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β-Asarone modulate adipokines and attenuates high fat diet-induced metabolic abnormalities in Wistar rats
Pharmacological Research 103 (2016) 227–235
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Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs
-Asarone modulate adipokines and attenuates high fat diet-induced metabolic abnormalities in Wistar rats Malesh M. Thakare ∗ , Sanjay J. Surana Department of Pharmacology, RC Patel Institute of Pharmaceutical Education and Research, Shirpur 425 405, Dhule, Maharashtra, India
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Article history: Received 19 April 2015 Received in revised form 1 December 2015 Accepted 1 December 2015 Available online 7 December 2015 Keywords: -Asarone Adipokines High fat diet Obesity Diabetes Dyslipidemia
a b s t r a c t Here we investigated the effect of -asarone on food preference and its therapeutic potential against high fat diet (HFD) induced obesity in rats. In food preference study, free access to HFD was given only for 4 h in addition to standard laboratory chow in rats and the preferential intake between chow and HFD was measured. For obesity induction, HFD was administered for 12 weeks and the HFD fed rats were treated with -asarone in the last 4 weeks, starting from 9th week onwards. Food intake, body weight was measured biweekly. Glucose tolerance and the levels of glucose, lipids, free fatty acids, leptin, and adiponectin were assessed. HFD fed rats showed progressive increase in body weight and developed glucose intolerance and dyslipidemia. In addition, they showed increased adiposity and the disturbed pattern of adipokine levels In the food preference paradigm, -asarone produced selective decrease in HFD intake in rats. In obese rats, -asarone treatment not only reduced body weight but also prevented HFD-induced metabolic alterations, including glucose intolerance, dyslipidemia and adipokine imbalance. The observed beneficial effects of -asarone appear due its ability to reduce intake of energy dense food by affecting food palatability, and to normalize the levels of leptin and adiponectin in rats. Overall, our results suggest that -asarone is a novel candidate molecule with significant therapeutic potential in the management of obesity and associated abnormalities. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Obesity is a worldwide epidemic [1] and a heterogeneous complex disorder of multiple etiologies characterized by excessive deposition of body fat [2]. Obesity usually develops as a result of an imbalance between caloric intake and expenditure, which contribute to co-morbid conditions like hypertension, hyperlipidemia, type II diabetes, coronary heart disease, stroke and cancer [3–5]. In developed countries, highly palatable foods are available in almost unlimited abundance, and overconsumption undoubtedly contribute to the epidemic [6,7]. Sedentary lifestyles, high-fat, energy-dense diets, and a genetic predisposition all contribute to the obesity epidemic [8].
Abbreviations: HFD, high fat diet; FFA, free fatty acid; AC, Acorus calamus; IAEC, institutional animal ethics committee; INSA, indian national science academy; DTNB, 5,5 dithiobis (2-nitrobenzoic acid); HDL, high density lipoprotein; p.o., peroral; BMI, body mass index; OGTT, oral glucose tolerance test; ELISA, enzyme linked immunosorbent assay; PBS, phosphate buffer saline; TBARS, thiobarbituric acid reactive substances; BSA, bovine serum albumin; TC, total cholesterol; TG, triglycerides; MDA, malondialdehyde; GSH, reduced glutathione; ANOVA, analysis of variance. ∗ Corresponding author. E-mail address: [email protected] (M.M. Thakare). http://dx.doi.org/10.1016/j.phrs.2015.12.003 1043-6618/© 2015 Elsevier Ltd. All rights reserved.
Herbs and phytochemicals have been known to play a major role in drug discovery process including anti-obesity molecules [9,10]. In recent years, the extract of Acorus calamus (AC) has been reported to improve metabolic functions [11,12]. A. calamus L., family Araceae, has been used in the Indian and Chinese systems of medicine for its valuable role in several CNS disorders [13–15]. In recent years, the extract of AC has been reported to hold hypoglycemic, hypolipidemic, insulin sensitizing and ␣glucosidase inhibitory activities [11,12,16], suggesting that it may play a role in regulation of metabolism. Moreover, ethyl acetate fraction of AC has also been reported to improve insulin release and attenuate postprandial hyperglycemia and cardiovascular complications in mice [17]. -Asarone, a major constituent of AC has been reported to produce anti-adipogenic and lipolytic actions in 3T3L1 cells, an action most likely exerted by subduing the expression of adipogenic transcription factors [18]. Thus, -asarone might be useful to correct energy imbalance and may prove to be beneficial in the management of obesity. Obesity can be characterized into two main types, hyperplasia (cell number increase) and hypertrophic (cell size increase) adipocytes. As a metabolic and endocrine organ, adipose tissue plays a critical role in regulation of energy balance, lipid metabolism and insulin action [19]. A chronic imbalance between energy intake and energy expenditure can lead to adipocyte
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hypertrophy and adipocyte hyperplasia [20], the former being thought to be particularlyimportant in determination of fat mass in adults [21]. As discussed above, the anti-adipogenic potential of -asarone may be useful to limit energy imbalance and obesity. The emphasis of the present study was placed on detailed investigation of the effect of -asarone on food palatability in food preference paradigm and its therapeutic potential against high fat diet-induced metabolic abnormalities in rats. 2. Materials and method 2.1. Experimental animals The experiments were carried out in male Wistar rats (250–300 g) obtained from Central Animal House facility of RC Patel Institute of Pharmaceutical Education and Research, Shirpur425 405, Maharashtra, India. The rats were kept in polyacrylic cages (3/cage) and maintained under standard husbandry conditions (room temperature 22 ± 2 ◦ C and relative humidity of 50–55%) with 12 h light/dark cycle (lights on at 8AM). The food in the form of dry pallets and water were made available ad libitum . The protocol was reviewed and approved by the Institutional Animals Ethics Committee (IAEC) and the animal experiments were carried out in accordance with the Indian National Science Academy (INSA) guidelines for use and care of animals. 2.2. Drugs and chemicals -Asarone (1,2,4-trimethoxy-5-[(Z)-prop-1-enyl]benzene) and DTNB [5,5 dithiobis (2-nitrobenzoic acid)] etc., was procured from Sigma–Aldrich, USA. Casein (Modern Diaries Ltd., Karnal, India), Cholesterol (HIMEDIA Laboratories Pvt. Ltd., Mumbai, India) and dl-Methionine (CDH, New Delhi, India) was used to prepare high fat diet. The diagnostic kits for serum glucose, cholesterol, HDL, triglycerides were obtained from Coral Diagnostics Ltd., Mumbai, India. All other chemicals used in the present study were of analytical grade. 2.3. Methodology 2.3.1. Experiment No.1— food preference paradigm In the food preference paradigm, rats were fed with equal amounts of standard laboratory chow (Ashirwad Industries Ltd., India, providing 7.82% as fat, 20–21% of calories as protein, 47% of calories as carbohydrate and containing a total of 3.6 kcal/g) and additional high fat diet (HFD) (58% fat, 25% protein and 17% carbohydrate, and containing a total of 5.2 kcal/g) [22]. Both diets were presented in separate bowls placed in opposite corners and were administered for one week daily for 4 h in addition to normal chow to habituate the animals to them and to achieve the baseline value of food intake. During habituation, the placement of food was changed daily to avoid placement preference and intake measurements were corrected for spillage. Following establishment of stable baseline chow and HFD intake, animals were treated with either vehicle (2 ml/kg) or -asarone at different doses (25 and 50 mg/kg, p.o.) During the experiment a pre-weighed amount of powdered chow and HFD was placed in the home cage and the amount of chow and HFD consumed was recorded for 4 h [23]. 2.3.2. Experiment No.2—diet-Induced obesity model-experimental procedure A total of 30 male Wistar rats, (weighing 200–250 g) were used in the study. The rats were kept on two dietary interventions, one group of rats (n = 6) was supplied with standard laboratory chow (Ashirwad India) and assigned as normal control (NC-Group 1), the remaining rats were given free access high fat diet (n = 24) for 12 weeks. The composition and preparation of HFD was the same as
