Secretion of cholesterol-rich lipoproteins by perfused livers of hypercholesterolemic rats.

Rats maintained on a high-fat diet supplemented with propylthiouracil develop a hypercholesterolemia, an increased serum level of apolipoprotein (apo) E, abnormal very low density lipoproteins (VLDL) and low density lipoproteins (LDL), and a fatty liver which contains cholesterol ester as its major lipid. The fatty liver secretes apoE into a recirculating perfusate at a significantly higher rate and produces cholesterol ester-rich, apoC-deficient VLDL with slower electrophoretic mobility than the triacylglycerol-rich VLDL produced by perfused normal livers. LDL, secreted in significant quantities by the perfused fatty liver, but not by the normal liver, is also cholesterol rich and contains apoE as well as apoB. The incorporation of [(3)H]leucine into apoVLDL and apoLDL secreted by the livers of the hypercholesterolemic animals and the apoVLDL secreted by the normal liver corresponds to the pattern visualized when the apoproteins are separated by polyacrylamide gel electrophoresis. Similar patterns are noted when non-recirculating perfusates are studied. These results indicate that the cholesterol ester-rich, apoC-deficient VLDL and the apoE-containing LDL found in the serum of hypercholesterolemic rats are not solely catabolic remnants of VLDL and chylomicrons but are secreted by the liver. Separation of the perfusate lipoproteins by agarose gel filtration revealed that most of the apoE secreted by the livers of hypercholesterolemic rats is found in the VLDL and LDL, whereas apoE secreted by the normal livers is distributed equally between VLDL, high density lipoproteins, and a low molecular weight fraction which corresponds to the virtually delipidated apoprotein. Thus the distribution of apoE among the lipoprotein fractions may be related to the total amount of cholesterol being transported in the circulation.

Secretion of nascent lipoproteins by isolated hepatocytes from hypothyroid and hypothyroid, hypercholesterolemic rats.

The induction of hypothyroidism in the rat is necessary for the development of pronounced dietary-induced hypercholesterolemia. The nature of nascent lipoproteins secreted by isolated hepatocytes from euthyroid, hypothyroid and hypothyroid, cholesterol-fed rats was investigated to distinguish between these hormonal and dietary effects. Serum total lipids, apolipoproteins, B, E and A-I, were greatly elevated in hypercholesterolemia. In hypothyroidism, serum apolipoproteins B and E were elevated, triacylglycerols were reduced by 65% and free cholesterol was increased by 50%. The total lipid, apolipoprotein B and E, secreted by hypercholesterolemic rat hepatocytes was markedly elevated when compared to normal. Triacylglycerol and phospholipid secretion was slightly increased by hypothyroid rat hepatocytes; however, apolipoprotein B, E and A-I secretion rates were unaffected. Gel filtration of the nascent lipoproteins demonstrated that compared to normal, proportionately more apolipoprotein B and E from hypercholesterolemic rat hepatocytes and apolipoprotein E from hypothyroid rat hepatocytes was secreted as larger lipoproteins. Hypercholesterolemic rat hepatocytes secreted abnormal cholesterol-rich particles even after 24 h of incubation in a lipid-deficient medium. Hypothyroidism alone cannot account for this observation, as hypothyroid rat hepatocytes secreted a triacylglycerol-rich, cholesterol-deficient lipoprotein having a normal nascent lipoprotein lipid composition. These observations are consistent with the hypothesis that in hypothyroidism the accumulation of beta-migrating lipoproteins results from impaired removal of lipoprotein catabolites from the serum, a condition which would only promote hypercholesterolemia in cholesterol feeding where direct synthesis of abnormal lipoproteins occurs.

Human plasma lecithin:cholesterol acyltransferase. On the substrate efficiency of cholest-5-ene-3 beta-thiol as a fatty acyl acceptor.

