Jane Wilkinson

A novel method for the rapid separation of plasma lipoproteins using self-generating gradients of iodixanol

We describe a new method for the rapid fractionation of plasma lipoproteins, which makes use of a new non-ionic, iodinated, density gradient medium, iodixanol, commercially available as Optiprep(TM). The method is simple: plasma or serum is mixed with iodixanol followed by centrifugation in a vertical or near vertical rotor. Separation of VLDL, LDL and HDL can be achieved in 3 h and the lipoprotein fractions are comparable in density and composition with those prepared using conventional salt based gradients. Each class of lipoprotein can be removed in a single fraction, or a profile of lipoprotein distribution can be obtained using a gradient fractionator. Because the medium is inert, fractions from the gradient can be analysed by agarose gel electrophoresis or assayed for lipid content or apolipoprotein composition by SDS-PAGE without removing the iodixanol. Small differences in electrophoretic mobility of HDL and LDL across several gradient fractions suggest that subfractionation of these classes may occur. The new method is simple, rapid and versatile with potential application for preparation of lipoproteins and for analysis of lipoprotein profiles in the research or clinical laboratory.

A role for smooth endoplasmic reticulum membrane cholesterol ester in determining the intracellular location and regulation of sterol-regulatory-element-binding protein-2

Cellular cholesterol homoeostasis is regulated through proteolysis of the membrane-bound precursor sterol-regulatory-element-binding protein (SREBP) that releases the mature transcription factor form, which regulates gene expression. Our aim was to identify the nature and intracellular site of the putative sterol-regulatory pool which regulates SREBP proteolysis in hamster liver. Cholesterol metabolism was modulated by feeding hamsters control chow, or a cholesterol-enriched diet, or by treatment with simvastatin or with the oral acyl-CoA:cholesterol acyltransferase inhibitor C1-1011 plus cholesterol. The effects of the different treatments on SREBP activation were confirmed by determination of the mRNAs for the low-density lipoprotein receptor and hydroxymethylglutaryl-CoA (HMG-CoA) reductase and by measurement of HMG-CoA reductase activity. The endoplasmic reticulum was isolated from livers and separated into subfractions by centrifugation in self-generating iodixanol gradients. Immunodetectable SREBP-2 accumulated in the smooth endoplasmic reticulum of cholesterol-fed animals. Cholesterol ester levels of the smooth endoplasmic reticulum membrane (but not the cholesterol levels) increased after cholesterol feeding and fell after treatment with simvastatin or C1-1011. The results suggest that an increased cellular cholesterol load causes accumulation of SREBP-2 in the smooth endoplasmic reticulum and, therefore, that membrane cholesterol ester may be one signal allowing exit of the SREBP-2/SREBP-cleavage-regulating protein complex to the Golgi.

Membrane-bound apolipoprotein B is exposed at the cytosolic surface of liver microsomes.

We have used a competitive enzyme‐linked immunoassay with a panel of monoclonal antibodies to probe the topography of the membrane‐bound form of apolipoprotein B (apo B) in rabbit microsomes. All epitopes investigated were found to be expressed at the cytosolic side of the microsomal membrane under conditions in which the vesicles remained sealed. These results indicate that the membrane‐associated form of apolipoprotein B is either at the cytosolic side of the endoplasmic reticulum membrane or integrated into the membrane. From this site apo B may be translocated to the lumen for assembly into VLDL or may be degraded.

Post-translational events in the intracellular transit of apolipoprotein-B: modulation by dietary lipids

Triacylglycerol and cholesteryl esters are transported in the blood as components of the plasma lipoproteins. Endogenous triacylglycerol synthesized by the liver is secreted into the plasma in the form of VLDL, which consists of a droplet of non-polar lipid (predominantly triacylglycerol with a variable amount of cholesteryl ester), stabilized by an outer shell of phospholipid, cholesterol and protein. Apolipoprotein-B (apo-B) is the major protein of VLDL. Although apo-B is an essential component of VLDL, secretion is driven by provision of substrates to the liver for triacylglycerol synthesis. These include glucose and nonesterified fatty acids. The composition and size of VLDL is influenced by the substrate provided (Sniderman & Cianflone, 1993). Dietary experiments and experiments using isolated hepatocytes have shown that provision of carbohydrates increases synthesis of triacylglycerol-enriched, larger, lighter VLDL (VLDLl), while provision of fatty acids results in production of smaller, denser VLDL (VLDL2). Since VLDLl are cleared rapidly from the circulation, while VLDL2 are cleared more slowly and are converted to the atherogenic LDL, the nature of VLDL secreted by the liver is an important determinant of cardiovascular risk (Packard et al. 1984; Shepherd & Packard, 1987). An understanding of the mechanisms involved in regulation of the assembly of VLDL and the factors determining their composition is, therefore, extremely important.