition or physical structure, or in the apparent mechanism of their metabolic interconversion.     

Dietary supplementation with Pluronic L-81 modifies hepatic secretion of very low density lipoproteins in the rat

Supplementation of high fat/cholesterol-enriched diets with polyoxypropylene-polyoxyethylene copolymers containing 90% hydrophobic constituents has been found to impair enteric secretion of chylomicrons, lower plasma levels of very low density (VLDL) and low density (LDL) lipoprotein cholesterol and prevent diet-induced hypercholesterolemia and atherosclerosis. These agents are known to be absorbed from the gastrointestinal tract and excreted in bile. In order to determine whether dietary supplementation with this group of hydrophobic poloxalenes influences hepatic secretion of triglyceride-rich lipoproteins, groups of rats were maintained for 21-34 days on either standard chow, semisynthetic diet containing 10.0% safflower oil/1.0% cholesterol, or each of the above diets supplemented with the hydrophobic poloxalene Pluronic L-81. At the end of the feeding period, newly secreted hepatic VLDL were isolated from 2-hr recirculating liver perfusates, quantitated, and characterized. Compared to perfusions in chow-fed rats, perfusion experiments in rats fed the high fat/cholesterol-enriched semisynthetic diet revealed a 3.1-fold increased net hepatic VLDL secretion rate; enrichment of secretory VLDL in cholesteryl esters and in C18:2 core lipid fatty acids; and a shift in the size distribution of secretory VLDL towards larger particles. When the 0.5% Pluronic L-81 was included in the high fat/cholesterol-enriched semisynthetic diet, the net hepatic VLDL secretion rate fell significantly and the physicochemical properties of secretory VLDL in these rats were found to resemble those of chow-fed animals. Supplementation of the chow diet with L-81 resulted in a significant fall in the net hepatic VLDL secretion rate from that observed in rats fed chow alone. Compared to rats fed chow alone, perfusate VLDL from rats fed each of the other experimental diets contained markedly lower amounts of both apoB molecular weight variants, as analyzed by gradient gel electrophoresis and densitometric gel scanning. Since previous studies have demonstrated that VLDL are the major cholesterol transport lipoproteins following fat/cholesterol feeding; a precursor-product relationship exists between fat/cholesterol-induced hepatic VLDL and plasma VLDL; such particles are capable of delivering cholesterol to the arterial wall; and dietary supplementation with hydrophobic poloxalenes prevents both the increase in plasma VLDL-cholesterol and diet-induced atherosclerosis, it is possible that dietary supplementation with hydrophobic poloxalenes may influence the atherogenic process through direct and/or indirect effects on hepatic VLDL transport.     

Intestinal lipoprotein synthesis in control and hypercholesterolemic rats

Previous studies in our laboratory have shown that very-low-density lipoproteins (VLDL) synthesized by the intestine of the diet-induced hypercholesterolemic rat are enriched in cholesteryl esters and unesterified cholesterol compared with intestinal VLDL from control rats. In these studies, we isolated and characterized nascent intestinal Golgi intermediate-density lipoproteins (IDL, d 1.006-1.040 g/ml) and studied isotope incorporation into apoliproteins of Golgi VLDL from control and hypercholesterolemic rats. IDL were triacylglycerol-rich lipoproteins but contained more cholesteryl ester and protein than the corresponding Golgi VLDL fractions. IDL from hypercholesterolemic rats were enriched in cholesteryl esters to a greater extent than IDL from control rats. The apolipoprotein patterns of IDL fractions were the same as those of intestinal Golgi VLDL, consisting of apolipoproteins (apo) B-48, A-I and A-IV. Time-course isotope incorporation curves for apo A-I and A-IV in Golgi VLDL were similar, but they differed from curves for apo B-48. None of these curves was markedly altered in the hypercholesterolemic rat. We conclude that the major effect of increased dietary cholesterol on intestinal lipoprotein biosynthesis is to increase the percentage of cholesteryl esters in Golgi lipoproteins. Dietary cholesterol does not alter the apolipoprotein composition of Golgi lipoproteins, nor does it have a significant effect on the pattern of isotope incorporation into apolipoproteins of Golgi VLDL. The effect of cholesteryl ester enrichment on the subsequent metabolism of these particles in the circulation and the effect of these particles on hepatic lipoprotein production remain to be determined.     

