Obesity is now a major global epidemic [1
]. It not only predisposes to an array of risk factors such as insulin resistance (IR) and chronic low-grade inflammation but also poses a risk to several non-communicable chronic diseases like type 2 diabetes (T2D) and cardiovascular disease [2
], leading to increased morbidity and mortality amongst adults. The vascular effects of obesity may have a role in the development of a rapidly expanding disease in the elderly population, Alzheimer’s disease (AD) [3
]. Although the mechanisms are not precise, the detrimental impact of obesity on cognitive function may be, at least in part, due to vascular defects like IR and chronic low-grade inflammation, impaired insulin metabolism or insulin resistance [4
]. There are multiple risk factors for AD, including age, obesity, chronic inflammation, genetics and insulin resistance [3
]. A prospective study of >10,000 participants examining the association between body mass index (BMI) and dementia over 36 years reported that obese participants (BMI ≥ 30) at midlife had more than 3-fold increase in the risk of developing AD compared to those with a normal weight range (18.5–25 kg/m2
]. Blood-based kinases such as protein kinase R and c-Jun N-terminal kinase, and in particular glycogen synthase kinase-3 (GSK-3) implicated in both IR and Tau phosphorylation and Aβ plaques, were found to be elevated in individuals with mild cognitive impairment and AD [6
]. Interventions targeting such kinases in the high-risk groups might be beneficial as an early intervention to reduce the risk of T2D and AD.
Research studies on long-chain omega-3 polyunsaturated fatty acids (LCn-3PUFA), eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) have previously shown neuroprotective properties [7
]. A substantial amount of evidence is available on the effects of LCn-3PUFA, specific to DHA and EPA, over neuronal membrane properties, brain plasticity, inflammation and memory [9
]. DHA is quantitatively the most important omega-3 PUFA in the brain; however, the endogenous synthesis of LCn-3PUFA is low within the brain compared with uptake from the plasma pools [10
], thus suggesting that the brain maintains DHA levels via the uptake from dietary sources such as fish or LCn-3PUFA oils (fish or algae oils). At a cellular level, EPA and DHA have been shown to contribute to the modulation of gene expression and of kinases, as well as activation of signaling pathways and metabolite formation, involved in neuroprotection [11
LCn-3PUFA may play a role in the alleviation of obesity-induced IR by promoting the adipose tissue function and maintaining its integrity [12
]. Strong evidence from pre-clinical and ex vivo studies suggests mechanisms of EPA and DHA in ameliorating IR linked to obesity [14
]. Epidemiological evidence presents an inverse association between higher levels of circulating LCn-3PUFA and the incidence of type 2 diabetes [15
]. Cross-sectional studies [17
] and a meta-analysis [18
] from our research team have reported an inverse sex-dependent relationship between LCn-3PUFA, IR and type 2 diabetes, demonstrating favorable effects, only in females. These results suggest that sex may also be one of the potential confounders contributing to the inconsistent findings between LCn-3PUFA and diabetes. Interestingly, clinical trials have produced contrasting results to that of in vivo evidence from animal studies [19
]. Therefore, further research is required to elucidate the role of LCn-3PUFA in ameliorating IR.
In the current study, we aimed to determine the effect of fish oil enriched with DHA as a source of LCn-3PUFA, on GSK-3 and IR in a randomized controlled trial involving overweight and obese individuals.
2. Materials and Methods
Participants were recruited for the ‘Do omega-3 polyunsaturated fatty acids have a gender-specific effect on insulin resistance?’ double-blind randomized controlled trial (ACTRN: 12616000287437) from the Newcastle (Callaghan, NSW, Australia) community through advertisements, local media and social media, and via the Hunter Medical Research Institute Volunteer Register. Volunteers were eligible to participate if they were aged between 18 and 70 years, had a body mass index (BMI) between 25–45 kg/m2, had a waist circumference ≥88 cm (females) or ≥102 cm (males). Participants were excluded if they were diagnosed with diabetes, were taking medications to lower blood sugar levels or to influence insulin sensitivity (e.g., Metformin), if they were pregnant or breastfeeding, were intolerant to the study products, were taking anticoagulants such as aspirin or warfarin, had a history of severe gastrointestinal or neurological disorders, consumed >2 serves of oily fish per week or reported taking fish oil supplements.
