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Article

The Antioxidant Quercetin Affects Mitochondrial Function and Inhibits the Differentiation of Human Preadipocytes

by
Agnieszka Dziewońska
,
Anna Gruca
,
Anna Polus
,
Bogdan Solnica
and
Joanna Góralska
*
Department of Clinical Biochemistry, Jagiellonian University Medical College, Skawinska St. 8, 31-066 Krakow, Poland
*
Author to whom correspondence should be addressed.
Molecules 2026, 31(4), 725; https://doi.org/10.3390/molecules31040725
Submission received: 9 January 2026 / Revised: 3 February 2026 / Accepted: 13 February 2026 / Published: 20 February 2026
(This article belongs to the Special Issue Exploring the Natural Antioxidants in Foods)
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Abstract

Obesity is associated with numerous pathological processes in the body, including inflammation, oxidative stress, and consequently, mitochondrial dysfunction. In recent years, research in anti-obesity therapy has also focused on the function of adipocytes and the inhibition of adipogenesis. In this study, we investigated the effect of the well-known flavonoid quercetin on mitochondrial function, apoptosis and differentiation of human preadipocytes. The Chub-S7 cell line model was used in the in vitro studies. Mitochondrial function was measured by oxygen consumption rates, intracellular ATP content, mitochondrial membrane potential, apoptosis assay (Annexin-5, caspase-9 activity), and ROS generation. Chub-S7 cell differentiation was assessed by Oil Red O staining. The results showed that the quercetin inhibited differentiation of human Chub-S7 preadipocytes and reduced fat accumulation in lipid droplets. Additionally, quercetin influenced mitochondrial biogenesis and mitochondrial uncoupling by changes in mitochondrial respiratory states and also increased mitochondrial membrane potential. Quercetin decreased routine respiration, R/E and netROUTINE control ratio. Our results demonstrate that quercetin is a dietary component that may modulate mitochondrial bioenergetics and inhibit adipogenesis. If these results were confirmed in in vivo studies, quercetin could be considered a factor used to prevent obesity.

Graphical Abstract

1. Introduction

Obesity is a global health problem regardless of gender and age of patients [1]. Recent studies indicate that the number of people who are obese now exceeds 2 billion, representing approximately 30% of the world’s population [2]. With the growing pandemic of obesity and its associated complications, there is increasing interest in the development, function, and manipulation of adipose tissue. It has been proven that excess body fat increases the risk of insulin resistance, type 2 diabetes, dyslipidemia, metabolic syndrome, hypertension, coronary artery ease and stroke [3,4]. Adipose tissues serving both as an endocrine organ and energy storage are crucial for systemic metabolic homeostasis. This tissue is composed mainly of adipocytes, which may change their size and number in response to nutrients and metabolic status. In the process of adipogenesis, adipocyte precursor cells—preadipocytes—proliferate and differentiate into mature adipocytes [5]. On the other hand, adipocytes can undergo cell death through necrosis or apoptosis pathways. It is estimated that 8% of human subcutaneous adipocytes undergo turnover each year to maintain a given total cell number [6]. Recently, in the context of searching for new tools to treat obesity, intensive research on the inhibition of the adipogenesis process and the dedifferentiation of adipocytes has been conducted [7].
The development of obesity and adipose tissue dysfunction may also be associated with energy imbalance and mitochondrial dysfunction, which contributes to the pathogenesis of many metabolic disorders. Tissues involved in nutrient metabolism including adipose tissue and liver are particularly affected, leading to inflammatory and oxidative damage [8,9]. Mitochondria in adipocytes play key roles in metabolism. Excess nutrients supply, typically occurring in obesity and metabolic syndrome, can overwhelm the Krebs cycle and the mitochondrial respiratory chain, leading to a higher ROS formation, and consequently oxidative stress and inflammatory response [10]. Moreover, excessive ROS production overloads antioxidant defense systems [11]. It has been shown that oxidative stress in adipocytes plays a significant role in the pathogenesis of metabolic syndrome, insulin resistance and type 2 diabetes [12]. Furthermore, mitochondrial biogenesis and activity increase dramatically during adipocyte differentiation. Adipocyte differentiation involves changes in the abundance, morphology and organization of mitochondria, and abnormalities of these processes disrupt the balance between energy storage and expenditure [13,14]. TG accumulation in preadipocytes in response to mitochondrial activity inhibition involves both a reduction in fatty acid oxidation and glucose transporter 4 (GLUT4) translocation to the plasma membrane [15].
Synthetic anti-obesity drugs are effective, but not widely available due to cost as well as the fact that they can be associated with various side effects [16,17]. Therefore, research on the anti-obesity effects of various natural bioactive compounds, such as quercetin, is needed.
Quercetin (3,5,7,3′,4′-pentahydroxyflavone) is one of the most common bioactive flavonoids present in fruits and vegetables, such as onions, spinach, apples, cranberry and tea [18]. Quercetin is known not only for its antioxidant properties [19], but also its anti-inflammatory [20], antibacterial [21], antifungal [22] and antiviral [23] properties. It was reported that this flavonoid may be helpful in lowering blood sugar and increasing insulin sensitivity [24]; thus, it is a desirable component of the diet of diabetic patients. Quercetin has also been found to have some anti-cancer properties, promoting loss of cell viability, apoptosis and autophagy by modulating various signaling pathways [25].
Quercetin is also being explored as an effective therapy for obesity, though its mechanism remains understudied. Recent studies have demonstrated that a polyphenol-rich diet has anti-obesity effects [26,27]. Polyphenols, including quercetin, are considered to promote adipocyte browning, increase thermogenesis, and regulate adipocyte differentiation [26]. It has been suggested that quercetin may also play a role in the treatment of metabolic disorders through different mechanisms such as decreasing leptin, increasing adiponectin, antioxidant activity, reduction in insulin resistance, the elevation of insulin level and blocking of calcium channel [28]. Furthermore, recent studies indicate that quercetin may reduce adipogenesis via apoptosis [29] and reduce the expression of JNK in 3T3-L1 cells [30]. Soe et al. [31] reported that quercetin inhibited MAPK and inflammatory cytokines in 3T3-L1. However, it should be emphasized that most studies have been conducted using mouse cell lines. In our study we used the Chub-S7 human preadipocyte cell line. Moreover, due to the involvement of energy imbalance and mitochondrial dysfunction in the development of obesity, in our work we decided to investigate the effect of quercetin on various mitochondrial functions and the differentiation process of human preadipocytes.