Isolation and characterization of lipoprotein of density less than 1.006 g/ml from rat hepatic lymph

The lipoprotein composition of rat hepatic lymph was studied using an animal model. The hepatic lymph duct of the adult male rat was cannulated and the hepatic lymph was collected. Hepatic lymph contains less than 1% of the total triglyceride output from the liver. Agarose gel electrophoresis of hepatic lymph showed the presence of two major lipoproteins bands, the alpha-migrating HDL and a band moving between plasma beta and pre-beta bands. Lipoprotein of density p less than 1.006 g/ml was then isolated by ultracentrifugation and it was found to correspond to the slow-moving pre-beta band. There was no difference in the mean diameter of hepatic lymph VLDL (64.4 nm) and that of plasma VLDL (64.6 nm). Compared with plasma VLDL, hepatic lymph VLDL has significantly more phospholipid (40% by weight), a higher cholesterol/cholesteryl ester ratio, and a marked reduction in triglyceride content (40% by weight). Although both plasma VLDL and hepatic lymph VLDL have apoE and apoB as the major apolipoproteins, there are other marked differences in apolipoprotein composition. Hepatic lymph VLDL has significantly less apoC and the apoB of hepatic lymph VLDL is predominantly the apoB240k (mol wt 240,000), with a small amount of the apoB330k (mol wt 335,000). On the other hand, plasma VLDL has an equal proportion of both apoB240k and apoB330k. This study presents for the first time the lipid and protein composition of rat hepatic lymph VLDL. Furthermore it has provided evidence that the hepatic lymph duct-cannulated rat can be used as an in vivo model for studying the secretion of nascent hepatic lymph VLDL.     

Structural and compositional changes in very low density lipoprotein triacylglycerols during basal lipolysis.

Triacylglycerols secreted by liver and carried by very low density lipoprotein (VLDL) are hydrolysed in circulation by lipoprotein and hepatic lipases. These enzymes have been shown to have positional and fatty acid specificity in vitro. If there were specificity in basal lipolysis in vivo, triacylglycerol compositions of circulating and newly secreted VLDL would be different. To study this we compared the composition of normal fasting VLDL triacylglycerol of Wistar rats to that obtained after blocking lipolysis by Triton WR1339, which increased plasma VLDL triacylglycerol concentration about 4.7-fold in 2 h. Analyses of molecular species of sn-1,2- and sn-2,3-diacylglycerol moieties and stereospecific triacylglycerol analysis revealed major differences between the groups in the VLDL triacylglycerol composition. In nontreated rats, the proportion of 16:0 was higher and that of 18:2n-6 lower in the sn-1 position. The proportion of 14:0 was lower in all positions and that of 18:0 was lower in the sn-1 and sn-3 positions in nontreated rats whereas the proportions of 20:4n-6, 20:5n-3, 22:5n-3 and 22:6n-3 were higher in the sn-1 and lower in the sn-2 position. These results suggest that the fatty acid of the sn-1 position is the most decisive factor in determining the sensitivity for hydrolysis of the triacylglycerol. In addition, triacylglycerol species with highly unsaturated fatty acids in the sn-2 position also favoured hydrolysis. The in vivo substrate specificity followed only partly that obtained in in vitro studies indicating that the nature of molecular association of fatty acids in natural triacylglycerol affects its susceptibility to lipolysis. To conclude, our results indicate that preferential basal lipolysis leads to major structural differences between circulating and newly secreted VLDL triacylglycerol. These differences extend beyond those anticipated from analysis of total fatty acids and constitute a previously unrecognized feature of VLDL triacylglycerol metabolism.     

