Integrative Analysis of Transcriptomics, Proteomics, and Metabolomics Data of White Adipose and Liver Tissue of High-Fat Diet and Rosiglitazone-Treated Insulin-Resistant Mice Identified Pathway Alterations and Molecular Hubs

The incidences of obesity and type 2 diabetes are rapidly increasing and have evolved into a global epidemic. In this study, we analyzed the molecular effects of high-fat diet (HFD)-induced insulin-resistance on mice in two metabolic target tissues, the white adipose tissue (WAT) and the liver. Additionally, we analyzed the effects of drug treatment using the specific PPARγ ligand rosiglitazone. We integrated transcriptome, proteome, and metabolome data sets for a combined holistic view of molecular mechanisms in type 2 diabetes. Using network and pathway analyses, we identified hub proteins such as SDHB and SUCLG1 in WAT and deregulation of major metabolic pathways in the insulin-resistant state, including the TCA cycle, oxidative phosphorylation, and branched chain amino acid metabolism. Rosiglitazone treatment resulted mainly in modulation via PPAR signaling and oxidative phosphorylation in WAT only. Interestingly, in HFD liver, we could observe a decrease of proteins involved in vitamin B metabolism such as PDXDC1 and DHFR and the according metabolites. Furthermore, we could identify sphingosine (Sph) and sphingosine 1-phosphate (SP1) as a drug-specific marker pair in the liver. In summary, our data indicate physiological plasticity gained by interconnected molecular pathways to counteract metabolic dysregulation due to high calorie intake and drug treatment

A Common Building Block for the Syntheses of Amorfrutin and Cajaninstilbene Acid Libraries toward Efficient Binding with Peroxisome Proliferator-Activated Receptors

A common building block for the synthesis of amorfrutin and cajaninstilbene acid derivatives has been developed. The library of synthesized compounds has enabled identification of new nontoxic ligands of peroxisome proliferator-activated receptors (PPAR) and potential inhibitors of the transcriptional corepressor protein NCoR. The biological data holds promise in identification of new potential leads for the antidiabetic drug discovery process

Cytotoxicity test and evaluation of inhibition of tumor growth by treating HT-29 tumor xenograft mouse models with IAE.

<p>Athymic nude mice were ectopically implanted with 5 million HT-29 cells in the flank and orally gavaged bidaily by 50 mg/kg IAE or vehicle for 4 weeks. A, Cytotoxicity was tested for colon cancer and primary colon cells (IC₅₀ = 25.82 μg/mL (HT-29); IC₅₀ = 36.12 μg/mL (CCD 841 CoN)). Data are expressed as mean ± SD (n = 4). B, Body weight during entire experiment. C, Tumor volume during entire experiment and images of exemplary xenografts of untreated and IAE-treated mice at end point. D, Tumor weight at the end of the study. Data are expressed as mean ± SEM (n = 18). n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 vs. control (one-tailed t test).</p

Structural Characterization of Amorfrutins Bound to the Peroxisome Proliferator-Activated Receptor γ

Amorfrutins are a family of natural products with high affinity to the peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor regulating lipid and glucose metabolism. The PPARγ agonist rosiglitazone increases insulin sensitivity and is effective against type II diabetes but has severe adverse effects including weight gain. Amorfrutins improve insulin sensitivity and dyslipidemia but do not enhance undesired fat storage. They bear potential as therapeutics or prophylactic dietary supplements. We identified amorfrutin B as a novel partial agonist of PPARγ with a considerably higher affinity than that of previously reported amorfrutins, similar to that of rosiglitazone. Crystal structures reveal the geranyl side chain of amorfrutin B as the cause of its particularly high affinity. Typical for partial agonists, amorfrutins 1, 2, and B bind helix H3 and the β-sheet of PPARγ but not helix H12

Activation of apoptosis signaling pathway in colon carcinoma cells after treatment with IAE.

<p>A, HT-29 and T84 cells were treated for 24 h. Enzymatic activation of caspases 2, 3/7, 6, 8 and 9 was determined by use of luminescence-based assays. Data are normalized to control treatment and are expressed as mean ± SEM (n = 4). B, Whole cell lysates from HT-29 cells treated for 24 h were analyzed for the expression of total and cleaved proteins of caspase 3, caspase 9 and PARP by immunoblotting. Numbers indicate densitometric ratios of the cleaved to total proteins normalized to control treatments. C, Fluorescence microscopy of HT-29 cells treated for 24 h. Cleaved caspase 3 was labeled green, F-actin red and the nucleus blue. Scale bars, 25 μm. D, HT-29 cells were treated for 6 h. DNA fragmentation was detected through accumulation of cytoplasmic BrdU-labeled DNA by ELISA. Data are normalized to control treatment and are expressed as mean ± SEM (n = 5). n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 vs. control.</p

Effect of IAE and reference compounds on proliferation of HT-29 and T84 colon carcinoma cells, PC-3 prostate cancer, MCF7 breast cancer and normal CCD 841 CoN colon cells.

