Hassan Nazih

Butyrate stimulates ApoA‐IV‐containing lipoprotein secretion in differentiated Caco‐2 cells: Role in cholesterol efflux

The aim of this study was to determine: (1) whether the Short Chain Fatty Acids (SCFA) Acetate, Propionate, and Butyrate enhance the synthesis and secretion of intestinal apolipoprotein A‐IV‐containing lipoproteins and (2) if so, whether these particles are able to promote cholesterol efflux in vitro. For this purpose Caco‐2 cells were used for their functional properties of differentiated enterocytes. They were incubated with the three SCFA (2, 4, and 8 mM) for 48 h. Only butyrate stimulated apoA‐IV gene expression and this was associated with an increase in apoA‐IV secretion. A nondenaturing 2D‐PAGE (agarose gel was followed by PAGE) was used to identify apoA‐IV‐containing lipoproteins in various media, and showed that butyrate stimulated the secretion of two small HDL sized particles. The influence of these secreted particles on cholesterol efflux was investigated using incubation of media with 3H‐cholesterol‐labeled Fu5AH cells. The data indicate that conditioned media from Caco‐2 cells treated with butyrate resulted in an increase of 20–30% in cholesterol efflux. We conclude that butyrate may regulate apoA‐IV secretion and, therefore, modulate reverse cholesterol transport. J. Cell. Biochem. 83: 230–238, 2001.

The differential apoA-I enrichment of preβ1 and αHDL is detectable by gel filtration separation

The aim of the study was to assess the isolation of HDL by fast protein liquid chromatography (FPLC) to perform kinetics studies of apolipoprotein (apo)A-I-HDL labelled with a stable isotope. Comparison between FPLC and ultracentrifugation has been made. ApoA-I-HDL kinetics were studied by infusion of [5.5.5-2H3]leucine for 14 h in five subjects. Using FPLC, preβ1 HDL and αHDL (HDL2 and HDL3) were separated from 200 μl of plasma samples. Total HDL was isolated by sequential ultracentrifugation (HDL-UC). The tracer-to-tracee ratio was higher in preβ1 HDL than in total HDL-UC. The higher leucine enrichment found in total HDL-UC compared to αHDL suggested the existence of a mixture of apoA-I-HDL sub-classes. From this difference in enrichments, the turnover rate of total HDL-UC, usually assumed to be αHDL, was probably overestimated in previous studies. To our knowledge, this study is the first report which provides a convenient tool to distinguish enrichments of apoA-I in preβ1 HDL and αHDL from total HDL previously used for kinetic measurements. This original and new method should help to understand the kinetics of HDL in humans and the reverse cholesterol transport dynamics.

Effect of LPS on basal and induced apo E secretion by 25-OH chol and 9cRA in differentiated CaCo-2

The infection and inflammation process is associated with disturbances in lipid and lipoprotein metabolism. The apolipoprotein E (apo E) plays an important role in the lipoprotein metabolism and has been linked to inflammatory disease such as atherosclerosis and Alzheimer disease. An anti‐inflammatory effect has also been suggested. The heterodimer nuclear receptor Liver‐X‐Receptorα/Retinoid‐X‐Receptor (LXRα/RXR) is considered to be a transcription factor for apo E. The aim of this study was to determine whether lipopolysaccharide (LPS) (principal component of the outer membrane Gram‐negative bacteria) has an effect on apo E secretion by intestinal mucosa cells, using the Caco‐2 cell line. Differentiated Caco‐2 cells grown on filter inserts were incubated apically with LPS and/or 25‐hydroxycholesterol (25‐OH chol) and 9 cis retinoic acid (9cRA), ligands of LXR and RXR, respectively. The apical and basolateral media were separately collected. Apo E was detected by specific antibodies after protein separation by Two‐dimensional nondenaturing gradient gel electrophoresis and apo E secreted in the cell culture media was measured by enzyme linked immunosorbent assay (ELISA). Apo E mRNA was analyzed by reverse transcription‐polymerase chain reaction (RT‐PCR). LXRα and RXR mass was analyzed by Western Blot. We demonstrate here that CaCo‐2 cells secrete apo E, by either apical or basolateral sides, associated with a high‐density like lipoprotein, with a stokes diameter comprised between 7.10 and 8.16 nm. We show that only apical secretion is decreased by LPS in a dose and time dependent manner. This is associated with a decrease in apo E gene expression contrasting with an increase of Il‐8, a chemokine factor. Moreover, we demonstrate that only basolateral apo E secretion by CaCo‐2 is significantly increased by 25‐OH chol and 9cRA while apical secretion remains unchanged. LPS does not decrease the 25‐OH chol and 9cRA mediated apo E secretion in basolateral compartment, while apical secretion is diminished under these circumstances. Our results provide evidence for the polarized secretion of apo E by intestinal epithelium. They also demonstrate that apo E secretion by CaCo‐2 cell line is decreased by LPS through an LXRα/RXR independent signaling pathway.

Nicotinic Acid Accelerates HDL Cholesteryl Ester Turnover in Obese Insulin-Resistant Dogs

Aim Nicotinic acid (NA) treatment decreases plasma triglycerides and increases HDL cholesterol, but the mechanisms involved in these change are not fully understood. A reduction in cholesteryl ester transfer protein (CETP) activity has been advanced to explain most lipid-modulating effects of NA. However, due to the central role of CETP in reverse cholesterol transport in humans, other effects of NA may have been hidden. As dogs have no CETP activity, we conducted this study to examine the specific effects of extended-release niacin (NA) on lipids and high-density lipoprotein (HDL) cholesteryl ester (CE) turnover in obese Insulin-Resistant dogs with increase plasma triglycerides. Methods HDL kinetics were assessed in fasting dogs before and four weeks after NA treatment through endogenous labeling of cholesterol and apolipoprotein AI by simultaneous infusion of [1,2 13C2] acetate and [5,5,5 2H3] leucine for 8 h. Kinetic data were analyzed by compartmental modeling. In vitro cell cholesterol efflux of serum from NA-treated dogs was also measured. Results NA reduced plasma total cholesterol, low-density lipoprotein cholesterol, HDL cholesterol, triglycerides (TG), and very-low-density lipoprotein TG concentrations (p < 0.05). The kinetic study also showed a higher cholesterol esterification rate (p < 0.05). HDL-CE turnover was accelerated (p < 0.05) via HDL removal through endocytosis and selective CE uptake (p < 0.05). We measured an elevated in vitro cell cholesterol efflux (p < 0.05) with NA treatment in accordance with a higher cholesterol esterification. Conclusion NA decreased HDL cholesterol but promoted cholesterol efflux and esterification, leading to improved reverse cholesterol transport. These results highlight the CETP-independent effects of NA in changes of plasma lipid profile.