OR07-02 Single-nucleus RNA-seq Studies In Human White Adipose Tissue Reveal Novel Adipocyte Subtypes And TSHZ3 As A Transcriptional Regulator Of Adipogenesis

Abstract Disclosure: V. Efthymiou: None. S.D. Kodani: None. A. Gupta: None. F. Shamsi: None. W. Ali: None. L. Poulos: None. A. Streets: None. Y. Tseng: None. M. Patti: Consulting Fee; Self; AstraZeneca, MBX-Biosciences, Hanmi Pharmaceutical. Other; Self; DSMB: Fractyl Health, Inc. Background and aim: White adipose tissue (WAT) is characterized by substantial functional and cellular heterogeneity, which may contribute to depot-dependent differences in risk for type 2 diabetes (T2D). Methods: We isolated nuclei from human subcutaneous (SAT) and intraabdominal (IAT) adipose biopsies obtained during abdominal surgery for single-nucleus RNA sequencing (snucRNA-seq), and applied scVI and VISION to integrate and analyze data. Additionally, we used siRNA-mediated knockdown to validate candidate transcriptional regulators in human adipogenic progenitor cells. Results: We analyzed the transcriptome of 117,717 high-quality nuclei from 22 biopsies (10 SAT, 12 IAT, 3 paired from same subject) obtained from 16 females and 3 males, with or without T2D, with BMI 45.4±10.3 kg/m2 (range 23.6-60.9). Both SAT and IAT depots had substantial cellular heterogeneity with 17 distinct clusters of adipose and non-adipose cell types. Distinct clusters of adipocytes were distinguished by high vs. low adiponectin expression (ADIPOQhi vs. ADIPOQlo). Adipose samples from males and participants with T2D had a higher proportion of ADIPOQlo adipocytes. ADIPOQlo adipocytes have distinct expression patterns, including upregulation of transcriptional regulators NR4A1 and ATF3, previously linked to adipose metabolic dysfunction.We utilized CheA3 analyses to identify transcription factors predicted to regulate genes differentially expressed between ADIPOQlo vs. ADIPOQhi. Among upstream regulators of ADIPOQlo gene expression was TSHZ3, a zinc-finger transcription factor previously linked to developmental processes. In our dataset, TSHZ3 is expressed in adipogenic progenitors and is downregulated during differentiation ex vivo. To test the role of TSHZ3 in adipogenic differentiation, we performed siRNA-mediated knockdown in a human subcutaneous white adipogenic progenitor cell line. TSHZ3 knockdown dramatically reduced adipogenesis vs. scrambled control, with reduction in lipid droplets and significant downregulation of adiponectin, leptin, CIDEA, AP2, FAS, PPARγ, and PGC1α. This effect was partially rescued by the PPARγ agonist rosiglitazone. Conclusion and perspectives: Our single-nucleus analysis reveals two distinct subtypes of mature adipocytes: ADIPOQhi vs. ADIPOQlo. We identify TSHZ3 as a key transcriptional regulator of adipogenesis which may contribute to differences in expression phenotypes between these populations. Overall, integrative bioinformatics analysis reveals distinct transcriptomic signatures between depots, between clusters, and across a range of BMI, that may be promising targets for understanding mechanisms by which specific adipose-resident cell populations mediate metabolic risk. Presentation: Thursday, June 15, 2023