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. 2024 Oct 10;13(1):2411453. doi: 10.1080/21623945.2024.2411453

10-Gingerol reduces cytoplasmic lipid droplets and induces lipolysis in 3T3-L1 adipocytes

María Elizabeth Preciado-Ortiz a,b, Erika Martínez-López a, Trinidad García-Iglesias c, Gildardo Gembe-Olivarez a,d, Nathaly Torres-Castillo a, Iris Monserrat Llamas-Covarrubias a, Juan José Rivera-Valdés a,✉
PMCID: PMC11468046  PMID: 39385585

ABSTRACT

Obesity is a globally prevalent metabolic disorder characterized by an increased number of adipose cells and excessive fat in adipocytes. Herbal medicines, such as ginger, have shown potential in treating obesity by inhibiting adipogenesis and reducing adipocyte hypertrophy. Ginger contains bioactive compounds, particularly gingerols, which have demonstrated anti-adipogenic and/or lipolytic effects. However, research on the effects of 10-gingerol on adipose tissue remains limited. This study aimed to evaluate the effect of 10-gingerol on lipid content, lipolysis markers, and the expression of genes related to lipid metabolism in 3T3-L1 adipocytes. Three groups were analyzed: a negative control (preadipocytes), a positive control (mature adipocytes), and a group treated with 10-gingerol (10-G). Results showed that 10-G reduced lipid accumulation by 42.16% in mature adipocytes compared to the control, without affecting cell viability. Additionally, 10-G increased glycerol release and downregulated lipogenic genes such as Pparγ, Acaca, Fabp4, and Mtor, while upregulating genes related to fatty acid oxidation, including Cebpα, Cpt1a, Lipe, and Prkaa1. In conclusion, 10-gingerol reduces lipid content in mature adipocytes by downregulating lipogenesis, increasing lipolysis, and enhancing fatty acid oxidation.

KEYWORDS: Ginger, obesity, nutraceuticals, phytochemicals, lipid metabolism

Introduction

Obesity is defined as the excessive accumulation of fat in the body that endangers human health [1–3]. Since 1970, the prevalence of overweight and obesity has doubled, and currently, one-third of the world’s adult population is affected by this condition [1,4]. Obesity is closely associated with the development of metabolic disorders and diseases, such as insulin resistance, type 2 diabetes mellitus, coronary heart disease, non-alcoholic fatty liver, diseases of the nervous system, some types of cancer, among others [2,3,5,6].

Adipocytes are the main type of cells in adipose tissue and play a crucial role in the enlargement of this tissue [7]. Increased number of adipose cells (hyperplasia) and excessive fat content in adipocytes (hypertrophy) are characteristic events in the progression of obesity [3,7,8]. Adipocyte hypertrophy arises from a positive energy balance that favours increased lipogenesis to promote the storage of energy in the form of triglycerides (TG) [7], while also it decreases catabolic pathways such as lipolysis [9] and β-oxidation of fatty acids (FA) [10,11].

Therefore, reducing the accumulation of TG in adipocytes and increasing their degradation and use as an energy substrate in other organs, is one of the strategies for the treatment of obesity [3,7,10,11].

Herbal medicine could contribute to the control of obesity and associated metabolic diseases [12,13]. Ginger, Zingiber officinale Roscoe, is a medicinal plant recognized as a safe food supplement by the United States Food and Drug Administration (FDA) [12,14,15]. Ginger root presents a variety of bioactive compounds, particularly gingerols, that could help in the prevention and treatment of different diseases, including obesity [16–21]. 6-Gingerol and 6-shogaol are the most abundant phenolics in fresh and dried ginger root, respectively, and have shown antiadipogenic and/or lipolytic effects in cell culture [12,22–25] and in animal studies [17,18,20,26,27].