described by Srinivasan et al., [22]. The HFD fed rats were randomly divided into 4 groups (each consisting of n = 6). Group 2 rats were fed with HFD and received vehicle treatment for the last four weeks (propylene glycol); Group3, 4 and 5 - HFD fed rats received treatment of -asarone in the last 4 weeks [from 9th to 12th week] at 12.5, 25 and 50 mg/kg p.o, respectively. (A) Anthropometric parameters The increase in the body weight was regarded as an index of obesity. Body weight was assessed biweekly. For individual rats, food intake measurements were recorded biweekly and taken as an average value. At the end of the experiment the adipose tissues (epididymal, retroperitoneal and mesenteric fat depots) were isolated, freed from surrounding tissues, weighed individually and the total weight was calculated [24]. • Biochemical parameters [Blood, Serum & Plasma] i Oral glucose tolerance test An oral glucose tolerance test (OGTT) was performed at the end of the treatment or after 12 weeks. The rats were deprived of food for 16 h, glucose load (2 g/kg body weight of 50% (w/v) glucose solution) was administered, 30 min after -asarone administration. Blood samples were collected from the tail vein at different time points viz. 0 min (before glucose challenge) and 30, 60, 90, 120, 180, 210 and 240 min after glucose load for measurement of glucose concentration in all groups by using a Glucometer (AccuChek Advantage, Roche, GMBH, Germany). • Serum glucose and lipid levels Rats were fasted overnight, sacrificed and the blood was collected from the abdominal aorta. Blood was placed into sterile Vacutainer plastic tubes (BDVacutainer, Plymouth, UK). Serum was separated by centrifugation (4000 × g, 10 min) and transferred to Eppendorf tubes. All serum samples were stored at −80 ◦ C until analysis. The concentrations of triglycerides, glucose, total cholesterol and HDL-cholesterol in serum were measured with commercial kits (Coral Diagnostics Ltd., Mumbai, India). • Measurement of plasma free fatty acids, serum leptin and adiponectin levels The levels of plasma free fatty acids were measured using FFA quantification kit (Wako Pure Chemical Industries, Osaka, Japan) and quantification procedure was performed in accordance with the manufacturer’s instructions. Serum leptin and adiponectin levels were measured by using enzyme linked immunosorbent assay (ELISA) kits (Ray Biotech Inc., GA, USA) • Markers of oxidative stress in liver i Liver homogenate preparation The rat liver was excised and the liver samples were cut into small pieces and homogenized in phosphate buffer saline (PBS) to give a 10% (w/v) liver homogenate. The homogenate was then centrifuged at 12,000 rpm for 50 min. The supernatant was separated and used for determining lipid peroxidation, levels of reduced glutathione, and the total protein content [25].
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• Malondialdehyde levels Lipid peroxidation in the hepatic tissue was evaluated using the thiobarbituric acid reactive substances (TBARS) technique described by Bird and Draper [26] in which malondialdehyde and the final products of lipid peroxidation react with barbituric acid, forming a colored complex. The absorbance of the supernatant was measured at 535 nm. The concentration of TBARS in the sample was calculated from the malondialdehyde analytical curve and the results were expressed as nM/g of tissue. • Reduced glutathione The levels of reduced glutathione was assessed according to the method described by Ellman [27]. Briefly, 1 ml supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 4 ◦ C for 1hr. The samples were centrifuged at 1200 × g for 15 min. To 1 ml of the supernatant, 2.7 ml of phosphate buffer (0.1 M, pH 8) and 0.2 ml of 5,5 dithiobis (2-nitrobenzoic acid) (DTNB) were added. The yellow color that developed was measured immediately at 412 nm using a spectrophotometer (Simadzu, UV1700). The concentration of reduced glutathione in the supernatant was determined from a standard curve (1–50 nmol) and expressed as mol/mg protein. • Protein estimation Total protein was measured in the liver homogenate by a method described by Lowry et al. [28] using bovine serum albumin (BSA) (1 mg/ml) as a standard. • Histopathological analysis For histological examination, the liver and adipose tissues were collected and fixed in (10% v/v) formalin. The tissue samples were then processed routinely and embedded in paraffin. Longitudinal sections (5 m) were stained with hematoxylin and eosin (H & E) and slides were prepared. The tissue sections were examined and the photographs were captured using a light microscope (MoticBA310 E-LED) at 400×.
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with -asarone showed selective inhibition of HFD intake and consumed more of the normal chow as compared with HFD (p < 0.05), whereas, total food intake remained unaffected. However, clear differentiation between normal chow and HFD intake was observed only at 50 mg/kg dose of -asarone (Fig. 1). Moreover, we have also investigated the effect of -asarone on normal chow intake, which remained unaltered (data not shown), thus these results suggesting that the -asarone may affect taste in animals. 7.2. Diet-induced obesity model 7.2.1. Effect of ˇ-asarone on HFD intake in Wistar rats Normal chow fed rats showed stable feed intake (gm) throughout the entire observation period, whereas HFD intake was slightly high during 1st week, declining and stabilizing thereafter. Although the lowest investigated dose of -asarone (12.5 mg/kg) did not modify the eating behavior in HFD fed rats, higher doses of asarone (25 and 50 mg/kg) produced significant decrease in food intake as compared with that of HFD control (p < 0.001) and both the doses were equally effective in reducing intake of HFD in rats (Fig. 2). 7.2.2. Effect of ˇ-asarone on body weight in HFD fed rats Chronic HFD consumption in rats produced significant and progressive increase in body weight as compared to normal chow fed rats (p < 0.001). -Asarone treatment significantly and dosedependently attenuated HFD induced increase in body weight of rats, indicating its anti-obesity potential (Fig. 3). However, -asarone at 25 and 50 mg/kg/day was observed to be equally effective in attenuating HFD-induced increase in body weight. 7.2.3. Effect of ˇ-asarone on HFD-induced changes in fat pad weights in Wistar rats Increase in adiposity is the characteristic feature of obesity. In line with the results observed with the analysis of body weight, a significant increase in fat pad weights was also observed following 12 weeks of HFD administration (Fig. 4). Significant increase in epididymal, mesenteric, retroperitoneal fat pad weights and a consequent increase in total fat pad weight was observed in HFD fed rats as compared with that of normal chow fed rats (p < 0.001). However, -asarone treatment for four weeks dose-dependently attenuated HFD-induced increase in fat pad weights (p < 0.001).
