Mitochondrial complex I (NADH dehydrogenase, NADH:ubiquinone oxidoreductase), consisting of 45 subunits, is one of the largest known proteins, with a molecular weight of 980 kDa (Fiedorczuk and Sazanov, 2018). Complex I is located in the inner mitochondrial membrane and transfers electrons from NADH to CoQ while coupling the ejection of four protons from the mitochondrial matrix to the intermembrane space, generating a proton gradient across the membrane that drives ATP synthesis (Laube et al., 2024). Recent studies have shown that mitochondrial complex I also plays a critical role in sodium ion transport, thereby contributing to the formation of the mitochondrial membrane potential (Hernansanz-Agustín et al., 2024).

The references included in this review employed multiple experimental approaches to evaluate the function and status of mitochondrial respiratory chain complexes. Among these, enzyme activity assays, Seahorse analysis, and blue native polyacrylamide gel electrophoresis (BN-PAGE) reflect distinct aspects of mitochondrial complex properties and are therefore highly complementary (Jha et al., 2016; Arroum et al., 2023). Enzyme activity assays are typically performed using isolated mitochondria or tissue homogenates and provide a direct assessment of the catalytic activity of individual respiratory chain complexes, but they are limited in their ability to capture the functional contribution of these complexes to integrated cellular respiration (Wang et al., 2025). Seahorse analysis assesses global mitochondrial respiratory function and metabolic flexibility by real-time measurement of oxygen consumption and glycolytic activity, thereby reflecting the overall impact of complex dysfunction on cellular energy metabolism, while lacking resolution of the structural status of specific complexes (Desousa et al., 2023). In contrast, BN-PAGE is primarily used to analyze the assembly, organization, and stability of mitochondrial respiratory chain complexes and supercomplexes, revealing structural alterations, but does not directly measure enzymatic activity or dynamic respiratory function. Accordingly, each experimental approach provides distinct and complementary information.

Mitochondrial complex I is one of the most extensively studied complexes in mitochondrial research (Agip et al., 2019). A range of factors can modulate its activity, and alterations in complex I function contribute to mitochondrial dysfunction, thereby playing a critical role in the pathogenesis of NAFLD (Ding et al., 2022). The below summarizes the specific link between mitochondrial complex I dysfunction and NAFLD (Table 1). Most studies (Solsona-Vilarrasa et al., 2019; García-Berumen et al., 2019; Verbeek et al., 2015) indicate that high-fat, high-cholesterol, high-sugar, and choline-deficient diets induced NAFLD, which is associated with decreased complex I activity, leading to mitochondrial defects, suppressed respiration, increased lactate dehydrogenase (LDH) activity, ATP depletion, a reduced respiratory control ratio, and increased ROS production. However, some studies (Maseko et al., 2024; Yu et al., 2021; Sun et al., 2017) have also reported an increase or slight increase in complex I activity and expression in NAFLD models, leading to enhanced mitochondrial respiration and the generation of more ROS, which causes oxidative stress. Whether complex I activity is increased or decreased, mitochondrial dysfunction and oxidative damage are evident, which are key factors in the onset and progression of NAFLD.

