Search Results (46)

Doxorubicin (DOX) remains an effective chemotherapeutic agent, but its clinical use is limited by dose-dependent cardiotoxicity. Mitochondrial dysfunction and metabolic remodeling are central features of DOX-induced cardiac injury. ATP synthase inhibitory factor-1 (IF1) is an endogenous inhibitor of the hydrolytic activity of mitochondrial ATP synthase and has emerged as an important regulator of cellular bioenergetics. Cardiac IF1 expression is increased in multiple pathological conditions; however, its role in chemotherapy-induced cardiotoxicity remains unclear. Here, we investigated the contribution of IF1 to DOX-induced cardiotoxicity using male C57BL/6J wild-type (WT) and IF1 knockout (IF1KO) mice, isolated cardiac mitochondria, cultured neonatal cardiomyocytes, and AC16 human cardiomyocytes. Cardiac function was assessed by echocardiography, mitochondrial function by high-resolution respirometry and Seahorse metabolic flux analysis, and myocardial injury by histological and ultrastructural analyses. DOX treatment markedly increased cardiac IF1 protein levels despite reduced IF1 mRNA expression. IF1 deficiency enhanced mitochondrial respiration in isolated cardiac mitochondria and cultured cardiomyocytes under both basal and DOX-stressed conditions. IF1KO mice exhibited attenuated cardiac dysfunction and improved myocardial ultrastructure following DOX treatment compared with WT mice. In AC16 cardiomyocytes exposed to DOX, overexpression of WT IF1 improved cellular metabolic activity but provided only limited preservation of mitochondrial respiratory capacity. In contrast, overexpression of the dominant-negative IF1 mutant (IF1E30A) not only improved metabolic activity but also preserved mitochondrial respiration. These findings identify IF1 as a key regulator of metabolic adaptation during DOX stress. Upregulation of functional IF1 may represent an adaptive response that promotes glycolytic ATP production during mitochondrial stress, whereas inhibition of IF1 activity preserves metabolic activity primarily through maintenance of mitochondrial function. Collectively, these findings provide new insights into the role of IF1 in DOX-induced cardiomyopathy and highlight IF1 as a potential therapeutic target in cardio-oncology. Full article

Mitochondria play a central role in numerous physiological and pathological processes, and mitochondrial transplantation is emerging as a promising strategy to restore cellular function and mitigate disease. The success of this approach depends critically on the methods used to isolate, preserve, and retrieve intact, functional mitochondria. Objective: To optimize an isolation strategy that preserves mitochondrial integrity, dynamics, and metabolic activity and to evaluate conditions that enable short-term storage for future organelle biobanking applications. Methods: We compared a mitochondria isolation method developed in our laboratory (Protocol A) with a commercially available kit (Protocol B). Donor mitochondria were isolated from proximal tubular cells and transplanted into HEK293T recipient cells. Mitochondrial functionality was assessed following transfer into HEK293T cells by measuring reactive oxygen species (MitoSOX Red), oxygen consumption rate (OCR) using Seahorse XF analysis, and high-resolution imaging of mitochondrial morphology and dynamics. We further evaluated mitochondrial storage at low temperature and subsequent functional recovery. Results: Protocol A enabled faster isolation (~30 min) than Protocol B (~80 min) and yielded mitochondria with higher transplantation efficiency, greater OCR, preserved dynamic morphology, and lower oxidative stress. Mitochondria isolated using Protocol A remained metabolically active after transplantation and continued to exhibit fission and fusion, whereas those isolated using Protocol B showed reduced dynamic behavior. Importantly, mitochondria isolated with Protocol A retained functional integrity after low-temperature storage, supporting their potential for standardized preservation. Conclusions: This study presents a robust, efficient, and reproducible isolation and frozen-storage protocol that yields highly functional mitochondria suitable for transplantation. The ability to preserve mitochondrial function after storage further highlights the potential for developing organelle biobanks to support future research and therapeutic applications. Full article

