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DOI 10.1002/mnfr.201300466
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RESEARCH ARTICLE
Curative diet supplementation with a melon superoxide dismutase reduces adipose tissue in obese hamsters by improving insulin sensitivity 2 ´ Julie Carillon1,3 , Lucie Knabe1 , Anne Montalban1 , Marie Stevant , Mayoura Keophiphath2 , 3 1,4 1 Dominique Lacan , Jean-Paul Cristol and Jean-Max Rouanet 1
´ ´ ´ Nutrition & Metabolisme, UMR 204 NutriPass – Prevention des Malnutritions et des Pathologies Associees, Universite´ Montpellier Sud de France, Montpellier, France 2 AdipoPhYt SAS, Paris, France 3 Bionov Sarl, Avignon, France 4 ´ Departement de Biochimie, Centre Hospitalier Universitaire, Montpellier, France Scope: Obesity-related metabolic syndrome is often associated with a decrease of insulin sensitivity, inducing several modifications. However, dietary antioxidants could prevent insulin resistance. We have previously shown the preventive effects of a melon superoxide dismutase (SOD) in obese hamsters. However, its antioxidant effects have never been studied on adipose tissue. Methods and results: We evaluated the effects of a 1-month curative supplementation with SODB on the adipose tissue of obese hamsters. Animals received either a standard diet or a cafeteria diet for 15 wk. Cafeteria diet induced obesity and related disorders, including insulin resistance and oxidative stress, in the abdominal adipose tissue. After SODB supplementation, the adipose tissue weight was decreased, probably by activating adipocytes lipolysis and thus reducing their size. SODB treatment also resulted in abdominal adipose tissue fibrosis reduction. Finally, SODB administration increased the expression of endogenous antioxidant enzymes and thus reduced oxidative stress and insulin resistance. The improvement of insulin sensitivity observed after SODB treatment could explain adipocyte lipolysis activation and fibrosis reduction. Conclusion: These findings demonstrate that a dietary SOD supplementation could be a useful strategy against obesity-related modifications in adipose tissue.
Received: June 27, 2013 Revised: September 4, 2013 Accepted: September 4, 2013
Keywords: Adipocyte / Endogenous antioxidant defence / Insulin resistance / Lipolysis / Oxidative stress
1
Introduction
The metabolic syndrome is a combination of disorders, such as glucose intolerance, central obesity, dyslipidemia, and Correspondence: Professor Jean-Max Rouanet, UMR 204 Nutri` Pass, Universite´ Montpellier Sud de France, Place Eugene Bataillon, 34090 Montpellier, France E-mail: [email protected] Fax: +33-467-14-3521 Abbreviations: ARE, antioxidant response element; CAT, catalase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GPx, glutathione peroxidase; HOMA-IR, homeostatic model assessment for insulin resistance; HSL, hormone sensitive lipase; Nrf2, nuclear-factor-E2-related; ROS, reactive oxygen species; SOD, superoxide dismutase C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
insulin resistance [1], that increases the risk of development of cardiovascular disease, type 2 diabetes, and cancer [2–4]. The current lifestyle, called modern Western lifestyle [5], including stress, positive energy balance, low-quality food (rich in fat and energy but, in the meantime, poor in micronutrients), and the disruption of chronobiological function/rhythms, contributes to the increase of metabolic syndrome incidence. Indeed, human obesity does not arise simply from excessive saturated fat in the diet, but rather from a complex interaction of multiple nutritional and lifestyle-related factors directly linked to the excessive consumption of industrial era foods [6]. Therefore, modeling the metabolic disorders of human obesity in animals is better with diets consisting of palatable industrially processed foods (named cafeteria diets), compared to traditional high-fat diets. Indeed, these cafeteria models lead to a phenotype of exaggerated obesity and related www.mnf-journal.com
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disorders [7], particularly in white adipose tissue, which is the main organ involved in obesity. The pathogenesis of obesity-related metabolic syndrome is mainly associated with an increased risk of developing insulin resistance [8,9]. Indeed, in obese subjects, the adiposity which raises both by hyperplasia (cell number increase) and hypertrophy (cell size increase) [10] is associated with an increased release of several factors that are involved in the development of insulin resistance [11, 12]. Insulin resistance causes several physiological modifications in adipose tissue, such as the disruption of the lipogenesis [13] and the lipolysis [14]. Excess of insulin could inhibit lipolysis and induce adipocyte differentiation and lipogenesis. These two modifications lead to adipose tissue expansion and to an increase in fatty acids storage. Finally, the hormone sensitive lipase (HSL), the main lipase involved in adipocyte lipolysis, is also inhibited in insulin resistant states [14, 15]. Moreover, obesity is often accompanied by several modifications of adipose tissue extra cellular matrix constituents [16,17]. Some of them, such as osteopontin, are overexpressed in obese adipose tissue inducing profibrotic factors [18] and collagens overexpression [19]. Obesity is, therefore, associated with adipose tissue fibrosis and particularly pericellular fibrosis that may slow down fat mass loss [20]. Many factors, such as insulin sensitivity, are involved in the development of fibrosis [21]. Finally, Keaney et al. [22] have reported that obesity is a strong independent predictor of systemic oxidative stress. All the modifications of the adipose tissue in obesity, such as inflammation, hypoxia, and alteration of lipid metabolism, induce an overproduction of reactive oxygen species (ROS) and a diminishing of antioxidant defense [23, 24] leading to an increased oxidative stress. Several studies have shown that oxidative stress is involved in the development of insulin resistance [25–27]. Some studies have shown a negative correlation between obesity and tissue or plasma antioxidant capacity [23, 27]. Dietary antioxidant supplementation could, therefore, be a potential therapy. Some authors have demonstrated beneficial effects of antioxidants in obesity and related disorders [28–30]. Besides treatment with dietary antioxidants, such as selenium or vitamins, an original way to increase antioxidant capacity could be by supplying antioxidant enzymes, which have longer lasting effects because of their lower rate of exhaustion than mere metabolites. In this context, we have previously demonstrated the antioxidant properties of superoxide R (SODB, Avignon, France), a dismutase (SOD) by Bionov gastro-resistant encapsulated melon, concentrate particularly rich in SOD, in several models. First, we have observed in vitro that the antioxidant capacity of this melon concentrate is due to its high content of SOD rather than to the other compounds present [31]. We have also reported that SODB has a preventive effect on oxidative stress in a hamster model of obesity and insulin resistance induced by a high-fat diet [32], which more closely mimics lipoprotein metabolism in humans than rat models [33]. However, the mechanism of action of SODB C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
in these studies is still unknown, and information is lacking regarding the potential curative effects of this melon SOD on adipose tissue of obese animals. In this work, we investigated the influence of a 1-month curative supplementation with SODB, in another Golden Syrian hamster model of obesity. Obesity was induced using a diet consisting of high fat, high sugar, and high salt supermarket products. Histopathological analysis of adipose tissue was performed in order to describe its state (fibrosis and adipocyte size and number), and its lipolytic activity and oxidative status were evaluated.