2.2. Study Design and Intervention
Eligible participants were randomly allocated using a computer-generated sequence to one of two groups, DHA-rich fish oil or Placebo (corn oil). Participants were asked to consume either 2 × 1 g DHA-rich fish oil capsules/day containing 430 mg DHA + 60 mg EPA or 2× placebo capsules each containing 1 g of corn oil, for a period of 12 weeks. DHA-rich fish oil and placebo capsules were manufactured and provided in kind by EPAX, Norway. The dose of DHA was based on the previous studies that showed efficacy of DHA on IR [20
]. Participants were asked to maintain their habitual diet and usual physical activity levels for the study duration, with dietary intake (3-day food record) and physical activity (international physical activity questionnaire, IPAQ—long form questionnaire) measured at the beginning and end of the study period to ensure compliance. Compliance to the study intervention was measured using capsule log, return capsule count, and gas chromatography analysis of the fatty acid profile of erythrocyte membranes. The study was conducted in accordance to the Declaration of Helsinki and following Good Clinical Practice guidelines and had ethical approval from the University of Newcastle Human Research Ethics Committee (H-2015-0167). Written informed consent was obtained from all participants prior to enrolment in the study.
2.3. Outcome Measurements
All anthropometric measurements were conducted using bio-electrical impedance scales (InBody 230, Biospace Co., Ltd., Seoul, Korea), according to standard operating procedures. Participants’ height (cm) was measured using a wall-mounted stadiometer (SE206, Seca, Hamburg, Germany) and waist circumference (WC in cm) was measured using a non-tensible tape measure at the mid-point between the lower rib and the iliac crest. Body mass index (BMI; kg/m2
) was calculated using the formula BMI = weight (kg)/height (m2
). A 20 mL fasting venous blood samples were collected from the antecubital vein into pre-coated vacutainers for plasma glucose (mmol/L), serum insulin (μIU/L), high sensitivity C-reactive protein (CRP; mg/L) and total cholesterol (TC; mmol/L), and were analysed by a commercial pathology laboratory (Hunter New England Area Pathology Services). Erythrocyte fatty acid composition was analysed using a one-step transmethylation method followed by gas chromatography (Hewlett Packard 7890A Series GC with Chemstations Version A.04.02) [22
]. GSK-3β was analysed using enzyme-linked immunosorbent assay (ELISA) kits. This ELISA kit has high specificity for human GSK-3β and no detectable cross-reactivity with other proteins which might interfere with the assay process and results (manufacturer of the kit- Aviva systems biology, detection range: 0.625–40 ng/mL; mean intra-assay CV < 10%; mean inter-assay CV < 12%). HOMA-IR, used as an indicator of insulin resistance, was calculated using an established equation—fasting glucose (mmol/L) * fasting insulin (μIU/L)/22.5.
2.4. Statistical Analysis
A total of seventy-two participants were recruited for the main study. Required plasma samples were available for fifty-eight participants for the analysis of the current study outcomes. For this study, n = 58 (27 allocated to placebo group and 31 allocated to DHA-rich fish oil intervention group) were analysed for GSK-3β resulting in a study power of 98.9% for the detection of a 2.3 ng/mL reduction in serum GSK-3β level (1.8 ng/mL SD, alpha value set at 0.01). A power calculation was conducted using PS power and sample size calculator (Version 3.1.2, 2014). Data were tested for normality using Shapiro–Wilk’s test and histogram, and data described as the mean ± standard deviation (SD) or median (interquartile range; IQR) as appropriate. Effect sizes are presented as the mean difference [95% Confidence Interval; 95% CI]. Changes in the outcome measures within the placebo and the intervention group across the study period were assessed using paired t-test (parametric) or Wilcoxin Sign-rank test for non-parametric data. Treatment effects were assessed using regression to assess absolute change from baseline using the placebo group as a reference and adjusting for baseline levels. Pearson’s product–moment correlations with Bonferroni correction were used to assess linear relationships between continuous variables, with between-group differences assessed using the immediate command cortesti in Stata to test the equality of two correlation coefficients in independent samples. It considers the respective correlation co-efficient (i.e., r-value) and sample size, with p < 0.05 signifying a significant difference between correlations. Multiple regression models were used to investigate effects of baseline factors (age, sex, BMI, and physical activity) on results. Subgroup analysis on effects of intervention were carried out in people with high and low baseline CRP levels. All statistical tests were two tailed, and alpha was set at p < 0.05. All statistical analyses were conducted using Stata (Version 14.2, StataCorp LLC, Lakeway, TX, USA).
