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Am J Physiol Gastrointest Liver Physiol 295: G873-G885, 2008. First published September 4, 2008; doi:10.1152/ajpgi.90376.2008
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TRANSLATIONAL PHYSIOLOGY

Modulation of intestinal cholesterol absorption by high glucose levels: impact on cholesterol transporters, regulatory enzymes, and transcription factors

Z. Ravid,1,2 M. Bendayan,1,2 E. Delvin,1,3 A. T. Sane,1,4 M. Elchebly,1,3 J. Lafond,5 M. Lambert,1,6 G. Mailhot,1,4 and E. Levy1,4

1Research Center, CHU-Sainte-Justine, Departments of 2Pathology and Cell Biology, 3Biochemistry, 4Nutrition and 6Pediatrics, Université de Montréal; and 5Department of Biological Sciences, Université du Québec à Montréal, Montréal, Québec, Canada

Submitted 15 June 2008 ; accepted in final form 29 August 2008

ABSTRACT

Growing evidence suggests that the small intestine may contribute to excessive postprandial lipemia, which is highly prevalent in insulin-resistant/Type 2 diabetic individuals and substantially increases the risk of cardiovascular disease. The aim of the present study was to determine the role of high glucose levels on intestinal cholesterol absorption, cholesterol transporter expression, enzymes controlling cholesterol homeostasis, and the status of transcription factors. To this end, we employed highly differentiated and polarized cells (20 days of culture), plated on permeable polycarbonate filters. In the presence of [14C]cholesterol, glucose at 25 mM stimulated cholesterol uptake compared with Caco-2/15 cells supplemented with 5 mM glucose (P < 0.04). Because combination of 5 mM glucose with 20 mM of the structurally related mannitol or sorbitol did not change cholesterol uptake, we conclude that extracellular glucose concentration is uniquely involved in the regulation of intestinal cholesterol transport. The high concentration of glucose enhanced the protein expression of the critical cholesterol transporter NPC1L1 and that of CD36 (P < 0.02) and concomitantly decreased SR-BI protein mass (P < 0.02). No significant changes were observed in the protein expression of ABCA1 and ABCG8, which act as efflux pumps favoring cholesterol export out of absorptive cells. At the same time, 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity was decreased (P < 0.007), whereas ACAT activity remained unchanged. Finally, increases were noted in the transcription factors LXR-{alpha}, LXR-β, PPAR-β, and PPAR-{gamma} along with a drop in the protein expression of SREBP-2. Collectively, our data indicate that glucose at high concentrations may regulate intestinal cholesterol transport and metabolism in Caco-2/15 cells, thus suggesting a potential influence on the cholesterol absorption process in Type 2 diabetes.

ABCA1; ABCG5/G8; SR-BI; CD36; NPC1L1; PPAR; LXR; SREBP; ACAT; HMG-COA reductase

ELEVATED PLASMA CHOLESTEROL levels constitute a major risk factor for atherosclerosis and coronary heart diseases (CHD) (60). Whole body cholesterol balance is regulated by the net effects of dietary cholesterol absorption, de novo cholesterol biosynthesis, and biliary excretion from the liver (21, 49). Available evidence supports the concept that several proteins are involved in mediating intestinal cholesterol transport. Whereas various transporters, including fatty acid translocase/cluster determinant 36 (FAT/CD36), scavenger receptor class B type I (SR-BI), and Niemann-Pick C1-Like 1 (NPC1L1), may influence cholesterol uptake, the ATP binding cassette (ABC) transporter family, including several cholesterol carriers (ABCA1, ABCB1, ABCG5/G8), act as efflux pumps favoring cholesterol export out of absorptive cells into the lumen or basolateral compartment. Among all the cholesterol transporters, the enriched NPC1L1 protein in the apical membrane of polarized cells is considered essential for intestinal cholesterol absorption. To provide only a few examples, 1) mice deficient in NPC1L1 lack the ability to absorb cholesterol and exhibit prevailing protection against the rise in plasma and hepatic cholesterol associated with feeding mice high-cholesterol diets (5, 22, 23); 2) genetic modifications of NPC1L1 in cultured intestinal cells alter cholesterol uptake (73, 88, 90); and 3) variations in NPC1L1 were found associated with reduced sterol absorption, LDL-cholesterol levels (17) and LDL-cholesterol response to ezetimibe therapy (41, 75, 84). Although, various aspects of NPC1L1, as a cell surface transporter or an intracellular cholesterol transport protein needs clarification (43), intensive research is focused on drugs that interact with NPC1L1 given their potential to treat individuals with hypercholesterolemia and to reduce their risk of developing CHD.