2
Materials and methods
2.1 Preparation and characterization of SODB SODB (Avignon) is a melon (not GMO) concentrate, particularly rich in SOD, resulting from a patented extraction process. For nutraceutical applications, SODB is coated with palm oil in order to protect the SOD activity from digestive enzymes. In this study, it contains 14 U SOD/mg powder measured according to the method of Zhou and Prognon [34]. Detailed information about the antioxidant content of SODB has been published in a previous study [31].
2.2 Experimental design Seventeen 3-wk-old male golden Syrian hamsters (Janvier, Le Genest-St-Isle, France) were used. They were housed at 23⬚C, subjected to a 12-h light–dark cycle with free access to both food and water, and handled according to the guidelines of the Committee on Animal Care at the University of Montpellier (permission number C 34 249) and NIH guidelines [35]. After an 18-day adaptation period, the hamsters (75– 80 g) were randomly divided into three groups. Two groups of hamsters (n = 5) were assigned for 19 wk to a cafeteria diet consisting of nine types of palatable industrially processed foods designed for human consumption (cake, potato crisps, sweets, cheese, etc.) and selected for their high energy, fat, sugar, and/or salt content. The cafeteria items, which induced obesity, were weighed before being presented to the hamsters, and were provided in excess. Detailed information about the nutritional value and ingredients of all foodstuffs used in this diet are presented in our previous study [36]. After 15 wk, one of the two groups of obese animals was given SODB (10 U/day) orally for the last 4 wk (the OB-SODB group), while the other group was maintained on the cafeteria diet alone (the OB group). Another group of hamsters (n = 7) was fed a standard pelleted diet (EF Hamster Control E21000– 04, SSNIFF, Soest, Germany) and served as control (the STD group). Food intake and body weight were recorded daily. www.mnf-journal.com
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2.3 Plasma analysis At the end of the experimental period, the hamsters were deprived of food overnight. Fasting blood samples were collected by cardiac puncture. Plasma and erythrocytes were separated by centrifugation at 4000 rpm for 10 min and stored at –80⬚C until analysis. Insulin level was assessed using immunoassay kits from Mercodia (Uppsala, Sweden). The homeostatic model assessment for insulin resistance (HOMA-IR) was determined from fasting insulin and glucose values according to Matthews et al. [37]: HOMA-IR = (fasting glucose (mmol /L ) ×fasting insulin (mU/L ))/22.5.
2.4 Adipose tissue sampling The abdominal, perirenal, and epididymal adipose tissues were rinsed with 0.15 M NaCl to remove residual blood, rapidly excised, weighed, sectioned for analyses, and stored at –80⬚C. The left tibia was dissected and its length was measured in order to standardize the adipose tissue weight, for comparison.
2.5 Adipose tissue histology Before the abdominal adipose tissue was frozen, tissue samples from each group were removed and fixed in 10% neutral-buffered formaldehyde for histopathological analysis. Adipose tissue samples were blocked in paraffin wax and 3-m-thick sections were processed routinely for hematoxylin and eosin staining. The mean area of 40 cells was measured, and number of adipocytes was counted on five different sections for each adipose tissue sample. For fibrosis determination, adipose tissue sections were stained with 0.1% picrosirius red and mounted in Eukitt medium. Fibrosis was quantified in five to ten given fields (of 100 adipocytes), and expressed as the percentage of fibrous tissue area stained with picrosirius red. All analyses were performed using image analysis software (ImageJ).
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(Milpitas, CA, USA). Hydroxyproline content was expressed as milligrams per gram of adipose tissue.
2.7 Determination of adipose tissue SOD, catalase (CAT), glutathione peroxidase (GPx), and HSL expression by Western blot The abdominal adipose tissue protein extraction was carried out on ice-cold 20 mM Tris buffer (pH 6.8) containing 150 mM NaCl, 1 mM EDTA, 1% Triton 20%, 0.1% SDS, and 1% protease inhibitor cocktail. After centrifugation (1500 rpm, 15 min at 4⬚C), the supernatant was collected and extracted tissue proteins were then separated by SDS PAGE. Equal amounts of proteins were loaded onto a 15% acrylamide gel with a 4% stacking acrylamide gel. Migration was conducted in a Tris-glycine-SDS buffer from Sigma-Aldrich (Saint Quentin Fallavier, France). After separation, proteins were transferred onto nitrocellulose membranes (Whatman, Dassel, Germany). Protein expressions were detected using Western blotting. The primary antibodies against rodent Cu/Zn-SOD, Mn-SOD, CAT, GPx, HSL, and the control protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were provided by R&D Systems (Lille, France). Expression of GAPDH was used for checking the equal protein load across gel tracks. Secondary antibodies (Life Technologies, Carlsbad, CA, USA) coupled with alkaline phosphatase were used for revealing all the primary antibodies. Band densities were obtained by scanning the membranes. Data were standardized within membranes by expressing the density of each band of interest relative to that of GAPDH in the same lane.
2.8 Determination of adipose tissue NAD(P)H-dependent superoxide anion production As previously described [38], samples of abdominal adipose tissue were homogenized and centrifuged at 4000 rpm for 20 min. The supernatant was used to measure NAD(P)Hdependent superoxide production in the presence of 2 mM of KCN, by the intensity of lucigenin (10 M)-enhanced chemiluminescence. The intensity of luminescence was recorded on a microplate luminometer (Perkin Elmer Wallace, Victor, Turku, Finland). Results were expressed as relative light units per milligram of protein.
2.6 Determination of adipose tissue hydroxyproline content
2.9 Statistical analyses
Homogenates of abdominal adipose tissue (100 mg in 1 mL of water) were hydrolyzed in the presence of concentrated HCl in teflon-capped vials at 120⬚C for 3 h. Hydrolyzates were dried and the determination of hydroxyproline content was performed with a commercial assay kit from BioVision
Data are shown as means ± SEM. Statistical analysis of the data was carried out using StatView IV software (Abacus Concepts, Berkeley, CA, USA) by one-way analysis of variance followed by Fisher’s protected least significant difference test. Differences were considered significant at p < 0.05.
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Table 1. Body and adipose tissue weights, insulinemia, HOMA-IR, and adipose tissue superoxide anion production in hamsters
STD Body weight at 15 wk (g) Final body weight (g) at 19 wk Total adipose tissue weight/tibia length (g/cm) Insulin (g/L) HOMA-IR Superoxide anion production (RLU × 103 /mg protein)
111 114 1.5 1.07 11.88 299
OB ± ± ± ± ± ±
2a 2a 0.1a 0.11a 1.74a 39a
195 200 6.3 5.10 77.15 781
OB-SODB ± ± ± ± ± ±
3b
195 188 4.9 3.85 40.38 259
2b 0.3b 0.58b 4.30b 202b
± ± ± ± ± ±
2b 2c 0.1c 0.55c 8.12c 100a
Values are means ± SEM (n = 14). Means in a row with superscripts without a common letter differ significantly, p < 0.05.