In 3T3-L1 preadipocyte cell line, 6-gingerol [22,24,25,28], 6-shogaol [23,25] and gingerenone A [16] exhibited an inhibitory effect on adipogenesis and lipid accumulation. The mechanism of action that explains these results is the reduction in the protein levels of the peroxisome proliferator-activated receptor γ (PPARγ) and CAAT enhancer binding protein alpha (C/EBPα) [12,23,24] which are important regulators of adipogenesis [6,29] and regulate genes involved in lipogenesis, lipolysis and fatty acid transport [6,12]. Furthermore, the protein levels of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and adipocyte-specific fatty acid-binding protein 4 (FABP4) decreased after treatment with these ginger phenols [12,24]. Moreover, lipid droplets reduction in mature 3T3-L1 adipocytes has only been shown by 6-shogaol [23] and gingerenone A [16]. Notably, 6-shogaol presented a lipolytic effect evidenced by a higher concentration of glycerol in the supernatant [23].

On the other hand, in animal models of obesity induced by a high-fat diet, supplementation with 6-gingerol [18,26,27], with ginger powder [30] or with ginger extracts [17,20,31] have been shown to decrease adipocyte hypertrophy and the expression of lipogenic proteins. In addition, complementary results of these studies were the increase in the mRNA and protein levels of carnitine palmitoyl transferase 1 (CPT1), a protein related to the oxidation of fatty acids [30], and reduction in the expression of proinflammatory cytokines including interleukin 6 (IL-6), tumour necrosis factor α (TNFα) and chemokines such as monocyte chemoattractant protein (MCP)-1 [16,18,20,26,30]. As well, in a randomized, double-blind, placebo-controlled study, obese women who received 2 g of ginger powder per day experienced a decrease in body mass index (BMI) [32]. Furthermore, the intake of dried ginger powder could reduce respiratory exchange rates and promote fat utilization by increasing fat oxidation in humans [33].

The beneficial effects of ginger and some of its bioactive compounds are well described in obesity models. However, the effects and mechanism of action of other bioavailable phenols in ginger, such as 10-gingerol, have been poorly investigated in adipose tissue. Hence, the aim of this study was to evaluate the effect of 10-gingerol on lipid content, lipolysis markers, and the expression of genes related to lipid metabolism in 3T3-L1 adipocytes.

Results

Dose–response curve of cell viability and lipid content with 10-gingerol

To assess the impact of 10-gingerol on lipid metabolism in adipocytes, the initial step involved determining the appropriate dosage of this bioactive compound for these cells. Next, the cytotoxic effect of 10-gingerol on 3T3-L1 adipocytes was investigated. 10-Gingerol (0–35 µg/mL) was added for the cell viability assay. A higher percentage of viable cells was observed with 10 and 15 µg/mL of 10-gingerol; in contrast, there was a significant reduction in cell viability with 25 and 35 µg/mL 10-gingerol (Figure 1b).

Figure 1.

The figure is composed of three parts. (A) Microscopic images showing lipid droplets stained red after Oil Red O staining in cells treated with varying concentrations of 10-gingerol (Vehicle, 0 µg/ml, 10 µg/ml, 15 µg/ml, 25 µg/ml, 35 µg/ml), where a reduction in lipid accumulation is observed at higher concentrations. (B) Bar graph depicting cell viability (%) across different concentrations of 10-gingerol, with a significant increase in viability at 10 µg/ml and 15 µg/ml. (C) Bar graph showing lipid accumulation (%) with a significant decrease at concentrations of 10, 15, 25 and 35 µg/ml compared to the control.

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Dose response curve of 10-gingerol on cell viability and lipid content. (a) Cell viability by MTT assay; differentiated 3T3-L1 adipocytes were treated with 10-gingerol (0–35 µg/mL) for 48 h. Dimethyl sulphoxide (DMSO) was used as a vehicle. (b) Lipid accumulation in 3T3-L1 adipocytes visualized using oil Red O (ORO) staining (40× magnification). (c) ORO dye was extracted using isopropanol and quantified with optical density at 570 nm (OD570). Data are presented as the mean ± standard error of the mean (SEM) of three independent experiments. **p<.01, ***p<.001, ****p<.0001.