2. Results

2.1. Oxygen Consumption

2.1.1. Routine Respiration

Routine respiration reflects the aerobic metabolic activity under routine culture conditions, without the addition of inhibitors or uncouplers of the respiratory chain.
The analysis of our results indicates that quercetin at each tested concentration significantly inhibits cellular respiration of preadipocytes. Moreover, this process is concentration-dependent. With the increase in quercetin concentration, oxygen consumption decreased (Figure 1).

2.1.2. Respiratory Rates

Based on measurements of different respiratory states (ROUTINE, LEAK, ETS, ROX), we calculated selected respiratory rates: R/E, L/E and netR/E. ROUTINE control ratio (R/E) reflects the activation state of cellular respiration in accordance with the routine ATP requirement of cells and degree of coupling. R/E measures how close ROUTINE activity of cells approaches the upper limit of ETS capacity at optimum uncoupler concentration. LEAK control ratio (L/E) is measured as oligomycin-inhibited respiration in intact cells. L/E shows how close LEAK respiration approaches the upper limit of non-coupler respiration. NetROUTINE control ratio is the fraction of ETS capacity directly utilized to drive phosphorylation of ADP to ATP [32,33]. All fluxes used for calculation of the described control ratios were corrected for non-mitochondrial respiration, measured after inhibition of the ETS.
No significant effect of lower doses (10 μM, 30 µM, 50 µM) of quercetin on respiratory rate was observed (Figure 2a–c). The highest concentrations of quercetin (70 μM and 100 μM) decreased the netR/E ratio (Figure 2d,e). A similar trend was also observed at the 50 μM concentration (p = 0.08) (Figure 2c). After incubation of Chub-S7 cells with the highest concentration of antioxidants, the R/E ratio also decreased, which indicates a decrease in routine respiration in relation to the ETS condition (Figure 2e).

2.2. Intracellular ATP Content

The mean intracellular ATP level in untreated, Chub-S7 control cells was 65.4 ± 7.53 nmol ATP/mg protein. No significant changes in the intracellular ATP content were observed after incubation of the cells with any of the concentrations of quercetin used (Figure 3).

2.3. Mitochondrial Membrane Potential

Mitochondrial membrane potential was measured using the JC-1 fluorescent dye in flow cytometry. Analysis of emitted fluorescence indicated that with increasing quercetin concentration (from 50 μM), the mitochondrial membrane potential increased (Figure 4). On the other hand, the lowest antioxidant concentrations used (<30 μM) did not affect ∆Ψm changes.

2.4. Apoptosis Assay

2.4.1. Caspase-9 Activity

None of the quercetin concentrations tested showed a significant effect on caspase-9 activity (Figure 5a). For the comparison, hydrogen peroxide (0.2 mM H2O2) was used as positive control, which increased caspase-9 activity in Chub-S7 cells after 24 h of incubation.

2.4.2. Annexin 5

Different stages of cell death were determined using the commercially available FITC Annexin V/Dead Cell Apoptosis Kit with FITC Annexin V and PI and detected by flow cytometry. Analysis of the obtained results showed that most of the detected cells, after incubation with quercetin (10 µM, 30 µM, 50 µM, 70 µM,100 µM; 24 h) were in the early phase of apoptosis (Table 1). The percentage of these cells increased non-significantly with increasing quercetin concentration, from 2.97% (±1.00) in the control to 4.03% (±2.92) at the highest antioxidant concentration. After incubation with 100 μM quercetin, the number of cells in the late phase of apoptosis increased significantly [from 1.57% (±0.29) in the control to 2.40% (±0.14)] (Table 1 and Supplementary Material S1). However, no significant effect of quercetin was observed on the percentage of cells undergoing necrosis (Table 1; Figure 5b).

2.5. ROS Generation

To analyze the effect of quercetin on the production of reactive oxygen species in preadipocytes, the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) dye was used. To confirm the antioxidant and protective effects of quercetin, the experiments were repeated with the use of hydrogen peroxide (H2O2), which, when added to the cells, increased the amount of ROS by over 100% (Figure 6c). Although the lowest concentration of quercetin used (10 µM) did not show any protective effect against free radicals, the remaining concentrations of quercetin (30 µM, 50 µM, 70 µM, 100 µM) significantly inhibited ROS generation in Chub-S7 cells (Figure 6a,b). All selected concentrations of quercetin also significantly reduced the amount of ROS in preadipocyte cells exposed to hydrogen peroxide (Figure 6a).