3
Results
3.1 SODB supplementation decreased body weight, insulinemia, and insulin resistance Cafeteria diet induced a significant threefold increase in body weight compared with the standard diet (Table 1). SODB supplementation decreased body weight (5% lower than in the untreated OB group), although food intake was not affected. In comparison with standard diet, insulinemia and HOMA-IR were increased fivefold by the cafeteria diet, and were significantly attenuated by SODB treatment (∼25 and ∼50%, respectively) (Table 1). 3.2 SODB supplementation reduced adipose tissue weight by decreasing adipocyte size In the OB group, the corrected adipose tissue weight (i.e., the sum of abdominal, perirenal, and epididymal adipose tissues, in relation to the tibia length) was fourfold higher than in the STD group (Table 1), and decreased significantly after SODB treatment (by 22% compared to untreated OB group). As shown in Fig. 1, cafeteria diet induced hyperplasia and hypertrophy of abdominal adipocytes. Indeed, adipocytes number was more than twice higher and adipocytes area was more than fourfold increased in OB group, compared to STD group. SODB treatment had no effect on adipocytes hyperplasia (Fig. 1B). However, adipocytes size was significantly reduced by 54% after SODB supplementation, compared to untreated OB group (Fig. 1C). 3.3 SODB supplementation modulated obesity-induced fibrosis of adipose tissue Figure 2A and B represents the amount of total fibrosis, quantified by picrosirius red staining, which was twofold increased in abdominal adipose tissue of obese hamsters (OB group), compared to STD group. The determination of hydroxyproline content confirmed this increase in adipose tissue fibrosis of OB hamsters (Fig. 2C). SODB treatment corrected the percentage of fibrous tissue area stained with picrosirius red and reduced the adipose tissue hydroxylproline content by 52%, compared to untreated OB group (Fig. 2). C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.4 SODB supplementation improved adipocytes lipolysis Cafeteria diet seemed to alter lipolytic activity of abdominal adipose tissue by reducing the expression of HSL (by 43% compared to STD group). However, SODB supplementation improved lipolytic activity of adipose tissue by restoring HSL expression (Fig. 3).
3.5 SODB supplementation modulated adipose tissue oxidative status Total superoxide anion production in the abdominal adipose tissue was increased more than twofold in OB hamsters when compared with the STD group, but was fully corrected after SODB supplementation (Table 1). Adipose tissue expression of antioxidant enzymes was significantly impaired by the cafeteria diet, i.e., a 28% decrease in total SOD level, a 54% decrease in GPx level, and a 42% decrease in CAT level, when compared to STD (Fig. 4). SODB treatment raised the expression of all these enzymes. Indeed, expressions of antioxidant enzymes in OB-SODB adipose tissue did not differ from those of the STD group (Fig. 4).
4
Discussion
The cafeteria diet induced obesity and related disorders in hamsters, including insulin resistance and oxidative stress, in the abdominal adipose tissue. Here, we show that curative supplementation with SODB decreases adipose tissue weight probably by activating adipocytes lipolysis and thus reducing their size. SODB treatment also resulted in abdominal adipose tissue fibrosis reduction. Finally, we demonstrate that SODB administration increases the expression of endogenous antioxidant enzymes and thus reduces oxidative stress and insulin resistance, confirming and extending our previous results regarding the antioxidant effects of SODB supplementation [32, 36]. Several models of obesity display a decrease in insulin sensitivity. Indeed, visceral fat accumulation is a major contributor to the development of insulin resistance [39]. In this context, cafeteria diet induced obesity and related www.mnf-journal.com
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Figure 1. Abdominal adipocyte size and number. (A) Hematoxylin/eosin staining of abdominal adipose tissue sections from a representative hamster from each group. (B) Number of adipocytes counted on a determined field on at least five sections per animal. (C) Mean of adipocytes area of 40 cells determined on at least five sections per animal. Values with different letters are significantly different (p < 0.05).
insulin resistance, as shown by HOMA-IR level in OB group. As previously demonstrated, the oral administration of SODB could improve insulin sensitivity [32, 36]. The body weight gain induced by the high-fat, high-sugar, and high-salt diet (OB hamsters) was higher than by the standard diet, and considerably amplified when compared to that generated by traditional high-fat diets [7, 32]. Cafeteria diet induced a significant increase in abdominal adipose tissue weight by both hyperplasia and hypertrophy. Indeed, as in numerous animal models of obesity [10], both adipocyte size and number were increased in the abdominal adipose tissue of OB hamsters. SODB oral administration led to adipose tissue weight reduction, which is mainly due to a decrease of the adipocyte size, while adipocyte number remained at the elevated level achieved during the period of weight gain. Indeed, in case of weight loss, adipocytes can shrink but often remain constant in number [40]. SODB does not seem to have anti-adipogenic activity. The decrease of adipocyte size and thus the loss in abdominal fat could be explained by an increased lipolysis, as shown C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
by others authors. For example, taurine, a powerful antioxidant, could raise lipolysis in adipocytes [41] and decreases body weight and abdominal fat [28, 30]. In the same way, oligonol, a phenolic product from lychee fruit, could enhance lipolysis in primary rat adipocytes [42]. Lipolysis modulation seems to be closely linked to insulin signaling and the interplay between adipose tissue state and insulin sensitivity has been pointed out by numerous studies [11, 43], showing that insulin inhibits adipocyte lipolysis and enhances adipocyte differentiation [43]. The insulin-induced phosphorylation and activation of the insulin receptor and several kinases and phosphatases stimulate lipogenesis [15]. On the contrary, insulin impairs lipolysis by restricting PKA activation by cAMP [15]. Hence, HSL expression is decreased in the obese insulin resistant state [14]. Therefore, the decrease of adipocyte lipolysis in the OB hamsters could be explained by hyperinsulinemia and insulin resistance induced by cafeteria diet. Moreover, the increase of lipolysis after SODB supplementation is probably linked to the correction of insulin signaling in SODB-treated obese
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Figure 2. Abdominal adipose tissue fibrosis quantification. (A) Picrosirius red staining of abdominal tissue sections from a representative hamster from each group. (B) Percentage of fibrotic tissue in a given field determined after picrosirius red staining observation. (C) Hydroxyproline content determination, expressed as milligrams per gram of adipose tissue. Values with different letters are significantly different (p < 0.05).
