Alpha-ketoglutarate enhances adipose-derived stem cells survival in wound healing by hypoxia-inducible factor 1-alpha-mediated redox homeostasis and glycogen-dependent bioenergetics.

Abstract

Background: Adipose-derived stem cells (ADSCs) hold significant therapeutic potential for regenerative medicine, particularly in wound healing, owing to their multipotency, paracrine activity, and relative abundance. However, the clinical application of ADSC-based therapies is substantially limited by the harsh microenvironment of acute wounds, characterized by hypoxia, nutrient deprivation, and oxidative stress, which leads to massive apoptotic cell death post-transplantation. Preconditioning strategies to enhance cellular resilience have thus gained considerable interest. Recent insights from cancer biology highlight the crucial role of metabolic reprogramming, orchestrated by hypoxia-inducible factor-1α (HIF-1α), in promoting survival under stress. Our previous work demonstrated that preconditioning with α-ketoglutarate (α-KG) enhances ADSC survival and accelerates wound healing, purportedly through HIF-1α upregulation. Nevertheless, the precise metabolic mechanisms by which α-KG preconditioning confers cytoprotection remain incompletely elucidated.

Aim: To investigate the mechanistic role of HIF-1α in mediating the enhanced survival and regenerative capacity of α-KG-preconditioned ADSCs in an acid burn wound model. Specifically, we sought to determine whether HIF-1α activation drives complementary adaptations in glutamine and glycogen metabolism to maintain redox and energy homeostasis, respectively, under the multifactorial stress conditions of a wound.

Methods: Human ADSCs were isolated from lipoaspirates and preconditioned with dimethyl-α-KG. In vitro, cells were subjected to single or combined stressors (hypoxia, glucose deprivation, H₂O₂-induced oxidative stress). Genetic modulation was performed using lentiviral shRNAs targeting HIF-1α, GLS1, and PYGL, or an overexpression vector for GLS1. Metabolic profiling included assessments of glycolytic flux, glucose oxidation, fatty acid β-oxidation, oxygen consumption, and glycogen content. Redox status was evaluated via glutathione (GSH) (GSH/GSH disulfide) ratios and reactive oxygen species (ROS) levels. In vivo, a murine acid burn wound model was established, and pre-labeled ADSCs were implanted. Cell survival was tracked via flow cytometry (Annexin V-PI) and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Wound healing was assessed histologically.

Results: α-KG preconditioning significantly enhanced the survival of ADSCs both in vitro under stress and in vivo in burn wounds. This was concomitant with HIF-1α stabilization. Mechanistically, HIF-1α orchestrated a dual metabolic adaptation: (1) It promoted glutaminolysis via GLS1, increasing glutamate and GSH synthesis, which enhanced antioxidant capacity and reduced ROS levels; and (2) It simultaneously stimulated glycogen storage (Gys1 upregulation) and mobilization (Pygl upregulation), preserving energy (ATP:AMP ratio) during glucose deprivation. Genetic inhibition of GLS1 abrogated the ROS detoxification benefit, while PYGL knockdown abolished the energy maintenance advantage, both reducing survival. Crucially, combined inhibition of both pathways completely negated the prosurvival effect of α-KG, confirming their synergistic role. In vivo, α-KG-preconditioned ADSCs accelerated wound closure, improved re-epithelialization, and enhanced angiogenesis compared to controls, effects that were HIF-1α-dependent.

Conclusion: This study demonstrates that α-KG preconditioning significantly enhances ADSC survival and therapeutic efficacy in burn wound healing through HIF-1α-mediated metabolic reprogramming. HIF-1α activation coordinately upregulates glutamine-driven GSH synthesis for redox homeostasis and glycogen storage for bioenergetic resilience, providing a dual mechanism of cytoprotection. These findings establish metabolic preconditioning as a potent, translatable strategy to improve the efficacy of stem cell-based therapies not only in wound healing but potentially in other ischemic and inflammatory conditions characterized by poor cell survival.