A Novel Splicing Variant of Peroxisome Proliferator-Activated Receptor-γ (<i>Pparγ1sv</i>) Cooperatively Regulates Adipocyte Differentiation with <i>Pparγ2</i>

<div><p>Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate expression of a number of genes associated with the cellular differentiation and development. Here, we show the abundant and ubiquitous expression of a newly identified splicing variant of mouse <i>Pparγ</i> (<i>Pparγ1sv</i>) that encodes PPARγ1 protein, and its importance in adipogenesis. The novel splicing variant has a unique 5′-UTR sequence, relative to those of <i>Pparγ1</i> and <i>Pparγ2</i> mRNAs, indicating the presence of a novel transcriptional initiation site and promoter for <i>Pparγ</i> expression. <i>Pparγ1sv</i> was highly expressed in the white and brown adipose tissues at levels comparable to <i>Pparγ2</i>. <i>Pparγ1sv</i> was synergistically up-regulated with <i>Pparγ2</i> during adipocyte differentiation of 3T3-L1 cells and mouse primary cultured preadipocytes. Inhibition of <i>Pparγ1sv</i> by specific siRNAs completely abolished the induced adipogenesis in 3T3-L1 cells. C/EBPβ and C/EBPδ activated both the <i>Pparγ1sv</i> and <i>Pparγ2</i> promoters in 3T3-L1 preadipocytes. These findings suggest that <i>Pparγ1sv</i> and <i>Pparγ2</i> synergistically regulate the early stage of the adipocyte differentiation.</p></div

Ezetimibe Promotes Brush Border Membrane-to-Lumen Cholesterol Efflux in the Small Intestine

<div><p>Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen ³H-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given ³H-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given ³H-cholesterol in mice, we found that lumen-to-BBM ³H-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux to dispose of endogenous cholesterol efficiently for therapeutic purposes.</p></div

Schematic model for the transcriptional control of <i>Pparγ</i> in adipocyte differentiation.

<p>C/EBPβ and C/EBPδ directly transactivate both <i>Pparγ1sv</i> and <i>Pparγ2</i> genes, which are in turn translated to PPARγ1 and PPARγ2 proteins, respectively. PPARγ ensures the expression of downstream genes involved in adipogenesis, with forming a positive feedback loop with C/EBPα. Arrows with an asterisk and a sharp are speculative feedback pathways for <i>Pparγ</i> up-regulation by PPARγ proteins.</p

Cholesterol efflux in ABCG5 and ABCG8 double knockout (DKO) mice.

<p><i>A</i>, Quantitative RT-PCR showed that <i>NPC1L1</i> gene expression level did not differ apparently with ABCG5/G8 deletions in mice. Gene expressions were normalized against <i>18s</i> expression levels. Then the relative expression levels were compared between the two genotypes, WT (n 10) and ABCG5/G8 DKO (n 6) with the levels in the WT mice as the references. Data are shown as mean ± SEM. <i>B</i>, Ezetimibe (50 μg)-induced increase in cholesterol efflux was partially abolished in ABCG5/G8 DKO mice. <i>Upper</i>, Plots for ³H-decay per minute (DPM) counts in the perfusate (<i>right</i>, <i>gray circles</i>) and the perfused intestinal segment (<i>left</i>, <i>open circles</i>). <i>Lower</i>; ³H-DPM in the perfusate was divided by that in the intestinal segment perfused to obtain a ratio (%), which was then converted to a common logarithm. Bars indicate medians. The difference between wild-type (WT) and DKO mice was compared using the Student’s <i>t</i>-test. Each plot shows an individual assay result. Alphabetical differences (in parentheses) indicate significant difference between the groups (<i>p</i> < 0.05) using Tukey's Honestly Significant Difference test. <i>C</i>, ABCG5/G8 DKO partially abolished the inhibitory effect of ezetimibe on the intestinal cholesterol transit. Bars indicate medians. Each plot shows an individual assay result. The difference between the WT and the DKO mice was compared using the Mann—Whitney <i>U</i>-test. <i>D</i>, Comparison of mRNA abundance by quantitative RT-PCR between the jejunal samples of C57BL/6J and 129^<i>+Ter</i>/SvJ. Data are shown as mean ± SEM (n 5). <i>E</i>, Characteristic comparisons of C57BL/6J and 129^<i>+Ter</i>/SvJ mice in luminal perfusion assay. <i>Upper</i>, efflux efficiency; <i>lower</i>, intestinal ³H-DPM abundance. The difference between the two strains was compared using the Mann—Whitney <i>U</i>-test. Bars indicate medians. Each plot shows an individual assay result.</p

Cholesterol enters HepG2 and differentiated Caco-2 cells by NPC1L1-independent manner mainly.

