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Abstract Background Androgen deprivation therapy (ADT) is widely used in the treatment of testosterone-dependent prostate carcinomas. ADT often increases plasma LDL and HDL cholesterol and triglycerides. The aim was to test whether ADT changes the transfer of lipids to HDL, an important aspect of this metabolism and HDL protective functions, and related parameters. Methods Sixteen volunteers with advanced prostate carcinoma submitted to pharmacological ADT or orchiectomy had plasma collected shortly before and after 6 months of ADT. In vitro transfer of lipids to HDL was performed by incubating plasma with donor emulsion containing radioactive lipids by 1 h at 37 °C. After chemical precipitation of apolipoprotein B-containing lipoprotein, the radioactivity of HDL fraction was counted. Results ADT reduced testosterone to nearly undetectable levels and markedly diminished PSA. ADT increased the body weight but glycemia, triglycerides, LDL and HDL cholesterol, HDL lipid composition and CETP concentration were unchanged. However, ADT increased the plasma unesterified cholesterol concentration (48 ± 12 vs 56 ± 12 mg/dL, p = 0.019) and LCAT concentration (7.15 ± 1.81 vs 8.01 ± 1.55μg/mL, p = 0.020). Transfer of unesterified (7.32 ± 1.09 vs 8.18 ± 1.52%, p

Introduction: The uptake of low- and high-density lipoproteins (LDL and HDL) through the LDL receptor (LDLR) and the scavenger receptor class B type I (SR-BI) mediates maternal to fetal cholesterol transfer in syncytiotrophoblast (STB) cells. STB cells deliver cholesterol via cholesterol efflux through the ATP-binding cassette transporters A1 (ABCA1, to ApoA-I), G1 (ABCG1, to HDL), and SR-BI (to HDL). In the human placenta, these proteins are localized in the apical (LDLR, SR-BI, ABCA1) and basal (SR-BI, ABCA1, ABCG1) membrane of STB cells. However, whether these proteins in polarized primary culture models of STB show a similar localization to those in the human placenta is currently unknown.Methods: Primary human trophoblasts (PHT) were isolated from normal placentas and cultured in Transwells® with Matrigel to obtain a polarized STB monolayer, proteins were determined by immunofluorescence and cholesterol efflux determined to different acceptors.Results: At day 5, LDLR and ABCA1 localized mainly in the apical membrane, ABCG1 in the basal membrane, and SR-BI in both. Cholesterol efflux towards the apical compartment was higher to adult and neonatal HDL compared to ApoA-I. When acceptors were added in the basal compartment, cholesterol was retained in the Matrigel.Discussion: Polarized STB monolayers express LDLR, SR-BI, ABCA1 and ABCG1, and their apical/basal localization resembles the one described in human placental tissue. This study confirms the high physiological value and suitability of this model for use in functional studies. Our findings also suggest that ABCA1 and SR-BI participate in cholesterol efflux to the maternal side of the cells. [ABSTRACT FROM AUTHOR]