Background: Autoimmune diseases such as primary Sjögren’s syndrome and type 1 diabetes are frequently complicated by hepatic injury, yet therapies that simultaneously target inflammation and parenchymal damage remain limited. Mitochondrial dysfunction with excessive reactive oxygen species (ROS) production drives a self-amplifying pathogenic loop by activating the cGAS-STING innate immune pathway. We previously observed that a Chinese herbal formula preserved mitochondrial ultrastructure in autoimmune NOD mice, and computational screening identified trifolirhizin—a natural pterocarpan flavonoid—as the candidate active constituent mediating this protection. Here, we investigated the hepatoprotective effects and underlying mechanisms of trifolirhizin in autoimmune-associated liver injury. Methods: Female NOD mice received trifolirhizin (5, 10, or 20 mg/kg/day) for four weeks, with C57BL/6J mice as healthy controls. Hepatic histopathology, inflammatory cytokines, mitochondrial ultrastructure (TEM), mitochondrial membrane potential (ΔΨm), and ROS levels were evaluated. Integrated transcriptomic and metabolomic profiling was performed to unbiasedly characterize protective mechanisms. In vitro, H₂O₂-induced oxidative stress was established in HepG2 cells. Cells were treated with trifolirhizin (15–25 µM) and assessed for antioxidant enzyme activities, ΔΨm, ROS production, glycolytic and mitochondrial respiration (Seahorse analysis), and cGAS-STING pathway protein expression. Pharmacological rescue experiments using the cGAS agonist cGAMP were conducted to test pathway dependency. Results: Trifolirhizin dose-dependently alleviated hepatic pathological damage and reduced pro-inflammatory cytokine levels in NOD mice. Multi-omics profiling revealed that oxidative stress responses, the mitochondrial electron transport chain, and glutathione metabolism were the most significantly restored pathways. Trifolirhizin preserved mitochondrial ultrastructure, restored ΔΨm, and attenuated ROS accumulation both in vivo and in vitro. Functionally, Seahorse analysis demonstrated that trifolirhizin rescued overall cellular bioenergetics, restoring both glycolytic capacity and mitochondrial respiratory parameters (basal respiration, ATP production, maximal respiration, and spare respiratory capacity). Mechanistically, trifolirhizin suppressed the cGAS-STING-TBK1-IRF3 axis, as evidenced by reduced expression of cGAS, p-STING, ZBP1, p-TBK1, and p-IRF3. Importantly, the cGAS agonist cGAMP abrogated the protective effects of trifolirhizin, confirming that the cGAS-STING pathway is functionally required for its action downstream of mitochondrial protection. Conclusion: Trifolirhizin attenuates liver injury in the nod mouse by preserving mitochondrial integrity, maintaining cellular energy metabolism, and thereby suppressing the ROS/cGAS-STING inflammatory cascade. These findings position trifolirhizin as a promising mitochondria-targeted therapeutic candidate for pSS-related hepatic complications and provide a mechanistic framework for discovering active compounds from mitochondrially active herbal formulations. Full article

The C-terminal binding protein 1 (CTBP1) is a transcriptional corepressor with a major role in nervous system growth and development. There are only 20 published cases with CTBP1 mutations, displaying a phenotype of Hypotonia, Ataxia, Developmental Delay and Tooth enamel defect Syndrome (HADDTS). Histochemical evidence of decreased mitochondrial respiratory chain activity has been previously reported, but comprehensive data on the metabolic phenotype assessed by various cellular respiration parameters are still missing. We present a 10-year-old female with typical HADDTS features, harboring the most reported de novo heterozygous CTBP1 mutation c.991C>T. To elucidate her metabolic phenotype, we quantified mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) utilizing an analyzer for assessing mitochondrial function (Seahorse XFp). Real-time metabolic assays revealed profound mitochondrial dysfunction with significantly attenuated maximal respiration and spare respiratory capacity compared to neurotypical controls. Following mitochondria-targeted nutritional support for one-year measurable bioenergetic improvements and reduced number of respiratory infections were registered. However, neurological recovery and new skill acquisition were not observed. We present a novel case of CTBP1-related neurodevelopmental disorder and demonstrate, for the first time, the application of non-invasive, real-time mitochondrial functional assessment in this setting, providing additional evidence for mitochondrial dysfunction in HADDTS. Full article

