No abstract

Selective uptake of cholesteryl esters (CE) from lipoproteins by cells has been extensively studied with high density lipoproteins (HDL). It is only recently that such a mechanism has been attributed to intermediate and low density lipoproteins (IDL and LDL). Here, we compare the association of proteins and CE from very low density lipoproteins (VLDL), IDL, LDL and HDL3 to HepG2 cells. These lipoproteins were either labelled in proteins with 125I or in CE with 3H-cholesteryl oleate. We show that, at any lipoprotein concentration, protein association to the cells is significantly smaller for IDL, LDL, and HDL3 than CE association, but not for VLDL. At a concentration of 20 microg lipoprotein/mL, these associations reveal CE-selective uptake in the order of 2-, 4-, and 11-fold for IDL, LDL, and HDL3, respectively. These studies reveal that LDL and HDL3 are good selective donors of CE to HepG2 cells, while IDL is a poor donor and VLDL is not a donor. A significant inverse correlation (r2 = 0.973) was found between the total lipid/protein ratios of the four classes of lipoproteins and the extent of CE-selective uptake by HepG2 cells. The fate of 3H-CE of the two best CE donors (LDL and HDL3) was followed in HepG2 cells after 3 h of incubation. Cells were shown to hydrolyze approximately 25% of the 3H-CE of both lipoproteins. However, when the cells were treated with 100 microM of chloroquine, a lysosomotropic agent, 85 and 40% of 3H-CE hydrolysis was lost for LDL and HDL3, respectively. The fate of LDL and HDL3-CE in HepG2 cells deficient in LDL-receptor was found to be the same, indicating that the portion of CE hydrolysis sensitive to chloroquine is not significantly linked to LDL-receptor activity. Thus, in HepG2 cells, the magnitude of CE-selective uptake is inversely correlated with the total lipid/protein ratios of the lipoproteins and CE-selective uptake from the two best CE donors (LDL and HDL3) appears to follow different pathways.

The objective of this study was to determine the distribution of creatine phosphokinase (CPK) into its three isoenzymes, MM, MB, and BB, in human prostatic tissue, in patients with benign hyperplasia (BPH) and adenocarcinoma. Specimens were obtained from 23 patients with adenocarcinoma of the prostate and 25 patients with benign hyperplasia. We also had the opportunity to analyze the CPK content in two normal prostates, the first from a 16 1/2-year-old boy and the second from a 9 1/2-year-old child. Our results showed prostate tissue to contain almost exclusively the BB isoenzyme with traces of the MB and MM dimers in both cancer and BPH as well as the specimen of normal prostate from the 16 1/2-year-old boy. As for the 9 1/2-year-old child, we found the following distribution: 39% MM, 21% MB, and 40% BB dimer. A comparison of the CPK-BB content in benign hyperplasia and adenocarcinoma revealed no significant difference between the two groups. Furthermore, we tried to correlate prostatic tissue CPK-BB levels with another possible tumor marker of the prostate, prostatic acid phosphatase (PAP) measured in the cytosol. No correlation was found between these two markers. We also studied the relationship of CPK-BB and PAP content in prostatic tissue to nuclear and cytosolic androgen receptor content in human prostatic tissue. We found some correlation between CPK-BB and androgen cytosolic receptors as well as between PAP content and androgen cytosolic receptors in patients with benign hyperplasia. No such correlation was found in the group with adenocarcinoma. In conclusion, this study does not show that the measurement of CPK-BB in the prostatic tissue could be used as an index of tissue malignancy.