6.1. Statistical analysis 7.3. Biochemical parameters The food preference in animals was analysed by paired t-test. The biochemical data for random glucose, lipid profile, oxidative stress markers, leptin, adiponectin and fat pad weights were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. Effects of -asarone on food intake, body weight, and OGTT at different time points were analysed using repeated measure two-way ANOVA followed by Bonferroni multiple comparison tests. p < 0.05 was set to be statistically significant.
7.1. Food preference paradigm
7.3.1. Effect of ˇ-asarone on oral glucose tolerance (OGTT) in HFD fed rats At the end of 12 weeks, rats were challenged with oral glucose load. HFD fed rats showed significantly higher levels of glucose, which declined progressively with time but did not reach toward initial values (122.2 mg/dl) even after 240 (155.3 mg/dl) min as compared with that of normal (94 mg/dl) chow fed rats (p < 0.001). This indicates development of insulin resistance/glucose intolerance in HFD fed rats (Fig. 6). -Asarone treatment decreased glucose levels following oral glucose load and normalized the glucose levels in HFD fed rats as compared to HFD control (p < 0.001), suggesting improvement in glucose tolerance (Fig. 5).
7.1.1. Effect of ˇ-asarone on food preference in rats (Chow vs. HFD) Rats are known to have preference for palatable foods like diets high in carbohydrate or fat or both. In the present study, vehicle treated rats showed preference for HFD when given food choice between normal chow and HFD, showing passive behavior of animals following vehicle administration. However, animals treated
7.3.2. Effect of ˇ-asarone on serum glucose levels and lipid profile in HFD-fed rats Chronic HFD administration in rats significantly altered serum biochemistry, the rats were slightly hyperglycemic and showed significant dyslipidemic changes as compared with those in chow fed rats (p < 0.001). Significant increases in serum total cholesterol (TC), triglyceride (TG) and a decrease in HDL-cholesterol were observed
7. Results
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Fig. 1. Effect of -asarone on cumulative HFD vs normal chow intake in rats.Values are expressed as mean ± SEM * p < 0.01HFD vs chow intake in rats.
Fig. 2. Effect of -asarone on cumulative HFD intake on weekly intervals in rats. Values are expressed as mean ± SEM ∗ p < 0.001 vs HFD- control rats.
@
p < 0.001 vs chow fed rats (NC-normal control),
in HFD fed rats as compared with chow control (p < 0.001). by contrast -asarone treated rats were normo-glycemic and showed significant attenuation in HFD-induced increase in TC and TG levels and restoration of HDL levels as compared with that of HFD control rats (Fig. 6).
HFD fed rats as compared with chow fed rats (p < 0.001). -Asarone treatment dose-dependently attenuated HFD-induced alterations in leptin and adiponectin levels and restored their values toward normal (p < 0.001) in HFD-fed rats as compared with HFD-control (Figs. 8 and 9).
7.3.3. Effect of ˇ-asarone on HFD-induced changes in FFA levels in rats The levels of plasma free fatty acids rose significantly following chronic HFD administration in rats as compared with rats maintained on normal chow (p < 0.001). -Asarone at the lowest investigated dose (12.5 mg/kg) was ineffective, whereas at higher doses (25 and 50 mg/kg) it attenuated significantly the HFDinduced increase in FFA levels (p < 0.001) (Fig. 7).
7.3.5. Effect of ˇ-asarone on markers of oxidative stress in liver of HFD-fed rats Chronic consumption of HFD in rats produced oxidative stress as evidenced by significant elevation in liver TBARS and a decrease in reduced glutathione as compared with that of chow fed rats (p < 0.001). However, -asarone dose-dependently attenuated HFD-induced oxidative stress and restored GSH levels toward normal values (p < 0.001) (Table 1).
7.3.4. Effect of ˇ-asarone on serum adiponectin and leptin levels in HFD fed rats Adipokines are known to play critical roles in overall energy homeostasis and insulin action. Significant elevation in serum leptin and decrease in serum adiponectin level were observed in
8. Discussion The observations made in the present investigation reveal the therapeutic potential of -asarone in HFD-induced obesity and other associated metabolic abnormalities in rats. In particular, the current study, clearly indicates that -asarone reduces
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Fig. 3. Effect of -asarone on body weight on weekly intervals in HFD fed rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
Fig. 4. Effect of -asarone on HFD-induced changes in retroperitoneal, epididymal and mesenteric fat pad weight of rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
spontaneous uptake of HFD in rats. It was experimentally observed that between HFD (limited access to only 4 h/day) and standard chow, rats preferred HFD and consumed large amounts of it (Fig. 1). Our observations are comparable with those in other studies [29–31], indicating that limited access to highly palatable
Table 1 Effect of -asarone on markers of oxidative stress in liver of HFD-fed rats. Groups
Normal control HFD -asarone (12.5 mg/kg) –asarone (25 mg/kg) –asarone (50 mg/kg)
Oxidative stress parameters GSH (nmol/mg protein)
MDA (mol /mg protein)
26.3 ± 0.44 8.44 ± 0.04@ 25.66 ± 0.31 27.71 ± 0.53* 29.36 ± 0.43*
0.25 ± 0.03 0.69 ± 0.08@ 0.26 ± 0.03 0.24 ± 0.02* 0.22 ± 0.02*
Values are expressed as mean ± SEM. @ p < 0.001 vs chow fed rats (NC-Normal control). * p < 0.001 vs HFD-fed rats.
food produces a “binge-type” pattern of consumption in rats. This paradigm models a clinical phenomenon of an excessive, bingetype food consumption, where individuals repeatedly seek out and consume large amounts of highly palatable food for a brief and discrete period of time [32]. In the present study, overconsumption of HFD in food preference paradigm was inhibited by -asarone (Fig. 1). It was interesting to note that -asarone did not modify standard chow intake (data not shown) but selectively reduced HFD intake when rats were given opportunity to consume HFD or standard chow. These results clearly indicate that the -asarone may affect palatability of food by influencing taste sensations. Similar inhibition of fat intake following administration of serotonin agonists, enterostatin etc. was also demonstrated in previous reports [33–36]. Diet induced obesity is the most widely used experimental animal model for testing anti-obesity potential of novel drugs [37,38]. Metabolic dysregulation in rats developed by chronic HFD consumption closely resembles the obesity in humans [22]. It has been
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Fig. 5. Effect of -asarone on oral glucose tolerance (OGTT) in HFD fed rats. Values are expressed as mean ± SD @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
Fig. 6. Effect of -asarone on serum glucose and lipids in HFD-fed rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), * p < 0.001 vs HFD-fed rats. Note: Glu = glucose; TC = total cholesterol; TG = triglyceride and HDL = high density lipoprotein.
