In cultured human and rat cells, the lipolysis-stimulated receptor (LSR), when activated by free fatty acids (FFA), mediates the binding of apoprotein B- and apoprotein E-containing lipoproteins and their subsequent internalization and degradation. To better understand the physiological role of LSR, we developed a biochemical assay that optimizes both the activation and binding steps and, thus, allows the estimation of the number of LSR binding sites expressed in the livers of living animals. With this technique, a strong inverse correlation was found in rats between the apparent number of LSR binding sites in liver and the postprandial plasma triglyceride concentration (r = -0.828, p < 0.001, n = 12). No correlation existed between the number of LSR and plasma triglycerides measured in the same animals after 24 h of fasting. The same membrane binding assay was used to elucidate the mechanism by which FFA induce lipoprotein binding to LSR. The LSR activation step was mediated by direct interaction of FFA with LSR candidate proteins of apparent molecular masses of 115 and 90 kDa and occurred independently of the membrane lipid environment. The FFA-induced conformational shift that revealed the lipoprotein binding site remained fully reversible upon removal of the FFA. However, occupancy of the site by the apoprotein ligand stabilized the active form of LSR. Comparison of the effect of different FFA alone or in combination indicated that the same binding site is revealed by different FFA and that the length and saturation of the FFA monomeric carbon chain are critical in determining the potency of the FFA activating effect. We propose that the LSR pathway represents a limiting step for the cellular uptake of intestinally derived triglyceride-rich lipoproteins and speculate that FFA liberated by lipolysis initiate this process by altering the conformation of LSR to reveal the lipoprotein binding site.

The effect of increasing amounts of a cyclic oligosaccharide, beta-cyclodextrin (BCD), included in the diet on plasma cholesterol and triglycerides, was investigated in two animal models, namely in male genetically hypercholesterolemic Rico rats and in male Syrian hamsters. The distribution of bile acids in the gastrointestinal tract and in the feces of hamsters was also determined. In the Rico rats and hamsters, plasma cholesterol and triglycerides decreased linearly with increasing doses of BCD. In these two species, 20% BCD as compared to control diet lowered cholesterolemia (-35%) and triglyceridemia (-70%). In the hamster, the BCD diet caused a marked decrease in cholesterol and triglycerides in chylomicrons and very low density lipoprotein, and in high density lipoproteins cholesterol. Composition and amounts of bile acids were modified in the gastrointestinal tract of hamsters receiving 10% BCD as compared to the control group. The total bile acid content of the gallbladder of treated hamsters was fourfold higher than in the control group, and the bile contained a large amount of hydrophilic bile acids. This trend was also observed in the small intestine, in which percentages and total quantities of cholic plus deoxycholic acids (cholic pathway) were higher than those of chenodeoxycholic plus ursodeoxycholic plus lithocholic acids (chenodeoxycholic pathway). The bile acid contents of the cecum and colon of treated hamsters were 2.7-fold higher than those of control animals, but the bile acid composition was similar in the two groups of hamsters.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenovirus vector-mediated transfer of the receptor-associated protein (RAP) gene into low density lipoprotein (LDL) receptor-deficient mice was shown to achieve plasma concentrations ranging between 20 and 200 micrograms/ml and to result in the accumulation of remnant lipoproteins (Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J. (1994) Science 264, 1471-1474). Both this finding and the observation that in addition to various other members of the LDL receptor gene family, RAP binds to a yet unidentified protein of apparent molecular mass of 105 kDa prompted us to examine the effect of high concentrations of RAP on the lipolysis-stimulated receptor (LSR). LSR is a receptor distinct from the LDL receptor and the LDL receptor-related protein and is capable of binding apoB and apoE when activated by free fatty acids. Data reported here show that in fibroblasts isolated from a subject homozygous for familial hypercholesterolemia, RAP fusion protein inhibited LSR-mediated binding of 125I-LDL and the subsequent internalization and degradation of the particles. Studies on the interaction of RAP with LSR in isolated rat liver membranes revealed that at concentrations > or = 10 micrograms/ml, RAP inhibited in a dose-dependent manner the binding of LDL to LSR; half-maximum inhibition was obtained with 20 micrograms/ml RAP. Ligand blotting studies revealed that RAP bound directly to two rat liver membrane proteins of apparent molecular masses identical to those that bind 125I-LDL after preincubation with oleate. However, unlike LDL, binding of 125I-RAP to LSR did not require preincubation with oleate. Preincubation of nitrocellulose membranes with an excess of unlabeled RAP fusion protein decreased oleate-induced binding of 125I-LDL to LSR candidate proteins, whereas preincubation with excess unlabeled LDL was unable to prevent the subsequent binding of 125I-RAP to the LSR proteins. Both the latter data and analysis of the mechanism of inhibition were consistent with the RAP inhibitory effect on LSR being achieved by interference with a site distinct from the oleate-induced LDL binding site. In conclusion, this study shows that at concentrations reported to delay chylomicron remnant removal in LDL receptor-deficient mice, RAP exerted a significant inhibitory effect on LSR.

