Essential Oil of Carvone Chemotype Lippia alba (Verbenaceae) Regulates Lipid Mobilization and Adipogenesis in Adipocytes

Obesity is characterized by an expansion of adipose tissue due to excessive accumulation of triglycerides in adipocytes, causing hypertrophy and hyperplasia, followed by hypoxia, alterations in adipocyte functionality, and chronic inflammation. However, current treatments require changes in lifestyle that are difficult to achieve and some treatments do not generate sustained weight loss over time. Therefore, we evaluated the effect of the essential oil (EO) of Lippia alba (Verbenaceae) carvone chemotype on viability, lipid mobilization, and adipogenesis of adipocytes in two normal and pathological cellular models in vitro. In 3T3-L1 adipocytes, a normal and a pathological model of obesity were induced, and then the cells were treated with L. alba carvone chemotype EO to evaluate cell viability, lipid mobilization, and adipogenesis. L. alba carvone chemotype EO does not decrease adipocyte viability at concentrations of 0.1, 1, and 5 µg/mL; furthermore, there was evidence of changes in lipid mobilization and adipogenesis, leading to a reversal of adipocyte hypertrophy. These results could be due to effects produced by EO on lipogenic and lipolytic pathways, as well as modifications in the expression of adipogenesis genes. L. alba carvone chemotype EO could be considered as a possible treatment for obesity, using the adipocyte as a therapeutic target.


Introduction
Adipocytes are endocrine cells of adipose tissue (AT) that store energy in the form of triglycerides within lipid droplets and release adipokines that participate in homeostatic balance, which are crucial in vascularization and immune response. However, when there is excess energy, adipocytes increase in size (hypertrophy) and quantity (hyperplasia), and adipogenesis and lipid mobilization are altered, causing changes that compromise adipocyte functionality and lead to the development of obesity [1,2].

GC/MS Analysis
Analysis was performed on a GC 6890 Plus gas chromatograph (Agilent Technologies, Palo Alto, CA, USA), equipped with a mass selective detector MSD 5973 Network (Agilent Technologies, Palo Alto, CA, USA) that used electron ionization [(ionization with electrons (IE), 70 eV]. Helium (99.995%, AP gas, Messer, Bogotá, Colombia) was used as carrier gas, with initial inlet pressure of 113.5 kPa; its volumetric flow rate was kept constant (1 mL/min) during the chromatographic run. The injection volume was 2 µL in split mode (30:1) and the injector temperature was kept at 250 • C.
Compound separation was carried out in two capillary columns, one with polar stationary phase of poly (ethylene glycol) ( With the non-polar column (DB-5MS), the temperature of the chromatographic oven was programmed from 45 • C (5 min) to 150 • C (2 min) at 4 • C/min, then up to 300 • C (10 min), at 5 • C/min. The temperature of the GC-MS transfer line was set at 230 • C when the polar column was used, and at 300 • C for the non-polar column. The temperatures of the ionization chamber and the quadrupole were 250 • C and 150 • C, respectively. The mass range for the acquisition of ionic currents was mass/charge ratio (m/z) 45-450 µ, with an acquisition speed of 3.58 scan/s. Data were processed with MSDChemStation G1701DA software (Agilent Technologies, Palo Alto, CA, USA). Compound identification was based on their linear retention indices (LRI), calculated from the retention times of the compound of interest and those of the C 6 -C 25 and C 8 -C 40 n-alkanes present in commercial mixtures (Sigma-Aldrich, San Luis, MO, USA), according to Equation (1): LRI = Linear retention index for the compound of interest. n, N = Number of carbon atoms of the n-alkane that elutes before (n), or after (N) the compound of interest (x). t RX = Retention time of the compound of interest (min). t RN , t Rn = Retention times of the n-alkanes that elute before (n) or after (N) the compound of interest (x) (min).
For tentative identification, experimental mass spectra were compared to those in Adams (2007), NIST (2017), and Wiley (2008) spectral databases. Confirmatory identification of some compounds was obtained by comparison of their LRIs and mass spectra with those of available standard substances.
Mature adipocytes were treated or not with different concentrations (0.1, 1, 5, and 10 µg/mL) of EOs distilled from carvone chemotype L. alba. Simultaneously, the in vitro obesity model (high glucose and high insulin-HGHI) characterized and described before [35] was used to evaluate the same parameters as a pathological condition mimicking obesity in vitro with or without the EO.