2.6. Cell Differentiation

The changes in lipid droplet accumulation in Chub-S7 cells were observed with light microscopy. The first lipid droplets could be observed after 7 days of differentiation. After 21 days of the differentiation process, the cells were stained with Oil Red O. Analysis of the results showed that quercetin significantly inhibited the accumulation of lipid droplets by Chub-S7 cells. This process was mainly dependent on the length of incubation time and quercetin concentration. Even short-term incubation (24 h) of Chub-S7 cells with quercetin significantly reduced the number of lipid droplets formed (Figure 7a), especially in the cases of concentrations of 10 and 70 μM. A similar tendency towards a reduction in the number of lipid droplets was observed at concentrations of 30 µM and 50 µM; however, these results are not statistically significant (p = 0.06 and p = 0.07). Extending the incubation time to 48 h additionally enhanced the inhibitory effect of quercetin (Figure 7b). Inhibition of the adipogenesis process in quercetin-treated cells was most visible after the longest incubation time (21 days). This process was strongly dependent on the quercetin concentration (Figure 7c). However, in this case the highest concentrations (50 μM, 70 μM and 100 μM) caused apoptosis of Chub-S7 cells.

3. Discussion

Quercetin is a well-known antioxidant, but its anti-obesity properties require further research [19,34]. In this study, special attention was paid to the effects of quercetin on adipocyte differentiation and apoptosis as well as mitochondrial function. Adipocytes, by regulating both triglyceride storage and the release of free fatty acids, are involved in maintaining lipid homeostasis and energy balance [35]. Previous studies have reported that human obesity is associated with mitochondrial dysfunction, mostly attributed to an imbalance between fatty acid supply and oxidation [36].
There are many reports on the effects of flavonoids on mitochondrial function, some of which indicate a protective effect of quercetin [37]. However, controversy surrounding this topic persists, as flavonoids may have both protective and damage-inducing effects. Therefore, this study aimed to investigate whether quercetin influences mitochondrial function in human adipocyte Chub-S7 cells, with special examination of mitochondrial respiration during selected respiratory states—LEAK, OXPHOS and ETS—and Coupling Control Ratios. The relative level of ROUTINE activity used to drive phosphorylation of ADP to ATP was significantly decreased after incubation with quercetin, even at the lowest concentration (10 µM). The highest concentration (100 μM) of quercetin additionally caused a decrease in R/E (ROUTINE control ratio), which reflects the state of activation of cellular respiration according to routine ATP demand and degree of coupling [32]. R/E decrease may indicate partial uncoupling and limitation of oxidative capacity by substrate supply or defects of the ETS [33]. Moreover, incubation of Chub-S7 cells with higher concentrations of quercetin (70 µM and 100 µM) decreased the netROUTINE control ratio, which additionally confirms full or complete cell metabolic arrest. The observed effect was accompanied by an increase in mitochondrial membrane potential. Despite the reduction in oxygen consumption and changes in mitochondrial membrane potential, quercetin did not affect ATP production. Reduced mitochondrial respiration in the presence of normal ATP levels may indicate increased mitochondrial uncoupling, reduced ATP demand, or changes in metabolic status. The mitochondrial changes toward mild uncoupling observed in our study may increase energy expenditure without negatively impacting ATP synthesis. Ortega and Garcia [38] reported that quercetin diminished the respiratory control and uncoupled oxidative phosphorylation, depending on the concentration. Some researchers explain that flavonoids may act similarly to classical uncouplers of oxidative phosphorylation due to their hydrophobic character [39,40]. Similar results were obtained by Dorta et al. [41] in the experiment on isolated rat liver mitochondria, showing that quercetin inhibited the respiratory chain of mitochondria.
Our results showed that the higher the quercetin concentration, the lower the accumulation of lipid droplets in Chub-S7 preadipocytes during differentiation towards mature adipocytes. The inhibition of preadipocyte differentiation in the presence of quercetin may be related to reduced cellular demand for ATP, which may explain decreased oxygen consumption and the low percentage of active respiration necessary for ATP synthesis. In our study, we observed a change in mitochondrial activity, which was associated with a reduction in the differentiation of Chub-S7 preadipocytes. Mitochondrial biogenesis is an integral part of adipocyte differentiation because mitochondria play a crucial role in the metabolism of intracellular lipids. Ducluzeau P-H. et al. [42] demonstrated that during adipogenesis of 3T3-L1 cells, mitochondria develop uncoupled metabolism and undergo fragmentation of their network, resulting in punctuate structures surrounding lipid droplets. They suggested that the coordinated increase in fatty acid β-oxidation gene activity and ROS production indicates a specialized metabolism, allowing lipogenesis and droplet expansion. Impairment of mitochondrial activity affects lipid-metabolizing tissues, and mild mitochondrial uncoupling is being investigated as a possible strategy to combat obesity and related diseases [43,44]. The mitochondrial changes observed in our studies, towards mild uncoupling and energy dissipation, may constitute a limiting impulse for preadipocyte differentiation. An increase in mitochondrial membrane potential after incubation with quercetin indicates stabilization of the mitochondrial membrane and may be related to the protection of membrane lipids by quercetin against oxidative changes. Our data showed that this flavonoid inhibited the production of reactive oxygen species in Chub-S7, also in the presence of hydrogen peroxide, which induces oxidative stress. Wang et al. [45] reported that quercetin can relieve cell damage and apoptosis from H2O2-induced human keratinocyte injury by antioxidation and mitochondrial protection. Margina et al. [46] reported the ability of quercetin to stabilize cell membranes, which correlated with a reduction in lipid peroxidation. Polyphenols, including flavonoids, can modify plasma membrane structure and physical characteristics, such as fluidity and electrical properties. These effects can result in functional changes in the