In research on mitochondrial complexes and NAFLD, several key subunits of mitochondrial complex I play critical roles. NDUFS1 (NADH: ubiquinone oxidoreductase core subunit S1), the largest core subunit of complex I, can modulate the activity of the mitochondrial respiratory chain complex I and the formation of mitochondrial ROS (Qi et al., 2022). NDUFS1 participates in electron transfer from NADH to coenzyme Q and is functionally coupled to proton translocation from the mitochondrial matrix to the intermembrane space, a process that is essential for preserving the structural integrity and proper function of complex I (Ni et al., 2019). Its overexpression promotes the formation of supercomplexes by complex I, effectively reducing the production of reactive oxygen species (ROS). This NDUFS1-mediated assembly of complex I into supercomplexes plays an important role in regulating ROS production and oxidative stress (Lopez-Fabuel et al., 2016). In patients with non-alcoholic fatty liver disease (NAFLD), the expression of NDUFS1 is reduced. Overexpression of NDUFS1 can reduce complex I activity and alleviate the progression of NAFLD (Wan et al., 2023). NDUFS2 (NADH: ubiquinone oxidoreductase core subunit S2) and NDUFA10 (NADH: ubiquinone oxidoreductase core subunit A10) are key components of mitochondrial respiratory complex I (Ding et al., 2022). NDUFS2 is located in the Q module 27, while NDUFA10 resides in the hydrophobic protein ND2 module of complex I. Both are involved in the assembly of complex I. The enzymatic activity of complex I is a rate-limiting step in oxidative phosphorylation and is an important regulatory factor. When complex I activity is reduced, it blocks electron transfer in the respiratory chain, leading to an imbalance between electron input and output (Dunham-Snary et al., 2019; Hoefs et al., 2011). This imbalance stimulates ROS production, which can potentially trigger severe liver disease (Ding et al., 2022). The NDUFB8 subunit of NADH:ubiquinone oxidoreductase contributes to the assembly of mitochondrial respiratory complex I, with this process occurring in both the endoplasmic reticulum and mitochondria (Piekutowska-Abramczuk et al., 2018). The NDUFB9 subunit of NADH: ubiquinone oxidoreductase B9 is localized to the mitochondrial inner membrane and is responsible for dehydrogenating nicotinamide adenine dinucleotide (NADH) and transferring electrons to coenzyme Q (Wu et al., 2016). NDUFB9 plays a key role in the oxidative phosphorylation reaction and is involved in the electron transfer process of the mitochondrial respiratory chain. Studies have shown that during the lipogenesis process of NAFLD, NDUFB9 expression is significantly upregulated, and silencing NDUFB9 (with an 83% silencing efficiency) inhibits lipogenesis (Zhu et al., 2022). As part of complex I, NDUFB8 and NDUFB9 may serve as biomarkers reflecting mitochondrial status, playing a role in the diagnosis and monitoring of NAFLD (Piekutowska-Abramczuk et al., 2018; Li et al., 2015).

In addition, it is important to note the differences among commonly used dietary NAFLD models (Ning et al., 2025; Tsuchida et al., 2018). As summarized in the tables of this review, HFD and WD models primarily mimic metabolic syndrome–associated NAFLD, characterized by pronounced obesity and insulin resistance, with only mild impairment of mitochondrial complexes and potential compensatory enhancement. In contrast, CDAHFD and MCD models rapidly induce severe steatohepatitis and fibrosis, though their metabolic contexts differ (MCD does not cause obesity or insulin resistance) (Yang, 2024; Suzuki-Kemuriyama et al., 2021). Mitochondrial complexes are markedly impaired in these models, with decreased ATP production and increased ROS generation. The differential effects of these models on complexes I–V help explain the variability in mitochondrial activity reported across studies.

Changes in mitochondrial complex activity affect the onset and progression of NAFLD, and the factors influencing complex I function, as described above, offer insights into the prevention and treatment of NAFLD. Most current studies using Seahorse focus on the overall activity of mitochondrial complexes I and II, but there is limited research on how specific subunits and the interactions between subunits impact the function of mitochondrial complex I.

Mitochondrial complex II (Complex II), also known as succinate dehydrogenase (SDH), is an important functional complex in the mitochondrial electron transport chain (Guo et al., 2017). It connects two essential physiological metabolic processes, the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) (Sharma et al., 2020). Complex II is a transmembrane protein complex that plays a key role in cellular respiration. It is responsible for oxidizing succinate to fumarate and transferring electrons to ubiquinone (coenzyme Q), thus participating in the electron transport chain (Sharma et al., 2024). In mammals, the electron transport chain (ETC) complexes I, III, and IV contain 45, 11, and 13 subunits, respectively (Zhu et al., 2016), composed of proteins encoded by both the mitochondrial and nuclear genomes. In contrast, complex II (SDH) is composed of only four nuclear-encoded subunits: SDHA, SDHB, SDHC, and SDHD. The SDHA subunit contains covalently bound flavin adenine dinucleotide (FAD) and is responsible for removing electrons from succinate (Sharma et al., 2024). The SDHB subunit contains three iron-sulfur clusters, which mediate the transfer of electrons to the ubiquinone binding site (Q site) on ubiquinone. The SDHC and SDHD subunits are embedded in the mitochondrial inner membrane, anchoring the entire complex to the membrane (Sharma et al., 2020; Wang et al., 2023).