Afterwards, 10-gingerol (0–35 µg/mL) was added to mature 3T3-L1 adipocytes and maintained for 48 h. The presence of 10-gingerol caused a significant reduction in cytoplasmic lipid accumulation determined by Oil Red O (ORO) staining (Figure 1a,c). The concentration of 10-gingerol selected for subsequent assays was 15 µg/mL, because it did not decrease cell viability, but significantly reduced cytoplasmic lipid content.

10-Gingerol reduced lipid accumulation in mature 3T3-L1 adipocytes

The study groups were formed: NC (3T3-L1 preadipocytes), PC (mature 3T3-L1 adipocytes) and 10-G (mature 3T3-L1 adipocytes treated with 10-gingerol). In the three groups, cell viability was assessed in triplicate using the MTT assay. No significant differences were observed in the percentage of live cells among the groups (Figure 2a).

Figure 2.

The figure is composed of three parts. (a) Bar graph showing cell viability (%) for three groups: NC (negative control), PC (positive control) and 10-G (10-gingerol treated group), with no significant differences between groups. (b) Bar graph depicting lipid accumulation (%), with a significantly higher accumulation in the PC group compared to the NC and 10-G groups. (c) Microscopic images showing lipid droplets stained red after Oil Red O staining for the NC, PC and 10-G groups, with minimal lipid accumulation in the NC group, extensive accumulation in the PC group, and reduced accumulation in the 10-G group.

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Effect of 10-gingerol on cell viability and lipid content in the study groups: NC, PC and 10-G. (a) 3T3-L1 adipocytes were treated with 15 μg/mL 10-gingerol, and cell viability was determined by MTT assay. (b) Lipid accumulation in 3T3-L1 adipocytes visualized by Oil Red O (ORO) staining (40× magnification). (c) ORO dye was extracted using isopropanol and measured spectrophotometrically (OD515). Data are presented as the mean ± standard error of the mean (SEM) of three independent experiments. **p<.0001. NC, negative control; PC, positive control; 10‑G, 10‑gingerol group.

Subsequently, the cytoplasmic lipid content was analysed with ORO staining in the study groups. The 10-G group showed less red staining compared to the PC group, indicating lower levels of stored fat (Figure 2c). When the dye was extracted, the lipid content was estimated to be 29.33% ± 0.53 in the NC group, 100% ± 3.52 in the PC group and 57.84% ± 3.01 in the 10-G group. The 10-G group presented a statistically significant reduction in cytoplasmic lipid content compared to the PC group (p < 0.0001) (Figure 2b).

10-Gingerol induced lipolysis in mature 3T3-L1 adipocytes

To assess the lipolytic effect of 10-gingerol on 3T3-L1 adipocytes, the concentrations of glycerol in the supernatants were measured. The 10-G group showed an increase in the percentage of glycerol released into the supernatant compared to the PC group (p < 0.01) (Figure 3).

Figure 3.

The bar graph showing glycerol release (%) in three groups: NC (negative control), PC (positive control) and 10-G (10-gingerol treated group). The 10-G group exhibits significantly higher glycerol release compared to the PC group (**p < 0.01), while the NC group shows minimal glycerol release.

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Effects of 10-G on glycerol release activity in 3T3-L1 adipocytes. The concentration of glycerol in the supernatant was determined using the VITROS 350 chemistry system with TRIG slides. Data are presented as the mean ± standard error of the mean (SEM) of three independent experiments. **p<.01, NC, negative control; PC, positive control, 10‑G, 10‑gingerol group.

Effects of 10-gingerol on the expression of genes related to lipid metabolism in 3T3-L1 adipocytes

The expression levels of genes related to lipid metabolism, mainly with the synthesis and oxidation of fatty acids, were evaluated at the mRNA. The 10-G group showed a significant reduction of Pparγ, Acaca, Fabp4 and Mtor expression compared with the PC group. In contrast, the mRNA levels of Cebpα, Cpt1a, Lipe and Prkaa1 were significantly increased in the 10-G group compared with the PC group (p < 0.05) (Figure 4 and 5).