activity of membrane-associated enzymes, ligand–receptor interactions, ion and/or metabolite fluxes, and the modulation of signal transduction [47]. However, our observations indicate that the effect of quercetin on human adipocytes is dose- and time-dependent, as long-term exposure of Chub-S7 cells to high doses of quercetin may lead to their apoptosis. Chou et al. [48] suggested that quercetin may induce apoptosis through the mitochondrial pathway by activation of caspase-6, -8 and -9 in breast cancer MCF-7 cells. Moreover, they showed that quercetin may increase the AIF protein released from mitochondria to nuclei and the GADD153 protein translocation from endoplasmic reticulum to the nuclei. However, in the cited experiments, a higher concentration of quercetin (150 μM) was used. In our study we generally did not confirm the effect of quercetin on preadipocytes apoptosis, measured by Annexin V assay, except for an increased number of cells in the late phase of apoptosis after incubation with 100 μM quercetin. Moreover, no effects of quercetin on mitochondrial caspase-9 activity of Chub-S7 preadipocytes were observed. As the primary aim of our study was to investigate the effect of quercetin on the differentiation and functioning of human preadipocytes mitochondria, no extended studies on apoptosis were continued. Instead, we deepened the analysis and particularly emphasized the importance of changes in mitochondrial respiratory states.
Our results clearly show that quercetin inhibits the differentiation process of human preadipocytes Chub-S7. Previous research indicated that quercetin suppressed fat accumulation in mouse-derived preadipocyte 3T3-L1 cell lines and reduced expression of adipocyte differentiation-related C/EBPα, C/EBPβ, PPAR-γ, and aP2 genes [49]. Furthermore, they suggested that this process may be the result of inhibition of the angiogenic process associated with matrix metalloproteinases (MMPs). Results obtained by Song et al. [50] in the animal model confirmed that quercetin suppresses adipocyte differentiation and prevented body weight gain by inhibiting transcription factors related to adipogenesis and MMPs (MMP-2 and MMP-9) in C57BL/6J mice fed a high-fat diet. Furthermore, Kobori et al. [51] showed, in the same animal model, that chronic dietary intake of quercetin reduces liver fat accumulation and improves systemic parameters related to metabolic syndrome, probably through decreasing oxidative stress and reducing PPARα expression, in mice fed a Western diet. These findings suggest that quercetin can be considered a bioactive dietary component or supplement used to support weight loss and improve the metabolism of adipocyte mitochondria.
To summarize, the results of our study showed that quercetin inhibited adipogenesis in human preadipocytes Chub-S7 cells. Furthermore, quercetin influenced mitochondrial biogenesis, bioenergetics and uncoupling by changes in mitochondrial respiratory states. The relative level of ROUTINE activity used to drive phosphorylation of ADP (ROUTINE respiration) was significantly decreased after incubation with quercetin. The highest concentration of the flavonoid tested additionally caused a decrease in R/E and netROUTINE control ratio, which confirms mitochondrial uncoupling. Furthermore, an increase in mitochondrial membrane potential after incubation with quercetin indicates stabilization of the mitochondrial membrane. Our results also confirmed the strong antioxidant properties of quercetin. These results are the more important because only mitochondria can provide the key substrates and factors necessary to support the lipogenesis during adipogenesis [4]. Linking adipogenesis and mitochondria-related processes, including bioenergetics, oxidative stress, apoptosis, suggests that mitochondria play an important role in the differentiation and maturation of preadipocytes. However, the relationship between these two processes should be investigated in more detail and the results presented here should be considered preliminary, providing a basis for further studies. It would be particularly important to investigate changes in mitochondrial biogenesis during the process of adipogenesis.
Although the study yielded interesting results, it has some limitations. One of the shortcomings is that in our study we did not take into account the increased autoxidative rate of quercetin, which could have influenced the antioxidant properties of the flavonoid. Investigation of the radical-scavenging mechanism of flavones showed that their anionic forms are oxidized predominantly via the sequential proton-loss electron transfer (SPLET) mechanism, while the neutral species is oxidized via a hydrogen atom transfer (HAT)/proton-coupled electron transfer (PCET) mechanism. The SPLET mechanism was found to be orders of magnitude faster than the HAT/PCET mechanism for many of the flavones studied, with the rate enhancement being dependent on type of the flavonoid [52,53]. The chemistry of flavonoids is very complex and difficult to control. These challenging chemical properties include poor solubility, formation of complex protic equilibria, auto-oxidation, and redox cycling with other experimental components [52].
Another limitation is that the in vitro Chub-S7 model derived from preadipocytes of a single obese individual does not reflect biological variability. It would be worthwhile to repeat the study for example on progenitor stromal vascular fraction (SVF) cells from different donors, but this is difficult due to invasiveness and ethical considerations. Recently, many studies have been published on the effect of quercetin on adipogenesis, but most of them used mouse 3T3-L1 [49] or other mouse cell lines [35,54]. Therefore, the results of our research based on human preadipocytes are all the more important. Further research on quercetin, which acts in adipocytes not only as the antioxidant, may provide an alternative treatment strategy to combat obesity in the future. However, it should be emphasized that these are preliminary data, providing a basis for further studies, especially due to the chemistry of flavonoids, which is very complex and difficult to control. Furthermore, flavonoids, like quercetin, have poor bioavailability and are quickly metabolized in the enterocytes. Therefore, metabolites of quercetin, rather than quercetin itself, will act in a living organism, and the effects may be slightly different. Many studies show that in vitro studies with flavonoids are often poorly reproduced in vivo. However, the results of our study may certainly be helpful in research on the use of quercetin in anti-obesity therapy.