animals (as shown by decreased insulinemia and insulin resistance). Adipocyte lipolysis leads to an increase in fatty acids release that can cause ectopic fat storage. However, several studies suggest that antioxidants can promote fat burning by removing ROS and thus stimulating O2 consumption in adipocytes [44]. Cafeteria diet also induced fibrosis in abdominal adipose tissue of OB hamsters, as already shown by Divoux et al. [20] and Henegar et al. [16]. Moreover, a positive relationship between insulin resistance and fibrosis was also demonstrated [45]. Indeed, fibrosis and collagen VI accumulation in human adipose tissue are inversely correlated with insulin sensitivity. Therefore, the increase of fibrosis in adipose tis-
sue of OB hamsters could be due to the cafeteria diet-induced insulin resistance. And the increase of adipose tissue insulin sensitivity could explain the decrease of fibrosis observed after SODB supplementation. Lowering in adipose tissue fibrosis and activation of adipocyte lipolysis, both beneficial effects observed after SODB treatment, appear to be linked to the decrease in insulin resistance. Overproduction of ROS has been implicated as an important contributor to the pathogenesis of obesityassociated insulin resistance [25,26]. As expected by excess of glucose and some saturated fatty acids [46], cafeteria diet induced over production of ROS in adipose tissue, as shown by the raise in O2 ·− production. Moreover, a decrease of
Figure 3. Expression of abdominal adipose tissue hormone sensitive lipase (HSL). Expression levels were quantified by standardizing the density of each band of interest to that of GAPDH in the same lane. Results are then expressed relative to levels in the STD group. Values with different letters are significantly different (p < 0.05). C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 4. Abdominal adipose tissue antioxidant enzyme expressions. (A) Total SOD expression (Cu/Zn-SOD plus Mn-SOD expression). (B) GPx expression. (C) CAT expression. Expression levels were quantified by standardizing the density of each band of interest to that of GAPDH in the same lane. Results are then expressed relative to levels in the STD group. Values with different letters are significantly different (p < 0.05).
antioxidant enzymes expression has been observed in adipose tissue of OB hamsters, as did Furukawa et al. [23], who also demonstrated an impairment of antioxidant defense in obese mice. This emergence of an oxidative stress could lead to an impaired insulin sensitivity in OB animals. Adversely, a fall of ROS production is linked to a decrease in insulin resistance. Yan et al. [29] reported that antioxidants, such as catechins, could improve adipose tissue insulin resistance, and exact this effect on their ROS scavenging functions. These findings suggest that the decrease of adipose tissue O2 ·− production after SODB supplementation, could restore insulin sensitivity, and then improve adipocyte lipolysis and fibrosis. The main result of this study is the induction of endogenous antioxidant enzymes expression after SODB supplementation. Indeed, the expression of total SOD, GPx, and CAT was corrected in SODB-treated OB group. Such an induction could explain the decrease in ROS production, and then the improvement of insulin sensitivity. Houstis et al. [26] observed a decrease in adipocyte insulin resistance after ROS C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
diminishing by SOD’s induction. This suggests that SODB treatment could enhance adipocyte lipolysis and decrease fibrosis in abdominal adipose tissue by inducing endogenous antioxidant defense. But the precise mechanism of action, which explains how an exogenous melon SOD could induce endogenous antioxidant enzymes, remains to be investigated. Although SOD cannot be absorbed, our results suggest that SODB could act by triggering a cascade of events from the intestine till the induction of antioxidant enzymes in other tissues. Other exogenous SODs seem to act by inducing endogenous antioxidant defense in the tissues, as reviewed by Carillon et al. [47], and some hypothesis have been proposed even if the precise mechanism is still unknown. Indeed, it could be hypothesized that the induction of antioxidant enzymes is regulated at the transcriptional level through the nuclear-factor-E2-related factor (Nrf2)/antioxidant response element (ARE) pathway [48–51]. Several studies have also demonstrated that dietary antioxidant supplements, such as 7,3 ,4 -trihydroxydihydroflavone (butin) and curcumin, exert
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their antioxidant effects by activating the Nrf2/ARE pathway [52, 53]. The induction of several antioxidant enzymes avoids imbalance, which could be involved in some diseases, such as Down syndrome [54, 55]. Indeed, CAT and GPx remove the H2 O2 produced after the dismutation of O2 ·− by SOD. This global induction of endogenous defense, possibly by the activation of Nrf2/ARE pathway, suggests that SODB could have potential applications in several situations in which oxidative stress is enhanced. Julie Carillon was supported by a “CIFRE grant” (Convention Industrielle de Formation par la REcherche, n⬚ 0417/2010) from Bionov (Avignon, France) and the French “Association Nationale de la Recherche et de la Technologie”. Potential conflict of interest statement: Dominique Lacan is Bionov R&D director. The other authors declare no conflicts of interest.
5
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[29] Yan, J., Zhao, Y., Suo, S., Liu, Y. et al., Green tea catechins ameliorate adipose insulin resistance by improving oxidative stress. Free Radic. Biol. Med. 2012, 52, 1648–1657. [30] Zhang, M., Bi, L. F., Fang, J. H., Su, X. L. et al., Beneficial effects of taurine on serum lipids in overweight or obese non-diabetic subjects. Amino Acids 2004, 26, 267–271. [31] Carillon, J., Del Rio, D., Teissedre, P. L., Cristol, J. P. et al., Antioxidant capacity and angiotensin I converting enzyme inhibitory activity of a melon concentrate rich in superoxide dismutase. Food Chem. 2012, 135, 1298–1302. ´ ´ K., Agne, A., Lacan, D., Ramos, J. et al., Preven[32] Decord e, tive effect of a melon extract rich in superoxide scavenging activity on abdominal and liver fat and adipokine imbalance in high-fat-fed hamsters. J. Agric. Food Chem. 2009, 57, 6461–6467. [33] Kasim-Karakas, S. E., Vriend, H., Almario, R., Chow, L.