Succinate accumulation exerts a dual role in inflammation (Myint et al., 2023): it functions both as a metabolic intermediate and as an immunological signaling molecule, promoting pro-inflammatory responses through HIF-1α stabilization, NLRP3 inflammasome activation, SUCNR1 signaling, and mitochondrial ROS production, particularly in M1 macrophages and acute inflammatory contexts (Huang et al., 2024; Chen et al., 2024). Moreover, when succinate feeds electrons into the mitochondrial electron transport chain via succinate dehydrogenase (SDH), reverse electron transport (RET) can occur to Complex I under high membrane potential, further enhancing ROS generation (Schönfeld et al., 2010; Schönfeld and Wojtczak, 2007). These ROS not only induce oxidative stress but also amplify the secretion of pro-inflammatory cytokines such as IL-1β, TNF-α, and IL-6 via the aforementioned pathways, driving metabolic reprogramming and exacerbating inflammatory responses (Keiran et al., 2019; Gobelli et al., 2023).

The primary function of mitochondrial complex II is to catalyze the oxidation of succinate to fumarate, while directly transferring electrons to the ubiquinone pool (Goetz et al., 2023). This process is crucial for maintaining the integrity of the mitochondrial electron transport chain and cellular energy metabolism. Additionally, complex II is involved in regulating the cell’s metabolic and respiratory adaptation to various intrinsic and extrinsic stimuli, influencing mitochondrial function and cellular energy metabolism (Iverson et al., 2023). Mitochondrial complex II plays a central role in cellular energy metabolism and mitochondrial function, and abnormalities in its structure and function are associated with various diseases (Hadrava Vanova et al., 2020). SDH creates the unique direct functional link between the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS), and it is ideally positioned to coordinate flux through both pathways. Unlike complex I, the transfer of electrons from SDH to the ubiquinone pool is not limited by the high proton gradient (Yu et al., 2023).

The table below summarizes the specific link between mitochondrial complex II dysfunction and NAFLD (Table 2). Researchers have found that in NAFLD, the expression of the SDHA and SDHB genes is reduced, and SDH activity is decreased, while protein expression of SDH subunits is increased (Staňková et al., 2021). However, other studies observed that Western diet (WD) did not significantly affect the gene and protein expression of the SDHA subunit (Staňková et al., 2020). This discrepancy may be related to factors involved in the assembly of subunits into functional complexes, which mediate the stability of intermediates during the assembly of individual subunits and the multimeric complex (Van Vranken et al., 2015). In NAFLD mice, mitochondrial damage resulting from oxidative stress and/or impaired mitochondrial autophagy mechanisms can lead to blockage in the later stages of the TCA cycle and failure in energy conversion (Liu et al., 2020). This may be due to mitochondrial dysfunction, leading to inhibition of mitochondrial SDHA enzyme levels and/or activity, causing succinate accumulation in hepatocytes. These studies highlight the close relationship between mitochondrial complex II and NAFLD. Whether considering the overall complex II or its subunits SDHA and SDHB, they can serve as targets for monitoring and treatment of NAFLD. Both the entire mitochondrial complex II and the SDHA and SDHB subunits have been extensively studied as potential therapeutic targets. Most studies (Staňková et al., 2021; Staňková et al., 2020; Nilsson et al., 2023) show that in NAFLD models, complex II activity is reduced, oxidative phosphorylation levels are decreased, and ketogenesis is enhanced. A few studies (Sangineto et al., 2021; Aghayev et al., 2023), however, show increased complex II activity and enhanced fatty acid oxidation. This discrepancy warrants further in-depth investigation.

