Because the potential of yerba maté (Ilex paraguariensis) has been suggested in the management of obesity, the aim of the present study was to evaluate the effects of yerba maté extract on weight loss, obesity-related biochemical parameters, and the regulation of adipose tissue gene expression in high-fat diet–induced obesity in mice. Thirty animals were randomly assigned to three groups. The mice were introduced to standard or high-fat diets. After 12 weeks on a high-fat diet, mice were randomly assigned according to the treatment (water or yerba maté extract 1.0 g/kg). After treatment intervention, plasma concentrations of total cholesterol, high-density lipoprotein cholesterol, triglyceride, low-density lipoprotein (LDL) cholesterol, and glucose were evaluated. Adipose tissue was examined to determine the mRNA levels of several genes such as tumor necrosis factor-α (TNF-α), leptin, interleukin-6 (IL-6), C-C motif chemokine ligand-2 (CCL2), CCL receptor-2 (CCR2), angiotensinogen, plasminogen activator inhibitor-1 (PAI-1), adiponectin, resistin, peroxisome proliferator-activated receptor-γ2 (PPAR-γ2), uncoupling protein-1 (UCP1), and PPAR-γ coactivator-1α (PGC-1α). The F4/80 levels were determined by immunoblotting. We found that obese mice treated with yerba maté exhibited marked attenuation of weight gain, adiposity, a decrease in epididymal fat-pad weight, and restoration of the serum levels of cholesterol, triglycerides, LDL cholesterol, and glucose. The gene and protein expression levels were directly regulated by the high-fat diet. After treatment with yerba maté extract, we observed a recovery of the expression levels. In conclusion, our data show that yerba maté extract has potent antiobesity activity in vivo. Additionally, we observed that the treatment had a modulatory effect on the expression of several genes related to obesity.
Yerba maté (Ilex paraguariensis) is one of the most widely consumed plants in South America. It grows naturally or is cultivated in Argentina, Brazil, Uruguay, and Paraguay. Maté beverages have been reported to have various biological activities, which have been attributed to the high polyphenol content of yerba maté. The phenolic compounds have long been known to possess biological functions. In addition to polyphenols such as flavonoids (quercetin and rutin) and phenolic acids (chlorogenic and caffeic acids), yerba maté is also rich in caffeine and saponins (1).
Recently published evidence have shown some beneficial effects of I. paraguariensis, which include antioxidant activity (2), a protective effect against induced DNA damage (2), vasodilatation effects (3), inhibition of glycation and atherosclerosis (4), thermogenic effects (5), an improvement in glucose tolerance (6), and antiobesity effects (7).
Obesity is a growing problem, resulting in significant morbidity and mortality due to weight-related disease as well as a reduced quality of life. The defect in energy balance that causes obesity and visceral adiposity is a serious problem that predisposes individuals to complications such as atherosclerosis, hepatic steatosis, and type 2 diabetes (8). The increasing incidence of obesity suggests that this epidemic will only worsen in the future (9).
It is recognized that the obesity is associated with chronic mild inflammation in which the metabolism of adipose tissue plays an important role (10). The adipose tissue is an endocrine organ which has a fundamental role in metabolism and homeostasis regulation through the secretion of several biologically active adipokines with different protein structures and functions including cytokines (leptin, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6)), chemoattractant proteins (CCL receptor-2 (CCR2) and C-C motif chemokine ligand-2 (CCL2)), proteins involved in the regulation of blood pressure, vascular homeostasis or angiogenesis (angiotensinogen and plasminogen activator inhibitor-1 (PAI-1)), molecules involved in glucose and lipid metabolism (adiponectin and resistin), adipogenesis peroxisome proliferator-activated receptor-γ (PPAR-γ), and thermogenesis (PPAR-γ coactivator-1α (PGC-1) and uncoupling proteins (UCPs)) (10).
There are data showing that animal models are a useful tool to evaluate the efficacy of potential compounds in the prevention and treatment of obesity. It has been reported that rodents fed high-fat diet are an excellent model of obesity where dietary environment is a major contributor (11). Additionally, in animal models, chronic exposure to a high-fat diet induces adipogenesis and the metabolic syndrome, and may modulate the inflammatory responses. Thus, the aim of the present study was to evaluate the effects of yerba maté extract on weight loss, obesity-related biochemical parameters, and the regulation of adipose tissue gene expression in high-fat diet–induced obesity in mice.
Methods and Procedures
Maté tea preparation
The roasted yerba maté extract was prepared by dissolving instant maté tea powder (Leao Jr, Curitiba-PR, Brazil) in pure water (25 mg/ml) using a homogenizer and was prepared fresh each day. The yerba maté extract and vehicle (pure water) were administrated by intragastric gavage, to guarantee total ingestion. The animals were treated for 8 weeks and received 1 g of instant yerba maté/kg body weight.