4. Materials and Methods

4.1. Cells Culture

Chub-S7 is an immortalized human preadipocytes line that is able to accumulate intracellular triglycerides and to express adipocyte markers. These transformed adipocyte precursors are derived from subcutaneous abdominal adipose tissue. The Chub-S7 cell line (Nestle Research Center, Lausanne, Switzerland) [55] were cultured in a mixture of DMEM/Ham’s F12 1:1 (v:v) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA). Quercetin (Sigma-Aldrich, St. Louis, MO, USA) was freshly dissolved in 1 M NaOH and then diluted in culture medium (100×) to a final concentration of 1 mM. Subsequent quercetin concentrations were also obtained by dilution in medium. The cells were incubated with five different concentrations of quercetin (10 µM, 30 µM, 50 µM, 70 µM and 100 µM) for 24 h. Control cells were incubated with an appropriate concentration of solvent.
None of the concentrations of quercetin selected for further analysis were toxic to Chub-S7 cells. Potential cytotoxic effect of quercetin on Chub-S7 cells was determined by the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega GmbH, Mannheim, Germany) following the manufacturer’s instructions. The assay measures the lactate dehydrogenase (LDH) activity in the cell culture. The concentration of quercetin was considered to be cytotoxic when the amount of LDH was increased by 20% when compared to the control. In the used experimental conditions, quercetin exerted a cytotoxic effect at the highest concentration of 200 µM (Supplementary Material S2).

4.2. Differentiation Procedure

The Chub-S7 cells were differentiated into mature adipocytes by incubating in a serum free DMEM/F12 medium, supplemented with 17 µM D-panthotenic acid (Sigma-Aldrich, St. Louis, MO, USA), 33 µM d-biotin (Sigma-Aldrich, St. Louis, MO, USA), 5 µg/mL transferrin (Sigma-Aldrich, St. Louis, MO, USA), 1 nM triiodothyronine (Sigma-Aldrich, St. Louis, MO, USA), 5 µg/mL insulin (Sigma-Aldrich, St. Louis, MO, USA), 500 µg/mL fetuin (Sigma-Aldrich, St. Louis, MO, USA), 5 ng/mL selenium (Sigma-Aldrich, St. Louis, MO, USA), 1 µM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) and 1 µM rosiglithazone (Cayman Chemical, Ann Arbor, MI, USA). For the first 48 h of differentiation, 500 µM IBMX (Sigma-Aldrich, St. Louis, MO, USA) was additionally added. The differentiation process lasted 21 days (according to protocol supplied by Nestle Research Center). Quercetin was added to the differentiation medium to obtain all five investigated concentrations on the first day of differentiation for 24 h, 48 h and 21 days.

4.3. Oxygen Consumption

Mitochondrial respiration was determined by high-resolution respirometry using an Oxygraph-2k (OROBOROS Instruments, Innsbruck, Austria) according to the coupling control protocol for intact cells. Mitochondrial oxygen consumption was monitored at 37 °C in a thermostatically controlled chamber. The cells were suspended in culture medium (DMEM/F12) at a concentration of 106 cells/mL and 2 mL samples of cells suspension were added to the Oxygraph chambers. Data were digitally recorded using Dat Lab v.4 software, where oxygen flux was calculated as the negative time derivative of oxygen concentration. Before each experiment, the polarographic oxygen sensors were calibrated by a two-point calibration, routinely achieved at air saturation and zero oxygen concentration according to the protocol by [56].
The protocol used, developed by OROBOROS INSTRUMENTS, started with a period of ROUTINE respiration, which illustrates the physiological respiratory function of cells with physiological substrates in the culture medium. The addition of oligomycin (ATP synthase inhibitor, final concentration of 2 µg/mL) (Sigma-Aldrich, St. Louis, MO, USA) was associated with a significant reduction in mitochondrial respiration due to the inhibition of ATP synthesis and induction of a LEAK state of respiration. The residual LEAK respiration is mainly associated with the compensation of proton leak, proton slip, and cation cycling across the inner mitochondrial membrane. Subsequently, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) (Sigma-Aldrich, St. Louis, MO, USA) as the proton gradient uncoupler was titrated to estimate electron transfer system (ETS) capacity at uncoupled state. Uncoupler titration (increments of 0.5 µM up to 2.5 µM FCCP) gradually increased mitochondrial respiration. The greatest increase was observed at 2.5 µM FCCP. Finally, inhibition of Complex I by rotenone (1.5 µM) (Sigma-Aldrich, St. Louis, MO, USA) and Complex III by antimycin A (7.5 µM) (Sigma-Aldrich, St. Louis, MO, USA) reflects Residual Oxygen Consumption (ROX) state, which is linked to other cellular oxygen-consuming processes besides the respiration chain (Figure 8). The remaining oxygen consumption is due to non-mitochondrial respiration such as oxidases and other cellular enzymes. All inhibitors and the uncoupler applied in this protocol are freely permeable through the intact plasma membrane and do not require cell membrane permeabilization [33]. All experiments were repeated three times and all obtained data were ROX-corrected.
On the basis of using the coupling control protocol, the following respiration rates were calculated: R/E (ROUTINE control ratio)—the ratio of routine respiration and ETS capacity, an expression of how far routine respiration operates from ETS capacity; L/E (LEAK control ratio)—the ratio of LEAK respiration and ETS capacity; netR/E [(R-L)/E]—the phosphorylation-related respiration as a fraction of ETS capacity.