Current studies indicate that mitochondrial respiratory chain complex function in high-fat–related NAFLD models can be either enhanced or impaired, resulting in inconsistencies among reported findings (Wang et al., 2025; Tsuchida et al., 2018; Yang, 2024). Overall, this variability can be attributed to several factors. First, differences in modeling strategies play a decisive role: commonly used dietary models—including high-fat diet (HFD), Western diet (WD), choline-deficient high-fat diet (CDAHFD), and methionine- and choline-deficient diet (MCD)—differ markedly in metabolic background and disease severity. HFD and WD primarily recapitulate metabolic syndrome–associated NAFLD, with relatively mild mitochondrial damage and, in some cases, compensatory functional enhancement, whereas CDAHFD and MCD rapidly induce severe steatohepatitis and fibrosis, causing pronounced impairment of complexes I–V (Suzuki-Kemuriyama et al., 2021). Second, discrepancies between in vivo and in vitro models are significant, as hepatocyte-based in vitro systems cannot fully replicate the complex hepatic microenvironment or intercellular mitochondrial interactions present in vivo. Third, NAFLD is a dynamic disease, and mitochondrial function may shift from early compensatory enhancement to late-stage decline; however, most studies assess mitochondria at a single time point. Fourth, species-specific differences in mitochondrial regulation and metabolic adaptation further limit the direct comparability of findings across studies (Guo et al., 2022).

Complex III plays a central role in the electron transport chain, where it is primarily responsible for oxidizing ubiquinol generated by complexes I and II (Zheng et al., 2024). This oxidation process is accompanied by the pumping of two protons into the mitochondrial intermembrane space, while electrons are transferred to cytochrome c in the intermembrane space, facilitating electron transfer in the electron transport chain and the reduction of cytochrome c (Letts and Sazanov, 2017). The core structure of complex III is composed of three key protein subunits: cytochrome b, which contains two heme groups, b562/bH and b566/bL. Cytochrome b is a highly hydrophobic transmembrane protein, and its precise role in the electron transport chain is not yet fully understood (Flores-Mireles et al., 2023). Cytochrome c1 contains a heme c1 group. It consists of a larger hydrophilic region and a smaller hydrophobic region. The hydrophilic region carries the heme cofactor and extends into the aqueous phase outside the membrane, where it binds with cytochrome c (Guo et al., 2017). Additionally, there is the iron-sulfur protein (FeS protein), which participates in various redox reactions. Structural and functional abnormalities in FeS proteins may lead to metabolic disorders or dysfunctions. The main function of FeS proteins is to participate in redox reactions, as the iron ions in their cofactors can switch between Fe2+ and Fe3+ states (Lill and Freibert, 2020).

Complex III plays a central role in the mitochondrial electron transport chain through the Q cycle, which mediates electron transfer from ubiquinol to cytochrome c (Murphy, 2009). During this process, the Qo site of Complex III is a major site of mitochondrial ROS generation, particularly when electron flow is impaired or the CoQ pool is over-reduced (Hernansanz-Agustín and Enríquez, 2021). In the context of NAFLD, dysfunction at Complex III can lead to excessive ROS production, contributing to oxidative stress, lipid peroxidation, and inflammatory signaling, thereby exacerbating hepatocellular injury and disease progression. Understanding the Q cycle and ROS generation at the Qo site is therefore critical for elucidating the mechanistic role of Complex III in NAFLD (Moreno-Loshuertos and Enríquez, 2016). UQCRC2, a subunit of mitochondrial respiratory chain complex III, has become a target of several studies (Fernandez-Vizarra and Zeviani, 2018). Among the five articles (García-Berumen et al., 2022; Sarr et al., 2019; Lee et al., 2018; Abdelmegeed et al., 2016; Ronis et al., 2013) related to NAFLD (see Table 3), four report a decrease in the activity of complex III as a whole or its subunits, leading to mitochondrial respiratory dysfunction, followed by increased ROS production and oxidative stress. Another study (Ronis et al., 2013), however, observed an enhancement in complex III activity, which subsequently improved mitochondrial respiration. Given the significant differences in these findings, it is worth further investigating the underlying mechanisms and potential impacts.

It is noteworthy that the tables in this review incorporate both cellular and animal models of NAFLD, necessitating a clear distinction between hepatocyte mitochondria and whole-liver mitochondria (Brenner et al., 2013). Hepatocytes, as the predominant parenchymal cell type in the liver, possess a high mitochondrial density to support their extensive metabolic demands, including glucose and lipid metabolism, protein biosynthesis, and detoxification. By contrast, assessments of whole-liver mitochondria unavoidably encompass mitochondrial populations derived from non-parenchymal cells, such as hepatic stellate cells, Kupffer cells, and liver sinusoidal endothelial cells, which differ from hepatocytes in mitochondrial abundance, morphology, and metabolic activity (Zhao et al., 2023). Accordingly, mitochondrial function measured in hepatocytes may not directly correspond to the averaged functional state of whole-liver mitochondria. This distinction is particularly critical in NAFLD models, as hepatocyte-specific mitochondrial alterations are key drivers of disease-associated metabolic dysfunction (Hunt et al., 2019).

