SREBP2 mediates the modulation of intestinal NPC1L1 expression by curcumin

Curcumin, the major phenolic compound in the spice turmeric, exhibits numerous biological effects, including lowering plasma cholesterol and preventing diet-induced hypercholesterolemia. The mechanisms underlying the hypocholesterolemic effect of curcumin are not fully understood. In this regard, intestinal Niemann-Pick C1-like 1 (NPC1L1) cholesterol transporter, the molecular target of intestinal cholesterol absorption inhibitor ezetimibe, plays an essential role in the maintenance of cholesterol homeostasis. The current studies were designed to investigate the effect of curcumin on NPC1L1 function, expression, and promoter activity in intestinal Caco-2 monolayers. NPC1L1 function was evaluated by the measurement of ezetimibe-sensitive [3H]cholesterol esterification. Relative abundance of NPC1L1 mRNA and protein was evaluated by real-time PCR and Western blotting, respectively. Luciferase assays were used to measure NPC1L1 promoter activity. Our results showed that curcumin significantly inhibited ezetimibe-sensitive cholesterol esterification in a dose-dependent manner with a maximum decrease (by 52% compared with control) occurring at 50 μM concentration. Curcumin treatment of Caco-2 monolayers also significantly decreased NPC1L1 mRNA and protein expression. Similarly, the promoter activity of the NPC1L1 gene was inhibited significantly (55%) by 50 μM curcumin. The decrease in NPC1L1 promoter activity by curcumin was associated with a reduction in the expression and the DNA-binding activity of the sterol response element-binding protein 2 (SREBP2) transcription factor. Furthermore, the overexpression of active SREBP2 protected NPC1L1 from the inhibitory effect of curcumin. Our studies demonstrate that curcumin directly modulates intestinal NPC1L1 expression via transcriptional regulation and the involvement of SREBP2 transcription factor

Overactivation of Intestinal SREBP2 in Mice Increases Serum Cholesterol

Sterol Response Element Binding Protein 2 (SREBP2) transcription factor is a master regulator of cholesterol homeostasis. Treatment with statins, inhibitors of cholesterol synthesis, activates intestinal SREBP2, which may hinder their cholesterol-lowering effects. Overactivation of SREBP2 in mouse liver was shown to have no effect on plasma cholesterol. However, the influence of activating intestinal SREBP2 on plasma cholesterol is not known. We have generated a novel transgenic mouse model with intestine specific overexpression of active SREBP2 (ISR2) driven by villin promoter. ISR2 mice showed overexpression of active SREBP2 specifically in the intestine. Microarray analysis of jejunal RNA from ISR2 mice showed a significant increase in genes involved in fatty acid and cholesterol synthesis. Cholesterol and triglyceride (TG) in jejunum and liver (mg/g protein) were significantly increased in ISR2 vs wild type mice. Serum Cholesterol was significantly increased in VLDL and LDL fractions whereas the level of serum triglycerides was decreased in ISR2 vs wild type mice. In conclusion, activation of intestinal SREBP2 alone seems to be sufficient to increase plasma cholesterol, highlighting the essential role of intestine in maintaining cholesterol homeostasis in the body.</p

Overactivation of intestinal SREBP2 in mice increases serum cholesterol.

Sterol Response Element Binding Protein 2 (SREBP2) transcription factor is a master regulator of cholesterol homeostasis. Treatment with statins, inhibitors of cholesterol synthesis, activates intestinal SREBP2, which may hinder their cholesterol-lowering effects. Overactivation of SREBP2 in mouse liver was shown to have no effect on plasma cholesterol. However, the influence of activating intestinal SREBP2 on plasma cholesterol is not known. We have generated a novel transgenic mouse model with intestine specific overexpression of active SREBP2 (ISR2) driven by villin promoter. ISR2 mice showed overexpression of active SREBP2 specifically in the intestine. Microarray analysis of jejunal RNA from ISR2 mice showed a significant increase in genes involved in fatty acid and cholesterol synthesis. Cholesterol and triglyceride (TG) in jejunum and liver (mg/g protein) were significantly increased in ISR2 vs wild type mice. Serum Cholesterol was significantly increased in VLDL and LDL fractions whereas the level of serum triglycerides was decreased in ISR2 vs wild type mice. In conclusion, activation of intestinal SREBP2 alone seems to be sufficient to increase plasma cholesterol, highlighting the essential role of intestine in maintaining cholesterol homeostasis in the body

Expression of genes involved in lipid metabolism in ISR2 mice.