-C. et al., Effects of dietary carbohydrates on glucose and lipid metabolism in golden Syrian hamsters. J. Lab. Clin. Med. 1996, 128, 208–213. [34] Zhou, J. Y., Prognon, P., Raw material enzymatic activity determination: a specific case for validation and comparison of analytical methods—the example of superoxide dismutase (SOD). J. Pharm. Biomed. Anal. 2006, 40, 1143–1148. [35] NIH, National Research Council Guide for the Care and the Use of Laboratory Animals. National Institutes of Health Publication no. 85–123 (rev.), U.S. Government Printing Office, Washington, DC 1985. [36] Carillon, J., Romain, C., Bardy, G., Fouret, G. et al., Cafeteria diet induces obesity and insulin resistance associated with oxidative stress but not with inflammation. Improvement by dietary supplementation of a melon superoxide dismutase. Free Radic. Biol. Med. 2013, 65, 254–261. [37] Matthews, D. R., Hosker, J. P., Rudenski, A. S., Naylor, B. A. et al., Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985, 28, 412–419. ´ ´ K., Riss, J., Dallas, C. et al., A commercial [38] Sutra, T., Decord e, extract of fruits and vegetables, Oxxynea, acts as a powerful antiatherosclerotic supplement in an animal model by reducing cholesterolemia, oxidative stress, and NADPH oxidase expression. J. Agric. Food Chem. 2007, 55, 4258–4263. [39] Nagaretani, H., Nakamura, T., Funahashi, T., Kotani, K. et al., Visceral fat is a major contributor for multiple risk factor clustering in Japanese men with impaired glucose tolerance. Diabetes Care 2001, 24, 2127–2133. [40] Spalding, K. L., Arner, E., Westermark, P. O., Bernard, S. et al., Dynamics of fat cell turnover in humans. Nature 2008, 453, 783–787. [41] Zentella de Pina, G., de la Rosa-Cuevas, G., Vazquez-Meza, H., Pina, E. et al., Taurine in adipocytes prevents insulin-
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[43] Schroder, K., Wandzioch, K., Helmcke, I., Brandes, R. P., NOX4 acts as a switch between differentiation and proliferation in preadipocytes. Arterioscler. Thromb. Vasc. Biol. 2009, 29, 239–245. [44] Wang, T., Si, Y., Shirihai, O. S., Si, H. et al., Respiration in adipocytes is inhibited by reactive oxygen species. Obesity 2010, 18, 1493–1502. [45] Pessin, J. E., Kwon, H., How does high-fat diet induce adipose tissue fibrosis? J. Invest. Med. 2012, 60, 1147–1150. [46] Han, C. Y., Umemoto, T., Omer, M., Hartigh, L. J. D. et al., NADPH oxidase-derived reactive oxygen species increases expression of monocyte chemotactic factor genes in cultured adipocytes. J. Biol. Chem. 2012, 287, 10379–10393. [47] Carillon, J., Rouanet, J. M., Cristol, J. P., Brion, R., Superoxide dismutase administration, a potential therapy against oxidative stress related diseases: several routes of supplementation and proposal of an original mechanism of action. Pharm. Res. 2013, doi:10.1007/s11095-013-1113-5. [48] Juurlink, B. H. J., Dietary Nrf2 activators inhibit atherogenic processes. Atherosclerosis 2012, 225, 29–33. [49] Negi, G., Kumar, A., Joshi, R. P., Sharma, S. S., Oxidative stress and Nrf2 in the pathophysiology of diabetic neuropathy: old perspective with a new angle. Biochem. Biophys. Res. Commun. 2011, 408, 1–5. [50] Nguyen, T., Sherratt, P. J., Pickett, C. B., Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu. Rev. Pharmacol. Toxicol. 2003, 43, 233–260. [51] Pedruzzi, L. M., Stockler-Pinto, M. B., Leite, M. Jr., Mafra, D., Nrf2ekeap1 system versus NF-kB: the good and the evil in chronic kidney disease? Biochimie 2012, 94, 2461–2466. [52] Carmona Ramirez, I., Santamaria, A., Tobon-Velascob, J. C., Orozco-Ibarra, M. et al., Curcumin restores Nrf2 levels and prevents quinolinic acid-induced neurotoxicity. J. Nutr. Biochem. 2013, 24, 14–24. [53] Zhang, R., Chae, S., Lee, J. H., Hyun, J. W., The cytoprotective effect of butin against oxidative stress is mediated by the up-regulation of manganese superoxide dismutase expression through a PI3K/Akt/Nrf2-dependent pathway. J. Cell. Biochem. 2012, 113, 1987–1997. [54] Muchova, J., Sustrova, M., Garaiova, I., Liptakova, A. et al. Influence of age on activities of antioxidant enzymes and lipid peroxidation products in erythrocytes and neutrophils of Down syndrome patients. Free Radic. Biol. Med. 2001, 31, 499–508. [55] Perluigi, M., Butterfield, A., Oxidative stress and down syndrome: a route toward Alzheimer-like dementia. Curr. Gerontol. Geriatr. Res. 2012, 2012, 724904.
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DOI 10.1002/mnfr.201300466
Mol. Nutr. Food Res. 2014, 58, 842–850
RESEARCH ARTICLE
Curative diet supplementation with a melon superoxide dismutase reduces adipose tissue in obese hamsters by improving insulin sensitivity 2 ´ Julie Carillon1,3 , Lucie Knabe1 , Anne Montalban1 , Marie Stevant , Mayoura Keophiphath2 , 3 1,4 1 Dominique Lacan , Jean-Paul Cristol and Jean-Max Rouanet 1
´ ´ ´ Nutrition & Metabolisme, UMR 204 NutriPass – Prevention des Malnutritions et des Pathologies Associees, Universite´ Montpellier Sud de France, Montpellier, France 2 AdipoPhYt SAS, Paris, France 3 Bionov Sarl, Avignon, France 4 ´ Departement de Biochimie, Centre Hospitalier Universitaire, Montpellier, France Scope: Obesity-related metabolic syndrome is often associated with a decrease of insulin sensitivity, inducing several modifications. However, dietary antioxidants could prevent insulin resistance. We have previously shown the preventive effects of a melon superoxide dismutase (SOD) in obese hamsters. However, its antioxidant effects have never been studied on adipose tissue. Methods and results: We evaluated the effects of a 1-month curative supplementation with SODB on the adipose tissue of obese hamsters. Animals received either a standard diet or a cafeteria diet for 15 wk. Cafeteria diet induced obesity and related disorders, including insulin resistance and oxidative stress, in the abdominal adipose tissue. After SODB supplementation, the adipose tissue weight was decreased, probably by activating adipocytes lipolysis and thus reducing their size. SODB treatment also resulted in abdominal adipose tissue fibrosis reduction. Finally, SODB administration increased the expression of endogenous antioxidant enzymes and thus reduced oxidative stress and insulin resistance. The improvement of insulin sensitivity observed after SODB treatment could explain adipocyte lipolysis activation and fibrosis reduction. Conclusion: These findings demonstrate that a dietary SOD supplementation could be a useful strategy against obesity-related modifications in adipose tissue.