<p><i>A</i>, (a) Transient overexpression of NPC1L1 in the plasma membranes examined by Western blotting analysis. <i>Upper</i>, QuickBlue staining; <i>lower</i>, immunostaining for NPC1L1. (b) ABCG5 (<i>upper</i>) and ABCG8 (<i>lower</i>) protein expressions in HepG2 cells. <i>Insets</i>, these two images were adjusted to increase visibility of the bands. <i>B</i>, Medium-to-cell cholesterol transit in HepG2 cells. Transfection and ezetimibe treatment were performed as indicated as shown in the bottom. Medium-to-cell ³H-cholesterol transit efficiency (%) was estimated as described in the “Materials and Methods”. Each plot shows an individual assay result. Bars indicate means. Alphabetical differences among the groups (in parentheses) indicate significant difference between the groups (<i>p</i> < 0.05) using Tukey's Honestly Significant Difference test. <i>C</i>, Cholesterol (<i>upper</i>) and protein (<i>lower</i>) abundance in HepG2 cells. No significant difference was observed among the groups using Tukey's Honestly Significant Difference test. Bars show mean ± SEM (<i>n</i> = 4). <i>D</i>, (a) An illustration of Caco-2 cell culture system used in this study. Caco-2 cells were grown on filter membranes to allow the development to an enterocyte-like phenotype (see text for the detailed methods). The supernatants in culture inserts and wells were designated as apical and basolateral media, respectively. Lipid micelles were added to the apical medium. (b) Absorptive epithelial cell morphology of differentiated Caco-2 cell monolayers with a cylinder-like cell shape, the development of microvilli, and the distal localization of nuclei demonstrated by an electron microscopic analysis. (c) NPC1L1 was localized to the apical membrane in differentiated Caco-2 cells. Confocal microscopic analysis showed that NPC1L1 colored in <i>green</i> was localized to the plasma membrane (the upper image), especially in the brush border area (the lower image). 7-amino-actinomycin D was used for counterstaining of nuclei (<i>red</i>). <i>E</i>, Ezetimibe had little effect on medium-to-cell ³H-cholesterol transit in differentiated Caco-2 cells. <i>Open circles</i>, vehicle (1% ethanol); <i>closed circles</i>, 50 μmol/l ezetimibe. Data were shown as mean ± SEM of triplicate assays. <i>F</i>, In contrast to human duodenum (a) and murine jejunum (b), the gene expressions of <i>ABCG5</i> and <i>ABCG8</i> were absent in differentiated Caco-2 cells (c). Gene expression levels of five major membrane sterol transporters were analyzed by quantitative RT-PCR. An RNA sample of human duodenum was assayed in triplicate. Murine jejunal RNA samples were obtained from three C57BL/6J mice and assayed in duplicate. Three separate total RNA samples were obtained from independent wells of differentiated Caco-2 cells and assayed in duplicate. Data were shown as mean ± SEM of analytical triplicate (a) or biological triplicate (b, c) assays.</p

Gene and cDNA structures of the novel splicing variant of mouse <i>Pparγ</i>.

<p>(A) Alignment of 5′-end sequences of <i>Pparγ1sv</i>, <i>Pparγ1</i>, and <i>Pparγ2</i> cDNAs. Each initiation codon is outlined, and exon 1, which is common to all three, is underlined. (B) Gene structure of N-terminal exons and common exon 1 of mouse <i>Pparγ</i> on chromosome 6. Distances between two exons and exon lengths are indicated as numbers of nucleotides. Arrows indicate the positions of the transcription initiation site of each transcription variant. (C) Multiple alignment of nucleotide sequences of mouse exon C and corresponding exons in other mammals. Distances between the 5′-end of each cDNA and exon C or corresponding exons of other mammals are indicated as numbers of base pairs.</p

Effect of ezetimibe on cholesterol transit in mice.

