Microvascular disorders in obese Zucker rats are restored by a rice bran diet

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is an accepted manuscript of a paper published in: Nutrition Metabolism & Cardiovascular Diseases: May (2014)  DOI: https://doi.org/10.1016/j.numecd.2013.10.032 Copyright: © 2025 Elsevier Inc., its licensors, and contributors. El acceso a la versión publicada del artículo puede requerir la suscripción de la revista. Access to the published version may require subscription. “This is an Accepted Manuscript of an article published by Elsevier in Nutrition Metabolism & Cardiovascular Diseases 2014, available at: https://doi.org/10.1016/j.numecd.2013.10.032”. Elsevier Editorial System(tm) for Nutrition, Metabolism & Cardiovascular Diseases Manuscript Draft Manuscript Number: NMCD-D-13-00371R1 Title: Microvascular disorders in obese Zucker rats are restored by a Rice bran diet Article Type: Research Paper Keywords: rice bran; mesenteric artery; obese Zucker rat; microvascular function; obesity Corresponding Author: Dr. Rosalia Rodriguez-Rodriguez, Corresponding Author's Institution: School of Pharmacy, University of Seville First Author: Maria Luisa Justo Order of Authors: Maria Luisa Justo; Carmen Claro; Elisabet Vila; Maria Dolores Herrera; Rosalia Rodriguez-Rodriguez Abstract: Background and aim: Nutritional-based approaches aimed to prevent microvascular dysfunction associated to obesity present potential advantages over pharmacological strategies. Our aim was to test whether a rice bran enzymatic extract (RBEE)-supplemented diet could attenuate microvascular alterations in obese rats. Methods and Results: Lean and obese Zucker rats were fed standard diet supplemented or not with 1% and 5% RBEE for 20 weeks. Functional studies were performed in small mesenteric arteries in isometric myograph. Immunoblotting and fluorescence studies were made in arterial homogenates and arterial sections, respectively. RBEE-supplementation restored microvascular funtion in obese rats through a marked increase in NO and endothelial-derived hyperpolarizing factor contribution by up-regulation of eNOS and calcium-activated potassium channels expression, respectively, in association to a substantial reduction of microvascular inflammation and superoxide anion formation. These data agrees with the beneficial actions of RBEE on dyslipidemia, hyperinsulinemia and hypertension in obesity. Conclusion: The multi-factorial properties of RBEE-diet, especially restoring function of small resistance arteries reveal this dietary-based approach as a promising candidate for prevention of microvascular alterations in obesity, which are crucial in cardiovascular events in obese subjects. 22nd October 2013 Dear Editor-in-Chief, Please find enclosed the revised version of our manuscript reference NMCD-D-13-00371. We certainly appreciate the thorough review of this manuscript. We have carefully addressed the comments raised by the editor and the reviewers. We have outlined our thought processes and the new data (some of them included as supplementary tables) and changes have been made as suggested by the reviewers. The modifications in the manuscript are highlighted in yellow. Also, a separate list of changes and answers to the reviewers are included in this letter. Yours sincerely, Dr. Rosalia Rodriguez-Rodriguez On behalf of all authors Department of Pharmacology, School of Pharmacy, University of Seville. C/ Profesor García González 2. 41012 Seville, Spain. Tel: +34 954557443; Fax: +34 954556074 e-mail: [email protected] Cover Letter ANSWERS TO REVIEWERS Editor's comment: the editor strongly suggests to modify the title to make clear that this is an animal experimental study. One possibility could be "A diet supplemented with rice bran enzymatic extract improves microvascular disorders in obese Zucker rats", but of course the authors can suggest another title, taking into account the above suggestion. We agree with the editor’s comment and the proposed title is absolutely correct and clear. However, we would like to suggest the following title: “Microvascular disorders in obese Zucker rats are restored by a Rice bran diet”. *Response to Reviewers Reviewer #1: In the paper submitted by Claro et al., the authors investigated the effect of the rice bran enzymatic extract (RBEE)-supplemented diet on microvascular alterations in obese rats. The paper is well