Corresponding author: Sang Hyun Kim Tel +82-63-270-2853 Fax +82-63-270-4234 E-mail INTRODUCTION
Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance, impaired glucose uptake, and systemic energy imbalance [1]. Skeletal muscle, which accounts for nearly 40% of total body mass, plays a central role in maintaining glucose homeostasis and regulating energy metabolism [2]. Moreover, previous studies have identified that mitochondria within skeletal muscle are essential for adenosine triphosphate (ATP) generation through oxidative phosphorylation, and their dysfunction is closely associated with reduced insulin sensitivity, excessive reactive oxygen species (ROS) production, and metabolic inflexibility [1]. Therefore, abnormal skeletal muscle mitochondrial function in type 2 diabetes, leading to impaired energy production and skeletal muscle weakness, is considered one of the hallmark pathological features of diabetes [3].
Following the advancement of modern industrialization, fine particulate matter (PM2.5) pollution in the atmospheric environment has become a primary environmental risk factor for metabolic disorders [4]. The majority of developed countries (such as the United States and South Korea) set a threshold of 35 μg/m³ for the single-day average concentration of PM2.5 as the level for issuing air quality alerts or health risk advisories for sensitive populations (According to the recommendations of the World Health Organization) [5]. Studies show that PM2.5 can penetrate deeply into the respiratory tract, enter systemic circulation, and reach peripheral organs, including the liver, heart, and skeletal muscle. Once in circulation, PM2.5 particles provoke systemic oxidative stress and vascular inflammation, contributing to multi-organ dysfunction [6]. Chronic exposure to PM2.5 promotes oxidative stress, inflammation, and mitochondrial damage, disrupting normal energy metabolism [7]. Several studies have reported that PM2.5 impairs mitochondrial respiration, induces cristae disruption and swelling, and increases ROS generation, suggesting that mitochondria are primary targets of PM2.5-induced cytotoxicity [8].
Although both T2DM and PM2.5 exposure individually contribute to mitochondrial dysfunction [9], the combined effects of these two stressors on skeletal muscle mitochondria remain poorly understood. Recent studies have demonstrated that chronic PM2.5 exposure worsens insulin resistance and mitochondrial dysfunction, suggesting a synergistic interaction between metabolic and environmental stressors. Acute PM2.5 exposure in particular causes chronic damage to skeletal muscle mitochondrial morphology, disrupting the mitochondrial network homeostasis [8]. Under diabetic conditions, skeletal muscle mitochondria bear immense metabolic and oxidative stress [10]. Whether PM2.5 exposure further exacerbates their structural and functional damage remains to be confirmed.
Therefore, this study aimed to investigate how PM2.5 exposure affects mitochondrial structure and protein expression in skeletal muscle under type 2 diabetic conditions. Therefore, we hypothesize that under type 2 diabetes conditions, PM2.5 exposure will further exacerbate mitochondrial structural damage and activate apoptosis-related signaling pathways while simultaneously reducing the expression of enzymes in the mitochondrial electron transport chain. This study will provide an important reference for protecting patients with insulin resistance-related metabolic diseases from further harm caused by atmospheric environmental pollution.
METHODS
1. Experimental procedures
In this study, male Nagoya-Shibata-Yasuda (NSY).B6-Tyr⁺Ay mice (Agouti yellow strain) [11] were crossed with female C57BL/6 mice to obtain wild-type (WT, n=12) and diabetic model (DM, n=12) male offspring. This protocol established a novel mouse model of type 2 diabetes characterized by early-onset disease and persistent hyperglycemia [11]. From 4 to 16 weeks of age, the WT was fed with a normal diet, and the DM was fed with a 60% high-fat diet (HFD) for 12 weeks to induce more stable type 2 diabetes status.
At 16 weeks, blood glucose levels were measured to confirm the diabetic phenotype. Each group was further divided into PM2.5-exposed or non-exposed subgroups, resulting in four experimental groups: Wild-type mice exposed to filtered air (WT-CON, n=6), Wild-type mice exposed to PM2.5 (WT-PM2.5, n=6), Diabetic mice exposed to filtered air (DM-CON, n=6), Diabetic mice exposed to PM2.5 (DM-PM2.5, n=6).
