JOURNAL OF MEDICINAL FOOD J Med Food 18 (1) 2015, 67–75 # Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition DOI: 10.1089/jmf.2013.0132

Fermented Soy Permeate Reduces Cytokine Level and Oxidative Stress in Streptozotocin-Induced Diabetic Rats Ludivine Malarde´,1 Carole Groussard,1 Luz Lefeuvre-Orfila,1 Sophie Vincent,1 The´o Efstathiou,2 and Arlette Gratas-Delamarche1 1

Laboratory M2S, Universite´ Rennes 2-ENS Cachan, Rennes, France. 2 Sojasun Technologies, Noyal sur Vilaine, France.

ABSTRACT Oxidative stress and inflammation are involved in the development of type 1 diabetes and its complications. Because two compounds found in soy, that is, isoflavones and alpha-galactooligosaccharides, have been shown to exert antioxidant and anti-inflammatory effects, this study aimed to assess the effects of a dietary supplement containing these two active compounds, the fermented soy permeate (FSP). We hypothesized that FSP would be able to reduce in vivo oxidative stress and inflammation in streptozotocin (STZ)–induced type 1 diabetic rats. Thirty male Wistar rats were divided into the control placebo, diabetic placebo, and diabetic FSP-supplemented groups. They received daily, by oral gavage, water (placebo groups) or diluted FSP (0.1 g/day; FSP-supplemented group). After 3 weeks, glycemic regulation (glycemia and fructosamine level); the plasma level of carboxymethyllysine (CML), a marker of systemic oxidative stress in diabetes; and the plasma levels of inflammatory markers (CRP, IL-1b, IL-6, and uric acid) were evaluated. Markers of oxidative damage (isoprostanes and GSH/GSSG), antioxidant enzymatic activity (SOD and GPX), and Mn-SOD content were determined in skeletal muscle (gastrocnemius). Diabetic placebo rats exhibited higher CML levels, lower SOD and GPX activities, and decreased Mn-SOD contents. FSP supplementation in diabetic animals normalized the CML and antioxidant enzymatic activity levels and tended to increase Mn-SOD expression. The markers of inflammation whose levels were increased in the diabetic placebo group were markedly decreased by FSP (IL-1b: - 75%, IL-6: - 46%, and uric acid: - 17%), except for CRP. Our results demonstrate that FSP exhibited antioxidant and anti-inflammatory properties in vivo in STZ-induced diabetic rats.

KEY WORDS:  alpha-galactooligosaccharides  inflammation  oxidative stress  rat  soy isoflavones  type 1 diabetes

damage.3,4,8,11 Moreover, genistein, one of the isoflavones in soy, is also able to inhibit inflammatory processes by suppressing NF-kB activation12,13 and the production of TNFalpha, IL-1, and IL-6.14,15 Type 1 diabetes mellitus is an autoimmune disease that is mediated by oxidant and inflammatory mechanisms, leading to beta-cell destruction.16 Among a number of assumptions explaining the origin of this disease, the pathogenesis of type 1 diabetes has recently been attributed to multiple alterations to the gut microflora,16 which trigger inflammatory processes via an abnormal immune response. Oxidative stress and inflammation are implicated in the onset and progression of diabetes-induced complications, such as neuropathy, nephropathy, and cardiovascular diseases.17,18 Despite the widespread use of insulin treatment, these complications induced by oxidative stress and inflammation still occur, requiring adjuvant therapies, such as physical activity or nutritional strategy. Recently, the beneficial effects of soy consumption, such as the glucoregulatory effects of isoflavones, on the therapeutic management of type 1 diabetes have been highlighted.2 Moreover, a diet designed for diabetic subjects, specifically rich in soy compound, allowed to reduce inflammation in patients.19 In a

