ABSTRACT: Background and aims: Developmental exposure to an elevated ratio of omega-6 (n6) to omega-3 (n3) fatty acids (FA) is linked to increased infant body fat and risk of future childhood obesity. We demonstrated in mice that the high n6/n3 developmental exposure reduced nuclear receptor subfamily 2 group F member 2 (NR2F2) in Adipocyte Stem Cells (ASCs), coincident with an altered ASC mitochondrial expression profile and increased white adipose accumulation in pups. This suggested that NR2F2- low ASCs might adopt a nutrient-storage phenotype. Here, we tested the hypothesis that NR2F2 is required in ASCs to undergo beige adipogenesis and metabolism needed during postnatal life for energy and thermogenesis. Methods: C57BL/6J dams were randomized to either n6-rich or balanced n6/n3 control diets at the time of mating and underwent normal gestation and parturition. On postnatal day 12 (PND12), whole-body offspring metabolism was quantified by indirect calorimetry in conjunction with 13C-palmitate and 13Cglucose tracing. Inguinal fat pad ASCs were isolated by flow cytometry to assess adipocyte differentiation potential, global gene expression and proteomics, and mitochondrial oxidation. NR2F2 was transiently re-activated in vitro in ASCs with its ligand, 1-deoxysphingosine (1-DSO), and NR2F2 was ablated in ASCs ex vivo using homozygous floxed Nr2f2 pups to determine loss of function, ensure specificity of 1-DSO treatment during gain of function. Results: Excess developmental n6-FA exposure reduced whole-body 13C-palmitate and 13C-glucose oxidation, diminished PND12 pup energy expenditure, and increased triacylglyceride accumulation in inguinal adipose. In ASCs isolated from n6-FA exposed pups, NR2F2 was decreased. These NR2F2-low ASCs formed downstream adipocytes with decreased beige metabolic regulators peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), peroxisome proliferator-activated receptor gamma (PPARγ), PR domain containing 16 (PRDM16), and uncoupling protein 1 (UCP1), had lower glycolysis and lipid metabolism enzymes, oxidized lipid and glucose at lower rates, and had increased lipogenic enzymes. Ex vivo deletion of Nr2f2 from ASCs recapitulated the metabolic deficits observed in the adipocytes derived from NR2F2-low ASCs isolated from n6-FA exposed pups. NR2F2 loss disrupted beige regulator induction, reduced adipocyte FAO, and promoted lipogenesis pathways, mirroring the n6-FA phenotype. Transient NR2F2 activation of ASCs from n6-FA pups using 1-DSO restored induction of beige regulators, increased mitochondrial oxidative phosphorylation enzymes, reduced lipogenic/storage pathways, ultimately enhancing nutrient oxidation. Conclusions: These findings demonstrate that excess n6-FA developmental exposure disrupts NR2F2- mediated ASC fate determination, leading to formation of nutrient-storing, lipogenic adipocytes. This work highlights NR2F2 as an important upstream or parallel regulator necessary for beige adipogenesis and underscores its activation as a potential therapeutic approach to mitigate early-life obesity risk.