written and clear. The minor point is: The quantities if RBEE used (1% and 5%) are nutritionally relevant? Could these quantities be used at nutritional level in human? If not, are there data suggesting its beneficial effect with lower quantities? The authors must clear this point. We are thankful to the reviewer for this important appreciation. The selected concentrations of RBEE in the experimental diets (1% and 5%) have been selected according to different statements: (i) considering the reported effective concentrations of other rice bran extracts or rice bran oil in humans and animal models of dyslipidemia or diabetes; (ii) considering the bioavailable concentrations of -oryzanol (the characteristic bioactive component in rice bran) reported to be effective in humans and animal models of dyslipidemia or diabetes. Regarding to the first statement, it is important to mention that the comparison between the nutritional concentrations of water-soluble RBEE and other rice bran extracts or oils is very delicate, since physicochemical and major composition (especially in protein or peptides) as well as consistency of these extracts strongly depends on the extraction process used, and therefore this could affect to the supplementation of the diet, bioavailability of the components and finally biological activity. In this sense, a wide range of concentrations of rice bran extract has been reported to be nutritionally relevant in animal models of dyslipidemia (Wilson et al., J Nutr Biochem 2007, 18: 105-112. Ardiansyah et al. Br J Nutr 2007, 97: 67-76. Revilla et al. Food Res Int 2009, 42: 387-393. Park et al. J Clin Biochem Nutr 2012, 51: 235-240) or diabetes (Chen et al. J Nutr 2006, 136: 1472-76. Chou et al., J Clin Biochem Nutr 2009, 45: 29-36. Kozuka et al. Diabetes 2012, 61: 3084-3093. Belobrajdic et al. Nutrition J 2013, 12: 62) as well as in diabetic or hyperlipidemic humans (Pereira et al. Am J Clin Nutr 2002, 75: 848-855. Panlasigui et al. Int J Food Sci Nutr 2006, 57: 151-158. Sun et al. Arch Intern Med 2010, 170: 961-969. Karupaiah et al. J Sci Food Agric 2011, 91: 1951-1956. Zavoshy et al. J Res Med Sci 2012, 17: 626-631. Kozuka et al. Obesity Res Clin Pract 2013, 7: e165-e172). The concentrations of RBEE used in our study are in the range of these investigations using other rice bran extract in either animal models or humans. Also, our previous studies with RBEE (1% and 5%) have demonstrated the beneficial effects of this supplementation in metabolic alterations of obese Zucker rats (Justo et al., Eur J Nutr 2013, 52: 789-797. Justo et al., J Nutr Biochem 2013, 24: 1456-1461) and dietinduced obese mice (Justo et al., unpublished data). Regarding to the second statement, the reported concentration range of -oryzanol that are effective in hyperlipidemic or diabetic animals and humans has been a stronger support to establish the nutritional administration of 1% and 5% RBEE. According to human studies, it has been reported that administration of -oryzanol in the diet in the range between 40-80 mg provides lipid-lowering and antidiabetic properties (Accinni et al. Nutr Metab Cardiov Dis 2006, 16: 121-127. Berger et al. Eur J Nutr 2005, 44: 163-173. Kozuka et al. Obesity Res Clin Pract 2013, 7: e165-e172). In our investigation, considering the content of -oryzanol in RBEE, we have administered 25 mg (in 1% RBEE) and 50 mg (in 5% RBEE) of -oryzanol, which is in the range of previous dose in humans. Therefore, we could suggest that the concentrations of RBEE used in this study could be nutritionally relevant in humans. Nevertheless, a more precise investigation about bioavailability of -oryzanol should be carried out in humans, since these studies are limited to animals. According to the latter, administration of 20mg/kg -oryzanol to rats have shown bioavailable plasmatic concentrations (1,9 µg/ml) (Kim et al. New Biotech 2010, 27: 368- 373). This point is now mentioned in the manuscript (page 5, lines 7-9), and some of these references are now included. Reviewer #2: In this manuscript, Justo et al. studied the effects of RBEE-supplemented diet on the eNOS pathway and lipid profile of obese Zucker rats. Their results supported their conclusion, and provided interesting insights of this potential therapeutic diet. However, the authors should consider adding more data about the secreted anti- or proinflammatory cytokines (eg. TNFa, MCP- 