Background: Tumor cells can reprogram their metabolism, constituting a hallmark of cancer that plays a crucial role in tumor progression. As tumor cells exhibit an increased demand for nutrients, e.g., amino acids, they rely on extracellular sources and show deregulation of transport proteins. Among these, SNAT1 (SLC38A1) is described as the loader for glutamine that is responsible for the main influx of this amino acid. The aim of this study was to assess the molecular function of SNAT1 in melanoma regarding its role in amino acid transport and regulation of cellular metabolism. Methods: siPool-mediated downregulation of SNAT1 expression in melanoma cell lines was used to investigate the molecular function of this protein. Glutamine transport was assessed by measuring the intracellular and extracellular concentrations of glutamine. Regulation of downstream effectors was evaluated with qRT-PCR and Western Blot. Metabolism was investigated by performing Seahorse flux analysis. Mitochondrial staining was examined via flow cytometry. Protein interaction was assessed with Co-IP, and in silico modeling of protein interaction was performed with AlphaFold3. Results: In this study, we uncovered the new finding that SNAT1 is not primarily implicated in glutamine influx into melanoma cells but in signaling in response to extracellular glutamine. We identified P62 and cMYC as downstream effectors of SNAT1. By activating the P62/cMYC-axis and target genes of cMYC, SNAT1 modulates the metabolism of melanoma cells depending on the glutamine level. SNAT1 and P62 are interaction partners. Conclusions: This finding newly suggests that SNAT1 may function as a sensor or receptor (“transceptor”) for glutamine rather than being a direct and primary glutamine transporter, and could open up new therapeutic options targeting melanoma cells. Full article

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by rapid progression, early metastasis, and high relapse rates due to acquired chemoresistance. The human DEAD-box RNA helicase DDX5 is overexpressed in SCLC and has recently gained attention as a viable therapeutic target. Supinoxin (RX-5902), a selective small-molecule inhibitor of DDX5, exhibits strong anti-tumor activity. Recent evidence suggests that its cytotoxic effects are mediated through the disruption of mitochondrial respiration. In this study, transcriptomic profiling via RNA sequencing (RNA-seq) revealed significant downregulation of genes involved in cellular respiration following Supinoxin treatment and DDX5 knockdown in chemoresistant H69AR cells. To functionally validate these findings, we employed the Seahorse XF Cell Mito Stress Test, which measures key parameters of mitochondrial bioenergetics through oxygen consumption rate (OCR) analysis. Supinoxin-treated cells exhibited marked mitochondrial dysfunction, supporting the hypothesis that DDX5 inhibition disrupts cellular energy metabolism. These findings illuminate a previously underappreciated role of DDX5 in mitochondrial regulation and offer mechanistic insights into Supinoxin’s cytotoxic effects, underscoring its potential as a targeted therapy in SCLC. Full article

Background/Objectives: Chondrosarcoma, glioblastoma, acute myeloid leukemia, chronic lymphocytic leukemia, and cholangiocarcinoma cancers all contain mutations in the gene isocitrate dehydrogenase 2 (IDH2). The mutant IDH2 enzyme metabolizes alpha-ketoglutarate (αKG) into the potent oncometabolite D-2-hydroxyglutarate (D2HG) in the mitochondria of these cancers, leading to altered cellular metabolism. Emerging evidence suggests that mitochondrial transfer between cancer and recipient cells represents an important form of intercellular communication that may influence cellular metabolism. The presence of intercellular TNTs between IDH2-mutant chondrosarcoma cells motivated an investigation into mitochondria-associated physiological changes occurring during an intercellular exchange with immune cells. A mitochondrial transfer is a two-way process, and we hypothesized that mitochondria-associated material derived from IDH2-mutant chondrosarcoma cells is exchanged with normal cells through TNTs. We further hypothesized that disruption of the actin cytoskeleton will inhibit this transfer. Accordingly, our objectives were to (1) quantify the extent and directionality of the mitochondrial exchange between IDH2-mutant cells and wild-type cells and to modulate this process via cytoskeletal inhibitors, and (2) measure the metabolic changes associated with the coculture and mitochondrial exchange. Methods: IDH2-mutant chondrosarcoma cells were cocultured with immune cells in vitro to quantify the extent and directionality of the mitochondrial exchange, and cytochalasin B was used as a cytoskeletal inhibitor to disrupt actin-dependent transfer. Metabolic changes associated with coculture and mitochondrial exchange were assessed using Seahorse extracellular flux analysis. Results: The experimental data presented here demonstrate a bidirectional exchange of mitochondria-associated material between IDH2-mutant chondrosarcoma cells and immune cells in vitro, accompanied by metabolic alterations in both cell types. Conclusions: These findings advance our understanding of intercellular communication in the tumor microenvironment and provide a foundation for future studies examining the functional and therapeutic relevance of a mitochondrial exchange in IDH2-mutant cancers. Full article

