Introduction 1 2 3 4 1 5 6 7 8 9 10 6 1 10 11 ob/ob 12 13 14 15 16 17 18 The above discrepancy could be due to different effects on preadipocytes and mature adipocytes in adipose tissue. It is clear that TZDs stimulate preadipocytes into the process of differentiation. However, their effect on mature adipocytes is less clear. In vivo studies do not allow us to distinguish between the effects on these two cell types. Therefore, to study the effects of TZDs on mature adipocytes, we used in vitro differentiated mature 3T3-L1 adipocytes. In addition to the assessment of lipid accumulation and adipokine secretion, we used transcriptomics to obtain insight into the effects of rosiglitazone. Although high insulin and high glucose levels mimic the situation in vivo when rosiglitazone is administered, we also included low insulin and low glucose levels for comparison. Materials and methods Cell culture and sample preparation 19 19 20 Oil red O staining 21 Glycerol assay N 2 Microarray 19 19 Microarray data analysis 19 t p http://www.ingenuity.com p Quantitative real-time RT-PCR  1 19 Rps15 One- and two-dimensional gel electrophoresis 19 20 22 Western blotting 20 Results Effect of rosiglitazone on the lipid content of mature adipocytes p n 1 Fig. 1 ORO n n Blank bars filled bars p t p t  Effect of rosiglitazone on gene expression 2 Pparg p Fig. 2 Blank bars filled bars Bold line  1 Ucp2 Fasn Scd1 1 Table 1 Key enzymes of metabolism pathways regulated by rosiglitazone Pathway Change Gene symbol Gene ID Name Rosi/Basal RosiIG/IG Fold-change p Fold- change p TCA cycle Up Idh3a 67834 Isocitrate dehydrogenase 3 (NAD+) alpha 2.53 0.000 2.55 0.000   Cs 12974 Citrate synthase 1.95 0.000 1.47 0.000 Oxidative  phosphorylation Up Ndufa5 68202 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5 1.72 0.009 1.20 0.235   Sdhd 66925 Succinate dehydrogenase complex, subunit D 1.83 0.001 1.51 0.039   Uqcrc1 22273 Ubiquinol-cytochrome c reductase core protein I 1.47 0.007 1.33 0.017   Cox7a1 12865 Cytochrome c oxidase subunit VIIa polypeptide 1 1.78 0.002 1.55 0.003   Atp5h 71679 ATP synthase, H+ transporting, mitochondrial  F0 complex, subunit d 1.71 0.006 1.49 0.005 Fatty acid uptake  and transport Up Cd36 12491 CD36 antigen, fatty acid translocase 2.54 0.000 2.27 0.006   Fabp4 11770 Fatty acid bind protein 4, adipocyte 1.64 0.088 1.57 0.142 Fatty acid  activation Up Acsl1 14081 Acyl-CoA synthetase long-chain family member 1 2.50 0.000 1.97 0.000 Fatty acid beta  oxidation Up Crat 12908 Carnitine acetyltransferase 1.45 0.006 1.47 0.007   Slc25a20 57279 Solute carrier family 25 (mitochondrial carnitine/acylcarnitine translocase), member 20 1.63 0.003 1.22 0.092   Acox1 11430 Acyl-coenzyme A oxidase 1, palmitoyl 2.70 0.000 2.54 0.000   Acaa1 113868 Acetyl-coenzyme A acyltransferase 1 (peroxisomal) 1.67 0.002 1.29 0.017   Acaa2 52538 Acetyl-coenzyme A acyltransferase 2 (mitochondrial) 2.70 0.000 1.94 0.000 Fatty acid  synthesis Down Fasn 14104 Fatty acid synthase −1.52 0.000 −1.55 0.001   Scd1 20249 Stearoyl-CoA desaturase 1 −3.65 0,000 −4.31 0.000 Triacylglycerol  synthesis Down Ppap2b 67916 Phosphatidic acid phosphatase type 2B −1.38 0.001 −1.48 0.000   Dgat2 67800 O −1.31 0.003 −1.37 0.006 Lipid droplet  formation Up/down Adfp 11520 Adipose differentiation related protein 5.97 0.000 6.60 0.000   Cav1 12389 Caveolin, caveolae protein 1 −2.20 0.000 −2.30 0.001 Glycerolipid lysis Up Pnpla2 66853 Patatin-like phospholipase domain  containing 2 1.52 0.006 1.67 0.007 Glycerol metabolism Up Gk 14933 Glycerol kinase 1.86 0.007 1.88 0.003   Gpd1 