PM2.5 exposure was conducted for five consecutive days, followed by a 4-week recovery period to assess post-exposure adaptation and persistent effects (Previous studies have shown that structural damage to skeletal muscle mitochondria persists even four weeks after exposure to PM2.5 at the same dose levels used in this study [8]). After the recovery period, the gastrocnemius muscles were collected for subsequent analyses. The overall experimental timeline is illustrated in Fig. 1. All experimental procedures were conducted in accordance with the guidelines and regulations set by the Institutional Animal Care and Use Committee of Jeonbuk National University (IACUC approval no. CBNU-2022-0067).
2. Atmospheric simulation chamber (ASC) system
The PM2.5 exposure method is consistent with previous studies [12]. The ASC system is a whole-body exposure device designed to replicate the inhalation of PM2.5 in the atmosphere while maintaining a consistent average concentration. The PM solution was formulated by mixing 10 organic and inorganic compounds, including oxalic acid, malonic acid, glutaric acid, sucrose, 2,5-dihydroxybenzoic acid, glycine, ammonium sulfate, ammonium nitrate, acetate, and glycerol, into distilled water (Table 1). The PM was aerosolized using a nebulizer (TQ-50-C0,5; Mein-hard, USA) and, subsequent to passing through a polypropylene melt-blown filter, particles larger than 2.5 μm are sieved out and enter the chamber. The concentration of PM2.5 within the chamber is continuously monitored in real-time using a particle counter (BT-610; Met One, USA), and a predetermined concentration is automatically maintained through a flow controller program. The WT-PM2.5 and DM-PM2.5 groups were exposed to PM2.5 for 2 hours per day for five consecutive days at a concentration of 52.7±8.9 μg/m3 (Table 2). The chamber maintained a humidity level of 55-60% and a temperature range of 23-25°C.
Table 1.
List of the chemical compositions, formula, and dry mass fractions of organic and inorganic species used in this study
| Functional Group | Components | Formula | Density (g/cm3) at 295 K* | Dry mass fraction (%) |
|---|---|---|---|---|
| Monocarboxylic acid | Acetate | C2H3O2 | 1.05 | 6.25 |
| Dicarboxylic acid | Oxalic acid | C2H2O4 | 1.90 | 6.25 |
| Malonic acid | C3H4O4 | 1.62 | 6.25 | |
| Glutaric acid | C5H8O4 | 1.35 | 6.25 | |
| Polyols | Glycerol | C3H8O3 | 1.26 | 6.25 |
| Sugars | Sucrose | C12H22O11 | 1.59 | 6.25 |
| Aromatics | 2,5-Dihydroxybenzoic acid | C7H6O3 | 1.55 | 6.25 |
| Amino acid | Glycine | C2H5O2N | 1.61 | 6.25 |
| Inorganic salts | Ammonium sulfate | (NH4)2SO4 | 1.77 | 25 |
| Ammonium nitrate | NH4NO3 | 1.72 | 25 |
* Values of measured densities are from www.chemicalbook.com.
3. Chemical composition
The PM2.5 chemical composition is consistent with previous studies [12]. The organic components and inorganic salts for the preparation of artificial PM2.5 are listed in Table 1. Eight organic compounds with carboxylic acid, polyol, sugar, aromatic, and amino acid functional groups were investigated. Ammonium sulfate and ammonium nitrate were used as models for inorganic salts due to their abundance in the air [13-16]. The 10 compounds in Table 1 were mixed at an organic-to-inorganic dry mass ratio of 1:1 to mimic the chemical complexity of atmospheric aerosols. The compounds were purchased from Sigma-Aldrich (purity ≥98%) and were used without further purification. The mixture of 10 components was dissolved in purified water.
4. Blood glucose analysis
After 16 weeks of age, blood glucose was measured from tail tip samples using a glucometer (Roche, ACCU-CHEK, Germany).
5. Western blot analysis
The analysis of protein expression was conducted using the gastrocnemius (GAS; A composite sample containing Type I, Type IIa, and Type IIb/x muscle fibers to represent the reactive effects across the whole skeletal muscle system), which was rapidly frozen in liquid nitrogen upon extraction and subsequently stored at −80°C. The GAS samples were homogenized in a cold lysis buffer [50 mM Tris·HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA, pH 7.4), 1 mM Pefabloc (Roche, Basel, Switzerland), 1 mM NaF, 1 μg/mL aprotinin, 1 μg/mL leupeptin, 1 μg/mL pepstatin, 0.1 mM bpV (phen), and 2 mg/mL β-glycerophosphate] kept on ice.