INTRODUCTION

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oy consumption is well known to exert some health benefits in Asian individuals and is associated with reductions in the prevalence of cardiovascular diseases and cancer.1 Because soy intake is very low in Western countries, primarily because of cultural differences in diet,2 numerous studies have aimed to identify the biologically active components in soy so that these components can be included in dietary supplements for this population. Among all of the active compounds in soy, isoflavones are the most widely studied. They exhibit a number of biological activities, including antioxidant and anti-inflammatory properties. Indeed, soy isoflavones are known to increase the activities of antioxidant enzymes3–6 and upregulate the expression levels of these genes,7 increase the total antioxidant capacity,8 decrease reactive oxygen species (ROS) production,9,10 and reduce the level of oxidative Manuscript received 10 July 2013. Revision accepted 18 June 2014 Address correspondence to: Ludivine Malarde´, PhD, Laboratoire M2S, ENS Cachan, Avenue Robert Schuman, 35170 Bruz, France, Email: [email protected]

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streptozotocin (STZ)–induced diabetic animal model, genistein has been shown to restore altered antioxidant enzyme activities (SOD, GPX, and CAT), decrease the TBARS level, and normalize the concentrations of proinflammatory cytokines (IL-1, IL-6, and TNF-alpha), which are increased in individuals with diabetes.20 Other compounds found in soybeans, namely, galactooligosaccharides (GOSs), have the ability to correct alterations to the gut microflora21 and thus decrease the inflammatory state in a different way. Moreover, GOSs potentiate the absorption of isoflavones.22 Nevertheless, the potential benefits of GOSs in type 1 diabetes have rarely been studied, unlike the effects of soy isoflavones. A French company, Sojasun Technologies, has developed a powder extracted from soybeans called fermented soy permeate (FSP). The novelty and originality of this product is due to the presence of both types of active molecules from soybeans: isoflavones and alpha-linked galactooligosaccharides (alpha-GOSs). The presence of both types of compounds allows the preservation of the synergistic effects of soy components, as the benefits of whole grains versus isolated components have recently been highlighted.23 This patented compound has been shown to exhibit strong antioxidant and anti-inflammatory effects in vitro.24 We hypothesized that, in vivo, FSP would be able to decrease oxidative stress and inflammation observed in diabetic rats. To test our hypothesis, the objective of the present study was therefore to examine the effects of 3 weeks of FSP supplementation (supplemented vs. placebo) on oxidative damages (isoprostanes and GSH/GSSG), antioxidant systems (GPX and SOD activities and Mn-SOD content), classical inflammation markers (CRP, IL-1, and IL-6), and uric acid, a specific marker of inflammation in our model of STZ-induced type 1 diabetes. MATERIALS AND METHODS Animals The experimental protocol was approved by the ethics committee of the University of Rennes and was in accordance with the Guide for the Care and Use of the Laboratory Animals. Thirty male Wistar rats, aged of 9 weeks, were purchased from Janvier and were housed individually in an air-conditioned room with a controlled temperature of 23– 25C and a 12/12-h light/dark cycle. Tap water and food (Table 1; 2020X Teklad Global Soy Protein-Free Extruded Rodent Diet; Harlan) were available ad libitum. During all the experimental protocols, body weight and morphological changes of the animals were daily noted to monitor adaptation of rats to the protocol. Diabetes induction After 1 week of acclimatization, diabetes was induced by intraperitoneal STZ injection (Sigma Aldrich Chemical). STZ was freshly dissolved in citrate buffer (0.1 M, pH 4.5) and administered at a dose of 45 mg/kg of body weight. An additional control healthy group received citrate buffer only. The blood glucose concentration was measured 48 h later

Table 1. Ingredient and Nutrient Composition of the Standard Control Diet Control diet 2020 Protein Fat (acid hydrolysis) Crude fiber Ash Starch Sugar Metabolizable energy Calories from protein Calories from fat Calories from carbohydrate

19.1% 6.5% 2.7% 5.1% 46% 4% 3.1 kcal/g (13.0 kJ/g) 24% 16% 60%

The list of ingredients (first five) for the Harlan-Teklad 2020 diet were ground wheat, ground corn, corn gluten meal, wheat middlings, and brewer’s yeast.

