Up-regulation of PXR target genes by PCN treatment.

<p>At end of the 7-week treatment with PCN, mice were sacrificed 4 h later and livers were taken and frozen at -80°C. Hepatic expressions of selected genes were measured by real-time PCR analysis. #P<0.05, ##P<0.01 compared to chow-DMSO group; *P<0.05, **P<0.01 compared to high-fat diet-DMSO group. Abbreviations: <i>Cyp3a11</i>, cytochrome P450, family 3, subfamily a, polypeptide 11 gene; <i>Cyp2b10</i>, cytochrome P450, family 2, subfamily b, polypeptide 10 gene; <i>Sult2a1</i>, cytosolic sulfotransferase 2A1 gene; and <i>Mdr1a</i>, multi-drug-resistance 1a gene. Each data point represents the average ± SD of 4 animals in each group.</p

Effect of PCN treatment on hepatic accumulation of lipids.

<p>At the end of the 7-week treatment, animals were sacrificed; liver sections were made and stained with H&E (<b>A–D</b>) or Oil Red O (<b>E–H</b>). <b>A</b> and <b>E</b>, animals on regular chow and treated with DMSO; <b>B</b> and <b>F</b>, animals on regular chow and treated with PCN; <b>C</b> and <b>G,</b> animals on high-fat diet and treated with DMSO; and <b>D</b> and <b>H</b>; animals on high-fat diet and treated with PCN. Scale bar, 20 µm.</p

PCN treatment protected mice against high-fat diet–induced obesity.

<p>Four-week-old male AKR/J mice were fed with high-fat diet or regular chow for 7 weeks with twice weekly injections of PCN (50 mg/kg, IP) or DMSO (carrier solution). <b>A,</b> growth curve; <b>B</b>, fat and lean mass; <b>C,</b> food intake; and <b>D</b>, calculated ratio of caloric intake/body weight. Each data point represents the average ± SD of 4 animals in each group. #P<0.05, ##P<0.01 compared to chow-DMSO group; *P<0.05, **P<0.01 compared to high-fat diet-DMSO group.</p

Effect of PCN treatment on energy expenditure of brown adipose tissue.

<p>Six-week-old male AKR/J mice were fed with high-fat diet for two weeks with three injections per week of PCN (50 mg/kg) or DMSO (control). By the end of the feeding period, mice were exposed at 4°C and the rectal temperature measured at different times. <b>A</b>, time-dependent rectal temperature. For determination of the relative mRNA level of genes involved in thermogenesis, animals at the end of 7-week treatment were injected with PCN or carrier solution and sacrificed 4 h later. BAT tissues were harvested and total RNA was extracted. <b>B.</b> Relative mRNA levels of genes involved in thermogenesis. Each data point represents the average ± SD of 4 animals in each group. #P<0.05, ##P<0.01 compared to chow-DMSO group; *P<0.05, **P<0.01 compared to high-fat diet-DMSO group. Abbreviations: PGC-1, peroxisome proliferator-activated receptor gamma coactivator 1 gene; Dio2, Type II iodothyronine deiodinase gene; Cidea, cell death-inducing DNA fragmentation factor alpha-like effector A gene; and UCP, uncoupling protein gene.</p

Primer sets for real time RT-PCR analysis of gene expression.

<p>Primer sets for real time RT-PCR analysis of gene expression.</p

Effect of PCN treatment on glucose tolerance, insulin sensitivity, serum concentration of insulin, and mRNA level of <i>G6Pase</i> and <i>Pepck</i>.

<p>Animals at the end of the 7-week treatments were fasted overnight for glucose tolerance tests, or fasted for 4 h for insulin sensitivity tests. <b>A</b>, time-dependent blood concentration of glucose upon IP injection of glucose (2 g/kg); <b>B</b>, area under the curve from glucose tolerance test in A; <b>C</b>, time dependent glucose concentration upon IP injection of insulin; <b>D</b>, insulin levels at the end of the 7-week feeding with high-fat diet and regular chow food with or without PCN treatment; <b>E</b>, relative mRNA level of <i>G6Pase</i> and <i>Pepck</i> in mouse liver at the end of animal feeding and PCN treatment; and <b>F</b>, HOMA-IR values calculated based on formula: Glucose (mg/dl) × Insulin ( µU/ml)/405. Each data point represents the average ± SD of 4 animals in each group. #P<0.05, ##P<0.01 compared to chow-DMSO group; *P<0.05, **P<0.01 compared to high-fat diet-DMSO group.</p

Effect of PCN treatment on expression of genes involved in adipocyte differentiation and lipid metabolism.