The prevalence of diabetes is increasing worldwide, and CHD is the leading cause of death in Type 2 diabetes mellitus (T2DM) (35). Patients with T2DM are two to three times more likely to die from CHD than nondiabetic individuals (31, 77). Therefore, considerable attention has been focused on the dyslipidemia accompanying diabetes and metabolic syndrome. Elevated liver very-low-density lipoprotein production represents a major pathway of the hypertriglyceridemia that characterizes the diabetic condition. On the other hand, a significant relationship has been shown between intestinally derived triacylglycerol (TG)-rich lipoproteins and the progression of atherosclerosis. Increased cholesterol absorption has also been described in patients with T2DM (36, 52). Although numerous investigations have attempted to elucidate the abnormal mechanisms of intestinal cholesterol absorption process in diabetic dyslipidemia, they have not given full consideration of nutrients other than TG and cholesterol. However, it has been reported that changing the carbohydrate content of a mixed meal altered the postprandial accumulation of chylomicrons (37). Furthermore, a high glucose level alters the genetic expression of various genes involved in high-density lipoprotein (HDL) metabolism in HepG2 cells, including human ABCA1, SR-BI, and hepatic lipase (79). The aims of the present study were 1) to evaluate the effect of a high glucose concentration on cholesterol absorption and 2) to explore the influence of an elevated glucose level on the expression of genes regulating cholesterol synthesis and absorption in a cell culture system.

MATERIALS AND METHODS

Cell culture. The Caco-2/15 cell line was obtained from Dr. J. F. Beaulieu (Department of Cellular Biology, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Quebec, Canada). This clone of the parent Caco-2 cell line (HTB37; American Type Culture Collection, Manassas, VA) has been extensively characterized (3, 6, 80) and was originally selected for expressing the highest level of sucrase-isomaltase among 16 clones obtained by random cloning. Caco-2/15 cells were grown at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) (GIBCO-BRL, Grand Island, NY) containing 1% penicillin-streptomycin and 1% Glutamax (GIBCO-BRL) and supplemented with 10% decomplemented fetal bovine serum (FBS) (Flow, McLean, VA). Caco-2/15 cells (passages 40–60) were maintained in T-75-cm2 flasks (Corning Glass Works, Corning, NY). Cultures were split (1:6) when they reached 70–90% confluence, by use of 0.05% trypsin-0.5 mM EDTA (GIBCO-BRL). For individual experiments, cells were plated at a density of 1 x 106 cells/well on 24.5-mm polycarbonate Transwell filter inserts with 0.4-µm pores (Costar, Cambridge, MA), in DMEM (as described above) supplemented with 5% FBS. The inserts were placed into six-well culture plates, permitting separate access to the upper and lower compartments of the monolayers. Cells were cultured for various periods, including 21 days, at which the Caco-2 cells are highly differentiated and appropriate for lipid metabolism (34, 54, 55, 61, 73). The medium was refreshed every second day. At day 21, Caco-2/15 cells were washed twice with phosphate-buffered saline (PBS) (Invitrogen) and incubated in a serum-free supplemented DMEM (Invitrogen) (5 mM or 25 mM glucose), added to the apical and basolateral compartments, for 24 h.

Cholesterol absorption by Caco-2/15 cells. To study cholesterol uptake by the cells, a solution containing 0.113 µCi [14C]cholesterol and 100 µM cholesterol bound to albumin was prepared. The differentiated cells were incubated at 37°C for 30 min and 4 h in DMEM containing 5 or 25 mM glucose, as well as cholesterol solution. At the end of the treatment, cells were washed twice with PBS, scrapped in 1 ml lysis buffer (5 mM Tris, 15 mM NaCl, EDTA 5 mM, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate) and homogenized by sonication followed by a 5-min at 13,800 g centrifugation to remove cell debris. An aliquot of 0.1 ml was placed in a scintillation vial with Ready Safe counting fluid (Beckman, Fullerton, CA). Radioactivity was measured by scintillation counting (LS 5000 TD, Beckman). Cell protein was quantified by the Bradford method (Bio-Rad).

ACAT activity assay. The activity of acyl-coenzyme A:cholesterol acyltransferase (ACAT) was determined at initial rates by adding 5 nmol of [14C]oleoyl-CoA (specific activity ~167 Bq/nmol) to the mixture containing 190 µg of cellular protein to initiate the reaction in a buffer solution (pH 7.5) consisting of cholesterol, 0.04 M KH2PO4, 50 mM NaF, 0.25 M sucrose, and 1 mM EDTA (73). After incubation for 10 min at 37°C, the reaction was stopped by adding chloroform-methanol (2:1, vol/vol) followed by free cholesterol (FC) and cholesteryl ester (CE) as carriers. The FC and CE formed were isolated by TLC and counted.

