M.T.R. Subbiah

The characteristics and metabolism of a genetically hypercholesterolemic strain of rats (RICO)

A genetically hypercholesterolemic strain of rats was selectively bred, starting from an ordinary albino mutant of Rattus norvegicus. The new strain was given the designation RICO, standing for rats with increased cholesterol. In these animals, hypercholesterolemia is established, in both sexes, one day after weaning, and it increases progressively thereafter. It is due to elevated concentrations of LDL- and HDL-cholesterol. As in the ordinary rat, the HDL fraction makes up the main part of the serum cholesterol in the RICO rat. Metabolic studies revealed that in the RICO strain the overall rate of hepatic cholesterol synthesis is accelerated, as a result of higher than normal activity of 3-hydroxy-3-methylglutaryl-CoA reductase. The activity of cholesterol-7 alpha-hydroxylase is decreased in RICO rats, indicating a lower than normal rate of cholesterol catabolism. No difference was found between RICO and ordinary rats with respect to fecal excretion of bile acids and cholesterol.

Cholestyramine treatment in early life of low-density lipoprotein receptor deficient Watanabe rabbits: decreased aortic cholesteryl ester accumulation and atherosclerosis in adult life.

Effect of cholestyramine treatment in early life of Watanabe heritable hyperlipidemic rabbits (an animal model lacking low-density lipoprotein receptor activity) on subsequent (6 months recovery) occurrence of natural atherosclerotic lesion and arterial cholesterol metabolism was investigated. Initial cholestyramine treatment decreased both plasma total cholesterol and HDL-cholesterol levels which normalized within 4 weeks after treatment was discontinued. At 9 months of age (age of occurrence of spontaneous atherosclerotic lesions), the extent of aortic atherosclerosis in cholestyramine pre-treated animals was modestly lower (P less than 0.05), as compared to controls, with a significant (P less than 0.05) decrease in aortic cholesteryl ester content. Furthermore, at the end of the recovery period aortic activity of acyl-CoA: cholesterol acyltransferase and neutral cholesterol esterase activity was significantly (P less than 0.05) lower in cholestyramine-pretreated animals. These studies show that early cholestyramine pre-treatment in a low-density lipoprotein receptor-deficient animal model causes persistent changes which might influence cholesteryl ester accumulation and atherogenesis in adult life, even after cholestyramine treatment is discontinued.

Persistent enhancement of bile acid synthesis in guinea pigs following stimulation of cholesterol catabolism in neonatal life.

Cholesterol catabolism to bile acids was stimulated in neonatal guinea pigs by feeding 1.11% cholestyramine (CT)-containing diet for 8 weeks. The animals were then switched to standard laboratory diet for an additional 4 weeks. At the end of the laboratory diet period: a) CT-pre-treated guinea pigs continued to excrete significantly higher (p less than 0.05) amounts of bile acids, b) the activity of hepatic 7 alpha-hydroxylase was significantly elevated (p less than 0.01) in CT-pre-treated animals, and c) isolated hepatocytes from CT-pre-treated guinea pigs secreted significantly higher (p less than 0.05) amounts of bile acid when compared to controls during a 4-hour incubation. These data provide biochemical support for our contention that stimulation of cholesterol catabolism during neonatal life can have effects that persist into adult life.

Abnormal bile acid pool and composition in neonates of spontaneously diabetic Wistar BB rats and its change during development.

Streptozotocin-induced diabetes during pregnancy in rats causes a decrease in primary bile acid pool in neonates. To rule out direct drug effect on the fetus as the basis for this change, studies of bile acid pool and composition at birth and during subsequent development was carried out in neonates of spontaneously diabetic Wistar BB rats and compared to control neonates. The cholic acid pool in neonates of diabetic rats was lower when compared to control neonates at birth. The pool of secondary bile acids was markedly increased in neonates of diabetic rats, with increases in lithocholic and 3 beta,12 alpha-dihydroxycholanoic acid. With age, the cholic acid pool of neonates from diabetic rats was increased and at 3 months of age it was actually higher than in control neonates. The pool of chenodeoxycholic at diabetes onset age was lower in neonates of diabetic rats. HDL-cholesterol was lower in neonates of diabetic rats at 1 week, but this reversed at 3 months of age. These studies firmly establish that neonates of diabetic rats have abnormal bile acid pool and composition at birth which changes to adult diabetic pattern with age.

Studies on aorta during development. II. Differences in ontogeny of the key enzymes involved in cholesteryl ester synthesis and hydrolysis in rabbit aorta.

