A randomized control trial of the impact of LCPUFA-ω3 supplementation on body weight and insulin resistance in pubertal children with obesity

Summary

Background:

Paediatric obesity and insulin resistance (IR) are potentially reversible inflammatory conditions. Long chain polyunsaturated fatty acids omega-3 (LCPUFA-ω3) show anti-inflammatory and metabolic properties, but their clinical efficacy is unclear.

Objective:

The objective of this study is to evaluate whether supplementation with LCPUFA-ω3 for 3 months reduces insulin resistance and weight to adolescents with obesity.

Methods:

Double-blind trial of 366 adolescents with obesity randomly assigned to 1.2-g LCPUFA-ω3 (DO3) or 1-g sunflower oil (DP) daily for 3 months; both groups received an energy-restricted diet. Children attended monthly for anthropometric, dietary, and clinical measurements. Basal and final blood samples were obtained to measure metabolic markers and erythrocytes fatty acids. Regression models were used for analysis.

Results:

A total of 119 DO3 and 126 DP children completed follow-up. At baseline, 92% of children presented IR, 66% hypertriglyceridemia, 37% low-grade inflammation, and 32% metabolic syndrome. Despite erythrocytes LCPUFA-ω3 increased more in DO3 (Median differences = 0.984 w/w%; 95 IC = 0.47, 1.53, P < 0.001), body weight, insulin, and HOMA changed similarly in both groups at the end of intervention. Adjusting for basal values, changes in weight, insulin, and HOMA was not related with supplementation.

Conclusions:

Supplementation with LCPUFA-ω3 does not affect body weight or insulin in adolescents with obesity.

1 |. INTRODUCTION

Paediatric obesity is one of the most serious public health problems in the XXI century, because it is the underlying cause of metabolic alterations that eventually lead to pathological conditions as metabolic syndrome, type 2 diabetes, and cardiovascular disease.¹ Insulin resistance (IR) is a transitional stage between obesity and its associated pathological comorbidities. Both obesity and IR are potentially reversible,^2,3 but the strategies used to reverse them have been disappointing; therefore, the search for effective therapies continues. This is crucial to prevent children with obesity progressing to chronic diseases.

Simplistically, the pathophysiological events linking obesity with IR include augmented production of adipokines that generate oxidative stress, inflammation, and IR.³ Thus, the use of anti-inflammatory agents as adjuvants in the treatment of obesity seems appropriate. Experimental studies have demonstrated that the long-chain polyunsaturated fatty acids omega-3 (LCPUFA-ω3), eicosapentaenoic (EPA), and docosahexaenoic (DHA) exert anti-inflammatory properties and stimulate the expression of genes involved in the metabolic pathways of insulin action,^4,5 making them potential candidates in the treatment of obesity and IR, but their effectiveness is not well established.

As in adults with obesity, the benefits of ω3 fatty acids have been studied in paediatric populations with inconsistent results, emphasizing the need for well-designed studies.⁶ Overall, the preventive role of dietary or circulating ω3 fatty acids has been documented in epidemiological studies through their associations with low body weight and less IR.⁷ However, results from intervention studies are variable. While some have found small decreases in HOMA or fasting insulin in children with obesity or IR,^8–10 another study reported borderline effects on insulin sensitivity but only in girls,¹¹ and others report no effects even after using high doses of ω3.^12,13 Interestingly, none of these intervention studies used any blood measurements of PUFA to evaluate adherence to intervention, proposing that the inconsistency of published results may be due to the lack of adherence to supplementation.

Thus, the interpretation of the evidence is that dietary or supplemented ω3 fatty acids may prevent weight gain and IR in healthy children, but nothing can be concluded about potential therapeutic effects in children who already have developed obesity and metabolic alterations.The main aim of this study was to evaluate whether supplementation with LCPUFA-ω3 administered together with a hypocaloric diet for 3 months reduces IR and weight to adolescents with obesity. We hypothesized that LCPUFA-ω3 supplementation decreases insulin and body weight in a magnitude sufficient to revert pathological conditions.

2 |. METHODS

2.1 |. Study population

A double blind, randomized, placebo-controlled, parallel study was conducted in the Unit of Research in Medical Nutrition of the Mexican Institute of Social Security (IMSS) in Mexico City. The research protocol was authorized by the IMSS National Committee of Scientific Research (R-2011-785-037) and registered in ClinicalTrials.gov (clinicaltrials.gov #: NCT01456221). Written informed parental consent and adolescents assent were obtained from all participants. Adolescents were recruited between July 2012 and December 2015 from three Family Medicine clinics of the IMSS in Mexico City. Field workers provided information of the study protocol to physicians in a session and daily to parents in the waiting rooms, inviting to participate. Eligible children were aged 12 to 18 years, both sexes, BMI ≥ 95th percentile, and without clinical evidence of chronic diseases. We invited 494 children, 366 met the selection criteria, 245 completed follow-up (Figure 1).