HMG-CoA reductase activity assay. Enzymatic activity was assayed as described previously (19, 73). The reaction mixture contained 100 mM potassium phosphate (pH 7.4), 200 µg of cellular protein, 20 mM glucose-6-phosphate, 12.5 mM dithiothreitol, 2.5 M NADP, and 1.2 units of glucose-6-phosphate dehydrogenase. Initiation of the reaction was done by the addition of [14C]3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) (200 Bq/nmol) for 30 min at 37°C. The [14C]mevalonate formed was converted into lactone by the addition of 10 N HCl, isolated by TLC, and counted by using an internal standard to correct for incomplete recovery.

Western blot. To assess the presence of NPC1L1, SR-B1, CD36, ABCA1, ABCG8, ACAT, and HMG-CoA reductase, Caco-2/15 cells were homogenized and prepared for Western blotting as described previously (56). The Bradford assay (Bio-Rad) was used to determine protein concentration. Proteins were denatured in sample buffer containing SDS and β-mercaptoethanol, separated on a 7.5% SDS-PAGE gel, and blotted onto nitrocellulose membranes. Nonspecific binding sites of the membranes were blocked with 5% defatted milk proteins. Reactions took place by the addition of primary antibodies directed against targeted proteins. Reaction was revealed with species-specific horseradish peroxidase-conjugated secondary antibody. β-Actin was used as an internal control to confirm equal loading protein on SDS-PAGE. Blots were developed and proteins were quantified by use of a Hewlett-Packard scanner equipped with a transparency adaptor and UN-SCAN-IT (Silk Scientific) software.

Immunocytochemical analysis. Caco-2/15 cells grown for 21 days and exposed to either low (5 mM) or high (25 mM) glucose medium were fixed with 1% glutaraldehyde in 0.1 M phosphate buffer for 2 h and processed for embedding in Lowicryl at –30°C as described in details previously (7, 55, 58, 76). Thin sections were mounted on Parlodion-carbon coated grids and processed for the immunogold labeling. Various proteins were studied, namely SR-B1, NPC1L1, ABCA1, ABCG8, and CD36. The thin sections of the cells were first treated with a saturated solution of sodium metaperiodate for 10 min, followed by 1% ovalbumin, and then incubated overnight at 4°C with the corresponding antibody. Grids were thoroughly rinsed with PBS and incubated with the protein A-gold or an anti-rabbit IgG-gold complex for 30 min at room temperature. Upon counterstaining with uranyl acetate, the sections were examined with a Philips 410 electron microscope. The antibodies were used at the following dilutions: NPC1L1 at 1:10, SRB1 at 1:50, CD36 at 1:10, ABCA1 at 1:10, and ABCG8 at 1:10. To assess specificity on the labeling, control experiments were performed omitting the incubation with the primary antibody. Grids were only exposed to the protein A-gold or the anti-rabbit IgG-gold complex for 30 min. For morphometrical evaluations, a large number of photographs were recorded at the original magnification of x14,000; they were scanned and printed to the final magnification of x28,000. The specific membrane domain was selected for morphometrical evaluation according to the specific localization of the transporters, i.e., the apical membrane with its large number of microvilli or the basolateral membrane with its deep invaginations. First, the length of the membrane was measured and then the number of gold particles delineating the same membranes was counted. Results are expressed in number of gold particles per micrometer (mean values ± SD). An image processing system (Videoplan 2, Carl Zeiss Toronto) was used. For each of the experiments and for each protein studied, the length of apical membrane evaluated was in the range of ~800 µm, whereas that of the basolateral membrane was in the range of ~250 µm. The major difference in membrane length evaluated between apical and basolateral membranes is due to the presence of the large number of microvilli in the apical membrane. Morphometrical evaluations were also performed on the control experiments.

RNA isolation. Total RNA was isolated from Caco-2/15 cells by using the TRIzol reagent according to the manufacturer's instructions (Sigma Chemical). Concentrations of RNA were determined by spectrophotometer analysis and the integrity of total RNA was assessed by electrophoresis.

RT-PCR. PCR experiments for transcription factors [liver X receptors (LXRs), retinoid X receptors (RXRs), peroxisome proliferator-activated receptors (PPARs), sterol regulatory element binding protein-2 (SREBP-2)] genes, ACAT, and HMG-CoA reductase, as well as GAPDH (as a control gene) were performed by using the mastercycler gradient (Eppendorf). Specific primers were designed to bind to regions with minimal homology, to span at least one intron for distinction from genomic DNA and to avoid nonspecific annealing (Table 1). All primers were Blast searched to confirm specificity for each individual isoform. Approximately 30–40 cycles of amplification were used at 95°C for 30 s, 53–62°C for 30 s, and 72°C for 30 s. Amplicons were visualized on standard ethidium bromide-stained agarose gels. For all RT-PCRs, analysis of mRNA expression was carried out during the exponential phase of the amplification, which was assessed in preliminary experiments for each pair of primers.