Fig. 7. Effect of -asarone on HFD-induced changes in plasma free fatty acid levels in rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
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Fig. 8. Effect of -asarone on serum leptin levels in HFD fed rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
Fig. 9. Effect of -asarone on serum adiponectin levels in HFD fed rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
observed clinically that overconsumption of high-caloric diet such as high-fat, low fiber foods and sweetened beverages cause excessive accumulation of fats in adipose tissue, eventually leading to development of obesity in humans [6,40]. The fact that -asarone dose-dependently reduced HFD intake (Fig. 2) and attenuated HFDinduced increase in body weight and total body fat, suggest its anti-obesity potential. Overconsumption of high fat diet has been reported to cause increase in the levels of circulating lipids & FFAs, leading to fat accumulation in adipocytes [41]. Moreover, the increased levels of serum lipids and circulating free fatty acids has been reported to contribute to development of oxidative stress and disturbance of the overall metabolic functions [42,43]. We found that chronic administration of HFD produced significant increases in serum lipids (increase in triglycerides, total cholesterol and a decrease in HDL-cholesterol) and FFAs levels (Figs. 6 and 7). -Asarone treatment in HFD fed rats, produced significant decrease in circulating lipids and FFAs and restored HDL levels. Liver is a major metabolic organ and the oxidative damage to hepatocytes has been reported to down-regulate various metabolic functions and favor dyslipidemia [44]. Herein, a significant increase in malondialdehyde (end product of lipid peroxidation) and reduction in depleted glutathione levels (Table 1) were observed in HFD fed rats, indicating the development of oxidative stress, accompanied by fatty depots in liver, adipocyte hypertrophy and fat accumulation (Fig. 10). -Asarone significantly attenuated these actions
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of HFD reducing oxidative stress and fat deposition in liver and adipose tissue and decreasing adipocyte hypertrophy. -Asarone has been shown to hold anti-adipogenic and hypolipidemic potential and reported to inhibit intracellular accumulation of lipids in 3T3-L1 cells [18]. Recently, the anti-oxidant potential of -asarone has been reported also in brain disorders [45]. Indeed, anti-oxidant mechanisms have been demonstrated to play important role in hepatoprotection in fatty liver disorders [46]. Thus, both the antioxidant potential and hypolipidemic actions may play important role in the observed anti-obesity effect of -asarone. Obesity has been associated with numerous co-morbidities in association with body weight and fat including systemic insulin resistance, hyperleptinemia, diabetes mellitus, dyslipidemia and hyperinsulinemia [47,48]. The majority of these co-morbidities including insulin resistance (as evidenced by glucose intolerance), hyperleptinemia, decrease in adiponectin levels and dyslipidemia occurred during the obese state in the present study. The interplay between two major adipokines, leptin and adiponectin, are thought to play important role in the regulation of metabolic homeostasis [49].The most recognized function of adiponectin is activation of fatty acid and glucose metabolism, as well as enhancement of insulin sensitivity, which contributes to anti-obesity and anti-diabetic effects [50]. Herein, HFD fed rats were glucose intolerant, indicating insulin resistance along with a significant decrease in adiponectin levels. A large amount of clinical data demonstrates the association between circulating adiponectin levels and metabolic functions. Decrease in blood adiponectin levels have been reported in cases of obesity, insulin resistance, diabetes, heptosteatosis, atherosclerosis and coronary artery disease [50,51]. Indeed, the association between insulin resistance and lower plasma adiponectin levels has been demonstrated in the tissues of HFD fed rats as well as in diet-induced form of human obesity [50,52]. Low adiponectin levels have also been strongly implicated in the development of insulin resistance of in mouse models of both obesity and lipoatrophy [52]. Moreover, lower plasma levels of adiponectin have been reported in insulin-resistant states including type 2 diabetes in humans [53]. the observation thjat -asarone dose-dependently attenuated HFD -induced glucose intolerance and restored adiponectin levels (Figs. 5 and 9) further points to its potential for therapeutic treatment of obesity. Leptin is known to regulate body weight by reducing food intake (FI) and increasing energy expenditure by actions primarily within the hypothalamus [54,55]. However, blood leptin levels increase with increasing adiposity and are directly proportional to fat mass [56–58]. In the present study, significant increase in total body fat, adipocyte hypertrophy and hyperleptinemia was observed following chronic HFD administration in rats. Since leptin is synthesized and secreted by adipose tissue and has been strongly associated with increased fat depots [57], the observed increase in leptin levels may be due to higher body fat (Fig. 8). It may also be due to loss of effectiveness of endogenous leptin on weight maintenance due to development of leptin resistance [54–59]. It has been suggested that the HFD-induced leptin resistance may be due to elevated plasma leptin levels, which result in chronic overstimulation of the leptin receptors and activation of negative feedback pathways that block further leptin signaling. In addition fat can either directly block leptin signaling or activate cellular processes which impair leptin actions and its weight reducing effects [60]. it has been suggested that the function of obesity-induced hyperleptinemia is not to prevent or inhibit diet-induced obesity, but rather to permit the stockpiling of calories within certain upper limits [61]. We found that -asarone significantly reduced HFD induced increment in body fat and adipocyte hypertrophy and restored elevated levels of leptin, indicating improvement in leptin sensitivity. -Asarone may improve leptin sensitivity either by decreasing adiposity or through its hypolipidemic actions.
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Fig. 10. Effect of -asarone on HFD-induced histological changes in liver and adipose tissue of rats (400×).