Essential fatty acid deficiency in mice is associated with hepatic steatosis and secretion of large VLDL particles

Essential fatty acid (EFA) deficiency in mice decreases plasma triglyceride (TG) concentrations and increases hepatic TG content. We evaluated in vivo and in vitro whether decreased hepatic secretion of TG-rich very low-density lipoprotein (VLDL) contributes to this consequence of EFA deficiency. EFA deficiency was induced in mice by feeding an EFA-deficient (EFAD) diet for 8 wk. Hepatic VLDL secretion was quantified in fasted EFAD and EFA-sufficient (EFAS) mice using the Triton WR-1339 method. In cultured hepatocytes from EFAD and EFAS mice, VLDL secretion into medium was measured by quantifying [(3)H]-labeled glycerol incorporation into TG and phospholipids. Hepatic expression of genes involved in VLDL synthesis and clearance was measured, as were plasma activities of lipolytic enzymes. TG secretion rates were quantitatively similar in EFAD and EFAS mice in vivo and in primary hepatocytes from EFAD and EFAS mice in vitro. However, EFA deficiency increased the size of secreted VLDL particles, as determined by calculation of particle diameter, particle sizing by light scattering, and evaluation of the TG-to-apoB ratio. EFA deficiency did not inhibit hepatic lipase and lipoprotein lipase activities in plasma, but increased hepatic mRNA levels of apoAV and apoCII, both involved in control of lipolytic degradation of TG-rich lipoproteins. EFA deficiency does not affect hepatic TG secretion rate in mice, but increases the size of secreted VLDL particles. Present data suggest that hypotriglyceridemia during EFA deficiency is related to enhanced clearance of altered VLDL particles.     

Guinea pig very low density lipoproteins are a good substrate for lipoprotein lipase.

In contrast to plasma from most other animals, guinea pig plasma causes little or no stimulation of lipoprotein lipase activity. Very low density lipoproteins (VLDL) isolated by ultracentrifugation of guinea pig serum caused a definite stimulation of lipase activity, whereas the infranatant inhibited the activity. Gel filtration in 5 M guanidinium hydrochloride of delipidated VLDL demonstrated that the activation was caused by a low molecular weight protein. The VLDL themselves were hydrolized at similar rates as human VLDL both by guinea pig and by bovine lipoprotein lipases. Thus, guinea pig VLDL contain an activator for lipoprotein lipase analogous to that in other animals and there is enough of the activator to support rapid hydrolysis of the VLDL lipids by the lipase.     

Distribution of oleoyl-estrone in rat plasma lipoproteins.

Pooled adult normal rat plasma was used for the separation of lipoprotein fractions: VLDL, LDL and HDL, from which a total lipids extract was obtained. The presence of fragments with the MW of estrone and oleoyl-estrone in the lipoprotein fractions was analyzed by HPLC-MS. The results show that oleoyl-estrone is the major estrone component in lipoproteins; this molecular species was present in all three lipoprotein lipid extracts. The lipoprotein fractions were used for the analysis of protein and lipid classes: triacylglycerols, total and esterified cholesterol and phospholipids as well as acyl-estrone. About half of the total acyl-estrone was in the HDL fraction and only about 10% in the VLDL fraction. HDLs contained about one molecule in 50 particles, LDLs one molecule per particle and VLDLs 15 molecules per particle, i.e. given their size, the larger lipoproteins contained more oleoyl-estrone than the HDLs. The distribution of this hormone suggests that oleoyl-estrone is lost with other lipids as the lipoproteins shrink. The results presented show that oleoyl-estrone is a molecule found naturally in rat lipoproteins in low concentrations - the lowest in HDLs - that are consistent with its postulated role in the control of body weight.     