Received: June 27, 2013 Revised: September 4, 2013 Accepted: September 4, 2013
Keywords: Adipocyte / Endogenous antioxidant defence / Insulin resistance / Lipolysis / Oxidative stress
1
Introduction
The metabolic syndrome is a combination of disorders, such as glucose intolerance, central obesity, dyslipidemia, and Correspondence: Professor Jean-Max Rouanet, UMR 204 Nutri` Pass, Universite´ Montpellier Sud de France, Place Eugene Bataillon, 34090 Montpellier, France E-mail: [email protected] Fax: +33-467-14-3521 Abbreviations: ARE, antioxidant response element; CAT, catalase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GPx, glutathione peroxidase; HOMA-IR, homeostatic model assessment for insulin resistance; HSL, hormone sensitive lipase; Nrf2, nuclear-factor-E2-related; ROS, reactive oxygen species; SOD, superoxide dismutase C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
insulin resistance [1], that increases the risk of development of cardiovascular disease, type 2 diabetes, and cancer [2–4]. The current lifestyle, called modern Western lifestyle [5], including stress, positive energy balance, low-quality food (rich in fat and energy but, in the meantime, poor in micronutrients), and the disruption of chronobiological function/rhythms, contributes to the increase of metabolic syndrome incidence. Indeed, human obesity does not arise simply from excessive saturated fat in the diet, but rather from a complex interaction of multiple nutritional and lifestyle-related factors directly linked to the excessive consumption of industrial era foods [6]. Therefore, modeling the metabolic disorders of human obesity in animals is better with diets consisting of palatable industrially processed foods (named cafeteria diets), compared to traditional high-fat diets. Indeed, these cafeteria models lead to a phenotype of exaggerated obesity and related www.mnf-journal.com
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disorders [7], particularly in white adipose tissue, which is the main organ involved in obesity. The pathogenesis of obesity-related metabolic syndrome is mainly associated with an increased risk of developing insulin resistance [8,9]. Indeed, in obese subjects, the adiposity which raises both by hyperplasia (cell number increase) and hypertrophy (cell size increase) [10] is associated with an increased release of several factors that are involved in the development of insulin resistance [11, 12]. Insulin resistance causes several physiological modifications in adipose tissue, such as the disruption of the lipogenesis [13] and the lipolysis [14]. Excess of insulin could inhibit lipolysis and induce adipocyte differentiation and lipogenesis. These two modifications lead to adipose tissue expansion and to an increase in fatty acids storage. Finally, the hormone sensitive lipase (HSL), the main lipase involved in adipocyte lipolysis, is also inhibited in insulin resistant states [14, 15]. Moreover, obesity is often accompanied by several modifications of adipose tissue extra cellular matrix constituents [16,17]. Some of them, such as osteopontin, are overexpressed in obese adipose tissue inducing profibrotic factors [18] and collagens overexpression [19]. Obesity is, therefore, associated with adipose tissue fibrosis and particularly pericellular fibrosis that may slow down fat mass loss [20]. Many factors, such as insulin sensitivity, are involved in the development of fibrosis [21]. Finally, Keaney et al. [22] have reported that obesity is a strong independent predictor of systemic oxidative stress. All the modifications of the adipose tissue in obesity, such as inflammation, hypoxia, and alteration of lipid metabolism, induce an overproduction of reactive oxygen species (ROS) and a diminishing of antioxidant defense [23, 24] leading to an increased oxidative stress. Several studies have shown that oxidative stress is involved in the development of insulin resistance [25–27]. Some studies have shown a negative correlation between obesity and tissue or plasma antioxidant capacity [23, 27]. Dietary antioxidant supplementation could, therefore, be a potential therapy. Some authors have demonstrated beneficial effects of antioxidants in obesity and related disorders [28–30]. Besides treatment with dietary antioxidants, such as selenium or vitamins, an original way to increase antioxidant capacity could be by supplying antioxidant enzymes, which have longer lasting effects because of their lower rate of exhaustion than mere metabolites. In this context, we have previously demonstrated the antioxidant properties of superoxide R (SODB, Avignon, France), a dismutase (SOD) by Bionov gastro-resistant encapsulated melon, concentrate particularly rich in SOD, in several models. First, we have observed in vitro that the antioxidant capacity of this melon concentrate is due to its high content of SOD rather than to the other compounds present [31]. We have also reported that SODB has a preventive effect on oxidative stress in a hamster model of obesity and insulin resistance induced by a high-fat diet [32], which more closely mimics lipoprotein metabolism in humans than rat models [33]. However, the mechanism of action of SODB C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
in these studies is still unknown, and information is lacking regarding the potential curative effects of this melon SOD on adipose tissue of obese animals. In this work, we investigated the influence of a 1-month curative supplementation with SODB, in another Golden Syrian hamster model of obesity. Obesity was induced using a diet consisting of high fat, high sugar, and high salt supermarket products. Histopathological analysis of adipose tissue was performed in order to describe its state (fibrosis and adipocyte size and number), and its lipolytic activity and oxidative status were evaluated.
2
Materials and methods
2.1 Preparation and characterization of SODB SODB (Avignon) is a melon (not GMO) concentrate, particularly rich in SOD, resulting from a patented extraction process. For nutraceutical applications, SODB is coated with palm oil in order to protect the SOD activity from digestive enzymes. In this study, it contains 14 U SOD/mg powder measured according to the method of Zhou and Prognon [34]. Detailed information about the antioxidant content of SODB has been published in a previous study [31].
2.2 Experimental design Seventeen 3-wk-old male golden Syrian hamsters (Janvier, Le Genest-St-Isle, France) were used. They were housed at 23⬚C, subjected to a 12-h light–dark cycle with free access to both food and water, and handled according to the guidelines of the Committee on Animal Care at the University of Montpellier (permission number C 34 249) and NIH guidelines [35]. After an 18-day adaptation period, the hamsters (75– 80 g) were randomly divided into three groups. Two groups of hamsters (n = 5) were assigned for 19 wk to a cafeteria diet consisting of nine types of palatable industrially processed foods designed for human consumption (cake, potato crisps, sweets, cheese, etc.) and selected for their high energy, fat, sugar, and/or salt content. The cafeteria items, which induced obesity, were weighed before being presented to the hamsters, and were provided in excess. Detailed information about the nutritional value and ingredients of all foodstuffs used in this diet are presented in our previous study [36]. After 15 wk, one of the two groups of obese animals was given SODB (10 U/day) orally for the last 4 wk (the OB-SODB group), while the other group was maintained on the cafeteria diet alone (the OB group). Another group of hamsters (n = 7) was fed a standard pelleted diet (EF Hamster Control E21000– 04, SSNIFF, Soest, Germany) and served as control (the STD group). Food intake and body weight were recorded daily. www.mnf-journal.com
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2.3 Plasma analysis At the end of the experimental period, the hamsters were deprived of food overnight. Fasting blood samples were collected by cardiac puncture. Plasma and erythrocytes were separated by centrifugation at 4000 rpm for 10 min and stored at –80⬚C until analysis. Insulin level was assessed using immunoassay kits from Mercodia (Uppsala, Sweden). The homeostatic model assessment for insulin resistance (HOMA-IR) was determined from fasting insulin and glucose values according to Matthews et al. [37]: HOMA-IR = (fasting glucose (mmol /L ) ×fasting insulin (mU/L ))/22.5.
2.4 Adipose tissue sampling The abdominal, perirenal, and epididymal adipose tissues were rinsed with 0.15 M NaCl to remove residual blood, rapidly excised, weighed, sectioned for analyses, and stored at –80⬚C. The left tibia was dissected and its length was measured in order to standardize the adipose tissue weight, for comparison.
2.5 Adipose tissue histology Before the abdominal adipose tissue was frozen, tissue samples from each group were removed and fixed in 10% neutral-buffered formaldehyde for histopathological analysis. Adipose tissue samples were blocked in paraffin wax and 3-m-thick sections were processed routinely for hematoxylin and eosin staining. The mean area of 40 cells was measured, and number of adipocytes was counted on five different sections for each adipose tissue sample. For fibrosis determination, adipose tissue sections were stained with 0.1% picrosirius red and mounted in Eukitt medium. Fibrosis was quantified in five to ten given fields (of 100 adipocytes), and expressed as the percentage of fibrous tissue area stained with picrosirius red. All analyses were performed using image analysis software (ImageJ).
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(Milpitas, CA, USA). Hydroxyproline content was expressed as milligrams per gram of adipose tissue.
