Excess glucose shapes mitochondrial metabolism and redox state in human primary white adipocytes

Mitochondrial dysfunction in white adipose tissue (WAT) is a hallmark of obesity, yet nutrient-driven responses in adipocytes remain poorly defined, partly due to widespread use of supra-physiological glucose-rich media in in vitro adipocyte models. We used integrated transcriptomics, fluxomics, and functional analyses to assess how glucose availability shapes mitochondrial metabolism and redox status during human adipocyte differentiation. Primary human adipocytes (n = 6 donors) were differentiated in commonly used media containing high glucose (DMEM/F12, 17.6 mM; DMEM/HG, 25 mM), physiological glucose (LG, 5.5 mM), or galactose (Gal, 25 mM). High-glucose conditions were associated with a shift from oxidative phosphorylation toward glycolysis, reduced mitochondrial biogenesis, NADH accumulation, and elevated mitochondrial reactive oxygen species, accompanied by impaired insulin sensitivity, reduced adiponectin secretion, together with transcriptional signatures of inflammatory and stress-associated responses. Fluxomics revealed altered pyruvate flux, enhanced anaplerotic pathways, and upregulated anabolic programs. In contrast, LG and Gal conditions preserved mitochondrial and redox features, more closely resembling characteristics of healthy WAT. Collectively, these data define a metabolic phenotype, in which supra-physiological glucose is associated with redox imbalance and metabolic reprogramming in human adipocytes under defined in vitro conditions. Our results highlight the importance of physiological glucose for adipocyte metabolism modeling and provide a framework for interpreting nutrient effects on mitochondrial and redox phenotypes.