Adult diabetes 2

AbsTrACT

Introduction Metabolic syndrome comprises a set of risk factors for chronic diseases including abdominal obesity, increased fasting blood glucose (FBG), altered lipid profile and elevated blood pressure (BP). Due to high prevalence of metabolic syndrome and its complications in the military personnel, the relevant problems should be identified and controlled. Therefore, the present study was conducted to determine the effect of synbiotic supplements on the components of metabolic syndrome in the military personnel with metabolic syndrome.

Methodology Sixty military personnel with metabolic syndrome were included in this double- blind randomised controlled clinical trial. During the intervention, they were asked to consume one capsule of synbiotic supplement or placebo per day for 8 weeks. Body Mass Index (BMI), waist circumference, BP, FBG and lipid profile were measured before and after the intervention.

results The results of the study showed that the synbiotic supplementation had a large significant adjusted effect on the BMI (Cohen’s d=0.82 (95% CI 0.29 to 1.34)). It also had a medium significant adjusted effect on the FBG (Cohen’s d=0.52 (95% CI 0.004 to 1.03)) as well as triglyceride (Cohen’s d=0.65 (95% CI 0.13 to 1.17)). Conclusion Findings of the study revealed that synbiotic supplementation may lead to a significant improvement in the BMI, triglyceride and FBG levels in the military personnel. Thus, consumption of synbiotic supplements is recommended as an adjuvant therapy in the military personnel with metabolic syndrome.

InTrOduCTIOn

Metabolic syndrome is referred to as the simultaneous occurrence of risk factors for cardiovascular diseases (CVDs) such as abdominal obesity, hypertension, glucose intolerance and lipid disorder.1 Metabolic syndrome increases the risk of type 2 diabetes and CVDs, and has become epidemic worldwide. People with metabolic syndrome are two times more likely to die and three times more likely to have a stroke or heart attack compared with the healthy ones.2 Due to the differences in diagnostic criteria, the prevalence of metabolic syndrome has been reported in varying degrees in different communities. Several studies have shown that the prevalence of metabolic syndrome is increasing in both high- income and low- income and middle-income countries. 3 The International Diabetes Federation (IDF) has presented a definition for metabolic syndrome, which is shown in Box 1.4 The prevalence of metabolic syndrome

Key messages

► Considering the occupational conditions of the military personnel and the importance of physical health, it is recommended to identify, prevent and control the metabolic syndrome in these personnel.

► Administration of the synbiotic supplements in the military personnel with metabolic syndrome resulted in a significant decrease in the fasting blood glucose and triglyceride levels.

► Administration of the synbiotic supplements had a large significant effect on the Body Mass Index.

► Synbiotic supplements may be helpful in improving the components of the metabolic syndrome in the military personnel with the metabolic syndrome.

has been reported by 19.6% in the Iranian military personnel.5 Some studies have shown the prevalence of coronary artery disease (CAD) in young military personnel.6 Occupational stress is among the most important factors in increasing the risks of death from coronary heart disease.7 The specific occupational conditions of the military personnel may lead to the development of CAD among them.8 Obesity and overweight are highly prevalent among the Iranian military personnel and given the association between the obesity and CVD, military personnel are at greater risk of developing the CVD.9 Studies have shown the high prevalence of hypertension and hyperlipidaemia, as two major risk factors for CVD in the military personnel.10 11 Mirzaeipour et al in a study on the Iranian military personnel found a significant complications in the military personnel and given that the risk factors of CVD have devastating effects on the combat readiness in military personnel, the metabolic syndrome components should be identified and controlled in the military personnel. Several factors are involved in the aetiology of the metabolic syndrome, but the evidence shows the insulin resistance and obesity as major causes of the metabolic syndrome.13

Various studies have shown that the gut microbiota is involved in the development of obesity and insulin resistance.14 Due to the potential effects of the gut microbiota on the metabolism and

Effects of synbiotic supplementation on the components of metabolic syndrome in military personnel: a double- blind randomised controlled trial

box 1 The International diabetes Federation definition for metabolic syndrome

Central obesity: it is referred to a waist circumference (WC) of

≥94 cm in men and ≥80 cm in women and meeting two of the four following criteria:

Systolic blood pressure of ≥130 mm Hg, diastolic blood pressure (BP) of ≥85 mm Hg or taking antihypertensive drugs.

Fasting blood glucose of ≥5.6 mmol/L or previously diagnosed with type 2 diabetes.

Triglyceride level of 1.7 mmol/L, or taking drug for triglyceride abnormality.