Diurnal variations of plasma lipids, tissue and plasma lipoprotein lipase, and VLDL secretion rates in the rat. A model for studies of VLDL metabolism

Circadian rhythms of plasma lipids and lipoproteins, lipoprotein lipase activities and VLDL secretion rates were studied in fed and food-deprived (12 h) male rats after a light/dark synchronization of 14 days. In ad libitum fed rats, a circadian rhythm of plasma triacylglycerol, blood glucose and liver glycogen was clearly identified. A rhythm was also identified for plasma cholesterol, but not phospholipids. The peak of plasma triacylglycerol occurred 2 h after the beginning of the light period (7.00 a.m.), and the nadir, 2 h after the beginning of the dark period (7.00 p.m.). The differences of plasma triacylglycerol at these two circadian stages were even more pronounced in food-deprived rats and were confined to the very-low-density lipoprotein (VLDL) fraction. Plasma post-heparin and heart and muscle lipoprotein lipase activities were 50-100% higher at 7.00 p.m., the time when plasma triacylglycerol were lowest, as compared to 7.00 a.m. Plasma post-heparin hepatic lipase and adipose tissue lipoprotein lipase activities, in contrast, did not change. VLDL secretion rates were somewhat higher at 7.00 a.m. compared to 7.00 p.m., but this difference was not significant. It is concluded that physiological variation of heart and muscle lipoprotein lipase together with small differences of VLDL secretion rates are responsible for normal range oscillations of plasma VLDL triacylglycerol levels.     

Inhibition by chloroquine of the secretion of very low density lipoproteins by cultured rat hepatocytes

Cultured rat hepatocytes were incubated in medium containing 1.0 mM oleic acid. The incorporation of [3H]glycerol into cell-associated and medium triacylglycerols was measured after 2 h incubation. More than 95% of the secreted [3H]triacylglycerols were recovered in the very low density lipoprotein (VLDL) fraction (d less than 1.006). Chloroquine and other lysosomotropic amines promoted a marked decrease in [3H]triacylglycerol secretion from the hepatocytes while the synthesis was unaffected. At 50-200 microM final concentration, chloroquine inhibited secretion of triacylglycerols by 70-90% of the control. Similar results were obtained when the mass of secreted triacylglycerols was measured. Chloroquine caused decreased secretion of [3H]triacylglycerols after 15-30 min incubation and the inhibitory effect was completely reversible within 1-2 h after washout of chloroquine. The reduced triacylglycerol secretion was not due to increased reuptake of secreted lipoproteins or decreased protein synthesis caused by chloroquine. Electron microscopy of chloroquine-treated cells showed that the inhibition of VLDL secretion occurs at or prior to the level of the Golgi apparatus. These results suggest that chloroquine interferes with crucial steps in the secretory process and/or that lysosomal function could be essential for secretion of VLDL.     

Nascent very low density lipoproteins from rat hepatocytic Golgi fractions are enriched in phosphatidylethanolamine

The phospholipid composition of nascent very low density lipoproteins (VLDL) of rat hepatocytic Golgi fractions differs greatly from that of plasma VLDL. The phospholipids of nascent VLDL contain about four times more phosphatidylethanolamine (PE) than plasma VLDL, whereas plasma VLDL contain considerably more sphingomyelin. Thus, the ratio of PE to sphingomyelin differs by a factor of about 12 between nascent Golgi VLDL and circulating plasma VLDL. It is evident from these data that the PE/sphingomyelin ratio of VLDL can be used to estimate endosomal contamination of hepatocytic Golgi fractions.     

