NPC1L1 mRNA Research Articles

The Hypocholesterolemic Potential of the Edible Algae Fucus vesiculosus: Proteomic and Quantitative PCR Analysis.

A brown seaweed consumed worldwide, Fucus vesiculosus, has been used to prevent atherosclerosis and hypercholesterolemia, among other uses. However, the mechanisms of action that lead to these effects are not yet fully understood. This work aims to study the in vitro effect of an aqueous extract of F. vesiculosus, previously characterized as rich in phlorotannins and peptides, on the expression of different proteins involved in the synthesis and transport of cholesterol. A proteomic analysis, Western blot, and qRT-PCR analysis were performed to identify protein changes in HepG2 cells exposed to 0.25 mg/mL of the F. vesiculosus extract for 24 h. The proteomic results demonstrated that, in liver cells, the extract decreases the expression of four proteins involved in the cholesterol biosynthesis process (CYP51A1, DHCR24, HMGCS1 and HSD17B7). Additionally, a 12.76% and 18.40% decrease in the expression of two important transporters proteins of cholesterol, NPC1L1 and ABCG5, respectively, was also observed, as well as a 30% decrease in NPC1L1 mRNA levels in the cells exposed to the extract compared to control cells. Our study reveals some of the mechanisms underlying the actions of bioactive compounds from F. vesiculosus that may explain its previously reported hypocholesterolemic effect, future prospecting its use as a functional food.

M6A-related bioinformatics analysis and functional characterization reveals that METTL3-mediated NPC1L1 mRNA hypermethylation facilitates progression of atherosclerosis via inactivation of the MAPK pathway.

Accumulating evidence has demonstrated that N6-methyladenosine (m6A) plays important roles in many major diseases, including atherosclerosis (AS). In the present study, we aimed to explore the transcriptomic m6A landscape of endothelial function-associated genes and identify potential regulators in AS progression. The GEO data (GSE142386) from MeRIP-seq in human umbilical vein endothelial cells (HUVECs) with METTL3 knocked down or not were analyzed. RNA-seq was performed to identify differences in gene expression. Gene ontology (GO) functional and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were conducted to evaluate the potential functions of the differentially expressed genes. MeRIP-qPCR was used to measure the m6A and mRNA levels of the top 8 downregulated genes, and NPC1L1 was selected as the candidate gene. Oxidized low-density lipoprotein (ox-LDL) was used to stimulate HUVECs, and METTL3 or NPC1L1 was silenced in ox-LDL-treated cells. And Transwell, ELISA, and cell apoptosis assays were performed to assess cell functional injury. ApoE-/- mice were fed with high-fat diet for 8 weeks to establish an AS model, and adenovirus-mediated NPC1L1 shRNA or NC shRNA was injected into the mice through the tail vein. Mouse aortic tissue damage and plaque deposition were evaluated by H&E, Oil Red O, and TUNEL staining. One hundred and ninety-four hypermethylated m6A peaks and 222 hypomethylated peaks were detected in response to knockdown of METTL3. Genes with altered m6A peaks were significantly involved in the histone modification, enzyme activity, and formation of multiple complexes and were predominantly enriched in the MAPK pathway. NPC1L1 was a most significantly downregulated transcript in response to knockdown of METTL3. Moreover, knockdown of NPC1L1 or de-m6A (METTL3 knockdown)-mediated downregulation of NPC1L1 could improve ox-LDL-induced dysfunction of HUVECs in vitro and high-fat diet-induced atherosclerotic plaque in vivo, which was associated with the inactivation of the MAPK pathway. METTL3-mediated NPC1L1 mRNA hypermethylation facilitates AS progression by regulating the MAPK pathway, and NPC1L1 may be a novel target for the treatment of AS.

Co-Amorphous Formation of Simvastatin-Ezetimibe: Enhanced Physical Stability, Bioavailability and Cholesterol-Lowering Effects in LDLr-/-Mice.