1, IL-10, IL-1Ra) in the rats before and after the diet administration. We would like to thank the reviewer for the time taken to revise the manuscript. We agree with the suggestion of including more data about levels of inflammatory cytokines. Therefore, in Table 1, we have included new data regarding the levels of the anti-inflammatory adipokine adiponectin in plasma. Obese Zucker rats fed a RBEE-enriched diet showed a significant restoration of plasmatic levels of adiponectin compared to non-treated obese rats. This is commented in the manuscript (page 7, lines 24-25; page 8, lines 1-2). Regarding to the anti-inflammatory cytokines, we have analyzed mRNA expression levels of TNF-α, IL- 1β and IL-6 in adipose tissue. These data are now shown in Table 1 and commented in the manuscript (page 7, lines 24-25; page 8, lines 1-2). RBEE treatment significantly reduced expression levels of these cytokines compared to non-treated obese rats. We have tried to measure the levels of these cytokines in plasmatic samples of the rats. However, we could not get reliable values of the production of the cytokines in the samples with the kit assay used, so we did not get trustworthy data. Nevertheless, the significant and highly reproductive results obtained for mRNA expression of cytokines in adipose tissue, and clear changes due to obesity (especially for TNF-α and IL-1β) and the substantial modifications by the RBEE treatment, led us to consider these measurements as a representative production of the proinflammatory cytokines. Reviewer #3: We would like to thank the reviewer for the time taken to prepare the comments. On the whole we have found the comments very constructive and have tried to address each suggestion in turn.  Introduction is too long. First paragraph can be deleted. We agree with this comment. This first paragraph has been mostly deleted.  The RBEE should be nutritionally well characterized and described in the text all the composition. We agree with this suggestion. Despite the specific composition of the RBEE used in this study has been previously determined and published by Parrado et al. (Food Chemistry 2006; 98: 742-748) (Ref. [13] in the revised manuscript), a brief section about the composition of this extract, indicating the main constituents of RBEE is included in the manuscript (page 4, lines 17-24) and the detailed composition and characterization is now included in the Supplementary material (Table S.I, Table S. II, Table S. III).  I think a biomarker of compliance should be measured in biological samples. One of the most characteristic biomarker of obesity is adiponectin level. Regarding to this, we have now included new data regarding the levels of the anti-inflammatory adipokine adiponectin in plasma (Table 1). Obese Zucker rats fed a RBEE-enriched diet showed a significant restoration of plasmatic levels of adiponectin compared to non-treated obese rats. This is commented in the manuscript (page 7, lines 24- 25; page 8, lines 1-2). Also, we have analyzed mRNA expression levels of the proinflammatory cytokines TNF-α, IL-1β and IL-6 in adipose tissue. These data are now shown in Table 1 and commented in the manuscript (page 7, lines 24-25; page 8, lines 1-2). RBEE treatment significantly reduced expression levels of these cytokines compared to non-treated obese rats. 1 1 2 Microvascular disorders in obese Zucker rats are restored by a Rice bran diet 3 Justo M.L.1, Claro C.1, Vila E.2, Herrera M.D.1, Rodriguez-Rodriguez R.1,* 4 5 1Department of Pharmacology, School of Pharmacy, University of Seville, Seville, Spain 6 2Departament de Farmacologia, Terapèutica i Toxicologia, Facultat de Medicina, Universitat 7 Autònoma de Barcelona, Barcelona, Spain. 8 9 *corresponding author: 10 Dr. Rosalia Rodriguez-Rodriguez 11 Department of Pharmacology, School of Pharmacy, University of Seville 12 Postal address: C/ Profesor Garcia-Gonzalez 2. 41012 Seville, Spain. 13 Phone: +34 954557443; Fax: +34 954556074. 