FIGURE 1.

Flow diagram of the progress through the phases of the clinical trial

Given that a decrease in insulin of 8 ± 35% after 1-month LCPUFA-ω3 supplementation was previously documented in the literature,⁸ we estimated the sample size necessary to detect an 8% difference in reduction of insulin after intervention, plus a 20% to replace dropouts. Considering an 80% power and an alpha of 0.05, and a difference of 8% the estimated sample was n = 119.

2.2 |. Design

Children were randomly assigned to receive daily doses of 800 mg EPA + 400 mg DHA (Pulse; Merck Laboratory) (DO3) or 1 g sunflower oil as placebo (DP) for 3 months, together with a hypocaloric diet consisting in 700 kcal restriction from usual daily intake.¹⁴ Randomization was conducted by one team member who was not involved in the selection and follow-up of children (LBC) using a computerized random numbers table.¹⁵ Placebo capsules were manufactured in a single lot; their appearance was comparable to that of the supplements. Treatments were prepared and offered to the children in white containers with an identification code to assure double-blindness. Identification codes were opened when the follow-up of the last child was completed. Random samples of supplement and placebo were analysed every 6 months to confirm fatty acids concentration.

2.3 |. Data collection

The field workers team was integrated with two physicians and four dietitians previously trained and standardized in anthropometric and dietary methods. Selected children were asked to attend the hospital at inclusion and monthly during 3 months to register anthropometry, blood pressure, dietary information, handed a format to register the pills consumed during that period, and getting the selected treatment. Blood samples were obtained at baseline and at the end of the 3-month period. Glucose, triglycerides, HDL-C, insulin, leptin, C-reactive protein (CRP), TNFα, adiponectin, and erythrocytes fatty acids were measured at baseline by qualified technicians. Except CRP and TNFα, biomarkers were measured also at the end of follow-up.

2.4 |. Anthropometric, clinical, and dietary information

Weight, height, and waist circumference were measured using standard procedures. Electronic balances (BWB-700, Tanita Corporation, Tokyo, Japan), fixed stadiometers (Holtain Limited), and fibre glass tapes were utilized. Body mass index (BMI) z-scores were calculated with the Epi-Info software (EPI-INFO 2000, release 3.2.2). Measured waist circumferences were compared with the Fernandez reference chart to estimate percentiles.¹⁶

Systolic (SBP) and diastolic blood pressure (DBP) were measured twice after sitting for 5 minutes, using a sphygmomanometer with an appropriate sized cuff on the right arm. Hypertension was defined when the measured figure exceeded the 90th percentile for sex, age, and height.¹⁷ Pubertal stage was established based on Tanner classification, asking children to identify themselves in printed pictures representing the different stages.^18,19 Children were classified as pubertal if Tanner is 3 or older or prepubertal otherwise.

Dietary information was obtained at baseline using the average of two multiple-pass 24-hour recalls.²⁰ Trained dietitians applied in-person recalls to children using food models and portion estimation tools. Dietitians, blinded to treatment groups, provided dietary support on monthly evaluations. Energy content was analysed with the Food Processor software (version 8.0, 2000, ESHA Research Inc, Salem, OR), which includes Mexican foods information. Missing foods were added to the database using the Mexican Food Exchange System. Energy intake was calculated for each child using the average obtained in the 24-hour recalls, and 700 kcal were subtracted for recommendation.

2.5 |. Laboratory determinations

Morning peripheral blood samples were obtained after 10-hour fasting and centrifuged at 3000 rpm. Serum and erythrocyte pellets were preserved at −70°C until laboratory determinations. Glucose, triglycerides, and HDL-C were determined with enzymatic methods (SPIN-React 120, Sant Esteve De Bas, Spain); insulin (Millipore, Billerica, Massachusetts) and leptin (Linco Research, Inc, St. Charles, Missouri) by radioimmunoassay; high molecular weight adiponectin, CRP (DSL UK Ltd, Oxon, UK), and high sensitivity TNFα (Quantikine R&D Systems, Minneapolis, Minnesota) with ELISA methods. Coefficients of variation were 7% to 10% for enzymatic analyses, 10% for radioimmunoassay, and 9.5% for ELISA assays.