Figure 4.

The bar chart displaying relative mRNA levels for four genes: Pparγ, Cebpα, Acaca and Fabp4 across three experimental groups (NC, PC, 10-G). Each bar represents one of these groups, with NC in solid black, PC in diagonal stripes and 10-G in dotted patterns. Significant differences between the conditions are indicated by asterisks, where * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and **** represents p < 0.0001. Specifically, the mRNA levels of Pparγ and Acaca are lower, while the levels of C/ebpα and Fabp4 are significantly higher in the 10-G group compared to the NC group.

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Effect of 10‑gingerol on genes related to lipid anabolism. Pparγ, Cebpα, Acaca and Fabp4 mRNA levels in 3T3‑L1 adipocytes were examined using quantitative PCR. The relative gene expression of each sample was normalized to Rn18s. Data are presented as the mean ± standard error of the mean (SEM) of three independent experiments. **p<.05, ***p<.001, ****p<.0001. NC, negative control; PC, positive control; 10‑G, 10‑gingerol group.

Figure 5.

The bar chart displaying relative mRNA levels for four genes: Cpt1a, Mtor, Lipe and Prkaa1 across three experimental groups (NC, PC, 10-G). Each bar represents one of these groups, with NC in solid black, PC in diagonal stripes and 10-G in dotted patterns. Significant differences between the conditions are indicated by asterisks, where * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001 and **** represents p < 0.0001. Specifically, the mRNA level of Mtor is lower, while the levels of Cpt1a, Lipe and Prkaa1 are significantly higher in the 10-G group compared to the NC group.

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Effect of 10‑gingerol on genes related to lipid catabolism. Cpt1a, Mtor, Lipe and Prkaa1 mRNA levels in 3T3‑L1 adipocytes were examined using quantitative PCR. The relative gene expression of each sample was normalized to Rn18s. Data are presented as the mean ± standard error of the mean (SEM) of three independent experiments. **p < .05, ***p < .001, ****p < .0001. NC, negative control; PC, positive control; 10‑G, 10‑gingerol group.

Discussion

Since obesity is the most prevalent metabolic disorder globally [3], it has prompted a search for effective treatments. Herbal medicines have attracted attention for their potential to address obesity by inhibiting adipogenesis and reducing adipocyte hypertrophy, the main characteristics of this condition [12]. In this context, ginger has emerged as a promising candidate, traditionally used to treat various ailments and recently metabolic diseases such as obesity [34]. Some studies have clarified the anti-adipogenic and anti-lipogenic effects of 6-gingerol [18,22,24] and 6-shogaol [23], the most abundant compounds in ginger, within obesity models. Nonetheless, the impact of other phenols found in ginger, such as 10-gingerol, on adipose tissue remains poorly understood.

Our previous study demonstrated 10-gingerol reduced adipogenesis and lipid droplet accumulation in 3T3-L1 cells by downregulating the mRNA of adipogenic transcriptional factors and genes associated with lipid metabolism [35]. Therefore, on this occasion, we investigated the impact of 10-gingerol on mature adipocytes, a condition commonly found in mammals, and additionally explored its possible mechanisms of action, using a culture of 3T3-L1 mature adipocytes.

In the present study, our data indicated that 10-gingerol reduces the accumulation of cytoplasmic lipids in 3T3-L1 adipocytes and promotes lipolysis by increasing the release of glycerol into the supernatant. This effect could partially be explained through the regulation at the mRNA level of key transcriptional factors of the adipogenic phenotype, the decrease in the mRNA expression of Acaca and Fabp4, genes that encode lipogenic enzymes, as well as increased expression of Cpt1α, a gene related to the oxidation of FA, Prkaa1 and Lipe genes involved in the regulation of lipolysis [36].