Lecithin:cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyses cholesteryl ester formation from lecithin and cholesterol present in the surface of plasma lipoproteins. Sterol fatty acid acceptors have previously been shown to require the presence of a trans conformation of the A/B ring and a 3 beta-OH group. Our laboratory has, however, demonstrated that two thiol sites within LCAT can become fatty acylated following lecithin cleavage although this does not appear to be essential for catalysis. In order to assess the ability of LCAT to donate a fatty acid derived from the sn-2 position of lecithin and present as an acyl enzyme intermediate (linked via an oxyester bond to Ser-181) to a sulfhydryl residue, we evaluated the ability of cholest-5-ene-3 beta-thiol to act as a substrate for cholesterol ester formation by LCAT. Thiocholesterol was a good terminal fatty acyl acceptor when incorporated into synthetic proteoliposomes containing lecithin/thiocholesterol/apo A-I in the molar ratios of 250:15:0.8. The Km for thiocholesterol was 203.6 microM with a Vmax of 5.3 nmol thiocholesteryl ester formed/h per microgram. The Km for cholesterol when substituted for thiocholesterol in the proteoliposomes was 29.5 microM with a Vmax of 8.8 nmol cholesteryl ester formed/h per microgram. Thiocholesterol and cholesterol were shown to occupy the same catalytic site in LCAT. Thus, thiocholesterol exhibits approx. 10% of the substrate efficiency of cholesterol when incubated with pure human LCAT. We conclude that LCAT can transacylate a fatty acyl moiety from the sn-2 position of lecithin to the 3 beta-SH group of thiocholesterol forming a cholesteryl thioester. Although the 3 beta-SH group is not as good a terminal acceptor as the 3 beta-OH group of cholesterol, LCAT is clearly capable of transacylating a fatty acid esterified via an oxyester linkage to one containing a thioester.

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.

Aromatic boronic acids as probes of the catalytic site of human plasma lecithin-cholesterol acyltransferase.

We have recently proposed a catalytic mechanism for human plasma lecithin-cholesterol acyltransferase (EC 2.3.1.43) (J. Biol. Chem. (1986) 261, 7032-7043), implicating single serine and histidine residues in phosphatidylcholine cleavage and two cysteine residues in cholesterol esterification. We now confirm the involvement of serine and histidine in catalysing the phospholipase A2 action of lecithin-cholesterol acyltransferase by demonstrating the inhibition of this activity by phenylboronic acid (Ki = 1.23 mM) and m-aminophenylboronic acid (Ki = 2.32 mM), inhibitors of known serine/histidine hydrolases. The specificity of the interaction of aromatic boronic acids with catalytic serine and histidine residues and the putative formation of a tetrahedral adduct between boron and the lecithin-cholesterol acyltransferase serine hydroxyl group which is similar to the transition-state intermediate formed between phosphatidylcholine and the catalytic serine residue was suggested by: substrate protection against inhibition by phenylboronic acids; a much reduced incorporation of phenylmethane[35S]sulphonyl fluoride into the enzyme in the presence of phenylboronic acid; the lack of interaction of histidine- or serine-modified enzyme with immobilized phenylboronic acid in the presence of glycerol (Ve/Vo = 2.7 and 2.3 respectively) when compared to the native enzyme (Ve/Vo = 5.25). Fatty acyl-lecithin-cholesterol acyltransferase, produced by incubation of the enzyme with a lecithin-apolipoprotein A-I proteoliposome substrate, was not retarded upon the sorbent column (Ve/Vo = 1.5). Modification of the enzyme's two free cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) or potassium ferricyanide reduced (Ve/Vo = 3.5) but did not abolish retardation on the sorbent column, indicating that these modifications resulted in steric hinderance of the interaction of the boron atom with the lecithin-cholesterol acyltransferase serine hydroxyl group. These data suggest that the serine and histidine residues are proximal within the enzyme catalytic site and that both cysteine thiol groups are close to the serine hydroxyl group. The presence of significant amino-acid sequence homologies between lecithin-cholesterol acyltransferase, triacylglycerol lipases and the transacylases of fatty acid synthase is also reported.

Sexual dimorphism in the metabolic response to the calcium channel antagonists, diltiazem and clentiazem, by hyperlipidemic JCR:LA-cp rats.

The JCR:LA-cp rat is obese, insulin resistant, and hypertriglyceridemic. The obese male rats spontaneously develop atherosclerosis and ischemic myocardial lesions that are prevented by treatment with the calcium channel antagonist, nifedipine. Male and female JCR:LA-cp rats were treated with the calcium channel antagonist, diltiazem, and a closely related compound, clentiazem (at 30 mg/kg). Clentiazem, but not diltiazem, caused a significant increase in body weight of both sexes in the presence of decreased food consumption. Serum triacylglycerols were decreased by half by both drugs in male rats only, reflecting decreased very-low-density lipoprotein (VLDL) secretion. Females did not respond with lower concentrations of triacylglycerol (although VLDL secretion rate was decreased) and showed increased concentrations of cholesterol in the high-density lipoprotein (HDL) fraction. Diltiazem-treated male rats showed decreased VLDL particle size, together with a shift to shorter-chain fatty acids in the triacylglycerols. This effect was not seen with clentiazem treatment. There was no effect on insulin and glucose metabolism in these insulin-resistant animals. Calcium channel antagonists have complex metabolic effects in the hypertriglyceridemic rats, with highly beneficial hypolipidemic effects in the males that are not seen in the females. The sexual dimorphism of these responses is sex linked, but appears not to be due to the steroid sex hormones. These results suggest caution in the chronic treatment of human females with these agents and the importance of detailed human studies in females and individuals with the insulin-resistant/hypertriglyceridemic/obese syndrome.

