SUN-639 A Novel Truncating EBF2 Variant Impairs White Adipocyte Function in A Dominant Negative Manner

Abstract Disclosure: D. Gilio: None. M.C. Foss de Freitas: None. E.A. Oral: Regeneron Pharmaceuticals, Amryt Pharmaceuticals, Ionis Pharmaceuticals Inc., Third Rock Ventures, Rejuvenate Bio, Novo Nordisk, Rhythm Pharmaceuticals, Fractyl Laboratories, Morphic Medical. T. Chun: None. Lipodystrophy syndromes are characterized by adipose tissue loss/dysfunction, causing insulin resistance and metabolic abnormalities. We identified a novel nonsense variant of early B cell factor 2 (EBF2; c.493G>T, p.E165X) in a patient with atypical partial lipodystrophy. EBF2 is a member of the EBF family, which plays a crucial role in the differentiation of multiple cell lineages. To investigate the pathological significance of this variant, we performed in vitro and in vivo studies. Newly generated heterozygous knock-in (Ebf2E165X/+, KI) mice exhibited defective postnatal white adipose tissue (WAT) expansion with mitochondrial dysfunction and fibrosis. We hypothesized that the EBF2 variant interferes with adipocyte function in a dominant negative manner. Methods: To determine if cell-autonomous adipocyte defects underlie the WAT phenotype of KI mice, we isolated the stromal vascular fraction (SVF) from WAT of 8-week-old male and female Ebf2+/+ (WT) and KI mice. We performed loss-of-function and gene-rescue experiments using lentiviral constructs encoding full-length and truncated EBF2 (1-164). Adipogenesis was induced with dexamethasone 0.25 μM, porcine insulin 10 μg/ml, rosiglitazone 10 μg/ml, and triiodo-L-thyronine 1.5 μg/ml. Lipid accumulation was quantified with BODIPY staining. RNA was extracted, and real-time qPCR was performed with TaqMan probes (Pparg, Fabp4, and Adipoq) and SYBR Green primers (Lep, Lpl, Ebf1, Ebf2, and Ebf3). Results/Discussion: The SVF isolated from WT mice differentiated efficiently into lipid-filled adipocytes, whereas SVF from KI mice showed significantly impaired lipid accumulation (by >80%, n=6, P=0.0002). Despite this, Pparg and Fabp4 expressions were comparable between WT and KI (n=5, P=0.92 and P=0.98). However, Adipoq and Lpl were significantly reduced in KI cells (n=5, by 39%, P=0.03, by 48%, P=0.02, respectively). Lep expression was too low to be reliably assessed. Transduction of WT SVF with EBF2 (1-164) significantly reduced lipid accumulation (by 76%, n=3, P=0.02), supporting the defective adipocyte maturation of KI SVF. The overexpression of full-length EBF2 failed to restore the adipogenic potential of KI cells. EBF2 (1-164) suppressed Lpl expression in WT cells (by 63%, n=3, P=0.01) and the overexpression of full-length EBF2 could not reverse the reduced expression of Lpl in KI cells (n=3, P=0.67). No differences in the expression of other Ebf family members (Ebf1, Eb3) were observed. These findings suggest that the truncated EBF2 product, EBF2 (1-164), impairs white adipocyte function through a cell-autonomous and dominant negative mechanism, potentially interfering with the EBF family. Reduced Lpl expression and compromised mitochondrial function may have contributed to the defective lipogenesis and adipocyte maturation. Presentation: Sunday, July 13, 2025