<p>Expression of HMG-CoA reductase, CYP51, PCSK9 and scd1 were assessed by real time PCR using gene specific primers and total RNA extracted from jejunum of ISR2 mice (TG) and wild type littermates (Control). The results are expressed as arbitrary unit (A.U.) and represent the Mean ± SE using 10–12 animals of each group. * P<0.05 as compared to WT mice.</p

Serum levels of cholesterol and triglycerides in ISR2 mice.

<p><b>A</b>: Serum was collected from ISR2 (TG) and wild type (WT) mice. The levels of cholesterol and triglycerides were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084221#s2" target="_blank">Materials and Methods</a>. The data are expressed as mg/dl and represent the Mean ± SE from 4 animals per group. * P<0.05 as compared to WT. <b>B</b>: Triglyceride levels were measured in different lipoprotein fractions prepared from ISR2 transgenic mice and their wild type littermates. The figure shows representative data showing a decrease in triglycerides of the VLDL from the ISR2 (open circles) mice as compared to their wild type littermates (closed triangles). Serum samples from three different ISR2 transgenic mice and three wild type littermates were pooled into one sample and lipoproteins were then obtained by the method of FPLC. <b>C</b>: Serum was collected from ISR2 (open circles) and wild type mice (closed triangles) for a total of 12 animals per group, and three animals were pooled into one aliquot for subsequent cholesterol measurement. Cholesterol levels were assessed in different lipoprotein fractions by the FPLC methods. The levels of cholesterol in different lipoprotein fractions are expressed as µg/fraction.</p

Intestine-specific overexpression of active SREBP2.

<p>Total RNA was extracted from different tissues from ISR2 mice (TG) and their wild type littermates (WT) and the expression of SREBP2 was then assessed by real time PCR using gene specific primers. <b>A</b>: The expression of active SREBP2 transgene evaluated using a set of primers specific for the N-terminal of SREBP2 mRNA in the jejunum, ileum and colon. <b>B</b>: The expression of endogenous SREBP2 mRNA in jejunum, ileum and colon assessed using primers specific for the C-terminal of the SREBP2 gene. <b>C</b>: The expression of SREBP2 in the kidney and lung utilizing N- and C-terminal specific primers. <b>D</b>: The expression of SREBP1c in jejunum and ileum of ISR2 and WT mice. The presented data represent the expression of respective gene relative to the expression of GAPDH, used as an internal control. The results are expressed as arbitrary unit (A.U.) and represent Mean ± SE using 10–12 animals of each group. * P<0.05 as compared to WT mice.</p

The levels of triglycerides and cholesterol in the jejunum of ISR2 mice.

<p>Total lipids were extracted from jejnunal mucosal scrapings from ISR2 (TG) and wild type (WT) mice and the levels of triglycerides, total cholesterol and cholesterol ester were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084221#s2" target="_blank">Materials and Methods</a>. Data are presented as mg lipid/g of tissues and expressed as Mean ± SE from 8 mice per group. * P<0.05 as compared to WT.</p

Generation of intestine-specific active SREBP2 mice.

<p>The coding sequence for the N-terminal of SREBP2 (representing the active transcription factor) was cloned down-stream of villin promoter. <b>A</b>: A schematic representation of the transgene and the location of the G1 and G2 primers used for genotyping and the identification of positive transgenic mice (designated as ISR2) <b>B</b>: A representative of genotyping results showing the expected amplified PCR fragment from genomic DNA extracted from ISR2 mice (+) but not their wild type littermates (−).</p

Distribution of active SREBP2 in ISR2 mice.

<p>Total protein lysates were prepared form intestinal mucosal scraping as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084221#s2" target="_blank">Materials and Methods</a>. <b>A</b>: A representative blot depicting the bands for active SREBP2 in ISR2 mice and their wild type littermates. Villin was used as a loading control. <b>B</b>: Villin staining (green) of the jejunum showing similar epithelial structure in ISR2 and wild type mice. <b>C</b>: immuno fluorescence staining of SREBP2 in jejunum of ISR2 and wild type mice. SREBP2 is stained with red and the nuclei with blue. The figure shows predominant cytoplasmic staining in wild type mice and increased colocalization of SREBP2 with the nuclei in ISR2 mice (white arrow).</p