Peter J. Dolphin

Inhibition of Atherosclerosis and Myocardial Lesions in the JCR:LA-cp Rat by β,β′-Tetramethylhexadecanedioic Acid (MEDICA 16)

Abstract Atherosclerosis-prone, insulin-resistant JCR:LA-cp male rats were treated from 6 weeks to 39 weeks of age with β,β′-tetramethylhexadecanedioic acid (MEDICA 16). Body weights were reduced (13%, P P P P P

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.

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.

Human plasma lecithin:cholesterol acyltransferase. Preparation and use of immobilized p-aminophenylarsenoxide as a catalytic site-directed covalent ligand in enzyme purification.

A method is described for the preparation of p-aminophenylarsenoxide-linked carboxymethyl (CM) Bio-Gel A and its use as a specific, catalytic site-directed affinity chromatography ligand in the final stages of the purification of human plasma lecithin:cholesterol acyltransferase (LCAT) (EC 2.3.1.43). Previous mechanistic studies by us demonstrated that phenylarsenoxide derivatives, which are highly specific for vicinal thiols, could inhibit LCAT via a covalent interaction with the sulphydryl groups of the two catalytic cysteine residues and that this inhibition could be rapidly and completely reversed upon addition of 2,3-dimercaptopropanesulphonic acid. The ligand was covalently linked to CM Bio-Gel A activated through an N-hydroxysuccinyl ester formed by N-hydroxysuccinimide and dicyclohexylcarbodiimide in dry dimethyl sulphoxide; 87% of the added p-aminophenylarsenoxide was coupled to the CM Bio-Gel A in 3 h at 25 degrees C giving 2.3 mg of p-aminophenylarsenoxide per ml of gel. Homogeneous LCAT free of apo A-I, apo E, apo D and albumin was obtained in an 11% yield and purified 15,013-fold overall. A 13-fold purification was obtained by chromatography upon p-aminophenylarsenoxide-CM Bio-Gel A. This method is a useful final step in LCAT purification and will prove valuable in the purification of other proteins and compounds containing vicinal thiols.

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.

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).

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.

Improvement of insulin sensitivity and cardiovascular outcomes in the JCR:LA-cp rat by D-fenfluramine

Summary Obese male rats of the JCR:LA-cp strain are insulin resistant, normoglycaemic, hypertriglyceridaemic, and atherosclerosis-prone. Such rats were treated from 6 to 39 weeks of age with 5 mg · kg–1· day–1 of d-fenfluramine. The treatment normalised food intake, after 20 weeks of age, to that of lean control animals. At 39 weeks, treated rats weighed about 650 g compared to 800 g for untreated cp/cp rats and 400 g for + / + controls. Fasting plasma glucose and triglyceride levels were not significantly affected; however, fasting insulin concentrations were lower and the size and volume density of the hyperplastic islets of Langerhans were markedly reduced. The severity of raised atherosclerotic lesions on the aortic arch was decreased by 39 % (p < 0.01). Concomitantly, the occurrence of mature, scarred ischaemic myocardial lesions was virtually abolished (p < 0.01). Severe food restriction of the obese rats to normalise body weights to those of lean controls reduced plasma insulin and triglyceride concentrations at 26 weeks of age, but without a significant reduction in the frequency of myocardial lesions. Rats (with established insulin resistance) were treated from 6 to 12 weeks of age with 2.5 mg · kg–1· day–1 of d-fenfluramine. Insulin-mediated glucose turnover during a euglycaemic insulin clamp was strongly increased (p < 0.05). Rats treated from 3 weeks of age (before development of the insulin resistance) showed a significant delay in the development of hyperinsulinaemia and a reduced postprandial increase in plasma insulin. In contrast, restriction of food to that consumed by rats treated with d-fenfluramine did not decrease post-absorptive hyperinsulinaemia. d-fenfluramine treatment markedly improved the maximum relaxant response of aortic rings to acetylcholine, indicating improvement of the defective endothelium-derived relaxation factor system. A matched-food restriction regimen had no effect on vascular relaxation. d-fenfluramine treatment thus improved insulin sensitivity and had anti-atherosclerotic and cardioprotective effects in the presence of continuing obesity and hyperlipidaemia. The results are consistent with the protection of the function and integrity of the vessel wall associated with a decreased hyperinsulinaemia. The results emphasise the importance of focussing treatment of the metabolic syndrome (obesity/insulin resistance/hyperlipidaemia) on improving insulin sensitivity and glycaemic control rather than on the simple normalisation of body weight. [Diabetologia (1998) 41: 380–389]

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.

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.