Similar results were reported by Suk and collaborators in 2016, where they demonstrated that 6-shogaol decreases the fat content of mature adipocytes and promotes lipolysis by increasing the concentrations of glycerol in the culture media [23]. Although the previous study did not explore any potential mechanisms to explain the lipolytic effect observed, our study evaluated the expression of Lipe gene, which encodes hormone-sensitive lipase (HSL), the enzyme responsible for degrading triglycerides into free FA and glycerol. The increase in Lipe expression suggests a stimulation of lipolytic activity, which could explain the rise in glycerol release [36]. This result is consistent with a report where the upregulated expression of Lipe increased the phosphorylation of perilipin and stimulated fragmentation of lipid droplets and led to lipolysis in 3T3-L1 adipocytes treated with ginger oil [37].

On the other hand, other ginger phenols, such as 6-gingerol and galanolactone, inhibit adipogenesis, evidenced by a lower lipid content at the end of the differentiation process, but their effect on mature adipocytes in vitro is not well known [22,24,38].

It has been described that in adipocytes, the expression levels of the transcriptional factors PPARγ and C/EBPα increase as the adipogenesis process progresses and subsequently maintain a constant expression [29]. This constant expression plays a fundamental role in selective gene expression of the adipocyte, and thus, metabolic homoeostasis in adipose tissue [29,39]. Therefore, we analysed whether 10-gingerol affects the mRNA expression levels of Pparγ and Cebpα in mature adipocytes. Our results demonstrated that 10-gingerol stimulation decreased the expression of Pparγ but showed contradictory results in Cebpα. The decrease in Pparγ expression in mature adipocytes coincided with what was reported for gingerone A, another ginger phenol [16]. Moreover, similar results were reported in animal models of obesity supplemented with 6-gingerol [17,18] and with an ethanolic extract of ginger [28,31,40], where it was demonstrated a downregulation in the mRNA levels of these transcriptional factors, resulting in smaller adipocyte size. On the other hand, our results indicated that 10-gingerol upregulated Cebpα expression. Many reports have suggested that 6-gingerol treatments significantly downregulated both C/EBPα and PPARγ expressions [22,24]. However, a study in obese mice supplemented with a ginger extract for 4 weeks, reported an increase at the mRNA levels of these transcriptional factors, but a decrease at the protein level [30]. This suggests that 10-gingerol could have an effect at the protein level of the analysed genes, but this was not evaluated in our study. In addition, another study has shown that ginger extract did not affect Cebpα expression, but suppressed Cebpβ and Cebpδ expressions, which may suggest a possible pathway that could be related to lipid decrease in 3T3-L1 adipocytes [34].

Subsequently, the mRNA expression of genes related to lipogenesis and FA transport in mature adipocytes was analysed. Stimulation with 10-gingerol significantly decreased the expression levels of Acaca and Fabp4. Acaca encodes ACC, which regulates endogenous fatty acid synthesis and triglyceride storage [22,27]. The FABP4 facilitates cellular uptake of long-chain fatty acids for their metabolism [16]. Downregulation of Pparγ in adipocytes stimulated with 10-gingerol could justify the downregulation in the expression of Acaca and Fabp4. Similar results have been reported in in vivo obesity studies supplemented with 6-gingerol [20] and with an ethanolic of ginger [12], where the downregulation of this transcriptional factor coincides with the decrease in the expression of Acaca and Fabp4, resulting in adipose tissue reduction and smaller adipocyte size.

Furthermore, lipogenesis and lipolysis are antagonistic pathways. Activation of lipolytic pathways, which in our results is demonstrated by the increase in glycerol in the supernatant and the increased expression of Lipe, promotes the downregulation of lipogenic pathways [7,41]. This could also explain the decrease in the expression of Acaca and Fabp4, as well as the lower lipid content [7].