Accumulation of cholestatic lipoproteins in ANIT-treated human apolipoprotein A-I transgenic rats is diminished through dose-dependent apolipoprotein A-I activation of LCAT.

Administration of alpha-naphthylisothiocyanate (ANIT) to rats induces changes to plasma lipids consistent with cholestasis. We have previously shown (J. Lipid Res. 37 (1996) 1086) that animals treated with ANIT accumulate large amounts of free cholesterol (FC) and phospholipid (PL)-rich cholestatic lipoproteins in the LDL density range by 48 h. This lipid was cleared by 120 h through apparent movement into HDL with concomitant cholesteryl ester (CE) production. It was hypothesised that the clearance was mediated through the movement of the PL and FC into apolipoprotein A-I (apo A-I) containing lipoproteins followed by LCAT esterification to form CE. To test this hypothesis, rats overexpressing various amounts of human apo A-I (TgR[HuAI] rats) were treated with ANIT (100 mg/kg) and the effect of plasma apo A-I concentration on plasma lipids and lipoprotein distribution was examined. In untreated TgR[HuAI] rats, human apo A-I levels were strongly correlated to plasma PL (r(2)=0. 94), FC (r(2)=0.93) and CE (r(2)=0.90), whereas in ANIT-treated TgR[HuAI] rats, human apo A-I levels were most strongly correlated to CE levels (r(2)=0.80) and an increased CE/FC ratio (r(2)=0.62) and the movement of cholestatic lipid in the LDL to HDL. Since LCAT activity was not affected by ANIT treatment, these results demonstrate that the ability of LCAT to esterify the plasma FC present in cholestatic liver disease is limited by in vivo apo A-I activation of the cholestatic lipid and not by the catalytic capacity of LCAT.

Post-secretory acquisition of apolipoprotein E by nascent rat hepatic very-low-density lipoproteins in the absence of cholesteryl ester transfer.

Previous work has shown that nascent hepatic very-low-density lipoproteins (VLDL) in the rat are biosynthesized without the obligatory co-factor (apolipoprotein C-II) for lipoprotein lipase-mediated hydrolysis of their core triacylglycerols. Upon secretion, apolipoproteins C-II and C-III are rapidly transferred to the particles from high-density lipoprotein (HDL) within the space of Disse and upon the entry into the plasma. Here we extend those studies to include observations on the apolipoprotein E content and lipid composition of nascent hepatic VLDL before and after exposure to plasma components. We have elected to use hepatic secretory vesicle VLDL rather than liver perfusate VLDL as truly representative of the nascent lipoproteins. Nascent VLDL from fed rats has an apolipoprotein B/E ratio of 6.6 +/- 0.5, whereas that from fasted animals is 13.9 +/- 2.3. Incubation of nascent VLDL from fed and fasted rats with d greater than 1.063 g/ml rat serum, HDL or the d greater than 1.21 g/ml fraction resulted in a mass transfer of apolipoprotein E to the VLDL such that the apolipoprotein B/E ratio decreased to at least that of serum VLDL (3.4 +/- 0.3). The d greater than 1.21 g/ml fraction appeared to contain a species of apolipoprotein E which most actively transferred to VLDL. The acquisition of apolipoprotein E by nascent secretory vesicle VLDL was attended by a loss of phospholipids, particularly the C40 (stearoylarachidonyl) molecular species, and an increase in the cholesterol-to-phospholipid ratio from 0.11 +/- 0.01 to 0.18 +/- 0.03. No evidence was obtained to suggest a simultaneous acquisition of cholesteryl esters upon incubation of nascent VLDL with VLDL-free serum. We conclude that nascent hepatic VLDL is modified after secretion by acquisition of apolipoproteins C-II, C-III and E with a concomitant loss of phospholipids.

Serum lipids and lipoproteins in the atherosclerosis prone LA/N corpulent rat.