2.7 Determination of adipose tissue SOD, catalase (CAT), glutathione peroxidase (GPx), and HSL expression by Western blot The abdominal adipose tissue protein extraction was carried out on ice-cold 20 mM Tris buffer (pH 6.8) containing 150 mM NaCl, 1 mM EDTA, 1% Triton 20%, 0.1% SDS, and 1% protease inhibitor cocktail. After centrifugation (1500 rpm, 15 min at 4⬚C), the supernatant was collected and extracted tissue proteins were then separated by SDS PAGE. Equal amounts of proteins were loaded onto a 15% acrylamide gel with a 4% stacking acrylamide gel. Migration was conducted in a Tris-glycine-SDS buffer from Sigma-Aldrich (Saint Quentin Fallavier, France). After separation, proteins were transferred onto nitrocellulose membranes (Whatman, Dassel, Germany). Protein expressions were detected using Western blotting. The primary antibodies against rodent Cu/Zn-SOD, Mn-SOD, CAT, GPx, HSL, and the control protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were provided by R&D Systems (Lille, France). Expression of GAPDH was used for checking the equal protein load across gel tracks. Secondary antibodies (Life Technologies, Carlsbad, CA, USA) coupled with alkaline phosphatase were used for revealing all the primary antibodies. Band densities were obtained by scanning the membranes. Data were standardized within membranes by expressing the density of each band of interest relative to that of GAPDH in the same lane.
2.8 Determination of adipose tissue NAD(P)H-dependent superoxide anion production As previously described [38], samples of abdominal adipose tissue were homogenized and centrifuged at 4000 rpm for 20 min. The supernatant was used to measure NAD(P)Hdependent superoxide production in the presence of 2 mM of KCN, by the intensity of lucigenin (10 M)-enhanced chemiluminescence. The intensity of luminescence was recorded on a microplate luminometer (Perkin Elmer Wallace, Victor, Turku, Finland). Results were expressed as relative light units per milligram of protein.
2.6 Determination of adipose tissue hydroxyproline content
2.9 Statistical analyses
Homogenates of abdominal adipose tissue (100 mg in 1 mL of water) were hydrolyzed in the presence of concentrated HCl in teflon-capped vials at 120⬚C for 3 h. Hydrolyzates were dried and the determination of hydroxyproline content was performed with a commercial assay kit from BioVision
Data are shown as means ± SEM. Statistical analysis of the data was carried out using StatView IV software (Abacus Concepts, Berkeley, CA, USA) by one-way analysis of variance followed by Fisher’s protected least significant difference test. Differences were considered significant at p < 0.05.
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Table 1. Body and adipose tissue weights, insulinemia, HOMA-IR, and adipose tissue superoxide anion production in hamsters
STD Body weight at 15 wk (g) Final body weight (g) at 19 wk Total adipose tissue weight/tibia length (g/cm) Insulin (g/L) HOMA-IR Superoxide anion production (RLU × 103 /mg protein)
111 114 1.5 1.07 11.88 299
OB ± ± ± ± ± ±
2a 2a 0.1a 0.11a 1.74a 39a
195 200 6.3 5.10 77.15 781
OB-SODB ± ± ± ± ± ±
3b
195 188 4.9 3.85 40.38 259
2b 0.3b 0.58b 4.30b 202b
± ± ± ± ± ±
2b 2c 0.1c 0.55c 8.12c 100a
Values are means ± SEM (n = 14). Means in a row with superscripts without a common letter differ significantly, p < 0.05.
3
Results
3.1 SODB supplementation decreased body weight, insulinemia, and insulin resistance Cafeteria diet induced a significant threefold increase in body weight compared with the standard diet (Table 1). SODB supplementation decreased body weight (5% lower than in the untreated OB group), although food intake was not affected. In comparison with standard diet, insulinemia and HOMA-IR were increased fivefold by the cafeteria diet, and were significantly attenuated by SODB treatment (∼25 and ∼50%, respectively) (Table 1). 3.2 SODB supplementation reduced adipose tissue weight by decreasing adipocyte size In the OB group, the corrected adipose tissue weight (i.e., the sum of abdominal, perirenal, and epididymal adipose tissues, in relation to the tibia length) was fourfold higher than in the STD group (Table 1), and decreased significantly after SODB treatment (by 22% compared to untreated OB group). As shown in Fig. 1, cafeteria diet induced hyperplasia and hypertrophy of abdominal adipocytes. Indeed, adipocytes number was more than twice higher and adipocytes area was more than fourfold increased in OB group, compared to STD group. SODB treatment had no effect on adipocytes hyperplasia (Fig. 1B). However, adipocytes size was significantly reduced by 54% after SODB supplementation, compared to untreated OB group (Fig. 1C). 3.3 SODB supplementation modulated obesity-induced fibrosis of adipose tissue Figure 2A and B represents the amount of total fibrosis, quantified by picrosirius red staining, which was twofold increased in abdominal adipose tissue of obese hamsters (OB group), compared to STD group. The determination of hydroxyproline content confirmed this increase in adipose tissue fibrosis of OB hamsters (Fig. 2C). SODB treatment corrected the percentage of fibrous tissue area stained with picrosirius red and reduced the adipose tissue hydroxylproline content by 52%, compared to untreated OB group (Fig. 2). C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
3.4 SODB supplementation improved adipocytes lipolysis Cafeteria diet seemed to alter lipolytic activity of abdominal adipose tissue by reducing the expression of HSL (by 43% compared to STD group). However, SODB supplementation improved lipolytic activity of adipose tissue by restoring HSL expression (Fig. 3).
3.5 SODB supplementation modulated adipose tissue oxidative status Total superoxide anion production in the abdominal adipose tissue was increased more than twofold in OB hamsters when compared with the STD group, but was fully corrected after SODB supplementation (Table 1). Adipose tissue expression of antioxidant enzymes was significantly impaired by the cafeteria diet, i.e., a 28% decrease in total SOD level, a 54% decrease in GPx level, and a 42% decrease in CAT level, when compared to STD (Fig. 4). SODB treatment raised the expression of all these enzymes. Indeed, expressions of antioxidant enzymes in OB-SODB adipose tissue did not differ from those of the STD group (Fig. 4).
4
Discussion
The cafeteria diet induced obesity and related disorders in hamsters, including insulin resistance and oxidative stress, in the abdominal adipose tissue. Here, we show that curative supplementation with SODB decreases adipose tissue weight probably by activating adipocytes lipolysis and thus reducing their size. SODB treatment also resulted in abdominal adipose tissue fibrosis reduction. Finally, we demonstrate that SODB administration increases the expression of endogenous antioxidant enzymes and thus reduces oxidative stress and insulin resistance, confirming and extending our previous results regarding the antioxidant effects of SODB supplementation [32, 36]. Several models of obesity display a decrease in insulin sensitivity. Indeed, visceral fat accumulation is a major contributor to the development of insulin resistance [39]. In this context, cafeteria diet induced obesity and related www.mnf-journal.com
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Figure 1. Abdominal adipocyte size and number. (A) Hematoxylin/eosin staining of abdominal adipose tissue sections from a representative hamster from each group. (B) Number of adipocytes counted on a determined field on at least five sections per animal. (C) Mean of adipocytes area of 40 cells determined on at least five sections per animal. Values with different letters are significantly different (p < 0.05).