High-density lipoprotein cholesterol of <1.29 mmol/L in women and <1.03 mmol/L in men, or taking drug for triglyceride abnormality.

especially weight regulation, there are an increasing number of researches on the gut microbiota variations by the dietary interventions. Recent studies have suggested that the alteration of the gut microbiota by the probiotics, prebiotics and synbiotics may be effective in the management of metabolic syndrome.15 Box 2 presents the definitions of the above-mentioned terms. Since there is no study about the effect of synbiotics on the metabolic syndrome components in the military personnel with this syndrome and considering the mixed results of the studies regarding the effects of probiotics and prebiotics on the metabolic syndrome components,16 17 the present study is carried out to determine the effect of synbiotic supplements on the components of metabolic syndrome in the military personnel with metabolic syndrome who are at risk for CVDs and diabetes.

MeThOdOlOgy Participants

Sixty military personnel with metabolic syndrome who referred to the Vali Asr Subspecialty Hospital in Tehran, between April 2019 and October 2019 were included in this double-blind randomised controlled clinical trial. Given the sample size formula suggested for comparing the two means, a minimum sample size of 25 military personnel with metabolic syndrome were determined considering a type I error of 5% (α=0.05) and type II error of 20% (β=0.20, power=80%) and serum triglyceride (TG) concentration was considered as a key variable.18 However, 60 patients were enrolled in the study taking

box 2 The definition of the probiotics, prebiotics and synbiotics

Probiotics

It is referred to the specific and living microorganisms exerting the beneficial effects on the health of the host including the humans or animals after application by influencing the microbial flora of the body.40

Prebiotics

It is referred to the non- digestible compounds fermented naturally by the microorganisms in the gut, and improving the growth of guts’ microorganisms.41 synbiotics

It is referred to a combination of the probiotics and prebiotics that can exert more beneficial effects than either prebiotics or probiotics alone.42

into account a loss to follow -up rate of 20%. Inclusion criteria included the age range between 20 and 50 years and being diagnosed with the metabolic syndrome according to IDF criteria. Exclusion criteria were the history of CVDs, liver and kidney failure, alcohol abuse, smoking and postmenopausal women.

Sixty participants were randomly assigned into intervention (n=30) and placebo (n=30) groups. The randomisation (15 blocks with sizes of 6) sequence was generated using a computer- generated list of random numbers to create a series of sequentially numbered envelopes containing equal assignments of synbiotic or placebo. The random numbers were placed in the sealed and opaque envelopes. For applying the blinding procedure in the study, synbiotic and placebo were packed in identical capsules and labelled as A and B by the producer. Therefore, the participants, researchers and outcome assessors were not aware of the assignments.

Participants were advised to take one supplement daily for 8 weeks and also were contacted through making the phone calls once a week for follow- up by a trained nutritionist. The participants were asked to avoid changing their physical activity as well as dietary intake during the study as much as possible in order to control the effect of physical activity and dietary intake on the study results. At the beginning and end of the study, a 3-day 24 hours dietary recall questionnaire (1 day off and 2 days on) was completed by all the participants.

Characteristics of the supplement

The synbiotic supplement used in this study was FamiLact (Zist Takhmir Company, Tehran, Iran). Active ingredients in each composition included seven living species: Lactobacillus casei (3.5×109 CFU), Lactobacillus acidophilus (1×109 CFU), Lactobacillus rhamnosus (7.5×108 CFU), Lactobacillus bulgaricus (1×108 CFU), Bifidobacterium breve (1×1010 CFU), Bifidobacterium longum (3.5×109 CFU) and Streptococcus thermophiles (1×108 CFU) and fructo- oligosaccharide (FOS), lactose, magnesium stearate, talc and silicon dioxide as adjuvants. The placebo (containing the same materials plus starch and no bacteria) was packaged in similar capsules. Supplements and placebo were coded by the manufacturer to blind the study.

Anthropometric and blood pressure measurements

Weight, height and waist circumference (WC)were measured in all the participants at the beginning and end of the study to determine the anthropometric indices and Body Mass Index (BMI). Weight was measured using the Tefal digital scale (Tefal, France) with minimal clothes, without shoes and 100 g precision. Height was measured using the Seca stadiometer (Seca, Germany) with 0.5 cm precision. The WC was measured in the narrowest area between the last rib and the iliac crest using a non- elastic tape measure. BMI was calculated by dividing the weight in kg by height in m2.