<p><i>A</i>, An illustrated protocol for the ³H-cholesterol distribution assay. <i>B</i>, <i>Upper</i>; ³H-decay per minute (DPM) count distribution 3 h after ³H-cholesterol was given orally to C57BL/6J mice. Bars indicate mean and the standard deviation (<i>n</i> = 5 for vehicle and <i>n</i> = 4 for ezetimibe). <i>Open bars</i>, vehicle; <i>gray bars</i>, ezetimibe (50 μg). The ³H-DPM abundance in each portion was shown as % as given ³H-DPM as 100% (<i>left</i>, <i>black bar</i>). In the vehicle treatment, almost all the tracer infused was recovered; thus, we did not measure the tracer in the cecum and data of the cecum is absent for the vehicle. In the ezetimibe treatment, the given tracer count was not sufficiently recovered. We then measured the cecum and detected approximately 20% of the given tracer in them. <i>B</i>, <i>Lower</i>; the pie charts show summaries of ³H distribution in the small intestine (tissue), intestinal tract (lumen), and absorbed (the sum of the serum and the liver). <i>C</i>, Dose-dependent inhibitory effect of ezetimibe on fractional cholesterol transit into the serum and the liver. The reduction in tracer activity in enterocytes reached a plateau at 5 μg ezetimibe, whereas reductions in the liver and serum were greater than that in enterocytes with 5 μg and 50 μg ezetimibe. Changes in the distribution are shown with vehicle treatment as 100%. The significance of individual differences was evaluated by using Dunnett’s test. * <i>p</i> < 0.05; ** <i>p</i> < 0.01 vs. vehicle. Bars indicate mean ± SEM (<i>n</i> = 6).</p

Inhibition of NPC1L1 increases trans-intestinal cholesterol efflux (TICE).

<p><i>A</i>, An example of the TICE assay settings. <i>B</i>, An illustrated protocol for TICE assay. Assay time course is indicated from left to right. Arrow heads show the time points when reagents were given to mice. <i>C</i>, Decay per minute (DPM) counts in 5 μl blood obtained at the indicated time points after ³H-cholesterol intravenous infusion via the jugular vein. The DPM counts for individual mice were normalized against that at 60 min. Each symbol indicates an individual assay result. Infused tracer seemed to be equivalent in the circulation approximately 30 min after the infusion. <i>Open symbols</i>, vehicle controls; <i>closed symbols</i>, mice treated with ezetimibe. <i>D</i>, Ezetimibe treatment increased TICE. <i>Open circles</i>, vehicle; <i>closed circles</i>, 50 μg ezetimibe treated. Plots and error bars show mean and SEM (n = 8), respectively. <i>E</i>, Plots for ³H-DPM counts in sera (<i>left</i>, <i>open circles</i>) and those in total perfusates (<i>right</i>, <i>gray circles</i>). <i>F</i>, Comparison of the area under the concentration-time curve (AUC) of <i>D</i>. Each plot shows an individual assay result. <i>P</i> value was obtained using the Student’s <i>t</i>-test.</p

Luciferase reporter assay for the <i>Pparγ1sv</i> promoter in 3T3-L1 cells.

<p>(A) Western blotting of 3T3-L1 cells during adipocyte differentiation (days 0–9) and NIH/3T3 cells transfected with the overexpression construct containing no insert (control) or coding region of C/EBPβ isoforms (p34 and p30) detected by anti-C/EBPβ antibody. (B) The luciferase reporter construct, the pGL3-Basic, containing the <i>Pparγ1sv</i>, <i>Pparγ1</i>, or <i>Pparγ2</i> promoter was co-transfected with the C/EBP overexpression construct to 3T3-L1 cells. Cells were harvested 2 days after transfection and assayed using Dual-luciferase reporter assay reagents. The values represent the mean of triplicate measurements. The activity obtained from cells transfected with empty vector is defined as 1. (C) Effect of C/EBPβ depletion by siRNA on <i>Pparγ</i> expression. Each of two discrete C/EBPβ siRNAs (siC/EBPβ #1 and #2) was transfected to 3T3-L1 cells. Cells were harvested at days 0 and 3 of differentiation, and expression of <i>Pparγ1sv</i> (black bar) and <i>Pparγ2</i> (gray bar) mRNAs was evaluated by qPCR. Values were normalized to those of 18S rRNA. The RNA expression of siControl cells at day 3 is defined as 100%. (D) Immunoblotting of the nuclear extracts of siC/EBPβ #1-treated cells detected by antibodies specific to each of C/EBPβ, PPARγ, or Lamin B1 (control).</p

Expression of three <i>Pparγ</i> transcripts during adipogenesis of 3T3-L1 cells and mouse primary preadipocytes.

<p>3T3-L1 cells (A) and mouse primary preadipocytes (B) were cultured in the differentiation medium described in the “Materials and Methods”, harvested at the indicated periods, and analyzed by real-time RT-PCR. The values obtained for <i>Pparγ1sv</i>, <i>Pparγ1</i>, and <i>Pparγ2</i> were normalized to those of 18S rRNA. Data represent the mean obtained from cells of three independent wells. Immunoblotting of nuclear extract of 3T3-L1 (C) and primary cultured cells (D) during adipocyte differentiation using the anti-PPARγ and anti-Lamin B1 (loading control) antibodies. Oil Red O staining of 3T3-L1 (E) and primary cultured cells (F) at indicated periods of adipocyte induction.</p