Hypercholesterolemia is one of the independent risk factors for the development of cardiovascular diseases such as atherosclerosis. The treatment of hypercholesterolemia is of great significance to reduce clinical cardiovascular events and patient mortality. Simvastatin (SIM) and ezetimibe (EZE) are commonly used clinically as cholesterol-lowering drugs; however, their treatment efficacy is severely affected by their poor water solubility and low bioavailability. In this study, SIM and EZE were made into a co-amorphous system to improve their dissolution, oral bioavailability, storage stability, and cholesterol-lowering effects. The SIM-EZE co-amorphous solids (CO) were prepared successfully using the melt-quenched technique, and the physicochemical properties of CO were characterized accordingly, which exhibited improved physical stability and faster dissolution release profiles than their physical mixture (PM). In the pharmacokinetic study, the SIM-EZE CO or PM was given once by oral gavage, and mouse blood samples were collected retro-orbitally at multiple time points to determine the plasma drug concentrations. In the pharmacodynamic study, low-density lipoprotein receptor-deficient (LDLr−/−) mice were fed with a high-fat diet (HFD) for two weeks to establish a mouse model of hypercholesterolemia. Using PM as a control, we investigated the regulation of CO on plasma lipid levels in mice. Furthermore, the mice feces were collected to determine the cholesterol contents. Besides, the effect of EZE on the NPC1L1 mRNA expression level in the mouse intestines was also investigated. The pharmacokinetics results showed that the SIM-EZE CO has improved bioavailability compared to the PM. The pharmacodynamic studies showed that SIM-EZE CO significantly increased the cholesterol-lowering effects of the drugs compared to their PM. The total cholesterol excretion in the mouse feces and inhibitory effect on NCP1L1 gene expression in the mouse intestines after being given the SIM-EZE CO were more dramatic than the PM. Our study shows that the SIM-EZE CO prepared by the melt-quenched method can significantly improve the stability, bioavailability, and cholesterol-lowering efficacy with excellent development potential as a new drug formulation.

In vitro Evaluation of the Anti-hypercholesterolemic Effect of Lactobacillus Isolates From Various Sources.

The anti-hypercholesterolemic effect of 11 Lactobacillus isolates was investigated in vitro by measuring remaining cholesterol in growth media, growth ability in media supplemented with cholesterol, and BSH activity. Among the selected isolates, DLBSH104, DLBSH122, and DLBSK207 have demonstrated outstanding potential as cholesterol-lowering cultures. The three isolates showed high cholesterol removal by growing cells, whereas resting and dead cells showed less cholesterol removal. Furthermore, visualization of those isolates in growing and non-growing states by SEM showed the ability of DLBSH104 to attach cholesterol to their cell surface. In contrast, alteration of DLBSH122 and DLBSK207 cells did not involve surface attachment of cholesterol. Thus, the isolates’ ability to remove cholesterol is mainly attributed to the cells’ metabolically active state that assimilates and incorporates cholesterol into the cell membrane as reflected by a significantly higher cholesterol removal in a growing state than a non-growing state. Only in DLBSH104 did cholesterol removal also involve attachment on the cell surface. Moreover, DLBSH104 has beneficially affected the host cell by a significant reduction of NPC1L1 mRNA levels that are responsible for intestinal cholesterol absorption. In hepatic cells, cell-free supernatant (CFS) from DLBSH104 and DLBSK207 were able to reduce LDLR and HMGCR mRNA at the transcription level. To sum up, L. helveticus DLBSH104 and L. plantarum DLBSK207 are confirmed as isolates with an anti-hypercholesterolemic effect.

The Effects of a High-Fat/Cholesterol Diet on the Intestine of Rats Were Attenuated by Sparassis latifolia Polysaccharides.

