1. IntroductionAdipocytes 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].Specifically, alteration of adipocyte functionality such as lipid mobilization leads to alterations in triglyceride accumulation (lipogenesis) and its positive stimulation pathways such as lipoprotein lipase (Lpl), insulin, and leptin, as well as its inhibition pathways such as AMPK [3,4,5,6]. Additionally, triglyceride hydrolysis (lipolysis) and its activation pathways such as hormone-sensitive lipase (HSL), triglyceride lipase (ATGL), and monoglyceride lipase (MGL), and the inhibitory pathways such as lipase, insulin, and AMPK, are altered as well [4,5,6,7]. In this context, altered lipid mobilization favors the adipocyte disbalance which, together with hypertrophy and hyperplasia, induces reticulum and mitochondrial stress in adipocytes [8], together leading to a state of chronic inflammation that evokes altered adipokines secretion, fibrosis, hypoxia, and induction of cell death [1].All these modifications lead to the development of obesity pathogenesis. It is considered an epidemic disease with great importance in public health due to the frequency of its presence in the world population and the economic cost it represents for the health systems of different countries [9]. In addition, obesity is currently considered an epidemic of the 21st century since it is expected to maintain a constant increase in its rates in children, adolescents, and adults [9]. Therapies currently used to reduce obesity are mainly based on lifestyle modifications, medications, and bariatric surgery. However, most of them do not induce a sustained weight reduction over time, generating high costs for different countries, individuals, and public health [8]. In this line, it is important to search for new methods that can reduce the effects on adipocyte morphology and functionality as a target to treat obesity. Therefore, natural pharmacological interventions for obesity are necessary and well recognized by the clinical community. In this sense, therapies aimed at reversing hypertrophy, hyperplasia, and reversing the adipocyte effects of obesity may potentially affect obesity management.The essential oils (EOs) distilled from aromatic plants have been confirmed to provide an effect like current therapeutic approaches, and previous studies demonstrated the reversal of obesity in Wistar rats fed high carbohydrate diets [10]. EOs have many biological properties, including antioxidant activity from having redox properties, allowing them to neutralize free radicals, and anti-inflammatory, by inhibiting the release of histamine and the activation of inflammatory mediators [11,12,13,14]. Recently, the effects of some compounds present in medicinal plants on adipocytes have been evaluated with a favorable effect on these cells [15,16,17,18,19,20,21,22,23,24,25,26,27], adipocyte number, and oxidative stress [28,29,30,31]. Moreover, recent data indicate that aqueous extracts of some EOs could improve metabolic alterations [15,32]. Some of these plants have been shown to have antidiabetic, antioxidant, and anti-inflammatory properties [3,6,10,11,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33], including Lippia spp, which has been confirmed in previous studies to influence adipocyte functionality by decreasing lipogenesis, reversing the hypertrophic state, and generating changes in lipolysis and adipogenesis [33]. Specifically, Lippia citriodora showed an effect on some important genes in adipogenesis (CCAAT/enhancer-binding protein Alpha (Cebpa) and Peroxisome Proliferator- activated receptor gamma (Pparg)) and, therefore, modifies lipid accumulation, decreases adipocyte hypertrophy, and affects adipokine release after treatment for 48 h at concentrations of 50 to 400 µg/mL in 3T3-L1 adipocytes [33]. In addition, Lippia alba is known in traditional medicine for its antidiarrheal, antifungal, anticholesterolemic, and analgesic properties [33]. Previous studies have also confirmed its anti-inflammatory/antioxidant properties [32], and hypocholesterolemia properties with high anti-obesity potential have been demonstrated [15]. Likewise, previous research has shown that L. alba chemotypes present many monoterpenes, which have been shown to affect the mevalonate pathway through which different molecules such as cholesterol are produced and, therefore, could have a hypolipidemic capacity [33]. Therefore, considering the properties of L. alba and the importance of adipocytes in the development of the pathophysiology of obesity, this study proposes for the first-time adipocytes as a target using L. alba carvone chemotype for obesity treatment. 4. DiscussionObesity 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 pre-adipocyte 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.