To elucidate the effects of the new antidiabetic agent, imeglimin (Ime, 2 mM), on high-glucose-induced cellular stress in cardiac cells, its effects were compared with those of the conventional antidiabetic agent metformin (Met, 2 mM) based on various cellular pathophysiological functions. H9c2 cardiomyoblasts were cultured under normal-glucose (5.5 mM, N-Glu) or high-glucose (50 mM, H-Glu) conditions. Cellular metabolic function was evaluated using a Seahorse XFe96 Bioanalyzer, along with measurements of reactive oxygen species (ROS) production, expression levels of the autophagy-related marker LC3, and intercellular adhesion properties measured based on transepithelial electrical resistance (TEER). Cells cultured under H-Glu conditions showed enhanced mitochondrial and glycolytic activities, which were suppressed by Met or Ime. Under H-Glu conditions, total cellular ROS (t-ROS) levels were significantly increased. Met had little effect on t-ROS under H-Glu conditions, whereas Ime markedly reduced both t-ROS and mitochondrial ROS (m-ROS) levels under H-Glu conditions. The LC3-II/LC3-I ratio, a marker of autophagic activity, decreased under H-Glu conditions; however, this reduction was not significantly affected by treatment with either Met or Ime. Regarding intercellular adhesion properties, TEER values were elevated under H-Glu conditions compared to N-Glu conditions, and those under H-Glu conditions were further increased by Ime but not Met. In support of these results, the mRNA levels of cell-adhesion-related molecules, including β-catenin and N-cadherin, were also altered by Ime. Collectively, Ime modulated high-glucose-induced alterations in the biological properties of H9c2 cardiomyoblasts, independent of changes in autophagic activity. Full article

Background: Mitochondrial substrate switching plays an important role in aging. The substrate metabolic rate is closely related to mitochondrial activity, as mitochondria are the primary site for substrate oxidation and ATP production. Different substrates (glucose, amino acids, and fatty acids) enter the mitochondria through distinct pathways and are metabolized at different rates, depending on the energy demand and cellular conditions. However, it remains unclear how the mitochondrial metabolic rate of these substrates affects auditory cellular function. This study aimed to characterize the substrate-dependent mitochondrial respiratory responses of cochlear cells under varying energy supply conditions and metabolic stress, focusing on glucose, amino acids, and fatty acids as representative energy sources. Methods: The oxygen consumption rate (OCR) was measured after substrate addition using an Agilent Seahorse XF24 Flux Analyzer In-House Ear Institute-Organ of Corti 1 (HEI-OC1) cells, and the maximum OCR (MOCR) was determined as part of the mitochondrial stress test. Statistical analyses were performed using analysis of variance (ANOVA). Results: The OCR increased significantly after glutamine (L-Gln) or palmitate addition. The MOCR after L-Gln addition was significantly higher than that after glutamic acid, glycine, and phenylalanine addition. The MOCR after pyruvate addition was significantly higher than that after glucose addition. However, there was no significant increase in the MOCR after fatty acid addition. Conclusions: Glucose is essential for basal metabolism but cannot rapidly meet sudden energy demands. Pyruvate and L-Gln serve as effective substrates for short-term, high-intensity energy demands. Fatty acids increase OCR through mitochondrial uncoupling effects, though their role may be limited in inner ear cells. These findings provide a foundation for exploring metabolic interventions to support cochlear function and hearing health. Full article

Background: Gold nanoparticles (AuNPs) have several beneficial properties that make them effective as intracellular drug carriers, and their potential for various diagnostic and therapeutic applications is gaining recognition. Depending on their size and shape, AuNPs can cross the central nervous system (CNS) through the blood–brain barrier (BBB). In the CNS, they can exert a variety of influences on neuronal and glial cells, which can be both supportive—promoting cell health and function—and cytotoxic, potentially leading to cellular damage. The hypothalamus (HT) is the first region where nanoparticles (NPs) interact, as this neuroendocrine center is particularly sensitive to factors in the systemic circulation due to its function and location. This area is affected by systemic factors, including pro-opiomelanocortin (POMC) neurons, which regulate metabolic function and maintain homeostasis. The activity of mitochondria within these cells influences their response to both external factors and the presence of AuNPs, thereby facilitating a complex interplay between nanoparticle interactions and cellular metabolism in this vital brain region. Aims: This study investigates how AuNPs, at different concentrations and exposure times under in vitro conditions, affect the mitochondrial activity of POMC neurons, aiming to provide a comprehensive understanding of the mechanisms in the HT. Methods: The study investigates the effect of varying gold nanoparticle concentrations on the mitochondrial activity of POMC neurons over treatment periods of 1, 15, 24, and 48 h. Mitochondrial activity was measured using a Seahorse XFp Analyzer to provide high-resolution insights. Additionally, mitochondrial functionality was assessed through the detection of reactive oxygen species (ROS) and cell viability. Results: The findings indicated that the effects of gold nanoparticles on mitochondrial activity depend significantly on their concentration and exposure time. Specifically, exposure leads to an increase in early response systems, the citric acid cycle, and proton efflux, ultimately resulting in the inhibition of mitochondrial function and ATP production in POMC cells. This disruption may affect hypothalamic regulation and energy metabolism. Full article

