Effect of increasing dietary fat on body weight and composition in male rats.

<p>LFD, low fat diet (control pelleted AIN-93G 14% fat diet); MFD, medium fat TEN diet (25% fat diet); HFD, high fat TEN diet (45% fat diet). (A), growth curves from three different diet groups. (B), visceral (gonadal fat) % body weight and (C), abdominal fat % body weight (D), Representative pseudocolored images of tibial peripheral quantitative CT scans (pQCT) (slice 3) from LFD, MFD and HFD rats. Color changes from blue, yellow, red, to gray represent decreases in bone density. Decreased bone mass in male rats fed HFD can be visualized. (E), total tibial bone mineral content (BMC). (F), tibial trabecular bone mineral density (BMD). (G), total tibial BMD. (H), tibial cortical BMD. Data bars are expressed as mean ± SEM (n = 6/group). Means with different letters differ significantly from each other at P<0.05, a</p

Increased adipogenesis in bone and bone marrow in HFD-induced obese animals.

<p>(A), Represented H&E staining picture of increased bone marrow adiposity in tibial bone section from HFD-induced obese animals (10x). (B) and (C), osteocalcin and Runx2 gene expression measured using real-time RT-PCR. (D) and (E), PPARγ and aP2 gene expression measured using real time RT-PCR. Total RNA was isolated from femur of each animal after bone marrow aspiration for RT-PCR. LFD, low fat diet (control pelleted AIN-93G 14% fat diet); MFD, medium fat diet (25% fat diet); HFD, high fat TEN diet (45% fat diet). Data bars are expressed as mean ± SEM (n = 6/group). Means with different letters differ significantly from each other at p<0.05, a</p

Activation of PPARγ and regulation of its target gene.

<p>(A), nuclear proteins were extracted from rat tibia and from ST2 cells treated with rat serum and FAs mixture using 10 cm dish. TransAM was performed for activity of PPARγ. (B) ST2 cells were treated for 24 h with FAs mixture according to the concentrations that appeared in animal circulation. ChIP of mouse aP2 enhancer elements by specific anti PPARγ antibody. Control, normal cell culture medium; LFD, low fat diet (control pelleted AIN-93G 14% fat diet); MFD, medium fat TEN diet (25% fat diet); HFD, high fat TEN diet (45% fat diet). Bars are expressed as mean ± SEM in triplicates. *, P<0.05, <i>versus</i> LFD by ANOVA followed by Student-Newman-Keuls post hoc analysis for multiple pairwise comparisons. #, P<0.05, <i>versus</i> control by t-test.</p

Effects of feeding HFD on serum leptin, non-esterified fatty acids (NEFA) and bone turnover markers.

<p>Serum bone formation marker osteocalcin (A), resorption marker RatLaps (B), leptin levels (C), and NEFA levels (D) were measured using standard ELISA methods. Data bars are expressed as mean ± SEM (n = 6/group). LFD, low fat diet (control pelleted AIN-93G 14% fat diet); MFD, medium fat TEN diet (25% fat diet); HFD, high fat TEN diet (45% fat diet). Means with different letters differ significantly from each other at p<0.05, a</p

Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Primer Sequences.

<p>Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Primer Sequences.</p

Increased PPARγ but decreased β-catenin protein expression in bone from HFD-induced obese rats.

<p>(A) Representative pictures of immunostained tibial bone sections using an anti-mouse β-catenin polyclonal antibody. White arrows indicate immunostained β-catenin, and black arrows indicate bone tissues. (B), Western blots of PPARγ (from total protein lysates, nuclear and cytoplasmic fractions), β-catenin and β-actin are depicted for four samples from LFD, MFD and HFD groups. LFD, low fat diet (control pelleted AIN-93G 14% fat diet); MFD, medium fat TEN diet (25% fat diet); HFD, high fat TEN diet (45% fat diet). Proteins were isolated from long bone after aspiration of bone marrow cells.</p

SPI stimulates osteoblast differentiation via down-regulation of caveolin-1.

<p>ST2 cells were treated with 2% serum either from control, 10 µg/kg/d E2 treated, SPI diet rats and 5 µM of purified genistein for 48 h. RNA and protein were collected. (A), Real-time PCR showing relative caveolin-1 and Runx2 mRNA expression. Data are Means ± SEM, triplicates, * p<0.05 versus control. (B), Western blotting showing caveolin-1 protein, nuclear SMD and cytosolic versus membrane BMP2 expression. Quantitation of the intensity of the caveolin-1 in the autoradiograms was performed relative to expression of β-actin.</p

Caveolin-1, membrane versus cytoplasm BMP-2 protein expression in bone of pre-pubertal female rats from Control, standard casein diet group; E2, 10 µg/kg/d E2 treated group; SPI, soy protein isolate diet group; E2+SPI, combination of 10 µg/kg/d E2 treated and SPI diet group.

<p>Quantitation of the intensity of the caveolin-1 bands in the autoradiograms was performed relative to expression of β-actin, membrane BMP-2 (M-BMP-2) and nuclear BMP-2 (N-BMP-2) were performed relative to total BMP-2 (T-BMP-2). Data are Means ± S.E.M, n = 3, with different letters differ significantly from each other at p<0.05, a</p

Real-time PCR verification of expression of genes of caveolin-1, ATF-3, calcitonin and Sost (sclerostin) in bone.

<p>Black bars represent fold changes with microarray analyses and gray bars represent relative mRNA expression from real-time PCR analyses. Control, standard casein diet group; E2, 10 µg/kg/d E2 treated group; SPI, soy protein isolate diet group; E2+SPI, combination of 10 µg/kg/d E2 treated and SPI diet group. Means ± S.E.M of real-time PCR results with different letters differ significantly from each other at p<0.05, a</p

Cell signaling transduction pathway after activation of BMP-2 by SPI and E2 in bone.

<p>(A), Protein was isolated from bone after aspiration of bone marrow cells and performed Western blots. Smad and ERK1/2 phosphorylation and Runx2 protein expression in pre-pubertal female rat bone from Control, standard casein diet group; E2, 10 µg/kg/d E2 treated group; SPI, soy protein isolate diet group; E2+SPI, combination of 10 µg/kg/d E2 treated and SPI diet group. Quantitation of the intensity of the phosphorylated bands of p-Smad1, 5, 8 and p-ERK1/2 in the autoradiograms were performed relative to expression of total Smad4 and ERK1/2. Blot of Ponceau S staining showing protein loading control for western blotting. (B), Showing real-time PCR analyses for Runx2. Data are Means ± S.E.M, n = 3, with different letters differ significantly from each other at p<0.05, a</p