Additionally, our results demonstrated that 10-gingerol stimulation decreased the expression of Mtor, which encodes a mammalian target of rapamycin (mTOR). mTOR has been implicated in the regulation of triglyceride synthesis [42,43] through the activation of sterol regulatory element-binding protein (SREBP), which promotes the transcription of lipogenic genes, such as ACC, FAS and FABP4 [44]. In this sense, decreased mTOR expression downregulates lipogenic pathways [43,44]. Therefore, the decrease in mRNA Mtor levels could justify the decrease in Acaca and Fabp4, which decreased the synthesis and transport of fatty acids and, consequently, lipid content in 3T3-L1 cells. Notably, some reports have shown that mTOR downregulation is often accompanied by an increase of catabolic pathways, such as lipolysis and FA oxidation [45,46].

It has been described that both mTOR and SREBP expression were controlled through the activation of AMP activated protein kinase (AMPK) pathway by a ginger extract in a high-fat diet mouse model [40]. The activation of AMPK stimulates the activation of lipolytic pathways and FA oxidation [40,45]. Although our study did not evaluate AMPK activation, we did analyse the expression of the Prkaa1 gene, which encodes the catalytic subunit alpha 1 of AMPK, whose expression was increased in response to treatment with 10-gingerol.

Our results showed a rise in glycerol concentrations in supernatant, a marker of lipolysis, as well as an increase in the levels of Cpt1a, which encodes the protein carnitine palmitoyl transferase 1, transporter necessary for the initiation and regulation of FA oxidation in the mitochondria [45,46]. Similar findings were reported by Seo et al., who demonstrated that a dry ginger extract significantly increased Cpt1a expression and was associated with a reduction in adipocyte hypertrophy in obese mice [30]. Likewise, Brahma Naidu et al. noted that 30 days of supplementation with a gingerol extract reduced the total body weight of obese rats [20]. This effect was attributed to an increase in the expression of CPT1 at both the mRNA and protein levels, accompanied by a decrease in the expression of lipogenic enzymes and enzymes related to cholesterol metabolism [20]. Therefore, the rise in these markers of lipolysis and FA oxidation could also help us to explain the lower content of cytoplasmic lipids found in mature adipocytes stimulated with 10-gingerol.

Among the strengths of this study include its pioneering evaluation of the effects of 10-gingerol on mature hypertrophic adipocytes, alongside the exploration of candidate genes within potential pathways. However, some of the limitations include the inability to determine protein expression for the assessed genes, as well as their activation or translocation to the nucleus in the case of transcription factors. This highlights an area for future research, which could elucidate the mechanism of action behind the observed effects of 10-gingerol on adipose tissue.

Conclusion

These results demonstrate that 10-gingerol inhibited the expression of Pparγ and Mtor in 3T3-L1 adipocytes, which could be related to the decrease in the expression of Acaca and Fabp4, reducing the synthesis and transport of fatty acids and consequently, the lipid content in these fat cells [27]. Additionally, 10-gingerol increased the concentrations of glycerol in the supernatant, marker of lipolysis, in addition to elevating the expression levels of Prkaa1 and Lipe, genes that encode proteins that promote lipolysis. 10-Gingerol also upregulated Cpt1a, which suggests a potential involvement of fatty acid oxidation in the observed effects [38,43]. This adds to the explanation of the decrease in cytoplasmic lipids in mature 3T3-L1 adipocytes.

The current data suggest that 10-gingerol exhibits anti-obesogenic potential by reducing lipid content in mature adipocytes. This could potentially be achieved through downregulation in the expression of lipogenic genes, increase in lipolysis markers and upregulation of genes related to FA oxidation. Further in vivo studies are necessary to validate the effects of 10-gingerol in hypertrophic adipose tissue, as well as clinical trials to corroborate its beneficial effects in humans and to determine its potential use as part of obesity treatment.

Materials and methods

Ethical approval

This research was approved by the institutional ethics, research and safety committees of the University Center for Health Sciences, University of Guadalajara (registration number CI-03821).