Legg–Calvé–Perthes disease is a juvenile ischemic osteonecrosis (ON) of the femoral head. A disruption of blood supply to the femoral head produces extensive cell death and necrotic debris. Macrophages are innate immune cells recruited to the necrotic bone to orchestrate the repair process. However, the role macrophages play in the ON repair process is still not elucidated. The purpose of this study was to determine the effect of artificial necrotic bone fluid (NBF) on porcine peripheral blood mononuclear cell (PBMC)-derived macrophages. Monocytes were positively selected by CD14 MicroBeads from pig PBMCs. After maturation, cells were treated with no stimulant (Con), LPS + IFNγ (M1), IL4 + IL13 (M2), or NBF. All culture supernatants and cells were harvested for ELISA, Western blot, FACS, RT-qPCR and bulk RNAseq. The Western blot and ELISA showed that only the M1 condition elevated the protein level of pro-inflammatory cytokines. The FACS results indicated that percentage of CD8086+ (M1 marker) cells was significantly lower in the M2 vs. other conditions, whereas the relative median fluorescence intensity of CD8086 was significantly higher in the M1 vs. other conditions. The NBF did not show any significant change compared to the Con. mRNA analysis showed significantly increased IL1β and IL8 expression in the M1 vs. Con scenario. TNFα expression was significantly decreased in the M2 vs. Con scenario. Interestingly, the NBF did not induce pro-inflammatory gene expression. For bulk RNAseq, the Gene Set Enrichment Analyses of the M1-stimulated cells revealed the enrichment of pro-inflammatory gene sets. For the M2, most of the enriched categories were related to the down-regulation of inflammation. For the NBF, the most enriched categories were related to the down-regulation of protein translation and mitochondrial metabolism. We further confirmed the suppressive effects of NBF on macrophage functions using Seahorse Cell Mito Stress Tests, ¹³C-glucose metabolic flux analysis, mitochondrial ROS detection via MitoSOX^TM staining, and phagocytosis assay. Taken together, these results revealed that the artificial NBF down-regulates the overall cellular activity and energy metabolism of macrophages. Full article

Mitochondrial metabolism in the trabecular meshwork (TM) plays a critical role in maintaining intraocular pressure homeostasis by supporting the energy-demanding processes involved in aqueous humour outflow. In primary open-angle glaucoma, oxidative stress impairs mitochondrial function, leading to TM dysfunction. Therefore, understanding and targeting mitochondrial health in TM cells could offer a novel therapeutic strategy. Pyrroloquinoline quinone (PQQ) is a redox cofactor with antioxidant and mitochondrial-enhancing properties. However, its effects on human TM (HTM) cells remain largely unexplored. This study examined PQQ cytoprotective effects against H₂O₂-induced oxidative stress in HTM cells. Seahorse analyses revealed that PQQ alone improves mitochondrial respiration and ATP production. Moreover, PQQ mitigates H₂O₂-induced cellular damage and preserves mitochondrial function by normalising proton leak and increasing ATP levels. Furthermore, TEM and confocal microscopy showed that PQQ can partially alleviate structural damage, restoring mitochondrial network morphology, thereby leading to reduced cell death. Although these protective effects seem not to be mediated by changes in mitochondrial content or activation of the SIRT1/PGC1-α pathway, they may involve modulation of SIRT3, a key factor of mitochondrial metabolism and homeostasis. Overall, these results suggest that PQQ may represent a promising candidate for restoring mitochondrial function and reversing oxidative damage in HTM cells. Full article