Sparassis latifolia polysaccharides can regulate lipids and cholesterol in serum and liver. However, little is known about the regulation mechanism of the polysaccharides on cholesterol metabolism and especially the causal relationship with gut microbiota regulation. This study will provide a theoretical basis for the cholesterol-lowering mechanism of S. latifolia polysaccharides and further development of functional foods. In this study, we investigated how the regulation mechanism of Sparassis latifolia polysaccharides affects intestinal cholesterol metabolism in high-fat and high-cholesterol diet-fed rats. Briefly, enzymatic colorimetric microplate assay was used to determine the concentration of faecal bile acid. Gas chromatography-mass spectrometry was used to detect the content of cholesterol and alcohol in faeces. Haematoxylin and eosin staining method was applied to observe the changes in the structure of the small intestine tissue. The related gene expressions in jejunum and ileum were detected by real-time fluorescent quantitative polymerase chain reaction. The related protein expressions in jejunum were studied by using Western blot. High-throughput sequencing was used to detect the intestinal flora changes of the caecal contents. Gas chromatography-mass spectrometry was applied to detect the concentration of short-chain fatty acids in the caecal content. The results showed that Sparassis latifolia polysaccharides could improve the intestinal morphological structure and physiological indices in rats fed high-fat and high-cholesterol diet. Moreover, it could improve intestinal cholesterol metabolism disorder induced by high-fat and high-cholesterol diets via the reduction of the expression of HMGCR, NPC1L1, ACAT2, MTP, ASBT and IBABP mRNA or protein, increasing ABCG8 mRNA expression. In addition, it could also increase the relative abundance of Bacteroides, Butyricicoccus, Parabacteroides, Parasutteerella and Alloprevotella and the short-chain fatty acid concentration, to comprehensively regulate the intestinal cholesterol metabolism. The metabolomics analysis found that Sparassis latifolia polysaccharides could affect lipid, carbohydrate and other related metabolites. Some biomarkers associated with cholesterol metabolism correlated significantly with the abundance of specific intestinal microbiota. These findings indicate that Sparassis latifolia polysaccharides could attenuate intestinal cholesterol metabolism disorder, correlating with modulating gut microbiota and improving host metabolism. They provide theoretical support for the development of Sparassis latifolia as a new food resource.

Regulation of the expression of cholesterol transporters by lipid-lowering drugs ezetimibe and pemafibrate in rat liver and intestine

Ezetimibe and pemafibrate are lipid-lowering drugs and promote reverse cholesterol transport. However, it is unknown whether cholesterol is mainly excreted by hepatobiliary excretion or by non-biliary transintestinal cholesterol efflux (TICE). We evaluated the effects of ezetimibe and pemafibrate on hepatic and intestinal cholesterol transporter regulation in Sham-operated rats, and examined the effects of these drugs on TICE in bile duct-ligated rats. Seven-week-old male Sprague-Dawley rats were treated as follows for two weeks: 1) Sham, Sham operation; 2) BDL, bile duct ligation; 3) E-Sham, Sham + ezetimibe; 4) E-BDL, BDL + ezetimibe; 5) P-Sham, Sham + pemafibrate; and 6) P-BDL, BDL + pemafibrate. Blood, liver, jejunum, and feces were collected 72 h post-surgery. Hepatic cholesterol levels were decreased in P-Sham and E-Sham, and were lower in E-BDL and P-BDL than in BDL. Fecal cholesterol levels increased in E-Sham and P-Sham compared with Sham, and were higher in E-BDL and P-BDL than in BDL. In liver, Abcg5 mRNA showed induction in E-Sham, Abcg5 and Abca1 mRNA were induced in P-Sham, Abcg5 mRNA was reduced in E-BDL, and Abca1 mRNA was increased in P-BDL. In jejunum, Abcg5 mRNA was induced in E-Sham. Abcg8 mRNA was induced in E-Sham and P-Sham. NPC1L1 mRNA showed reduced expression in P-Sham and P-BDL. SR-B1 mRNA was reduced in P-Sham, and the expression decreased in P-BDL. LDL receptor mRNA was induced in BDL and P-BDL. Ezetimibe and pemafibrate may promote TICE by increasing Abcg5/g8, while pemafibrate may inhibit intestinal cholesterol absorption by decreasing SR-B1 and NPC1L1.

Identification of a Novel Potential Probiotic Lactobacillus plantarum FB003 Isolated from Salted-Fermented Shrimp and its Effect on Cholesterol Absorption by Regulation of NPC1L1 and PPARα.

Cholesterol-lowering activity is an important health benefit of lactic acid bacteria (LAB). This study aimed to screen LAB strains with cholesterol-lowering activities from salted fermented shrimp and evaluate probiotic characteristics and cholesterol-lowering potentials of these LAB isolates. Among 191 lactic acid strains isolated from traditional salted-fermented shrimp food, FB003 isolate showed the highest cholesterol-lowering activity and investigated as probiotics with cholesterol-lowering ability. Biochemical analysis and 16S rRNA sequencing revealed that this LAB isolate was Lactobacillus plantarum FB003. To screen probiotic trait, L. plantarum FB003 was found to be susceptible to six antibiotics tested and broad-spectrum antimicrobial activity. It also produced various enzymes such as galactosidase, glucosidase, and mannosidase. In addition, this strain showed autoaggregation, and coaggregation capacity for various pathogens. Moreover, it could adhere to Caco-2 cells and be exerted lowering cholesterol effects in Caco-2 cells via an upregulation of PPARα to inhibit NPC1L1 mRNA expression. Strain L. plantarum FB003 might be effective as a candidate probiotic with high cholesterol-lowering activity. The results of the present study suggest that L. plantarum FB003 have an impact on preventing high cholesterol level and may be used as starter culture for shrimp fermentation.