The LA/N rat is one of two congenic strains bred from the original obese, hyperphagic and hypertensive rats of Koletsky. With the exception of hypertension the LA/N strain, when homozygous for the corpulent gene, is phenotypically similar to the parent Koletsky strain and prone to the development of vascular and myocardial lesions. Here we report a detailed analysis of the serum lipids, lipoproteins and apolipoproteins B, E and A-I levels in young adult homozygous corpulent (cp/cp) rats of both sexes and in lean males of the same age which were demonstrable non-carriers (+/+) of the cp gene. Both male and female cp/cp rats were hypertriglyceridemic (282-512 mg/100 ml) and moderately hypercholesterolemic (74-84 mg/100 ml). Elevations in these lipids reflected the presence of large (622 A), triacylglycerol-rich and apoprotein-poor VLDL containing both apolipoproteins Bh and B1 and increased phospholipid-rich HDL. Similar, but less pronounced, elevations in serum apolipoproteins B and E in the cp/cp rats when compared to the +/+ animals were also noted. Apolipoproteins A-I levels were 2.7-3-fold higher in cp/cp rats. The levels of VLDL were significantly higher in female cp/cp rats; however, the levels of IDL (intermediate-density lipoproteins), LDL and HDL were significantly lower than in the more atherosclerosis prone male cp/cp rats. Similarly, apolipoprotein A-I was higher and apolipoprotein B lower in the male cp/cp than in the female cp/cp rats. The LDL (d = 1.030-1.063 g/ml) in cp/cp rats, like that in normal animals, was heterogeneous and contained apolipoproteins Bh, E, A-I and C. This fraction was significantly elevated in male cp/cp rats when compared to females but still represented less than 13% of the total serum cholesterol and less than 6% of the total serum lipids in 3-month-old cp/cp animals. The ratio of cholesterol to phospholipids was significantly lower for all lipoproteins in cp/cp rats when compared to +/+ males and these ratios for female cp/cp rats were in all cases lower than those of male cp/cp animals.

Effect of age on serum lipids and lipoproteins of male and female JCR:LA-corpulent rats.

The JCR:LA-cp rat is a strain incorporating the corpulent (cp) gene. When homozygous for the cp gene, the rats are hyperphagous, hyperinsulinemic, hyperlipidemic and obese. The corpulent male rats develop atherosclerotic and myocardial lesions from an early age, while corpulent female and lean rats do not develop lesions. The hyperlipidemia is due to elevated levels of VLDL resulting in moderately raised cholesterol levels and markedly elevated triacylglycerol levels. The VLDL concentrations are similar in corpulent male and female rats at an early age with both having much higher levels than lean rats. As the animals age, the VLDL hyperlipidemia in the corpulent male increases at 3 months and then decreases slowly and rises again at 12 months of age. The corpulent female rats show higher triacylglycerol and phospholipid concentrations than the males at 3 months age and reach values over 1000 mg/100 ml by 9 months of age, then decrease at 12 months of age. The cholesterol concentrations of the corpulent females are greater than those of the males from 9 months of age. Thus, in the period of life up to middle age, the cardiovascular disease incidence does not correlate with the degree of hyperlipidemia. The disease progression does correlate with the severity of insulin resistance and glucose intolerance, which is more severe in the corpulent male than female rats. The results suggest that the hyperlipidemia must be a necessary condition for development of atherosclerotic disease in this strain of rats, but it is not a sufficient condition.

Reduction of hyperlipidemia in the LA/N-corpulent rat by dietary fish oil containing n-3 fatty acids.

The LA/N rat, when homozygous for the corpulent gene (cp/cp), is obese, hyperphageous, hyperinsulinemic, hypertriglyceridemic and prone to the development of vascular and myocardial lesions. The hypertriglyceridemia, which in 3-month-old cp/cp males is 282 +/- 42 mg/dl and in females, 512 +/- 83 mg/dl, results from the presence of a large triacylglycerol-rich VLDL. The moderate hypercholesterolemia in these animals is largely due to markedly elevated HDL levels, which reach 172 +/- 21 mg total lipid/dl in males and 154 +/- 22 mg total lipid/dl in females. The LA/N-cp rat is thus an interesting animal model of endogenous hypertriglyceridemia in which to examine the hypolipidemic effects of pharmacological agents and also dietary oil supplements containing the n-3 fatty acids. In this study, 1-month-old male and female cp/cp rats were fed a normal low fat laboratory chow supplemented with either 10% olive oil or 10% redfish (Sebastes marinus) oil ad libitum for a period of 2 months. The redfish oil contained 4.9 +/- 0.1% of its total fatty acids as eicosapentaenoic (20:5(n-3)) and 2.3 +/- 0.5% as docosahexaenoic acid (22:6(n-3)), the predominant fatty acids being gondoic (20:1(n-3)), 21.9 +/- 0.9% and cetoleic acid (22:1(n-11)), 21.7 +/- 1.7%, which are of dietary origin. Daily caloric intake was similar to the oil-fed versus control rats. However, the oil-fed animals weighed significantly more than the controls after 2 months of oil supplementation. Redfish oil reduced serum triacylglycerols by 54% in males and 45% in females after 2 months. VLDL levels, after the same time period, were reduced by 44% in males and 39% in females. HDL lipid mass was significantly reduced in both sexes (by 27% in males and 49% in females). However, the levels remained above those of male LA/N +/+ rats of the same age and Long-Evens rats. Olive oil feeding significantly reduced serum cholesterol, triacyglycerols and phospholipids in male but only cholesterol and phospholipids in female animals. This oil had no significant effect upon VLDL total lipid levels in either sex, but significantly increased the particle diameter with a concomitant reduction in the cholesterol and phospholipid content. HDL total lipid levels were unaffected: However, HDL total cholesterol increased significantly in males only. Both oils markedly reduced serum LDL levels in both sexes.(ABSTRACT TRUNCATED AT 400 WORDS)