By using the isotopic equilibrium method in the young male Syrian hamster, the rates of cholesterol turnover processes, i.e. dietary cholesterol absorption, cholesterol synthesis, cholesterol excretion in the faeces and urine and cholesterol transformation into bile acids, were determined in the hamster receiving a control (C) or a lithogenic diet (L) for 7 weeks. At the end of this period the gall bladder of all animals in group L contained cholesterol gallstones. The coefficient of dietary cholesterol absorption was reduced by 26 %, cholesterol synthesis and cholesterol faecal excretion were twofold higher in group L than in group C. Bile acid content in the small intestine was diminished in group L, but bile acid composition was similar in the two groups. The increase in cholesterogenesis in lithiasic animals essentially took place in the liver. Bile acid biosynthesis did not significantly differ in the two groups, but represented only 35 % of total cholesterol input (dietary absorption + internal secretion) in group L v. 52% in group C. Thus, in the lithiasic hamster, hepatic synthesis of cholesterol and bile acids are not coupled. The molar percentage of cholesterol in bile was twofold higher in group L than in group C but those of bile acids and of phospholipids were not modified. In the lithiasic hamster the specific activity of biliary cholesterol was similar to that in plasma and liver. Consequently, biliary cholesterol does not derive directly from cholesterol newly synthesized in the liver but from hepatic cholesterol rapidly exchangeable with plasma cholesterol. Lithiasis: Cholesterol : Bile acids: Gallstones: Hamster Cholesterol gallstones were induced in young golden Syrian hamsters maintained on a high-sucrose fat-free diet (Dam, 1969). The mechanisms of gallstone formation in this animal model are not fully understood but seem to be associated with an increase in hepatic cholesterol synthesis (Ginsberg et al. 1977;Turley & Spady, 1983) and biliary cholesterol secretion (Robins & Fasulo, 1973 ;Wheeler, 1973). In patients with cholesterol gallstones, alterations of these two mechanisms have also been observed (Salen et al. 1975; Reuben er al. 1985). Other mechanisms of cholesterol metabolism and particularly changes in cholesterol turnover processes induced by a fat-free diet in the hamster might explain gallstone formation. These turnover processes of cholesterol, i.e. the input (absorption and synthesis) and the output (faecal cholesterol and bile acid excretion) studied by an isotopic equilibrium method have not been documented in this animal (Lutton, 1990). In man and the hamster the role of the liver in endogenous cholesterol synthesis has been well described (Salen et al. 1975;Ginsberg et al. 1977), but the extent to which extrahepatic sterol synthesis was affected under this nutritional condition remained unclear. Moreover, in the lithiasic hamster, it is also not clear whether the increase in biliary cholesterol secretion is related to the transport of larger quantities of hepatic newly-sy...