Simple SummaryThe enzymes, G9a and G9a-like protein (GLP), add methyl groups to protein targets. They can regulate gene transcription by modifying the proteins that organize DNA within cells, but they can also modify other targets independent of their enzyme activity. Studies show that G9a and GLP are important proteins in the development of metabolic disease, but their role in fat cell function is not well understood. Fat cells, known as adipocytes, help to regulate whole-body metabolic health by safely storing fat and by secreting molecules that communicate with other tissues, including the brain. In this study, we decreased the levels of G9a, GLP, or both enzymes in fat cells and examined the effects on adipocyte gene expression and fat breakdown, known as lipolysis, when fat cells were exposed to a proinflammatory protein, TNFα, that is associated with metabolic diseases states, such as Type 2 diabetes. The loss of both G9a and GLP enhanced TNFα’s ability to induce lipolysis and regulate gene expression. Our data also show that these enzymes form a complex with NF-κB, which is a signaling molecule induced by TNFα. These novel observations provide specific details on how G9a and GLP expression in adipocytes is associated with systemic metabolic health.AbstractImpaired adipocyte function contributes to systemic metabolic dysregulation, and altered fat mass or function increases the risk of Type 2 diabetes. EHMTs 1 and 2 (euchromatic histone lysine methyltransferases 1 and 2), also known as the G9a-like protein (GLP) and G9a, respectively, catalyze the mono- and di-methylation of histone 3 lysine 9 (H3K9) and also methylate nonhistone substrates; in addition, they can act as transcriptional coactivators independent of their methyltransferase activity. These enzymes are known to contribute to adipocyte development and function, and in vivo data indicate a role for G9a and GLP in metabolic disease states; however, the mechanisms involved in the cell-autonomous functions of G9a and GLP in adipocytes are largely unknown. Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine typically induced in adipose tissue in conditions of insulin resistance and Type 2 diabetes. Using an siRNA approach, we have determined that the loss of G9a and GLP enhances TNFα-induced lipolysis and inflammatory gene expression in adipocytes. Furthermore, we show that G9a and GLP are present in a protein complex with nuclear factor kappa B (NF-κB) in TNFα-treated adipocytes. These novel observations provide mechanistic insights into the association between adipocyte G9a and GLP expression and systemic metabolic health.