High Glucose-High Insulin Induction Model (HGHI)
To induce the pathological model of obesity and insulin resistance in adipocytes in vitro, we used the protocol previously established in Bonilla-Carvajal et al., 2022 [35]. Briefly, DMEM medium was added with 450 mg/dL glucose (v/v) and insulin at 10 6 pmol/L (m/v) for 24 h.

Evaluation of Cytotoxicity by MTT
Mature adipocytes that were untreated (normal model) and those with a pathological state-HGHI (HGHI model)-were exposed to carvone chemotype L. alba EO at different concentrations of 0.1, 1, 5, and 10 µg/mL during 8 h; once the cells were treated with EO, the MTT [3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazole bromide] assay was performed as previously described [34,36]. Briefly, the cells were treated with an MTT solution at a concentration of 0.5 g/L (m/v) for 5 h at 37 • C, then the MTT solution was removed and dimethyl sulfoxide (DMSO) was added. Finally, the absorbance was read at 570 nm in the spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Cell Size
Briefly, following the protocol previously established by Bonilla-Carvajal et al., 2022 [35], pathological and normal model cells were exposed to carvone chemotype L. alba EO at different concentrations of 0.1, 1, and 5 µg/mL, and then the samples were stained with Oil Red O to measure the diameter by light microscopy. At least 30 adipocytes were selected by each experiment and per each replicate for cell size measurement. According to the protocol described above, cells were fixed in 4% (m/v) paraformaldehyde (PFA) at room temperature in phosphate-buffered saline [PBS 0.01 mmol/l, pH 7.4] for 15 min, washed with 60% (v/v) isopropanol (Sigma-Aldrich, San Luis, MO, USA) and stained with Oil Red O (Sigma-Aldrich, San Luis, MO, USA) for 30 min. The cells were observed with a Leica DM500 optical microscope (Leica, Wetzlar, Alemania) and the images obtained were analyzed with Image J software.

Lipogenesis (Lipid Synthesis) and Lipolysis (Lipid Breakdown)
Triglyceride content (lipogenesis) and glycerol release (lipolysis) were measured in mature adipocytes treated or not with the HGHI model, following the previously described protocol [34]. Specifically, to evaluate lipogenesis, mature adipocytes previously exposed to the normal or pathological model and stimuli or not with carvone chemotype L. alba EO at different concentrations of 0.1, 1, and 5 µg/mL were used to measure lipolysis, and the cell culture medium was used to measure lipolysis. Once treated with the EO, the culture medium was removed and stored for lipolysis quantification. To evaluate lipogenesis, the adipocytes were washed with PBS 1X, then fixed with PFA at 4% (m/v), and once removed, with isopropanol at 60% (v/v), and were allowed to dry for 15 min. Finally, Oil Red O (Sigma-Aldrich, San Luis, MO, USA) (v/v) was added for 30 min, to be revealed with isopropanol at 100% (v/v). Finally, the samples were read at 540 nm (Thermo Fisher, Waltham, MA, USA).
Regarding lipolysis, in all the samples extracellular glycerol was measured using a colorimetric technique where a Free Glycerol Reagent (Sigma-Aldrich, San Luis, MO, USA) (v/v) kit (Sigma-Aldrich, San Luis, MO, USA) was used, as previously employed [22]. Finally, the glycerol reagent was added, incubated for 15 min at room temperature, and finally read at 540 nm (Thermo Fisher, Waltham, MA, USA).

