F. Jeffrey Field

Polyunsaturated fatty acids decrease the expression of sterol regulatory element-binding protein-1 in CaCo-2 cells: effect on fatty acid synthesis and triacylglycerol transport.

Regulation of sterol regulatory element-binding proteins (SREBPs) by fatty acid flux was investigated in CaCo-2 cells. Cells were incubated with 1 mM taurocholate with or without 250 microM 18:0, 18:1, 18:2, 20:4, 20:5 or 22:6 fatty acids. Fatty acid synthase (FAS) and acetyl-CoA carboxylase mRNA levels and gene and protein expression of SREBPs were estimated. 18:2, 20:4, 20:5 and 22:6 fatty acids decreased the amount of mature SREBP-1 and mRNA levels of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase. SREBP-2 gene or mature protein expression was not altered. Liver X receptor (LXR) activation by T0901317 increased gene expression of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase without altering SREBP-2. 20:5, but not 18:1, prevented the full expression of SREBP-1c mRNA by T0901317. T0901317 increased SREBP-1 mass without altering the mass of mature SREBP-2. Although only 18:2, 20:4, 20:5 and 22:6 suppressed SREBP-1, acetyl-CoA carboxylase and FAS expression, all fatty acids decreased the rate of fatty acid synthesis. T0901317 increased endogenous fatty acid synthesis yet did not increase secretion of triacylglycerol-rich lipoproteins. In CaCo-2 cells, polyunsaturated fatty acids decrease gene and protein expression of SREBP-1 and FAS mRNA, probably through interference with LXR activity. Since all fatty acids decreased fatty acid synthesis, mechanisms other than changes in SREBP-1c expression must be entertained. Increased endogenous fatty acid synthesis does not promote triacylglycerol-rich lipoprotein secretion.

Effect of dietary fat saturation, cholesterol and cholestyramine on acyl-CoA: Cholesterol acyltransferase activity in rabbit intestinal microsomes

The regulation of intestinal acyl-CoA: cholesterol acyltransferase was investigated by dietary manipulation. Rabbits were fed the following diets: normal rabbit chow, 10% safflower oil, safflower oil plus 1% cholesterol, coconut oil plus 1% cholesterol, or cholestyramine. Acyl-CoA: cholesterol acyltransferase activity was increased in intestinal microsomes from animals fed safflower oil but not from animals fed coconut oil. Both diets containing cholesterol increased acyl-CoA: cholesterol acyltransferase activity; however, the safflower oil plus cholesterol diet was a more potent stimulator than coconut oil plus cholesterol. Cholestyramine decreased microsomal acyl-CoA: cholesterol acyltransferase activity. The different diets significantly modified microsomal lipid content in these groups. The two cholesterol diets resulted in equal increments in microsomal cholesterol. Microsomal cholesterol was unchanged in animals on the safflower oil diet; however, coconut oil and cholestyramine decreased the cholesterol content. Linoleic acid content increased in microsomes from animals on both the safflower oil-containing diets. Myristic acid accumulated and linoleic acid was decreased in microsomes from animals on both diets containing coconut oil. Subcellular fractionation of the intestine yielded a 4-fold enhancement of acyl-CoA: cholesterol acyltransferase activity in the rough endoplasmic reticulum. The lipid modifications and the subsequent changes in acyl-CoA: cholesterol acyltransferase activity in the rough endoplasmic reticulum from animals on normal, safflower oil, and safflower oil plus cholesterol diets paralleled that observed in the crude microsomal preparations. The changes in acyl-CoA: cholesterol acyltransferase activity observed with the different diets were not due to changes in microsomal fatty acyl-CoA pool size. It is concluded that dietary manipulation can alter microsomal lipid content. Microsomal fat saturation, independent of microsomal cholesterol content, regulates intestinal acyl-CoA: cholesterol acyltransferase and modifies the stimulatory effect of exogenous cholesterol on this enzyme.

