Prevention of type 2 diabetes in prediabetic patients by using functional olive oil enriched in oleanolic acid: The PREDIABOLE study, a randomized controlled trial

Depósito de investigación de la Universidad de Sevilla https://idus.us.es/ Esta es la versión aceptada del artículo publicado en: This is a accepted manuscript of a paper published in: Diabetes, Obesity and Metabolism (2019): 31 July 2019 DOI: https://doi.org/10.1111/dom.13838 Copyright: © 2019 John Wiley & Sons Ltd. 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 the peer reviewed version of the following article: Santos-Lozano JM, Rada M, Lapetra J, Guinda Á, Jiménez-Rodríguez MC, Cayuela JA, Ángel-Lugo A, Vilches-Arenas Á, Gómez-Martín AM, Ortega-Calvo M, Castellano JM. Prevention of type 2 diabetes in prediabetic patients by using functional olive oil enriched in oleanolic acid: The PREDIABOLE study, a randomized controlled trial. Diabetes Obes Metab. 2019 Nov;21(11):2526-2534., which has been published in final form at https://doi.org/10.1111/dom.13838 . 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Please cite this article as doi: 10.1111/dom.13838 PREVENTION OF TYPE 2 DIABETES IN PREDIABETIC PATIENTS BY USING FUNCTIONAL OLIVE OIL ENRICHED IN OLEANOLIC ACID: THE PREDIABOLE STUDY, A RANDOMISED CONTROLLED TRIAL Running title: Prevention of diabetes with oleanolic acid José M. Santos-Lozanoa,b,c Mirela Radad, José Lapetraa,e, Ángeles Guindad, María C. Jiménez-Rodrígueza, José A. Cayuelab, Antonio Ángel-Lugoa, Ángel Vilches-Arenasf, Ana M. Gómez-Martínb, Manuel Ortega-Calvoa, José M. Castellanod*. aCIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain bDepartment of Family Medicine, Primary Health District Seville, Seville, Spain cDepartment of Medicine, Faculty of Medicine, University of Seville, Spain d Food and Health Department, Instituto de la Grasa, Spanish National Research Council (CSIC), Seville, Spain e Andalusian Public Foundation for the Management of Health Research in Seville (FISEVI), Seville, Spain. fDepartment of Preventive Medicine and Public Health, Faculty of Medicine, University of Seville, Spain *Corresponding author: José M. Castellano. Institute de la Grasa-CSIC. Campus University Pablo de Olavide, Building 46. 41013-Seville (SPAIN). phone number +34654611550. e-mail: [email protected] This article is protected by copyright. All rights reserved. ABSTRACT Aims/hypothesis Oleanolic acid (OA), a natural component of olive (Olea europaea L.), has demonstrated antidiabetic action in vitro and in experimental animals. However, a similar action has not been proved in humans. The PREDIABOLE (PREvention of DIABetes with OLEanolic acid) Study is a randomised and controlled trial, performed in primary care, designed to assess whether the regular intake of an OA-enriched olive oil is effective in the prevention of diabetes. Methods Prediabetic individuals (IFG + IGT) of both sex (176 patients, 30-80 years old) were randomised to receive 55 mL/day of OA-enriched olive oil (equivalent-dose 30 mg OA/day) (intervention group; IG) or the same oil not enriched (control group; CG). Main outcome was the incidence of new onset type 2 diabetes in both groups. Results Forty-eight new diabetes cases occurred, 31 in the CG and 17 in the IG. Multivariateadjusted hazard ratio was 0.45 (95% CI, 0.24-0.83) for the IG when compared with the CG. Intervention-related adverse effects were not reported. Conclusion/perspectives The intake of OA-enriched olive oil reduces the risk of developing diabetes in prediabetic patients. The results of the PREDIABOLE trial promotes the use of OA in new functional foods and drugs for the prevention of diabetes in individuals at risk of developing it. Clinical Trial Registration: Current Controlled Trials number ISRCTN03372660 This article is protected by copyright. All rights reserved. INTRODUCTION Diabetes, one of the most prevalent chronic diseases, represents a major public health problem worldwide. According to the International Federation of Diabetes, there were 425 million diabetic people in 2017, and a significant increase in the