(MediSense Optium glucometer; Abbott); animals with blood glucose levels > 250 mg$dL - 1 were considered diabetic. The other animals received a second STZ injection with further blood glucose measurement 48 h after injection.25 Animals with glycemia < 250 mg$dL - 1 after the two injections were not included in the protocol. Production of FSP and supplementation protocol FSP was provided by Sojasun Technologies, which patented the manufacturing process. In this process, soybeans are dehulled and cooked in water, and then the soy milk is extracted and condensed. The soy extract is then subjected to a fermentation step with baker’s yeast (Saccharomyces cerevisiae) and desiccated to obtain a dry powder. The final product is a soluble powder containing 29% alpha-GOSs and 0.5% soy isoflavones (daidzein and genistein). One week after the confirmation of diabetes, oral supplementation began, and the diabetic rats were randomly assigned to one of two groups (n = 10 rats per group): diabetic rats supplemented with the placebo (group 2) and diabetic rats supplemented with FSP (group 3). Moreover, an additional control group of healthy rats supplemented with the placebo was studied to confirm the pro-oxidant and inflammatory states induced by diabetes (group 1). FSP powder was diluted in water (10%) and administered each day by oral gavage for a period of 3 weeks. Water was used as the placebo. The volume administered was adjusted daily according to the body weight of each animal. Each rat received an FSP dose equivalent to 1 mg of soy isoflavones and 70 mg of alpha-GOSs per day per kg of body weight, according to the recommendations of the French Agency of Food Safety. Preparation of blood and tissue samples At the end of the intervention protocol, the animals were fasted for 24 h and then anesthetized with an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (3 mg/kg).

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Whole blood was collected by cardiac puncture in EDTA tubes, the blood glucose concentration was measured, and the blood samples were immediately centrifuged at 2000 g for 10 min at 4C to obtain the plasma. Aliquots were stored at - 80C until the analysis of biochemical parameters and systemic inflammation. Before decapitation, skeletal muscle (gastrocnemius) was quickly removed, extensively washed in physiological saline buffer, frozen in liquid nitrogen, and stored at - 80C until analysis. After slow thawing, the tissue samples were homogenized in ice-cold 50 mM phosphate buffer (pH 7.4, 1:10 w/w) and centrifuged at 10,000 g for 10 min at 4C. Aliquoted supernatant was used to determine the levels of oxidative stress markers and antioxidant system activity. Free-radical scavengers were added in appropriate amounts to prevent artificial oxidation: 10 lL of butylated hydroxytoluene (10 mM) was added to aliquots used for isoprostane analysis, and 10 lL of MV2P (the scavenger provided in the kit) was added to aliquots used for GSH/ GSSG ratio analysis. The results obtained from skeletal muscle were normalized by protein content to account for individual variations in the body weights of animal during the experiment. Biochemical analysis Biochemical parameters (glycemic regulation and glycooxidation). The plasma fructosamine level was determined by Cerba Laboratory, using a colorimetric method. Carboxymethyllysine (CML) level was determined using a commercially available Circulex ELISA kit (CycLex). Markers of oxidative damage in skeletal muscle. The isoprostane analysis consisted of isoprostane extraction from tissue homogenates by thermolysis and liquidchromatography/mass-spectrometry (LC/MS) analysis, as described previously.26 The extraction protocol was slightly modified for use with tissue homogenates. A volume of 990 lL of plasma, 10 lL of deuterated 8-isoprostane as an internal standard (80 ng/mL), 10 lL of 20 mM deferiprone, 10 lL of 20 mM Desferal, and 1 mL of 15% KOH was added, and the sample was incubated for 60 min at 37C. The alkali was neutralized by the addition of 3.5 mL of 1 M KH2PO4 and 2 mL of 0.1 M phosphate buffer (pH 7) to adjust the pH to a value between 7.2 and 7.4. The samples were loaded into isoprostane affinity columns (Cayman Chemical Company) that had been prepared according to the manufacturer’s instructions. The column was washed twice with 2 mL of 0.1 M phosphate buffer (pH 7) and twice with 2 mL of ultrapure water. Isoprostanes were eluted with 2 mL of 95% ethanol. The eluate was evaporated to dryness under vacuum in a SpeedVac (Sc110A-UVS400A; Savant). The sample was redissolved in 20 lL of solution containing two solvents: solvent B, accounting for 60% (H2O and 0.5% of NH3), and solvent C, accounting for 40% (55% acetonitrile, 45% methanol, and 0.5% of NH4OH). An aliquot of 20 lL of the sample was injected into the high-performance liquid chromatography system and analyzed as previously described.26 The levels of reduced and oxidized glutathione were measured with a Bioxytech GSH/GSSH-412 kit (Bioxytech;