4.4. Intracellular ATP Content

The intracellular ATP content was measured using an ATPliteTM Luminescence ATP Detection Assay System (PerkinElmer, Waltham, MA, USA), according to the protocol provided by the manufacturer. This system is based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin. Briefly, cells were washed 2× with PBS, treated with mammalian cell lysis solution, and shaken for 5 min in an orbital shaker at 700 rpm to lyse cells and stabilize ATP content. Then, 150 µL of cell lysate was transferred to a 96-well plate and 50 µL of substrate solution, containing luciferase/luciferin, was added to each well. The plate was dark-adapted for 10 min. The luminescence was measured using a Tecan Genios microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Results were calculated with Magellan v.6 software as nmol ATP and then adjusted for protein content (measured in the cell lysates using the Total Protein Kit, Micro Lowry, Peterson’s Modification, Sigma-Aldrich, St. Louis, MO, USA) and reported as nmol ATP/mg of protein. As a positive control, we used FCCP, the uncoupler of oxidative phosphorylation, which caused a reduction in ATP content. The control cells were incubated with 2.5 µM FCCP for 10 min.

4.5. Mitochondrial Membrane Potential

Mitochondrial membrane potential was measured by using a Mito Probe TM JC-1 Assay Kit (Molecular Probes, Eugene, OR, USA) on flow cytometry. The cells were incubated with selected factors for 24 h and after this time JC-1 dye was added to the fresh medium (final concentration 2 µM) and incubated in 37 °C in the dark for 45 min. Briefly, cells were trypsinizated, washed 2× with PBS, centrifuged (500× g) and suspended in 200 µL PBS. JC-1-induced fluorescence changes were recorded with FACSCanto flow cytometry (BD Biosciences Discovery Labware, Bedford, MA, USA) using 488 nm excitation with 530/30 nm (FLI, green) and 585/42 nm (FL2, orange) band pass emission filters. The fluorescence of 1 × 104 cells was collected during a single instrument run. The data were analyzed using the FacsDIVA software v.6.1.2 (BD Biosciences Discovery Labware, Bedford, MA, USA). Changes in mitochondrial membrane potential (depolarization) were identified by the red/green fluorescence intensity ratio. This ratio is dependent only on the mitochondrial membrane potential and not on other factors such as mitochondrial size, shape and density [57,58].
In this analysis, as positive control for the JC-1 sensitivity characterizing changes in the mitochondrial membrane potential, we used the protonophoric uncoupler of oxidative phosphorylation carbonylcyanide m-chlorophenylhydrazone (CCCP) (1 µM) (Sigma-Aldrich, St. Louis, MO, USA).

4.6. Caspase-9 Activity

Caspase-9 activity was measured by a Caspase-9 Colorimetric Assay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Briefly, cells were trypsinized (0.25% trypsin with 0.02% EDTA) and washed 2× with PBS; 2 × 106 cells were transferred to each tube and centrifuged for 10 min at 250× g. Then, the supernatant was removed, and 50 μL of cold lysis buffer was added to the pellets. The cells were incubated on ice for 10 min and centrifuged for 1 min at 10,000× g. Then, 50 μL of the supernatant was transferred to a 96-well plate. To each well, 50 μL of 2× Reaction Buffer 9 (previously prepared by adding 10 μL of fresh DTT) and 5 μL of caspase-9 colorimetric substrate were added. The plate was incubated for 1–2 h at 37 °C. After this time, absorbance was read using an ELISA microplate reader at 405 nm (Thermo Fisher Scientific, Waltham, MA, USA). The level of caspase-9 enzymatic activity in cell lysates is directly proportional to the absorbance intensity. The changes in activity of caspase-9 were determined by comparing the results with the control. H2O2 at concentration of 0.2 mM was used as a positive control [59].

4.7. Apoptosis Assay—Annexin 5

Cell apoptosis was analyzed using an FITC Annexin V/Dead Cell Apoptosis Kit with FITC Annexin V and PI (Invitrogen, Carlsbad, CA, USA) and detected on flow cytometry (BD Canto II, Becton Dickinson, Franklin Lakes, NJ, USA). The cells were incubated with different concentrations of quercetin for 24 h, trypsinizated (0.25% trypsin with 0.02% EDTA), and washed 2× with PBS. Then, 5 µL of FITC Annexin V and 1 µL of the 100 µg/mL PI were added to each 100 µL of the cell suspension 1 × 106/mL in PBS. Cells were incubated for 15 min in the dark, and then the suspension was diluted with 400 µL of Annexin V-binding buffer. The stained cells were analyzed by flow cytometry, and the fluorescence emission was measured at 530 nm and >575 nm. The cell population was separated into three groups: apoptotic cells showing green fluorescence, dead cells showing both red and green fluorescence, and live cells showing no fluorescence.

4.8. ROS Generation

Changes in reactive oxygen species (ROS) generation were measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Sigma-Aldrich, St. Louis, MO, USA). DCFH-DA is a lipophilic compound that freely crosses the cell membrane. In a living cell, DCFH-DA is degraded to dichlorofluorescein (DCFH), which, upon oxidation in the presence of ROS, emits fluorescence. This fluorescence intensity is proportional to the amount of ROS. The cells were cultured in 12-well plates. After incubation with quercetin (24 h), the cells were washed Krebs-Ringer-Hepes buffer (KRH) and incubated with DCFH-DA (100 µM) (37 °C in the dark, 40 min). After this, the cells were washed once with KRH buffer, 2× with PBS, and then trypsinized (0.25% trypsin with 0.02% EDTA). Then, cells were transferred to tubes, washed 2× with PBS, and centrifuged for 5 min at 500× g. The supernatant was removed, and the pellets were dissolved in 200 μL of PBS. Fluorescence readings were performed on a flow cytometer (BD Canto II, Becton Dickinson, Franklin Lakes, NJ, USA). Changes in ROS generation were also illustrated by fluorescence microscopy (Nikon ECLIPSE TS100, Nikon Corporation, Tokyo, Japan). The effect of quercetin on ROS production induced by 1 mM H2O2 was also analyzed. For this purpose, during incubation with DCFH-DA, an additional 400 µL 1 mM H2O2 was added to the cells.

