Saturated fat-induced changes in S-f 60-400 particle composition reduces uptake of LDL by HepG2 cells

The ability of human postprandial triacylglycerol-rich lipoproteins (TRLs), isolated after meals enriched in saturated fatty acids (SFAs), n-6 PUFAs, and MUFAs, to inhibit the uptake of I-125-labeled LDL by the LDL receptor was investigated in HepG2 cells. Addition of TRLs resulted in a dose-dependent inhibition of heparin-releasable binding, cell-associated radioactivity, and degradation products of I-125-labeled LDL (P < 0.001). SFA-rich Svedberg flotation rate (S-f) 60-400 resulted in significantly greater inhibition of cell-associated radioactivity than PUFA-rich particles (P = 0.016) and total uptake of I-125-labeled LDL compared with PUFA- and MUFA-rich particles (P = 0.02). Normalization of the apolipoprotein (apo)E but not apoC-III content of the TRLs removed the effect of meal fatty acid composition, and addition of an anti-apoE antibody reversed the inhibitory effect of TRLs on the total uptake of I-125-labeled LDL. Real time RT-PCR showed that the SFA-rich Sf 60-400 increased the expression of genes involved in hepatic lipid synthesis (P < 0.05) and decreased the expression of the LDL receptor-related protein 1 compared with MUFAs (P = 0.008). In conclusion, these findings suggest an alternative or additional mechanism whereby acute fat ingestion can influence LDL clearance via competitive apoE-dependent effects of TRL on the LDL receptor.-Jackson, K. G., V. Maitin, D. S. Leake, P. Yaqoob, and C. M. Williams. Saturated fat-induced changes in Sf 60 400 particle composition reduces uptake of LDL by HepG2 cells.

Contribution of apolipoprotein E genotype and docosahexaenoic acid to the LDL-cholesterol response to fish oil

Objectives: To investigate the impact of apolipoprotein E (apoE) genotype on the response of the plasma lipoprotein profile to eicosapentaenoic acid (EPA) versus docosahexaenoic acid (DHA) intervention in humans. Methods and results: 38 healthy normolipidaemic males, prospectively recruited on the basis of apoE genotype (n = 20 E3/E3 and n = 18 E3/E4), completed a double-blind placebo-controlled cross-over trial, consisting of 3 × 4 week intervention arms of either control oil, EPA-rich oil (ERO, 3.3 g EPA/day) or DHA-rich oil (DRO, 3.7 g DHA/day) in random order, separated by 10 week wash-out periods. A significant genotype-independent 28% and 19% reduction in plasma triglycerides in response to ERO and DRO was observed. For total cholesterol (TC), no significant treatment effects were evident; however a significant genotype by treatment interaction emerged (P = 0.045), with a differential response to ERO and DRO in E4 carriers. Although the genotype × treatment interaction for LDL-cholesterol (P = 0.089) did not reach significance, within DRO treatment analysis indicated a 10% increase in LDL (P = 0.029) in E4 carriers with a non-significant 4% reduction in E3/E3 individuals. A genotype-independent increase in LDL mass was observed following DRO intervention (P = 0.018). Competitive uptake studies in HepG2 cells using plasma very low density lipoproteins (VLDL) from the human trial, indicated that following DRO treatment, VLDL2 fractions obtained from E3/E4 individuals resulted in a significant 32% (P = 0.002) reduction in LDL uptake relative to the control. Conclusions: High dose DHA supplementation is associated with increases in total cholesterol in E4 carriers, which appears to be due to an increase in LDL-C and may in part negate the cardioprotective action of DHA in this population subgroup.

Characterisation of chicken riboflavin carrier protein gene structure and promoter regulation by estrogen

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2),which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.