An evaluation of the marmoset Callithrix jacchus (sagüi) as an experimental model for the dyslipoproteinemia of human Schistosomiasis mansoni.

Human infection with the parasite Schistosoma mansoni is a relatively common occurrence in regions of South America and is associated with liver dysfunction and dyslipoproteinemia. Specifically, the activity of plasma lecithin:cholesterol acyltransferase (LCAT) activity is reduced, the concentration of plasma cholesterol esters falls, phospholipid concentrations are elevated and erythrocyte membranes become cholesterol enriched. Previous studies have utilized rodents (rats and mice) as experimental models to study the dyslipoproteinemia induced by S. mansoni infection. However, the plasma lipoprotein profiles in these animals is very different from humans and infection is not accompanied by decreases in LCAT activity or cholesterol enrichment of their erythrocyte membranes. Here we have evaluated the suitability of the marmoset Callithrix jacchus (sagüi) which is small and readily available in Brazil, as a potential animal model for the study of the dyslipoproteinemia of S. mansoni infections. The plasma lipoprotein compositions and distributions in sagüi, unlike rats or mice, approximate those of man with the LDL representing a major lipoprotein species. The molecular species of phospholipids, cholesterol esters and triglycerides present in sagüi plasma are also very similar to man, whereas those of rats and mice favor the longer chain more unsaturated species, Sagüi, like rodents, can be successfully infected with S. mansoni and after 60 days, this results in a 50% reduction in plasma LCAT activity, an 11% reduction in plasma cholesterol esters, an absolute increase of 46% in plasma phospholipids and an 18% increase in the cholesterol content of erythrocyte membranes. These changes are qualitatively and quantitatively very similar to those previously reported following human infections. Based upon these changes, and the observation that the plasma lipoprotein profile of sagüi and human is similar, we conclude that C. jacchus (sagüi) is an appropriate animal model for the study of dyslipoproteinemia associated with S. mansoni infections.

Development of insulin resistance in the JCR:LA-cp rat: role of triacylglycerols and effects of MEDICA 16.

The JCR:LA-cp rat develops an extreme obese/insulin-resistant syndrome such that by 12 weeks of age, there is no longer any insulin-mediated glucose turnover. At 4 weeks of age, obese and lean rats have essentially identical basal and insulin-mediated glucose uptake in skeletal muscle. By 8 weeks of age, however, the obese rats no longer exhibit such intake. Plasma insulin concentrations in the normal fed state show only small increases up to 4 weeks, with a rapid rise to a marked hyperinsulinemia thereafter, with an age at half-development of 5.5 weeks. Plasma triacylglycerol concentrations in fed obese rats are elevated at 3 weeks and rise rapidly thereafter. The triacylglycerol content of skeletal muscle is significantly elevated in the obese rats at 4 weeks of age. Histological examination of Oil Red O-stained muscle tissue and transmission electron microscopy shows the presence of intracellular lipid droplets. Treatment with the potent triacylglycerol-lowering agent MEDICA 16 (beta,beta'-tetramethylhexadecanedioic acid) from 6 weeks of age reduces plasma lipids markedly, but it reduces body weight and insulin resistance only modestly. In contrast, treatment with MEDICA 16 from the time of weaning at 3 weeks of age results in the normalization of food intake and body weight to over 8 weeks of age. The development of hyperinsulinemia is also delayed until 8.5 weeks of age, and insulin levels remain strongly reduced. Plasma triacylglycerol concentrations remain at the same level as in lean rats, and neither an elevated muscle triacylglycerol content nor intracellular lipid droplets are found at 4 weeks of age. The results indicate that insulin resistance develops in the young animals and is not directly due to a genetically determined defect in insulin metabolism. The mechanism of induction instead appears to be related to an exaggerated triacylglycerol metabolism.