Oxis International, Inc.) with some modifications to adapt the protocol to muscle homogenates, as previously described.27 Activity of antioxidant enzymes (SOD and GPX). Ransod and Ransel kits (Randox) were used to evaluate the SOD and GPX activities, respectively, according to the manufacturer’s instructions. Expression of Mn-SOD. Western blot analyses were used to determine relative levels of Mn-SOD in gastrocnemius muscle samples. Briefly, samples were solubilized in a buffer containing Tris-HCl (pH 6.8), SDS, bromophenol blue, glycerol, and 2-b-mercaptoethanol. Proteins were separated on an SDS-polyacrylamide gel (12.5%) and then transferred overnight onto nitrocellulose membranes (Millipore) in a transfer buffer (25 mM Tris, 192 mM glycine, 0.01% SDS, and 10% ethanol). For normalization, a selected protein sample was run on each gel. The membrane was then washed in Tris-buffered saline/Tween 20 (TTBS 0.1%) for 10 min. After blocking nonspecific binding sites for 1 h at room temperature using 5% bovine serum albumin diluted in 0.1% TTBS, the membranes were incubated overnight at 4C with anti-Mn-SOD antibodies (Enzo Life Sciences). The membranes were washed three times with TTBS and then incubated for 1 h with infrared-labeled secondary goat antimouse IRDye 800 antibodies (LI-COR Biosciences) that bound to the primary antibody. The bound complex was detected using the Odyssey Infrared Imaging System (LI-COR Biosciences). The images were analyzed using Odyssey Application Software, version 1.2 (LI-COR), to obtain the integrated intensities. Western blots were normalized using the level of a housekeeping protein (HSC70: the constitutive isoform of heat shock protein 70). Inflammatory markers. The uric acid, IL-1b, IL-6, and C-reactive protein levels in serum were determined using commercial enzyme-linked assay kits according to the manufacturer’s instructions. The kits were purchased from Cayman Chemical (Enzo Life Sciences). Statistical analyses Data are expressed as means – SEM. Data were analyzed by performing a one-way analysis of variance followed by a Student-Newman-Keuls post-hoc test to determine the significance of the differences between the three groups. P < .05 was considered significant. RESULTS Effects of FSP supplementation on glycemic regulation and glycoxidation markers As expected, at the end of the protocol, the blood glucose, plasma fructosamine, and CML levels were higher in diabetic placebo rats (group 2) than in controls (group 1) (Fig. 1A–C).

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FIG. 1. Effects of diabetes and fermented soy permeate (FSP) supplementation (3 weeks) on biochemical parameters (A, B: glycemic regulation and C: glycoxidation). Values are the means – SEM. **P < .01 when compared with group 1 (i.e., the effects of diabetes). xxP < .01 when compared with group 2 (i.e., the effects of FSP supplementation in diabetic rats).

FSP supplementation had no effect on the blood glucose or plasma fructosamine level (Fig. 1A, B). In contrast, the level of plasma CML, a glycoxidative marker, was significantly lower after 3 weeks of supplementation ( - 36%) (Fig. 1C).

Effects of FSP supplementation on muscle pro/antioxidant status As shown in Figure 2A and B, the muscle markers of oxidative damages, isoprostanes and GSH/GSSG ratio,

FIG. 2. Effects of diabetes and FSP supplementation (3 weeks) on markers of oxidative damages (A, B) and antioxidant enzymatic activities (C, D) in diabetic rats. Values are the means – SEM. *P < .05 when compared with group 1 (i.e., the effects of diabetes). x P < .05 when compared with group 2 (i.e., the effects of FSP supplementation in diabetic rats).

