ANKRD53 is downregulated in human obesity and coordinates lipolysis with mitochondrial oxidative metabolism in adipocytes

Human adipose tissue is central to obesity‐associated metabolic dysfunction. ANKRD53 is a human‐specific, adipocyte‐enriched ankyrin repeat scaffold protein with largely unknown function. We investigated its role in human adipocyte metabolism and the underlying mechanism. RNA-seq analysis of subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) from 236 individuals quantified ANKRD53 expression and its association with metabolic traits. In human primary adipocytes, we assessed lipolysis (free fatty acid and glycerol release) and mitochondrial respiration (oxygen consumption rate) after ANKRD53 overexpression or knockdown. An AAV was used to overexpress ANKRD53 in mouse inguinal white adipose tissue (iWAT). Protein interactors were identified by immunoprecipitation–mass spectrometry, and knockdown experiments confirmed a functional role of ACSL1. ANKRD53 expression in both adipose depots was markedly reduced in obesity and inversely correlated with BMI, adiposity measures, insulin resistance indices, and circulating triglycerides, while positively associated with adiponectin and HDL. In human adipocytes, ANKRD53 overexpression enhanced forskolin-stimulated lipolysis and mitochondrial respiration, whereas silencing impaired these processes. Adipose-targeted ANKRD53 overexpression in mice increased lipolysis in vivo. Mechanistically, ANKRD53 interacted with ACSL1 and promoted its mitochondrial localization, channeling lipolysis-derived FFAs into β-oxidation; silencing ACSL1 abrogated ANKRD53’s effects. ANKRD53 is reduced in obesity and coordinates lipolysis with mitochondrial oxidative metabolism in human adipocytes, promoting efficient use of lipolysis-derived FFAs via ACSL1. These findings establish ANKRD53 as a key regulator of adipocyte energy metabolism and a potential therapeutic target for improving metabolic health in obesity. • ANKRD53 expression is reduced in obesity and inversely correlated with adiposity and metabolic risk measures. • ANKRD53 is uniquely expressed in human adipocytes but undetectable in mouse adipose tissue. • In human primary adipocytes, ANKRD53 enhances lipolysis and mitochondrial respiration. • ANKRD53 interacts with ACSL1 to channel lipolysis-derived fatty acids into mitochondria, linking lipid mobilization to mitochondrial oxidative metabolism.