A. calamus is one of the plants used in Traditional Medicine, whereas the rhizomes of this plant are used extensively worldwide. The most important ingredients of A. calamus are the ␣ and -asarones [62]. The role of -asarone has been much less investigated and its therapeutic potential is hampered by its genotoxic potential, and issue, however that is still debated. Both positive and negative results have been reported for -asarone in various in-vitro studies [63–67]. The genotoxic potential of -asarone was seen under certain conditions, and it requires metabolic activation, whereas its metabolite did not showed any sign of genotoxicity or only in certain strains with larger doses [63,64,67]. According to a recent report, in-vivo micronuclei test in mice bone marrow demonstrated that -asarone did not have any genotoxic or mutagenic effects [68]. Thus, it could be considered safe for therapeutic use, even if further investigations is required to conclusively assess this. Nonetheless, our results showed promising therapeutic potential of -asarone in metabolic disorders such as obesity. In summary, the current study for the first time demonstrates interesting outcomes of -asarone against HFD induced obesity and associated co-morbidities. -Asarone did not inhibit direclty afood intake but affected food palatability; in addition it reduced HFD-induced obesity and associated co-morbidities including hypoadiponectemia, hyperleptinemia, dyslipidemia and insulin resistance. The observations made in the present investigation conclusively suggest that the anti-obesity potential of
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-Asarone modulate adipokines and attenuates high fat diet-induced metabolic abnormalities in Wistar rats Malesh M. Thakare ∗ , Sanjay J. Surana Department of Pharmacology, RC Patel Institute of Pharmaceutical Education and Research, Shirpur 425 405, Dhule, Maharashtra, India
a r t i c l e
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Article history: Received 19 April 2015 Received in revised form 1 December 2015 Accepted 1 December 2015 Available online 7 December 2015 Keywords: -Asarone Adipokines High fat diet Obesity Diabetes Dyslipidemia
a b s t r a c t Here we investigated the effect of -asarone on food preference and its therapeutic potential against high fat diet (HFD) induced obesity in rats. In food preference study, free access to HFD was given only for 4 h in addition to standard laboratory chow in rats and the preferential intake between chow and HFD was measured. For obesity induction, HFD was administered for 12 weeks and the HFD fed rats were treated with -asarone in the last 4 weeks, starting from 9th week onwards. Food intake, body weight was measured biweekly. Glucose tolerance and the levels of glucose, lipids, free fatty acids, leptin, and adiponectin were assessed. HFD fed rats showed progressive increase in body weight and developed glucose intolerance and dyslipidemia. In addition, they showed increased adiposity and the disturbed pattern of adipokine levels In the food preference paradigm, -asarone produced selective decrease in HFD intake in rats. In obese rats, -asarone treatment not only reduced body weight but also prevented HFD-induced metabolic alterations, including glucose intolerance, dyslipidemia and adipokine imbalance. The observed beneficial effects of -asarone appear due its ability to reduce intake of energy dense food by affecting food palatability, and to normalize the levels of leptin and adiponectin in rats. Overall, our results suggest that -asarone is a novel candidate molecule with significant therapeutic potential in the management of obesity and associated abnormalities. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction Obesity is a worldwide epidemic [1] and a heterogeneous complex disorder of multiple etiologies characterized by excessive deposition of body fat [2]. Obesity usually develops as a result of an imbalance between caloric intake and expenditure, which contribute to co-morbid conditions like hypertension, hyperlipidemia, type II diabetes, coronary heart disease, stroke and cancer [3–5]. In developed countries, highly palatable foods are available in almost unlimited abundance, and overconsumption undoubtedly contribute to the epidemic [6,7]. Sedentary lifestyles, high-fat, energy-dense diets, and a genetic predisposition all contribute to the obesity epidemic [8].
Abbreviations: HFD, high fat diet; FFA, free fatty acid; AC, Acorus calamus; IAEC, institutional animal ethics committee; INSA, indian national science academy; DTNB, 5,5 dithiobis (2-nitrobenzoic acid); HDL, high density lipoprotein; p.o., peroral; BMI, body mass index; OGTT, oral glucose tolerance test; ELISA, enzyme linked immunosorbent assay; PBS, phosphate buffer saline; TBARS, thiobarbituric acid reactive substances; BSA, bovine serum albumin; TC, total cholesterol; TG, triglycerides; MDA, malondialdehyde; GSH, reduced glutathione; ANOVA, analysis of variance. ∗ Corresponding author. E-mail address: [email protected] (M.M. Thakare). http://dx.doi.org/10.1016/j.phrs.2015.12.003 1043-6618/© 2015 Elsevier Ltd. All rights reserved.
Herbs and phytochemicals have been known to play a major role in drug discovery process including anti-obesity molecules [9,10]. In recent years, the extract of Acorus calamus (AC) has been reported to improve metabolic functions [11,12]. A. calamus L., family Araceae, has been used in the Indian and Chinese systems of medicine for its valuable role in several CNS disorders [13–15]. In recent years, the extract of AC has been reported to hold hypoglycemic, hypolipidemic, insulin sensitizing and ␣glucosidase inhibitory activities [11,12,16], suggesting that it may play a role in regulation of metabolism. Moreover, ethyl acetate fraction of AC has also been reported to improve insulin release and attenuate postprandial hyperglycemia and cardiovascular complications in mice [17]. -Asarone, a major constituent of AC has been reported to produce anti-adipogenic and lipolytic actions in 3T3L1 cells, an action most likely exerted by subduing the expression of adipogenic transcription factors [18]. Thus, -asarone might be useful to correct energy imbalance and may prove to be beneficial in the management of obesity. Obesity can be characterized into two main types, hyperplasia (cell number increase) and hypertrophic (cell size increase) adipocytes. As a metabolic and endocrine organ, adipose tissue plays a critical role in regulation of energy balance, lipid metabolism and insulin action [19]. A chronic imbalance between energy intake and energy expenditure can lead to adipocyte
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hypertrophy and adipocyte hyperplasia [20], the former being thought to be particularlyimportant in determination of fat mass in adults [21]. As discussed above, the anti-adipogenic potential of -asarone may be useful to limit energy imbalance and obesity. The emphasis of the present study was placed on detailed investigation of the effect of -asarone on food palatability in food preference paradigm and its therapeutic potential against high fat diet-induced metabolic abnormalities in rats. 2. Materials and method 2.1. Experimental animals The experiments were carried out in male Wistar rats (250–300 g) obtained from Central Animal House facility of RC Patel Institute of Pharmaceutical Education and Research, Shirpur425 405, Maharashtra, India. The rats were kept in polyacrylic cages (3/cage) and maintained under standard husbandry conditions (room temperature 22 ± 2 ◦ C and relative humidity of 50–55%) with 12 h light/dark cycle (lights on at 8AM). The food in the form of dry pallets and water were made available ad libitum . The protocol was reviewed and approved by the Institutional Animals Ethics Committee (IAEC) and the animal experiments were carried out in accordance with the Indian National Science Academy (INSA) guidelines for use and care of animals. 2.2. Drugs and chemicals -Asarone (1,2,4-trimethoxy-5-[(Z)-prop-1-enyl]benzene) and DTNB [5,5 dithiobis (2-nitrobenzoic acid)] etc., was procured from Sigma–Aldrich, USA. Casein (Modern Diaries Ltd., Karnal, India), Cholesterol (HIMEDIA Laboratories Pvt. Ltd., Mumbai, India) and dl-Methionine (CDH, New Delhi, India) was used to prepare high fat diet. The diagnostic kits for serum glucose, cholesterol, HDL, triglycerides were obtained from Coral Diagnostics Ltd., Mumbai, India. All other chemicals used in the present study were of analytical grade. 