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FSP-supplemented group (nonsignificant), although the levels were not restored to the control levels (Fig. 3). Effects of FSP supplementation on systemic inflammatory markers Figure 4 illustrates the levels of inflammatory markers (uric acid, IL-1b, IL-6, and CRP) in the three groups. Diabetic placebo rats exhibited elevated CRP and uric acid levels compared with controls, but the IL-1b and IL-6 levels were increased in the control rats. FSP supplementation significantly reduced the plasma levels of uric acid, IL-1b, and IL-6 but did not change the level of CRP. DISCUSSION

FIG. 3. Western blot analysis of the Mn-SOD protein in skeletal muscle (gastrocnemius). The bars represent the optical density of the bands for Mn-SOD at 25 kDa (n = 6 rats per group), normalized with respect to the level of HSC70. *P < .05 when compared with group 1 (i.e., the effects of diabetes).

were not modified by the diabetic state or by FSP supplementation. The low activities of antioxidant enzymes (SOD and GPX) in the diabetic placebo group normalized after 3 weeks of FSP supplementation (Fig. 2C, D). The Mn-SOD content, decreased by diabetes, tended to increase in the

In this study, we investigated the effect of FSP supplementation on in vivo inflammation and oxidative stress in diabetic rats. Diabetes induced by STZ injection resulted in a proinflammatory state and an altered redox balance, which are characteristics of type 1 diabetes mellitus. FSP exhibited strong anti-inflammatory activity and reinforced the antioxidant system. These findings are important because oxidative stress and inflammation are responsible for diabetic complications, especially cardiovascular complications. Thus, the interventions that are able to protect against oxidative stress and inflammation are essential and must be encouraged. Moreover, this study allows to better understand the synergistic effects of soy compounds and offers new perspectives in therapeutic management of diabetic patient. Antioxidant properties of FSP in diabetic rats Hyperglycemia, which is characteristic of STZ-induced diabetes, has been linked with the development of oxidative

FIG. 4. Effects of diabetes and FSP supplementation (3 weeks) on systemic inflammatory markers in diabetic rats. (A: CRP; B: Uric acid; C: IL-1B; D: IL-6) Values are the means – SEM. *P < .05 when compared with group 1 (i.e., the effects of diabetes). xP < .05 when compared with group 2 (i.e., the effects of FSP supplementation in diabetic rats).

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stress.28 Overproduction of ROS, associated with alterations in the antioxidant defense system, is triggered by multiple mechanisms resulting from the excess of glucose, such as glucose autoxidation,29 protein glycation, and formation of advanced glycated end products (AGE),30 and activation of the polyol pathway.28 The elevated fructosamine levels indicated that hyperglycemia was sustained for the entire duration of the experiment, and these alterations in the glucose metabolism were associated with a pro-oxidant state, as evidenced by the increase in the CML level. In diabetes, N(e)-(carboxymethyl)lysine is considered a general marker of oxidative stress because it is a major product of the oxidative modifications of glycated proteins.31 The onset of oxidative stress could be due to a decrease in the activities of the antioxidant enzymes SOD and GPX, as previously shown in various tissues (liver,32 pancreas,33 and kidney34). Nevertheless, other studies reported unchanged antioxidant enzymatic activities.35,36 These discrepant results could be explained by the duration and/or the severity of the disease.37 When a decrease of the antioxidant enzymatic system is observed, this alteration was attributed to a decrease in the expression/content of antioxidant enzymes32 or to their glycation (post-transcriptional inactivation of enzymes in high-glucose medium).38,39 Our study showed that in skeletal muscle, the decrease in SOD activity resulted from a decrease in the level of the protein. This result is in accordance with previous study conducted in liver, which reported a decrease of the antioxidant enzyme activities induced by a lower antioxidant enzyme content, both for SOD and GPX.32 However, we cannot exclude a concomitant glycation of the antioxidant enzymes. Nevertheless, these alterations of antioxidant system were not associated with oxidative damages, as attested by the lack of differences in isoprostane level and GSH/GSSG ratio between healthy and diabetic placebo groups (groups 1 and 2, respectively), possibly due to a short diabetes history (3 weeks). Indeed, an increase of pro-oxidative markers (isoprostanes and GSH/GSSG ratio) has been previously reported (especially in our laboratory40), but after a longer period of diabetes (8 to 10 weeks after diabetes induction by STZ). We can assume that oxidative damages were preceded by the alterations of antioxidant system. This result points out the importance of an early therapeutic management of oxidative stress in diabetic patients to avoid the development of complications. In the diabetic rats, in which the antioxidant system was altered, FSP tended to upregulate the Mn-SOD level and increased the antioxidant enzyme activities to levels similar to those in the control rats. Our study confirmed our hypothesis of the in vivo antioxidant properties of FSP, which had been previously observed in vitro.24 According to the literature data, the antioxidant effect of FSP could be attributed to soy isoflavones, especially genistein, which increases Mn-SOD expression, decreases superoxide anion production by mitochondria, and increases GPX activity.7,10,41 Nevertheless, the improvement of SOD activity could not be fully attributed to a higher Mn-SOD content, as the beneficial effect of FSP on Mn-SOD content was not