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  		Table 1. Sequences of the specific primers, as confirmed by BLAST sequence analysis, designed to amplify human HMG-CoA reductase, ACAT-2, LXRs, PPARs, RXRs and SREBP-2 isoforms by RT-PCR

 
Statistical analysis. Unless otherwise stated, all values are given as mean values ± SD. Data were assessed by Student's two-tailed t-test. A P value <0.05 was considered statistically significant.

RESULTS

Cholesterol absorption. Following preincubation (24 h) of Caco-2/15 cells with medium containing 5 or 25 mM glucose, cholesterol uptake was determined at short- and long-term incubation times. As illustrated by Fig. 1, cells exposed to 25 mM glucose displayed a higher capacity to incorporate cholesterol compared with cells treated with 5 mM glucose (P < 0.04). Furthermore, compared with control cells, the output of cholesterol at 6 h (23%, P < 0.04) was also augmented at the long-term incubation time (data not shown).


Figure 1
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  		Fig. 1. Effect of glucose concentrations on cholesterol (chol) uptake in Caco-2/15 cells. Differentiated Caco-2/15 cells were cultured for 24 h in DMEM containing 5 or 25 mM glucose. At the end of this preincubation, cells were exposed to 100 µM cholesterol containing 250,000 dpm [14C]cholesterol in the presence of the same concentrations of glucose for 30 min. Results are expressed as mean values ± SD, in nmol/mg cell protein (prot). N = 4. *P < 0.04 vs. 5 mM glucose condition.

 
To determine whether the influence of glucose on cholesterol transport could possibly be explained by the difference in the osmolarity of the 5 and 25 mM glucose solutions, we incubated Caco-2/15 cells with mannitol and sorbitol combined to 5 mM glucose. The cholesterol uptake of cell monolayers maintained in 5 mM glucose plus 20 mM mannitol or sorbitol did not differ from cells maintained in physiological glucose (5 mM) media (Fig. 2). We therefore conclude that extracellular glucose concentration is uniquely involved in the regulation of intestinal cholesterol transport.


Figure 2
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  		Fig. 2. Combinatory effect of glucose (gluc) and mannitol (mann) or sorbitol (sorb) on cholesterol uptake in Caco-2/15 cells. Differentiated Caco-2/15 cells were cultured for 24 h in medium DMEM containing 5 mM, 5 mM glucose+20 mM mannitol, 5 mM glucose+20 mM sorbitol, or 25 mM glucose. At the end of this preincubation, cells were exposed to 100 µM cholesterol containing 250,000 dpm [14C]cholesterol in the presence of the same concentrations of sugars for 30 min. Results are expressed as mean values ± SD, in nmol/mg cell protein. N = 4. *P < 0.04 vs. 5 mM glucose condition.

 
Protein expression of cholesterol transporters assessed by Western blot. The enhanced cholesterol uptake exhibited by Caco-2/15 cells incubated with the high concentration (25 mM) of glucose may be due to differences in the expression of cholesterol transporters. To test this hypothesis, the protein expression of cholesterol transporters present in intestinal epithelial cells was examined. We assessed NPC1L1, CD36, and SR-B1 that transport cholesterol into the enterocyte, as well as ABCA1 and ABCG8 that are presumed to be involved in cholesterol efflux from the enterocyte toward plasma HDL or back into the intestinal lumen, respectively. Exposure to 25 mM glucose compared with 5 mM glucose resulted in a significant increase in the protein expression of NPC1L1 and CD36 along with a decrease in the protein expression of SR-B1 (Fig. 3). On the other hand, the protein expression of both ABCA1 and ABCG8 was not affected by the different glucose concentrations (Fig. 4).


Figure 3
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  		Fig. 3. Effect of glucose concentrations on the protein expression of transporters mediating cholesterol influx. Caco-2/15 cells were cultured for 24 h in DMEM containing 5 or 25 mM glucose. Western blot was used to analyze the protein expression of Niemann-Pick C1-Like 1 (NPC1L1; A), cluster determinant 36 (CD36; B), and scavenger receptor class B type I (SR-B1; C). Values are means ± SD. N = 4. *P < 0.02 vs. 5 mM glucose condition.

 

Figure 4
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  		Fig. 4. Effect of glucose concentrations on the protein expression of transporters mediating cholesterol efflux. Caco-2/15 cells were cultured for 24 h in DMEM containing 5 or 25 mM glucose. Western blot was used to analyze the protein expression of ABCA1 (A) and ABCG8 (B). Values are means ± SD. N = 4.