Background: Androgen deprivation therapy (ADT) is widely used in the treatment of testosterone-dependent prostate carcinomas. ADT often increases plasma LDL and HDL cholesterol and triglycerides. The aim was to test whether ADT changes the transfer of lipids to HDL, an important aspect of this metabolism and HDL protective functions, and related parameters. Methods: Sixteen volunteers with advanced prostate carcinoma submitted to pharmacological ADT or orchiectomy had plasma collected shortly before and after 6 months of ADT. In vitro transfer of lipids to HDL was performed by incubating plasma with donor emulsion containing radioactive lipids by 1 h at 37 °C. After chemical precipitation of apolipoprotein B-containing lipoprotein, the radioactivity of HDL fraction was counted. Results: ADT reduced testosterone to nearly undetectable levels and markedly diminished PSA. ADT increased the body weight but glycemia, triglycerides, LDL and HDL cholesterol, HDL lipid composition and CETP concentration were unchanged. However, ADT increased the plasma unesterified cholesterol concentration (48 ± 12 vs 56 ± 12 mg/dL, p = 0.019) and LCAT concentration (7.15 ± 1.81 vs 8.01 ± 1.55μg/mL, p = 0.020). Transfer of unesterified (7.32 ± 1.09 vs 8.18 ± 1.52%, p < 0.05) and esterified cholesterol (6.15 ± 0.69 vs 6.94 ± 1.29%, p < 0.01) and of triglycerides (6.37 ± 0.43 vs 7.18 ± 0.91%, p < 0.001) to HDL were increased after ADT. Phospholipid transfer was unchanged. Conclusion: Increase in transfer of unesterified and esterified cholesterol protects against cardiovascular disease, as shown previously, and increased LCAT favors cholesterol esterification and facilitates the reverse cholesterol transport. Thus, our results suggest that ADT may offer anti-atherosclerosis protection by improving HDL functional properties. This could counteract, at least partially, the eventual worse effects on plasma lipids. [ABSTRACT FROM AUTHOR]

The fundamental task of lipoprotein particles is extracellular transport of cholesterol, lipids, and fatty acids. Besides, cholesterol-rich apoB-containing lipoprotein particles (i.e., low density lipoprotein LDL) are key players in progression of atherosclerotic cardiovascular disease and are associated with familial hypercholesterolemia (FH). So far, lipoprotein particle binding to the cell membrane and subsequent cargo transfer is directly linked to the lipoprotein receptors on the target cell surface. However, our observations showed that lipoprotein particle cargo transport takes place even in the absence of the receptor. This finding suggests that an alternative mechanism for lipoprotein-particle/membrane interaction, besides the receptor-mediated one, exists. Here, we combined several complementary biophysical techniques to obtain a comprehensive view on the nonreceptor mediated LDL-particle/membrane. We applied a combination of atomic force and single-molecule-sensitive fluorescence microscopy (AFM and SMFM) to investigate the LDL particle interaction with membranes of increasing complexity. We observed direct transfer of fluorescently labeled amphiphilic lipid molecules from LDL particles into the pure lipid bilayer. We further confirmed cargo transfer by fluorescence cross-correlation spectroscopy (FCCS) and spectral imaging of environment-sensitive probes. Moreover, the integration of the LDL particle into the membranes was directly visualized by high-speed atomic force microscopy (HS-AFM) and cryo-electron microscopy (cryo-EM). Overall, our data show that lipoprotein particles are able to incorporate into lipid membranes upon contact to transfer their cargo in the absence of specific receptors.

Niemann-Pick C1 (NPC1) is a membrane protein required for the transport of low-density lipoprotein (LDL)-derived cholesterol from endosomes and lysosomes to the other organelles. Here, we describe the recombinant protein expression, purification, and characterization of the human NPC1. The protein is transiently expressed in human embryonic kidney (HEK) cells. Our purification protocol describes the steps to obtain a pure and homogeneous NPC1 protein. Niemann-Pick C2 (NPC2) is a small soluble protein, which mediates cholesterol transport in tandem with NPC1. Finally, we also describe two biochemical approaches to characterize NPC1 function in vitro-a cholesterol transfer assay from purified NPC2 to NPC1 and a binding assay between NPC1 and NPC2.

Low-density lipoproteins (LDLs) are taken up by the cell mainly through receptor-mediated endocytosis. LDL-derived cholesterol leaves lysosome and further transports to downstream organelles for specific cellular needs. We recently report that cholesterol transfers from lysosome to peroxisome through lysosome-peroxisome membrane contact (LPMC). Here, we use iodixanol density gradient centrifugation to isolate lysosomes and peroxisomes separately for the in vitro reconstitution of LPMC. We also apply 3 H-cholesterol-labeled lysosomes and peroxisomes in vitro to measure 3 H-cholesterol transfer through LPMC.