<p>At the end of the 7-week treatment animals were treated with PCN or DMSO (carrier solution) and sacrificed 4 h later. WAT and BAT tissues were harvested and total RNA was extracted. Relative mRNA level of genes involved in adipose differentiation in WAT (<b>A</b>) and BAT (<b>D</b>) tissue; genes involved in the lipogenesis in WAT (<b>B</b>) and BAT (<b>E</b>); and genes involved in lipids uptake and β-oxidation in WAT (<b>C</b>) and BAT (<b>F</b>)<b>.</b> Each data point represents the average ± SD of 4 animals in each group. #P<0.05, ##P<0.01 compared to chow-DMSO group; *P<0.05, **P<0.01 compared to high-fat diet-DMSO group. Abbreviations: aP2, adipocyte protein 2 gene; C<i>/</i>EBPα, CCAAT/enhancer binding protein alpha gene; PPARγ, peroxisome proliferator-activated receptor gamma gene; ACC1, acetyl-CoA carboxylases 1 gene; CPT1b, carnitine palmitoyltransferase 1b gene; and HSL, hormone-sensitive lipase gene.</p

Effect of PCN treatment on expression of genes involved in lipogenesis and lipids uptake.

<p>Four h after treatment, mice were sacrificed and livers were harvested. The expression of genes involved in the cholesterol metabolism (<b>A</b>, <b>B</b>), lipids metabolism (<b>C</b>) and lipids uptake (<b>D</b>) were measured by real-time PCR. Each data point represents the average ± SD of 4 animals in each group. #P<0.05, ##P<0.01 compared to chow-DMSO group; *P<0.05, **P<0.01 compared to high-fat diet-DMSO group. Abbreviations: Cyp7a1, cholesterol 7 alpha-hydroxylase gene; HMGCR, 3-hydroxy-3-methylglutaryl coenzyme A reductase gene; Cyp27a1, sterol 27-hydroxylase; Abca1, Abcg1, ATP-binding cassette transporter A1 and G1; SREBP<i>-</i>1c, sterol regulatory element-binding protein 1c; FAS, fatty acid synthase; Scd-1, stearoyl CoA desaturase 1; and L-FABP, liver fatty-acid-binding protein.</p

High-Fat Diet-Induced Adiposity, Adipose Inflammation, Hepatic Steatosis and Hyperinsulinemia in Outbred CD-1 Mice

<div><p>High-fat diet (HFD) has been applied to a variety of inbred mouse strains to induce obesity and obesity related metabolic complications. In this study, we determined HFD induced development of metabolic disorders on outbred female CD-1 mice in a time dependent manner. Compared to mice on regular chow, HFD-fed CD-1 mice gradually gained more fat mass and consequently exhibited accelerated body weight gain, which was associated with adipocyte hypertrophy and up-regulated expression of adipose inflammatory chemokines and cytokines such as <i>Mcp-1</i> and <i>Tnf-α</i>. Increased fat accumulation in white adipose tissue subsequently led to ectopic fat deposition in brown adipose tissue, giving rise to whitening of brown adipose tissue without altering plasma level of triglyceride. Ectopic fat deposition was also observed in the liver, which was associated with elevated expression of key genes involved in hepatic lipid sequestration, including <i>Ppar-γ2</i>, <i>Cd36</i> and <i>Mgat1</i>. Notably, adipose chronic inflammation and ectopic lipid deposition in the liver and brown fat were accompanied by glucose intolerance and insulin resistance, which was correlated with hyperinsulinemia and pancreatic islet hypertrophy. Collectively, these results demonstrate sequentially the events that HFD induces physiological changes leading to metabolic disorders in an outbred mouse model more closely resembling heterogeneity of the human population.</p></div

HFD impaired glucose homeostasis, which subsequently gave rise to hyperinsulinemia and pancreatic islet hypertrophy.

<p>(A) Profiles of blood glucose concentration as function of time upon intraperitoneal injection of glucose (n = 5). (B) Profiles of glucose concentration (percentage of initial value) as a function of time upon intraperitoneal injection of insulin (n = 5). (C) Blood insulin (n = 4). (D) Representative images of pancreas histological examinations. Values in (A), (B) and (C) represent average ± SD. * <i>P</i> < 0.05 compared with mice on chow, ** <i>P</i> < 0.01 compared with mice on chow.</p