To measure erythrocytes fatty acid profile, serum was removed immediately after centrifugation and erythrocyte pellets washed twice with 0.9% saline and stored at −20°C until analysis. Total fat was extracted from 0.5 g of frozen erythrocytes with 10.5 mL of isopropanol:hexane (4.5:6); butylated hydroxytoluene was added as an antioxidant (10 μg/mL final volume). Tubes were shaken for 10 minutes and centrifuged for 5 minutes at 1200 rpm at 4°C, the clear supernatant was poured off and dried at 37°C under a stream of ultrahigh purity (UHP) nitrogen. Fatty acids were methylated with methanolic HCl 3 N in methyl alcohol and hexane at 90°C during 1 hour. The upper organic layer was recovered, and solvent was removed by a UHP nitrogen stream. Total fatty acid methyl esters were dissolved in 2-mL vials with an appropriate volume of isooctane to reach a concentration of 2 mg/mL; 1 μL sample was injected to a gas chromatograph (7820A, Agilent Technologies, INC, Delaware) with flame ionization detector. Fatty acids (FAs) were separated using CP-Sil 88 capillary column (100 m × 0.25 mm ID; Varian BV, The Netherlands). Ultrahigh purity helium was used as carrier gas at a flow rate of 1.2 mL/min. Detector temperature was fixed at 270°C and the injector temperature at 250°C. The following oven temperature programme was used: 70°C with an 8-minute hold; ramp: 30°C/min to 175°C with 1-minute hold, 1.2°C/min to 230°C with 5-minute hold. We identified FAs ranging from C12 to C24 chain lengths; peaks were identified by comparing their retention times against those of high purity (<99%) standard mixtures (Sigma-Aldrich Chemie GmbH, 37 FAs mixture).

2.6 |. Clinical diagnoses

Fasting insulin was used to identify IR using 15 μU/mL as cut-off point. HOMA was calculated using the following formula: (insulin [μU/mL] * glucose [mmol/L]/22.5), a cut-off point of 3.16 was used.²¹ Inflammation was defined as CRP > 3 mg/dL.²² To diagnose metabolic syndrome (MS), we combined the International Diabetes Federation criteria²³ and those of the Heart, Lung, and Blood Institute.²⁴ MS was diagnosed if at least three of the following five criteria were present: (a) waist circumference ≥ 90th percentile, age, and sex specific; (b) fasting glucose ≥ 100 mg/dL; (c) HDL-C ≤ 40 mg/dL for boys and girls younger than 16 years old, and ≤ 50 mg/dL for girls 16 years or older; (d) SBP or DBP greater than or equal to 90 percentile for age, sex, and height; (e) TAG ≥ 110 mg/dL.

2.7 |. Statistical analysis

The Minitab statistical package (v17, State College, Pennsylvania) was used for analysis. P value ≤ 0.05 was considered for statistical significance. Descriptive statistics is presented as mean or median, and 95% confidence intervals for quantitative variables, and as count and percentages for categorical variables. Student t test or Mann-Whitney U test were used to compare anthropometric, clinical, and biochemical variables between groups. Frequencies of IR and MS by treatment groups were analysed with χ² test.

Linear regression analysis was used to assess the influence of supplementation on weight, insulin, or HOMA, adjusting for their corresponding baseline values, introducing treatment as covariates. Linear regression analysis was also used to evaluate the potential independent effect of changes in body weight on predicting changes in insulin concentration or HOMA after intervention. To analyse changes in weight, insulin, and HOMA, delta values were calculated by subtracting the amount obtained at baseline from that obtained at the end of follow-up.

3 |. RESULTS

From 494 eligible children, 183 were assigned to each treatment group. Analysis of 119 DO3 and 126 DP is presented, causes for dropout are indicated in Figure 1. Most of the children who discontinued the study did so because they did not allow taking the final blood sample.

3.1 |. Baseline

The proportion of children with IR was 92%, 28% presented hypertension, 6% prediabetes, 66% hypertriglyceridemia, 21% low HDL-C, and 37% inflammation. Despite only 32% of children meeting the criteria for MS, an additional 42% presented two of the diagnosis criteria. Age, gender, pubertal status, as well as clinical, anthropometric, biochemical, and dietary characteristics are presented stratified by treatment groups (Table 1).

TABLE 1.