Physiological changes in animals induced by environmental shifts in aquatic ecosystems can be studied using fish cell lines derived from vulnerable species. Therefore, we investigated how environmental shifts—specifically, an increase of 5 °C in temperature—impact the physiology of the cell line CMAfin1 derived from maraena whitefish (Coregonus maraena). Cellular growth, morphology, and metabolic responses were examined under two growth conditions: a control temperature of 20 °C and an elevated temperature of 25 °C. Using trypan blue staining, automated cell counting, phase contrast microscopy, and actin staining, we observed morphological changes in the cells. Metabolic functions were assessed using a Seahorse XFe96 Flux Analyzer, focusing on the bioenergetic capacities of mitochondrial respiration and glycolytic activity. Hyperthermia resulted in faster growth rates but reduced cell size in the CMAfin1 cell line. The cells’ metabolic activity (mitochondrial respiration and glycolytic activity) was inhibited, leading to a quiescent energy state. Our findings indicate reduced motility and altered intercellular communication at higher temperatures. The results highlight the potential of in vitro models to study environmental stress on fish physiology and emphasize the value of fish cell lines for understanding metabolic responses. Full article

Background/Objectives: Colorectal cancer (CRC) remains one of the most common and deadly malignancies worldwide, driven by metabolic reprogramming and mitochondrial dysfunction, which support tumor growth and progression. Several studies showed that nutrition is a contributing factor in the prevention and management of CRC. In this context, carnitines, amino acid derivatives abundant in food of animal origin, such as meat and milk, are crucial for mitochondrial function. Recently, l-carnitine and acetyl-l-carnitine have received particular attention due to their antioxidant, anti-inflammatory, and antitumor properties. However, to date, there is no conclusive evidence on the effects of l-carnitine and acetyl-l-carnitine in CRC or the underlying molecular mechanism. Methods: In this study, we investigated in HCT 116 and HT-29 CRC cells the effects of l-carnitine and acetyl-l-carnitine on mitochondrial homeostasis by XF HS Seahorse Bioanalyzer and cell death pathways by flow cytometry and western blot assays. Results: Data showed that l-carnitine and acetyl-l-carnitine reduced cell viability (p < 0.001), modulated cellular bioenergetics, and induced oxidative stress (p < 0.001). These phenomena promoted autophagic flux and the mitophagy process via PINK1 and Parkin modulation after 72 h of treatment. Of note, the combined treatment with l-carnitine and acetyl-l-carnitine showed a synergistic effect and enhanced the effect of single carnitines on tumor cell growth and metabolic dysfunction (p < 0.05). Moreover, exposure to l-carnitine and acetyl-l-carnitine promoted CRC cell apoptosis, suggesting a mechanism involving mitophagy-related cell death. These data were associated with increased SIRT4 expression levels (p < 0.01) and the activation of AMPK signaling (p < 0.01). Conclusions: Overall, the results, by supporting the importance of nutritional factors in CRC management, highlight l-carnitine and acetyl-l-carnitine as promising agents to target CRC metabolic vulnerabilities. Full article

Diabetes wound healing presents several significant challenges, which can complicate recovery and lead to severe consequences. Polyethylene glycol loxenatide (PEG-loxe), a long-acting glucagon-like peptide-1 receptor agonist (GLP-1RA), shows cardiovascular benefits, yet its role in diabetic wound healing remains unclear. Diabetic mice received PEG-loxe (0.03 mg/kg/week, i.p.) for three months. Glucose metabolism was evaluated using the insulin tolerance test (ITT) and oral glucose tolerance test (OGTT). Wound closure rates and angiogenesis-related proteins were analyzed. Serum proteomics was performed using the Olink assay to evaluate systemic inflammation. In vitro, human endothelial progenitor cells (EPCs) were exposed to high glucose and palmitic acid, with or without PEG-loxe treatment. EPC tube formation and migratory capacity were evaluated using the tube formation assay and migration assay, respectively. Levels of nitric oxide (NO) and phosphorylated endothelial nitric oxide synthase (p-eNOS) were quantified. Mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential were assessed using MitoSOX and JC-1 staining. Cellular respiratory function was analyzed via the Seahorse XF assay. Autophagy was evaluated by examining the expression of autophagy-related proteins and the colocalization of mitochondria with lysosomes. PEG-loxe improved glucose tolerance, accelerated wound closure, and upregulated the hypoxia-inducible factor-1α/vascular endothelial growth factor/stromal cell-derived factor-1 axis (HIF-1α/VEGF/SDF-1) in diabetic mice. Serum proteomics revealed reduced pro-inflammatory markers and elevated anti-inflammatory IL-5. In vitro, PEG-loxe restored EPC function by enhancing NO production, reducing mitochondrial ROS, improving cellular respiratory function, and restoring autophagic flux. These findings suggest that PEG-loxe offers therapeutic benefits for diabetic wound healing by downregulating systemic inflammation, enhancing angiogenesis, and improving mitochondrial quality control in EPCs, highlighting GLP-1RAs as potential therapies for diabetic vascular complications. Full article