Background
Obesity is a serious health issue of global concern that not only decreases life span but is also associated with the development of many chronic illnesses, such as cardiovascular diseases, type 2 diabetes, hypertension, fatty liver disease and cancer. It is considered the fifth risk of death worldwide [
1,
2].
In 2017, Mexico had the highest global prevalence of obesity in the adult population (15–74 years, 32.4%), while countries like Japan or Korea had the lowest incidence (below 6%). Accelerated urbanisation, the improvement in socioeconomic conditions, and dietary changes are considered responsible for the dramatic and rapid increase in obesity prevalence. Traditional food consumption has decreased in favour of increased consumption of refined foods, as well as sweets and fat/sugar-rich diets [
3]. With obesity rates projected to increase further by 2030, new strategies and cost-effective actions are needed for the prevention and control of obesity in children, adolescents and adults [
4]. Promoting healthy lifestyles in the population is a major challenge for the health sector. Comprehensive public policies aimed at reversing the trend worldwide are required, particularly in Mexico.
Current anti-obesity agents often present disadvantages, while disappointing results may be observed after the arrest of lifestyle modification or pharmacotherapy, indicating a need for alternative treatment modalities that produce better and long-term results of obesity prevention or weight management [
5]. Thus, identifying efficient and easy-to-use agents is a priority for medical research. Herbal supplements and diet-based therapies for weight loss are among the most common, complementary and alternative therapeutic modalities [
6‐
8]. In Mexico, numerous plants have been identified and used in folk medicine to prevent and cure chronic diseases. Among them,
Opuntia spp., including the fruit, stems, seeds and cladodes, exhibit diverse health benefits and high biotechnological potential.
Opuntia cladodes are a good source of dietary fibers, which contributes to reducing body weight [
5,
9‐
11], and the presence of antioxidants could be responsible for the nutritional and protective benefits of
Opuntia-enriched diets in chronic diseases [
6].
Opuntia is a species of cactus native to Mexico. Besides its medicinal purposes, it has been domesticated or naturally selected for food and ornamental use [
6]. Scarce information is available concerning the effects of domestication on the biological properties of
Opuntia, at the molecular and biochemical levels. In previous studies, we highlighted the variations in chemical composition and the anti-atherogenic and anti-tumoral properties of various wild and domesticated
Opuntia varieties [
12‐
14], including
O. streptacantha (OSC),the wildest variety, followed by
O. hyptiacantha,
O. megacantha,
O. albicarpaand
O. ficus-indica (OFI), which is the most known and cultivated species, with the highest degree of domestication.
Rodent studies have shown that
Opuntia extracts modify obesity biomarkers. In Zucker obese rats, Nopal consumption attenuated hepatic steatosis related to obesity and reduced obesity-related metabolic abnormalities. Vinegar or isolated molecules present in
Opuntia cladodes, such as kaempferol or isorhamnetin, used in obese mice models, corroborated the anti-obesity and anti-diabetic potentials of these molecules [
15‐
21]. These studies suggest that remarkable effects could be observed in rat/mouse models of obesity induced by a high-fat diet (HFD), supplemented with
Opuntia cladode powders (OCP). Further results with animal models are required to understand the underlying molecular mechanisms for these effects.
Adipose tissue growth occurs because of an increase in the size of existing adipocytes or the number of adipocytes. An imbalance between energy intake and energy expenditure generates an excess in adipose tissue resulting in obesity. Differentiation of pre-adipocytes into adipocytes involves a comprehensive network involving transcription factors responsible for the expression of key proteins that induce mature adipocyte formation. Adipogenesis also involves changes in cell morphology, induction of insulin sensitivity, and changes in β-cell secretory capacity. Deciphering the mechanism of how certain nutrients affect adipocyte differentiation and adipogenesis is important for the prevention of obesity and related diseases [
22].