 
Protein levels of cholesterol transporters assessed by high-resolution quantitative immunogold approach. Since Western blotting measures the total protein mass without being able to distinguish the cellular localization of cholesterol transporters, we employed the protein A-gold immunocytochemical technique, to determine whether alterations in cholesterol transporters, as a function of glucose concentrations, were associated with their specific membrane domain. Electron microscopic immunocytochemical experiments mostly confirmed the findings obtained by Western blot. They revealed significant increases in immunogold labelings for NPC1L1 and CD36 in the luminal region of enterocytes, particularly associated with the apical plasma membrane lined by the microvilli (Fig. 5). A representative illustration documents the immunochemical detection of CD36 in Caco-2/15 cells following exposure to different glucose levels (Fig. 6A: 5 mM; Fig. 6B: 25 mM). However, the labeling of ABCA1 by gold particles was decreased in the basolateral membrane following the addition of 25 mM glucose (Fig. 5). Furthermore, no significant alterations were noted in the intensity of labeling of SR-BI and ABCG8 in the apical membrane (Fig. 5). Importantly, under control conditions, the labeling was negligible and the few gold particles present over the cells were rather randomly distributed.


Figure 5
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  		Fig. 5. Immunocytochemical evaluation of cholesterol transporters following incubation of Caco-2/15 cells were cultured for 24 h in DMEM containing 5 or 25 mM. Thereafter, cells were fixed with 1% glutaraldehyde and embedded in Lowicryl. Immunogold labelings were carried out on thin sections. Immunogold labeling for NPC1L1 (A), CD36 (B), SR-B1 (C), ABCA1 (D), and ABCG8 (E) were quantified. For each of the proteins, the total length of apical and basolateral membranes evaluated was in the range of 800 and 250 µm, respectively. Under control conditions, the labeling was negligible with few gold particles randomly distributed over the cells. For the 5 and 25 mM glucose experiments, the control protein A-gold density (gold particles/µm) on apical membrane was 0.037 ± 0.006 and 0.023 ± 0.012, respectively, and on basolateral region 0.076 ± 0.048 and 0.063 ± 0.012, respectively. For the control IgG gold, the density (gold particles/µm) on the apical membrane was 0.021 ± 0.005 and 0.015 ± 0.007 for 5 and 25 mM glucose, respectively, and on basolateral membrane 0.037 ± 0.018 and 0.050 ± 0.015, respectively. Values are means ± SE. *P < 0.05.

 

Figure 6
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  		Fig. 6. CD36 detection in Caco-2/15 cells as a representative illustration of the immunocytochemical detection. Protein A-gold immunocytochemical technique was applied with the specific polyclonal antibody directed against CD36 to reveal it in the apical membrane. A: Caco-2/15 cells cultured with 5 mM glucose. B: Caco-2/15 cells cultured with 25 mM glucose. MV, microvilli. Bars = 0.5 µm.

 
Involvement of NPC1L1 in cholesterol uptake. To elucidate the specific contribution of NPC1L1 to cholesterol uptake in the presence of glucose, we employed ezetimibe, a selective hypocholesterolemic drug, which has been reported to bind NPC1L1 and substantially block cholesterol absorption. We could observe a lessened action of glucose when ezetimibe was added to the culture medium (Fig. 7), highlighting the input of NPC1L1.


Figure 7
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  		Fig. 7. Interference of ezetimibe with the uptake of cholesterol in the presence of glucose. To delineate the role of NPC1L1 in the increased glucose-mediated cholesterol absorption, ezetimibe (100 µM) was added to the medium of Caco-2/15 cells cultured at the experimental conditions described in the legend of Fig. 2. Results are expressed as % of controls. Values are means ± SD. N = 3. *P < 0.05 vs. 5 mM glucose condition.

 
Regulatory enzymes of cholesterol metabolism. Next, we determined the impact of glucose on the regulatory sterol enzymes: HMG-CoA reductase (EC 1.1.1.34 [EC] ), the rate-limiting step in cholesterol synthesis, and ACAT (EC 2.3.1.26 [EC] ), an integral protein present in the rough endoplasmic reticulum (ER) that catalyzes the formation of CE from FC and fatty acyl-CoA. HMG-CoA reductase mRNA and protein expression remained unchanged following increase of glucose to 25 mM glucose to Caco-2/15 cells (Fig. 8). On the other hand, the higher glucose concentration led to a significant reduction in the enzymatic activity of HMG-CoA reductase (Fig. 8). As to ACAT, no significant changes were recorded in the gene expression and activity after the exposure to 25 mM glucose (Fig. 9).


Figure 8
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  		Fig. 8. Effect of glucose concentrations on 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase gene expression, protein mass, and activity. Caco-2/15 cells were treated as described in the legend of Fig. 3 and tested for transcript levels (A) and protein mass (B) by RT-PCR and Western blotting, respectively. Cell homogenates were assayed for HMG-CoA reductase activity (C). Values are means ± SD. N = 4. *P < 0.007 vs. 5 mM glucose condition.

 

Figure 9
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  		Fig. 9. Effect of glucose concentrations on acyl-coenzyme A:cholesterol acyltransferase (ACAT) gene expression and activity. Caco-2/15 cells were treated as described in the legend of Fig. 3 and tested for transcript levels (A) by RT-PCR. Cell homogenates were assayed for ACAT activity (B). Values are means ± SD. N = 4.

