Effects of eicosapentaenoic acid supplementation on heat shock protein 27, glycemic status and anthropometric indices in type 2 diabetes patients

Abstract

Purpose

Heat shock proteins (HSP-27) are reported to be involved in the pathophysiology of diabetes complications. The purpose of the current study is to assess the effects of eicosapentaenoic acid (EPA) supplementation on serum HSP-27, glycemic status and anthropometric indices in type 2 diabetes mellitus (T2DM) patients.

Methods

Thirty-six patients with T2DM were randomly allocated to obtain 2 g per day EPA (n = 18) or placebo (n = 18) for 8 weeks in a randomized, double-blind, placebo-controlled clinical trial. Fasting serum levels of HSP 27, fasting blood sugar, hemoglobin A1C, as well as anthropometric indices were measured.

Results

EPA supplementation reduces the serum level of HSP 27 in the EPA group compared with the placebo (P < 0.03). Although waist circumference (WC) decreased significantly in the EPA group at the end of the trial (P < 0.02), there was no significant difference in weight, WC, body mass index (BMI), and glycemic markers in both groups after intervention (P > 0.05).

Conclusions

We found that EPA supplementation reduces HSP 27 serum level in T2DM patients. However, future large-scale trials are needed.

Introduction

Diabetes mellitus (DM) is a metabolic disorder defined as higher than normal blood glucose levels during a long time period. The insulin becomes disabled to perform the main physiological function because of impaired insulin production or body resistance to the insulin [1–3]. The prevalence of diabetes is increasing globally. It is estimated that the prevalence of type 2 diabetes mellitus (T2DM) increase to more than 366 million people worldwide in 2030 [4, 5]. Excessive production of free radicals and impaired antioxidant defenses have been indicated in diabetes [6–8]. Free radicals are the main culprits in diabetes complications such as inflammation and vascular complication. DM and long-term hyperglycemia are contributed to chronic end-organ damage in the eyes, kidneys, and the brain and other complications including cardiovascular diseases (CVDs) that can increase morbidity and mortality among patients with DM [9–12]. The cardiovascular complication of diabetes comprises vascular effects and direct cardiomyopathy [10].

Heat shock proteins (HSPs) are a family of highly conserved proteins expressed by cells in response to environmental stressors, including free radicals, hypoxia, chemical exposures, tumorigenesis, and toxins such as oxidized low-density lipoprotein (oxLDL) [13–15]. Several studies suggest that the HSP protects the cells against the free radicals. HSP expressions reduce oxidative stress either via activation of antioxidants or direct scavenging of free radicals [16] HSPs are organized by molecular weight. HSPs` role in the pathophysiology of diabetes complications such as diabetic neuropathy has been proposed [17]. Heat shock protein 27 (HSP 27), is a 27 kDa conserved peptide associated with cytoskeletal actin[18]. Numerous studies in experimental models have shown overexpression of HSP27 in glomeruli, retina, and atherosclerotic plaque, representative HSP27 induction in target tissues of diabetes complications[18]. An increased level of HSP 27 is found in subjects with coronary artery disease (CAD), acute coronary syndrome, and metabolic syndrome[19]. Furthermore, HSP 27 seems to be associated with cardiovascular complications in subjects with glucose intolerance [20].

The beneficial effects of n-3 polyunsaturated fatty acids for cardiovascular complications and chronic inflammatory conditions have been shown [21]. Stimulating the immune system, reducing platelet aggregation, clotting, smooth muscle contraction, leukocyte chemotaxis, and modulating inflammatory cytokine production have been proposed as plausible mechanisms [22]. Elevated expression of HSPs because of dietary saturated fatty acid is found in experimental models[23].

Here in the present randomized controlled trial (RCT) we have investigated the effect of eicosapentaenoic acid (EPA) supplementation on serum HSP-27, glycemic status and anthropometric indices in patients with T2DM.

Materials and methods

Patients and study design

As shown in Figs. 1 and 36 patients with T2DM were included in the present study. Ten patients were excluded before beginning the trial. Patients were chosen based on available American Diabetes Association guidelines [24]. Inclusion criteria for the participants were: having a history of at least one year of T2DM affliction before the participation in the study, aged 35–50 years, 25 ≤ BMI < 30 kg/m2, and consumption of antidiabetic drugs for at least three months. Subjects excluded based on exclusion criteria: reluctance to continue the study, need for insulin, change in the dose and type of medication, change in physical activity level, low compliance for using supplements (< 10%), history of hypertension, arrhythmia, renal, hepatic, gastrointestinal, endocrinological, inflammatory or hematological disease.

Fig. 1.