It is well known that cholesteryl ester accumulation is dramatically increased in the atherosclerotic artery. The enzymes acyl-CoA: cholesterol acyltransferase (ACAT), acid cholesteryl esterase (ACE) and neutral cholesteryl esterase (NCE) may play key roles in the accumulation of cholesteryl esters in the arterial wall. However, very little is known regarding the developmental pattern of the key enzymes involved in cholesteryl ester synthesis and hydrolysis. The total activities of ACAT, ACE and NCE were measured by radioassay using liposomal substrates in rabbit aortic homogenates. Our results indicate that ACAT activity decreases as a quadratic function with age (P less than 0.05). ACAT activity (pmol/100 mg protein/min) decreased from a high value in the fetus at term (63.3 +/- 7.4) to gradually lower values with increasing age. On the other hand, ACE activity (pmol/mg protein/min) was low in the fetus at term, and changed as a quadratic function with age (P less than 0.05) increasing gradually to higher activities with age up to a maximum at 12 weeks then decreased at 21 weeks. NCE activity (pmol/mg protein/min) increased dramatically from a low value in the fetus at term (3.34 +/- 0.48) to a maximum value at 1.5 weeks (14.65 +/- 2.73) then decreased as a linear function with increasing age up to 21 weeks (P less than 0.05). Plasma total cholesterol (mg/dl) also increased sharply from the fetal value at term of 98.5 +/- 5.2 to a maximum value at 1.5 weeks of 666.4 +/- 33.4, then decreased as a quadratic function with increasing age up to 21 weeks (40.8 +/- 6.7) (P less than 0.05). The free cholesterol content (microgram/mg protein) of the aortic tissue was initially high in the fetus (24.8 +/- 5.9) then increased with age. Examination of the ratio of synthesis to hydrolysis of cholesteryl esters as an index of enzyme activity units demonstrated a very high index in the fetus of 6.1 that rapidly decreased with increasing age in the young adult rabbit down to a value of 0.4 by 21 weeks of age. Correlation coefficients between enzyme activities, plasma cholesterol levels and aortic cholesterol levels indicated (a) a positive correlation of NCE activity with plasma cholesterol, (b) a negative correlation of NCE and ACE with aortic-cholesteryl ester content, and (c) no significant correlation of ACAT activity with either plasma cholesterol or aortic cholesterol content, indicating other factors are involved.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of Testosterone and Oxandrolone on Thrombocyte Aggregation and Synthesis of Prostaglandins in Thrombocytes and Aorta of Atherosclerosis-susceptible Pigeons*

Effect of intramuscular administration (10 mg/kg/week) of testosterone and oxandrolone on a) thrombocyte aggregation and b) synthesis of prostaglandins from [14C]‐arachidonic acid in thrombocytes and aorta of atherosclerosis‐susceptible White Carneau pigeon was examined. Neither testosterone nor oxandrolone influenced collagen, ADP and arachidonic acid induced aggregation or the synthesis of prostaglandins in thrombocytes. However, both testosterone and oxandrolone stimulated (p < 0.05) the synthesis of 6‐keto‐PGF1α (stable product of prostacyclin) und PGE2 in aorta.

Fetal aortic cholesterol concentration and metabolism: relationship to plasma cholesterol and potential role of placental factors.

The relationship of fetal plasma cholesterol levels to aortic free and esterified cholesterol concentration was determined in the rabbit during gestation. Plasma cholesterol levels (mg/dl) in early fetuses were markedly high and decreased at term. While aortic free cholesterol content increased with fetal age, cholesteryl ester concentration did not change significantly and no pathological accumulation was evident. This occurred despite high fetal aortic cholesterol esterification activity noted in earlier studies. We evaluated the potential effect of rabbit placental extracts on lipid metabolism and cellular proliferation in fetal aortic explants to explore the role of placental factors in affecting lipid metabolism. Labeled [14C]oleate and [3H]thymidine were used to investigate the rates of incorporation of (a) oleate into lipids, and (b) thymidine into DNA, respectively. Placental extracts at term (but not from 22 days gestation) significantly decreased oleate incorporation (P less than 0.05) into cholesteryl esters, phospholipids and diglycerides. This effect of placental extracts was noted both in absence or presence of serum in the culture medium, and was predominantly found in fraction of Mr greater than 100,000. [3H]Thymidine incorporation (dpm/g protein) into DNA was significantly decreased (P less than 0.01) by placental extracts. These studies suggest that placental extracts contain factor(s) influencing fetal aortic lipid metabolism in culture.