14 e-mail: [email protected] 15 16 Keywords: rice bran, mesenteric artery, obese Zucker rat, microvascular function, obesity 17 18 Word count: 2814 19 Number of references: 30 20 Number of Tables and Figures: 1 Table and 4 Figures 21 22 23 24 *Manuscript Click here to view linked References 2 Abstract 1 2 Background and aim: Nutritional-based approaches aimed to prevent microvascular 3 dysfunction associated to obesity present potential advantages over pharmacological 4 strategies. Our aim was to test whether a rice bran enzymatic extract (RBEE)-supplemented 5 diet could attenuate microvascular alterations in obese rats. 6 Methods and Results: Lean and obese Zucker rats were fed standard diet supplemented or not 7 with 1% and 5% RBEE for 20 weeks. Functional studies were performed in small mesenteric 8 arteries in isometric myograph. Immunoblotting and fluorescence studies were made in 9 arterial homogenates and arterial sections, respectively. RBEE-supplementation restored 10 microvascular funtion in obese rats through a marked increase in NO and endothelial-derived 11 hyperpolarizing factor contribution by up-regulation of eNOS and calcium-activated 12 potassium channels expression, respectively, in association to a substantial reduction of 13 microvascular inflammation and superoxide anion formation. These data agrees with the 14 beneficial actions of RBEE on dyslipidemia, hyperinsulinemia and hypertension in obesity. 15 Conclusion: The multi-factorial properties of RBEE-diet, especially restoring function of 16 small resistance arteries reveal this dietary-based approach as a promising candidate for 17 prevention of microvascular alterations in obesity, which are crucial in cardiovascular events 18 in obese subjects. 19 20 9 SMA from obese rats exhibited a significant decrease in the expression of both SKCa and IKCa 1 (Fig. 2A and B). Administration of RBEE-enriched diets induced a marked increase in 2 protein expression of SKCa (Fig. 2A). IKCa expression was only restored by diets enriched in 3 5% RBEE (Fig. 2B). Protein expression of eNOS was also ameliorated in SMA from obese 4 Zucker rats and this was significantly up-regulated by a 5% RBEE administration (Fig. 2C). 5 6 iNOS immunofluorescence 7 SMA sections from obese control rats showed an increased protein expression of iNOS, 8 particularly in the smooth muscle cells, when compared to lean groups (Fig. 2D). Treatment 9 with RBEE induced an important attenuation of arterial iNOS immunofluorescence in the 10 vascular wall (Fig. 2D). 11 12 Superoxide anion production 13 To investigate vascular O2●− production, SMA sections were exposed to DHE, which is 14 oxidized by O2●− to yield the red fluorescent DNA stain ethidium. SMA from obese rats 15 showed a marked increase staining throughout the vessel wall compared to the lean control 16 group (Fig. 3). Red fluorescence from all vascular cell layers was significantly reduced in 17 mesenteric preparations from obese animals treated with RBEE (Fig. 3). 18 19 Discussion 20 This study demonstrates that administration of a rice bran enriched-diet attenuates 21 microvascular disorders in obesity through a marked restoration on the contribution of 22 endothelium-derived mediators to the arterial function in association to a substantial 23 reduction of inflammation and oxidative stress in SMA from obese Zucker rats. These results 24 are in agreement with our observations showing the ability of rice bran supplementation to 25 10 ameliorate disorders related to metabolic syndrome such as dyslipidemia, insulin resistance, 1 hypertension and vascular impairment of large conductance arteries [14, 15]. 2 Microvascular dysfunction may affect both peripheral vascular resistance and insulin-3 mediated glucose disposal, thereby contributing to hypertension and insulin resistance, 4 respectively, in obesity [3]. Although several studies in humans and animal models have 5 evidenced the beneficial daily intake of rice bran and other wholegrain derivatives in 6 metabolic disorders associated to obesity and diabetes [15-19, 21], their specific effects on 7 microvascular complications linked to these disorders remain unknown. In our investigation 8 we have evidenced for the first time the beneficial effect of a rice bran diet administration on 9 obesity-related alterations in small resistance arteries. 10 Several studies have shown a blunted vasorelaxation in obese subjects, with a reduced 11 contribution of NO [22] and EDHF [23]. In our study, although endothelial-dependent 12 dilatation to ACh was similar between the experimental groups, there was an evident 13 impaired NO contribution to the vasodilatation in SMA from obese rats. We observed that 14 arteries from obese animals treated with 5% RBEE showed an improved vasorelaxation 15 similar to the lean control group in the presence of the NO-independent inhibitors. This fact 16 could be related to the up-regulation that RBEE administration induced in eNOS vascular 17 expression. Our results agree with previous investigations where, despite the lack of changes 18 in vasodilatation to ACh between lean and obese Zucker rats, there was a reduced inhibitory 19 effect in the dilatation of SMA from obese rats after simultaneous inhibition of COX and 20 EDHF-responses compared to lean rats [11]. 