Flow diagram of patient enrollment in the study

Present study was a randomized, double-blind, and placebo-controlled clinical trial. Study protocols were conducted in keeping with the Helsinki declaration and CONSORT checklist. Patents selected from Iran Diabetes Association (Tehran, Iran). After the ethics committee of Tehran University of Medical Sciences (TUMS) approved the study protocol, and informed consent was attained based on Ethics Committee on Human Experimentation from TUMS guidelines, patients were randomly allocated to receive 2 g/day of the softgel of EPA (eicosapentaenoic acid ethyl ester (75%), supplied as 1-g softgel) or placebo (edible paraffin) for 8 weeks. All patients and personnel were blinded until the end of the study as the containers were coded with A or B by the manufacturer before the study. Subjects were stratified by sex and randomly allocated. Blocked randomization was performed for allocation with block sizes of four hidden in a bowl by one of the researchers. The blocks were composed of A and B demonstrating containers of capsules coded with A or B to ensure concealment. The other researcher randomly allocated patients to groups. EPA and placebo were in the same appearance and all patients and researchers were uninformed about allocation until the main analysis. They were advised not to change their physical activity level, nutritional habits, and their medication dose during the study. At last, the compliance was judged by counting the number of capsules used and the number of capsules returned. Phone contact was done for all patients almost weekly. The trial was registered in the Clinical Trials (www.clinicaltrials.gov) for registration of clinical trials (NCT03258840).

The sample size was calculated according to type one (α) and type two errors (β) as 0.05 and 0.20 (power = 80%), respectively, and according to the previous study. Standard deviation (SD) and difference in mean or effect size (d) of serum paraoxonase are considered 11.61 U/ml and 11.4U/ml respectively as the key variables [25]. Although we needed 16 subjects in each group because of possible dropouts 36 subjects have participated in the study.

At the start and the end of the study, each participant was assessed with a general questionnaire. The questions include demographic variables (age, sex), family history of diseases (diabetes, hyperlipidemia, and hypertension, cardiovascular, etc.), age at the diagnosis of T2DM, type of the medication used, and daily life habits (including the history of smoking, alcohol consumption).

Anthropometric Assessment

Anthropometric data including weight, height, waist, and hip circumference were assessed without shoes and in a minimal clothing state. Body weight and height were evaluated to the nearest 0.1 kg and 0.1 cm respectively by using available equipment (Seca, Hamburg, Germany). Waist circumference (WC) was calculated by tape measure (Seca; Germany) based on previous studies. BMI was calculated by dividing body mass (kg) by squared height (m2).

Dietary intake assessment

At the beginning and at the end of the trial, nutrient intakes were estimated using a 24-hour diet recall questionnaire for 3 days, including 2 working days and 1 weekend day. The dietary recalls were analyzed using Nutritionist IV software (First Databank, San Bruno, CA, USA). With the purpose of knowing accurate portion sizes, subjects were asked to report their intake based on household measures. The portion sizes were converted to grams.

Biochemical assays

Fasting blood samples (10 ml) (overnight fast for 10–12 h) were drawn from the antecubital vein at the beginning and the end of the study then centrifuged (3000 rpm for 10 min at 4 °C for serum acquisition) and preserved at − 80 °C until analysis. The glycated Hb (HbA1c) test was performed on the whole blood sample by means of Biosystem commercial kits (Biosystems) using the chromatography method on the day of taking the blood samples.

Hsp27 serum concentration was measured by Bioassay Technology Laboratory kit by ELISA method. Fasting blood sugar (FBS) was determined using a commercial kit and HbA1c was measured by D-10 kites (BioRad laboratories, Schilitigheim, France).

Statistical analysis

The data were analyzed using SPSS software (version 16.0 for Windows; SPSS Inc., Chicago, IL, USA), and the results are detailed as mean ± SD or mean ± standard error. The Independent t-test was used for the comparison of variables between the two groups. Within-group, comparisons were performed by paired-sample t-test. The analysis of covariance (ANCOVA) was performed to identify differences between the two groups independently adjusting for baseline values. P values of < 0.05 were regarded as statistically significant.

Results

At baseline, there were no significant differences between the two groups in measured factors as shown in Table 1. Also, no significant differences were reported in physical activity level and medication dose (Data not shown), total energy intake, or macronutrient intake between the two groups of patients at the baseline (Table 1), and end of the trial.

Table 1.