prevalence is expected in the next years, estimating that the number of diabetics in the world will achieve 629 million in 2045.1 An essential topic of diabetes approach is prevention, especially in people at high risk. Diabetes is preceded by a period of dysglycemia, known as prediabetes,2 in which plasma glucose is higher than normal but not meet the criteria for diabetes. Prediabetes is defined by impaired fasting glucose (IFG), and/or impaired glucose tolerance (IGT) and/or a glycated hemoglobin (HbA1C) in the range 5.7–6.4% (39–47 mmol/mol).3 Prediabetes is associated with obesity, dyslipidemia with hypertriglyceridemia and/or low HDL-cholesterol, and hypertension. Compared to euglycemics, people with both IFG and IGT have 20-fold higher risk of developing type 2 diabetes.4,5 Therefore, they are ideal target population to investigate new preventive strategies against diabetes at short time. Oleanolic acid (OA; 3b-hydroxy-olean-12-en-28-oic acid) is a plant secondary metabolite, widely distributed as free acid or aglycone of triterpenoid saponins.6 It is especially abundant in the olive tree (Olea europaea L.), as component of the cuticle waxes that cover fruit and leaf epidermis.7 OA is also natural component of olive oils.8 This triterpene has a wide range of biological activities,9-12 including an antidiabetic action.13,14 OA improves insulin action, preserves functionality and survival of β-cells, and protects against micro- and macrovascular complications of diabetes, throughout a complex and multifactorial mechanism.14 Pharmacological potential of OA has been demonstrated in experimental models, but studies in humans are very scarce.12,15-17 A likely reason could be the belief of its poor bioavailability.18- This article is protected by copyright. All rights reserved. 20 A recent pharmacokinetic study in humans,21 administering OA dissolved in olive-pomace oil, has proved good bioavailability for the triterpene, 35-50 folds higher than when administered as micronized powder.22,23 Lifestyle changes have revealed effective in preventing or delaying diabetes, and the inclusion in the diet of functional foods containing bioavailable OA could be an interesting strategy. With this aim, we have performed the PREDIABOLE Study, a randomised trial designed to assess whether the regular intake of an OA-enriched olive oil may be effective in preventing the progression to diabetes in prediabetic individuals with IFG and ITG. SUBJECTS AND METHODS Design Overview The PREDIABOLE (PREvention of DIABetes with OLEanolic acid) Study is a parallel-group, randomised, controlled, double-blind, and multicentre trial, entirely performed in primary care from June 2010 to November 2016. Participants in the trial were assigned in a 1:1 ratio to one of the two study groups: diet with OA-enriched olive oil (intervention group, IG), or the same diet with not enriched olive oil (control group, CG). Settings and Participants Eligible participants were community-dwelling men and women (aged 30 to 80 years), diagnosed of IFG + IGT, according to ADA’s criteria at 2008,24 with overweight/obesity (bodymass index 25.0-39.9 kg/m²), that provided written informed consent to participate in the study. Pregnant women and those within 3 months postpartum or breastfeeding were not included. Exclusion criteria were a history of IFG and/or IGT longer than seven years, diabetes or the use of hypoglycemic agents. People with alcohol dependence, drug addiction, and physical or This article is protected by copyright. All rights reserved. intellectual limitations were also excluded. Other exclusion criteria were fasting triglycerides >600 mg/dL despite treatment, Grade 3 Hypertension (SBP ≥ 180 mm Hg and/or DBP ≥ 110 mm Hg) according to 2007 guidelines by the European Society of Hypertension, suboptimaltreated thyroid disease or others endocrine diseases and cancer treatment. Finally, people using systemic glucocorticoids, selective reuptake inhibitors of serotonin or other drugs for weight reduction were also excluded. Recruitment and randomised allocation Twenty-five primary care centres