14555 Glycerol-3-phosphate dehydrogenase 1 (soluble) 2.07 0.009 1.68 0.032 Energy expenditure Up Ucp2 22228 Uncoupling protein 2 (mitochondrial,  proton carrier) 1.78 0.001 1.50 0.003 Glutathione  metabolism Up/down Mgst3 66447 S 2.11 0.001 1.88 0.001   Gsta4 14860 S 1.36 0.046 −1.01 0.924   Gstm1 14862 S −1.88 0.000 −2.01 0.010   Gstt1 14871 S −1.38 0.060 −1.51 0.044   Gstz1 14874 Glutathione transferase zeta 1 −1.44 0.001 −1.57 0.015 Prostaglandin  metabolism Up/down Cbr3 109857 Carbonyl reductase 3 3.04 0.000 3.96 0.000   Ptges2 96979 Prostaglandin E synthase 2 1.45 0.012 1.61 0.000   Ptgis 19223 2 −1.71 0.002 −1.41 0.004 Steroids  metabolism Up/down Hsd17b7 15490 Hydroxysteroid 17-beta dehydrogenase 7 1.63 0.003 1.73 0.002   Nsdhl 18194 NAD(P) dependent steroid dehydrogenase-like 1.51 0.039 1.37 0.017   Hsd11b1 15483 Hydroxysteroid 11-beta dehydrogenase 1 −2.73 0.001 −2.05 0.004 Glyconeogenesis Up Fbp2 14120 Fructose-1,6-bisphosphatase 2 2.92 0.003 2.82 0.014 Glycogen synthesis Up Gyg 27357 Glycogenin 1.52 0.001 1.34 0.008 Pyruvate  metabolism Up Pdha1 18597 Pyruvate dehydrogenase E1 alpha 1 1.38 0.001 1.33 0.001   Pdhb 68263 Pyruvate dehydrogenase (lipoamide) beta 1.59 0.005 1.61 0.004 Pentose phosphate Up H6pd 100198 Hexose-6-phosphate dehydrogenase (glucose  1-dehydrogenase) 1.60 0.000 1.47 0.001   Taldo1 21531 Transaldolase 1 1.57 0.001 1.33 0.036 Leu catabolism Down Ivd 56357 Isovaleryl coenzyme A dehydrogenase −1.42 0.004 −1.72 0.000   Mccc1 72039 Methylcrotonoyl-coenzyme A carboxylase 1  (alpha) −1.26 0.026 −1.35 0.031 n Rosi/basal: the effect with low insulin/glucose; RosiIG/IG: the effect with high insulin/glucose 1 S S S S S 2 2 2 2 2 1 23 Effect of rosiglitazone on the expression of genes encoding adipocyte-secreted proteins 24 27 20 Il1b Il6 Il10 Apoe 2 Table 2 Effect of rosiglitazone on the expression of genes encoding adipocyte-secreted proteins Function Gene symbol Gene ID Name Rosi/Basal RosiIG/IG Fold-change p Fold-change p Lipid  metabolism Adn 11537 Complement factor D (adipsin) −3.55 0.000 −4.23 0.000 C3 12266 Complement component 3 −2.25 0.000 −2.68 0.001 Lpl 16956 Lipoprotein lipase 1.12 0.166 −1.02 0.627 Apoe 11816 Apolipoprotein E 1.34 0.026 1.18 0.145 Insulin  sensitivity Retn 57264 Resistin −3.07 0.000 −3.17 0.001 Igf1 16000 Insulin-like growth factor 1 −1.55 0.012 −1.58 0.001 Pbef1 59027 Pre-B-cell colony-enhancing factor 1 −1.24 0.050 −1.28 0.085 Adipoq 11450 Adiponectin −1.09 0.462 −1.25 0.108 Inflammation Hp 15439 Haptoglobin −2.93 0.000 −4.42 0.000 Ptx3 19288 Pentaxin-related gene −1.68 0.001 −1.58 0.000 Tgfb3 21809 Transforming growth factor, beta 3 −1.58 0.008 −1.44 0.056 B2m 12010 Beta-2 microglobulin −1.54 0.018 −1.64 0.006 Ccl2 20296 Chemokine (C-C motif) ligand 2 (previously known as monocyte chemoattractant protein-1) −1.54 0.012 −1.48 0.023 Saa3 20210 Serum amyloid A 3 −1.49 0.047 −1.38 0.001 Cxcl12 20315 Chemokine (C-X-C motif) ligand 12 −1.45 0.006 −1.42 0.046 Il6ra 16194 Interleukin 6 receptor, alpha −1.16 0.128 −1.15 0.037 Tgfb1 21803 Transforming growth factor, beta 1 −1.15 0.163 1.02 0.914 Tgfb2 21808 Transforming growth factor, beta 2 −1.15 0.306 −1.07 0.584 Il18 16173 Interleukin 18 −1.15 0.224 −1.01 0.937 D17Wsu104e 28106 DNA segment, Chr 17, Wayne State University 104,  expressed (previously known as IL25) −1.03 0.794 −1.16 0.088 Lgals1 16852 Lectin, galactose binding, soluble 1 1.05 0.774 1.08 0.590 Lcn2 16819 Lipocalin 2 1.11 0.228 1.20 0.179 Mif 17319 Macrophage migration inhibitory factor 1.18 0.076 1.17 0.137 Vascular  function Adm 11535 Adrenomedullin −1.41 