2.3. Methodology 2.3.1. Experiment No.1— food preference paradigm In the food preference paradigm, rats were fed with equal amounts of standard laboratory chow (Ashirwad Industries Ltd., India, providing 7.82% as fat, 20–21% of calories as protein, 47% of calories as carbohydrate and containing a total of 3.6 kcal/g) and additional high fat diet (HFD) (58% fat, 25% protein and 17% carbohydrate, and containing a total of 5.2 kcal/g) [22]. Both diets were presented in separate bowls placed in opposite corners and were administered for one week daily for 4 h in addition to normal chow to habituate the animals to them and to achieve the baseline value of food intake. During habituation, the placement of food was changed daily to avoid placement preference and intake measurements were corrected for spillage. Following establishment of stable baseline chow and HFD intake, animals were treated with either vehicle (2 ml/kg) or -asarone at different doses (25 and 50 mg/kg, p.o.) During the experiment a pre-weighed amount of powdered chow and HFD was placed in the home cage and the amount of chow and HFD consumed was recorded for 4 h [23]. 2.3.2. Experiment No.2—diet-Induced obesity model-experimental procedure A total of 30 male Wistar rats, (weighing 200–250 g) were used in the study. The rats were kept on two dietary interventions, one group of rats (n = 6) was supplied with standard laboratory chow (Ashirwad India) and assigned as normal control (NC-Group 1), the remaining rats were given free access high fat diet (n = 24) for 12 weeks. The composition and preparation of HFD was the same as
described by Srinivasan et al., [22]. The HFD fed rats were randomly divided into 4 groups (each consisting of n = 6). Group 2 rats were fed with HFD and received vehicle treatment for the last four weeks (propylene glycol); Group3, 4 and 5 - HFD fed rats received treatment of -asarone in the last 4 weeks [from 9th to 12th week] at 12.5, 25 and 50 mg/kg p.o, respectively. (A) Anthropometric parameters The increase in the body weight was regarded as an index of obesity. Body weight was assessed biweekly. For individual rats, food intake measurements were recorded biweekly and taken as an average value. At the end of the experiment the adipose tissues (epididymal, retroperitoneal and mesenteric fat depots) were isolated, freed from surrounding tissues, weighed individually and the total weight was calculated [24]. • Biochemical parameters [Blood, Serum & Plasma] i Oral glucose tolerance test An oral glucose tolerance test (OGTT) was performed at the end of the treatment or after 12 weeks. The rats were deprived of food for 16 h, glucose load (2 g/kg body weight of 50% (w/v) glucose solution) was administered, 30 min after -asarone administration. Blood samples were collected from the tail vein at different time points viz. 0 min (before glucose challenge) and 30, 60, 90, 120, 180, 210 and 240 min after glucose load for measurement of glucose concentration in all groups by using a Glucometer (AccuChek Advantage, Roche, GMBH, Germany). • Serum glucose and lipid levels Rats were fasted overnight, sacrificed and the blood was collected from the abdominal aorta. Blood was placed into sterile Vacutainer plastic tubes (BDVacutainer, Plymouth, UK). Serum was separated by centrifugation (4000 × g, 10 min) and transferred to Eppendorf tubes. All serum samples were stored at −80 ◦ C until analysis. The concentrations of triglycerides, glucose, total cholesterol and HDL-cholesterol in serum were measured with commercial kits (Coral Diagnostics Ltd., Mumbai, India). • Measurement of plasma free fatty acids, serum leptin and adiponectin levels The levels of plasma free fatty acids were measured using FFA quantification kit (Wako Pure Chemical Industries, Osaka, Japan) and quantification procedure was performed in accordance with the manufacturer’s instructions. Serum leptin and adiponectin levels were measured by using enzyme linked immunosorbent assay (ELISA) kits (Ray Biotech Inc., GA, USA) • Markers of oxidative stress in liver i Liver homogenate preparation The rat liver was excised and the liver samples were cut into small pieces and homogenized in phosphate buffer saline (PBS) to give a 10% (w/v) liver homogenate. The homogenate was then centrifuged at 12,000 rpm for 50 min. The supernatant was separated and used for determining lipid peroxidation, levels of reduced glutathione, and the total protein content [25].
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• Malondialdehyde levels Lipid peroxidation in the hepatic tissue was evaluated using the thiobarbituric acid reactive substances (TBARS) technique described by Bird and Draper [26] in which malondialdehyde and the final products of lipid peroxidation react with barbituric acid, forming a colored complex. The absorbance of the supernatant was measured at 535 nm. The concentration of TBARS in the sample was calculated from the malondialdehyde analytical curve and the results were expressed as nM/g of tissue. • Reduced glutathione The levels of reduced glutathione was assessed according to the method described by Ellman [27]. Briefly, 1 ml supernatant was precipitated with 1 ml of 4% sulfosalicylic acid and cold digested at 4 ◦ C for 1hr. The samples were centrifuged at 1200 × g for 15 min. To 1 ml of the supernatant, 2.7 ml of phosphate buffer (0.1 M, pH 8) and 0.2 ml of 5,5 dithiobis (2-nitrobenzoic acid) (DTNB) were added. The yellow color that developed was measured immediately at 412 nm using a spectrophotometer (Simadzu, UV1700). The concentration of reduced glutathione in the supernatant was determined from a standard curve (1–50 nmol) and expressed as mol/mg protein. • Protein estimation Total protein was measured in the liver homogenate by a method described by Lowry et al. [28] using bovine serum albumin (BSA) (1 mg/ml) as a standard. • Histopathological analysis For histological examination, the liver and adipose tissues were collected and fixed in (10% v/v) formalin. The tissue samples were then processed routinely and embedded in paraffin. Longitudinal sections (5 m) were stained with hematoxylin and eosin (H & E) and slides were prepared. The tissue sections were examined and the photographs were captured using a light microscope (MoticBA310 E-LED) at 400×.
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with -asarone showed selective inhibition of HFD intake and consumed more of the normal chow as compared with HFD (p < 0.05), whereas, total food intake remained unaffected. However, clear differentiation between normal chow and HFD intake was observed only at 50 mg/kg dose of -asarone (Fig. 1). Moreover, we have also investigated the effect of -asarone on normal chow intake, which remained unaltered (data not shown), thus these results suggesting that the -asarone may affect taste in animals. 7.2. Diet-induced obesity model 7.2.1. Effect of ˇ-asarone on HFD intake in Wistar rats Normal chow fed rats showed stable feed intake (gm) throughout the entire observation period, whereas HFD intake was slightly high during 1st week, declining and stabilizing thereafter. Although the lowest investigated dose of -asarone (12.5 mg/kg) did not modify the eating behavior in HFD fed rats, higher doses of asarone (25 and 50 mg/kg) produced significant decrease in food intake as compared with that of HFD control (p < 0.001) and both the doses were equally effective in reducing intake of HFD in rats (Fig. 2). 7.2.2. Effect of ˇ-asarone on body weight in HFD fed rats Chronic HFD consumption in rats produced significant and progressive increase in body weight as compared to normal chow fed rats (p < 0.001). -Asarone treatment significantly and dosedependently attenuated HFD induced increase in body weight of rats, indicating its anti-obesity potential (Fig. 3). However, -asarone at 25 and 50 mg/kg/day was observed to be equally effective in attenuating HFD-induced increase in body weight. 7.2.3. Effect of ˇ-asarone on HFD-induced changes in fat pad weights in Wistar rats Increase in adiposity is the characteristic feature of obesity. In line with the results observed with the analysis of body weight, a significant increase in fat pad weights was also observed following 12 weeks of HFD administration (Fig. 4). Significant increase in epididymal, mesenteric, retroperitoneal fat pad weights and a consequent increase in total fat pad weight was observed in HFD fed rats as compared with that of normal chow fed rats (p < 0.001). However, -asarone treatment for four weeks dose-dependently attenuated HFD-induced increase in fat pad weights (p < 0.001).