Adipogenesis
Mature adipocytes were exposed to both cellular models (normal and pathological) and subsequently treated with the carvone chemotype L. alba EO at different concentrations of 0.1, 1, and 5 µg/mL, and were used to perform adipogenesis evaluation. For this purpose, RNA extraction from adipocytes was performed using Trizol (Ambion-Invitrogen, Waltham, MA, USA) and purified with the RQ1 RNase-Free DNase kit (Promega, Madison, WI, USA). Integrity and quantification were performed using NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA), and gene expression of factors important in adipocyte phenotype was determined by real-time PCR. Primers and probes were designed using Biosearch Technologies software, using Forward (F) and Reverse (R) for preadipocyte factor 1 (Pref-1) F: TGCGTGGACCTGGAGAAAG, and R: TGGCAGGGAGAACCATTGATC, for Lipoproteinlipase (Lpl) F: GGCTCTCTGCCTGAGTTGTAGAAAG and R: TCTTGGCTCTCTCTGAC-CTTGTTGA, for Peroxisome Proliferator-activated receptor gamma (Pparg) F: GAAC-CCAGAGTCTGCTGCTGATCTCTG and R: TCAGCGGGAAGGACTTTATGTATATG, and, finally, for CCAAT/enhancer-binding protein Alpha (Cebpa) F: GCGGGAACGCAACG-CAACAACATC and R: CGGTCATTGTCACTGGTCAAC. Measurements were performed on days 0, 3, 6, and 10 of adipocyte differentiation using both cell models and treated or not with the EO. Cells were harvested for real-time PCR (BIORAD, CFX 96, Hercules, CA, USA) gene expression studies of the different markers of importance during adipocyte differentiation (Lpl, Pref-1, Cebpa, Pparg). All results were normalized for the expression of 18S rRNA (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative data analysis was performed with Gen5 software (Agilent, Santa Clara, CA, USA).

Statistical Analysis
Data are expressed as the mean ± SEM of six replicates of at least three independent experiments. Statistical differences between mean values were analyzed using one-way ANOVA followed by Tukey's post hoc test. A p value < 0.05 was considered statistically significant. The statistical analyses were performed using the SPSS/Windows software (v15.0, Chicago, IL, USA) and GraphPad prism software (v7.0, La Jolla, CA, USA).

Characterization of the Essential Oil of Carvone Chemotype L. alba EO
The typical chromatograms of the L. alba carvone chemotype essential oil, obtained by GC/MS on both non-polar DB-5MS (60 m) and polar DB-WAX (60 m) columns, appear in Figures S1 and S2 of the Supplementary Materials. The relative GC (%) amount of each L. alba EO component is registered in Table 1, from which one can observe that the main EO compound was carvone (37.3%), followed by limonene (31.5%), germacrene (11.8%), and β-bourbonene (2.6%), as described elsewhere [19].

Viability
In our study we found, as evidenced in Figure 1, that cells treated with the EO studied at concentrations of 0.1, 1, and 5 µg/mL have cell viability higher than 96% compared to the control (not treated with EO). However, the concentration of 10 µg/mL produced in adipocytes a significant cell viability reduction (p = 0.0001), decreasing up to 40% compared to the control. Therefore, according to ISO 10993-5 [40], a moderate decrease in viability was obtained in adipocytes treated with a concentration of 10 µg/mL. However, with the concentrations of 0.1, 1, and 5 µg/mL, we did not identify a cytotoxic effect on adipocytes.

Cell Size
To identify changes after the EO treatment, cell size was measured (Figure 2a). Normal adipocytes treated with the EO showed a diameter of 0.12 mm (84%), while the control of the normal model without EO treatment had a diameter of 0.15 mm (100%). Therefore, the cell diameter in adipocytes treated with the carvone chemotype L. alba EO significantly decreased (p = <0.0001) up to 16% with respect to the control. Likewise, pathologi-

Cell Size
To identify changes after the EO treatment, cell size was measured (Figure 2a). Normal adipocytes treated with the EO showed a diameter of 0.12 mm (84%), while the control of the normal model without EO treatment had a diameter of 0.15 mm (100%). Therefore, the cell diameter in adipocytes treated with the carvone chemotype L. alba EO significantly decreased (p = <0.0001) up to 16% with respect to the control. Likewise, pathological adipocytes (stimulus with the HGHI model) showed a diameter of 0.14 mm (99%) after treatment with the EO, while the control for pathological adipocytes (not treated with the EO) obtained a diameter of 0.19 mm (128%). Therefore, the cell diameter decreased significantly (p = <0.0001) in the pathological adipocytes (HGHI model) by 28% after carvone treatment compared to the control of the HGHI model.

