Regulating Adipocyte Oxidative Stress: The Effects of Exogenous Ketones on Human Adipocytes in a Hypoxic Environment

Abstract

Background: Ketone bodies, particularly β-hydroxybutyrate (βOHB), have been proposed to exert antioxidant and cytoprotective effects by modulating mitochondrial function and antioxidant gene expression. In adipocytes, redox homeostasis is tightly regulated and strongly influenced by oxygen availability. Hypoxia, a condition of low oxygen pressure in the cell environment, has been associated with increased reactive oxygen species (ROS) production and impaired antioxidant defenses. However, findings remain inconsistent, particularly in human adipocyte models. The potential for exogenous ketones to mitigate hypoxia-induced oxidative stress in human adipocytes remains poorly characterized. Accordingly, the overall objective was to determine the impact of exogenous ketones on adipocyte redox homeostasis under varying oxygen conditions, with a focus on ROS production and expression of key antioxidant genes. We hypothesized that hypoxia would increase ROS and suppress antioxidant gene expression in a dose-dependent manner in human differentiated adipocytes, whereas exogenous βOHB would dose-dependently reduce ROS and increase antioxidant gene expression, thereby attenuating hypoxia-induced oxidative stress.

Methods: Human subcutaneous preadipocytes were differentiated into mature adipocytes and exposed for 48 hours to varying oxygen concentrations (21%, 10% and 3%) followed by a 24-hour treatment with increasing concentrations of exogenous βOHB (0 to 10 mM). Intracellular ROS levels were assessed using a DCFDA assay, while lipid accumulation was measured by Oil Red O staining. Gene expression of key antioxidants (catalase, FOXO3, Mt2, SOD1 and SOD2) was quantified by rt-qPCR. Statistical analyses were performed using linear mixed-effects models or ANOVA, where appropriate.

Results: Hypoxic exposure did not significantly increase intracellular ROS levels in differentiated human adipocytes (p=0.307), regardless of glucose or βOHB concentration. Despite the absence of elevated ROS, hypoxia induced selective, oxygen-dependent changes in antioxidant expression, with FOXO3 (p<.001) and Mt2 (p<.001) significantly upregulated under severe hypoxia (3% oxygen). In contrast, catalase (p<.001) and SOD1 (p=0.033) expression decreased under hypoxic conditions, while SOD2 expression remained unchanged (p=0.409). Exogenous βOHB did not significantly modulate ROS levels (p=0.361) or antioxidant gene expression (p values between 0.547 and 0.944 for all genes) under normoxic or hypoxic conditions.

Conclusion: These findings indicate that hypoxia elicits adaptive transcriptional responses in human adipocytes without inducing sustained oxidative stress. Contrary to our initial hypothesis, exogenous βOHB did not attenuate hypoxia-associated redox responses under the conditions tested. Together, the results suggest that human adipocytes may possess robust mechanisms to maintain redox homeostasis under reduced oxygen availability, and that ketone-mediated antioxidant effects are highly context-dependent.

Contribution to advancement of knowledge: This thesis provides evidence that challenges the assumption that hypoxia universally induces oxidative stress in adipocytes, and that exogenous ketones act as broad antioxidant modulators. By demonstrating selective redox adaptation in the absence of increased ROS, our work refines current models of adipocyte hypoxia and highlights the importance of temporal dynamics, cellular context, and experimental design when evaluating ketone-mediated redox effects.