KC Hayes - US grants

Gallstones continue to be a major concern of affluent populations. Ample evidence suggests a dietary basis for the problem, i.e. highest prevalence in obese persons with a high-calorie, high-fat intake. Further, altered lipoprotein (LP) metabolism appears implicated and an increased triglyceride (TG) and cholesterol synthesis and VLDL secretion are commonly associated with the disease. It appears that disposition of hepatic cholesterol, originating from de novo synthesis vs that from exogenous (chylomicron remnants) or other lipoprotein cholesterol (LDL, HDL) returning to the liver, dictate bile lithogenicity. The latter depends on whether the liver secretes the cholesterol in bile as free biliary cholesterol (BC) or converts it to bile acids (BAs) via 7-Alpha-OHase. Although reports continue to describe these relationships, a cohesive link between diet, LP metabolism, and bile lipid secretion has not been made in a systematic manner in the same model. The hamster has been widely studied as an effective model of cholelithiasis due to the similarity of its bile lipid and BA patterns with human bile. In the proposed studies we will define specific interrelationships between dietary fats (2 saturated, 2 unsaturated) and cholesterol and their interaction on relevant aspects of lipid and LP metabolism on lithogenic bile formation. To this end, a semipurified diet will be developed for Syrian hamsters with dietary cholesterol and estrogen to induce gallstone formation under physiological dietary conditions (i.e., normal growth). Utilizing variations of this diet we will characterize bile lipids, plasma LPs, VLDL secretion rate, relative rates of TG and PL synthesis, and the relative synthesis and incorporation of newly-synthesized vs LP cholesterol into biliary cholesterol and BAs. From these studies we hope to perfect hamster cholelithiasis to generate an ideal animal model for resolution of mechanisms of cholelithiasis as it relates to individual dietary components and their interactions. Specifically, what role do EFAs play in regulating bile lithogenicity and what is the nature of the genetic variant in this process that distinguishes hypo- vs hyperresponder hamsters in their susceptibility to gallstone formation?

The objective of these studies is to understand the mechanism and implications of tocopherol, cholesterol, and phospholipid delivery to the retina as factors contributing to the pathogenesis of various retinal degenerations, such as retinitis pigmentosa, retrolental fibroplasia, vitamin E deficiency. Accordingly the specific aims are 1) to determine the nature of the retinal pigment epithelial (RPE) cell lipoprotein receptor activity 2) to assess LP uptake by RPE using LPs from a variety of physiological and nutritional circumstances 3) to determine the correlates (apoprotein and lipids) of preferred tocopherol transport by lipoproteins 4) to measure the cholesterol synthesis in RPE as affected by LP uptake. The hypothesis to be tested is that tocopherol and EFA transport across the blood brain barrier (e.g., the RPE) depends on adequate receptor activity and appropriately "charged" LPs. To this end we will study cultured bovine and human RPE to measure normal receptor binding and uptake of normal LPs (chylo remnants, VLDL, LDL, HDL) and debris (photoreceptor outersegments, damaged LDL) on both apical and basilar surfaces of RPE. This will be followed by studies of LPs from infants (cord blood) and adults (normo and hyperlipemic, patients with RP) or animals fed different dietary fats (saturated vs PUFA), cholesterol, and with or without vitamin E deficiency. In this way we will generate a wide variety of LPs to examine the physiology of LP uptake and lipid delivery to the retina on the assumption that the results will enhance our understanding RPE transport of lipids and associated retinal lipid metabolism.

Cholesterol gallstones are a serious and expensive complication of the Western diet, commonly occurring in overweight women consuming too many calories and expressing a high plasma lipid, hyperinsulinemic profile. An elevated HDL2 pool seems conducive to gallstone induction for reasons that are unclear. The hamster also develops which the same metabolic profile, including an exaggerated HDL2 level. Thus, chronic feeding (postprandial condition) seems critical to biliary cholesterol saturation. Accordingly this research will investigate the nutritional aspects of this phenomenon by investigating diet-induced modulations in hamster biliary secretion. The specific aims will be to identify the normal circadian flux (fed/fast cycle) of newly synthesized vs. performed cholesterol into biliary lipids in the hamster fed purified diets; to determine how this flux is altered by specific dietary components manipulated to enhance gallstone formation; and to examine specific lipoproteins contribution to that sterol flux in hamsters and compare these results to similar data to be generated in the human hepatoma cell line (HEP G2 cells) as a potential model for investigating mechanisms of lithogenesis. Methods will include mass measure of cholesterol synthesis with 3H2O, assay of biliary lipids collected by bile fistulas, in addition to the lithogenic index, and gallstones in gallbladder bile and in nascent biliary secretions. An index of hepatic cholesterol metabolism will be based on mRNA abundance for key apoliproteins, AI, E, B and LDL receptor. Collectively these data will provide new insights on the mechanisms of diet-induced gallstone formation.

The overall objective of these studies is to clearly define the role of dietary fat (fatty acids) in the regulation of plasma lipoproteins and platelet sensitivity to aggregation, i.e., the two key elements initiating atherogenesis. Although saturated fatty acids are known to be the major dietary factor causing elevation in the plasma cholesterol concentration, the specific contribution of each saturated fatty acid is controversial. Furthermore, our preliminary studies suggest that the contribution of each fatty acid varies depending on the balance of other fatty acids present. How this interplay of fatty acids affects platelet aggregation is only superficially explored to date. To address these issues we have established three specific aims that will be applied in experimental model systems including whole animals (gerbils, monkeys) and tissue culture (hepatocytes). First, gerbils and monkeys will be fed various blends of fats to determine the impact of specific fatty acids on lipoprotein (LDL, HDL) kinetics, including cholesteryl ester transfer activity between lipoproteins. Second, this regulatory control by fatty acids will be further documented in a tightly controlled in vitro system with cultured hepatocytes during incubation with lipoproteins from monkeys or gerbils fed specific fat blends or with albumin-bound fatty acids, either as single fatty acids or blends of fatty acids that mimic those in the diet. The third aim will establish the effect of dietary fatty acids on platelets exposed to collagen ex vivo. By manipulating the source of platelets and plasma in cross-over experiments, we will separate the individual impact of platelet membrane fatty acid changes (affecting eicosanoid metabolism) from plasma lipoprotein-induced modulation of platelet aggregation (LDL/HDL) as influenced by the fatty acids consumed. In the final analysis we would suggest that these carefully designed experiments in highly selective models will provide the most in-depth scrutiny of the dietary (fatty acid) impact on lipoprotein metabolism and platelet aggregation ever undertaken in well-controlled nutritionally- defined experiments.