Mitochondrial complex IV, also known as cytochrome c oxidase, is the final enzyme in the mitochondrial respiratory chain (Kadenbach, 2021; Dietrich et al., 2024). Its primary function is to transfer electrons from cytochrome c to molecular oxygen (O2), generating water (H2O) (Brzezinski et al., 2021). Complex IV consists of 13 subunits. Among these, subunits I, II, and III are encoded by mitochondrial DNA (mtDNA) and form the catalytic center of complex IV. These three subunits are essential for electron transfer and proton pumping in the mitochondrial respiratory chain. The remaining 10 subunits are encoded by nuclear genes, synthesized in the cytoplasm, and then imported into the mitochondria (Brischigliaro and Zeviani, 2021). Mitochondrial complex V, also known as ATP synthase, is the last complex in the mitochondrial respiratory chain (Fernandez-Vizarra and Zeviani, 2021). Complex V is composed of 16 subunits, with these subunits encoded by both mitochondrial and nuclear genes. It consists of two main subunits: F1 and F0. The F1 subunit is located in the mitochondrial matrix, while the F0 subunit is embedded in the mitochondrial inner membrane (Singh, 2023). The main function of complex V is to catalyze ATP synthesis by utilizing the proton electrochemical gradient generated by the respiratory chain. It can also hydrolyze ATP through the reverse process (Del Dotto et al., 2024). Complex V synthesizes ATP from ADP, inorganic phosphate (Pi), and Mg2+, providing energy for the cell. In this process, complexes I, III, and IV transfer electrons while pumping protons from the mitochondrial matrix across the mitochondrial inner membrane into the intermembrane space, establishing the electrochemical gradient (Althaher and Alwahsh, 2023).

Electron leakage arises from reduced efficiency of terminal electron transfer at complex IV, leading to passive upstream electron escape and ROS generation, whereas electron overload results from functional constraints of complex V that induce a high membrane potential state, in which electron input exceeds ATP utilization capacity and triggers RET-dependent ROS bursts (Tabata Fukushima et al., 2024). Complex IV is the terminal electron acceptor of the mitochondrial respiratory chain, catalyzing the transfer of electrons from cytochrome c to molecular oxygen to form water, while simultaneously driving proton translocation across the membrane to maintain the membrane potential. In NAFLD, lipid overload can impair the assembly and stability of cytochrome c oxidase (COX), resulting in decreased complex activity. Lipid accumulation alters the lipid environment of the mitochondrial inner membrane, disrupting proper folding of COX subunits and supercomplex formation, thereby reducing electron transfer efficiency and ATP production while increasing ROS generation, ultimately promoting oxidative stress and hepatocellular injury (Gu et al., 2016; Durand et al., 2021). These protons are ultimately utilized by complex V to generate ATP. Therefore, complex V is a key enzyme in mitochondrial oxidative phosphorylation and chloroplast photophosphorylation for ATP synthesis (Cogliati et al., 2021). Complex V is responsible for synthesizing ATP using the proton motive force; however, under pathological conditions it may also operate in reverse, hydrolyzing ATP to maintain the membrane potential (Salewskij et al., 2020). In NAFLD models, mitochondrial damage and alterations in the proton gradient may increase ATP hydrolysis activity, thereby reducing energy efficiency and exacerbating metabolic stress. Moreover, impairment of complex V function can feed back to influence electron flow in upstream complexes, enhancing ROS production and further promoting lipid peroxidation and inflammatory responses (Nesci et al., 2015).