6.1. Statistical analysis 7.3. Biochemical parameters The food preference in animals was analysed by paired t-test. The biochemical data for random glucose, lipid profile, oxidative stress markers, leptin, adiponectin and fat pad weights were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. Effects of -asarone on food intake, body weight, and OGTT at different time points were analysed using repeated measure two-way ANOVA followed by Bonferroni multiple comparison tests. p < 0.05 was set to be statistically significant.
7.1. Food preference paradigm
7.3.1. Effect of ˇ-asarone on oral glucose tolerance (OGTT) in HFD fed rats At the end of 12 weeks, rats were challenged with oral glucose load. HFD fed rats showed significantly higher levels of glucose, which declined progressively with time but did not reach toward initial values (122.2 mg/dl) even after 240 (155.3 mg/dl) min as compared with that of normal (94 mg/dl) chow fed rats (p < 0.001). This indicates development of insulin resistance/glucose intolerance in HFD fed rats (Fig. 6). -Asarone treatment decreased glucose levels following oral glucose load and normalized the glucose levels in HFD fed rats as compared to HFD control (p < 0.001), suggesting improvement in glucose tolerance (Fig. 5).
7.1.1. Effect of ˇ-asarone on food preference in rats (Chow vs. HFD) Rats are known to have preference for palatable foods like diets high in carbohydrate or fat or both. In the present study, vehicle treated rats showed preference for HFD when given food choice between normal chow and HFD, showing passive behavior of animals following vehicle administration. However, animals treated
7.3.2. Effect of ˇ-asarone on serum glucose levels and lipid profile in HFD-fed rats Chronic HFD administration in rats significantly altered serum biochemistry, the rats were slightly hyperglycemic and showed significant dyslipidemic changes as compared with those in chow fed rats (p < 0.001). Significant increases in serum total cholesterol (TC), triglyceride (TG) and a decrease in HDL-cholesterol were observed
7. Results
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Fig. 1. Effect of -asarone on cumulative HFD vs normal chow intake in rats.Values are expressed as mean ± SEM * p < 0.01HFD vs chow intake in rats.
Fig. 2. Effect of -asarone on cumulative HFD intake on weekly intervals in rats. Values are expressed as mean ± SEM ∗ p < 0.001 vs HFD- control rats.
@
p < 0.001 vs chow fed rats (NC-normal control),
in HFD fed rats as compared with chow control (p < 0.001). by contrast -asarone treated rats were normo-glycemic and showed significant attenuation in HFD-induced increase in TC and TG levels and restoration of HDL levels as compared with that of HFD control rats (Fig. 6).
HFD fed rats as compared with chow fed rats (p < 0.001). -Asarone treatment dose-dependently attenuated HFD-induced alterations in leptin and adiponectin levels and restored their values toward normal (p < 0.001) in HFD-fed rats as compared with HFD-control (Figs. 8 and 9).
7.3.3. Effect of ˇ-asarone on HFD-induced changes in FFA levels in rats The levels of plasma free fatty acids rose significantly following chronic HFD administration in rats as compared with rats maintained on normal chow (p < 0.001). -Asarone at the lowest investigated dose (12.5 mg/kg) was ineffective, whereas at higher doses (25 and 50 mg/kg) it attenuated significantly the HFDinduced increase in FFA levels (p < 0.001) (Fig. 7).
7.3.5. Effect of ˇ-asarone on markers of oxidative stress in liver of HFD-fed rats Chronic consumption of HFD in rats produced oxidative stress as evidenced by significant elevation in liver TBARS and a decrease in reduced glutathione as compared with that of chow fed rats (p < 0.001). However, -asarone dose-dependently attenuated HFD-induced oxidative stress and restored GSH levels toward normal values (p < 0.001) (Table 1).
7.3.4. Effect of ˇ-asarone on serum adiponectin and leptin levels in HFD fed rats Adipokines are known to play critical roles in overall energy homeostasis and insulin action. Significant elevation in serum leptin and decrease in serum adiponectin level were observed in
8. Discussion The observations made in the present investigation reveal the therapeutic potential of -asarone in HFD-induced obesity and other associated metabolic abnormalities in rats. In particular, the current study, clearly indicates that -asarone reduces
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Fig. 3. Effect of -asarone on body weight on weekly intervals in HFD fed rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
Fig. 4. Effect of -asarone on HFD-induced changes in retroperitoneal, epididymal and mesenteric fat pad weight of rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
spontaneous uptake of HFD in rats. It was experimentally observed that between HFD (limited access to only 4 h/day) and standard chow, rats preferred HFD and consumed large amounts of it (Fig. 1). Our observations are comparable with those in other studies [29–31], indicating that limited access to highly palatable
Table 1 Effect of -asarone on markers of oxidative stress in liver of HFD-fed rats. Groups
Normal control HFD -asarone (12.5 mg/kg) –asarone (25 mg/kg) –asarone (50 mg/kg)
Oxidative stress parameters GSH (nmol/mg protein)
MDA (mol /mg protein)
26.3 ± 0.44 8.44 ± 0.04@ 25.66 ± 0.31 27.71 ± 0.53* 29.36 ± 0.43*
0.25 ± 0.03 0.69 ± 0.08@ 0.26 ± 0.03 0.24 ± 0.02* 0.22 ± 0.02*
Values are expressed as mean ± SEM. @ p < 0.001 vs chow fed rats (NC-Normal control). * p < 0.001 vs HFD-fed rats.
food produces a “binge-type” pattern of consumption in rats. This paradigm models a clinical phenomenon of an excessive, bingetype food consumption, where individuals repeatedly seek out and consume large amounts of highly palatable food for a brief and discrete period of time [32]. In the present study, overconsumption of HFD in food preference paradigm was inhibited by -asarone (Fig. 1). It was interesting to note that -asarone did not modify standard chow intake (data not shown) but selectively reduced HFD intake when rats were given opportunity to consume HFD or standard chow. These results clearly indicate that the -asarone may affect palatability of food by influencing taste sensations. Similar inhibition of fat intake following administration of serotonin agonists, enterostatin etc. was also demonstrated in previous reports [33–36]. Diet induced obesity is the most widely used experimental animal model for testing anti-obesity potential of novel drugs [37,38]. Metabolic dysregulation in rats developed by chronic HFD consumption closely resembles the obesity in humans [22]. It has been
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Fig. 5. Effect of -asarone on oral glucose tolerance (OGTT) in HFD fed rats. Values are expressed as mean ± SD @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
Fig. 6. Effect of -asarone on serum glucose and lipids in HFD-fed rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), * p < 0.001 vs HFD-fed rats. Note: Glu = glucose; TC = total cholesterol; TG = triglyceride and HDL = high density lipoprotein.