0.003 −1.50 0.000 Agt 11606 Angiotensinogen −1.22 0.161 −1.15 0.150 Serpinf1 20317 Serine (or cysteine) peptidase inhibitor, clade F, member 1  (previously known as pigment epithelium derived factor) −1.15 0.237 −1.25 0.187 Vegfa 22339 Vascular endothelial growth factor A −1.03 0.724 −1.08 0.644 Apln 30878 Apelin 1.01 0.969 1.02 0.790 Serpine1 18787 Serine (or cysteine) peptidase inhibitor, clade E, member 1  (previously known as plasminogen activator inhibitor-1) −1.01 0.897 1.27 0.013 Antioxidant Sod3 20657 Superoxide dismutase 3, extracellular −1.09 0.500 −1.39 0.036 Mt1 17748 Metallothionein 1 −1.11 0.577 −1.12 0.417 Extracellular  matrix  component Nid2 18074 Nidogen 2 −1.64 0.002 −1.96 0.003 Sparc 20692 Secreted acidic cysteine rich glycoprotein −1.63 0.000 −1.56 0.000 Col5a2 12832 Procollagen, type V, alpha 2 −1.62 0.000 −1.62 0.003 Col5a1 12831 Procollagen, type V, alpha 1 −1.59 0.003 −1.48 0.013 Col1a2 12843 Procollagen, type I, alpha 2 −1.64 0.020 −1.40 0.002 Col1a1 12842 Procollagen, type I, alpha 1 −1.60 0.065 −1.38 0.020 Col3a1 12825 Procollagen, type III, alpha 1 −1.52 0.024 −1.59 0.002 Col4a5 12830 Procollagen, type IV, alpha 5 −1.44 0.034 −1.37 0.019 Col6a2 12834 Procollagen, type VI, alpha 2 −1.32 0.011 −1.19 0.108 Col6a1 12833 Procollagen, type VI, alpha 1 −1.31 0.117 1.05 0.740 Col5a3 53867 Procollagen, type V, alpha 3 −1.20 0.046 −1.29 0.076 Col4a1 12826 Procollagen, type IV, alpha 1 −1.16 0.114 −1.16 0.069 Col4a2 12827 Procollagen, type IV, alpha 2 −1.16 0.153 −1.29 0.003 Nid1 18073 Nidogen 1 −1.04 0.723 1.04 0.767 Col6a3 12835 Procollagen, type VI, alpha 3 −1.03 0.690 −1.01 0.960 Extracellular  matrix  processing Mmp3 17392 Matrix metalloproteinase 3 −2.01 0.003 −2.02 0.013 Lox 16948 Lysyl oxidase −1.87 0.005 −2.17 0.002 Timp2 21858 Tissue inhibitor of metalloproteinase 2 −1.81 0.002 −1.92 0.000 Mmp2 17390 Matrix metalloproteinase 2 −1.44 0.054 −1.30 0.100 Timp1 21857 Tissue inhibitor of metalloproteinase 1 −1.32 0.095 −1.30 0.055 Pcolce 18542 Procollagen C-endopeptidase enhancer protein −1.31 0.029 −1.48 0.002 Mmp19 58223 Matrix metalloproteinase 19 −1.01 0.848 −1.08 0.347 Mmp11 17385 Matrix metalloproteinase 11 1.05 0.860 −1.12 0.413 Timp4 110595 Tissue inhibitor of metalloproteinase 4 1.06 0.672 1.25 0.055 Miscellaneous Gal 14419 Galanin −3.04 0.000 −2.89 0.000 Gsn 227753 Gelsolin −1.63 0.000 −1.52 0.000 Ppic 19038 Peptidylprolyl isomerase C −1.27 0.049 −1.38 0.001 n Rosi/basal: the effect with low insulin/glucose; RosiIG/IG: the effect with high insulin/glucose www.foodbioactives.nl 3 3 Fig. 3 a, c b, d a, b c, d  4 5 6 Fig. 4 marked bands (arrows)  Fig. 5 squares a b c d e  Fig. 6 n p t p t  General confirmation of the microarray data by quantitative real-time RT-PCR analysis Rps15 Rps15 1 The effect of rosiglitazone on lipolysis 7 Fig. 7 n p t p t  Discussion As a PPARG agonist, rosiglitazone stimulates adipogenesis, which is accompanied by an increasing lipid content of the differentiating cells. However, our results showed that rosiglitazone does not induce an increase of the lipid content, but decreases it in mature adipocytes at high insulin/glucose level. The pathway analysis based on transcriptome data points out that an altered energy metabolism may underlie this phenomenon. Ucp2 Scd1 28 29 30 31 12 12 30 12 32 12 Fasn 33 34 Adfp 1 35 35 Adfp 36 37 10 38 9 1 Apoe 39 19 40 41 16 Pparg 42 44 Cav1 Scd1 , , 45 1 2 Scd1 46 Cav1 47 48 49 50 13 Electronic Supplementary Material Below is the link to the electronic supplementary material 
 Table 1 Q-PCR primer sequences (DOC 26 kb) Fig. 1 n n