Selective hepatic enrichment of polyunsaturated phosphatidylcholines after intravenous administration of dimethylethanolamine in the rat

The content of polyunsaturated phosphatidylcholines (PCs) is one of the parameters which regulate membrane functions. Polyunsaturated PCs are preferentially synthesized in the liver by the microsomal enzyme phosphatidylethanolamine N-methyltransferase. The activity of this enzyme may be stimulated in vitro in isolated rat hepatocytes by supplementation with dimethylethanolamine (DME), the polar head group of the precursor of PC along this pathway. The aim of this study was to evaluate in vivo the effect of an intravenous infusion of DME in the rat on the hepatic phospholipid composition. Bile fistula rats were intravenously infused for 15 h with sodium taurocholate (1 mumol/kg per min), with or without the addition of 0.3 mg/kg per min of [14C]DME. The concentration per gram of wet liver of individual phospholipid classes, PC molecular species and of total triacylglycerols, as well as the distribution of radioactivity in liver phospholipids, in rat tissues and body fluids were analyzed. A significant (P less than 0.01) enrichment in PC was found in the liver of DME-infused rats with respect to controls. No differences in the other phospholipid classes were found. DME-infused rats showed a significant (P less than 0.01) decrease in the hepatic concentration of triacylglycerols. At HPLC analysis, the enrichment in PC in DME-infused rats was found to be selectively due to three molecular species (i.e., sn-1 stearoyl/sn-2 arachidonoyl, sn-1 stearoyl/sn-2 linoleoyl, sn-1 stearoyl/sn-2 docosahexanoyl molecular species). In agreement with quantitative data, more than 70% of hepatic radioactivity was recovered in polyunsaturated PC species, with the highest specific activity in the sn-1 stearoyl PCs. The specific activity of hepatic PC approximates that of phosphatidyldimethylethanolamine. This finding together with the effective incorporation of DME in PC suggests that this amino base is methylated after its incorporation into phosphatidyldimethylethanolamine, throughout the stimulation of hepatic N-methyltransferase activity. The selective hepatic enrichment with polyunsaturated PC species after DME infusion may offer a new experimental tool for studying hepatic membrane metabolism.     

Hepatic overexpression of sterol carrier protein-2 inhibits VLDL production and reciprocally enhances biliary lipid secretion

We examined in vivo a role for sterol carrier protein-2 (SCP-2) in the regulation of lipid secretion across the hepatic sinusoidal and canalicular membranes. Recombinant adenovirus Ad.rSCP2 was used to overexpress SCP-2 in livers of mice. We determined plasma, hepatic, and biliary lipid concentrations; hepatic fatty acid (FA) and cholesterol synthesis; hepatic and biliary phosphatidylcholine (PC) molecular species; and VLDL triglyceride production. In Ad.rSCP2 mice, there was marked inhibition of hepatic fatty acids and cholesterol synthesis to <62% of control mice. Hepatic triglyceride contents were decreased, while cholesterol and phospholipids concentrations were elevated in Ad.rSCP2 mice. Hepatic VLDL triglyceride production fell in Ad.rSCP2 mice to 39% of control values. As expected, biliary cholesterol, phospholipids, bile acids outputs, and biliary PC hydrophobic index were significantly increased in Ad.rSCP2 mice. These studies indicate that SCP-2 overexpression in the liver markedly inhibits lipid synthesis as well as VLDL production, and alters hepatic lipid contents. In contrast, SCP-2 increased biliary lipid secretion and the proportion of hydrophobic PC molecular species in bile. These effects suggest a key regulatory role for SCP-2 in hepatic lipid metabolism and the existence of a reciprocal relationship between the fluxes of lipids across the sinusoidal and canalicular membranes.     

