In the present study, we used the human EA.hy926 endothelial cell line as the model system to investigate the effect of human serum albumin (HSA) and its structural variants on cholesterol efflux. Initial studies showed that HSA promoted cholesterol efflux in a dose- and time-dependent manner, reaching a plateau at 10 mg/ml at 90 min. As a control, gelatin displayed no significant effect on efflux, while HSA was significantly more efficient than ovalbumin and bovine serum albumin (BSA) in promoting cholesterol efflux. Equal molar concentrations of HSA and apolipoprotein A-I (apoA-I) showed that apoA-I had considerably higher efficiency in efflux. However, the prevailing high plasma concentrations of HSA may compensate for its lower efflux rate compared to apoA-I. To characterize the mechanism of HSA-mediated cholesterol efflux, we studied the effects of cAMP and temperature on efflux using both EA.hy926 endothelial cells and murine RAW 264.7 macrophages. We found that HSA-mediated efflux occurred via a cAMP-independent and relatively temperature-insensitive pathway. We next examined the nature of HSA-cholesterol interaction by comparing the effects of various HSA mutants to wild-type HSA on cholesterol efflux. We found specific interactions between subdomains 2A and 3A and cholesterol, as indicated by the changes in the efflux rate of various HSA mutants. In conclusion, our study provides evidence for the role of HSA in cholesterol efflux, and shows that the substitution of specific amino acid residues in subdomains of 2A and 3A may be important structural determinants in its ability to bind to cholesterol and participate in cholesterol efflux.

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Statins reduce cholesterol biosynthesis by inhibiting HMG-CoA reductase and thereby lower total cholesterol and LDL cholesterol levels in serum, which in turn lower the incidence of cardiovascular disease (CVD). Statins are also known to modulate various cellular functions such as gene expression, cell proliferation, and programmed cell death through inhibition of downstream intermediates in cholesterol synthesis. In this study, we have investigated the possible effects of statins on the secretion of serum albumin from cultured HepG2 cells since high levels of serum albumin are associated with reduced risks for CVD and statins are effective in lowering the risk of CVD through other effects in addition to their effects on serum total cholesterol and LDL cholesterol levels, known as pleiotropic effects. Our results showed that simvastatin increased HSA secretion up to 32.3% compared to the control group. Among 3 statin analogs we tested, simvastatin exhibited the highest stimulatory effects on HSA secretion compared to the control group. Our study also showed that the increased HSA secretions from HepG2 cells by simvastatin treatments were due to the increased rate of HSA synthesis, not due to the reduced posttranslational degradation rate of HSA. Our finding suggests another added benefit of statins' treatments in preventing CVD through stimulation of HSA biosynthesis.

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There is mounting evidence that free fatty acids (FFAs) plays a role in the pathogenesis of diabetes and cardiovascular diseases. However, the effects of human serum albumin (HSA)/FFA complexes on ß‐cells as well as on HSA‐mediated cholesterol efflux are not well known. Thus, we exposed the insulinoma ß‐cells, HIT‐TI5 to different HSA/FFAs complexes for 24hrs followed by assessment of insulin secretion and cell viability. Also, a human endothelial cell line, EA.hy926 was used as the model system to study cholesterol efflux. In the presence of HSA, palmitate and stearate induced significant cell death at 0.1mM or higher, which was not significantly inhibited by the two apoptosis inhibitors, cyclosporin A and ZVAD‐FMK. In contrast, myristate, palmitoleate, oleate, and elaidate bound to HSA showed minimal effects on cell viability. Additionally, insulin secretion was significantly reduced by HSA/oleate complexes. We also found differential effects on cell viability by HSA mutants/FFAs complexes. For efflux studies, HSA complexed to oleate or palmitate at 1:5.3 molar ratios showed significant reduction in cholesterol efflux but not with arachidonate. In summary, this study showed that FFA‐induced changes of cellular functions are dependent upon the types and concentrations of FFAs which might be modified by HSA variants.[Funding: Hawaii EXPORT Centre, HCF, and an award from the American Heart Association].