Lipogenesis
We evaluated lipogenesis for which triglyceride accumulation in adipocyte lipid droplets was quantified. As shown in Figure 3, adipocyte lipogenesis significantly decreased after treatment with the carvone chemotype L. alba EO at concentrations of 1 (p = <0.0001; 73%) and 5 (p = <0.0001; 64%) µ g/mL with respect to the control. Similarly, in pathological adipocytes (HGHI model), a significant decrease in lipogenesis was evidenced after treatment with the EOs at concentrations 0.1 (p = <0.0001; 35%), 1 (p = <0.0001; 36%), and 5 (p = <0.0001; 61%) µ g/mL, with respect to the control. Furthermore, this significant decrease in adipocyte diameter was also evident in Oil Red O-stained images (Figure 2b), where changes in the size and number of lipid droplets can be observed in both cell models after the L. alba EO treatment. Therefore, it was evident that treatment with carvone chemotype L. alba chemotype EO decreases adipocyte diameter in the normal model and the HGHI model. However, in the HGHI model, a greater reversal of adipocyte hypertrophy was evident.

Lipolysis
To evaluate the lipid breakdown process, lipolysis was determined. We found that after treatment with the carvone chemotype L. alba EO, there was a significant decrease in lipolysis in normal adipocytes. Specifically, a dose-dependent lipolysis value of 89% at 0.1 µ g/mL concentration (p = 0.0009), 82% at 1 µ g/mL concentration (p = <0.0001), and 81% at 5 µ g/mL concentration (p = <0.0001) was obtained, as shown in Figure 4. Furthermore, in pathological adipocytes (HGHI model), a reduction of lipolysis was also evidenced, obtaining a value of 51% at a concentration of 0.1 (p = 0.0013), compared to control cells ( Figure 4). Therefore, the carvone chemotype L. alba EO generates a dose-dependent decrease in lipolysis both in normal and pathological adipocytes.  Furthermore, to confirm this decrease in triglyceride accumulation, the relationship between cell diameter and triglyceride accumulation was calculated (Figure 3b). We found a significant decrease after treatment with the carvone chemotype L. alba EO in normal adipocytes (p = 0.0007; 84%) and in pathological adipocytes (HGHI model) (p = 0.0006; 78%) with respect to the controls of each cell model.
The above results suggest that treatment with the carvone chemotype L. alba EO produces a decrease in triglyceride accumulation in the adipocytes of the normal model and the pathological model of hypertrophic adipocytes (HGHI).

Lipolysis
To evaluate the lipid breakdown process, lipolysis was determined. We found that after treatment with the carvone chemotype L. alba EO, there was a significant decrease in lipolysis in normal adipocytes. Specifically, a dose-dependent lipolysis value of 89% at 0.1 µg/mL concentration (p = 0.0009), 82% at 1 µg/mL concentration (p = <0.0001), and 81% at 5 µg/mL concentration (p = <0.0001) was obtained, as shown in Figure 4. Furthermore, in pathological adipocytes (HGHI model), a reduction of lipolysis was also evidenced, obtaining a value of 51% at a concentration of 0.1 (p = 0.0013), compared to control cells ( Figure 4). Therefore, the carvone chemotype L. alba EO generates a dose-dependent decrease in lipolysis both in normal and pathological adipocytes. µ g/mL concentration (p = 0.0009), 82% at 1 µ g/mL concentration (p = <0.0001), and 81% at 5 µ g/mL concentration (p = <0.0001) was obtained, as shown in Figure 4. Furthermore, in pathological adipocytes (HGHI model), a reduction of lipolysis was also evidenced, obtaining a value of 51% at a concentration of 0.1 (p = 0.0013), compared to control cells ( Figure 4). Therefore, the carvone chemotype L. alba EO generates a dose-dependent decrease in lipolysis both in normal and pathological adipocytes.