Fig. 7. Effect of -asarone on HFD-induced changes in plasma free fatty acid levels in rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
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Fig. 8. Effect of -asarone on serum leptin levels in HFD fed rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
Fig. 9. Effect of -asarone on serum adiponectin levels in HFD fed rats. Values are expressed as mean ± SEM @ p < 0.001 vs chow fed rats (NC-normal control), ∗ p < 0.001 vs HFD-fed rats.
observed clinically that overconsumption of high-caloric diet such as high-fat, low fiber foods and sweetened beverages cause excessive accumulation of fats in adipose tissue, eventually leading to development of obesity in humans [6,40]. The fact that -asarone dose-dependently reduced HFD intake (Fig. 2) and attenuated HFDinduced increase in body weight and total body fat, suggest its anti-obesity potential. Overconsumption of high fat diet has been reported to cause increase in the levels of circulating lipids & FFAs, leading to fat accumulation in adipocytes [41]. Moreover, the increased levels of serum lipids and circulating free fatty acids has been reported to contribute to development of oxidative stress and disturbance of the overall metabolic functions [42,43]. We found that chronic administration of HFD produced significant increases in serum lipids (increase in triglycerides, total cholesterol and a decrease in HDL-cholesterol) and FFAs levels (Figs. 6 and 7). -Asarone treatment in HFD fed rats, produced significant decrease in circulating lipids and FFAs and restored HDL levels. Liver is a major metabolic organ and the oxidative damage to hepatocytes has been reported to down-regulate various metabolic functions and favor dyslipidemia [44]. Herein, a significant increase in malondialdehyde (end product of lipid peroxidation) and reduction in depleted glutathione levels (Table 1) were observed in HFD fed rats, indicating the development of oxidative stress, accompanied by fatty depots in liver, adipocyte hypertrophy and fat accumulation (Fig. 10). -Asarone significantly attenuated these actions
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of HFD reducing oxidative stress and fat deposition in liver and adipose tissue and decreasing adipocyte hypertrophy. -Asarone has been shown to hold anti-adipogenic and hypolipidemic potential and reported to inhibit intracellular accumulation of lipids in 3T3-L1 cells [18]. Recently, the anti-oxidant potential of -asarone has been reported also in brain disorders [45]. Indeed, anti-oxidant mechanisms have been demonstrated to play important role in hepatoprotection in fatty liver disorders [46]. Thus, both the antioxidant potential and hypolipidemic actions may play important role in the observed anti-obesity effect of -asarone. Obesity has been associated with numerous co-morbidities in association with body weight and fat including systemic insulin resistance, hyperleptinemia, diabetes mellitus, dyslipidemia and hyperinsulinemia [47,48]. The majority of these co-morbidities including insulin resistance (as evidenced by glucose intolerance), hyperleptinemia, decrease in adiponectin levels and dyslipidemia occurred during the obese state in the present study. The interplay between two major adipokines, leptin and adiponectin, are thought to play important role in the regulation of metabolic homeostasis [49].The most recognized function of adiponectin is activation of fatty acid and glucose metabolism, as well as enhancement of insulin sensitivity, which contributes to anti-obesity and anti-diabetic effects [50]. Herein, HFD fed rats were glucose intolerant, indicating insulin resistance along with a significant decrease in adiponectin levels. A large amount of clinical data demonstrates the association between circulating adiponectin levels and metabolic functions. Decrease in blood adiponectin levels have been reported in cases of obesity, insulin resistance, diabetes, heptosteatosis, atherosclerosis and coronary artery disease [50,51]. Indeed, the association between insulin resistance and lower plasma adiponectin levels has been demonstrated in the tissues of HFD fed rats as well as in diet-induced form of human obesity [50,52]. Low adiponectin levels have also been strongly implicated in the development of insulin resistance of in mouse models of both obesity and lipoatrophy [52]. Moreover, lower plasma levels of adiponectin have been reported in insulin-resistant states including type 2 diabetes in humans [53]. the observation thjat -asarone dose-dependently attenuated HFD -induced glucose intolerance and restored adiponectin levels (Figs. 5 and 9) further points to its potential for therapeutic treatment of obesity. Leptin is known to regulate body weight by reducing food intake (FI) and increasing energy expenditure by actions primarily within the hypothalamus [54,55]. However, blood leptin levels increase with increasing adiposity and are directly proportional to fat mass [56–58]. In the present study, significant increase in total body fat, adipocyte hypertrophy and hyperleptinemia was observed following chronic HFD administration in rats. Since leptin is synthesized and secreted by adipose tissue and has been strongly associated with increased fat depots [57], the observed increase in leptin levels may be due to higher body fat (Fig. 8). It may also be due to loss of effectiveness of endogenous leptin on weight maintenance due to development of leptin resistance [54–59]. It has been suggested that the HFD-induced leptin resistance may be due to elevated plasma leptin levels, which result in chronic overstimulation of the leptin receptors and activation of negative feedback pathways that block further leptin signaling. In addition fat can either directly block leptin signaling or activate cellular processes which impair leptin actions and its weight reducing effects [60]. it has been suggested that the function of obesity-induced hyperleptinemia is not to prevent or inhibit diet-induced obesity, but rather to permit the stockpiling of calories within certain upper limits [61]. We found that -asarone significantly reduced HFD induced increment in body fat and adipocyte hypertrophy and restored elevated levels of leptin, indicating improvement in leptin sensitivity. -Asarone may improve leptin sensitivity either by decreasing adiposity or through its hypolipidemic actions.
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Fig. 10. Effect of -asarone on HFD-induced histological changes in liver and adipose tissue of rats (400×).
A. calamus is one of the plants used in Traditional Medicine, whereas the rhizomes of this plant are used extensively worldwide. The most important ingredients of A. calamus are the ␣ and -asarones [62]. The role of -asarone has been much less investigated and its therapeutic potential is hampered by its genotoxic potential, and issue, however that is still debated. Both positive and negative results have been reported for -asarone in various in-vitro studies [63–67]. The genotoxic potential of -asarone was seen under certain conditions, and it requires metabolic activation, whereas its metabolite did not showed any sign of genotoxicity or only in certain strains with larger doses [63,64,67]. According to a recent report, in-vivo micronuclei test in mice bone marrow demonstrated that -asarone did not have any genotoxic or mutagenic effects [68]. Thus, it could be considered safe for therapeutic use, even if further investigations is required to conclusively assess this. Nonetheless, our results showed promising therapeutic potential of -asarone in metabolic disorders such as obesity. In summary, the current study for the first time demonstrates interesting outcomes of -asarone against HFD induced obesity and associated co-morbidities. -Asarone did not inhibit direclty afood intake but affected food palatability; in addition it reduced HFD-induced obesity and associated co-morbidities including hypoadiponectemia, hyperleptinemia, dyslipidemia and insulin resistance. The observations made in the present investigation conclusively suggest that the anti-obesity potential of
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