21 Experimental studies suggest that the contribution of EDHF increases as the vessel size 22 decreases, with predominant activity in resistance arteries and a compensatory up-regulation 23 of EDHF in states characterized by reduced NO availability [24]. For this reason, the restored 24 EDHF contribution observed in SMA from obese animals fed RBEE-supplemented diets 25 11 could be an alternative resource to counteract the loss of NO availability in these animals. 1 EDHF has been shown particularly important in the microcirculation, where systemic 2 vascular resistance is regulated [24, 25]. Despite the on-going dispute over its identity, it is 3 generally accepted that EDHF-dependent dilatation depends on endothelial SKCa channels 4 [25, 26]. Several authors have described how functional SKCa channels are down-regulated in 5 resistance arteries from obese animals [4, 23, 27], as observed in the present investigation. 6 This EDHF impairment has been related to the metabolic disorders characteristic in obese 7 Zucker rats, especially glucose and insulin alterations [23, 27]. In our study, a significant 8 increase in SKCa expression was induced by RBEE-enriched diets in obese animals compared 9 to the obese control group, reaching similar expression levels as the lean group. Also, 5% 10 RBEE induced an upward trend in both SKCa and IKCa expression. These results could be 11 associated to the beneficial effects of RBEE in insulin resistance demonstrated in obese 12 Zucker rats [14]. Also, the effect of RBEE-diets on NO and EDHF contribution could 13 contribute to the improvement in systolic blood pressure levels observed in obese Zucker rats 14 fed rice bran diets by an enhanced peripheral vascular function [5, 27]. 15 Many lines of evidence suggest that increased vascular oxidative stress plays an important 16 role in endothelial dysfunction and, in turn, in the impairment of NO/EDHF-mediated 17 relaxation and the development of cardiovascular disease [11, 26]. Vascular depolarization 18 leads to O2●− production and NO bioavailability reduction [28]. Blockade of SKCa and IKCa 19 converts the ACh-induced hyperpolarization into depolarization, evidencing the role of these 20 channels in the control of ROS signaling in vascular cells [28]. RBEE-enriched diet induces a 21 significant attenuation of O2●− production in SMA from obese Zucker rats that could be 22 closely linked to the significant restoration of KCa channels and EDHF contribution induced 23 by this dietary supplement. 24 12 In obesity exists an increased production of pro-inflammatory cytokines released from 1 adipose tissue, such as tumor necrosis factor (TNF)-α and interleukin-6, and a decreased 2 production of the anti-inflammatory adipokine adiponectin [29]. Abdominal obesity is closely 3 associated with microvascular dysfunction, through indirect mechanisms, such as insulin-4 resistance, and directly, because of the production by the perivascular adipose tissue of 5 adipokines and pro-inflammatory cytokines which in turns induce oxidative stress [1, 3, 30]. 6 Enhanced inflammatory markers in obesity are not limited to adipose tissue and circulating 7 blood, but also to the vascular wall. In this sense, obese Zucker rats present increased levels 8 of pro-inflammatory markers such as TNF-α and iNOS expression in visceral adipose tissue 9 and aorta [15, 29]. Also, plasmatic levels of adiponectin are attenuated in these obese rats 10 [14]. Administration of RBEE-enriched diet restored levels of these inflammatory indicators 11 in both adipose tissue and aorta from obese Zucker rats [15]. In the present investigation, 12 resistance arteries from obese rats fed a RBEE diet showed decreased levels of iNOS 13 expression, suggesting the anti-inflammatory potential of this treatment in obesity. The 14 beneficial effect of RBEE against inflammation in adipose tissue might be related to the 15 improved profile of inflammatory indicators in microvessels. 