Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes

<div><p>Background</p><p>While screening for an inhibitor of the peptidyl prolyl cis/trans isomerase, Pin1, we came across a brown algae polyphenol that blocks the differentiation of fibroblasts into adipocytes. However, its effectiveness on the accumulation of fat in the body has never been studied.</p><p>Methodology/Principal Findings</p><p>Oral administration of brown algae polyphenol to mice fed with a high fat diet, suppressed the increase in fat volume to a level observed in mice fed with a normal diet. We speculate that Pin1 might be required for the differentiation of stem cell to adipocytes. We established wild type (WT) and <i>Pin1</i>^<i>-/-</i> (Pin1-KO) adipose-derived mesenchymal stem cell (ASC) lines and found that WT ASCs differentiate to adipocytes but Pin1-KO ASCs do not.</p><p>Conclusion and Significance</p><p>Oral administration of brown algae polyphenol, a Pin1 inhibitor, reduced fat buildup in mice. We showed that Pin1 is required for the differentiation of stem cells into adipocytes. We propose that oral intake of brown algae polyphenol is useful for the treatment of obesity.</p></div

Comparison of ASC differentiation to adipocytes.

<p>Comparison of adipocyte differentiation between the WT (<i>Pin1</i>^<i>+/+</i><i>; p53</i>^<i>-/-</i>) ASC and Pin1-KO (<i>Pin1</i>^<i>-/-</i><i>; p53</i>^<i>-/-</i>) ASC, and Pin1-KO ASC rescued with the lentiviral Pin1 cDNA. The wild type ASC, the Pin1-KO ASC- infected with Mock and the Pin1-KO ASC- infected with lentiviral Pin1 cDNA were cultured in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin for 0–16 days. ASCs were treated with 4% paraformaldehyde and 60% 2-propanol, and then stained with Oil Red O. The images of the oil red O-stained cells on 0, 4, 7, 10, 13, and 16 days after treatment with the differentiation reagent are shown.</p

Primer List for the Real-time PCR

<p>Primer List for the Real-time PCR</p

Effect of brown algae polyphenol on the differentiation of NIH3T3-L1 cells to adipocytes.

<p><b>A) The amount of the intracellular lipid stained with oil red O.</b> NIH3T3-L1 cells were cultured in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin with 0 (diamond), 50 (square), 100 (triangle), and 150 μg/ml (x) brown algae polyphenol for 0–8 days. NIH3T3-L1 cells were treated with 4% paraformaldehyde and 60% 2-propanol and then stained with Oil Red O. The amount of oil red O extracted from the cells was determined three times by measuring absorbance at 550 nm (means ± SEM). <b>B) PCR analysis of adipocyte biomarker</b> mRNA levels. NIH3T3-L1 cells were cultured with 0, 50, 100, and 150 μg/ml of brown algae polyphenol for 0–8 days. The mRNA levels of PPARγ, C/EBPα, Glut4, FABP4, and LPL in these cells were compared by PCR.</p

Real time PCR analysis of adipocyte biomarkers.

<p>The mRNA levels of adipocyte biomarker molecules, such as C/EBPα, Glut4, PPARγ, IRS1, and LPL in the WT and Pin1-KO ASC treated with the differentiation reagent were quantitatively measured with real time PCR three times using the primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168830#pone.0168830.t001" target="_blank">Table 1</a> (means ± SEM). **p<0.01, * p<0.05.</p

Comparison of obesity marker levels in sera between mouse groups (n = 5 each).

<p>Leptin (ng/ml), total cholesterol (mg/dl), free fatty acids (mEq/L), neutral fat (mg/dl), and adiponectin (ng/ml) in the serum of each mouse, 1: high fat diet + brown algae polyphenol, 2: high fat diet, 3: normal diet.</p

Pin1-KO ASC- transfected with Lentiviral Pin1 cDNA.

<p>Transfection was verified by observing GFP with fluorescence microscope and the western blot analysis of Pin1 and α-tubulin (1st: anti-h/m Pin1 antibody (Santa Cruz), anti-α-tubulin antibody (SIGMA), 2nd anti-mouse IgG HRP-linked antibody (Cell Signaling), All antibodies were diluted to 1/1000).</p

Paraffin-embedded sections of visceral and subcutaneous fat tissue from mice fed with a normal diet, high fat diet (HFD) and high fat diet plus brown algae polyphenol (HFD + BAP) were stained with hematoxylin and eosin.

<p>Paraffin-embedded sections of visceral and subcutaneous fat tissue from mice fed with a normal diet, high fat diet (HFD) and high fat diet plus brown algae polyphenol (HFD + BAP) were stained with hematoxylin and eosin.</p

Association of Pin1 and CREB.

<p>(A) Lysates of COS-7 cells, transfected with <i>HA-CREB</i> (<i>wild-type; T172A; S271A; S80A; S80A/T172A; T172A/S271A; S80A/S271A</i>) and stimulated with 10 µM forskolin for 6 hr, were pulled down with GST-Sepharose (control), GST-Pin1-Sepharose, and input (upper panel). CREB levels in the supernatant are shown in the lower panel. (B) 10⁶ of COS-7 were co-transfected with pCRE-Luc and CMV-Pin1 (wt; wild type, W34A; mutation at WW domain, R68,69A; prolyl isomerase mutant). The cells were treated with 100 µM of forskolin (CST) for 6 hours and luciferase activities were determined using the Dual-Luciferase Reporter Assay System (Promega).</p

Relationship between quantity of fat and Pin1 expression.

<p>High fat diets were fed to wild-type (n = 8) and Pin1^−/− (n = 5) mice between the ages of 4–28 weeks and the amount of food intake (A) and body weight (B) were recorded. Weights of subcutaneous and genital fat tissues were measured (C). The total weight of all fat tissues removed was also measured (D). The amount of food intake per mouse weight was monitored. Student t-test *p<0.05, **p<0.01, ***p<0.001.</p