The present study was designed to investigate whether
Opuntia species exert anti-obesity properties by examining the anti-adipogenic effect of two
Opuntia cladode powders (OCP) and elucidating the mechanisms underlying such effects. For this purpose, we used powders from the wildest (OSC) and the most domesticated (OFI)
Opuntia varieties. We chose these two species for their highest domestication gradient difference (from the ancestor specie (OSC, growing in wild habitat) to the most propagated
Opuntia for commercial production (OFI). The differentiation of pre-adipocytes into adipocytes was examined by treating 3 T3-F442A cells [
22] with OCP to investigate the effects at the cellular level. The same powders were also tested on an animal model of obesity by feeding Sprague–Dawley rats with a high-fat diet (HFD) supplemented or not with the powders. This animal model allowed us to examine the whole-body level, as the first step towards human trials.
Discussion
In the present study, we investigated the effects on obesity of two OCP, the wildest OSC and the most domesticated OFI, using 3 T3-F442A adipocytes cells and HFD obese rats.
Adipocytes play a central role in the maintenance of lipid homeostasis and energy balance by storing TG or releasing free fatty acids in response to changes in energy demand. These cells represent a good model for investigating molecules able to reduce obesity via an impairment of differentiation and adipogenesis. In this work, we used the well-characterised murine pre-adipose 3 T3-F442A cell line for exploring the effects of OCP on adipogenesis [
29,
30]. Our results indicate that cladode powders from two different species effectively alter adipogenesis by reducing TG accumulation during the differentiation process, without generating cytotoxicity. This observation is in agreement with previous studies showing that OCP (powders or ethanol extracts) may reduce adipocyte differentiation and adipogenesis [
22,
27,
31,
32]. The differentiation of 3 T3-F442A pre-adipocytes in mature adipocytes by insulin includes an increase in both TG content and glucose uptake [
33]. Our data show that high OCP concentrations inhibited the uptake of glucose elicited by insulin in differentiating cells. It is important to emphasize that this low glucose uptake was not associated with an increased TG storage, which would be indicative of insulin resistance. In contrast, the association of low TG content and low glucose uptake elicited by OCP in conditions of preadipocyte differentiation by insulin, supports an inhibitory effect of these agents on the differentiating process into mature adipocytes. Accordingly, it could be hypothesised that OCP and especially OFI treatment, may reduce adipocyte storage of TG and consequently adipocyte hypertrophy. Our data show that both OCP (with OFI being the most effective) exert anti-adipogenic effects in the 3 T3-F442A cell line, at concentrations effective against low-density lipoprotein oxidation, foam cell formation, and atherogenesis in apoE-knockout mice, and in cellular models for colon cancer studies in vitro [
13,
14]. OCP effects could be compared with those of resveratrol, which exerts anti-obesity effects by inhibiting glucose utilisation in 3 T3-F442A cell line [
34].
The chemical composition and the presence of phenolic compounds in the different
Opuntia species has been previously reported [
12]. Among the molecules identified in OCP, flavonoids, quercetin, kaempferols and isorhamnetin, could be implicated in weight loss [
15,
16,
22,
35,
36]. Our previous studies indicated that the levels of flavonoids, quercetin, kaempferols and isorhamnetin detected by mass spectroscopy (LC-MS/MS), are higher in OFI than in OSC [
12], which may explain the higher efficacy of this OCP in reducing HFD-induced weight gain. It is to note that low OCP concentrations were not active, possibly due to very low concentrations of the different phenolic compounds in the powders. These data are in agreement with studies reported by Lee et al. [
16,
36], who showed that purified molecules (isorhamnetin or kampferol) may inhibit adipocyte differentiation and lipid accumulation. Kampferol blocked the phosphorylation of AKT and mTOR, acting on early adipogenic factors, which resulted in attenuation of late adipogenic factors such as C/EBP-α and PPARγ. The same genes (C/EBP-α and PPARγ) and their target genes (LPL, aP2, LXR) were identified as isorhamnetin targets. Thus, these active compounds being present in O
puntia cladodes, they could act at the molecular level by regulating lipid metabolism. As OFI is the most domesticated cultivar, it could be of interest to select OFI species with higher phenolic compound content to improve their anti-obesity properties.