3T3-L1 cell culture and differentiation

Mouse 3T3-L1 preadipocytes were obtained from the Immunology Laboratory of the University Center of Health Sciences, University of Guadalajara (Guadalajara, Mexico). Dulbecco’s Modified Eagle Medium (DMEM; Sigma‑Aldrich; Merck KGaA; cat. no. SIG‑D6429) supplemented with 10% calf bovine serum (CBS; Cytiva; HyClone; cat. no. 12389812) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; Gibco; Thermo Fisher Scientific, Inc.; cat. no. 15140122) was used for the proliferation and maintenance of the cell line. The incubation was performed at 37°C in a humidified atmosphere with 5% CO2 in a cell incubator (cat. no. SCO6AD; Sheldon Manufacturing, Inc.). 3T3‑L1 preadipocytes were seeded in Petri dishes at a density of 1 × 105 cells/dish and the medium was replaced every 2–3 days until the cells reached 100% confluence. The day after cells reached confluence (day 0), the differentiation process began by replacing the culture media with differentiation medium, which was composed of DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc.; cat. no. 21041025) supplemented with 10% foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.; cat. no. 16000044), 1% antibiotics and an adipogenic cocktail including 500 μM 3‑Isobutyl‑1‑methylxanthine, 1 μM dexamethasone, 1.5 μg/mL insulin and 1 μM rosiglitazone (3T3‑L1 Differentiation kit; Sigma‑Aldrich; Merck KGaA; cat. no. SIG‑DIF001‑1KT) for 3 days at 37°C and 5% CO2. Then, differentiation media were replaced with a maintenance medium composed of DMEM/F12 supplemented with 10% FBS, 1% antibiotics and 5 μg/mL insulin. The maintenance medium was replaced every 2 days for a total of 8 days of differentiation.

10‑G preparation

10‑Gingerol was acquired from Merck KGaA (Sigma‑Aldrich; cat. no. G5798) at a standard concentration of 5 mg/mL. A stock solution was prepared by dissolving 10‑gingerol in methanol. Subsequently, dilutions were made to 50 μg/mL in dimethyl sulphoxide (DMSO; Sigma‑Aldrich; Merck KGaA; cat. no. 276855‑1 L).

Dosage determination and treatment

A dose‑response curve assessment was performed with 0, 10, 15, 25 and 35 μg/mL of 10‑G. 3T3-L1 cells were differentiated into adipocytes as previously described. On day 8 of differentiation, concentrations of 10-gingerol were added to adipocytes for a period of 48 h. DMSO was used as vehicle.

Subsequently, three study groups were formed: the negative control (NC) comprised 3T3-L1 preadipocytes, positive control (PC) group consisted of 3T3-L1 preadipocytes differentiated for 8 days into adipocytes and the 10-gingerol group (10-G), formed by adipocytes (8 days of differentiation) treated for 48 h with a medium containing 10-gingerol. Supernatants from the study groups were collected in tubes and stored at −80°C. Finally, 3T3‑L1 preadipocytes and adipocytes were cryopreserved at −80°C for further experimental analysis.

MTT assay

3T3‑L1 cells were seeded at a concentration of 5 × 103 cells/well in 96‑well plates. Cells were differentiated as described earlier. Fully differentiated 3T3‑L1 cells were treated with 10‑G or vehicle for 48 h. Next, 1 mg/mL MTT (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. M6494) solution was added to the wells and the cells were incubated for 1 h at 37°C. After incubating the cells, the formazan crystals were dissolved using an extraction buffer (20% SDS and 50% dimethylformamide) followed by spectrophotometric measurement at 570 nm using a microplate reader (MultiScan GO; Thermo Fisher Scientific, Inc.; cat. no. 51119300). All measurements were taken in triplicate.

Oil Red O staining

The lipid accumulation in differentiated 3T3‑L1 adipocyte was determined by Oil Red O staining. Fully differentiated cells were fixed with 10% (v/v) formaldehyde solution for 60 min at room temperature and washed with distilled water. Then, lipid content in mature adipocytes was stained with oil Red O (Sigma‑Aldrich; Merck KGaA; cat. no. O0625) solution for 15 min and washed with distilled water at room temperature. Briefly, the stained Oil Red O was dissolved in 100% 2‑propanol (Sigma‑Aldrich; Merck KGaA; cat. no. I9516‑1 L) followed by spectrophotometric measurement at 515 nm using a microplate reader (MultiScan GO; Thermo Fisher Scientific, Inc.; cat. no. 51119300). All experiments were repeated three times.