insulin resistance, as shown by HOMA-IR level in OB group. As previously demonstrated, the oral administration of SODB could improve insulin sensitivity [32, 36]. The body weight gain induced by the high-fat, high-sugar, and high-salt diet (OB hamsters) was higher than by the standard diet, and considerably amplified when compared to that generated by traditional high-fat diets [7, 32]. Cafeteria diet induced a significant increase in abdominal adipose tissue weight by both hyperplasia and hypertrophy. Indeed, as in numerous animal models of obesity [10], both adipocyte size and number were increased in the abdominal adipose tissue of OB hamsters. SODB oral administration led to adipose tissue weight reduction, which is mainly due to a decrease of the adipocyte size, while adipocyte number remained at the elevated level achieved during the period of weight gain. Indeed, in case of weight loss, adipocytes can shrink but often remain constant in number [40]. SODB does not seem to have anti-adipogenic activity. The decrease of adipocyte size and thus the loss in abdominal fat could be explained by an increased lipolysis, as shown C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
by others authors. For example, taurine, a powerful antioxidant, could raise lipolysis in adipocytes [41] and decreases body weight and abdominal fat [28, 30]. In the same way, oligonol, a phenolic product from lychee fruit, could enhance lipolysis in primary rat adipocytes [42]. Lipolysis modulation seems to be closely linked to insulin signaling and the interplay between adipose tissue state and insulin sensitivity has been pointed out by numerous studies [11, 43], showing that insulin inhibits adipocyte lipolysis and enhances adipocyte differentiation [43]. The insulin-induced phosphorylation and activation of the insulin receptor and several kinases and phosphatases stimulate lipogenesis [15]. On the contrary, insulin impairs lipolysis by restricting PKA activation by cAMP [15]. Hence, HSL expression is decreased in the obese insulin resistant state [14]. Therefore, the decrease of adipocyte lipolysis in the OB hamsters could be explained by hyperinsulinemia and insulin resistance induced by cafeteria diet. Moreover, the increase of lipolysis after SODB supplementation is probably linked to the correction of insulin signaling in SODB-treated obese
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Figure 2. Abdominal adipose tissue fibrosis quantification. (A) Picrosirius red staining of abdominal tissue sections from a representative hamster from each group. (B) Percentage of fibrotic tissue in a given field determined after picrosirius red staining observation. (C) Hydroxyproline content determination, expressed as milligrams per gram of adipose tissue. Values with different letters are significantly different (p < 0.05).
animals (as shown by decreased insulinemia and insulin resistance). Adipocyte lipolysis leads to an increase in fatty acids release that can cause ectopic fat storage. However, several studies suggest that antioxidants can promote fat burning by removing ROS and thus stimulating O2 consumption in adipocytes [44]. Cafeteria diet also induced fibrosis in abdominal adipose tissue of OB hamsters, as already shown by Divoux et al. [20] and Henegar et al. [16]. Moreover, a positive relationship between insulin resistance and fibrosis was also demonstrated [45]. Indeed, fibrosis and collagen VI accumulation in human adipose tissue are inversely correlated with insulin sensitivity. Therefore, the increase of fibrosis in adipose tis-
sue of OB hamsters could be due to the cafeteria diet-induced insulin resistance. And the increase of adipose tissue insulin sensitivity could explain the decrease of fibrosis observed after SODB supplementation. Lowering in adipose tissue fibrosis and activation of adipocyte lipolysis, both beneficial effects observed after SODB treatment, appear to be linked to the decrease in insulin resistance. Overproduction of ROS has been implicated as an important contributor to the pathogenesis of obesityassociated insulin resistance [25,26]. As expected by excess of glucose and some saturated fatty acids [46], cafeteria diet induced over production of ROS in adipose tissue, as shown by the raise in O2 ·− production. Moreover, a decrease of
Figure 3. Expression of abdominal adipose tissue hormone sensitive lipase (HSL). Expression levels were quantified by standardizing the density of each band of interest to that of GAPDH in the same lane. Results are then expressed relative to levels in the STD group. Values with different letters are significantly different (p < 0.05). C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Figure 4. Abdominal adipose tissue antioxidant enzyme expressions. (A) Total SOD expression (Cu/Zn-SOD plus Mn-SOD expression). (B) GPx expression. (C) CAT expression. Expression levels were quantified by standardizing the density of each band of interest to that of GAPDH in the same lane. Results are then expressed relative to levels in the STD group. Values with different letters are significantly different (p < 0.05).
antioxidant enzymes expression has been observed in adipose tissue of OB hamsters, as did Furukawa et al. [23], who also demonstrated an impairment of antioxidant defense in obese mice. This emergence of an oxidative stress could lead to an impaired insulin sensitivity in OB animals. Adversely, a fall of ROS production is linked to a decrease in insulin resistance. Yan et al. [29] reported that antioxidants, such as catechins, could improve adipose tissue insulin resistance, and exact this effect on their ROS scavenging functions. These findings suggest that the decrease of adipose tissue O2 ·− production after SODB supplementation, could restore insulin sensitivity, and then improve adipocyte lipolysis and fibrosis. The main result of this study is the induction of endogenous antioxidant enzymes expression after SODB supplementation. Indeed, the expression of total SOD, GPx, and CAT was corrected in SODB-treated OB group. Such an induction could explain the decrease in ROS production, and then the improvement of insulin sensitivity. Houstis et al. [26] observed a decrease in adipocyte insulin resistance after ROS C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
diminishing by SOD’s induction. This suggests that SODB treatment could enhance adipocyte lipolysis and decrease fibrosis in abdominal adipose tissue by inducing endogenous antioxidant defense. But the precise mechanism of action, which explains how an exogenous melon SOD could induce endogenous antioxidant enzymes, remains to be investigated. Although SOD cannot be absorbed, our results suggest that SODB could act by triggering a cascade of events from the intestine till the induction of antioxidant enzymes in other tissues. Other exogenous SODs seem to act by inducing endogenous antioxidant defense in the tissues, as reviewed by Carillon et al. [47], and some hypothesis have been proposed even if the precise mechanism is still unknown. Indeed, it could be hypothesized that the induction of antioxidant enzymes is regulated at the transcriptional level through the nuclear-factor-E2-related factor (Nrf2)/antioxidant response element (ARE) pathway [48–51]. Several studies have also demonstrated that dietary antioxidant supplements, such as 7,3 ,4 -trihydroxydihydroflavone (butin) and curcumin, exert
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their antioxidant effects by activating the Nrf2/ARE pathway [52, 53]. The induction of several antioxidant enzymes avoids imbalance, which could be involved in some diseases, such as Down syndrome [54, 55]. Indeed, CAT and GPx remove the H2 O2 produced after the dismutation of O2 ·− by SOD. This global induction of endogenous defense, possibly by the activation of Nrf2/ARE pathway, suggests that SODB could have potential applications in several situations in which oxidative stress is enhanced. Julie Carillon was supported by a “CIFRE grant” (Convention Industrielle de Formation par la REcherche, n⬚ 0417/2010) from Bionov (Avignon, France) and the French “Association Nationale de la Recherche et de la Technologie”. Potential conflict of interest statement: Dominique Lacan is Bionov R&D director. The other authors declare no conflicts of interest.
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