Dietary supplementation with DHA-rich fish oil was accompanied by a reduction in plasma levels of GSK3. DHA-rich fish oil also lowered IR and fasting insulin in individuals with abdominal obesity and hyperinsulinemia compared to the placebo group. The correlation analysis indicated a significant inverse relationship between fasting insulin and IR and changes in insulin and IR only in individuals with high baseline CRP values. No significant changes were observed in fasting glucose and CRP levels. In this study, baseline GSK-3β, insulin and IR were significant predictors of the response to the treatment. Therefore, a greater reduction in these parameters was observed in participants with higher baseline GSK-3β, IR and insulin values.
Hyperinsulinemia and IR are significantly associated with the development of both T2D and AD [23
]. Cross-sectional studies have demonstrated significant associations of HOMA-IR with mild cognitive impairment and AD [24
]. Presence of IR also accelerates the formation of neuritic plaques which are involved in the pathogenic process of AD [25
]. A large body of pre-clinical evidence is currently available on the effects of LCn-3PUFA over insulin signalling and insulin sensitivity [14
]. In line with these studies, epidemiological studies indicate a positive correlation between LCn-3PUFA status and insulin sensitivity [15
]. However, systematic reviews and meta-analysis of clinical trials indicate ambiguous and inconclusive results over efficacy of LCn-3PUFA on insulin sensitivity and glycaemic control [14
]. Results from this study suggest that these inconsistencies may be due to differences in the baseline levels of IR and insulin, as no benefit was apparent in participants with low levels of baseline IR or insulin.
GSK3 is widely expressed in two isoforms α and β. in the human tissues and can also be detected in the peripheral blood mononuclear cells [28
]. GSK-3 is involved in the downstream signalling pathway of kinases (phosphatidylinositol 3-kinase) that are involved in insulin signalling and IR [29
]. Dysregulation of insulin pathways, referred to as IR, is linked to increased levels of GSK3 [29
]. GSK-3 induce IR via enhanced phosphorylation of insulin receptor substrate-1 protein, resulting in defective GLUT translocation interfering with insulin actions and glucose uptake [29
]. Pre-clinical and clinical studies have shown that LCn-3PUFA improve insulin sensitivity by increasing insulin-stimulated disposal of amino acids and glucose and by improving insulin sensitivity [30
]. In the current study, LCn-3PUFA was shown to reduce GSK-3 levels in individuals at high risk of IR and hyperinsulinemia. DHA-rich fish oil associated reduction in IR and fasting insulin levels suggests a possible GSK-3 mediated improvement in the insulin levels.
GSK3 is also implicated in the pathogenesis of AD via α-form linking to precursor tau phosphorylation and β-form linking to Aβ precursor protein [28
]. Systematic reviews have concluded that dietary supplementation with LCn-3PUFA has the potential to ameliorate AD-related symptomology [8
]. The current study supports previous findings that LCn-3PUFA supplementation may interfere with GSK-3β activity. Further studies are required to substantiate the efficacy of LCn-3PUFA on reducing GSK3 in peripheral blood mononuclear cells obtained from individuals with cognitive impairment or with a high risk of AD.