 
Transcription factors. To approach the mechanisms triggered by glucose, we assessed the gene expression of several factors that affect the transcription of a variety of genes associated with lipid and cholesterol metabolism, including liver X receptors (LXR-{alpha}, β), peroxisome proliferator-activated receptors (PPAR-{alpha}, -β, -{gamma}), retinoid X receptors (RXR-{alpha}, -β), and protein and gene expression of sterol regulatory element binding protein-2 (SREBP-2). Data in Figs. 10–12 illustrate how glucose at the high concentration of 25 mM impacted on the expression of the different nuclear and transcription factors in Caco-2/15 cells. It did not cause any significant variation on the mRNA levels of RXR-{alpha} (Fig. 10C), RXR-β (Fig. 10D), PPAR-{alpha} (Fig. 11A), and SREBP-2 (Fig. 12 A) gene expression, whereas it produced a significant enhancement in gene expression of LXR-{alpha} (Fig. 10A) and LXR-β (Fig. 10B), as well as PPAR-β (Fig. 11B) and PPAR-{gamma} (Fig. 11C). Finally, when we explored the effect of glucose on SREBP-2 protein expression, we detected a significant reduction upon exposure to 25 mM glucose (Fig. 12B).


Figure 10
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  		Fig. 10. Effect of glucose concentrations on the gene expression of the nuclear receptors liver X receptor (LXR) and retinoid X receptor (RXR). Caco-2/15 cells were treated as described in the legend of Fig. 3. The transcript levels of LXR-{alpha} (A), LXR-β (B), RXR-{alpha} (C), and RXR-β (D) were assessed by RT-PCR. Representative autoradiograms of the different amplicons are shown. Values are means ± SD. N = 5. *P < 0.004 and **P < 0.001 vs. 5 mM glucose condition.

 

Figure 11
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  		Fig. 11. Effect of glucose concentrations on the gene expression of the nuclear receptors peroxisome proliferator-activated receptors (PPAR). Caco-2/15 cells were treated as described in the legend of Fig. 3. The transcripts of PPAR-{alpha} (A), PPAR-β (B) and PPAR-{gamma} (C) were assessed by RT-PCR. Representative autoradiograms of the different amplicons are shown. Values are means ± SD. N = 5. *P < 0.003 and **P < 0.002 vs. 5 mM glucose condition.

 

Figure 12
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  		Fig. 12. Effect of glucose concentrations on the gene and protein expression of sterol regulatory element binding protein-2 (SREBP-2). Caco-2/15 cells were treated as described in the legend of Fig. 3. The levels of transcripts (A) and protein expression (B) were determined by RT-PCR and Western blotting, respectively. Values are means ± SD. N = 5. *P < 0.04 vs. 5 mM glucose condition.

 
DISCUSSION

Numerous studies have dealt with the regulation of intestinal fat absorption by lipid components (27, 57, 61, 70). However, the role of carbohydrates has barely been investigated. In the present paper, we showed that high glucose concentrations 1) enhance cholesterol transport in Caco-2/15 cells by upregulating the protein expression of NPC1L1 and CD36 and 2) reduce SR-BI protein mass and HMG-CoA reductase activity without altering ABCA1 and ABCG8, involved in cholesterol efflux. A schematic diagram (Fig. 13) depicts the major players in cholesterol transport and metabolism. Moreover, our studies document that particular transcription factors are glucose sensors, which may explain the impact of glucose on cholesterol absorption via its action on specific cholesterol transporters.


Figure 13
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  		Fig. 13. Diagram of the main players influencing cholesterol transport in intestinal epithelial cells. Uptake of alimentary or biliary cholesterol is mediated by putative sterol transporters such as NPC1L1, SR-BI, and fatty acid translocase/cluster determinant 36 (FAT/CD36). Excessive cholesterol is secreted back to the intestinal lumen by the ABCG5/ABCG8 heterodimer localized at the apical membrane of the enterocyte. Similarly, ABCA1 promotes cholesterol efflux (through the basolateral membrane) to plasma apolipoprotein A-I, thereby enhancing the formation of nascent HDL. Key enzymes such as HMG-CoA reductase and ACAT contribute to cholesterol homeostasis by synthesizing and esterifying, respectively, intracellular cholesterol. Most of the processes, involved in cholesterol metabolism, are controlled by transcription factors (RXR, LXR, SREBP-2, and PPAR). According to our data, glucose-mediated cholesterol uptake may be sensed by transcription factors, which in turn altered the cholesterol transporters and downregulated cholesterol biosynthesis.

 
In the present study, we have found a relationship between glucose and cholesterol assimilation. To clarify whether this observation could be explained by differences in the osmolarity of the 5 and 25 mM glucose solutions, we incubated Caco-2/15 cells with 20 mM mannitol or sorbitol combined to 5 mM glucose. Our results indicate that the exposure of Caco-2/15 cells to high concentrations of glucose, but not to structurally related compounds such as mannitol and sorbitol, increased cholesterol transport capacity.

