This study determined the effects of apoA-I, HDL3, or hydroxy- -cyclodextrin on apoB-100 secretion and bile acid synthesis by HepG2 cells. The principal observations were that: 1 ) ApoB-100 secretion into the medium was significantly less after the addition of any of the three agents. 2 ) Triglyceride mass was not significantly changed from control in the medium but was significantly, although modestly, reduced in the cells. 3 ) Neither free cholesterol (FC) nor cholesteryl ester (CE) mass in the medium was changed; by contrast, CE mass was reduced within the cells although FC was not. 4 ) Although the total mass of cholesterol in the medium was unaffected, the proportion associated with apoB-100 was reduced, whereas the proportion associated with the non-apoB-100 fraction was increased. 5 ) There was also an unanticipated, but substantial, increase in bile acid synthesis induced by apoA-I, HDL3, or hydroxy- -cyclodextrin, which was time and concentration dependent, and which was associated with marked increases in cholesterol 7 ␣ -hydroxylase activity. There were no significant changes in ACAT activity and only modest increases in HMG-CoA reductase activity. These findings support previous clinical observations that an elevated apoB-100 can accompany a low HDL cholesterol in normotriglyceridemic subjects. They also point to physiologically important, although still only partially understood, metabolic relationships amongst hepatic apoB-100 secretion, cholesterol efflux, and bile acid synthesis. -Sniderman, A. D., Z. Zhang, J. Genest, and K. Cianflone. Effects on apoB-100 secretion and bile acid synthesis by redirecting cholesterol efflux from HepG2 cells. J. Lipid Res. 2003. 44: 527-532.
Abstract-Our understanding of the factors that regulate the secretion of apoB100 lipoproteins remains incomplete with considerable debate as to the role, if any, for cholesterol ester in this process. This study examines this issue in primary cultures of hamster hepatocytes, a species in which both cholesterol and apoB100 metabolism are very similar to man. Addition of oleate to medium increased the mass of triglyceride and cholesterol ester within the hepatocyte and also increased the secretion of triglycerides, cholesterol ester, and apoB100 into the medium. Next, the responses of hamster hepatocytes to addition of either an HMG-CoA reductase inhibitor (lovastatin) or an acyl-CoA cholesterol acyltransferase inhibitor (58-035) to the medium, with or without added oleate, were determined. Effects of either agent were only evident in the oleate-supplemented medium in which cholesterol ester mass had been increased above basal. If oleate was not added to the medium, neither agent reduced apoB100 secretion; equally important, over the 24-hour incubation, neither agent, at the concentration used, produced any detectable change in intracellular cholesterol ester mass. However, in contrast to the estimates of mass, which were unchanged, under the same conditions radioisotopic estimates of cholesterol ester synthesis were markedly reduced. Any conclusion as to the relation of cholesterol ester mass to apoB100 secretion would therefore depend on which of the 2 methods was used. Overall, the data indicate a close correlation between the mass of cholesterol ester within the hepatocyte and apoB100 secretion from it and they go far to explain previous apparently contradictory data as to this relation. More importantly, though, taken with other available data, they indicate that the primary response of the liver to increased delivery of lipid is increased secretion rather than decreased uptake. These results point, therefore, to a hierarchy of hepatic responses to increased flux of fatty acids and increased synthesis of cholesterol that in turn suggests a more dynamic model of cholesterol homeostasis in the liver than has been appreciated in the past. T he rate at which the apoB100 lipoprotein particles are secreted by the liver is a major determinant of IDL and LDL particle number in plasma. Accordingly, it is a major determinant of atherogenic risk. 1 Unfortunately, our understanding of the factors that regulate the rate of apoB100 secretion by the liver is recent and remains incomplete. In the case of apoB100 lipoprotein particles, variation in the rate at which they are secreted appears to be determined principally by variation in the proportion of newly synthesized apoB100 molecules, which are degraded compared with the proportion that are secreted, reflecting the complex coupling of multiple lipids to protein involved in the assembly and secretion of a lipoprotein particle. 2 There are now considerable in vitro and in vivo data that point to a direct relation between the rate of cholesterol synthesis or mass of cholest...
p16(INK4) is a specific cyclin D-dependent kinase inhibitor and a multiple tumor suppressor. Inactivation of p16 is frequent in both primary tumors and tumor-derived cell lines. We describe here the conformational properties and oligomerization state of seven mutant p16 proteins; all of them are deficient in function. Four of the seven proteins show significantly disrupted secondary structure and backbone folding. The other three adopt partially folded, molten globule-like conformations. These proteins have near-native levels of secondary structure, but lack the ability to undergo a cooperative thermal transition and are substantially less resistant to proteolysis than is wild type p16. At low concentrations, two of the seven proteins are monomers, three exhibit an apparent molecular weight between the value of a monomer and a dimer, and the other two aggregate significantly. Our results strongly suggest that defective protein folding and/or aggregation is a common mechanism for inactivation of p16.
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