of Seville (Spain) participated in the study. Through an opportunistic recruitment, individuals diagnosed of IFG [with at least two fasting glucose values in the range 100-125 mg/dL (5.6-6.9 mmol/L) in the last six months] were identified by their family physicians, and derived to the study coordination centre, where they underwent a standard 75g OGTT.25 Those individuals with IGT [2-h postload glucose 140–199 mg/dL (7.8– 11.1 mmol/L)], and meeting the other inclusion criteria and none of the exclusion ones were randomly assigned to the study groups. Allocation was performed following computergenerated random numbers prepared by the trial coordinator. Four strata of randomization were built by sex and age (cut-off 60 years). The study nurse requested by phone the assignment of participants to the two study groups at the time of admission (centralized randomization). Elaboration and characteristics of the functional olive oil A commercial blend of virgin and refined olive oils (control oil) was selected by its low content of minor components (phenolics, phytosterols, tocopherols, etc.) in order to minimize the influence of their bioactivity. The functional olive oil enriched at 600 mg OA/kg was elaborated This article is protected by copyright. All rights reserved. from the control oil by adding OA (≥97 % purity) obtained from olive leaves.26,27 No other substance was added as adjuvant. Every six months, 500L-batches of control and functional oils were elaborated and bottled in 250 mL green-coloured flasks and labelled with a six alphanumeric characters’ code for blinding. Participants, physicians and nurses were unaware of it. Five bottles of both oil types were aleatory selected from each semiannually elaborated batches, and analysed for the chemical composition as well as the physico-chemical quality indexes, following official methods.28,29 Representative chemical composition of the oils are shown in Supplementary Appendix. In addition, the Official Panel of the Instituto de la Grasa- CSIC carried out the sensory evaluation,30 and did not find significant differences between both oil types in odour, taste and appearance. Intervention and follow-up Participants were instructed to intake 55 mL/day of the assigned oil, preferably raw and freely distributed among the three main meals. They were followed up for up to 30 months from recruitment, according to a programmed schedule of visits. At baseline and quarterly thereafter, all participants received six litres of the assigned olive oil for free, and were submitted to anthropometric and blood pressure measurements. They were also interviewed about gastrointestinal disarrays or other kind of discomfort, and filled in a questionnaire about lifestyle (diet, physical activity, alcohol and tobacco consumption), medical conditions, and use of medication. Participants participated also in Workshops about healthy foods, recipes, seasonal shopping lists, and the culinary use of olive oil. They were likewise advised for the avoiding of fast/highly processed food, pastries, as well as sweetened and alcoholic drinks. This article is protected by copyright. All rights reserved. At baseline and every six months afterwards, fasting blood samples were withdrawn from the cubital vein, and collected in sterile plastic tubes with vacuum system. The tubes were subsequently centrifuged at origin, to separate plasma from the blood cells. The plasma samples were transported at 4-7 ºC in insulated containers, within a gap of 4 hours, to the Laboratory of Analysis and Clinical Biochemistry of the Virgen del Rocio University Hospital of Seville, where they were analyzed for glucose, insulin, glycated hemoglobin (HbA1c), and hs- RCP, as well as for triglycerides, total cholesterol, HDL, and LDL. At each annual visit, an OGTT was done after a 12-hour fast, following the WHO recommendations. To see a detailed report on the laboratory techniques used, please address to the Supplementary Apendix 3, which shows a full version of the PREDIABOLE protocol. Compliance to allocation Adherence to the dietary intervention was assessed