Blood pressure (BP) of the participants was measured after a 15 min rest, at least at three 5 min intervals in a sitting position using a Welch Allyn digital BP monitor (Welch Allyn, USA) by a trained nurse and their mean value was recorded. laboratory tests

For measuring the biochemical parameters, blood samples were obtained from all the participants after 12 hours fasting at the beginning and end of the study, and fasting blood glucose (FBG) and lipid profile were measured. FBG was measured by the enzymatic glucose oxidase method using a kit (Pars Azmoon, Tehran, Iran). The levels of TG, total cholesterol (TC) and high-density

lipoprotein cholesterol (HDL- C) were measured enzymatically using the same kit (Pars Azmoon) by a Selectra ProM autoanalyzer (The Netherlands). All the intra- assay and inter- assay coefficients of variation were <5%. Low-density lipoprotein cholesterol (LDL-C) was calculated using the following formula analysis

Independent samples t-test and χ2 test were used to compare the variables between the two study groups at baseline. One- way analysis of variance as a crude model was used to compare the mean of variables in each group at the end of study period. Also, a one-way analysis of covariance (ANCOVA) was used to adjust the effect of intervention for the pre- intervention values of outcome variables (adjusted model 1) as well as an ANCOVA model adjusted for both pre- intervention values of outcome variables and age as confounding variable (adjusted model 2).

Paired-samples t- test was used to compare the nutritional intakes before and after intervention in each group. The results were presented as mean±SE/SD. P value of <0.05 was considered as statistically significant. Stata software V.13 (StataCorp, College Station, Texas, USA) was used for statistical analysis of the obtained data. The effect sizes and the 95% CIs were calculated using the Cohen’s d. Values of 0.2, 0.5 and 0.8 were generally interpreted as small, medium and large effects, respectively. Also, partial η2 was used as another effect size measure and values of 0.01, 0.06 and 0.14 were generally interpreted as small, medium and large effects, respectively.

resulTs

The present study was performed on 60 military personnel with metabolic syndrome whose disease was diagnosed based on IDF criteria and they were assigned into the intervention and placebo groups (30 individuals per each group). No important harm or unintended effect was detected during the study, so, the study ended at the expected date. No one left the study until the end of the study (Figure 1).

There was no significant difference in the demographic data and biochemical anthropometric characteristics between the two study groups in the beginning of the study (Table 1). The mean age of the participants in the intervention group was 42.33±8.1 years and the mean age of the participants in the control group was 40.6±6.2 years and there was no significant difference between the two study groups in this regard (p>0.05). There were 19 (63.3%) males in the intervention and 16 (53.3%) in the control groups (p>0.05). Dietary intake of the participants was evaluated before and after the intervention and there was no significant difference between the two study groups in terms of dietary intake (Table 2). The results showed no statistically significant difference in all the variables except BMI and TG level between the two study groups at the end of study.

Table 3 shows the estimated effects of the synbiotic supplementation on the outcome variables based on the crude and adjusted model analyses. The results obtained from the crude models as well as the models of baseline covariates and age were also reported. The synbiotic supplementation had a significant effect on the BMI after adjustment (adjusted model 1) for baseline BMI (p=0.003). The effect sizes were medium and significant at post-intervention:

Table 3 Outcome distribution in pre- intervention and post- intervention by study groups according to different models

Variable Model Time synbiotic supplements (n=30) Mean difference (95% CI)* Cohen’s d (95% CI) Partial η2 P value†

Body Mass Index (kg/ Crude m2‡) Pre

Data are estimated marginal means±SEs.

*Mean difference (95% CI)=placebo−synbiotic supplements.

†Calculated based on one- way analysis of variance. Significant findings are in bold.

‡Adjusted for baseline pre- intervention outcome and age (calculated based on one-way ANCOVA model).

§Adjusted for baseline pre- intervention outcome (calculated based on one- way ANCOVA model).

ANCOVA, one-w ay analysis of covariance; HDL- C, high- density lipoprotein cholesterol; LDL- C, low- density lipoprotein cholesterol.

Cohen’s d=0.78 (95% CI 0.25 to 1.30), partial η2=0.1398. After adjustment for baseline BMI and age (adjusted model 2), a significant effect was observed on BMI (p=0.002). The effect sizes were large and significant at post- intervention: Cohen’s d=0.82 (95% CI 0.29 to 1.34), partial η2=0.1534. The synbiotic supplementation had a non-significant effect on the WC before and after adjusting the covariates (p=0.15). The effect sizes were small and non-significant at post- intervention (adjusted model 2): Cohen’s d=0.38 (95% CI −0.13 to 0.89), partial η2=0.0365.