The homogenate was solubilized in Laemmli sample buffer after determining the protein concentration using the Bradford (BIO-RAD, Bio-Rad Protein Assay Dye Reagent Concentrate, CA, USA) [17]. Following gel electrophoresis, each sample was transferred onto nitrocellulose membranes and blocked with skim milk at room temperature. Subsequently, the membranes were incubated overnight at 4°C with primary antibodies [Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α; GTX37356, GeneTex, USA); Mitochondrial transcription factor A (Tfam; sc-166965, SCBT, USA); Total OXPHOS Rodent WB Antibody Cocktail (NADH-UO, SUO, COX-1 and ATPsyn; ab110413, abcam, USA); Glucose transporter protein type-4 (GLUT4, sc-53566, SCBT, USA); Hexokinase-2 (HK-2; sc-374091, SCBT, USA); phos-phofrucokinase-1 (PFK-1; sc-166722, SCBT, USA), lactate dehydrogenase (LDH; sc-133123, SCBT, USA); Bcl2-associated X protein (Bax; sc-7480, SCBT, USA); B-cell lymphoma 2 (Bcl-2; sc-7382, SCBT, USA); PINK- 1 (sc-517353, SCBT, USA); Parkin (sc-32282, SCBT, USA); β-actin (MA1-140, Invitrogen, USA); and then subjected to further incubation with appropriate secondary antibodies [mouse anti-goat (sc-2354, SCBT, USA); mouse anti-rabbit (sc-2357, SCBT, USA); goat anti-mouse (sc-2005, SCBT, USA)] for protein detection. Protein visualization was conducted using the ECL Western Blotting Detection Reagent (GE Healthcare, Chalfont St Giles, UK), and quantification was carried out using the ChemiDoc XRS+ system (BIO-RAD, Hercules, CA, USA).
6. Transmission electron microscopy
Transmission electron microscopy (TEM) was used to examine the mitochondrial structure. The gastrocnemius muscle was fixed in a solution containing 2.5% glutaraldehyde and 4% formaldehyde and a 0.1 M phosphate buffer at pH 7.4 for 2 hours immediately after extraction. The fixed muscles were post-fixed with 1% osmium tetroxide for 2 hours. The muscles were then dehydrated using a graded series of ethanol solutions and embedded in Epon-812 resin. Sections were obtained using a NOVA ultramicrotome (LKB, Vienna, Austria) and mounted on a 100-mesh grid. Sections approximately 80 nm thick were prepared for TEM and stained with 0.1% toluidine blue. To enhance visualization, the sections were stained with uranyl acetate and lead citrate and examined using an H7650 electron microscope (Hitachi, Japan) with an accelerating voltage of 80 kV to confirm the specimens. Samples were analyzed using a JEM-2010 TEM (JEOL) at the Center for University Wide Research Facilities at Jeonbuk National University.
The results for each group were derived from three distinct mouse samples (each sample underwent observation of 3-4 sections, with six representative images ultimately retained per sample). TEM images were independently reviewed by two examiners who were unaware of the sample information. The examination aimed to assess the following four indicators to verify the level of mitochondrial damage: (1) loss or disruption of cristae structure, (2) swelling of the mitochondrial matrix, (3) compromised outer membrane integrity and (4) the presence of small vacuoles within the mitochondria. These criteria were consistently applied to all samples.
7. Statistics analysis
All data were presented as mean±standard deviation (SD) and were analyzed using GraphPad Software (Prism 10, MA, USA). A two-way ANOVA was conducted to examine the main effects of diabetes (WT vs. DM), M2.5 exposure (CON vs. PM2.5), and their interaction. When significant effects were detected, Tukey's HSD post-hoc test was applied for multiple comparisons. Differences were considered statistically significant at p <.05.