In our study, HFD-fed rats gained markedly more weight than those fed a normal diet, confirming that diet-induced obesity was successful. No noticeable difference was observed in food intake among all groups. Interestingly, OCP supplementation (F-OSC, F-OFI) prevented the weight of gain of animals with comparable food intake. Furthermore, OCP tended to reduce abdominal fat development over the 8 weeks of the diet without reducing food intake. Thus, we can conclude that OCP supplement in food could decrease the body weight gain by repressing the expansion of adipose tissue mass.
Leptin is a secreted peptide encoded by the
obese gene and produced primarily by adipose cells. It plays a vital role in controlling body weight, presumably by acting in the hypothalamus to suppress appetite. Body fat is the most important determinant of circulating leptin levels, but other factors also acutely regulate the production and secretion of leptin, for instance, fasting decreases leptin, while refeeding restores the circulating leptin in both mice and humans [
37]. Adiponectin is also secreted from adipocytes, and low circulating levels have been epidemiologically associated with obesity, insulin resistance, type 2 diabetes and cardiovascular diseases. Adiponectin promotes cell proliferation and differentiation of pre-adipocytes into adipocytes, augmenting programmed gene expression responsible for adipogenesis, and increasing lipid content and insulin responsiveness of the glucose transport system in adipocytes [
38]. Circulating leptin levels are increased in HFD-fed animals, in parallel with a decrease in circulating adiponectin concentrations. In our study, serum adiponectin concentration was noticeably reduced in HFD-fed rats, which was reversed by OCP supplementation, suggesting that OCP treatment activated the adipocyte production of adiponectin. Moreover, serum leptin levels were increased in all HFD groups compared with the standard diet (SD), but the leptin levels were lower in the F-OSC and F-OFI groups. Leptin is known to regulate food intake and stimulates energy expenditure. As no differences were observed in food intake, the anti-obesity effects of OCP could be related to increased leptin sensitivity and modifications in energy expenditure. All these results are in agreement with an improvement in blood parameters related to obesity due to OCP supplementation in HFD. The same results were observed in a mouse model of diet-induced obesity, using isorhamnetin glycosides extracted from OFI [
15], or using different bioactive compounds extracted from seaweed or ginseng leaf or Korean red ginseng on insulin sensitivity [
8,
33].
A HFD is known to induce NAFLD in animal models and humans by causing fat deposition in the liver [
39,
40]. NAFLD is closely associated with obesity. In our HFD-fed rat model of obesity, we showed that OCP supplement in the diet tends to lower the liver weight, which can be correlated to less TG storage in the liver. Similarly, Moran-Ramos et al. demonstrated that
Opuntia cladode consumption attenuates hepatic steatosis in obese Zucker rats [
18], and other studies based on HFD supplemented with quercetin [
35] or
Vignanakashimae extracts (another flavonoid-rich plant) [
32] also resulted in a lowering of body weight gain and hepatic lipid accumulation. Taken together, these results suggest that OCP could be efficient against fatty liver in HFD obese rats. It is noteworthy that OCP treatment did not cause any detectable adverse toxic effects on the liver.
Uebelhack et al. [
9] and Chong et al. [
41] illustrated that the effects of
Opuntia-derived fibers act in reducing dietary fat absorption in human volunteers, by binding to dietary fat and increasing its excretion in faeces, probably by decreasing fat intestinal absorption. To further understand the mechanisms explaining the lower weight gain induced by
Opuntia supplementation in a HFD, we evaluated the faecal excretion of fats in our rat model, which was markedly increased in rats fed a HFD, and was much more pronounced when rats were supplemented with OFI. These findings support the hypothesis that the effects of OCP on weight are achieved by reducing dietary fat absorption, leading to lower energy intake and thus, to a lesser weight gain. Finally, nopal anti-inflammatory effects had been identified. Bouhini et al showed a reduction in low-grade chronic inflammation associated with obesity, this could be due to the effect of nopal fibers on the gut microbiota [
17,
20].
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