Regulation of Rabbit Intestinal Acyl Coenzyme A-Cholesterol Acyltransferase In Vivo and In Vitro

The rate of intestinal cholesterol esterification may be an important determinant of the rates of entry and exit of cholesterol from the body. Acyl coenzyme A-cholesterol acyltransferase, the intracellular cholesterol esterifying enzyme, may play a role in this process. To assess this, the response of rabbit intestinal acyl coenzyme A-cholesterol acyltransferase in vivo was studied. Animals were fed a diet containing cholesterol and corn oil, and they responded with an increase in acyl coenzyme A-cholesterol acyltransferase activity. The increase was apparent in all segments of the intestine proximal to the distal ileum, and it occurred specifically in the villus cells where the bulk of lipid absorption is believed to take place. In cultured intestinal explants, the activity responded rapidly to sterols (increased) and to fatty acids (decreased) when control intestine was used. If intestine from cholesterol-corn oil-fed animals was cultured, sterols still induced an increase, but fatty acids did not affect the enzyme activity. The acutely induced increases in acyl coenzyme A-cholesterol acyltransferase activity were not prevented by cycloheximide. The results show that acyl coenzyme A-cholesterol acyltransferase in the absorptive cells of intestine responds both acutely and chronically to dietary factors, supporting the hypothesis that acyl coenzyme A-cholesterol acyltransferase plays a role in cholesterol absorption.

Regulation of cholesterol metabolism in the intestine

The small intestine is a major site of cholesterol biosynthesis and lipoprotein degradation. It is also the organ responsible for absorbing dietary and endogenously produced biliary cholesterol. Cholesterol metabolism in the intestine is regulated by factors that will alter cellular cholesterol requirements. Thus, during increased cholesterol flux, which occurs by bile acid-faciliated cholesterol absorption or by lipoprotein-mediated uptake of cholesterol, cholesterol synthetic rates decrease and esterification rates increase. The mechanisms by which dietary fats regulate intestinal cholesterol metabolism are complex. Dietary fats alter membrane fatty acid composition. Simultaneously, they also promote lipoprotein secretion and alter cholesterol absorption. Intestinal 3-hydroxyl-3-methylglutaryl coenzyme. A reductase activity is regulated by enzyme phosphorylation-dephosphorylation. The regulation of acylcoenzyme A-cholesterol acyltransferase activity by this mechanism remains controversial. Data on hormone regulation of intestinal cholesterol metabolism are not conclusive, although progesterone seems to be a potent inhibitor of acylcoenzyme A-cholesterol acyltransferase activity in intestinal cell culture and isolated cells. In a manner similar to the regulation of cholesterol metabolism in other cells, the enterocyte responds appropriately to factors that alter cholesterol flux. Therefore, changes that occur in the rates of cholesterol synthesis and esterification will reflect the cholesterol requirements of the cell.

Inhibition of acylcoenzyme A: cholesterol acyltransferase activity by PD128O42: effect on cholesterol metabolism and secretion in CaCo-2 cells.

The regulation of cholesterol uptake and secretion by acylcoenzyme A:cholesterol acyltransferase (ACAT) was investigated in the human intestinal cell line, CaCo-2. A new ACAT inhibitor, PD128042 (CI-976), was first characterized. The addition of the fatty acid anilide to membranes prepared from CaCo-2 cells inhibited ACAT activity without altering the activities of HMG-CoA reductase, fatty acid Co-A hydrolase, or triglyceride synthetase. PD128042 was a competitive inhibitor of ACTA with 50% inhibition occurring at a concentration of 0.2 μg/mL. When added to the medium of CaCo-2 cells at a concentration of 5 μg/mL, PD128042 inhibited oleate incorporation into cholesteryl oleate by 92% and increased oleate incorporation into triglycerides and phospholipids by 51% and 38%, respectively. After incubating CaCo-2 cells with the ACAT inhibitor, the rate of newly synthesized cholesterol decreased by 75% and membranes prepared from these cells contained significantly less HMG-CoA reductase activity. PD128042 significantly decreased the basolateral secretion of newly synthesized cholesteryl esters without affecting the secretion of newly synthesized triglycerides or phospholipids. The inhibitor decreased the esterification of labeled exogenous cholesterol which was taken up by the cell from bile salt micelles. Moreover, after 16 hr of ACAT inhibition, less labeled unesterified micellar cholesterol was associated with the cell. The esterification of cholesterol in CaCo-2 cells plays an integral role in the uptake of cholesterol through the apical membrane and its eventual secretion at the basolateral membrane.