Total phenolic concentration and chromatographic analysis by high-performance liquid chromatography
The total phenolic concentration was measured by the Folin Ciocalteau methodology, as described elsewhere (12). Chlorogenic acid (Sigma, St Louis, MO) was used as a standard and total polyphenol concentration was expressed as equivalents to chlorogenic acid per g of instant maté tea.
The instant maté tea was analyzed with no other modification than the appropriate dilution in the mobile phase to fit the standard curves. A Thermo Separation Products high-performance liquid chromatography equipped with Spectra Series Gradient Pump, an automatic Spectra Series Autosampler and a UV/VIS SpectraSystem UV/VIS detector was used for the determinations. All the modules were controlled by a personal computer equipped with the high-performance liquid chromatography System Manager SN4000 (SpectraSystem, San Jose, CA). A 4.6 × 250 mm, 5 µm C18 Microsorb column was used for the separation. Injection volume was 20 µl. The analytical determination was carried out by means of high-performance liquid chromatography using a two-solvent isocratic elution. The composition of the solvents was: (A) water/acetic acid (99.5:0.5 v/v) and (B) methanol. The mobile phase composition was 75% of solvent A and 25% of solvent B. The flow rate was 0.8 ml/min. Data were obtained at 272 nm for caffeine and theobromine and 323 nm for phenolic acids. The identification of phenolic compounds and methylxantines was based on the comparison of the spectra obtained between 250 and 350 nm and the retention time of the unknown substances in relation to that of pure standards. Quantification was achieved by external calibration, using a five-point curve of different dilutions of a standard solution. Pearson's correlation coefficient (r) was always >0.99. The phytochemicals from maté tea were shown in Table 1.
Table 1. Phytochemicals from maté tea (mg/g/day)
Animals and diets
Thirty 6-week-old male Swiss strain mice (Sw/Uni) (27.3 ± 0.4 g) free of specific pathogens were obtained from Centro Multidisciplinar Para Investigacao Biologica (State University of Campinas, Campinas, Brazil). The experiments were performed in accordance with the principles outlined by the Brazilian College for Animal Experimentation (Colegio Brasileiro de Experimentacao Animal) and received an approval from Ethics Committee of the São Francisco University, Bragança Paulista, Brazil. The animals were maintained on a 12 h/12 h artificial light–dark cycle and housed in individual cages.
After random selection, mice were introduced to standard (s.d., n = 10) or high-fat diets (n = 20) for 16 weeks. After the first 8 weeks on a high-fat diet the obesity status was observed and the animals were randomly divided into two subgroups in accordance with the treatment: one group (HF-Maté, n = 10) received an aqueous extract of roasted yerba maté extract (1.0 mg/kg) and the other group received the vehicle (HF-C, n = 10) for 8 weeks. The compositions of the experimental diets are shown in Table 2. Body weight was measured twice a week during the feeding period. At the end of the experiment mice were deeply anaesthetized (1:1 xilazine–ketamine) and blood samples were collected from the heart. Then, transcardiac perfusion with 70 ml isotonic saline solution (4 ºC) was carried out over a period of 6 min. White adipose tissue (WAT) from the epididymal depot was dissected and weighed. The plasma, epididymal WAT, and interscapular brown adipose tissue (BAT) samples were stored at −80 °C until they were analyzed.
Table 2. Experimental diets composition
Plasma concentrations of total cholesterol (CHOL), high-density lipoprotein cholesterol, and triglyceride were determined using the Cobas-Mira System (Roche Diagnostics, Indianapolis, IN). Plasma LDL cholesterol concentration was calculated according to the formula: CHOL−(triglyceride/5 + high-density lipoprotein). The glucose levels in serum were also determined using the Cobas-Mira System (Roche Diagnostics).