Baseline characteristics by treatment group

	DO3, n = 119	DP, n = 126
	Mean ± SD
General
Gender male, n (%)	59 (49.58)	57 (45.24)
Pubertal, n (%)	92 (77.31)	107 (84.92)
Age, y	13.73 ± 1.97	13.58 ± 1.79
Anthropometric
Body weight, kg	80.94 ± 16.75	80.29 ± 13.77
Height, m	1.60 ± 0.08	1.61 ± 0.08
Body mass index, kg/m2	31.53 ± 4.92	31.16 ± 3.79
Body mass index, z-score	2.76 ± 0.68	2.70 ± 0.50
Waist circumference, cm	100.4 ± 12.37	99.41 ± 9.81
Clinical
Systolic blood pressure, mmHg	109.59 ± 10.80	108.46 ± 10.26
Diastolic blood pressure, mmHg	71.34 ± 7.99	70.94 ± 8.33
Metabolic syndrome, n (%)	38 (32.28)	41 (32.20)
Insulin Resistance, n (%)	107 (89.92)	118 (93.65)
Biochemical
Glucose, mg/dL	88.75 ± 8.38	88.43 ± 7.53
Insulin, μU/mL	33.05 ± 16.50	31.30 ± 15.05
HOMA	7.38 ± 4.21	6.89 ± 3.55
High density lipoprotein, mg/dL	46.59 ± 9.05	48.58 ± 9.95
Triglycerides, mg/dL	159.5 ± 88	152.87 ± 79.31
C-reactive protein, mg/mL	4.53 ± 6.08	2.90 ± 3.97
Tumour necrosis factor-α, pg/mL	3.08 ± 2.26	2.73 ± 1.63
Leptin, ng/mL	37.98 ± 19.19	38.8 ± 18.62
High molecular weight adiponectin, μg/mL	5.27 ± 3.13	6.26 ± 3.46
Arachidonic acid, w/w%	11.28 ± 6.49	11.32 ± 6.28
Eicosapentanoic acid, w/w%	0.431 ± 0.330	0.437 ± 0.275
Docosahexaenoic acid, w/w%	2.03 ± 1.35	2.01 ± 1.32
Eicosapentaenoic + docosahexaenoic, w/w%	2.47 ± 1.44	2.44 ± 1.40
Dietary
Energy, kcal	2192 ± 817	2208 ± 903
Protein, g	88.66 ± 40.0	89.35 ± 35.98
Carbohydrates, g	274.5 ± 100.5	272.6 ± 111.8
Lipids, g	79.94 ± 41.89	81.31 ± 48.98
Fibre, g	21.19 ± 10.38	19.73 ± 9.34
Calcium, mg	839 ± 458	704 ±408
Magnesium, mg	263 ± 135	250 ±133
Omega-3, mg	1.26 ± 1.68	1.03 ± 1.18

3.2 |. Follow-up

At the end of intervention, there were no differences between DO3 and DP groups in the percentage of reported ingested capsules (85 ± 22% vs 84 ± 24%) and the proportion of children who progressed to the next Tanner stage (17% vs 17%). The mean decrease of BMI z-score for DO3 and DP, respectively, (−0.64 [−0.82, −0.46] vs −0.75 [−0.93, −0.58], P = 0.777), and the proportion of children whose BMI dropped greater than or equal to 5% (48% vs 49%) were also comparable. Likewise, insulin concentration (−2.3 [−4.5, 0.06 μU/mL] vs −2.3 [−4.6, 0.01 μU/mL], P = 0.496) and HOMA (−0.62 [−1.2, −0.1] vs −0.63 [−1.2, −0.1] P = 0.516) decreased similarly in DO3 and DP. In contrast, the median decrease of SBP (−3.8 [−5.4, −2.2 mmHg] vs −1.9 [−4.0, −1.7 mmHg], P = 0.089) and triglycerides (−23 [−33, −12 mg/dL] vs −10 [−21, 0.7 mg/dL], P = 0.085) was greater in DO3 than in DP groups. EPA (0.39 [0.28, 0.52 w/w%], P < 0.001) and DHA (0.57 [0.11, 1.03 w/w%], P = 0.007] increased more in DO3 as compared with DP. An increase of at least 50% LCPUFA-ω3 concentration was observed in 55 DO3 and 32 DP children.

3.3 |. Multivariate analysis

No effect of LCPUFA-ω3 supplementation on weight, insulin, or HOMA was detected even after adjusting for their corresponding baseline values. No effect of supplementation was observed either on changes in insulin concentration after considering changes in body weight (Table 2).

TABLE 2.