Ezetimibe interferes with cholesterol trafficking from the plasma membrane to the endoplasmic reticulum in CaCo-2 cells

Niemann-Pick C1-like 1 protein (NPC1L1) is the putative intestinal sterol transporter and the molecular target of ezetimibe, a potent inhibitor of cholesterol absorption. To address the role of NPC1L1 in cholesterol trafficking in intestine, the regulation of cholesterol trafficking by ezetimibe was studied in the human intestinal cell line, CaCo-2. Ezetimibe caused only a modest decrease in the uptake of micellar cholesterol, but markedly prevented its esterification. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was profoundly disrupted by ezetimibe without altering the trafficking of cholesterol from the endoplasmic reticulum to the plasma membrane. Cholesterol oxidase-accessible cholesterol at the apical membrane was increased by ezetimibe. Cholesterol synthesis was modestly increased. Although the amount of cholesteryl esters secreted at the basolateral membrane was markedly decreased by ezetimibe, the transport of lipids and the number of lipoprotein particles secreted were not altered. NPC1L1 gene and protein expression were decreased by sterol influx, whereas cholesterol depletion enhanced NPC1L1 gene and protein expression. These results suggest that NPC1L1 plays a role in cholesterol uptake and cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. Interfering with its function will profoundly decrease the amount of cholesterol transported into lymph.

LXR/RXR ligand activation enhances basolateral efflux of β-sitosterol in CaCo-2 cells

To examine whether intestinal ABCA1 was responsible for the differences observed between cholesterol and β-sitosterol absorption, ABCA1-facilitated β-sitosterol efflux was investigated in CaCo-2 cells following liver X receptor/retinoid X receptor (LXR/RXR) activation. Both the LXR agonist T0901317 and the natural RXR/LXR agonists 22-hydroxycholesterol and 9-cis retinoic acid enhanced the basolateral efflux of β-sitosterol without altering apical efflux. LXR-mediated enhanced β-sitosterol efflux occurred between 6 h and 12 h after activation, suggesting that transcription, protein synthesis, and trafficking was likely necessary prior to facilitating efflux. The transcription inhibitor actinomycin D prevented the increase in β-sitosterol efflux by T0901317. Glybenclamide, an inhibitor of ABCA1 activity, and arachidonic acid, a fatty acid that interferes with LXR activation, also prevented β-sitosterol efflux in response to the LXR ligand activation. Influx of β-sitosterol mass did not alter the basolateral or apical efflux of the plant sterol, nor did it alter ABCA1, ABCG1, ABCG5, or ABCG8 gene expression or ABCA1 mass. Similar to results observed with intestinal ABCA1-facilitated cholesterol efflux, LXR/RXR ligand activation enhanced the basolateral efflux of β-sitosterol without affecting apical efflux. The results suggest that ABCA1 does not differentiate between cholesterol and β-sitosterol and thus is not responsible for the selectivity of sterol absorption by the intestine. ABCA1, however, may play a role in β-sitosterol absorption.

Effect of eicosapentaenoic acid on triacylglycerol transport in CaCo-2 cells.