Characteristics of the two groups at the baseline and end of study

Characteristics	Baseline			After 8 weeks’ supplementation
	Placebo group (n = 18)	EPA group (n = 18)	P value	Placebo group (n = 18)	EPA group (n = 18)	P value
No. of patients (female/male)	9/9	9/9	-	-	-	-
Age, year	44.72 ± 4.69	44.44 ± 3.79	0.846	-	-	-
Duration of DM, year	6.61 ± 3.68	6.44 ± 2.83	0.880	-	-	-
Physical activity			0.721			0.721
Low	3	5		3	5
Medium	14	12		14	12
High	1	1		1	1
Hip circumference, cm	106.00 ± 11.82	105.33 ± 6.69	0.836	105.61 ± 12.32	104.61 ± 7.59	0.771
Waist/hip ratio	0.92 ± 0.08	0 0.92 ± 0.06	0.922	0.92 ± 0.07	0.92 ± 0.06	0.940
FBS, mg/dL	138.06 ± 49.13	143.72 ± 53.53	0.743	157.06 ± 52.34	121.94 ± 23.56	0.016*
HbA1C, %	7.47 ± 1.67	7.89 ± 1.75	0.459	7.77 ± 1.42	7.86 ± 1.58	0.792
Total energy intake, kcal	2,070.67 ± 307.27	1,953.94 ± 297.12	0.254	1,961.56 ± 232.21	274.36 ± 1,973.61	0.325
Carbohydrates intake, g/d	270.63 ± 50.37	260.32 ± 35.44	0.483	35.44 ± 260.32	42.89 ± 260.82	0.756
Proteins intake, g/d	68.95 ± 20.26	63.19 ± 14.78	0.337	11.97 ± 70.09	14.06 ± 63.92	0.177
Lipids intake, g/d	92.71 ± 19.85	86.11 ± 22.68	0.359	16.56 ± 76.39	20.13 ± 76.86	0.543
Fibers intake, g/1,000 kcal	16.66 ± 4.99	14.75 ± 4.64	0.243	2.28 ± 14.64	4.64 ± 15.36	0.179

Data are shown as mean ± standard deviation or number (%)

Statistical analysis was performed using independent t-test

DM, diabetes mellitus; BMI, body mass index; FBS, fasting blood glucose; HbA1C, hemoglobin A1C.; EPA, eicosapentaenoic acid

All selected patients completed the trial; all participants have well tolerated the intervention for 8 weeks and no side effects throughout the study were reported.

At the end of the intervention, no significant differences in weight, height, BMI, WC, and HbA1c were not seen between the two groups (Table 1). FBS decreased significantly in the intervention group at the end of the trial.

Table 2 represents the effect of EPA supplementation on serum HSP 27 levels. The serum levels of Hsp27 reduced significantly in the EPA group at the end of the intervention (P < 0.008) and compared with the control group (P < 0.03).

Table 2.

The effect of EPA supplementation on anthropometric indices and serum HSP 27 levels

Variable		EPA (n = 18)	Placebo (n = 18)	P b
		mean ± SE	mean ± SE
Weight (kg)	Baseline	78.02 ± 2.98	78.30 ± 2.90	0.94
	End of trial	77.15 ± 2.98	78.23 ± 3.15	0.29d
	P c	0.08	0.89
	Change	-0.87 ± 0.48	-0.06 ± 0.49	0.45
BMI (kg/m2)	Baseline	28.48 ± 0.93	28.91 ± 1.27	0.77
	End of trial	28.16 ± 0.92	28.86 ± 1.32	0.25d
	P c	0.09	0.78
	Change	-0.32 ± 0.17	-0.04 ± 0.17	0.50
WC (cm)	Baseline	97.55 ± 2.27	97.47 ± 2.57	0.98
	End of trial	96.44 ± 2.39	97.08 ± 2.76	0.35d
	P c	0.02	0.55
	Change	-1.11 ± 0.44	-0.38 ± 0.63	0.18
HSP27 (ng/ml)	Baseline	39.77 ± 3.15	35.26 ± 2.23	0.24
	End of trial	33.28 ± 1.13	34.22 ± 1.67	0.03 d
	P c	0.008	0.45
	Change	-6.48 ± 2.16	-1.04 ± 1.34	0.03

EPA = Eicosapentaenoic Acid, SE = Standard Error, BMI = body mass index, WC = waist circumference, HSP 27 = Heat shock protein 27

^b Obtained from Independent sample t-test

^d Obtained from ANCOVA, adjusted for baseline values

^c Obtained from paired T test

There were no significant differences in BMI (P = 0.25), WC (P = 0.35) and weight (P = 0.29) between the two groups of patients at the end of intervention. Intervention group show significant change just in WC (P = < 0.02) but not in BMI (P = < 0.09) and weight (P = < 0.08).