Adipogenesis
When evaluating the differentiation of adipocytes after treatment with the carvone chemotype L. alba EO, as shown in Figure 5a, for the adipocytes of the normal model, the expression of Pref-1 decreased in all the concentrations of EO used (0.1, 1, and 5 µ g/mL)

Adipogenesis
When evaluating the differentiation of adipocytes after treatment with the carvone chemotype L. alba EO, as shown in Figure 5a, for the adipocytes of the normal model, the expression of Pref-1 decreased in all the concentrations of EO used (0.1, 1, and 5 µg/mL) in the different days of differentiation (0, 3, 6, and 10; p = <0.0001), compared to the control. Furthermore, in adipocytes previously treated with the HGHI model, there was also a decrease in Pref-1 expression on all differentiation days evaluated after treatment with the carvone chemotype L. alba EO chemotype at concentrations 0.1, 1, and 5 µg/mL (p = <0.0001 for all the concentrations). Thus, the carvone chemotype L. alba EO negatively affects dose-dependent Pref-1 expression in the normal model and the pathological model of obesity and insulin resistance.
Upon further evaluation of adipogenesis, we quantified Pparg expression (Figure 5b), finding that adipocytes in the normal model showed an increase in Pparg expression on day 3 of differentiation after treatment with the carvone chemotype L. alba EO in all concentrations used (0.1, 1, and 5 µg/mL; p = <0.0001). Likewise, in adipocytes previously treated with the HGHI pathological model, an increase in Pparg expression was evidenced at day 6 of differentiation in cells treated with the carvone chemotype L. alba EO at all concentrations used (0.1, 1, and 5 µg/mL; p = <0.0001) with respect to the control model. Therefore, the carvone chemotype L. alba EO increases Pparg expression at different days of adipogenesis in both cell models used.
The expression of Cebpa was measured and we found, as shown in Figure 5c, that in the adipocytes of the normal model, the highest Cebpa expression was evidenced on day 3 in cells treated with EO at all concentrations used (0.1, 1, and 5 µg/mL; p = <0.0001). In addition, in the adipocytes of the pathological HGHI model, on day 6 an increase in Cebpa expression was observed after treatment with the carvone chemotype L. alba EO at 0.1, 1, and 5 µg/mL (p = 0.0001 for all the concentrations). Therefore, the carvone chemotype L. alba EO increases Cebpa expression in the normal model and the pathological model of obesity and insulin resistance.
Finally, we determined the effect of the carvone chemotype L. alba EO on Lpl expression, finding, as shown in Figure 5d