NPC1L1 is a critical protein for cholesterol absorption by the small intestine, since NPC1L1 knockout mice exhibited a reduction in intestinal cholesterol absorption and are insensitive to ezetimibe (5, 23), a drug that lowers serum cholesterol by reducing cholesterol absorption. In addition, the use of genetically modified intestinal epithelial cells and ezetimibe support the central role for NPC1L1 in intestinal cholesterol absorption (28, 31, 73, 90). The high glucose level used in our investigation raised not only cholesterol uptake but also the protein expression of NPC1L1. Our results are in line with the findings in diabetic patients who displayed increased levels of NPC1L1 mRNA in intestinal tissue (52). Increased cholesterol absorption has also been shown in streptozotocin diabetic rats (89), in which NPC1L1 mRNA was found to be increased (51). Altogether, these findings suggest an important role for intestinal NPC1L1 in the delivery of cholesterol to the blood circulation in the presence of high glucose levels. However, additional studies are needed to examine whether glucose may influence the absorption of cholesterol via nonmediated passive uptake of cholesterol or other intestinal transporters since residual cholesterol absorption persisted in NPC1L1-deficient mice (5).

In the present investigation, the protein CD36 was highly expressed in intestinal luminal surface of enterocytes (82) and was found to be raised upon exposure to high glucose levels. Undoubtedly, CD36 does contribute to the intestinal transport of cholesterol since enterocytes isolated from Cd36–/– mice exhibit reduced uptake of cholesterol (60%) (68). From the present experiments, we can deduce that elevated glucose-mediated cholesterol uptake is likely related to the upregulation of NPC1L1 and CD36. Interestingly, the participation of NPC1L1 and CD36 was reinforced by the experiments with ezetimibe, although the former displayed more sensitivity to ezetimibe inhibition (32) than the latter (82). Of note is the modest decrease in cholesterol uptake from the apical side, in line with the studies of Field et al. (28), probably because the glucuronidated form of ezetimibe is more potent than the native unmodified drug in inhibiting cholesterol absorption by binding more avidly to enterocyte brush-border membranes (83).

SR-BI was originally identified as a novel scavenger receptor that mediates endocytosis of acetylated LDL (2). Subsequent studies revealed that SR-BI is a cell surface receptor that binds HDL with high affinity and mediates the selective uptake by liver and steroidogenic tissues of cholesterol esters without endocytic uptake of HDL apolipoproteins (1). Efflux of radiolabeled cholesterol on the cell surface to HDL particles is also promoted by SR-BI (45). Together, SR-BI accelerates reverse cholesterol transport by promoting cholesterol efflux from peripheral cells, including macrophages in vascular walls (16), and selective uptake of HDL-cholesterol by hepatocytes for excretion of cholesterol as bile acids. Therefore, SR-BI plays crucial roles in the atheroprotective functions of HDL (48). Additional investigations have reported that SR-BI is highly expressed in the luminal side of proximal small intestine villi where the bulk of cholesterol absorption takes place and may be responsible for the cholesterol uptake by enterocytes (4, 39, 55). Nevertheless, the involvement of SR-BI in cholesterol absorption has been questioned since variable results were obtained with genetically modified mice (8, 62). In the present investigation, glucose in high concentrations downregulates SR-BI protein expression. This suppressive effect has also been reported in hepatocytes HepG2 cells following exposure to high glucose concentrations (67). The use of inhibitors for select signal transduction pathways in HepG2 cells indicated that glucose suppression of SR-BI expression is partially mediated by the activation of the p38 MAPK-Sp1 pathway (67). Further studies are needed to determine the detailed regulatory mechanisms of intestinal SR-BI expression.

We reasonably hypothesized that increased NPC1L1- and CD36-mediated cholesterol uptake would lead to reduced HMG-CoA reductase activity. On the basis of the data in Fig. 8, this assumption turned out to be true. However, ACAT was insensitive to the accumulation of intracellular cholesterol, probably because the latter did not expand the finite cholesterol substrate pool for ACAT and manifested a high-order dependence on ER cholesterol concentration (11, 14).

The coordinated regulation of genes implicated in cholesterol homeostasis is governed by the actions of several transcription factors, such as LXRs and PPARs. Also, SREBPs are transcription factors and crucial regulators of cholesterol synthesis and metabolism. In response to specific effectors, LXRs form heterodimers with RXRs that regulate an integrated network of genes that control whole body cholesterol and lipid homeostasis assays (44, 53). In particular, LXR appears to serve as a safety valve to limit free cholesterol in tissues that are experiencing high cholesterol flux (20). Moreover, glucose activates LXR at physiological concentrations expected in the liver and induces expression of LXR target genes with efficacy similar to that of oxysterols, the known LXR ligands (66). Therefore, since these nuclear factors act as glucose sensors (66) and exhibit antidiabetic effects (13, 50), we first measured their gene expression. LXR-{alpha} and LXR-β mRNA were increased by the presence of 25 mM glucose in Caco-2/15 cells but were not accompanied with the expected induction of ABCA1 and ABCG8 protein expression. This may be due to the irresponsiveness of RXRs that work as partners with LXRs.