The synbiotic supplementation had a non- significant effect on the systolic BP and diastolic BP before and after adjusting the covariates (p=0.85, p=0.67, respectively). The effect sizes were trivial and non-significant at post- intervention (adjusted model 2). The synbiotic supplementation had a non-significant effect on the FBG after adjustment (adjusted model 1) for baseline FBG (p=0.06). The Cohen’s d was medium but non- significant at post- intervention (0.50 (95% CI −0.02 to 1.00)), partial η2 was small (0.0571). After adjustment for baseline FBG and age (adjusted model 2), a non- significant effect was observed on FBG (p=0.05). The effect sizes were medium and significant at post-intervention: Cohen’s d=0.52 (95% CI 0.004 to 1.03), partial η2=0.0647.

The synbiotic supplementation had a non-significant effect on the TC, LDL-C and HDL- C before and after adjusting the covariates (p=0.34, p=0.32, p=0.43, respectively). The effect sizes were small and non- significant at post- intervention (adjusted model 2). The synbiotic supplementation had a significant effect on the TG level after adjustment (adjusted model 1) for baseline TG level (p=0.02). The effect sizes were medium and significant at post- intervention: Cohen’s d=0.60 (95% CI 0.08 to 1.11), partial η2=0.0866. After adjustment for baseline TG level and age (adjusted model 2), a significant effect was observed on the TG level (p=0.01). The effect sizes were medium and significant at post- intervention: Cohen’s d=0.65 (95% CI 0.13 to 1.17), partial η2=0.1023. dIsCussIOn

In the present study, the effects of synbiotic supplementation on the metabolic syndrome components and other outcomes (ie, BMI, LDL-C and TC) were investigated among the Iranian military personnel. Administration of synbiotic supplementation in the Iranian military personnel was the novel aspect of the current study. Controlling the risk factors in the patients with metabolic syndrome can reduce the risk of complications such as cardiovascular diseases and diabetes. Although the first step in reducing the risk factors for metabolic syndrome is improving the lifestyle, in this regard, weight loss, blood lipid control, control of hypertension and treatment of underlying diseases are also very important in reducing the risk of metabolic syndrome. The synbiotic supplementation was found to have a significant effect on the BMI, TG and FBG levels.

The results of the present study showed a large significant adjusted effect on the BMI but there was a small insignificant adjusted effect on the WC at post- intervention. These results are in line with the findings of other studies.16 19 Ferolla et al showed that synbiotic supplementation in the patients with non-alcoholic fatty liver disease resulted in a significant reduc- tion in the BMI.19 Some studies have reported an association between the gut microbiota species and obesity and weight loss and have shown an association between increased bifidobacteria in the gut and weight loss.20 Specific strains of bacteria in the form of probiotics and prebiotics can be useful for patients with obesity.21 The mechanisms involved in weight loss are not well understood, but reduced calorie intake, reduced adipose cell size and prevention of adipose tissue synthesis may be involved in the weight loss.22 23 Studies have also shown that the use of synbiotics can decrease the appetite by increasing the glucagon-like peptide-1, and peptide YY, as appetite suppressant neurotransmitters causing the weight loss.16

Regarding the lipid profile, although synbiotic supplementation had a small and insignificant adjusted effect on the TC, LDL- C and HDL- C, there was a medium and significant adjusted effect on the TG level. Rabiei et al showed that synbiotic supplementation did not have a significant effect on the lipid profile.16 In another study, synbiotic supplementation (two capsules/day) for 28 weeks led to significant changes in the TG, TC and HDL- C levels, but LDL-C levels did not change. 13 The differences in the results of studies may be due to the duration of study, or dose of supplements. Dyslipidaemia in the patients with metabolic syndrome may contribute to the increased risk of atherosclerosis and in particular the risk of cardiovascular diseases. The microbial metabolism of lipids in the gut results in the generation of the products such as choline, trimethylamine N-oxide, and betaine that can cause atherosclerosis and cardio- vascular disease.24 Much attention has been paid to improve the human gut microbiome by the probiotic and prebiotic dietary supplements due to the effect of the gut microbiome on the lipid profile. Several mechanisms have been proposed for this effect. For instance, some studies have attributed this decrease in the lipid profile to the production of short- chain fatty acids from bacterial fermentation of the probiotics.25 The butyrate, acetate and propionate go to the liver. In the

liver, propionate results in decreased activity of HMG-CoA (3- hydroxy-3- methylglutaryl– coenzyme A (HMG-CoA)) reductase and thereby reduces