Discussion
Obesity is characterized by an expansion of adipose tissue due to hypertrophy and hyperplasia produced in adipocytes [1,2], causing alterations in adipocyte functionality and inflammation, which is directly related to metabolic disease [3]. Because of this, several studies focus their attention on natural treatments that have effects on the pathophysiology of the adipocyte and promote sustained weight loss over time [41]. Therefore, our study investigated the effects of the carvone chemotype L. alba EO on cell viability, lipid mobilization, and adipogenesis in normal adipocytes and in pathological adipocytes simulating a state of obesity and insulin resistance (in vitro HGHI model).
Previous studies have shown that carvone not only demonstrates antioxidant, antimicrobial, anticancer, and anti-inflammatory properties [20][21][22]42], but that the S-carvone isomer reduces obesity induced by a high-fat diet and insulin resistance; it inhibits the inflammation that causes obesity by blocking the expression of genes such as Pparg, transcriptional factor sterol regulatory element-binding protein 1 c (Srebp1c), acetyl-CoA carboxylase 1 (Acc1), and fatty acid synthase (Fas), which are important in the process of lipogenesis. [20].
Another important chemical component identified in the characterization of the carvone chemotype L. alba EO was limonene. In previous research, limonene has been shown to reverse hypertrophy in white and brown adipocytes, as well as to reduce the levels of triglycerides, total cholesterol, low-density lipoproteins (LDL), and glucose in the blood of mice [23]. It has also been shown that limonene improves mitochondrial biogenesis and lipolysis, and induces a phenotype like brown adipocytes, thus having effects on lipid metabolism [21][22][23][24]. Therefore, it could be possible that the chemical components of the carvone chemotype L. alba EO could be involved in the results obtained in this study by having effects on lipid mobilization and adipogenesis of adipocytes.
In our study, we found that a concentration of 10 µg/mL of EO significantly decreased viability. These results contrast with previous studies where L. alba was used and showing that higher concentrations (139.5, 164.9, and 375.5 µg/mL) maintain cell viability [43,44]. This could be explained by the cell lines used (HeLa and Vero cells) [44], which differ from the adipocytes used in this study. However, it is essential to note that lower doses of one of the carvone chemotypes (0.1, 1, and 5 µg/mL) do not affect adipocyte cell viability, this could be due to the major chemical components of the carvone chemotype L. alba EO, since previous studies using limonene (31.5%) [21] alone as a treatment in 3T3-L1 adipocytes did not affect cell viability [22,23].
Furthermore, recognizing that obesity produces a state of adipocyte hypertrophy, we evaluated the effects after treatment with the carvone chemotype L. alba EO, finding a decrease in adipocyte diameter and lipid droplet content, leading to the reversal of adipocyte hypertrophy. These changes in cell size could be because the amount of triacylglycerol (TAG) stored in the adipocyte lipid droplet depends on lipid mobilization, the balance between lipogenesis and lipolysis [4,5]. Therefore, it is evident that treatment with the carvone chemotype L. alba EO decreases adipocyte hypertrophy in both adipocyte models (normal and HGHI). It is notable that in the pathological model a greater reversal of adipocyte hypertrophy was evident. These results could be due to the chemical compounds of the carvone chemotype L. alba EO (37.3%) [21] because, in previous studies where monoterpenes such as limonene were used as treatment, inhibition of lipid accumulation and therefore a decrease in the size of adipocytes and lipid droplets was observed, by enhancing mitochondrial biogenesis, increasing HSL protein levels and causing changes in adipogenesis [4,21,24].
It is also important to note that when the intake of hypercaloric foods is exceeded, and other factors such as lack of physical activity are added, an excessive increase in lipogenesis is generated, leading to an imbalance of homeostasis and, in consequence, increasing adiposity, typical of obesity [4]. In our study, we observed that lipogenesis (lipid accumulation) of adipocytes of the normal and pathological HGHI model was significantly reduced; our results were like results previously described where the effect of different natural extracts such as Fraxinus rhynchophylla and Oroxylum indicum was evaluated, and where inhibition of lipogenesis and adipogenesis were observed [25]. This could be because L. alba EO causes chronic activation of adenosine monophosphate-activated kinase (AMPK) [26], which would decrease lipogenesis and promote adequate lipid mobilization [4]. This has already been demonstrated in previous studies where it was shown that limonene, an important chemical component of the carvone chemotype L. alba EO [21], has been shown to reduce the accumulation of triglycerides in adipocytes by increasing the expression of HSL (hormone-sensitive lipase), FAS, and AMPK [33]. Other plant extracts, such as Citrus paradisi, which has limonene among its active components, have been shown to regulate lipid metabolism through the expression of carnitine palmitoyl-transferase 1a (CPT1a) in an animal model [27]. Therefore, the above results suggest that the carvone chemotype L. alba EO induced a large decrease in triglyceride accumulation in both adipocyte models. However, it is relevant to note that in the lipogenesis study, a more significant decrease in triglyceride accumulation was observed in the pathological adipocytes after EO treatment. This could be explained by the fact that the carvone chemotype L. alba EO had a more substantial reduction of severe hypertrophy, as previously investigated with other extracts using in vivo and in vitro obesity models [28,29].