16 To summarize, RBEE-supplementation was able to restore microvascular function in obese 17 Zucker rats, by increasing the EDHF contribution throughout up-regulation of endothelial 18 SKCa and IKCa channels and a partial increase of NO bioavailability from eNOS (Fig. 4), both 19 mechanisms as a compensatory response against metabolic and vascular disorders in obesity. 20 This positive effect was associated to a significant reduction of superoxide production and 21 iNOS expression in the vascular wall. The beneficial effects of RBEE on inflammatory 22 markers from visceral adipose tissue could play a critical role on these microvascular 23 responses (Fig. 4). The management of obesity-related clinical disorders including 24 microvascular dysfunction is currently based on the treatment of their individual components, 25 13 e.g. hypertension or insulin resistance. Due to the multi-factorial properties demonstrated by 1 RBEE, especially restoring function of small resistance arteries, this dietary-based approach 2 may be a promising candidate for prevention of cardiovascular and metabolic complications 3 of obesity. Particularly, the present investigation provides evidence for the beneficial effects 4 of rice bran supplementation in the prevention of microvascular alterations associated to 5 obesity and the mechanisms mediating these actions, involving potential advantages over 6 pharmacological strategies to treat obesity-related complications in terms of health costs and 7 socio-economical impact. 8 9 Acknowledgements 10 This research was supported by the Spanish Ministry of Science and Innovation (AGL2009-11 1159). M.L. Justo is a recipient of an FPU Fellowship from the Spanish Government. We 12 thank Dr. Juan Parrado (University of Seville) for supplying the rice bran extract (RBEE). 13 Fluorescence experiments were performed at the “Servei de Microscopia” (Universitat 14 Autònoma de Barcelona). 15 16 17 18 14 References 1 [1] Bagi Z, Feher A, Cassuto J. Microvascular responsiveness in obesity: implications for 2 therapeutic intervention. Br J Pharmacol 2012; 165(3): 544-560. 3 [2] Jonk AM, Houben AJ, de Jongh RT, Serné EH, Schaper NC, Stehouwer CD. 4 Microvascular dysfunction in obesity: a potential mechanism in the pathogenesis of 5 obesity-associated insulin resistance and hypertension. Physiology (Bethesda) 2007; 22: 6 252-260. 7 [3] Hornstra JM, Serné EH, Eringa EC, Wijnker MC, de Boer MP, Yudkin JS, Smulders 8 YM. Insulin's microvascular vasodilatory effects are inversely related to peripheral 9 vascular resistance in overweight, but insulin-sensitive subjects. 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Concentration-response curves to acetylcholine (ACh) (A, C, E) in the presence of 3 the combination of inhibitors of NO synthase (L-NAME), COX (indomethacin, INDO) and 4 EDH response (Charybdotoxin and apamin; Ctx and Apa, respectively). Areas under the 5 curve (AUC) obtained from every cumulative curve to ACh in the presence of the 6 combinations of the inhibitors were graphically represented (B, D, F). AUC were expressed 7 as arbitrary units (A.U.). Data are mean ± S.E.M. (n=5-7). *P < 0.05, **P < 0.05 vs LC. #P < 8 0.05, ##P < 0.01, ###P < 0.001 vs OC. 9 10 Figure 2. Expression of small and intermediate conductance calcium-activated potassium 11 channels (SKCa and IKCa, respectively), endothelial NO synthase (eNOS) and inducible NO 12 synthase (iNOS) in small mesenteric arteries from Zucker rats. Western blots revealing the 13 expression of SKCa (A), IKCa (B) and eNOS (C). Results were quantified by densitometry and 14 presented as the ratio of density of SKCa, IKCa and eNOS bands versus that of -actin from 15 the sample (A-C). Immnunofluorescence images showing iNOS expression in arterial 16 sections and results of optical density quantification (D). Data are mean ± S.E.M. (n=3-5). *P 17 < 0.05 vs LC. #P < 0.05 vs OC. 18 19 Figure 3. Superoxide anion production by fluorescence DHE staining in sections of small 20 mesenteric arteries from Zucker rats. Graph (right) showing optical density of DHE 21 fluorescence. Data are mean ± S.E.M. (n=5). **P < 0.01 vs LC. ##P < 0.01 vs OC. 22 23 Figure 4. Representation of the mechanisms involved in the effects of RBEE in the 24 dysfunction of small resistance arteries in obesity. Administration of RBEE attenuates 25 e-components Click here to download e-components: Supplementary data_R1.docx