Transcriptional control of intestinal cholesterol absorption, adipose energy expenditure and lipid handling by Sortilin

The sorting receptor Sortilin functions in the regulation of glucose and lipid metabolism. Dysfunctional lipid uptake, storage, and metabolism contribute to several major human diseases including atherosclerosis and obesity. Sortilin associates with cardiovascular disease; however, the role of Sortilin in adipose tissue and lipid metabolism remains unclear. Here we show that in the low-density lipoprotein receptor-deficient (Ldlr−/−) atherosclerosis model, Sortilin deficiency (Sort1−/−) in female mice suppresses Niemann-Pick type C1-Like 1 (Npc1l1) mRNA levels, reduces body and white adipose tissue weight, and improves brown adipose tissue function partially via transcriptional downregulation of Krüppel-like factor 4 and Liver X receptor. Female Ldlr−/−Sort1−/− mice on a high-fat/cholesterol diet had elevated plasma Fibroblast growth factor 21 and Adiponectin, an adipokine that when reduced is associated with obesity and cardiovascular disease-related factors. Additionally, Sort1 deficiency suppressed cholesterol absorption in both female mice ex vivo intestinal tissue and human colon Caco-2 cells in a similar manner to treatment with the NPC1L1 inhibitor ezetimibe. Together our findings support a novel role of Sortilin in energy regulation and lipid homeostasis in female mice, which may be a potential therapeutic target for obesity and cardiovascular disease.

Wild Lonicera caerulea berry polyphenol extract reduces cholesterol accumulation and enhances antioxidant capacity in vitro and in vivo

The hypocholesterolemic effect of Lonicera caerulea berry extract rich in polyphenols (LCBP) on high cholesterol-induced hypercholesterolemia and lipoprotein metabolite changes was examined in Caco-2 cells and rats. Cyanidin-3-glucoside, catechin, and chlorogenic acid are the major phenolic components of LCBP. The cholesterol-reducing effect and antioxidant capacity of these components were compared in Caco-2 cells. LCBP (80 μg/mL) and cyanidin-3-glucoside, catechin, and chlorogenic acid (50 μM) were found to be effective (p < 0.05). Rats were fed a high cholesterol diet (HCD) with or without LCBP supplementation (75, 150, and 300 mg/kg body weight intragastrically once daily) for 12 weeks. Compared with the HCD control group, LCBP supplementation at 150 and 300 mg/kg decreased the levels of TC, TG, and LDL-C, but increased that of HDL-C. LCBP treatment promoted greater neutral and acidic sterol excretion (p < 0.05) and improved the antioxidant capacity of the colon tissue, colon contents, and blood. Moreover, trimethylamine N-oxide (TMAO) levels were decreased in serum (p < 0.05). NPC1L1, ACAT2, and MTP mRNA and protein expression were reduced and ABCG5/8 expression was increased (p < 0.05) after LCBP treatment. Our results suggest that LCBP could be used as a functional food for the prevention and treatment of diseases related to excessive cholesterol accumulation.

Abstract 601: Global and Liver Specific Bmal1 Deficient Mice Regulate Intestinal Lipid Absorption