RNA extraction and quantitative real-time PCR
WAT and BAT tissue fragments were collected, snap frozen, and stored at −80 °C in RNAlater (Qiagen, Valencia, CA). Total RNA was isolated using the RNeasy tissue kit (Qiagen). Single-stranded cDNA was synthesized using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. Quantitative real-time PCR was performed using a 7300 real-time PCR System (Applied Biosystems), and threshold cycle numbers were determined using RQ Study Software (Applied Biosystems). Reactions were performed in triplicate, and threshold cycle numbers were averaged. The 50 µl reaction mixture was prepared as follows: 25 µl of Platinum SYBR Green Quantitative PCR SuperMix-UDG (Invitrogen Life Technologies, Alemeda, CA), 10 µmol/l of each primer (Table 3), and 10 µl of cDNA (100 ng). The reaction was cycled with preliminary Uracil–DNA glycosylase treatment for 2 min at 50 °C and a denaturation step for 2 min at 95 °C, followed by 45 cycles of denaturation at 95 °C for 15 s, annealing for 15 s, and primer extension at 72 °C for 15 s. This was followed by melting point analysis of the double-stranded amplicons consisting of 40 cycles of 1 °C decrement (15 s each) beginning at 95 °C. The first derivative of this plot, dF/dT, is the rate of change of fluorescence in the reaction, and a significant change in fluorescence accompanies the melting curve of the double-stranded PCR products. A plot of –dF/dT vs. temperature displays these changes as distinct peaks. TNF-α, leptin, IL-6, CCL2, CCR2, angiotensinogen, PAI-1), adiponectin, resistin, PPAR-γ2, UCP1, and PGC-1α expression were examined and normalized to a constitutive gene (β-actin), and the relative fold induction was calculated according to the formula 2(−ΔΔCt) (13).
Table 3. Primers user in quantitative real-time PCR
Protein extraction and western immunoblot analysis
Epididymal fat tissue fragments were excised and immediately homogenized in solubilizing buffer at 4 °C (1% Triton X-100, 100 mmol/l Tris–HCl (pH 7.4), 100 mmol/l sodium pyrophosphate, 100 mmol/l sodium fluoride, 10 mmol/l EDTA, 10 mmol/l sodium orthovanadate, 2.0 mmol/l PMSF, and 0.1 mg aprotinin/ml). Insoluble material was removed by centrifugation for 20 min at 9,000 g at 4 °C. The protein concentration of the supernatants was determined by the Biuret method. The extracts were treated with Laemmli sample buffer containing 100 mmol/l dithiothreitol and heated in a boiling water bath for 5 min, after which they were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis in a Bio-Rad miniature slab gel apparatus (Mini-Protean). For immunoblot experiments, 0.15 mg of protein extracts from each tissue was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and blotted with anti-F4/80 or anti-β-actin. The nitrocellulose membranes were developed using commercial chemoluminescent kits. Band intensities were quantitated by optical densitometry (Scion Image software; ScionCorp, Frederick, MD) of the developed autoradiographs.
Data are expressed as the mean ± s.e.m. Comparisons among groups of data were done using one-way ANOVA followed by the Dunnett Multiple Comparisons test. An associated probability (P value) of <5% was considered significant.
Body weight and biochemical analysis
After 8 weeks on a high-fat diet there was a significant increase in body weight as well as in glucose blood levels compared to animals fed the standard diet (data not shown). At the end of the study the total body weight of animals fed with the standard diet was lower than those fed the high-fat diet (P < 0.01, Figure 1). Regular ingestion of yerba maté extract significantly decreased the final body weight (P < 0.05), when compared to the HF-C group. The yerba maté intervention did not affect the food intake. The epididymal fat weight of mice fed the high-fat diet was significantly higher than those fed the standard diet (P < 0.01) (Table 4).
Table 4. Anthropometric and biochemical parameters
The serum levels of cholesterol, high-density lipoprotein cholesterol, triglycerides, and LDL cholesterol were significantly higher among the HF-C group when compared to animals fed the standard diet. Additionally, the high-fat diet caused an increase in glucose serum levels (Table 4). The treatment with yerba maté extract for 8 weeks decreased serum levels of cholesterol, triglycerides, LDL cholesterol, and glucose, when compared to the HF-C group (Table 4).
Adipokines mRNA expression
The mRNA expression levels of TNF-α, leptin, IL-6, CCR2, CCL2, angiotensinogen, PAI-1, adiponectin, resistin, PPAR-γ2, and PGC-1α were determined in WAT isolated from the previously described animals. Quantitative real-time PCR analysis was also performed to evaluate the expression of PGC-1α and UCP2 in interscapular BAT. In WAT, the high-fat diet caused an upregulation of the TNF-α, IL-6, leptin, CCR2, CCL2, angiotensinogen, and PAI-1 genes, significantly downregulated adiponectin and PPAR-γ2, and did not affect the expression of resistin and PGC-1α (Table 5). In BAT, the high-fat diet significantly downregulated the expression of UCP1, PPAR-γ2, and PGC-1α. After treatment with yerba maté extract, the expression levels of cytokines (TNF-α, IL-6, and leptin), chemoattractant proteins (CCR2 and CCL2), and genes involved in the regulation of blood pressure, vascular homeostasis or angiogenesis (angiotensinogen and PAI-1) were significantly reduced. On the other hand, the downregulation of genes implicated in adipogenesis (PPAR-γ2) and glucose and lipid metabolism (adiponectin) were reversed (Table 4). In addition, the yerba maté treatment recovered the expression of genes implicated in thermogenesis (PGC-1α and UCP1) in BAT (Table 5).