Parastouei K, et al. BMJ Mil Health 2020;0:1–6. doi:10.1136/bmjmilitary-2020-001459

the cholesterol synthesis.26 Other suggested mechanisms for lowering the cholesterol levels are as follows: the bacteria absorb the cholesterol in the gut, or prevent the cholesterol absorption by binding it to the bacterial cell wall or by competing with it in absorption.27 28 Probiotics, on the other hand, can deconjugate the bile salts, thereby reducing the fat absorption.29

The results of the present study showed that the effect sizes of synbiotic supplementation on FBG were medium and significant at post- intervention, which is in line with the other previous studies.16 17 Also, the results of a systematic review study showed that the probiotic and synbiotic supplements can be effective in reducing the FBG, especially in the subjects with high blood glucose.30 Changes in the gut microbiota lead to increased permeability and immune response of the intestinal mucosa and are probably involved in insulin resistance and incidence of type 2 diabetes.31 Some studies have shown that the probiotics may be involved in the treatment of insulin resistance and type 2 diabetes by modulating the gut microbiota.32 Recent evidence shows that the lipopolysaccharides (LPS) and cell wall compositions of Gram- negative bacteria are correlated with the insulin and glucose levels, and insulin resistance.33 Increased level of LPS results in an elevated insulin resistance. Variations in the gut microbial flora by reducing the Gram- negative bacteria, and as a result, reduced level of LPS, decreased production and absorption of intestinal toxin, increased intestinal pH, decreased glucose absorption, decreased adipose tissue, altered intestinal permeability, increased proglucagon expression and also decreased production of proinflammatory cytokines can be among the possible mechanisms regarding the effect of synbiotics on decreasing the insulin resistance as well as lowering the blood glucose.34 35

The results of the present study showed that the treatment had a trivial insignificant effect on the systolic BP and diastolic BP before and after adjusting the covariates at post- intervention, which is consistent with some previous studies36 37 and inconsistent with other ones.38 In the study by Nabhani et al, daily consumption of one synbiotic supplement (each capsule contained Lactobacillus probiotic strains consisting of L. acidophilus, L. plantarum, L. fermentum, L. gasseri and FOS as prebiotic substance) for 6 weeks resulted in a significant reduction in systolic BP and diastolic BP in women with gestational diabetes mellitus.38 Some studies have shown that the gut microbiome is involved in regulating the BP. This beneficial effect of probiotics on BP can be due to the release of bioactive peptides such as inhibitory peptides of ACE.39 Given that the effect of probiotics on BP depends on the type of used strain,36 the inconsistency in the results of the studies may be due to the differences in the type of used strains.

To the best of our knowledge, there is no study on the effect of synbiotic supplementation on the military personnel with metabolic syndrome and this study is the first study in this context. Since, Vali Asr Subspecialty Hospital is a reference hospital for military personnel in Tehran metropolis, the results of this study could be generalised to military personnel with metabolic syndrome residing in urban areas of Iran.

There were several limitations in this study. First, the present study was conducted on the military personnel; therefore, one may not be able to generalise the results to general population. Second, the follow-up period was relatively short compared with the similar studies on the metabolic syndrome components. Third, the physical activity was not assessed using a standard questionnaire. Fourth, some variables including insulin, inflammatory factors, oxidative stress and gut microflora were not evaluated before and after the intervention due to the budget constraints.

COnClusIOn

Metabolic syndrome comprises a group of risk factors for chronic diseases including abdominal obesity, increased FBG, altered lipid profile and elevated BP. Considering that the military personnel experience the stressful conditions in their job due to specific occupational conditions and also as proper physical health is of paramount importance in the military personnel, then there is a need to improve their physical condition. The results of the present study showed that the synbiotic supplementation may have an effective role in improving the BMI, TG and FBG levels. Thus, consumption of synbiotic supplements is recommended as a complementary therapy in the military personnel with metabolic syndrome.

Contributors KP, SS, MS, MT and SA studied concept and designed the study. KP, SS and MT collected of data. KP, SS, MS, MT and SA analysed and interpreted of data. MT drafted of the manuscript. KP, SS, MS, MT and SA revised the manuscript. All authors read and approved the final manuscript.

Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not- for- profit sectors.

Competing interests None declared. Patient consent for publication Not required.

ethics approval Prior to starting the research, the objectives and stages of the study were explained to the participants and informed consent was obtained in writing. The study was approved by the Ethics Committee for Research at Baqiyatallah University of Medical Sciences (IR.Bmsu.REC.1397.148) and the Iranian Registry of