statistically significant when evaluated by western blot (despite a visual difference between the blots). Restoration of SOD activity in FSP-supplemented diabetic rats could then be also explained by the decrease of oxidant species production, thereby removing the inhibition of SOD observed in a pro-oxidant medium.42 In the same way, the increase of GPX activity could result from FSP-induced ROS decrease, since high oxidative stress level in type 1 diabetes has been shown to inhibit GPX activity.43 The reduced oxidative stress level was confirmed by the decrease of CML in FSP-supplemented diabetic rats; as FSP supplementation did not improve hyperglycemia, the decrease of CML (which results from combined action of hyperglycemia and oxidative stress) was due to a decrease in oxidative stress level. Anti-inflammatory properties of FSP in diabetic rats Besides to induce oxidative stress, hyperglycemia has also been associated with the development of inflammatory state.18,44 Indeed, as demonstrated by Hofmann et al.,45 high level of HbA1c (marker of chronic hyperglycemia) was correlated with elevated NF-kB level, which induced a significant increase in cytokine production by monocytes. Moreover, the release of ROS by activated monocytes and macrophages has been shown to be significantly increased in T1DM compared with healthy people,46 this ROS overproduction reinforcing in turn the inflammatory processes.47 Among the numerous markers used to study inflammation, IL-1 and IL-6 are of particular interest in type 1 diabetes, because these interleukins are involved in glucose homeostasis,48,49 and were found to be elevated during hyperglycemic events. Recently, a large clinical trial study has highlighted that the most relevant markers of inflammation in type 1 diabetes were IL1-RA, IL-6, and CRP.50 In our study, the inflammatory state associated with type 1 diabetes was characterized by elevated CRP and uric acid levels. Nevertheless, the IL-1b and IL-6 levels were unusually high in the control group, approximately twofold higher than the levels in the diabetic group. This abnormal increase could be due to the gavage itself. Because control rats were more active during the administration of water than the diabetic rats (because of a better general health), we hypothesize that the syringe used induced tissue damage in the esophagus, leading to inflammation. This hypothesis was confirmed by measuring the IL-1b and IL-6 levels in a group of healthy rats that did not receive any supplementation or placebo by gavage. In these healthy rats, the IL-1b and IL-6 levels were *100 pg/ mL versus 500 pg/mL and 1100 pg/mL, for IL-1b and IL-6, respectively, in the control group (data not shown). However, if the gavage was associated with increased levels of interleukins, then we would expect a similar increase of CRP. However, the CRP values were not influenced by the gavage, as we checked in the control group not submitted to the gavage that the values were similar as the rats were submitted to the gavage or not. This surprising result (divergent evolution of interleukins vs. CRP) was previously reported without being explained,51 and constitutes an interesting finding to