In addition, our study has also indicated that DHA-rich fish oil-associated reductions in IR and insulin levels were only significant in participants with high baseline CRP levels. As the previous evidence also strongly links inflammation and insulin resistance [32
], these two observations may be an important consideration in assessing the efficacy of DHA and EPA on IR. Results from this study are also consistent with a systematic review, which showed that LCn-3PUFA supplementation reduced IR in populations who displayed at least one symptom of metabolic disorder, while there was no effect seen in healthy populations [27
]. Our study population displayed at least one characteristic of metabolic dysfunction (i.e., either hyperinsulinemia or glucose levels in the range of impaired fasting glucose or abnormal CRP levels) and are either overweight or obese. The results in the present study indicate a narrow window for optimal efficacy of LCn-3PUFA on measures of IR when hyperinsulinemia and high inflammation are present before the onset of diabetes or AD. Targeting this window of opportunity may help optimize nutritional strategies aimed towards the reduction in IR and prevention of AD.
In this intervention, we used a DHA-enriched FO capsule. EPA and DHA have been shown to exhibit specific cellular and physiological functions. As mentioned above, DHA is more involved in the regulation of GSK3 than EPA. Further, DHA is more effective than EPA in stimulating peroxisome-proliferated activator receptor-gamma (PPAR-γ) and secretion of adiponectin in vitro; both of these are proposed mechanisms for the insulin-sensitising action of LCn-3PUFA [33
]. Furthermore, administration of DHA derived lipid mediators (D-series resolvins) in knockout-mice models of diabetes reduced glucose intolerance and macrophage infiltration in adipose tissue [34
]. Taken together, these suggest that DHA may be more effective than EPA in reducing IR.
In an intervention study with a similar intervention period (12 weeks), Browning et al. showed that LCn-3PUFA (1.3 g/day EPA + 2.6 g/day DHA) reduced IR in overweight women with a raised inflammatory profile at baseline, while no effect was observed in women with low levels of inflammation [35
]. These observations are consistent with our findings that baseline levels of inflammation may influence the response of LCn-3PUFA on insulin and IR levels. GSK-3-mediated kinase pathways are implicated in regulating the host IR and inflammatory response, playing a pivotal role in regulating the pro- and anti-inflammatory cytokines [36
]. In-vitro studies have shown inhibition of GSK-3, which may promote tolerance to inflammatory stimuli and suppress cytokine production [37
]. Abnormal function of GSK-3 is already indicated in high inflammatory conditions such as AD, diabetes, and cancer [38
]. Therefore, LCn-3PUFA-mediated reduction or lowering of the activity of GSK-3 could potentially explain the lowering in IR, and beneficial effects of LCn-3PUFA in people with high inflammation levels. Further research is warranted to delineate differential effects of EPA and DHA-rich formulations over these metabolic and inflammation parameters. In line with the observations from a meta-analysis with 68 randomized controlled trials [39
] and systematic reviews [40
], this study failed to report a significant effect of LCn-3PUFA on CRP.
Both log-based capsule count and fatty acid profiling exhibited a high level of compliance by participants in both the DHA-rich fish oil and the placebo groups. GSK-3β ELISA kits used to measure the primary outcome have high specificity for human GSK-3β and no detectable cross-reactivity with other relevant proteins that could potentially interfere with assay process. The study was adequately powered for GSK3β. The link between AD and IR is a relatively new and emerging area of research. In this study, we were able to provide basis for DHA-rich fish oil for use as a potential adjuvant for ameliorating common risk factors in early AD prevention interventions.
Though adequately powered, the results of this study remain a secondary analysis and are limited by their preliminary nature. A follow-up study with a narrow age and BMI range is required to substantiate effects of DHA-rich fish oil on GSK3β and determine whether it can affect neurological and metabolic parameters specific to T2D and AD. The preliminary results of this study may not be generalizable nor transferable to other populations. In relation to IR, we employed a surrogate marker (HOMA-IR) instead of a clamp technique, the gold-standard measure of IR. This may also be considered a limitation of the current study; however, HOMA-IR has been shown to have a relatively high correlation (R~0.77) with clamp technique and is currently the most used surrogate marker to measure longitudinal changes to IR [41
]. Therefore, long-term studies, stratified for inflammation, may be required for substantiating the results from the current study.