Circadian rhythms controlled by clock genes affect plasma lipids, known risk factors for atherosclerosis. Clock and Bmal1 are critical clock genes that positively regulate circadian rhythms. We have shown that Clock plays an important role in lipid absorption. Additionally, we showed that global ablation of Bmal1 in Apoe –/– and Ldlr –/– mice, and liver-specific ablation of Bmal1 in Apoe –/– (L -Bmal1 –/– Apoe –/– ) mice increases atherosclerosis. However, it is unknown whether Bmal1 regulates intestinal lipid absorption. We studied cholesterol and triglyceride absorption in normal and lipase-inhibited Bmal1 –/– and Bmal1 –/– Apoe –/– mice with global Bmal1 deficiency and compared it with Bmal1 +/+ and Bmal1 +/+ Apoe –/– , respectively. To study the effect of liver-specific Bmal1 deficiency, studies were performed in L- Bmal1 –/– and L- Bmal1 –/– Apoe –/– mice and data were compared with Bmal1 fl/fl and Bmal1 fl/fl Apoe –/– mice. In addition, enterocytes isolated from these mice were used to study uptake and secretion of cholesterol and oleic acid. Further, protein and mRNA levels of candidate genes involved in lipid uptake, lipoprotein assembly and secretion were quantified. Both normal and lipase-inhibited Bmal1 –/– mice absorbed significantly higher amounts of cholesterol and triglyceride. Further, uptake and secretion of cholesterol and fatty acids by enterocytes was increased in Bmal1 –/– mice. As reported previously, liver-specific Bmal1 ablation increased VLDL production. Surprisingly, postprandial plasma lipids were also significantly increased in L- Bmal1 –/– mice than in control Bmal1 fl/fl mice. Further, enterocytes isolated from L- Bmal1 –/– took up more lipids and secreted more lipoproteins. Moreover, in vivo lipid absorption in L- Bmal1 –/– was increased. Gene expression quantifications revealed that intestinal MTP, DGAT1, CD36 and NPC1L1 mRNA levels were increased in global and hepatic Bmal1 deficient animals. Global and liver-specific ablation of Bmal1 increases intestinal lipoprotein production by increasing the expression of genes involved in lipid uptake and lipoprotein assembly. These data for the first time suggest that intestinal lipoprotein assembly is regulated by hepatic Bmal1.

Combination of curcumin and piperine prevents formation of gallstones in C57BL6 mice fed on lithogenic diet: whether NPC1L1/SREBP2 participates in this process?

BackgroundA disruption of cholesterol homeostasis characterized by the physical-chemical imbalance of cholesterol solubility in bile often results in formation of cholesterol gallstones. Our earlier studies revealed that curcumin (1000 mg/kg) could prevent formation of gallstones. It has been proved that curcumin is poorly absorbed while piperine is a bioavailability-enhancer. Nevertheless, whether curcumin combined with piperine could enhance the effect of curcumin in preventing gallstones is still awaited.MethodC57BL6 mice were fed on a lithogenic diet concomitant with curcumin at 500 or 1000 mg/kg and/or piperine at 20 mg/kg for 4 weeks. The ratio of gallbladder stone formation was recorded and samples of blood, bile, gallbladder, liver and small intestine were also collected. The volume of gallbladder and weight of liver were calculated, and blood and bile samples were analyzed through biochemical methods. Intestinal NPC1L1 and SREBP2 mRNA and protein expression were detected by real-time PCR and Western blot.ResultCombining with piperine can significantly enhance the effect of curcumin, thus preventing the development of gallbladder stones, lowering the saturation of blood lipids and cholesterol in bile, as well as decreasing the expression of NPC1L1 and SREBP2 in both mRNA and protein levels.ConclusionCurcumin can prevent the formation of cholesterol gallstones induced by high fat diet in mice and SREBP2 and NPC1L1 may participate in this process. Piperine can increase curcumin’s bioavailability, thereby enhancing the effect of curcumin.

Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation

Intestinal NPC1L1 transporter is essential for cholesterol absorption and the maintenance of cholesterol homeostasis in the body. NPC1L1 is differentially expressed along the gastrointestinal tract with very low levels in the colon as compared with the small intestine. This study was undertaken to examine whether DNA methylation was responsible for segment-specific expression of NPC1L1. Treatment of mice with 5-azacytidine (i.p.) resulted in a significant dose-dependent increase in NPC1L1 mRNA expression in the colon. The lack of expression of NPC1L1 in the normal colon was associated with high levels of methylation in the area flanking the 3-kb fragment upstream of the initiation site of the mouse NPC1L1 gene in mouse colon as analyzed by EpiTYPER® MassARRAY®. The high level of methylation in the colon was observed in specific CpG dinucleotides and was significantly decreased in response to 5-azacytidine. Similar to mouse NPC1L1, 5-azacytidine treatment also increased the level of human NPC1L1 mRNA expression in the intestinal HuTu-80 cell line in a dose- and time-dependent manner. Silencing the expression of DNA methyltransferase DNMT1, -2, -3A, and -3B alone by siRNA did not affect NPC1L1 expression in HuTu-80 cells. However, the simultaneous attenuation of DNMT1 and -3B expression caused a significant increase in NPC1L1 mRNA expression as compared with control. Also, in vitro methylation of the human NPC1L1 promoter significantly decreased NPC1L1 promoter activity in human intestinal Caco2 cells. In conclusion, our data demonstrated for the first time that DNA methylation in the promoter region of the NPC1L1 gene appears to be a major mechanism underlying differential expression of NPC1L1 along the length of the gastrointestinal tract.

