Neutral lipids accumulate in cellular lipid droplets. These droplets vary remarkably in number and amount between cells. In the present studies, the variability in lipid content was quantified by comparing the coefficient of variation of fluorescence histograms of nile red lipid-stained cells to the variability of cell size or cell protein distributions. This measure of lipid droplet variability persisted through a wide range of cell lipid content and averaged 4.4-fold more variability than cell size and 2.6-fold more variability than cell protein content. While looking for possible explanations for this variability, it was determined that cell to cell variability could not be explained by multiple clonal populations of cells or the confluence of the cell monolayer. Analysis of lipid variability using a more droplet-specific fluorescent dye, bodipy, reduced variability by about 44%. Cell cycle analysis revealed that G2 + M cells contained more lipid than S-phase cells, which in turn contained more lipid than Go + GI cells, but that variability was equally large throughout the cell cycle. The cholesteryl ester hydrolase inhibitor, diethylumbelliferyl phosphate, inhibited hydrolysis of both cholesteryl esters and triglycerides. Lipid content of diethylumbelliferyl phosphate-treated cells increased while the variability in lipid staining decreased by an average of 72%. Thus, the excess lipid fluorescence variability compared to cell size or protein fluorescence could in part be explained by variability in cellular hydrolysis of triglyceride and cholesteryl ester. Excess lipid fluorescence variability could be reduced by an average of 44% when a more lipid droplet-specific stain was used instead of nile red. Key terms: Nile red, fluorescence, quantitative lipid staining, bodipy, diethylumbelliferyl phosphate, lipid dropletsThe mechanisms leading to lipid accumulation in cells are not well understood. Since lipid accumulation in one cell type, the macrophage, may act as the initiating event in the atherosclerotic process (2,19), considerable effort has been directed toward understanding this process. The lipophilic dye nile red has been used in some studies of macrophages (11,131. These studies suggest that nile red might act as a quantitative lipid stain since nile red fluorescence is increased in at least rough proportion to the amount of lipid fed to the macrophages (13). Compared to the variability of protein fluorescence in most cells (81, nile red fluorescence seems quite variable. The present studies were prompted by these two observations.The MA-10 cells are a clonal strain of Leydig tumor cells. These cells have been utilized extensively by this laboratory for studies of cholesterol metabolism (61, storage (4), and transport (5,15,16). Unlike macrophages, MA-10 cells are clonally selected and thus should represent a single genetic population of cells. This characteristic should prove useful in studies exploring the nature of lipid droplet variability. The MA-10 cells can also be manipulated in a manner lendi...

No abstract

No abstract

To reveal the well known effect of smoking on the incidence of early abortion, the possible effects of cigarette alkaloids on progesterone and estradiol synthesis were investigated. A suspected cause for early spontaneous abortion is corpus luteum insufficiency. The present experiments evaluate the effects of cigarette smoke alkaloids on progesterone and estradiol biosynthesis. Human granulosa cells were obtained from patients undergoing in vitro fertilization and embryo transfer treatment because of infertility. Incubation of the granulosa cells with cotinine, anabasine, with the combination of nicotine, cotinine and anabasine, or with an aqueous extract of cigarette smoke resulted in inhibition of progesterone synthesis. The alkaloids and smoke extract decreased the DNA content of the culture dish. These latter findings suggested a cytotoxic effect of the alkaloids. Both cotinine and anabasine slightly stimulated the synthesis of normalized estradiol. However, nicotine, combination of all three alkaloids, and cigarette smoke extract had no significant influence on estradiol production. Taken together, these data would suggest that cigarette alkaloids inhibit cellular progesterone synthesis both by inhibiting progesterone synthesis and by causing less specific toxic effects to the cell. In contrast, cigarette smoke alkaloids slightly stimulated or had no effect on estradiol production. These concomitant actions of cigarette alkaloids partly explain the higher incidence of early abortion in pregnant women who smoke.

No abstract