Familial lecithin:cholesterol acyltransferase deficiency: further resolution of lipoprotein particle heterogeneity in the low density interval.

Patients presenting with a familial deficiency of lecithin:cholesterol acyltransferase (LCAT) typically exhibit multiple quantitative and qualitative perturbations of apo B- and apo A-I-containing plasma lipoproteins. Marked particle heterogeneity has been detected over the low-density range (d = 1.019-1.063 g/ml), involving lipoprotein(X) (LP-X) and large molecular weight LDL (LM-LDL). We describe the chromatographic fractionation and characterization of the major particle species distributed within the low-density interval in a new French LCAT-deficient family. Detailed analyses of the plasma lipoprotein and apolipoprotein spectrum are reported. The plasma lipoproteins were enriched in unesterified cholesterol and phospholipids with markedly reduced concentrations of cholesteryl esters. By a combination of gel filtration and affinity chromatography on heparin-sepharose, the heterogeneous mixture of low-density particles was resolved into three distinct particle populations: LP-X (diameter 400 A) corresponding to LM-LDL, an apo A-I and albumin-containing particle similar to LP-X2 (diameter 300 A), and cholesteryl ester-deficient (0.9%) triglyceride-rich (58.4%) LDL containing apo B-100 (diameter 260-270 A). Use of affinity chromatography allowed separation of HDL-like particles (diameter 140-160 A) which were rich in free cholesterol (21.4%) and phospholipids (52.9%) and which were isolated in association with LP-X upon gel filtration chromatography. Ultracentrifugal density gradient analysis of plasma from the LCAT-deficient subject over a period of 3 years showed a net shift of the lipoprotein distribution in the low density range due to an increase in plasma LP-X levels. We propose that the presence of LP-X in the plasma is correlated with a progressive alteration in the renal function recently observed in this patient.

Effect of castration on hyperlipidemic, insulin resistant JCR:LA-corpulent rats.

The JCR:LA-cp rat exhibits an obese, insulin resistant, hyperlipidemic syndrome. Obese male rats, only, develop atherosclerosis and ischemic myocardial lesions. The obese males have a greater hyperinsulinemia, but the obese females have a much greater hypertriglyceridemia due to hypersecretion of very low density lipoprotein (VLDL). Obese rats of both sexes were surgically castrated at 6 weeks of age to study the influence of testosterone and estrogen secretion on the sexual dimorphism of metabolism and disease in this strain. Castration had no effect on body weight or food consumption up to 16 weeks of age. Castrated male rats had significantly improved glucose tolerance, but a doubled serum triglyceride concentration. Castrated female rats showed approximately halved triglyceride levels. The distribution of the triglyceride molecular species was altered in the castrated male rats to resemble that of the females in which there was no change with castration. The effects suggest that testosterone may inhibit hepatic triglyceride secretion and promotes insulin insensitivity. Estrogen appears to exacerbate hepatic hypersecretion of VLDL. Castration had no effect on myocardial lesion frequency in 9-month-old rats of either sex. This implies that estrogen does not exert a direct protective effect against cardiovascular disease in this animal model.

Familial lecithin:cholesterol acyltransferase deficiency: molecular analysis of a compound heterozygote: LCAT (Arg147 --> Trp) and LCAT (Tyr171 --> Stop).

