This observation was made in the last funding period that mono-unsaturated fat did not protect against coronary artery atherosclerosis in spite of an improved LDL/HDL ration. Due to the public health significance of this observation, we propose to confirm and extend this observation with further studies, while attempting to define mechanisms through which the outcome occurs. Groups of African green monkeys will be fed four different dietary fats (saturated, mono-unsaturated, and n-6 and n-3 polyunsaturated) to modify LDL particle composition. Plasma LDL cholesterol concentrations will be balanced among dietary fat groups so that the effect of LDL composition on the extend of coronary artery atherosclerosis can be assessed directly. The spectrum of plasma lipoprotein responses will be measured in each of the animals in the study, and the extend of coronary artery atherosclerosis that develops over a four year period of diet-induction will also be measured so that any lipoprotein-atherosclerosis relationship, and dietary fatty acid effect thereon, can be identified. The main hypothesis to be tested in this proposal is that compositionally modified, cholesteryl oleate enriched low density lipoproteins promote atherosclerosis out of proportion of their number through a predisposition to promote cholesterol accumulation in the coronary arteries. To examine how compositionally modified LDL could promote atherogenesis, LDL from each diet group will be examined for their binding to arterial proteoglycans (PG) and effects on PG production by arterial smooth muscle cells. Substitution of dietary n-3 polyunsaturated fatty acids into the diet results in less cholesteryl oleate accumulation and secretion by liver, as well as an increased cholesterol 7alpha-hydroxylase (C7H) activity and reduced free cholesterol content of liver. The two liver cholesterol -metabolizing enzymes, acyl- CoA: cholesterol acyltransferase (ACAT) and C7H appear to be key regulatory sites for determining cholesterol balance across the liver. Study of the regulation of hepatic ACAT(s) and C7H will be carried out in sequential liver biopsies taken from monkeys fed different diets, and that in isolated monkey hepatocytes incubated with varying fatty acid types and cholesterol concentrations, and during isolated liver perfusion with livers isolated from animals from each of the different diet groups. Evidence is accumulating that there are multiple ACAT enzymes in liver, and we will identify and characterize the hepatic ACAT(s) to be sure that regulation of the pertinent enzyme is document. Finally, we propose to examine a transgenic mouse with very high LDL cholesterol levels a model in which to test the dietary fatty acid effects on LDL composition and atherosclerosis, since genetic modifications in ACAT and/or C7H, for example can eventually be tested in mice.
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