Activation of dolichyl-phospho-mannose synthase by phospholipids

Dolichyl-phospho-mannose synthase, or GDPmannose:dolichyl-phosphate mannosyltransferase (EC 2.4.1.83), was solubilized from rat liver microsomes with 1.0% Nonidet P-40 and the enzyme was further purified by column chromatography on DEAE-cellulose in the presence of 0.1% Nonidet P-40. The purified enzyme preparation (880-fold over microsomes) was unstable in the presence of detergent and had no activity in the presence of Nonidet P-40, Triton X-100, octyl beta-glucoside, or deoxycholate. Detergent-free enzyme was active in the presence of phosphatidylethanolamine (PtdEtn) and in the presence of phospholipid mixtures of PtdEtn and phosphatidylcholine (PtdCho) when the molar proportion of PtdCho was 70% or less. The enzyme was inactive in the presence of PtdCho alone. Unsaturated species of PtdEtn have a tendency to destabilize membrane bilayers [Cullis, P. R. & de Kruijff, B. (1978) Biochim. Biophys. Acta 507, 207-218] and we have shown that dolichol promotes the destabilizing effect of PtdEtn on membranes composed of PtdCho and PtdEtn [Jensen, J. W. & Schutzbach, J. S. (1984) Biochemistry 23, 115-1119]. These results suggest that dolichyl-P-mannose synthase is optimally active in a phospholipid matrix that contains some component phospholipids that prefer non-bilayer structural organization in isolation. Heat-inactivation and sedimentation experiments demonstrated that the synthase associated with PtdEtn in the presence of dolichyl-P. The PtdEtn-reconstituted enzyme catalyzed the reversible transfer of mannose from GDP-mannose to dolichyl-P. The Km for GDP-mannose was found to be 0.69 microM and the apparent Km for dolichyl-P was 0.3 microM. GMP, GDP, and GTP inhibited mannosyl transfer 50% at concentrations of 16 microM, 1.3 microM and 3 microM respectively.     

Metabolism of two forms of apolipoprotein B of VLDL by rat liver

Apolipoprotein B (apoB) is composed of metabolically distinct fractions of higher molecular weight (apoBh) and lower molecular weight (apoBl). When 125I-labeled very low density lipoprotein (VLDL) prepared from recirculating liver perfusates was injected into rats, labeled apoBl was preferentially removed from the plasma and apoBh entered low density lipoprotein (LDL). The time-related movement of labeled apoBh into higher density fractions was independent of that of labeled apoBl. When 125I-labeled triglyceride-rich lipoprotein (TRL) prepared from sucrose-fed rats was incubated with plasma from rats injected with heparin and then studied in a recirculating liver perfusion, apoBl was preferentially removed compared to apoBh. Thus, the loss of apoBl of hepatic VLDL in vivo was similar to the loss of apoBl of lipase-treated TRL in vitro. In control perfusions where TRL was incubated with heat-treated postheparin plasma, not only was there less initial hepatic clearance of apoB but the early phase of preferential apoBl removal during 30 min of perfusion was not observed. ApoE removal from perfusates was the same whether or not the TRL had been treated with heparin-releasable lipases. Apoprotein degradation, as indicated by the appearance in the perfusate of labeled degradation products, occurred 30 min after the preferential phase of apoBl removal. These results suggest that hepatic clearance of VLDL and TRL remnants is favored by lipolysis and by the presence of apoBl on the particle that enhances their hepatic binding and degradation.     

Eritadenine-induced alterations of plasma lipoprotein lipid concentrations and phosphatidylcholine molecular species profile in rats fed cholesterol-free and cholesterol-enriched diets

The effects of dietary eritadenine on the concentration of plasma lipoprotein lipids and the molecular species profile of plasma lipoprotein phosphatidylcholine (PC) were investigated in rats fed cholesterol-free and cholesterol-enriched diets to obtain insights into the relationship between the changes in PC molecular species profile and the hypocholesterolemic action of eritadenine. The effect of eritadenine on the secretion rate of very low density lipoprotein (VLDL) from the liver was also estimated. Rats were fed the control or eritadenine-supplemented (50 mg/kg) diets with or without exogenous cholesterol for 14 d. Eritadenine supplementation significantly decreased the cholesterol of major plasma lipoproteins, high density lipoprotein and VLDL, in rats fed cholesterol-free and cholesterol-enriched diets, respectively. The ratio of PC to phosphatidylethanolamine, delta6-desaturase activity, and the ratio of arachidonic acid to linoleic acid in liver microsomes were markedly decreased by eritadenine irrespective of the presence or absence of exogenous cholesterol. Dietary eritadenine increased the proportion of 16:0-18:2 molecular species with a decrease in 18:0-20:4 in plasma lipoprotein PC in both rats fed cholesterol-free and cholesterol-enriched diets. Eritadenine did not depress the secretion rate of VLDL in rats fed a cholesterol-free diet containing a high level of choline. The results indicate that dietary eritadenine elicits its hypocholesterolemic action with modulations of the fatty acid and molecular species profiles of PC irrespective of the presence or absence of exogenous cholesterol. The eritadenine-induced alteration of PC molecular species profile is discussed in relation to the hypocholesterolemic action of eritadenine.     