Lecithin:cholesterol acyltransferase (LCAT) is responsible for the formation of the majority of plasma cholesteryl esters. Familial LCAT deficiency is associated with corneal opacity, anemia and proteinurea and typically results in renal failure in the 4-5th decade; this syndrome is equally characterized by the quasi-absence of plasma LCAT activity with variable enzyme mass and very low levels of plasma cholesteryl esters. In this study, we report detailed analyses of plasma lipids and lipoprotein profile in two sisters (CM and ML) presenting classical homozygous LCAT-deficiency; the younger sibling (CM) had proteinurea from an early age whereas the older sister (ML) has never exhibited renal dysfunction. We investigated the molecular defect in the 45 year-old woman (proband CM) exhibiting all clinical and biochemical features of familial LCAT deficiency: a plasma cholesterol level of 105 mg/dl, of which 95% was unesterified, an HDL-cholesterol of 6.5 mg/dl and an apo A-I level of 52 mg/dl. The proband (CM) displayed a plasma cholesterol esterification rate which corresponded to 2% of normal LCAT activity; plasma LCAT protein concentration was 0.56 microg/ml and equivalent to approximately 10% of normal LCAT mass. Analysis by single strand conformation polymorphism (SSCP) of the PCR products corresponding to exons 4 and 5 of the LCAT gene revealed a visible band shift. Sequence analyses of exons 4 + 5 revealed two separate single point mutations: a C --> T transition replacing Arg147 by Trp and a T --> G transition converting Tyr171 to a stop codon. The presence of these two point mutations was confirmed by restriction enzyme analyses: the C --> T transition abolished a MwoI site whereas the T --> G transition created an AvrII site. The Arg147 mutation was associated with a non-secreted protein. The Tyr171 mutation resulted in formation of a truncated protein lacking the catalytic site. In summary, we have identified an LCAT deficient patient corresponding to a compound heterozygote for the Arg147 --> Trp mutation and a new molecular defect involving a Tyr171 --> Stop mutation in the LCAT gene.

Atherogenesis in two strains of obese rats. The fatty Zucker and LA/N-corpulent.

Two strains of obese rats, the fatty Zucker and the LA/N-corpulent have been compared at 6 months age for the presence of vascular and myocardial disease. Both strains, when obese, exhibit a VLDL hyperlipidemia with elevated triglycerides and moderate elevations of plasma cholesterol concentrations compared to the lean rats of the same strain. The hyperlipidemia is more modest in the fatty Zucker than the corpulent LA/N, and the serum lipid concentrations of the lean Zucker are lower than those of the lean LA/N. Apolipoprotein concentrations were similar and elevated in the two obese genotypes compared to the lean genotypes which were also similar to each other. Male and female obese animals of both strains exhibited hyperinsulinemia under fasting conditions and after oral glucose, with obese male LA/N rats exhibiting the most severe hyperinsulinemia. Glucose tolerance was impaired in obese LA/N animals but was normal in lean rats of both strains and fatty Zucker rats of both sexes. The glucose intolerance observed in obese LA/N animals was more severe in the male than in the female rats. Unlike the corpulent rat, which develops atherosclerotic lesions, the fatty Zucker shows no evidence of advanced vascular lesions on scanning electron microscopy. The fatty Zucker also does not develop the myocardial lesions that are frequent in the male corpulent LA/N rat. It is suggested that the initiation of the atherogenic process is dependent upon elevated insulin levels or transient hyperglycemia. Development of the advanced lesions appears to require the presence of hyperlipidemia.

The lipoproteins of human umbilical cord blood apolipoprotein and lipid levels.

The levels of apolipoproteins B, E and A-1 and the molecular species of triacylglycerols, phospholipids and cholesteryl esters were individually quantitated by electroimmunoassay and gas chromatographic total lipid profiling in 50 fresh samples of umbilical cord sera obtained from full term, normal delivery, healthy human neonates. All samples were screened for IgA to eliminate those samples with maternal blood contamination. The whole serum apolipoprotein levels in mg/dl +/- SEM for all neonates were; Apo B = 25.4 +/- 1.2; Apo E = 5.0 +/- 0.3; Apo A-1 = 86.6 +/- 2.3. These values represented 25% and 60% of adult serum values for Apo B and A-1, respectively, with normal adult values for Apo E. Apo A-1 was higher (P less than 0.020) in sera from female when compared to male neonates. The whole serum lipid values in mg/dl +/- SEM for all neonates were: 20.8 +/- 2.0 for triacylglycerols; 74.2 +/- 2.6 for lecithin and sphingomyelin; 79.8 +/- 2.7 for cholesteryl esters and 20.4 +/- 0.8 for unesterified cholesterol. Phospholipids, cholesteryl esters and total cholesterol levels were higher (P less than 0.025) in sera from female neonates when compared to males. The proportion of unesterified cholesterol relative to cholesteryl esters was high in comparison to adult sera, however the total cholesterol to phospholipid ratios were similar. The molecular species of cord sera triacylglycerols indicated a decreased proportion of C16 fatty acids with increased C18 and C20 when compared to adult sera. The molecular species of cord sera phospholipids similarly contained a decreased proportion of C16/18 fatty acids with increased C18/20 or C16/22 fatty acid combinations when compared to adults. The cord sera cholesteryl esters contained a significantly higher proportion of cholesterol esterified to C16 fatty acids with decreased amounts of cholesterol esterified to C18 and C20 fatty acids when compared to adults. Good correlations were obtained between Apo B and total serum cholesterol (R = 0.77) and also between Apo B and total serum triacylglycerols (R = 0.78).