In this work, we were also able to determine the effect of the carvone chemotype L. alba EO on lipolysis, where we observed a decrease in lipolysis in both models. These results could be because treatment with the EO would influence the AMPK pathway, generating a decrease in lipogenesis and lipolysis [5,6] to maintain energy homeostasis, which is necessary for adipocyte survival. Briefly, AMPK activation would suppress lipolysis by inhibiting HSL, thus preventing diacylglycerol (DAG) hydrolysis [7]. Therefore, the carvone chemotype L. alba EO generates a dose-dependent decrease in lipolysis in adipocyte cell models. In addition, it is important to highlight that this decrease in lipolysis obtained in our study contrasts with other studies [21], where an increase in lipolysis in adipocytes after treatment with limonene was evidenced. However, this could be because limonene was only one of several chemical components that make up the carvone chemotype L. alba EO of this study, and therefore this effect would be caused by the synergistic behavior of all the components.
On the other hand, considering that adipogenesis is related to lipid mobilization in the adipocyte [41,45], we evaluated whether carvone treatment with the carvone chemotype L. alba EO influences adipogenesis in adipocytes from normal and pathological adipocytes in vitro. We evaluated whether carvone treatment with the carvone chemotype L. alba EO has an effect on adipogenesis in adipocytes from normal and pathological adipocytes in vitro, for which we quantified the expression of Pref 1, Pparg, Cebpa, and Lpl in normal and HGHI cell models.
When evaluating Pref-1 expression, we found that after treatment with the carvone chemotype L. alba EO, there is a decrease in Pref-1 expression in both models. This indicates that EO treatment could stimulate early adipocyte differentiation, both in the normal and the pathological state, as previous studies have shown that Pref-1 was a marker of the preadipocyte state [46]. To continue with the evaluation of adipogenesis in the adipocyte, we found an increase in Pparg expression since day 3, and in the pathological model since day 6, with respect to the control. Considering that Pparg expression activity positively feeds back on Pparg activity, and that these two factors enhance each other's expression and maintain the differentiated state [47,48], Cebpa expression was evaluated and had a similar behavior to Pparg. Previous studies have shown that not only is Pparg an activator of differentiation, but it also stimulates the expression of Cebpa [48] by performing a synergistic process in which, when expressed together, they stimulated adipogenesis [47,48]. Furthermore, this increased expression of Pparg induced adipocyte differentiation, while Cebpa participated in adipocyte proliferation and insulin sensitivity [49]. We concluded in our study that in pathological adipocytes there was a late differentiation of adipocytes compared to adipocytes of the normal model by increasing Pparg expression, which would stimulate Cebpa expression, which as previously demonstrated would lead to a higher activation of Pparg expression [47,49].
The above results were related to those observed when evaluating Lpl expression, where we found an increased expression of Lpl in the normal model since day 3 and in the pathological model since day 6, compared to the control. Taking into account that Lpl expression stimulates insulin sensitivity [50], this process of late differentiation of adipocytes in the pathological adipocytes is reflected in a decrease in lipogenesis and lipolysis; furthermore, in this process there could be involved the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling pathway of adipogenesis, which promotes the activity of Cebpa and Pparg activators, in addition to the AMPK pathway that has been shown to reduce Pparg and Cebpa expression.
Therefore, treatment with the carvone chemotype L. alba EO influences adipocyte differentiation by inducing an increase in Pparg, Cebpa, and Lpl expression at different days of differentiation. This could be related to limonene in the carvone chemotype L. alba EO. From the results obtained in previous studies, it was evident that this chemical compound could positively regulate Pparg [23] and Cebpa, triggering an increase in adipogenesis through the Akt signalling pathway in adipocytes [22], which would lead to the reversal of the dysfunctional state of adipose tissue, and thus prevent the state of cellular senescence and inflammation characteristic of obesity. This is in addition to normalizing lipid mobilization, thereby regulating the production of adipokines and cytokines, and thus preventing the onset of metabolic disease, insulin resistance, and diabetes [22].
For all the above-described reasons, the results obtained in our study make it evident that treatment with the carvone chemotype L. alba EO substantially favors a reversal of the pathological state of obesity by influencing lipid mobilization, reduced hypertrophy, and, change the differentiation state of adipocytes.

Conclusions
The carvone chemotype L. alba EO induces a reversion of the pathological state of the adipocyte, causing changes in lipid mobilization and adipogenesis. Therefore, the carvone chemotype L. alba EO could be considered as a possible treatment for obesity, using the adipocyte as a therapeutic target. These studies allow continuity to future in vivo studies, where the L. alba EO treatment could be evaluated in animal models of obesity, focusing on adipocytes.