Most studies (Meng et al., 2024; Xu et al., 2022; Chimienti et al., 2021; Zhang X. et al., 2021; Zhao et al., 2018; Liu et al., 2015) focus on the overall complex IV (Table 4), observing a decrease in its expression and activity in NAFLD, which leads to weakened mitochondrial respiratory function, impaired mitochondrial function, and increased oxidative stress levels. Some studies have shown that in obese subjects, the activity of mitochondrial ETS complex IV is higher, with enhanced mitochondrial oxidative metabolism, and the increased oxidative metabolism in peripheral blood mononuclear cells (PBMCs) is associated with hepatic steatosis in obese young individuals (Shirakawa et al., 2023). Although the direct relationship between complex IV and NAFLD is not mentioned, hepatic steatosis is closely related to NAFLD, and the interaction between obesity and complex IV warrants further attention. Compared to visceral adipose tissue (VAT) in non-alcoholic fatty liver participants, individuals with non-alcoholic steatohepatitis (NASH) exhibit lower expression of oxidative phosphorylation complex IV (Pafili et al., 2022). In a high-fat diet-induced C57BL/6J mouse model of NAFLD, compared to normal mice, the expression of mitochondrial cytochrome c oxidase subunit IV (COX IV) in brown adipose tissue was increased, while its expression in white adipose tissue was decreased. This differential expression of complex IV subunits in adipose tissues deserves further investigation (Yoneshiro et al., 2018).

Complex IV as a whole has been extensively studied, and most results show decreased complex IV activity in NAFLD models, leading to mitochondrial dysfunction, lipid accumulation, and increased ROS levels. One study targeting cytochrome c oxidase subunit IV isoform 1 (COX4I1) showed that increased COX4I1 activity leads to higher mitochondrial density and mass. Studies on complex V are relatively fewer (Table 4). One article (Murphy, 2009) showed that in the NAFLD model, the expression of subunit ATPA was reduced, and complex V activity decreased, triggering mitochondrial dysfunction. Overall, both complex IV and V likely undergo reduced activity and mitochondrial dysfunction under NAFLD conditions. Further research on complex V would enrich the understanding of the interaction between mitochondrial complexes and NAFLD, providing a more comprehensive reference for future studies.

PGC-1α is a key transcriptional coactivator of mitochondrial biogenesis and may promote the synthesis and assembly of respiratory chain complexes I–V by coordinating the expression of nuclear- and mitochondrial-encoded genes (Sukhorukov et al., 2024; Shi et al., 2024; Lou et al., 2024). Most existing studies are correlative, and the precise mechanisms by which PGC-1α and related factors specifically regulate the function and stability of complexes I–V remain to be elucidated. Studies have shown that Sanhuang decoction protects against obesity/diabetes-induced NAFLD in obesity and galr1 gene knockout mouse models by upregulating the PGC-1α/PEPCK signaling pathway (Fang et al., 2020). Further research has explored how Fenofibrate, through enhancing the PPARα/PGC-1α signaling pathway, promotes mitochondrial β-oxidation, enhances mitochondrial biogenesis and ATP production, and improves mitochondrial function to alleviate non-alcoholic fatty liver disease (NAFLD) (Wang et al., 2024). Under high-fat conditions, SIRT1 affects mitochondrial physiology and lipid autophagy through the PGC-1α pathway, thereby regulating various cellular characteristics (Jiang et al., 2021).

AMPK is an energy-sensing kinase that is activated under conditions of cellular energy deficiency, such as a decreased ATP/AMP ratio. Most existing studies are correlative and suggest that AMPK may indirectly enhance the expression and assembly of respiratory chain complexes I–V by phosphorylating PGC-1α or its upstream regulators (Jiang, 2024). AMPK has been shown to induce mitochondrial fission by directly phosphorylating MFF (mitochondrial fission factor) during energy stress, and regulate mitochondrial autophagy through DRP1 (Cheng et al., 2024). After sustained energy stress, AMPK promotes mitochondrial biogenesis through transcriptional activation via PGC-1α, TFEB, or TFE3 (Garcia and Shaw, 2017). Dysfunction of respiratory chain complexes directly leads to an imbalance in mitochondrial fission, fusion, and selective mitophagy by altering mitochondrial energetic and redox states (Lacombe and Scorrano, 2024). Damage to complexes I–V restricts electron transfer, causes a decline or instability in membrane potential, and increases ROS production, thereby activating mitochondrial quality-sensing pathways centered on PINK1–Parkin to label and eliminate mitochondria harboring defective complexes; mitochondrial fission facilitates the segregation of damaged components, whereas fusion can partially buffer mild functional defects (Li et al., 2023; R et al., 2017). In contrast, when mitophagy is impaired, dysfunctional mitochondria cannot be efficiently removed, leading to the accumulation of defective respiratory chain complexes, exacerbated ROS generation, energy deficiency, and further amplification of dysfunction and pathological signaling (Shadel and Horvath, 2015). Studies have shown that royal jelly proteins (MRJPs) activate the AMPK/SIRT3 pathway in vitro, upregulating the expression of cytochrome c oxidase subunit IV, promoting energy metabolism and mitochondrial biogenesis, and alleviating NAFLD (Zhang et al., 2021a).