Classical LCAT deficiency resulting from a novel homozygous dinucleotide deletion in exon 4 of the human lecithin: cholesterol acyltransferase gene causing a frameshift and stop codon at residue 144.

Lecithin: cholesterolacyltransferase (LCAT) transacylates the fatty acid at the sn-2 position of lecithin to the 3beta-OH group of cholesterol forming lysolecithin and the majority of cholesteryl ester found in plasma. LCAT participates in the reverse cholesterol transport pathway in man where it esterifies tissue-derived cholesterol following efflux from peripheral cells into HDL. Only 38 unique mutations in the human LCAT gene have been reported worldwide. Our French female proband presented with corneal opacity and no detectable plasma LCAT activity using either endogenous or exogenous assays. Her total plasma cholesterol and HDL cholesterol were low (2.34 mmol/l and 0.184 mmol/l, respectively) with a very high cholesterol/cholesteryl ester molar ratio (10.9:1). Plasma triglycerides were 0.470 mmol/l with low apo B (40.5 mg/dl), apo A-I (14.7 mg/dl), apo A-II (6.8 mg/dl) and apo E (2.1 mg/dl) levels. Plasma lipoprotein analysis by ultracentrifugation showed very low HDL concentrations and a characteristic shift of the lipoprotein profile towards larger, less dense particles. No proteinuria, renal dysfunction or signs of atherosclerosis were noted at age 45. Sequence analysis of her LCAT gene showed a novel homozygous TG-deletion at residues 138-139 that resulted in a frameshift causing the generation of a stop codon and premature termination of the LCAT protein at amino acid residue 144. Western blotting of the patient's plasma using a polyclonal IgY primary antibody against human LCAT failed to demonstrate the presence of a truncated LCAT protein. A 53 bp mismatched PCR primer was designed to generate an Fsp 1 restriction site in the wild type sequence of exon 4 where the mutation occurred. The 155 bp PCR product from the wild type allele produced a 103 bp and 52 bp fragment with Fsp 1 and no cleavage products with the mutant allele thus permitting rapid screening for this novel mutation.

Antiatherogenic effects of long-term benfluorex treatment in male insulin resistant JCR:LA-cp rats.

The JCR:LA-corpulent rat is an animal model that, if homozygous for the cp gene (cp/cp), spontaneously exhibits obesity and a severe insulin resistance, with a resultant hyperinsulinemia and hypertriglyceridemia. The obese male rats show defective nitric oxide-mediated vascular relaxation, advanced atherosclerosis, and ischemic myocardial lesions. Benfluorex has both anorectic and metabolic effects that lower body weight and improve insulin sensitivity in obesity and type 2 diabetes. Male cp/cp rats that were treated with benfluorex (or pair-fed to the treated animals) from the time of weaning, at 3 weeks of age, showed a marked delay in the development of postprandial hyperinsulinemia. At 12 weeks of age benfluorex-treated cp/cp rats did not show the extreme insulin response to a test meal that was observed in untreated or pair-fed rats. Both benfluorex-treated and pair-fed rats had a significant increase in sensitivity to acetylcholine-induced (nitric oxide-mediated) vascular relaxation. Corpulent male rats were also treated from 6 to 39 weeks of age with benfluorex in the feed at a dose of approximately 36 mg/kg/day at 12 weeks of age and decreasing to 23 mg/kg/day at 39 weeks to determine the effects on cardiovascular outcomes. The rats showed a sustained decrease in food consumption and body weight, although they exhibited 50% of the excess body weight of the controls and were grossly obese. Both fasting insulin concentrations and the hyperplasia of the islets of Langerhans were decreased by approximately 50%. Serum triglyceride concentrations were decreased by 44%, and free cholesterol and cholesteryl esters by 30%. The severity of the atherosclerotic lesions on the aortic arch was decreased (P < 0.05). There was also a decrease in the size of early ischemic myocardial lesions that are characterized by cell lysis and chronic inflammatory cell infiltration. Mature, scarred myocardial lesions were essentially absent in the hearts of 39-week-old benfluorex-treated rats. Long-term major food restriction (18 g/day) decreased the body weights of obese rats to essentially those of lean control animals, with similar beneficial effects on the insulin resistance and hyperlipidemia. While myocardial lesion frequency was reduced in these much thinner animals, lesions remained and the apparent effect was not statistically significant. This evidence shows that the beneficial metabolic effects of benfluorex are associated with long-term effects on the vessel wall and delay the onset of insulin resistance and cardiovascular disease in an animal model.