Mass‐spectrometric characterization of phospholipids and their primary peroxidation products in rat cortical neurons during staurosporine‐induced apoptosis

The molecular diversity of phospholipids is essential for their structural and signaling functions in cell membranes. In the current work, we present, the results of mass spectrometric characterization of individual molecular species in major classes of phospholipids – phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), sphingomyelin (CerPCho), and cardiolipin (Ptd₂Gro) – and their oxidation products during apoptosis induced in neurons by staurosporine (STS). The diversity of molecular species of phospholipids in rat cortical neurons followed the order Ptd₂Gro > PtdEtn >> PtdCho >> PtdSer > PtdIns > CerPCho. The number of polyunsaturated oxidizable species decreased in the order Ptd₂Gro >> PtdEtn > PtdCho > PtdSer > PtdIns > CerPCho. Thus a relatively minor class of phospholipids, Ptd₂Gro, was represented in cortical neurons by the greatest variety of both total and peroxidizable molecular species. Quantitative fluorescence HPLC analysis employed to assess the oxidation of different classes of phospholipids in neuronal cells during intrinsic apoptosis induced by STS revealed that three anionic phospholipids – Ptd₂Gro >> PtdSer > PtdIns – underwent robust oxidation. No significant oxidation in the most dominant phospholipid classes – PtdCho and PtdEtn – was detected. MS‐studies revealed the presence of hydroxy‐, hydroperoxy‐ as well as hydroxy‐/hydroperoxy‐species of Ptd₂Gro, PtdSer, and PtdIns. Experiments in model systems where total cortex Ptd₂Gro and PtdSer fractions were incubated in the presence of cytochrome c (cyt c) and H₂O₂, confirmed that molecular identities of the products formed were similar to the ones generated during STS‐induced neuronal apoptosis. The temporal sequence of biomarkers of STS‐induced apoptosis and phospholipid peroxidation combined with recently demonstrated redox catalytic properties of cyt c realized through its interactions with Ptd₂Gro and PtdSer suggest that cyt c acts as a catalyst of selective peroxidation of anionic phospholipids yielding Ptd₂Gro and PtdSer peroxidation products. These oxidation products participate in mitochondrial membrane permeability transition and in PtdSer externalization leading to recognition and uptake of apoptotic cells by professional phagocytes.     

Exchange of phospholipids between unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and plasma very low density lipoproteins.

Purified phosphatidylcholine exchange protein from bovine liver was used to exchange [14C]dipalmitoyl phosphatidylcholine from sonicated vesicles to human plasma very low density lipoproteins (VLDL). The exchange of [14C]-dipalmitoyl phosphatidylcholine for VLDL phospholipids was temperature dependent and linear with respect to time and amount of exchange protein. In the absence of the exchange protein, less than 10% of the [14C]dipalmitoyl phosphatidylcholine was transferred. At an initial weight ratio of [14C]-dipalmitoyl phosphatidylcholine vesicles to VLDL phospholipid (1.2 mg) of 2.2, the exchange protein (14 microgram) replaced 55% of the VLDL phospholipids with [14C]dipalmitoyl phosphatidylcholine in 15 min; VLDL protein and cholesterol content were unaltered. From these studies we conclude that the exchange protein is a useful method to alter the phospholipid composition of VLDL under conditions such that there is minimal perturbation of the lipoprotein.