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  • As mentioned above disruption of Tgif in mice

    2020-09-24

    As mentioned above, disruption of Tgif1 in mice is associated with dyslipidemia, and here we investigate the effects of overexpression of TGIF1 on expression of the Npc1l1 gene and markers of intestinal cholesterol absorption.
    Materials GSK1324726A sale and methods
    Results
    Discussion In this study, we sought to investigate effects by overexpression of TGIF1 on Npc1l1 and markers of intestinal cholesterol absorption. We found TGIF1 to function as a repressor of NPC1L1 and that intestinal-specific overexpression of TGIF1 in mice resulted in lower levels of markers of cholesterol GSK1324726A sale and reduced intestinal expression levels of the LXRα target genes Npc1l1, Abca1, Abcg5, and Abcg8. We also found TGIF1 to be able to oppose the induction of the promoter activity of NPC1L1 by SREBP2, HNF1α, and HNF4α. Thus, TGIF1 is a new player in intestinal cholesterol metabolism. The effects were specific to the intestine and compensated for by an increased whole-body cholesterol synthesis. SREBP2 has previously been reported to function as a positive regulator of the NPC1L1 promoter activity in human intestinal Caco2 cells [31]. Here we could confirm the induction by SREBP2 and also show that TGIF1 is able to oppose this induction of the NPC1L1 promoter activity. We have previously found HNF1α and HNF4α to function as positive regulators of another cholesterol regulated gene, SOAT2 [20,32], and that TGIF1 opposes the induction of the human SOAT2 promoter activity by these two transcription factors in hepatoma and intestinal cells [33]. Similarly, here we found HNF1α and HNF4α to function as positive regulators of NPC1L1 and that TGIF1 was able to oppose this induction of the NPC1L1 promoter activity in Caco2 cells. TGIF1 can bind to DNA via its consensus sequence CTGTCAA [4] but also to RXR binding sites [5]. We identified multiple putative TGIF and RXR binding sites in the human NPC1L1 promoter region and found several TGIF sites which upon mutation lowered the repression exerted by TGIF1; we also found TGIF1 to be able to bind to this region. Moreover, we identified a consensus half site motif AGGTCA, located at −895 to −890 bp upstream of the ATG transcription start site, which completely abolished the NPC1L1 promoter activity. However, this site do not seems to be important for the repression of the NPC1L1 promoter activity by TGIF1 nor was it 100% conserved in mice and we were thus not able to further investigate its importance. Serum levels of the two plant sterols campesterol and sitosterol to cholesterol are commonly used as surrogate markers of intestinal cholesterol absorption in humans. Igel et al. [30] reported a positive correlation between cholesterol absorption, assessed by the constant isotope feeding method using [2H6]cholesterol and [2H4]sitostanol and measurement of fecal sterols, and the ratio of campesterol to cholesterol in serum also in mice. Cholesterol and plant sterols are both absorbed via NPC1L1 in the intestine. In contrast to cholesterol, plant sterols cannot be endogenously synthesized and hence derive strictly from the diet. Synthetic diets, like the high-fat diet used in this study, contain trace amounts of plant sterols and thus we added 0.05% β-sitosterol (≥70% sitosterol; also ≤30% campesterol and β-sitostanol) to the high-fat diet. The added β-sitosterol also contained campesterol and we were thus able to measure both of these plant sterols in serum, although the absolute levels cannot be directly comparable to mice fed a standard chow diet. Differences in the composition of these two diets, with the chow diet containing more fibers than the high-fat diet, are also likely to contribute to differences in hepatic CYP7A1 activity. Mice fed a chow diet have higher CYP7A1 activity, which may be due to excretion of more bile acids in the feces, compared to mice fed a high-fat diet as dietary fibers affect reabsorption of bile acids.