Our laboratory applies state-of-the-art mass spectrometry and computa-59into adipose stromal cell heterogeneity.tional methods for proteome analysis in complex biological systems such as white adipose tissue (WAT). Upon energy excess, WAT expands through two primary mechanisms: hyperplasia, which involves differentiation of adipocyte precursors, and hypertrophy, which is due to the enlargement of existing adi-pocytes. We have developed two research efforts centered around these mecha-nisms, one focuses on the molecular and functional heterogeneity of adipocyte progenitor populations, and the other on the intercellular communication of adipocytes, precursors and immune cells in the WAT.To investigate sex- and depot (i.e. visceral or subcutaneous WAT)-dependent adipocyte progenitor cell heterogeneity, we performed a multilayered omics analysis for eight adipose stromal cell populations and quantified 4870 proteins and 15477 transcripts. The data are freely accessible as a resource at “Pread Pro-filer”. Both proteomic and transcriptomic data clearly separated the eight cell populations. However, gene expression at the protein level appeared to better reflect the functional heterogeneity of the different cell populations.Proteomic data revealed functional pathways that could discriminate cell populations. For example, expression of 32 proteins involved in glutathione metabolism distinguished male gonadal WAT (gWAT) adipocyte precursor cells (APCs) from all the other populations. Ingenuity pathway analysis (IPA) revealed that glutathione-mediated detoxification was enhanced in male gWAT APCs compared to fibro-inflammatory precursors (FIPs). Notably, GSTM1 was the most abundant at the protein level, while Gpx3 had the highest abundance at the transcript level. We then conducted CRISPR-Cas9 gene editing to inac-tivate Gstm1 in isolated APCs and FIPs. The differentiation-linked decrease in the GSH:GSSG ratio was almost completely blocked in APCs expression either of the two gRNAs, and Gstm1-deficiency also reduced the adipogenic potential of APCs. Interestingly, Gpx3 was functionally dispensable for the maintenance of cellular redox status and differentiation of APCs.Together, this multilayered omics analysis provides unprecedented insights Figure: A multilayered omics analysis reveals sex- and depot-dependent adipose stromal cell heterogeneity(a) An integrated transcriptomic and proteomic ap-proach to dissect molecular signatures of adipose stro-mal cell populations. (b) Gene expression at the protein and transcript levels across all 24 samples. Data were normalized to total abundance in each sample and z-score transformed. Red represents a z-score larger than 0 and blue represents a z-score smaller than 0. (c) Prin-cipal component analysis showing sample group sepa-ration based on proteomics (left) and transcriptomics data (right). (d) Expression of 32 proteins involved in glutathione metabolism clearly discriminated male gWAT APCs from all the other populations. (e) Expres-sion of genes involved in glutathione-mediated detoxi-fication between male gWAT APCs and FIPs at protein (left) and transcript (right) levels. (f) Glutathione exists in reduced (GSH) and oxidized (GSSG) forms, with the ratio of GSH:GSSG indicative of cellular redox status and oxidative stress. Glutathione-S-transferases (GSTs) cata-lyze the conjugation of GSH to xenobiotic substrates and regulate cellular GSH:GSSG ratios. (g) GSH:GSSG ratios in cell lysates from primary FIPs and APCs transduced with the indicated CRISPR lentivirus. Day 0 denotes the time when samples were harvested for cell differentiation analysis by Oil Red O staining. N = 4. Data are shown as the mean ± s.e.m. * p < 0.05 between APC and FIP by two-way ANOVA. (h) Representative bright field images of Oil Red O-stained cultures of differentiated primary FIPs and APCs transduced with the indicated CRISPR lentivirus. Scale bar = 200 uM.Recent Major PublicationsBo Shan, Clive S. Barker, Mengle Shao, Qianbin Zhang, Rana K. Gupta, Yibo Wu. Multilayered omics reveal Sex- and depot-dependent adipose progenitor cell heteroge-neity. Cell Metabolism (In revision)Shao M, Hepler C, Zhang Q, Shan B, Vishvanath L, Ger-vaise H. H, Zhao S, Yu A A, Wu Y, Strand W. D, and Rana K. Gupta. Pathologic HIF1α signaling drives adipose progenitor dysfunction in obesity. Cell Stem Cell 28, 1-17 (2021)Mostafa D, Yanagiya A, Georgiadou E, Wu E, Stylianides T, Rutter A G, Suzuki T, Yamamoto T. Loss of β-cell identity and diabetic phenotype in mice caused by disruption of CNOT3-dependent mRNA deadenylation. Commun Biol 3, 476 (2020)YCI Laboratory for Next-Generation ProteomicsYoung Chief Investigator: Yibo Wu
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