CRISPR/Cas9-mediated deletion of adipocyte genes associated with NAFLD alters adipocyte lipid handling and reduces steatosis in hepatocytes in vitro.

Obesity is a major risk factor for the development of nonalcoholic fatty liver disease (NAFLD), and the subcutaneous white adipose tissue (scWAT) is the primary lipid storage depot and regulates lipid fluxes to other organs. Our previous work identified genes upregulated in scWAT of patients with NAFLD: , , and . Herein, we knocked down (KD) their expression in human adipose-derived mesenchymal stem cells (hADMSCs) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterized their phenotype. We found that , , and expression in hADMSC-derived adipocytes was not critical for adipogenesis. However, the metabolic characterization of the cells suggested that the genes played important roles in lipid metabolism. Reduction of expression significantly increased both de novo lipogenesis (DNL) and palmitate-induced lipogenesis (PIL). Editing out reduced DNL while increasing isoproterenol-induced lipolysis and insulin-induced palmitate accumulation. Conversely, reduced PIL and DNL. Moreover, RNA-sequencing analysis of edited cells showed that these genes not only altered lipid metabolism but also other biological pathways related to inflammatory processes, in the case of , extracellular matrix remodeling for , or cellular transport for . Finally, to evaluate a possible adipocyte-hepatocyte axis, human hepatoma HepG2 cells were cocultured with edited hADMSCs-derived adipocytes in the presence of [3H]-palmitate. All HepG2 cells cultured with DUSP1-, SIK1-, or SOCS3-KD adipocytes decreased [3H]-palmitate accumulation compared with control adipocytes. These results support our hypotheses that , , and regulate multiple aspects of adipocyte function, which may play a role in the progression of obesity-associated comorbidities, such as NAFLD. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology successfully edited genomic DNA of human adipose-derived mesenchymal stem cells (hADMSC). , , and regulate adipocyte lipid handling. Silencing , , and expression in hADMSC-derived adipocytes reduces hepatocyte lipid storage in vitro.