Mo1803 Ileal Bile Acid Transporter and FGF19 Transcript Expression Related to SEHCAT Retention in Patients With Chronic Diarrhea

Intestinal NPC1L1 transporter is essential for cholesterol absorption and the increase in its expression is associated with high risk for atherosclerosis. NPC1L1 is differentially expressed along the gastrointestinal tract with high levels of expression in the small intestine as compared to colon. Also, NPC1L1 is expressed in villus but not in crypt cells. Recent studies have shown that epigenetic regulation may play a role in controlling the expression of intestinal transport proteins. Whether epigenetic mechanisms such as DNA methylation is involved in determining NPC1L1 expression along the GI tract is not known. Current studies were, therefore, undertaken to investigate the effect of DNA methylation on NPC1L1 gene expression utilizing both in vitro and in vivo models. Intestinal HUTU-80 cells have very low levels of NPC1L1 expression and, thus, were used to investigate the effects of changes in DNA methylation on its expression. HUTU-80 cells were incubated with 10 μM of 5azacytidine, inhibitor of DNA methylation, and NPC1L1 mRNA expression was assessed by real time PCR. As compared to control cells, treatment with 5-azacytidine significantly increased the NPC1L1 expression in a time dependent manner with a maximum induction occurring after 72 h (∼24 fold, P,0.005). We also examined the methylation status of the promoter region of the NPC1L1 gene utilizing methylation specific PCR (MSP) on bisulfite converted genomic DNA. MSP analysis demonstrated that NPC11L1 is highly methylated in HUTU-80 cells consistent with the low level of NPC1L1 mRNA expression. Treatment with 5-azacytidine (10 μM, 72 h) decreased the DNA methylation of NPC1L1 promoter region by 53.13 ± 11.67 % as compared to control cells concomitant with the increase in NPC1L1 expression. DNA methylation is mediated by the activity of DNAmethyltransferases: DNMT1, 3A and 3B. Silencing the expression of both DNMT1 and 3B simultaneously by specific siRNA in HUTU-80 cells significantly increased NPC1L1 mRNA expression by ∼2 fold (P,0.05) as compared to control. Similar to the observed changes in the cell culture model, intraperitoneal injections of 5-azacytidine (10 mg/kg body weight, 3 injections/week for three weeks) to C57BL/6 mice (8 weeks of age) resulted in a significant increase in NPC1L1 expression in the colon ( ∼32 fold, P,0.005) as compared to control mice. In conclusion, our data demonstrate that DNA methylation in the promoter region of NPC1L1 gene appears to be a major mechanism underlying differential expression of NPC1L1 along the GI tract. We speculate that harnessing alterations in DNA methylation by dietary manipulations may prove to be of potential benefit in decreasing cholesterol absorption and preventing atherosclerosis (Supported by NIDDK & VA).

Lecithin:Cholesterol Acyltransferase Deficiency Protects against Cholesterol-induced Hepatic Endoplasmic Reticulum Stress in Mice