RESULTS
1. Altered glucose metabolism in skeletal muscle following PM2.5 exposure under diabetic conditions
To assess how PM2.5 exposure affects glucose metabolism in skeletal muscle, we analyzed blood glucose levels and the expression of glucose transport and glycolytic enzymes (GLUT-4, HXK-2, PFK-1, and LDH; Fig. 2).
Fig. 2.
Fig. 2.Effects of PM2.5 Exposure on Glucose Metabolism-Related Protein Expression in Diabetic Mice. (A) Blood glucose levels after 12 weeks of normal diet and high-fat diet in WT and DM mice. (B) Representative western blot images of GLUT-4, HXK-2, PFK-1, LDH, and β-Actin protein expression in gastrocnemius muscle. Protein expression levels of (C) GLUT-4, (D) HKX-2, (E) PFK-1, and (F) LDH were quantified by densitometric analysis, normalized to β-Actin. WT-CON, wild type control; WT-PM2.5, wild type with PM2.5 exposure; DM-CON, diabetic control; DM-PM2.5, diabetic with PM2.5 exposure; All data are presented as Mean±SD. **p<.01, ***p<.001 between the groups; ns, not significant.
After 12 weeks of dietary intervention, blood glucose levels were significantly higher in the DM group than in the WT group (p <.001; Fig. 2A), confirming the successful establishment of the diabetic model.
Representative Western blot analysis showed that the expression level of GLUT-4 is regulated by both the independent effects and the interaction between DM and PM2.5 exposure (Fig. 2C). In contrast, LDH expression level was affected only by PM2.5 exposure (Fig. 2F).
GLUT-4 protein expression (Fig. 2B) was significantly elevated in diabetic groups compared with WT, and the DM+PM2.5 group exhibited a further increase compared with DM-CON (p <.01; Fig. 2C). In contrast, the expression levels of glycolytic enzymes—HXK-2 (Fig. 2D), PFK-1 (Fig. 2E), and LDH (Fig. 2F)—remained unchanged among all groups.
These results indicate that GLUT-4 upregulation represents a compensatory response to cellular energy deficiency under insulin-resistant conditions, yet glucose uptake and glycolytic flux were not effectively restored [18]. Persistent insulin resistance may have impeded the translocation of GLUT-4 to the plasma membrane [19], thereby limiting glucose utilization despite its increased protein abundance.
2. Effects of PM2.5 exposure on mitochondrial biogenesis regulators and respiratory chain enzymes
To evaluate mitochondrial biogenesis and oxidative phosphorylation activity, the protein expression of PGC-1α, Tfam, and representative subunits of the mitochondrial respiratory chain were analyzed in the gastrocnemius muscle (Fig. 3A).
Fig. 3.
Fig. 3.PM₂.₅ exposure promotes selective upregulation of mitochondrial regulatory proteins in diabetes. (A) Representative western blot images of PGC-1a, Tfam, ATPsyn, COX-1, SUO, NADH-UO, and β-Actin protein expression in gastrocnemius muscle. Protein expression levels of (B) PGC-1a, (C) Tfam, (D) NADH-UO, (E) SUO, (F) COX-1, and (G) ATPSyn were quantified by densitometric analysis, normalized to β-Actin. WT-CON, WT control; WT-PM₂.₅, WT with PM₂.₅; DM-CON, diabetic control; DM-PM₂.₅, diabetic with PM₂.₅; All data are presented as Mean±SD; *p<.05, **p<.01; ns, not significant.
Results indicate that the expression levels of PGC-1α, Tfam, NADH-UO, SUO, COX-1, and ATPsyn are all regulated by the independent effect of DM (Fig. 3). Among these, the expression levels of Tfam, SUO, and COX-1 are also independently affected by PM2.5 (Fig. 3C, E, and F).
PGC-1α levels showed no significant differences among groups (Fig. 3B), whereas Tfam expression was significantly elevated in the DM+ PM2.5 group (p <.05), indicating activation of mitochondrial transcriptional machinery (Fig. 3C). Among the oxidative phosphorylation complexes, SUO (complex II) and COX-1 (complex IV) were significantly increased in the PM2.5-exposed groups, particularly in DM+PM2.5. In contrast, NADH-UO (complex I) and ATP synthase (complex V) exhibited no significant changes (Fig. 3D-G).