The human intestinal cell line, CaCo-2, was used to study the effect of the n-3 fatty acid, eicosapentaenoic acid, on triacylglycerol secretion. In cells incubated with 250 microM eicosapentaenoic acid, the incorporation of [3H]glycerol into triacylglycerols secreted into the medium was decreased by 58% compared to cells incubated with 250 microM oleic acid. The incorporation of [3H]glycerol into cellular triacylglycerols was decreased 32% in cells incubated with eicosapentaenoic acid. In cells preincubated with [3H]glycerol to label existing triacylglycerols, the rates of secretion of preformed triacylglycerols were similar in response to the addition of either fatty acid. Initial uptake rates of the n-3 fatty acid were higher than for oleic acid. Both eicosapentaenoic acid and oleic acid were minimally oxidized to CO2. Oleic acid was predominantly incorporated into cellular triacylglycerols (62% vs. 47%), whereas more eicosapentaenoic acid was incorporated into cellular phospholipids (46% vs. 30%). Phospholipids of microsomes prepared from cells incubated with eicosapentaenoic acid were enriched in this fatty acid. The rate of synthesis of triacylglycerol and diacylglycerol acyltransferase activities were significantly less in microsomes prepared from cells incubated with eicosapentaenoic acid. Triacylglycerol mass secreted by CaCo-2 cells incubated with either fatty acid was similar. In CaCo-2 cells, eicosapentaenoic acid decreases the synthesis and secretion of newly synthesized triacylglycerol without decreasing the secretion of triacylglycerol mass. Modification of microsomal membrane phospholipid fatty acid composition is associated with a decrease in microsomal triacylglycerol synthesis and diacylglycerol acyltransferase activities.

TNF-alpha decreases ABCA1 expression and attenuates HDL cholesterol efflux in the human intestinal cell line Caco-2.

HDL cholesterol levels are decreased in Crohns disease, a tumor necrosis factor-α (TNF-α)–driven chronic inflammatory condition involving the gastrointestinal tract. ATP-binding cassette transporter A1 (ABCA1), one of several liver X receptor (LXR) target genes, is a cell surface transporter that mediates the rate-controlling step in HDL synthesis. The regulation of ABCA1 and HDL cholesterol efflux by TNF-α was investigated in the human intestinal cell line Caco-2. In response to cholesterol micelles or T0901317, an LXR nonsterol agonist, TNF-α decreased the basolateral efflux of cholesterol to apolipoprotein A1 (apoA1). TNF-α, by attenuating ABCA1 promoter activity, markedly decreased ABCA1 gene expression without attenuating the expression of LXR-α, LXR-β, and most other LXR target genes, such as ABCG1, FAS, ABCG8, scavenger receptor-B1 (SR-B1), and apoC1. TNF-α also decreased ABCA1 mass by markedly enhancing the rate of ABCA1 degradation and modestly inhibiting its rate of synthesis. Inhibitors of the nuclear factor-κB (NF-κB) pathway, which is activated by TNF-α, partially reverse the effect of TNF-α on ABCA1 protein expression. The results suggest that TNF-α, the major cytokine implicated in the inflammation of Crohns disease, decreases HDL cholesterol levels by attenuating the expression of intestinal ABCA1 and cholesterol efflux to apoA1.

Regulation of intestinal NPC1L1 expression by dietary fish oil and docosahexaenoic acid

To address the effect of the n-3 fatty acid, docosahexaenoic acid (22:6), on proteins that play a role in cholesterol absorption, CaCo-2 cells were incubated with taurocholate micelles alone or micelles containing 22:6 or oleic acid (18:1). Compared with controls or 18:1, 22:6 did not interfere with the cellular uptake of micellar cholesterol. Apical cholesterol efflux was enhanced in cells incubated with 22:6. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was decreased by 22:6. 22:6 decreased Niemann-Pick C1-Like 1 (NPC1L1) protein and mRNA levels without altering gene or protein expression of ACAT2, annexin-2, caveolin-1, or ABCG8. Peroxisome proliferator-activated receptor δ (PPARδ) activation decreased NPC1L1 mRNA levels and cholesterol trafficking to the endoplasmic reticulum, suggesting that 22:6 may act through PPARδ. Compared with hamsters fed a control diet or olive oil (enriched 18:1), NPC1L1 mRNA levels were decreased in duodenum and jejunum of hamsters ingesting fish oil (enriched 22:6). In an intestinal cell, independent of changes in ABCG8 expression, 22:6 increases the apical efflux of cholesterol. 22:6 interferes with cholesterol trafficking to the endoplasmic reticulum by the suppression of NPC1L1, perhaps through the activation of PPARδ. Moreover, a diet enriched in n-3 fatty acids decreases the gene expression of NPC1L1 in duodenum and jejunum of hamster.