4.9. Oil Red O Staining

To assess the differentiation of Chub-S7 cells into adipocytes, lipid droplets in cells were stained with red oil dye. For the staining, 0.5 g Oil Red O Solution (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 100 mL of isopropanol (POCH, Gliwice, Poland). Briefly, cells were fixed using a 4% solution of paraformaldehyde (POCH, Gliwice, Poland) for 1 h. After this, cells were washed with distilled water and isopropanol (POCH, Gliwice, Poland) and incubated for 1 h with the red oil dye (Sigma-Aldrich, St. Louis, MO, USA). Next, the Oil-Red-O-containing medium was removed and differentiation of cells was illustrated using the Nikon ECLIPSE TS100 microscope. Additionally, the differentiation level of Oil-Red-O-stained Chub-S7 cells was assessed by the colorimetric method. After staining the cells with red oil, 100 µL of isopropanol was added. The absorbance was measured using an ELISA microplate reader at 492 nm.

4.10. Statistical Analysis

All data were expressed as the mean + S.D. from five independent experiments measured in triplicate. Statistical significances for comparisons between treated and control cell samples were determined by an unpaired t-test using Statistica v.10.0 PL software.

5. Conclusions

In the presented in vitro model, quercetin inhibited differentiation of human Chub-S7 preadipocytes and reduced fat accumulation in lipid droplets. The effect was accompanied by mild mitochondrial uncoupling and changes in mitochondrial respiratory states. If the ability of quercetin to modulate mitochondrial bioenergetics and reduce lipid accumulation was confirmed in the in vivo studies, quercetin could be considered a factor used to prevent obesity.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31040725/s1, Supplementary Material S1: Apoptosis assay; Supplementary Material S2: Cytotoxic effect [46,60,61].