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explore in further studies. The administration of a dietary supplement by oral gavage with a syringe is commonly used to ensure the accuracy of this technique. We choose to use this method because the FSP dose must be carefully adjusted based on the body weight of each rat. Relative to the basal values of healthy control rats (*100 pg/mL), the IL-1b and IL-6 were markedly increased in the diabetic group. This cytokine release was most likely induced by an autoimmune process, which resulted from the disturbed microbiota, leaky gut, and altered mucosal immunity associated with type 1 diabetes.52 These processes trigger monocyte activation and IL-1b production. IL-1b then stimulates IL-6 and CRP production, leading to the elevated interleukin and CRP levels observed in this study. In addition, inflammatory state could also result from the hyperglycemia-induced oxidative stress, especially due to activation of NF-kB pathway by the binding of AGE to their specific receptors.53 Concerning uric acid levels, our results are in agreement with previous studies that reported an increase in type 1 diabetes.54 Although, in healthy people, uric acid contributes to antioxidant defenses, in pathological subjects (such as type 1 diabetes subjects), uric acid is considered as a marker of complications (the patients with increased uric acid level, soon after onset of type 1 diabetes, have higher risks to develop complications.54). Moreover, uric acid is now known to be directly implicated in the development of these complications.54 Indeed, it participates in monocyte activation and IL-1b secretion,55 and it induces the increased expression of the IL-1b precursor.56 These two mechanisms could also have contributed to the high levels of IL-1b (and thus the high levels of IL-6 and CRP) in the diabetic placebo group (with basal values considered to be *100 pg/mL). Our hypothesis of anti-inflammatory properties of FSP is confirmed by a strong decrease of interleukins ( - 70% and - 47% for IL-1 and IL-6, respectively), and by reduction of 17% of uric acid level. Nevertheless, FSP did not affect the CRP level. This surprising result has been reported previously,51 but no author has been able to provide a satisfactory explanation. Both active compounds of FSP (alpha-GOSs and isoflavones) were implicated in the anti-inflammatory activity of FSP. Alpha-GOSs exhibit bifidogenic effects on the intestinal microbiota of diabetic rats by stimulating nonpathogen commensal microflora, which participate in reinforcing the tight junctions of the intestinal barrier.21 The ‘‘leaky gut’’ phenomenon is thus decreased, together with the abnormal inflammatory immune response. Moreover, isoflavones were recently found to decrease uric acid levels and thus limit inflammatory processes in STZ-induced diabetic rats. Uric acid, when present at elevated levels as observed in the context of diabetes, activates the NLRP3 inflammasome system, which is responsible for the expression of the IL-1b precursor. Soy isoflavones, by decreasing the uric acid level, could inhibit the activation of the NLRP3 inflammasome and the subsequent cascade of inflammatory reactions.57 Finally, the anti-inflammatory effects of FSP could also result from its antioxidant effects; because antioxidant systems were reinforced by FSP, FSP

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allowed these systems to better prevent ROS-induced inflammatory processes. That is why the therapeutic management of diabetes should aim to reduce simultaneously oxidative stress and inflammation, to generate a virtuous circle (where oxidative stress and inflammation decreased each other). Therefore, the manufacturer of FSP included both isoflavones and alpha-GOSs, which exhibit antioxidant and anti-inflammatory effects, respectively, in their dietary supplement to improve the effects of this supplement. Methodological considerations regarding the effectiveness of isoflavones and alpha-GOSs in FSP Although the antioxidant effects of soy isoflavones have been well documented in vitro and in animal studies, numerous studies failed to confirm these effects in humans.58 This lack of conclusive results could be explained by the following. First, studies usually administered one compound isolated from soybeans, but it is well known that the consumption of whole grains results in synergistic effects of different compounds.23 Second, when whole grains are transformed by an industrial process, the concept of equivalence is not taken into account.{ The FSP-manufacturing process and administration protocol were designed to observe this concept. FSP is prepared from whole grains and preserved the natural association of the two main types of active compounds in soy: isoflavones and alpha-GOSs. Further, alpha-GOSs positively affect the intestinal microbiota to promote the conversion of glycosides into aglycones, allowing the absorption of larger amounts of isoflavones from the diet.59 The dose of FSP was also adjusted to achieve an optimal effect. At high doses, soy isoflavones, as phytoestrogen compounds, exhibit estrogenic effects that can be harmful.60 The physiological dose of soy isoflavones has been determined to be *5 lM (blood concentration resulting from all food intake),61 which corresponds to a daily dose of 1 mg/kg in the diet, according to the recommendations of the French Agency of Food Safety.62 Previous observations made by our team indicated that the equivalent oral dose provided by FSP (1 mg/kg of soy isoflavones associated with alpha-GOSs) corresponds to a plasma concentration of 4 lM after 1 week of gavage (unpublished observation, Malarde´ et al., M2S, Rennes). At this physiological dose, a plateau in the blood concentration appeared after 2 weeks of supplementation.11 This plateau was sustained over 24 h, supporting the administration of a single daily dose.63 Moreover, to account for individual variations in the kinetics of the blood isoflavone concentration, we extended the length of the supplementation protocol to 3 weeks. In conclusion, our hypothesis of antioxidant and antiinflammatory properties of FSP was confirmed in an in vivo diabetic rat model. FSP exhibited antioxidant properties, enhancing the antioxidant enzymatic defenses and reducing the CML concentration. FSP also exhibited anti-inflammatory properties. { This concept states that the blood level of the active compounds must be similar (to be effective), whether these compounds were supplied by natural diet or by a dietary supplement.