Table 5. Gene expression determined by quantitative real time PCR
Macrophage infiltration in adipose tissue
The level of F4/80 was increased in WAT from the HF-C group when compared with animals fed the standard diet and was significantly decreased after treatment with yerba maté extract (Figure 2). F4/80 is a specific marker of activated macrophage infiltration in the adipose tissue.
In this study, yerba maté was evaluated for its putative effects on weight loss, obesity-related biochemical parameters, and the regulation of adipose tissue gene expression in high-fat diet–induced obese mice. Our data clearly demonstrated that when treated with yerba maté, obese mice exhibited marked attenuation of weight gain adiposity, a decrease in epididymal fat-pad weight, and a restoration of the serum levels of cholesterol, triglycerides, LDL cholesterol, and glucose, as previously reported (4,5,6).
Yerba maté has been reported to have various biological activities which have been mainly attributed to its high polyphenol content (1). Chlorogenic acid, the main polyphenol in yerba maté, is thought to modulate the activity of glucose-6-phosphatase, which is involved in glucose metabolism (14), and reduce the risk of cardiovascular disease by decreasing the oxidation of LDL and cholesterol (15). In this sense, our results are in accordance with previous studies that have shown that I. paraguariensis treatment improves glucose tolerance in obese animals (4,7). In addition to chlorogenic acid, the presence of methylxantines is also thought to account for some of the pharmacological effects of yerba maté (6). The saponins, another important compound found in yerba maté, have been reported to interfere with cholesterol metabolism (16). Thus, the effects on cholesterol levels could be partially attributed to its saponin content. The data presented in this study suggested that the compounds found in yerba maté extract may act synergistically to suppress body weight gain, visceral fat accumulation, and decrease the serum levels of cholesterol, triglycerides, LDL cholesterol, and glucose.
The adipose tissue is an endocrine organ which has a fundamental role in metabolism and homeostasis regulation. The production and secretion of an excess or insufficient amount of adipokines greatly influence insulin sensitivity, glucose metabolism, inflammation, and atherosclerosis, and may provide a molecular link between increased adiposity and the development of diabetes mellitus, metabolic syndromes, and cardiovascular diseases (17). In the present study the expression of TNF-α, IL-6, leptin, CCR2, CCL2, angiotensinogen, PAI-1, adiponectin, PPAR-γ2, PGC-1α, and UCP1 in adipose tissue were directly regulated by high-fat diet. Additionally we described that treatment with yerba maté extract was able to recover the expression of these genes.
Obesity is associated with a state of chronic, low-grade inflammation characterized by abnormal cytokine production and the activation of inflammatory signaling pathways in WAT (18). Obesity leads to increased production of several inflammatory cytokines, which play a critical role in obesity-related inflammation and metabolic pathologies. TNF-α is a potent cytokine that induces the production of IL-6 (19), which is the major determinant of the acute phase response, and also has effects on glucose transport, lipid metabolism, and insulin action (20). It has been reported that in obese individuals and animal models, the levels of TNF-α and IL-6 are persistently elevated (20), and a reduction of adipose mass leads to a decrease in these expression levels (20). Additionally, TNF-α can also modulates leptin secretion by increasing its gene expression and circulating levels (21). Although leptin levels have been positively associated with the amount of body fat (22), a reduction in body weight could lead to a reduction in leptin levels, as previously reported (22). Our data showed that adipose tissue upregulates TNF-α, IL-6, and leptin mRNA expression, therefore it seems reasonable that the anti-inflammatory effects observed after yerba maté extract treatment could be attributed to the observed reduction on adipose mass, and to an intrinsic anti-inflammatory activity of yerba maté compounds (1).
Obesity also induces the accumulation of macrophages, through the monocyte chemoattractant protein-1 (CCL2)/CCR2 pathway, which produces some of the pro-inflammatory molecules, released by adipose tissue and have been implicated in the development and maintenance of obesity-induced adipose tissue inflammation (23). Adipose tissue expression of (CCL2) and its receptor (CCR2) are increased in proportion to obesity, as described in the present study (24). As CCR2 can regulate monocyte and macrophage chemotaxis as well as local inflammation responses (25), our data indicate that inhibition of CCL2/CCR2 could explain the low level of macrophage infiltration observed after yerba maté extract treatment, thus contributing to reduce the adipose tissue inflammation.