The family of SREBP regulates the coordinated expression of genes involved in lipid synthesis and uptake (10). Three SREBP isoforms are known in mammals: SREBP-1a, SREBP-1c, and SREBP-2. Whereas SREBP-1c preferentially activates genes required for fatty acid (FA) synthesis and their incorporation into TGs and phospholipids, SREBP-2 preferentially activates the LDL-receptor gene and various genes required for cholesterol synthesis such as HMG-CoA reductase (EC1.1.1.34) (42). SREBP-1a is an activator of both the cholesterol and the FA biosynthetic pathway, but it is present in much lower amounts (74). Since SREBP-2 plays a more important role in the regulation of cholesterol synthesis in the intestine (25, 26), we measured the gene and protein expression of SREBP-2. Our results highlighted a decline in the protein expression of SREBP-2 without any alteration of SREBP-2 mRNA. Similarly, the gene and protein expression of HMG-CoA reductase were not changed, but the activity was decreased, suggesting posttranscriptional mechanisms for SREBP-2 and HMG-CoA reductase.

PPARs have been shown to regulate the expression of genes involved in a variety of biological processes, including lipid metabolism and insulin sensitivity (18, 81). PPAR-{alpha} regulates numerous aspects of FA catabolism, whereas PPAR-{gamma} controls adipocyte differentiation, systemic glucose levels and lipid homeostasis (65, 87). PPAR-{delta} is also involved in development, lipid metabolism, and epidermal cell proliferation (59). The PPARs are ligand-dependent transcription factors that regulate target genes expression by binding to characteristic DNA sequences termed peroxisome proliferator response element (PPREs) located in the 5'-flanking region of target genes (33, 69). Each receptor binds to its PPRE as a heterodimer with the receptor for 9-cis-retinoic acid, the RXR. Upon binding a ligand, the conformation of a PPAR is altered and stabilized so that a binding cleft is created, and recruitment of transcriptional coactivators occurs. In the present study, treatment of Caco-2/15 cells with 25 mM glucose enhanced the gene expression of PPAR-β and PPAR-{gamma} and concomitantly decreased the protein concentration of SREBP-2. Similarly, PPAR-{gamma} activation by troglitazone downregulated cholesterol synthesis in Caco-2 cells by a reduced concentration of SREBP-2 protein (47).

Glucose absorption in the small intestine is mediated by the combined action of two glucose transporters in the enterocytes: the sodium-dependent D-glucose cotransporter SGLT1 in the brush-border membrane and the sodium-independent glucose transporter GLUT2 in the basolateral membrane (40, 46). Dietary glucose significantly influences blood glucose concentration (63), as well as glucose and fat metabolism in the liver (15, 78). Apparently mammals developed a highly complex regulatory network in which small intestinal sugar absorption steers the regulation of gastric and intestinal mobility (30, 72), liver metabolism (9, 29), and insulin secretion (64, 87). Both the glucose concentration in the small intestinal lumen and the activity of glucose uptake systems SGLT1 and GLUT2 determine the D-glucose concentration in the small intestinal submucosa and the portal blood. In the present study, glucose was effective in regulating cholesterol uptake and intracellular processing confirming the pioneer work by Play et al. (70a). Since the excessive consumption of diets containing high levels of carbohydrates enhances the absorption of monosaccharides and influences the risk of developing insulin resistance and T2DM (24), we reasonably propose that glucose-mediated intestinal cholesterol may contribute to increasing circulating cholesterol and, consequently, the risk of developing CHD, a feature of T2DM.

In conclusion, our experiments provide evidence that the process of intestinal cholesterol uptake is regulated by glucose concentrations, which modify important cholesterol transporters and transcription factors. In fact, high glucose concentrations may presumably modify the transcription factors, which in turn altered the cholesterol transporters and therefore cholesterol uptake.

GRANTS

The present work was supported by research grants from the Canadian Institutes of Health Research (E. Levy, MOP#49433), the Canadian Diabetes Association (E. Levy), and Diabète Québec (M. Bendayan). The authors thank Schohraya Spahis, Carole Garofalo, and Alice Bendayan for expert technical assistance.

FOOTNOTES


Address for reprint requests and other correspondence: E. Levy, Research Centre, CHU-Sainte-Justine, 3175 Côte Ste-Catherine, Montréal, Québec, Canada H3T 1C5 (e-mail: emile.levy{at}recherche-ste-justine.qc.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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