The mechanisms of the mitochondrial electron transport chain and mitochondrial dysfunction have been extensively documented in the literature (Vercellino and Sazanov, 2022). This review presents a schematic diagram that highlights the interactions between mitochondrial complexes and their effects on cellular energy production and overall health, while clearly illustrating the link between mitochondrial dysfunction and various diseases (See Figure 1). The arrangement of mitochondrial respiratory chain complexes into higher-order aggregates (supercomplexes) reduces ROS production by maintaining a high rate of electron transfer (Maranzana et al., 2013). In a cholesterol-fed NAFLD mouse model, the cholesterol load in vivo reduced the levels of Complex III and the assembly of respiratory supercomplexes (I1+III2+IV and I1+III2), leading to oxidative stress and liver damage (Solsona-Vilarrasa et al., 2019). The function of supercomplexes remains a topic of debate, but these superstructures may be related to the hypothesis of substrate oxidation efficiency and reduced ROS production in mitochondrial electron transport, making them potential therapeutic targets for NAFLD (Ramírez-Camacho et al., 2020). Most existing studies are primarily correlative analyses (Acin-Perez and Enriquez, 2014). In cardiac and renal mitochondria, ATP production primarily depends on long-chain fatty acid β-oxidation. Respiratory chain supercomplexes play a central role (Panov, 2024; Blaza et al., 2014): two large supercomplexes contain complex I, a dimer of complex III, and two dimers of complex IV, while a third smaller supercomplex contains a dimer of complex III and two dimers of complex IV. Fatty acid β-oxidation enzymes are physically associated with these supercomplexes, enabling efficient electron transfer. The generated QH₂ can reverse electron flow through complex II and promote succinate formation, further enhancing β-oxidation. Oxidation of QH₂ by the smaller supercomplex increases the NADH/NAD⁺ ratio and mitochondrial energy level, while transhydrogenase regulates NADP(H), ensuring ATP supply and energy homeostasis in the heart.

Mitochondrial Flashes are quantized signals within mitochondria that involve multiple changes, such as bursts of mitochondrial reactive oxygen species (ROS), transient alkalization of the matrix, and a temporary decline in membrane potential, occurring over a time scale of several tens of seconds (Feng et al., 2017). This phenomenon represents a novel form of fundamental mitochondrial activity, reflecting highly conserved mitochondrial functions. Mitochondrial Flashes are widely observed across multiple species and various cell types, manifesting whenever functional mitochondria are present (Hou et al., 2014).

Mitochondrial dysfunction plays a crucial role in the pathogenesis of NAFLD, contributing to oxidative stress, lipid accumulation, and hepatocyte apoptosis. Although Mitochondrial Flashes are indicative of mitochondrial activity, their direct implications in mitochondrial dysfunction, particularly in NAFLD, remain to be fully elucidated (Wang et al., 2012). The burst of superoxide anions associated with Mitochondrial Flashes could exacerbate oxidative stress, thereby influencing the progression of NAFLD. Additionally, these flashes may indicate an abnormal state of mitochondrial energy metabolism, linking them to metabolic dysfunction observed in NAFLD. Despite these associations, further research is needed to fully understand the specific relationship between Mitochondrial Flashes and NAFLD. Therefore, closely integrating mitochondrial flashes and advanced imaging technologies with the pathological context of NAFLD can enhance the biological interpretability of these emerging research areas.