Selective uptake of cholesteryl esters (CE) from lipoproteins by cells has been extensively studied with high density lipoproteins (HDL). It is only recently that such a mechanism has been attributed to intermediate and low density lipoproteins (IDL and LDL). Here, we compare the association of proteins and CE from very low density lipoproteins (VLDL), IDL, LDL and HDL3 to HepG2 cells. These lipoproteins were either labelled in proteins with 125I or in CE with 3H-cholesteryl oleate. We show that, at any lipoprotein concentration, protein association to the cells is significantly smaller for IDL, LDL, and HDL3 than CE association, but not for VLDL. At a concentration of 20 microg lipoprotein/mL, these associations reveal CE-selective uptake in the order of 2-, 4-, and 11-fold for IDL, LDL, and HDL3, respectively. These studies reveal that LDL and HDL3 are good selective donors of CE to HepG2 cells, while IDL is a poor donor and VLDL is not a donor. A significant inverse correlation (r2 = 0.973) was found between the total lipid/protein ratios of the four classes of lipoproteins and the extent of CE-selective uptake by HepG2 cells. The fate of 3H-CE of the two best CE donors (LDL and HDL3) was followed in HepG2 cells after 3 h of incubation. Cells were shown to hydrolyze approximately 25% of the 3H-CE of both lipoproteins. However, when the cells were treated with 100 microM of chloroquine, a lysosomotropic agent, 85 and 40% of 3H-CE hydrolysis was lost for LDL and HDL3, respectively. The fate of LDL and HDL3-CE in HepG2 cells deficient in LDL-receptor was found to be the same, indicating that the portion of CE hydrolysis sensitive to chloroquine is not significantly linked to LDL-receptor activity. Thus, in HepG2 cells, the magnitude of CE-selective uptake is inversely correlated with the total lipid/protein ratios of the lipoproteins and CE-selective uptake from the two best CE donors (LDL and HDL3) appears to follow different pathways.

Apolipoprotein E (apoE) plays a major role in lipoprotein metabolism by mediating the binding of apoE-containing lipoproteins to receptors. The role of hepatic apoE in the catabolism of apoE-free lipoproteins such as low density lipoprotein (LDL) and high density lipoprotein-3 (HDL 3 ) is however, unclear. We analyzed the importance of hepatic apoE by comparing human LDL and HDL 3 metabolism in primary cultures of hepatic cells from control C57BL/6J and apoE knockout (KO) mice. Binding analysis showed that the maximal binding capacity (B max ) of LDL, but not of HDL 3 , is increased by twofold in the absence of apoE synthesis/secretion. Compared to control hepatic cells, LDL and HDL 3 holoparticle uptake by apoE KO hepatic cells, as monitored by protein degradation, is reduced by 54 and 77%, respectively. Cleavage of heparan sulfate proteoglycans (HSPG) by treatment with heparinase I reduces LDL association by 21% in control hepatic cells. Thus, HSPG alone or a hepatic apoE-HSPG complex is partially involved in LDL association with mouse hepatic cells. In apoE KO, but not in normal hepatic cells, the same treatment increases LDL uptake/degradation by 2.4-fold suggesting that in normal hepatic cells, hepatic apoE increases LDL degradation by masking apoB-100 binding sites on proteoglycans. Cholesteryl ester (CE) association and CE selective uptake (CE/protein association ratio) from LDL and HDL 3 by mouse hepatic cells were not affected by the absence of apoE expression. We also show that 69 and 72% of LDL-CE hydrolysis in control and apoE KO hepatic cells, respectively, is sensitive to chloroquine revealing the importance of a pathway linked to lysosomes. In contrast, HDL 3 -CE hydrolysis is only mediated by a nonlysosomal pathway in both control and apoE KO hepatic cells. Overall, our results indicate that hepatic apoE increases the holoparticle uptake pathway of LDL and HDL 3 by mouse hepatic cells, that HSPG devoid of apoE favors LDL binding/association but impairs LDL uptake/degradation and that apoE plays no significant role in CE selective uptake from either human LDL or HDL 3 lipoproteins. [7]. The LDLr and the LRP are two receptors that mediate the endocytosis and degradation of lipoproteins particles (holoparticle uptake pathway), while the LBS mediates cholesteryl ester (CE) selective uptake [8]. CE selective uptake refers to a transfer of CE into the cells without the complete internalization of the lipoprotein. The LBS shares many characteristics with the scavenger receptor class B type I (SR-BI) identified in rodents [9] and in human as CD36-and LIMPII-analogous-1 (CLA-1) [10]. Localized at the cell-surface of hepatocytes, both receptors can bind various types of apolipoproteins and can mediate CE selective uptake from HDL [11,12]