through the self-reported compliance Haynes-Sackett test31 and the return of empty bottles presumably consumed. Moreover, plasma OA was analysed in a randomised collection of blood samples from 25 individuals per study group, corresponding to 0, 12, 18 and 30 months of follow-up, according to Rada et al.21 Outcomes The primary outcome was new-onset diabetes, diagnosed according to ADA’s criteria24 at 2008: fasting plasma glucose ≥126 mg/dL (7.0 mmol/L) or symptoms of hyperglycemia and a casual plasma glucose >200 mg/dL (11.1 mmol/L) or 2h plasma glucose ≥200 mg/dL (11.1 mmol/L) during a standard 75-g OGTT. In the absence of unequivocal hyperglycemia, these criteria were confirmed by repeat testing on a different day. The case ascertain was definitively done by the PREDIABOLE Clinical Event Committee, whose members were blinded to This article is protected by copyright. All rights reserved. allocation in the study groups. New-diabetic individuals were informed and referred to their family doctors, and subsequently left out the study. As secondary outcomes we obtained data about blood parameters, BMI, waist circumference, blood pressure, physical activity, alcohol and tobacco consumption, and the homeostasis model assessment [HOMA-IR=(serum insulin (μU/mL)×blood glucose (mmol/L))/22.5]. Safety of the dietary intervention At the quarterly interviews, the trial doctors questioned the participants to self-report their medical conditions and the appearance of acute affections that required medical assistance. Furthermore, at December 2018, a family physician collaborating with the trial, blinded to the assignment and not belonging to the trial staff, conducted a retrospective study of the electronic medical records of the participants. The period from recruitment up to two years after the completion of the trial was inspected, and all recorded analytical values of serum creatinine (marker of renal function) and alanine and aspartate aminotransferase (markers of hepatic disorder) were collected. In addition, the electronic medical records were also examined to gather all reported vascular events (cerebral, cardiac and peripheral) or other adverse findings (including pathologies as cancer or neurodegenerative diseases). Ethics The Ethics and Health Research Committee of the Primary Health Care District Seville (Andalusian Health Service, Andalusia, Spain) approved the protocol on May 20, 2008. All participants signed their consent, after being informed of the objectives and methodology of the trial. PREDIABOLE was conducted according to the recommendations of the Helsinki This article is protected by copyright. All rights reserved. those who dropout and those who complete the study, minimized the bias in the analysis of the results. Our study also has strengths. PREDIABOLE is a randomised trial designed and executed entirely in primary care, the gateway to the National Health System in Spain. This aspect supports the external validity and applicability of the results, and gives greater consistency to the evidence obtained, as it reproduces usual conditions of clinical practice. The randomization process for participants’ allocation yielded study groups very well balanced avoiding potential sources of confounding and standing out the causal association between OA administration and diabetes prevention. The PREDIABOLE trial observed the ITT principle, what kept randomization until the end of the trial and decreased the probability of biasing. Other strength of the trial was its double-blind design, in which both participants and health staff were unaware of the oils assigned to the study groups. This aspect is a clear advantage over studies in which the blinding of the dietary intervention was not feasible. Testing the effect of the OA-enriched olive oil not against a placebo or a low-fat diet, but against the same olive oil unreinforced in the triterpene should be also considered a strength of the trial. Meta-analysis of the effects of olive oil-supplemented diets indicate that the consumption of olive oil is associated both with a decreased risk to