These findings suggest that PM2.5 exposure induces a selective upregulation of Tfam and specific electron-transport-chain components (complex II and IV) rather than a uniform enhancement of the entire oxidative phosphorylation system. Under oxidative stress, disruption of the PGC-1α-Tfam interaction can lead to partial activation of mitochondrial biogenesis [20], consistent with the incomplete remodeling observed in this study. The results imply that, under diabetic conditions, PM2.5 elicits a partial or incomplete compensatory remodeling of mitochondrial biogenesis and respiration, possibly as a cellular response to persistent energy stress.
3. Activation of mitophagy and apoptotic signaling pathways in diabetic skeletal muscle after PM2.5 exposure
To determine whether PM2.5 exposure affects mitochondrial quality control in diabetic skeletal muscle, we examined the expression of mitophagy-and apoptosis-related proteins, including PINK1, Parkin, Bax, and Bcl-2 (Fig. 4A).
Fig. 4.
Fig. 4.PM₂.₅ exposure induces concurrent activation of mitophagy and apoptosis pathways in diabetic skeletal muscle. (A) Representative western blot images of PINK1, Parkin, Bax, Bcl-2, and β-Actin protein expression in gastrocnemius muscle. Protein expression levels of (B) PINK1, (C) Parkin, (D) Bax, and (E) Bcl-2 were quantified by densitometric analysis, normalized to β-Actin. (F) Bax/Bcl-2 ratio was calculated to evaluate the balance between pro- and anti-apoptotic signaling. WT-CON, wild type control; WT-PM₂.₅, wild type with PM₂.₅ exposure; DM-CON, diabetic control; DM-PM₂.₅, diabetic with PM₂.₅ exposure; All data are presented as Mean±SD. *p<.05, **p<.01 between the groups; ns, not significant.
The expression levels of PINK1, parkin, Bax, and Bcl-2 proteins were all independently affected by DM and PM2.5 (Fig. 4). Among these, only PINK1 and Bcl-2 showed an interaction effect between DM and PM2.5 (Fig. 4B and E).
The expression of PINK1 and Parkin—key regulators of mitophagy—was markedly increased in the DM+PM2.5 group compared with all other groups (p <.01; Fig. 4B, C), indicating the activation of PINK1/Parkin-mediated mitophagy under combined diabetic and PM2.5 stress. Similarly, both Bax (a pro-apoptotic protein) and Bcl-2 (an anti-apoptotic protein) were significantly elevated in the DM+PM2.5 group (p <.01; Fig. 4D, E). However, the Bax/Bcl-2 ratio showed no significant change among the groups (Fig. 4F), suggesting that although apoptotic signaling stress, the increase in Bcl-2 may reflect a compensatory anti-apoptotic response.
Collectively, these results demonstrate that PM2.5 exposure under diabetic conditions concurrently activates mitophagy and apoptosis-related pathways, indicating that mitochondria are in a state of persistent stress and turnover imbalance rather than complete recovery following exposure [21].
4. PM2.5 exposure aggravates ultrastructural mitochondrial damage in diabetic skeletal muscle
TEM was used to examine the ultrastructural morphology of mitochondria in skeletal muscle (Fig. 5). Mitochondria from WT-CON mice exhibited a normal elongated shape with densely packed and well-organized cristae, indicating healthy morphology.
Fig. 5.
Fig. 5.PM2.5-induced mitochondrial ultrastructural damage in skeletal muscle of diabetic mice. Representative TEM images of skeletal muscle mitochondria from WT-CON, WT-PM₂.₅, DM-CON, and DM-PM₂.₅ groups. WT-CON mitochondria exhibited normal elongated morphology with intact cristae. WT-PM₂.₅ and DM-CON showed mild swelling and irregular cristae, whereas DM-PM₂.₅ exhibited severe mitochondrial damage with disrupted cristae, matrix vacuolation, and swelling (yellow arrows). TEM images were taken at 20,000× and 40,000× magnification. Scale bars: 1 μm (upper), 0.5 μm (lower). WT-CON, wild type control; WT-PM₂.₅, wild type with PM₂.₅ exposure; DM-CON, diabetic control; DM-PM₂.₅, diabetic with PM₂.₅ exposure; All data are presented as Mean±SD. ***p<.001 between the groups; ns, not significant.
