Loren G. Fong

Use of an anti-low density lipoprotein receptor antibody to quantify the role of the LDL receptor in the removal of chylomicron remnants in the mouse in vivo.

Lipoproteins are removed from the plasma by LDL receptor-dependent and -independent pathways. The relative contribution of these has been established for LDL by using modified lipoproteins, but this has not been possible for apoE-rich lipoproteins, such as chylomicron remnants. To do this, we used a monospecific antibody to the rat LDL receptor. The antibody was injected intravenously into mice followed by 125I-lipoproteins. Blood samples were obtained sequentially and radioactivity measured to determine the plasma clearance of the lipoproteins. The animals were then sacrificed and the tissues removed, dried, and the radioactivity measured to determine tissue uptake. An albumin space was also measured to correct for blood trapping. With 125I-human LDL, approximately 50% of the injected dose was cleared in 180 min. This was reduced to 30% by the antibody and this was identical to the disappearance of reductively methylated LDL. This is a lower estimate of LDL-mediated uptake (40%) than in other species. LDL uptake per gram tissue was similar for the liver and the adrenal gland and was approximately 50% LDL receptor-dependent in both tissues. With 125I-chylomicron remnants, clearance was much more rapid with approximately 50% cleared in 5 min. By agarose gel electrophoresis, radioactivity was not transferred from chylomicron remnants to other lipoprotein classes. Chylomicron remnants with label on only apoB or in 3H-cholesterol esters showed a similar pattern. Combining the estimates of the three labeling procedures, approximately 35% of the 30 s and 25% of the 5 min chylomicron remnant disappearance was LDL receptor dependent. The liver, per gram tissue, took up five times as much radioactivity as the adrenal gland. At 5 min, at least 50% of this was LDL receptor-dependent in liver and 65% in adrenal gland. We conclude that the LDL receptor plays a major, and somewhat similar quantitative role in the clearance of both LDL and chylomicron remnants in the mouse. However, at least in the mouse, non-LDL receptor-mediated lipoprotein clearance is quantitatively important and is also very rapid for chylomicron remnants. Thus, for chylomicron remnants, it can easily compensate for LDL receptors if they are blocked or absent. Further, the tissue distribution of lipoprotein uptake may be directed by factors other than LDL receptor density.

Immunohistochemical localization of low density lipoprotein receptors in adrenal gland, liver, and intestine.

The localization of LDL receptors in adrenal gland, liver, and intestine was studied using immunohistochemistry. The anti-LDL receptor antibody used was shown to be monospecific and did not react with striated muscle, a tissue which has a very low level of LDL receptors. Similarly, cerebral cortex showed only faint reactivity and that was to an area previously demonstrated to have LDL receptors. Adrenal gland was intensely reactive with the zona fasciculata, having a greater density of receptors than the zona reticularis. In normal liver, LDL receptors were present on the sinusoidal membranes and were sparse in the areas of hepatocyte-to-hepatocyte contact without an obvious portal to central gradient. LDL receptors were present throughout the intestine. In jejunum, staining was most intense at the base of the villus and extended up toward the villus tip. At the base of the villus, the receptor was primarily at the basal lateral membrane, but toward the villus tip, there was appreciable intracellular staining. Staining in crypts was more faint; in duodenum, staining in crypts equaled that in the villus region in intensity. In colon, there was intense staining throughout the epithelial cells. These results provide new information about the cellular and subcellular localization of LDL receptors and raise the interesting possibility that there is a role for LDL-derived cholesterol in new lipoprotein formation.

LDL and cAMP cooperate to regulate the functional expression of the LRP in rat ovarian granulosa cells

Rat ovarian granulosa rely heavily on lipoprotein-derived cholesterol for steroidogenesis, which is principally supplied by the LDL receptor- and scavenger receptor class B type I (SR-BI)-mediated pathways. In this study, we characterized the hormonal and cholesterol regulation of another member of the LDL receptor superfamily, low density lipoprotein receptor-related protein (LRP), and its role in granulosa cell steroidogenesis. Coincubation of cultured granulosa cells with LDL and N6,O2′-dibutyryl adenosine 3′,5′-cyclic monophosphate (Bt2cAMP) greatly increased the mRNA/protein levels of LRP. Bt2cAMP and Bt2cAMP plus human (h)LDL also enhanced SR-BI mRNA levels. However, there was no change in the expression of receptor-associated protein, a chaperone for LRP, or another lipoprotein receptor, LRP8/apoER2, in response to Bt2cAMP plus hLDL, whereas the mRNA expression of LDL receptor was reduced significantly. The induced LRP was fully functional, mediating increased uptake of its ligand, α2-macroglobulin. The level of binding of another LRP ligand, chylomicron remnants, did not increase, although the extent of remnant degradation that could be attributed to the LRP doubled in cells with increased levels of LRP. The addition of lipoprotein-type LRP ligands such as chylomicron remnants and VLDL to the incubation medium significantly increased the progestin production under both basal and stimulated conditions. In summary, our studies demonstrate a role for LRP in lipoprotein-supported ovarian granulosa cell steroidogenesis.