Time dependent effects of rosiglitazone on osteoblast differentiation.

<p>Primary bone marrow cells were treated with rosiglitazone (10 μM) at the indicated times in the presence of DAG. Extents of osteoblast differentiation were determined by Alizarin red staining (A) and western blotting (B). Experiments were carried out three times and representative results are shown. Densitometric analysis was conducted using UN-SCAN-IT gel ver. 5.1 software (Silk Scientific) and is expressed as means ± SEMs of three experiments. #<i>P</i><0.05 vs differentiated (D). D, differentiated in DAG medium; DR, DAG in the presence of rosiglitazone.</p

Effects of PHD inhibitors on rosiglitazone-induced anti-osteoblast differentiation.

<p>Primary bone marrow cells were treated with either DMOG (1 mM) or EDHB (20 μM) in the presence or absence of rosiglitazone (10 μM) for 12 days (media were changed every 48 h). Extents of osteoblast differentiation were monitored by Alizarin red staining (A), and the mRNA levels of the PHD 1–3 and osteomarker marker genes were determined by RT-PCR (B). The protein levels of Runx2 and levels of Runx-2 ubiquitination were determined by western blotting (C) and by immunoprecipitation using polyubiquitination antibody (D). The effect of MG-132 (5 μM; an inhibitor of proteasomal degradation) on Runx-2 expression was examined by western blotting (E). Experiments were conducted three times and representative results are shown. Densitometric analysis was conducted using UN-SCAN-IT gel ver. 5.1 software (Silk Scientific) and is expressed as means ± SEMs of three experiments. *<i>P</i><0.05 vs control (C), #<i>P</i><0.05 vs differentiated (D), ✝<i>P</i><0.05 vs DR. C, control (no differentiation); D, differentiated in DAG medium; DR, DAG in the presence of rosiglitazone; DM, DMOG; ED, EDHB; MG, MG-132.</p

<i>In vivo</i> effects of rosiglitazone in female ICR mice.

<p>Rosiglitazone (10 mg/kg once daily) was administered orally to ICR mice (6 weeks old) for eight weeks. Primary bone marrow cells were isolated from the femora of both vehicle and rosiglitazone-treated animals, and extents of osteoblast differentiation were determined by Alizarin red staining (A). PHD expression and ubiquitinated Runx2 levels were determined by western blotting and immunoprecipitation, respectively (B). Immunohistochemistry of the PHD isoforms (C), and quantification of PHD1, PHD2, and PHD3 signal intensity in bone marrow section (D). Runx2 expression was determined by immunohistochemistry (E). Red arrow indicates each PHD and Runx2 expression. Experiments were conducted three times and representative results are shown. *<i>P</i><0.05 vs vehicle.</p

DEXA analysis of rosiglitazone-treated mice.

<p>DEXA analysis of rosiglitazone-treated mice.</p

Concentration dependent effects of rosiglitazone on osteoblast differentiation.

<p>Primary bone marrow cells were treated with the indicated concentrations of rosiglitazone for 12 days in the presence of DAG and extents of osteoblast differentiation were determined by Alizarin red staining (A) and western blotting (B). Experiments were conducted three times and representative results are shown. Densitometric analysis was conducted using UN-SCAN-IT gel ver. 5.1 software (Silk Scientific), and is expressed as means ± SEMs of three experiments. *<i>P</i><0.05 vs control (C), #<i>P</i><0.05 vs differentiated (D). C, control (no differentiation); D, differentiated in DAG medium; R, rosiglitazone.</p

Effects of PHD knockdown on the rosiglitazone-induced inhibition of osteoblast differentiation.

<p>The PHD1, 2, and 3 isoforms were knocked down by transfecting in primary bone marrow cells with isoform-specific shRNAs or siRNA on day 0 and day 4 of a 12-day differentiation period. Extents of knockdown were determined by RT-PCR (A), and osteoblast differentiation was monitored by Alizarin red staining (B). The protein levels of Runx2 were determined by western blotting (C). Experiments were conducted twice and representative results are shown. Densitometric analysis was conducted using UN-SCAN-IT gel ver. 5.1 software (Silk Scientific) and is expressed as means ± SEMs of two experiments. *<i>P</i><0.05 vs control shRNA in the absence of rosiglitazone, #<i>P</i><0.05 vs control shRNA in the presence of rosiglitazone.</p

Effects of PPARγ antagonists on rosiglitazone-induced adipocyte differentiation.

<p>Primary bone marrow cells were treated with BADGE (20 μM) or GW9662 (1 μM) in the presence of rosiglitazone (10 μM) for 12 days. Osteoblast differentiation was determined by Alizarin red staining (A), and the mRNA levels of PHD1, 2, and 3, and osteogenic marker genes were analyzed by RT-PCR (B). Densitometric analysis was conducted using UN-SCAN-IT gel ver. 5.1 software (Silk Scientific) and is expressed as means ± SEMs. *<i>P</i><0.05 vs control (C), #<i>P</i><0.05 vs differentiated (D), ✝<i>P</i><0.05 vs DR. Experiments were conducted three times and representative results are shown. C, control (no differentiation); D, differentiated in DAG medium; DR, DAG in the presence of rosiglitazone; BD, BADGE; GW, GW9662.</p

Metabolite marker signals contributing to the differentiation and prediction.

<p>a. S-plot analysis showing the correlation and covariation. Metabolites on the upper right corner contribute to the improved group and on the lower left corner contribute to the non-improved group. b. PLS loading plot showing the contribution to the prediction of the HbA1c. Metabolites signals were identified using Chenomx and in-house built metabolite libraries.</p

Prediction of HbA1c using PLS multivariate regression.

<p>PLS regression models were built with the NMR profile at 7-day time point and the 3-month post-operative HbA1c values. The observed (X-axis) values are actually measured values and the predicted (Y-axis) values are from the PLS regression model obtained with two PLS components. The diagonal dashed line represents the theoretical perfect match, and the dotted line represents the least-square fitted line. Comparison between the observed and predicted values obtained from the training dataset (a), leave-one-out analysis (b), and three-fold cross validation (c). The predicted values in (a) do not represent true prediction since all the data were used in the model building. One (b) or seven (c) samples were left out at a time, and the predictions were made using the model built without the test data to be predicted until every sample was left out once.</p

Change of Anthropometric and Metabolic Parameters in improved and non-improved groups.

<p>*Patients who had glycated hemoglobin percentage less than 7.0% without glucose-lowering agent in 3 months after metabolic surgery.</p>1<p>OGTT indicates oral glucose tolerance test at 30, 60, 90, or 120 min after 75 g glucose intake.</p>2<p>paired t-test.</p>3<p>Not available.</p><p>Change of Anthropometric and Metabolic Parameters in improved and non-improved groups.</p