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The well-known health benefits of soy consumption were thus retained in a dietary supplement that may improve the therapeutic management of diabetic individuals. This study has provided important baseline data and constitutes a first step that can be extended to human subjects in the future. The objective would be to improve therapeutic management of diabetic subject by reducing diabetic complications (by decreasing oxidative stress and inflammation), with nutritional strategy instead an additional drug therapy. In the same way, nutritional strategy could be associated with physical activity, another natural therapy that is well known to exhibit antioxidant and anti-inflammatory properties.

ACKNOWLEDGMENTS This research was part of the APAS-212-A project. The authors thank the Valorial research cluster for its precious assistance. AUTHOR DISCLOSURE STATEMENT Theo Efstathiou is an employee of Sojasun Technologies, the company that developed FSP. There are no potential conflicts of interest for the other authors. REFERENCES 1. Barnes S: Evolution of the health benefits of soy isoflavones. Proc Soc Exp Biol Med 1998;217:386–392. 2. Anderson JW, Smith BM, Washnock CS: Cardiovascular and renal benefits of dry bean and soybean intake. Am J Clin Nutr 1999;70(3 Suppl):464S–474S. 3. Ibrahim WH, Habib HM, Chow CK, Bruckner GG: Isoflavonerich soy isolate reduces lipid peroxidation in mouse liver. Int J Vitam Nutr Res 2008;78:217–222. 4. Takekawa S, Matsui T, Arakawa Y: The protective effect of the soybean polyphenol genistein against stress-induced gastric mucosal lesions in rats, and its hormonal mechanisms. J Nutr Sci Vitaminol (Tokyo) 2006;52:274–280. 5. Liu J, Chang SK, Wiesenborn D: Antioxidant properties of soybean isoflavone extract and tofu in vitro and in vivo. J Agric Food Chem 2005;53:2333–2340. 6. Cai Q, Wei H: Effect of dietary genistein on antioxidant enzyme activities in SENCAR mice. Nutr Cancer 1996;25:1–7. 7. Borras C, Gambini J, Gomez-Cabrera MC, Sastre J, Pallardo FV, Mann GE, Vina J: Genistein, a soy isoflavone, up-regulates expression of antioxidant genes: involvement of estrogen receptors, ERK1/2, and NFkappaB. FASEB J 2006;20:2136–2138. 8. Fang YC, Chen BH, Huang RF, Lu YF: Effect of genistein supplementation on tissue genistein and lipid peroxidation of serum, liver and low-density lipoprotein in hamsters. J Nutr Biochem 2004;15:142–148. 9. Wei H, Bowen R, Cai Q, Barnes S, Wang Y: Antioxidant and antipromotional effects of the soybean isoflavone genistein. Proc Soc Exp Biol Med 1995;208:124–130. 10. Borras C, Gambini J, Lopez-Grueso R, Pallardo FV, Vina J: Direct antioxidant and protective effect of estradiol on isolated mitochondria. Biochim Biophys Acta 2010;1802:205–211. 11. Djuric Z, Chen G, Doerge DR, Heilbrun LK, Kucuk O: Effect of soy isoflavone supplementation on markers of oxidative stress in men and women. Cancer Lett 2001;172:1–6.

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Fermented soy permeate reduces cytokine level and oxidative stress in streptozotocin-induced diabetic rats.

Oxidative stress and inflammation are involved in the development of type 1 diabetes and its complications. Because two compounds found in soy, that i...
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