We recently reported that lecithin:cholesterol acyltransferase (LCAT) knock-out mice, particularly in the LDL receptor knock-out background, are hypersensitive to insulin and resistant to high fat diet-induced insulin resistance (IR) and obesity. We demonstrated that chow-fed Ldlr-/-xLcat+/+ mice have elevated hepatic endoplasmic reticulum (ER) stress, which promotes IR, compared with wild-type controls, and this effect is normalized in Ldlr-/-xLcat-/- mice. In the present study, we tested the hypothesis that hepatic ER cholesterol metabolism differentially regulates ER stress using these models. We observed that the Ldlr-/-xLcat+/+ mice accumulate excess hepatic total and ER cholesterol primarily attributed to increased reuptake of biliary cholesterol as we observed reduced biliary cholesterol in conjunction with decreased hepatic Abcg5/g8 mRNA, increased Npc1l1 mRNA, and decreased Hmgr mRNA and nuclear SREBP2 protein. Intestinal NPC1L1 protein was induced. Expression of these genes was reversed in the Ldlr-/-xLcat-/- mice, accounting for the normalization of total and ER cholesterol and ER stress. Upon feeding a 2% high cholesterol diet (HCD), Ldlr-/-xLcat-/- mice accumulated a similar amount of total hepatic cholesterol compared with the Ldlr-/-xLcat+/+ mice, but the hepatic ER cholesterol levels remained low in conjunction with being protected from HCD-induced ER stress and IR. Hepatic ER stress correlates strongly with hepatic ER free cholesterol but poorly with hepatic tissue free cholesterol. The unexpectedly low ER cholesterol seen in HCD-fed Ldlr-/-xLcat-/- mice was attributable to a coordinated marked up-regulation of ACAT2 and suppressed SREBP2 processing. Thus, factors influencing the accumulation of ER cholesterol may be important for the development of hepatic insulin resistance.

Role of SGKs in Glucocorticoid-Mediated Regulation of NHE3

mechanisms not fully understood. Since high level of plasma glucose is the main feature of diabetes mellitus, we hypothesized that hyperglycemia induces NPC1L1 expression in diabetic patients via modulation of gene transcription. Therefore, current studies were undertaken to examine the transcriptional regulation of NPC1L1 in human intestinal Caco2 cells in response to glucose. Cells were transiently transfected with different promoter constructs of NPC1L1 gene. Relative promoter activity was measured by luciferase activity normalized to β-galactosidase. The relative abundance of NPC1L1 mRNA was evaluated by real-time PCR. Incubation of Caco2 cells with D-glucose (25 mM, 24 h) significantly increased the NPC1L1 mRNA expression by ~6 fold. In contrast, the removal of D-glucose from the culture medium of Caco2 cells for 24 h significantly decreased the NPC1L1 promoter activity (45.1±2.5 % of control, P<0.001). Consistent with increased mRNA expression, 24 h incubation with different concentrations of glucose (1, 5, 25 mM) significantly increased the promoter activity in a dose dependent manner with a maximum effect at 5 mM concentration (230±10.6 %, P<0.001) compared to control cells treated with 5 mM mannitol. Exposure of Caco2 cells to the non-metabolized form of glucose (3-O-methyl-D-glucopyranose, 5 mM, 24h) had no effect on the promoter activity of NPC1L1 indicating that observed effects are dependent on glucose metabolism. Also, pyruvate showed no effect on NPC1L1 promoter activity suggesting that metabolic products of glycolysis upstream of pyruvate mediate the effects of glucose. Deletion of NPC1L1 promoter construct to -291 nucleotide did not alter glucose-induced stimulation. These data indicated that glucose responsive elements are located in the region between -291 and +56 of NPC1L1 promoter. Conclusion: High levels of glucose stimulate NPC1L1 expression via transcriptional regulation. These data provide mechanistic insights into the increase in NPC1L1 expression observed in diabetesmellitus and may be exploited to improve therapeutic modalities for the associated hypercholesterolemia.

Oleic acid decreases the expression of a cholesterol transport-related protein (NPC1L1) by the induction of endoplasmic reticulum stress in CaCo-2 cells