develop the disease in healthy individuals and with an improvement of glucose homeostasis in diabetic patients.40 Most of those studies used virgin olive oil and focused their attention on the fatty acid composition or the presence of phenolics, overlooking the biological activity of other natural functional components of olive oils, such as OA. Therefore, the protective effect offered by the functional olive oil when compared with the control olive oil in the PREDIABOLE trial further stands out the antidiabetic action of OA and its therapeutic usefulness for humans. This article is protected by copyright. All rights reserved. In conclusion, the PREDIABOLE Study is a fully randomised, controlled, double-blind trial, well designed and executed, supporting that consumption of an OA-enriched olive oil results in a substantial risk reduction of developing type 2 diabetes in prediabetic patients. This dietary intervention is well accepted by the participants and also safe and palatable, showing high potential to be long-term sustainable. The burst of diabetes prevalence in the last decades, demands urgent measures delaying or avoiding the appearance of the disease. In this scenario, the use of OA-enriched functional foods is an interesting strategy. Nevertheless, further research is needed to consolidate the evidence of PREDIABOLE and to extend the use of OA in the design of new foods and drugs. Acknowledgments. The authors especially thank ACESUR Group (Dos Hermanas, Seville, Spain), which donated all the commercial olive oil for the trial. This collaborator had no role in the design, collection, analysis, or interpretation of the data or in the decision to submit the manuscript for publication. The authors also express their gratitude to Dr. Alberto Gil-Peralta, Neurology Service of the Virgen del Rocío University Hospital (Seville, Spain), for the critical reading of the manuscript and his helpful comments. CIBEROBN is an initiative of Instituto de Salud Carlos III (ISCIII), Madrid, Spain. Funding. The PREDIABOLE trial was supported by de official funding agency for biomedical research of the Spanish government, ISCIII, through the Fondo de Investigación para la Salud (FIS), which is co-funded by the European Regional Development Fund, including the following projects: PI10/00913 (led by J.M.S-L.) and PI10/01415 (led by J.A.C.). The study was also supported by grant of Consejería de Salud de la Junta de Andalucía (PI-0037/2008, led by J.M.S-L.). This article is protected by copyright. All rights reserved. None of the funding sources took part in the design, collection, analysis, or interpretation of the data or in the decision to submit the manuscript for publication. Duality of Interest. No potential conflicts of interest relevant to this article were reported. Author Contributions. JM Santos-Lozano, J Lapetra. and JM Castellano contributed to the study design. M Rada, Á Guinda, JA Cayuela, MC Jiménez-Rodríguez, A Ángel-Lugo, AM Gómez-Martín, J Lapetra and JM Castellano contributed to data acquisition. M Rada., M Ortega-Calvo, Á Vilches-Arenas, J Lapetra, JM Santos-Lozano and JM Castellano contributed to data analysis. JM Castellano wrote the manuscript. JM Santos-Lozano and JM Castellano are the guarantors of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. They were responsible for the integrity of the work as a whole. REFERENCES 1. 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Olive oil in the prevention and management of type 2 diabetes mellitus: a systematic review and meta-analysis of cohort studies and intervention trials. Nutr Diabetes 2017;7:e262. https://doi.org/10.1038/nutd.2017.12 This article is protected by copyright. All rights reserved. Table 1. Baseline characteristics of prediabetic participants according to study groups control oil OA-enriched oil n 92 84 age (years) 67.4 ± 9.2 67.9 ± 9.0 male sex (%) 53.2 48.8 weight (kg) 81.7 ± 14.9 81.4 ± 15.1 height (cm) 162.7 ± 9.7 163.8 ± 10.2 BMI (kg/cm 2 ) 30.7 ± 4.2 30.1 ± 4.1 waist circumference (cm) 110.5 ± 10.2 108.1 ± 9.1 SBP (mm Hg) † 127.9 ± 15.7 130.2 ± 16.6 DBP (mm Hg) 