Author Contributions

Conceptualization, A.D. and J.G.; methodology, A.D., J.G. and A.G.; formal analysis, A.D. and J.G.; investigation, A.D.; data curation, A.D., J.G. and A.G.; writing—original draft preparation, A.D.; writing—review and editing, J.G., A.P. and B.S.; visualization, A.D. and A.G.; supervision, A.D. and J.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by LIPIDOMICNET EU FP7 202272: 7th Framework Programme. The APC was funded by Jagiellonian University Medical College.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Routine respiration measured by high-resolution respirometry (Oxygraph-2k, OROBOROS Instruments, Innsbruck, Austria) in Chub-S7 control cells and cells incubated with different concentration of quercetin (10 µM, 30 µM, 50 µM, 70 µM and 100 µM) for 24 h. Results are presented as percentage of control, as mean + S.D. (n = 5); * p < 0.05 compared to control.
Figure 1. Routine respiration measured by high-resolution respirometry (Oxygraph-2k, OROBOROS Instruments, Innsbruck, Austria) in Chub-S7 control cells and cells incubated with different concentration of quercetin (10 µM, 30 µM, 50 µM, 70 µM and 100 µM) for 24 h. Results are presented as percentage of control, as mean + S.D. (n = 5); * p < 0.05 compared to control.
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Figure 2. Selected respiratory rates: R/E, L/E and netR/E of Chub-S7 cells after 24 h incubation with increasing concentrations of quercetin: 10 μM (a); 30 μM (b); 50 μM (c); 70 μM (d); 100 μM (e). Data presented as mean + S.D. (n = 5); * p < 0.05 compared to control.
Figure 2. Selected respiratory rates: R/E, L/E and netR/E of Chub-S7 cells after 24 h incubation with increasing concentrations of quercetin: 10 μM (a); 30 μM (b); 50 μM (c); 70 μM (d); 100 μM (e). Data presented as mean + S.D. (n = 5); * p < 0.05 compared to control.
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Figure 3. Intracellular ATP content in Chub-S7 control and quercetin (10 µM, 30 µM, 50 µM, 70 µM, 100 µM)-treated cells. ATP levels were measured by the luminometric method, normalized to protein content in cell lysate. Results are presented as percent of control, as mean + S.D. measured in triplicate, (n = 5).
Figure 3. Intracellular ATP content in Chub-S7 control and quercetin (10 µM, 30 µM, 50 µM, 70 µM, 100 µM)-treated cells. ATP levels were measured by the luminometric method, normalized to protein content in cell lysate. Results are presented as percent of control, as mean + S.D. measured in triplicate, (n = 5).
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Figure 4. Flow cytometry analysis of mitochondrial membrane potential of Chub-S7 control and quercetin (10 µM, 30 µM, 50 µM, 70 µM,100 µM, 24 h)-treated cells. Results are presented as % of control red-to-green fluorescence ratio (JC-1), as mean + S.D. (n = 5); * p < 0.05 compared to control.
Figure 4. Flow cytometry analysis of mitochondrial membrane potential of Chub-S7 control and quercetin (10 µM, 30 µM, 50 µM, 70 µM,100 µM, 24 h)-treated cells. Results are presented as % of control red-to-green fluorescence ratio (JC-1), as mean + S.D. (n = 5); * p < 0.05 compared to control.
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Figure 5. Apoptosis assay in Chub-S7 control and quercetin (10 µM, 30 µM, 50 µM, 70 µM, 100 µM, 24 h)-treated cells. (a) Changes in caspase-9 activity measured by the colorimetric method. Results are presented as % of control, as mean + S.D. (n = 5); (b) Annexin V assay. Results are presented as fluorescence intensity (% of cells), as mean + S.D. (n = 4). * p < 0.05 compared to control.
Figure 5. Apoptosis assay in Chub-S7 control and quercetin (10 µM, 30 µM, 50 µM, 70 µM, 100 µM, 24 h)-treated cells. (a) Changes in caspase-9 activity measured by the colorimetric method. Results are presented as % of control, as mean + S.D. (n = 5); (b) Annexin V assay. Results are presented as fluorescence intensity (% of cells), as mean + S.D. (n = 4). * p < 0.05 compared to control.
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Figure 6. Reactive oxygen species (ROS) production in Chub-S7 cells after 24 h incubation with quercetin in the absence or presence of hydrogen peroxide (H2O2). (a) Fluorescence microscope images; dichlorofluorescein staining; 10× magnification. (b) ROS production. Results are presented as fluorescence intensity (% of control), (n = 5); * p < 0.05 vs. control without H2O2; # p < 0.05 vs. control with H2O2. (c) ROS production assay in control Chub-S7 cells after hydrogen peroxide addition. Results are presented as fluorescence intensity (% of control), as mean + S.D. (n = 5). * p < 0.05 vs. control without H2O2.
Figure 6. Reactive oxygen species (ROS) production in Chub-S7 cells after 24 h incubation with quercetin in the absence or presence of hydrogen peroxide (H2O2). (a) Fluorescence microscope images; dichlorofluorescein staining; 10× magnification. (b) ROS production. Results are presented as fluorescence intensity (% of control), (n = 5); * p < 0.05 vs. control without H2O2; # p < 0.05 vs. control with H2O2. (c) ROS production assay in control Chub-S7 cells after hydrogen peroxide addition. Results are presented as fluorescence intensity (% of control), as mean + S.D. (n = 5). * p < 0.05 vs. control without H2O2.
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Figure 7. Lipid droplet accumulation during the differentiation process of Chub-S7 cells after incubation with quercetin (10 µM, 30 µM, 50 µM, 70 µM, 100 µM) for 24 h (a), 48 h (b) and 21 days (c); Oil Red O staining; 20× magnification and absorbance measurement (% of control), (n = 5); * p < 0.05 vs. control.
Figure 7. Lipid droplet accumulation during the differentiation process of Chub-S7 cells after incubation with quercetin (10 µM, 30 µM, 50 µM, 70 µM, 100 µM) for 24 h (a), 48 h (b) and 21 days (c); Oil Red O staining; 20× magnification and absorbance measurement (% of control), (n = 5); * p < 0.05 vs. control.
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Figure 8. Sample illustration of changes in mitochondrial respiration states analyzed by coupling control protocol in Chub-S7 control cells; measured by high-resolution respirometry (Oxygraph-2k, OROBOROS Instruments, Insbruck, Austria).
Figure 8. Sample illustration of changes in mitochondrial respiration states analyzed by coupling control protocol in Chub-S7 control cells; measured by high-resolution respirometry (Oxygraph-2k, OROBOROS Instruments, Insbruck, Austria).
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Table 1. Quercetin-induced necrosis and apoptosis of human preadipocyte cell line Chub-S7. Results are presented as % of control, as mean ± SD; from 4 experiments; * p < 0.05 vs. control.
Table 1. Quercetin-induced necrosis and apoptosis of human preadipocyte cell line Chub-S7. Results are presented as % of control, as mean ± SD; from 4 experiments; * p < 0.05 vs. control.
Q1
Necrotic Cells [%]		Q2
Cells in Late Phase of Apoptosis [%]		Q4
Cells in Early Phase of Apoptosis [%]
Control0.73 ± 0.151.57 ± 0.292.97 ± 1.00
Quercetin 10 µM0.47 ± 0.061.50 ± 0.422.63 ± 1.37
Quercetin 30 µM0.73 ± 0.242.10 ± 0.533.40 ± 1.36
Quercetin 50 µM0.50 ± 0.261.63 ± 0.564.00 ± 2.03
Quercetin 70 µM0.60 ± 0.181.58 ± 0.493.85 ± 1.86
Quercetin 100 µM0.53 ± 0.102.40 ± 0.14 *4.03 ± 2.92
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Dziewońska, A.; Gruca, A.; Polus, A.; Solnica, B.; Góralska, J. The Antioxidant Quercetin Affects Mitochondrial Function and Inhibits the Differentiation of Human Preadipocytes. Molecules 2026, 31, 725. https://doi.org/10.3390/molecules31040725

AMA Style

Dziewońska A, Gruca A, Polus A, Solnica B, Góralska J. The Antioxidant Quercetin Affects Mitochondrial Function and Inhibits the Differentiation of Human Preadipocytes. Molecules. 2026; 31(4):725. https://doi.org/10.3390/molecules31040725

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Dziewońska, Agnieszka, Anna Gruca, Anna Polus, Bogdan Solnica, and Joanna Góralska. 2026. "The Antioxidant Quercetin Affects Mitochondrial Function and Inhibits the Differentiation of Human Preadipocytes" Molecules 31, no. 4: 725. https://doi.org/10.3390/molecules31040725

APA Style

Dziewońska, A., Gruca, A., Polus, A., Solnica, B., & Góralska, J. (2026). The Antioxidant Quercetin Affects Mitochondrial Function and Inhibits the Differentiation of Human Preadipocytes. Molecules, 31(4), 725. https://doi.org/10.3390/molecules31040725

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