The reported data indicate that oleic acid (OA) decreases cholesterol absorption. To explore the underlying mechanisms, the effects of OA on the expression of cholesterol transport-related proteins (NPC1L1, ABCG5/8, ACAT2, MTP) and the unfolded protein response (UPR) pathway were studied in CaCo-2 enterocytes by incubating CaCo-2 cells with taurocholate micelles or taurocholate micelles containing different concentrations of OA (0.25-1.0 mM). We show that OA effectively induces XBP1 mRNA splicing, a key component of the UPR signaling, and the expression of BiP and mature ATF6 proteins in a concentration-dependent manner, leading to the induction of endoplasmic reticulum (ER) stress and activation of the UPR. Interestingly, OA decreases NPC1L1 expression in a dose-dependent manner while it has no effects on ABCG5 and MTP mRNA level or SREBP-2, ABCG8, and ACAT2 protein level. In CaCo-2 cells treated with 1.0 mM OA, both the NPC1L1 mRNA level and the NPC1L1 protein expression in brush-border membrane fractions were decreased by 39% and 37%, respectively (P < 0.01). A dose of 1 mM dithiothreitol (DTT), a positive control for ER stress induction, also decreases NPC1L1 mRNA and protein expression by 27% and 23%, respectively (P < 0.05). Furthermore, 4-phenyl-butyric acid, an UPR inhibitor, blocks OA- and DTT-induced reduction on NPC1L1 mRNA and protein levels. The results suggest that OA down-regulates NPC1L1 mRNA and protein expression via the induction of the UPR, which may play an important role in reducing intestinal cholesterol absorption.

Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression

BackgroundCurcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1) protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells.MethodsCaco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification.ResultsWe found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 μM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 μM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively.ConclusionCurcumin inhibits cholesterol uptake through suppression of NPC1L1 expression in the intestinal cells.

493 RESULTS OF LIVER RETRANSPLANTATION IN A MONOCENTRIC COHORT OF HCV INFECTED PATIENTS

quartile of LDLR mRNA expression in the PI biopsy survived with a significantly (Log-Rank test P < 0.05) better survival than those in the three lowest quartiles. Surviving graft (n = 34, median followup 44.8 months, range 8.6–72.3) had significantly higher LDLR (P < 0.01) and NPC1L1 (P < 0.05) mRNA expression in the PI biopsy and HMGCR (P < 0.05) mRNA expression in the PR biopsy than lost grafts (n = 13, median follow-up 6.0 months, range 0.03–43.7). Conclusions: In the settings of human liver transplantation: 1. hepatic LDLR mRNA is overexpressed immediately after graft reperfusion, suggesting an increased hepatocyte cholesterol uptake from blood; 2. hepatic upregulation of genes involved in cholesterol recruitment and synthesis is inversely related to the severity of IRI and is associated with better graft survival.

495 ARE LIVER TRANSPLANT HIV-1 INFECTED PATIENTS AT RISK TO DEVELOP HIV-1 DRUG RESISTANCE?

quartile of LDLR mRNA expression in the PI biopsy survived with a significantly (Log-Rank test P < 0.05) better survival than those in the three lowest quartiles. Surviving graft (n = 34, median followup 44.8 months, range 8.6–72.3) had significantly higher LDLR (P < 0.01) and NPC1L1 (P < 0.05) mRNA expression in the PI biopsy and HMGCR (P < 0.05) mRNA expression in the PR biopsy than lost grafts (n = 13, median follow-up 6.0 months, range 0.03–43.7). Conclusions: In the settings of human liver transplantation: 1. hepatic LDLR mRNA is overexpressed immediately after graft reperfusion, suggesting an increased hepatocyte cholesterol uptake from blood; 2. hepatic upregulation of genes involved in cholesterol recruitment and synthesis is inversely related to the severity of IRI and is associated with better graft survival.

Decreased NPC1L1 expression in the liver from Chinese female gallstone patients

BackgroundCholesterol gallstone disease is a very common disease in both industrialized and developing countries. Many studies have found that cholesterol gallstones are more common in women than men. The molecular mechanisms underlying the relationship between female gallstone disease and hepatic sterol transporters are still undergoing definition and have not been evaluated in humans.AimsThe aim of this study is to probe for underlying hepatic molecular defects associated with development of gallstones in female.Methods/ResultsFifty-seven nonobese, normolipidemic Chinese female gallstone patients (GS) were investigated with 12 age- and body mass index-matched female gallstone-free controls (GSF). The bile from the female GS had higher cholesterol saturation than that from the female GSF. The hepatic NPC1L1 mRNA levels were lower in female GS, correlated with SREBP2 mRNA. NPC1L1 downregulation was confirmed at protein levels. Consistently, immunohistochemistry showed decreased NPC1L1 expression in female GS.ConclusionsThe decreased hepatic NPC1L1 levels in female GS might indicate a downregulated reabsorption of biliary cholesterol in the liver, which, in turn, leads to the cholesterol supersaturation of bile. Our data are consistent with the possibility that hepatic NPC1L1 may be mediated by SREBP2.