In contrast, WT-PM2.5 and DM-CON groups displayed mild mitochondrial swelling, irregular cristae, and partial loss of inner membrane integrity, suggesting early signs of structural stress. Notably, the DM+ PM2.5 group showed the most severe mitochondrial damage, characterized by marked swelling, disrupted and fragmented cristae, vacuolation, and matrix disorganization (yellow arrows) [8,22]. These morphological abnormalities demonstrate that PM2.5 exposure under diabetic conditions markedly exacerbates mitochondrial ultrastructural damage, consistent with impaired mitochondrial homeostasis observed in biochemical analyses [8].
The TEM findings provide direct visual evidence that mitochondria fail to recover after combined metabolic (diabetes) and environmental (PM2.5) stress, remaining in a state of persistent structural degeneration [10].
DISCUSSION
This study investigated how fine particulate matter (PM2.5) exposure affects skeletal muscle mitochondria under diabetic conditions. Our findings demonstrate that PM2.5 exposure aggravates metabolic and mitochondrial dysfunction, leading to persistent structural damage even after a recovery period. This pathological process is characterized by four key responses: compensatory but ineffective GLUT-4 upregulation (Fig. 2), incomplete mitochondrial biogenesis (Fig. 3), concurrent activation of mitophagy and apoptosis signaling (Fig. 4), and severe ultrastructural degeneration observed by TEM (Fig. 5). Together, these findings indicate that the combination of metabolic (diabetes) and environmental (PM2.5) stress maintains mitochondria in a chronic state of imbalance between energy demand, biogenesis, and degradation.
In diabetic skeletal muscle, insulin resistance limits glucose uptake and utilization despite elevated circulating glucose levels [18]. Consistent with this, GLUT-4 expression was markedly increased in the DM+PM2.5 group, whereas glycolytic enzymes (HXK-2, PFK-1, LDH) remained unchanged (Fig. 2). This pattern suggests that GLUT-4 upregulation represents a compensatory attempt to overcome cellular ATP deficiency through AMPK activation [23]. However, due to insulin resistance, GLUT-4 fails to translocate to the plasma membrane, preventing effective glucose transport and glycolytic flux [20]. Thus, PM2.5 exposure exacerbates metabolic stress in diabetic muscle by maintaining high GLUT-4 levels without restoring energy metabolism.
At the mitochondrial level, PM2.5 exposure under diabetic conditions induced partial activation of the PGC-1α-Tfam pathway (Fig. 3). Tfam and COX-1 were significantly increased, whereas NADH-UO, ATP synthase, and PGC-1α showed no significant change. This reflects an incomplete mitochondrial biogenesis response, in which transcription and replication were initiated but respiratory function was not fully restored [20,24]. Previous studies have shown that oxidative stress uncouples the PGC-1α-Tfam signaling pathway, resulting in defective transcriptional control of mitochondrial biogenesis and impaired oxidative phosphorylation [20]. These results imply that mitochondrial remodeling was insufficient to re-establish oxidative phosphorylation, likely due to persistent oxidative stress and disrupted nuclear-mitochondrial communication.
Furthermore, the DM+PM2.5 group exhibited marked upregulation of PINK1 and Parkin (Fig. 4B, C), indicating activation of mitophagy, the quality-control pathway for eliminating damaged mitochondria [21]. Although PINK1/Parkin signaling is widely recognized as a hallmark of lysosomal phagocytosis initiation, increased expression alone is insufficient to establish functional lysosomal phagocytosis flux. Studies indicate that mitochondrial depolarization coupled with impaired autophagosome-lysosome fusion can lead to PINK1/Parkin accumulation without effective mitochondrial degradation [25]. Future studies incorporating autophagy flux analysis (e.g., LC3-II, p62 degradation, mitochondrial mass measurements) are needed to determine whether the observed PINK1/Parkin upregulation corresponds to effective mitochondrial clearance.