Discussion

The prevalence of diabetes is increasing, so a rising number of patients with diabetic kidney disease, end-stage renal disease (ESRD), and also cardiovascular diseases are anticipated [26]. The prolonged increased glucose level in diabetes leads to many chronic complications such as vascular disease, retinopathy, neuropathy, and kidney disorders. Furthermore, DM-related abnormal lipid metabolism is considered a major cause of the development of atherosclerosis and cardiovascular complication [27]. Oxidative stress has been implicated in diabetes complications. Overexpression of HSP27 in exposure to oxidative stress through different mechanisms like reduction of intracellular iron level has been reported [28]. Increased expression of HSPs, especially HSP27 inhibits apoptosis by blocking caspase activation or antioxidant capabilities[29]. However, cell damage may lead to the release of intracellular HSP and as a result increased HSP serum concentrations[30]. So because of systemic oxidative stress, an elevated serum HSP concentration in patients with diabetes complication is not unexpected. For instance, HSPs response by the kidneys in response to oxidative stress and inflammation could increase apoptosis, reduce kidney function, and initiate cardiovascular manifestation in uremic patients[29]. Elevated serum HSP27 levels in DM patients with diabetic nephropathy, distal symmetrical polyneuropathy, and DM-induced micro and macroalbuminuria have been reported compared to diabetic control subjects[18, 29]. A relationship between the presence of neuropathy and circulating HSP 27 has also been known in a cohort of patients with T1DM [18]. Other studies revealed a strong association between HSP expression and the manifestations of atherosclerosis[18]. DM may affect cardiac function independently of CAD and hypertension such as higher heart rate in DM patients which has been revealed in Framingham Heart Study[31]. In that case, a higher level of HSP27 is found in subjects with CAD, acute coronary syndrome, cardiovascular complications, and in subjects with glucose intolerance [19, 20].

Several studies suggest that fish oil supplementation may reduce the inflammatory response thorough decreasing lymphocyte proliferation, cytokine production, and antibody production[22]. In this study, we expected that HSP27 concentrations may be decreased following EPA supplementation in T2DM patients, so the relationship between serum HSP27 levels and EPA supplementation was investigated. Inter- and intra-group analysis revealed decreased serum HSP27 concentrations following EPA supplementation compared to diabetic control subjects. Because of the above-mentioned mechanisms which express HSP’s involvement in pathophysiological procedures in DM, this reduction could be considered an important effect of EPA supplementation. In the same study, 120 patients with metabolic syndrome were randomly allocated to use180 mg EPA and 120 mg docosahexaenoic acid (DHA) daily for 6 months. The control subjects did not receive a placebo. At the end of the study high-sensitivity C-reactive protein (hs-CRP), and HSP 27 antibody titers were reduced significantly [22].

It must be noted that anthropometric factors such as WC, weight and BMI did not change significantly. Previous studies have reported those parameters as an independent CVD risk factor [32]. The previous study also revealed that just waist to hip ratio (WHR) [33] and WC decreased [34] significantly in the omega-3 supplementation group but no changes were seen in weight, BMI, and WHR in intervention or placebo groups after the intervention similar to other reports [33].

The present study has several limitations. First, a relatively small sample size so must be confirmed in a larger sample size of patients. Next, the exact mechanisms by which EPA decreases the serum levels of HSP 27 have not been clarified, and further work is necessary to delineate the molecular mechanism. Thus, it is better than the serum levels of inflammatory markers, as well as the percentage of EPA in the membrane of red blood cell measured in further studies. For these reasons, additional studies will be necessary to determine the general applicability of our study results.

Conclusions

In conclusion, the supplementation of EPA was effective in the reduction of HSP 27 in patients with T2DM. However, further studies are required to investigate the underlying mechanisms and to confirm the effectiveness of EPA supplementation in T2DM.

Author contribution

E.G. and J.G. conducted the trial; MH.G. and M.Z. performed biochemical analysis and prepared a draft of the method and results; H.M. contributed to data analysis and results in approval; E.G. and A.H. prepared the main manuscript. All authors approved the final version of the manuscript. M. J and M.H.J approved the manuscript and were the supervisor.

Funding

Tehran University of Medical Sciences funded the present study. The ethics committee of the Tehran University of Medical Sciences (TUMS) approved the study.

Data Availability

The data used and/ or analyzed during the current study are available from the corresponding author on reasonable request.

Code Availability

NCT03258840.

Data Availability

The data used and/ or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

All the authors declared that they have no conflicts of interest.

Ethical considerations

Ethical issues (including plagiarism, misconduct, data fabrication, falsification, double publication or submission, and redundancy) have been completely observed by the authors.