The chronic and low level inflammation provided by adiposity has been associated with obesity-related health problems (18). PAI-1 is an important endogenous inhibitor of tissue plasminogen activator and is a major determinant of fibrinolytic activity. PAI-1 contributes to the pathogenesis of atherothrombosis and cardiovascular diseases (26). It has been shown that the elevation of PAI-1 levels can be attributed to its upregulation in WAT (27). Accordingly, the data presented in this and other studies have shown a positive correlation between PAI-1 levels and increased visceral fat depots (28). However, weight loss is associated with a reduced PAI-1 activity in obese subjects (28). Additionally, it has been reported that TNF-α can regulate PAI-1 expression (29). As it has been shown that I. paraguariensis extract can inhibit the progression of atherosclerosis (4), the data presented in this study showed that this could be due to the ability of yerba maté extract to restore the mRNA levels of PAI-1. Therefore, the downregulation of PAI-1 seems to be due to the decrease in visceral fat-pad weight and the downregulation of TNF-α, which might be a protective factor against cardiovascular diseases.
Additionally, hypertension is also a frequent complication of obesity and a major risk for the development of cardiovascular diseases. A positive correlation between blood pressure and circulating levels of angiotensinogen has been demonstrated (30). In animal models, the upregulation of angiotensinogen mRNA in WAT resulted in elevated plasma angiotensinogen, hypertension, and increased WAT mass (30). In addition, angiotensinogen-deficient mice are partially protected from diet-induced obesity (31). Our data showed that the mRNA levels of angiotensinogen were significantly increased in obese animals, and the yerba maté supplementation was able to decrease those levels, which could lead to a putative protection against hypertension.
Adiponectin is the most abundantly expressed adipokine in WAT (32). It is a multifunctional protein that exerts pleiotropic insulin-sensitizing effects. It lowers hepatic glucose production and increases glucose uptake and fatty acid oxidation in skeletal muscle (32). Additionally, an anti-inflammatory role of adiponectin has also been reported (32). Unlike most adipokines, adiponectin mRNA in WAT and its serum levels are decreased in obese individuals (33). It has been reported that in animal models adiponectin levels decreased with weight gain when the animals became obese (33). However, weight loss resulted in significant increases in circulating adiponectin levels (33). The results presented here demonstrated that yerba maté extract supplementation can recover the adiponectin mRNA levels, as an effect of weight loss.
Adipogenesis is the developmental process by which a multipotent mesenchymal stem cell differentiates into a mature adipocyte. PPARγ was found to be a master regulator of adipogenesis (34). PPAR-γ2, a splicing isoform of PPARγ, is expressed selectively in the adipose tissues and promotes the differentiation and proliferation of adipocytes causing an increase in adipose tissue (35). In the present study the mRNA levels of PPAR-γ2 were downregulated in visceral adipose tissue from mice fed the high-fat diet. Although it has been reported that a high-fat diet increases the expression levels of PPAR-γ2 (36), our data show that the high-fat diet significantly decreased the mRNA levels genes as previously described (7). Accordingly, it appears that chronic exposure to high-fat diets leads to an adaptive response that aims to limit the expansion of fat storage (7). The results presented in this study show that treatment with yerba maté extract recovered the mRNA levels of PPAR-γ2 as previously described (7).
In contrast to WAT, which is mainly used for fat storage, BAT uses lipids as a fuel for adaptative thermogenesis. BAT is characterized by small, multilocular lipid droplets and high numbers of mitochondria (37). Recently, a coactivator of PPARγ, PGC-1α has been identified and seems to stimulate mitochondrial biogenesis and respiration in muscle by inducing the expression of UCPs (38). Several studies have focused on adaptive thermogenesis by UCP families (UCP1, UCP2, and UCP3) as a physiological defense against obesity and diabetes (38). UCP-1 expression in BAT is known to be a significant component of whole body energy expenditure, and its dysfunction contributes to the development of obesity (39). Our data showed that a high-fat diet downregulates the expression of PGC-1α and UCP1 in BAT, which may have decreased energy expenditure and increased diet-induced obesity. The present study also showed that PGC-1α and UCP-1 mRNA levels in BAT were recovered after yerba maté treatment, which could be attributed to the methylxantine content of yerba maté, as suggested previously (7).
In conclusion, this study reports that yerba maté extract has potent antiobesity activity in vivo. Additionally, we observed that the treatment has a modulatory effect on the expression of several genes related with the obesity process.
We thank Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (06/61797-0), Financiadora de Estudos e Projetos (FINEP), and Leão Junior S/A for financial support.