Glycerol release

Measurement of glycerol released in the supernatants was conducted using VITROS 350 Chemistry System (Ortho-Clinical Diagnostics, Johnson & Johnson Services Inc., Rochester, NY, USA), with TRIG SLIDES (Vitros 350, cat. OCD-MS-1336544), following the manufacturer’s instructions.

Quantification of mRNA expression by qPCR

Total RNA from 3T3-L1 adipocytes was isolated using the RNeasy Mini Kit (Qiagen, cat. no. 74104) following to the manufacturer’s instructions. The quantity and quality of total RNA were determined by measuring absorbance with a spectrophotometer (MultiScan GO; Thermo Fisher Scientific, Inc.; cat. no. 51119300). First-strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the M-MLV Reverse Transcriptase Protocol (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. 28025013). Gene expression was analysed by qPCR (LightCycler 96 Thermocycler; Roche Diagnostics) using OneTaq® Hot Start Master Mix (NEB-R, Cat. N01-M0484L) and TaqMan® probes (Thermo Fisher Scientific): Rn18s (Thermo Fisher Scientific, Cat. MM03928990_G1), Cebpα (Thermo Fisher Scientific, Cat. MM00514283_S1), Pparγ (Thermo Fisher Scientific, Cat. MM00440940_M1), Fabp4 (Thermo Fisher Scientific, Cat. MM00445878_M1), Acaca (Thermo Fisher Scientific, Cat. MM01304257_M1), Cpt1a (Thermo Fisher Scientific, Cat. Mm01231183_m1), Mtor (Thermo Fisher Scientific, Cat. Mm00444968_m1), Lipe (Thermo Fisher Scientific, Cat. Mm00495359_m1) and Prkaa1 (Thermo Fisher Scientific, Cat. Mm01296700_m1). Thermocycler conditions for qPCR were as follows: Initial denaturation at 95°C for 300 sec, followed by 30 cycles of denaturation at 95°C for 20 sec and amplification at 60°C for 60 sec, and final extension at 68°C for 300 sec. Relative gene expression was determined using the 2-ΔΔCq method [47], and Cq values were normalized to the Rn18s gene. All results were obtained from at least three independent repeats.

Statistical analysis

Data were expressed as mean ± standard error of the mean (SEM). Data were analysed by one‑way ANOVA followed by Tukey’s post-hoc test to determine differences among group means. Data were analysed with GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). A value of p < 0.05 was established as a statistically significant difference.

Acknowledgments

We thank the Jalisco State Council of Science and Technology for the funding provided for this project through the Jalisco Scientific Development Fund (grant no. FODECIJAL-7944-2019).

Funding Statement

The present study was supported by Consejo Estatal de Ciencia y Tecnología del Estado de Jalisco through the Jalisco Scientific Development Fund [grant no. FODECIJAL-7944-2019]. In addition, this research was funded in part by a scholarship awarded by the Consejo Nacional de Ciencia y Tecnología (CONACYT) for the student M.E.P.-O. [1034591].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceptualization, JRV and EML; Experimental work, MPO, TGI and GGO; Data Analysis, NTC, ILC and EML; Investigation, MPO and GGO; Resources, TGI, EML and JRV; Writing, MPO, GGO and JRV; Review and Editing, EML, NTC, TGI and ILC; Funding Acquisition, JRV. All authors have read and approved the final manuscript.

Data availability statement

The datasets used and/or analysed during the current study are available via zenodo repository (https://zenodo.org/records/11272333).

Ethical approval

The ethical approval was obtained by the research committee, research ethics committee and biosafety committee of the University Center for Health Sciences of the University of Guadalajara (registration number CI-03821).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets used and/or analysed during the current study are available via zenodo repository (https://zenodo.org/records/11272333).

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