The influence of LCPUFA-ω3 supplementation on main outcomes was analysed with regression models adjusted for baseline values

	Coefficient	95 Confidence Interval	P value
	Final Insulin, μU/mL
Basal insulin, μU/mL	0.507	0.40–0.61	<0.001
DO3a	0.710	−2.60–4.02	0.674
	Final HOMA
Basal HOMA	0.475	0.37–0.58	<0.001
DO3a	0.246	−0.55–1.04	0.541
	Final body weight, kg
Basal body weight, kg	0.954	0.93–0.98	<0.001
DO3a	0.313	−0.45–1.07	0.418
	Delta insulin, μU/mLb
Delta body weight, kgb	1.425	0.83–2.02	<0.001
DO3a	−0.450	−4.14–3.24	0.811

^aCompared with DP.

^bDeltas were obtained by subtracting the amount obtained at baseline from the amount obtained at the end of follow-up.

4 |. DISCUSSION

This study demonstrates a null effect of LCPUFA-ω3 supplementation to adolescents with obesity, on insulin resistance and body weight. This observed lack of effect is inconsistent with the effects reported by others^8–11; therefore, we explored some probable explanations. First, it is possible that the influence of the restrictive diet recommended to our children may have masked a somewhat mild effect of supplementation, but the low magnitude of the observed weight lost and the similitude between groups makes this explanation unsupported. Second, probably the dose of LCPUFAs-ω3 we used was insufficient to provide significant effects because an important proportion of our children exhibited extreme obesity, which may have resulted in a low dose per kilogram body weight. However, our results are comparable to those of others who did not find any influence of supplementation using doses even higher than ours.^12,13 Third, we explored the likelihood of a lack of power to detect differences, but we think this is improbable because the observed decrease in insulin in DO3 group was almost identical to that detected in the placebo group, suggesting that a huge sample would be necessary to identify statistically significant differences between groups, which besides would not be biologically important. Fourth, DP children received a daily dose of sunflower oil, rich in linoleic acid known for its pro-inflammatory effects. At first glance, it might appear that the administration of a pro-inflammatory factor to the DP children could have had an impact in the outcomes of the study. However, the chemical analysis of placebo capsules reported by the manufacturer and confirmed in our laboratory demonstrated that each 1-g capsule contained 55.64% linoleic acid and no arachidonic acid. The recommendation of the IOM Food and Nutrition Board²⁵ for linoleic acid is 16 g/d for boys and 11 g/d for girls 14 to 18 years old. Therefore, our capsule provided only 3.5% to 5% of the daily requirement, which would not be expected to be sufficient to produce any significant effect. Thus, it seems that none of these potential confounders explain the lack of effect of LCPUFAs-ω3 supplementation on IR. Therefore, our results support the notion that such supplementation do not provide a clinical effect to children and adolescents who already have obesity and metabolic disturbances.

It is important to clarify that our results do not discount previous findings of the preventive effect of LCPUFAs-ω3 in healthy children or adolescents, because we studied an unhealthy sample. Consequently, our results are not contrary to the reported protective role, but instead, they show that there is no a therapeutic effect. This finding is in line with those of others obtained from intervention studies that failed to demonstrate the benefits of LCPUFAs-ω3 in individuals with diabetes or with insulin resistance.²⁶ Nevertheless, based on the scientific evidence from experimental studies, regarding the effect of LCPUFAs-ω3 in the expression of molecules involved in insulin sensitivity,⁵ we believe that even though such supplementation does not reverse the already stablished insulin resistance, other metabolic benefits might be provided.

Data collected as part of the present study are available for the further exploration of potential secondary outcomes. Preliminary analysis of these secondary outcomes suggest a contribution of LCPUFAs-ω3 supplementation on preventing progression to IR or metabolic syndrome in children who present obesity but have not yet developed a related morbidity (data not shown). These results, although are in need of further evaluation, may support a potential effect of LCPUFA-omega 3 supplementation on the progression of disease. Nonetheless, the exploration of potential secondary outcomes is beyond the scope of this paper, as it is the understanding of mechanisms that may be utilized by LCPUFAs-ω3 to delay the progression to a pathological manifestation of obesity. This is important because it is known that resolvins (lipidic mediators derived from the LCPUFAs-ω3 DHA) acutely turn off inflammation and stimulate a class of immune cells that limit or offset the cytokines effects on lipotoxicity.²⁷ Based on this, the efficacy of adding DHA or resolvins to the existing therapies used to prevent disease progression is under investigation in other inflammatory diseases such as eczema, periodontal disease, and cancer. The results of these ongoing investigations will be relevant to consider an LCPUFAs-ω3 as potential therapeutic agents to be used in children or adolescents who already have obesity or even a metabolic alteration, to prevent progression to a more severe pathological condition.

In conclusion, our results provide strong evidence that LCPUFA-ω3 supplementation does not exert therapeutic effects in children with obesity and insulin resistance.