74.8 ± 9.3 75.8 ± 10.0 plasma biomarkers fasting glucose (mg/dL) 112.5 ± 17.7 106.2 ± 14.6 HbA1c [% (mmol/mol IFCC)] 6.0 ± 0.4 (42.0 ± 4.4) 5.8 ± 0.5 (40.0 ± 5.5) fasting insulin (µU/mL) 15.4 ± 6.4 16.2 ± 5.2 HOMA-IR‡ 4.3 ± 2.2 3.9 ± 2.3 triglycerides (mg/dL) 144.5 ± 63.1 141.3 ± 67.4 total cholesterol (mg/dL) 204.0 ± 40.0 203.8 ± 36.1 LDL (mg/dL) 121.9 ± 33.8 121.9 ± 31.6 HDL (mg/dL) 54.7 ± 14.2 55.4 ± 17.5 hs-CRP(mg/L) § 2.4 ± 2.0 2.4 ± 2.5 medication use [n (%)] ACE inhibitors /ARB ¶ 42 (45.2 %) 39 (46.4 %) diuretics 22 (23.9 %) 18 (21.4%) other antihipertensive agents 26 (28.3 %) 20 (23.8%) statins 34 (37.0 %) 30 (35.7 %) others lipid lowering agents 3 (3.3 %) 4 (4.8 %) living habits tobacco (cigarettes/day) 20.1 ± 12.4 21.2 ± 16.3 alcohol (units/week) 9.6 ± 11.9 10.2 ± 14.2 sedentary lifestyle (%) 51.5 59.5 family antecedent of diabetes (%) 57.6 48.8 prediabetes evolution (months) 22.3 ± 17.9 19.8 ± 18.2 This article is protected by copyright. All rights reserved. Results are expressed as mean ± SD or percentages All variables passed the Mann-Whitney-Wilcoxon U-test to assess the population homogeneity † SBP and DBP means systolic and diastolic blood pressures, respectively. ‡ HOMA-IR=(serum insulin (μU/mL) × blood glucose (mmol/L))/22.5 § hs-CRP denotes high-sensitivity C-Reactive Protein ¶ ACE (angiotensin-converting enzyme) inhibitors /ARB (angiotensin receptor blocker) Table 2. Incidence of diabetes after a median follow-up of 27.5 months and relative risk of diabetes by allocation groups OA-enriched oil control oil p value This article is protected by copyright. All rights reserved. †Cox’s regression models were used to assess the relative risk of diabetes for allocation group, estimating HR and 95% CI. ‡ Individuals were considered as hypertensive if DBP>90 mmHg or SBP>140 mmHg. §Multivariable model adjusted for insulin resistance (HOMA-IR ≥3.8 yes; else no) and hypercholesterolemia (TC ≥200 mg/dL yes; else no). ¶Additionally adjusted for sedentary lifestyle (physical activity null/scarce = 0; moderate/intense = 1) participants n 84 92 months follow-up [median (IQR)] 25.0 (12.0-30.0) 30.0 (15.0-30.0) person-months of follow-up 1923 1903 events new onset diabetes 17 31 incidence rate per 1000 persons-months (95% CI) 8.8 (5.1-14.2) 16.3 (11.1-23.1) hazard ratio (95% CI)† a) univariable analysis olive oil type (crude model) 0.542 (0.300-0.980) 1 (ref.) 0.043 sex 1.066 (0.605-1.878) “ 0.825 age 0.991 (0.960-1.023) 0.595 BMI 1.489 (0.845-2.657) 0.166 waist circumference 1.004 (0.985-1.045) 0.345 fasting glucose 1.066 (1.048-1.085) <0.001 HbA1c 3.914 (2.068-7.406) <0.001 fasting insulin 1.054 (1.018-1.091) 0.003 HOMA-IR 2.724 (1.501-4.945) 0.001 TG 1.004 (0.999-1.008) 0.103 TC 1.994 (1.094-3.637) 0.024 LDL 1.008 (0.999-1.017) 0.081 HDL 1.007 (0.989-1.025) 0.441 hypertension‡ 1.229 (0.706-2.140) 0.465 sedentary lifestyle 0.901 (0.510-1.589) 0.718 alcohol 1.010 (0.994-1.026) 0.224 tobacco 1.802 (0.871-3.726) 0.112 b) multivariable analysis multivariable adjusted model-1§ 0.489 (0.269-0.891) 1 (ref.) 0.019 multivariable adjusted model-2¶ 0.448 (0.242-0.825) 1 (ref.) 0.011 This article is protected by copyright. All rights reserved. Table 3. Individuals with significant health events, appearing during their participation in the trial (during) and/or the two-years period after their completion (post). †AL T y AST mea n alani ne and aspa rtate amin otra nsfe rase enzy mes, resp ectiv ely case nº oil type event period other finding antecedents death 1 control elevated ALT+AST† post Cancer (lung) - + 2 control transient ischemic stroke post - - - 3 control ischemic heart disease post - - - 4 control elevated creatinine post Cancer (bladder) ischemic heart disease - 5 control stroke during - - - 6 control ischemic heart disease during and post angina - - 7 control peripheral arterial disease post - - - 8 OA-enriched elevated creatinine during Cancer (lung) - - elevated ALT+AST post - - - 9 OA-enriched chronic ischemia post - transient ischemic stroke - 10 OA-enriched effort angina during - - This article is protected by copyright. All rights reserved.