At the same time, Bax and Bcl-2 expression also increased (Fig. 4D, E), suggestive of apoptotic signaling stress. The unaltered Bax/Bcl-2 ratio (Fig. 4F) implies that anti-apoptotic defenses were simultaneously engaged to suppress excessive cell death [21]. These findings support that PM2.5 exposure triggers both mitochondrial degradation and survival mechanisms, reflecting a chronic stress state where mitophagy and apoptosis coexist. Such concurrent activation of degradation and survival pathways reflects persistent oxidative stress [6]. Notably, despite activation of the PINK1/Parkin axis, the Bax/Bcl-2 ratio remained unchanged. This finding suggests that pro-apoptotic signaling is not merely a downstream consequence of mitophagy induction, but rather represents a tightly regulated equilibrium between survival and death pathways. Under chronic oxidative stress, mitophagy may serve as a cellular protective mechanism, preventing mitochondrial outer membrane permeabilization and subsequent cytochrome c release [21]. In this context, efficient removal of damaged mitochondria may inhibit Bax translocation, stabilizing pro-apoptotic Bcl-2 family proteins and thereby maintaining an apparently stable Bax/Bcl-2 ratio [21]. Future studies should integrate downstream indicators such as cytochrome C release, caspase activation, and mitochondrial respiratory function to further elucidate the temporal and mechanistic relationship between mitochondrial autophagy and apoptotic progression following PM2.5 exposure.
Finally, TEM imaging (Fig. 5) provided direct morphological evidence of mitochondrial damage. Mitochondria from WT-CON mice displayed elongated shapes with intact cristae, while those from WT-PM2.5 and DM-CON groups showed mild swelling and cristae irregularities. In contrast, DM+PM2.5 mitochondria exhibited severe structural disruption, matrix vacuolization, and cristae loss, confirming persistent ultrastructural degeneration even after a 4-week recovery period [8,22]. This suggests that mitochondrial repair and turnover capacity were overwhelmed under combined diabetic and environmental stress.
Collectively, these results revealed that PM2.5 exposure amplifies mitochondrial stress and energy imbalance in diabetic skeletal muscle. Persistent insulin resistance and oxidative stress lead to compensatory GLUT-4 and Tfam upregulation, incomplete mitochondrial biogenesis, and simultaneous activation of mitophagy and apoptosis. These maladaptive responses culminate in structural collapse of mitochondria, as seen in TEM, preventing recovery of normal metabolic function [10]. Thus, PM2.5 acts as a potent secondary metabolic stressor in diabetes [4], accelerating mitochondrial deterioration and highlighting the critical need for environmental control to mitigate metabolic complications.
This study has several limitations. First, the sample size (n=6 per group) may have limited statistical power. Second, this study did not include direct indicators of mitochondrial function, such as mitochondrial oxygen consumption rate, ATP production capacity, or mitochondrial enzyme activity. Third, this study did not evaluate GLUT4 transport-related markers—a key determinant of glucose uptake. Changes in GLUT4 protein expression alone cannot determine specific functional effects. Fourth, although results indicate significant upregulation of Tfam expression, Tfam expression alone cannot confirm the precise effects of transcriptional mechanisms. Subsequent studies should examine mitochondrial DNA copy number, mitochondrial transcription rates, or changes in signaling pathways like nuclear respiratory factor-1/2 to clarify the specific mechanisms underlying these effects. Fifth, this study provides only qualitative descriptions based on transmission electron microscopy images without quantitative morphometric analysis (e.g., mitochondrial area, cristae density, roundness index). The aforementioned limitations warrant validation and in-depth discussion in future research to elucidate the specific effects and mechanisms of environmental PM2.5 exposure in populations with metabolic disorders such as diabetes.
CONCLUSION
In summary, this study significantly demonstrates that PM2.5 was associated with greater mitochondrial impairments in skeletal muscle under diabetic conditions, and compensatory upregulation of GLUT-4 and mitochondrial biogenesis-related proteins such as Tfam. Instead, concurrent activation of mitophagy and apoptotic pathways, together with severe ultrastructural mitochondrial damage, indicates that the cellular quality-control mechanisms were insufficient to counteract combined metabolic and environmental stress.
These findings suggest that fine particulate matter acts as a secondary metabolic stressor in diabetes, intensifying mitochondrial stress and impairing muscle energy homeostasis even after recovery. This highlights the importance of environmental exposure management and lifestyle interventions in preventing or mitigating the progression of diabetic myopathy and related metabolic complications.
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