<p>Effect of IAE and reference compounds on proliferation of HT-29 and T84 colon carcinoma cells, PC-3 prostate cancer, MCF7 breast cancer and normal CCD 841 CoN colon cells.</p

Extra- and intracellular formation of reactive oxygen species (ROS) and of lipid peroxides in HT-29 cells after treatment with IAE.

<p>A, Extracellular formation of ROS in full cell culture medium was kinetically detected using the ROS-sensitive CellROX Orange fluorogenic probe. Data are expressed as mean ± SEM (n = 8). B, Intracellular ROS was detected by flow cytometry of HT-29 cells stained with CellROX Orange after treatment for 24 h. Histograms (left) show one representative experiment for each treatment condition. Bar plots (right) show fluorescence intensities as mean ± SEM (n = 6). C, Intracellular lipid peroxidation was detected by flow cytometry of cells treated for 24 h using the Click-iT technology. Increasing fluorescence intensities are a result of enhanced lipid peroxidation upon treatment. Histograms (left) show one representative experiment for each treatment condition. Bar plots (right) show fluorescence intensities as mean ± SEM (n = 6). D, Intracellular lipid peroxidation was visualized by fluorescence microscopy (green, lipid peroxides; blue, nucleus). Scale bars, 25 μm. n.s. not significant, **p≤0.01, ***p≤0.001 vs. control.</p

Cell cycle analysis of HT-29 colon carcinoma cells after treatment with 30 μg/ml IAE for 24 h.

<p>A, Cell cycle was analyzed by flow cytometry of propidium iodide stained cells. Histograms (top) show one representative experiment for each treatment condition. Bar plots (bottom) show percent of cell population in apoptotic SubG1, G0/G1, S and G2/M phases of the cell cycle and are expressed as mean ± SEM (n = 3). *p≤0.05, ***p≤0.001 vs. control. B, Whole cell lysates were analyzed for the expression of cyclin A2, cyclin D3, CDK2, CDK4, CDK6 and GAPDH proteins by immunoblotting using specific antibodies.</p

Camomile flowers extract improves dyslipidemia in obese DIO mice.

<p>(A) Fasting plasma NEFA after 6 weeks of treatment. (B) Fasting plasma triacylglycerol after 6 weeks of treatment. (C) Fasting plasma total, HDL and LDL/VLDL cholesterol in DIO mice after 6 weeks of treatment. Data are expressed as mean ± SEM. *<i>p</i>≤0.05, **<i>p</i>≤0.01, ***<i>p</i>≤0.001, n.s. not significant vs. vehicle-treated HFD-fed mice. LFD, low-fat diet; HFD, high-fat diet; VEH, vehicle (<i>n</i>=13-14); RGZ, rosiglitazone (<i>n</i>=8-14) ; CFE, camomile flowers extract (<i>n</i>=12-14). </p

Camomile flowers extract (CFE) does not induce adverse effects commonly linked with PPAR agonists.

<p>(A) Effect of CFE on cellular viability in human HepG2 cells after treatment for 24 h. Data are expressed as mean ± SD (n=3/group). (B, C) Mouse body weight during treatment of DIO mice for 6 weeks with CFE or HFD alone (B) and during the preventive study by 20 weeks feeding of healthy C57BL/6 mice with LFD, HFD alone or HFD with CFE (C). Data are expressed as mean ± SEM (<i>n</i>=14/group). Data are shown as mean ± SEM. (D, E) Food intake during treatment of DIO mice for 6 weeks with CFE or HFD alone (D) and during the preventive study by 20 weeks feeding of healthy C57BL/6 mice with LFD, HFD alone or HFD with CFE (E). Data are expressed as mean ± SEM (<i>n</i>=14/group). (F) Hematocrit of treated DIO mice after 6 weeks (mean ± SEM, n=14/group). (G) Effect of CFE on plasma osteocalcin levels after treatment of DIO mice for 6 weeks (mean ± SEM, n=14/group). *<i>p</i>≤0.05, n.s. not significant vs. untreated HFD-fed mice. LFD, low-fat diet; HFD, high-fat diet; CFE, camomile flowers extract.</p