Exercise, omega-3 fatty acids and vitamin D for cognitive function

Exercise is a candidate to attenuate neuropathology and enhance brain health throughout life via the regulation of growth factors and neurotrophins such as insulin-like growth-factor 1 (IGF-1), brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor that are involved in angiogenesis, synaptogenesis and neurogenesis and the modulation of systemic inflammation.5 Exercise can positively influence cardiovascular health and insulin resistance, which are pathways relevant to cognitive decline.6 A large body of research has demonstrated that greater levels of physical activity are associated with slower rates of cognitive decline7 and a lower risk of dementia.8 Furthermore, randomised controlled trials (RCTs) have shown that even acute bouts of exercise can provide small benefits to cognition.9 Findings from recent meta-analyses have indicated that longer duration exercise interventions can enhance cognition in healthy p eople>50 years of age10 as well as individuals at risk of or diagnosed with AD.11

While questions still remain regarding the optimal mode and dose of exercise required for cognitive improvements,12 a recent systematic review and meta-analysis of 36 RCTs in community-dwelling adults, aged 50 years and above,10 provided the recommendation that combined aerobic and resistance exercise, at a moderate to high intensity, as many days of the week as feasible, is suitable to enhance cognitive function. Exercise interventions of 4–6 months’ duration that have used resistance or multimodal programmes, incorporating resistance training, have indicated that, in older people, a frequency of two sessions per week can benefit cognition.13–15 Importantly, cognitive benefits have been observed for resistance training when compared with ‘sham’ exercise or stretching conditions designed not to impact heart rate or aerobic capacity.13 14 16 The use of a control exercise condition that is matched for social interaction, including attention from the trainer delivering the programme, is necessary to account for possible benefits to cognition attributed to the social experience, rather than the exercise programme.

Accumulating evidence, largely from epidemiological studies, suggests that various dietary factors alone and in combination, particularly omega-3 fatty acids and vitamin D, are strongly linked with cognitive decline and dementia.17–20 Polyunsaturated fatty acids (PUFAs) serve an essential role in the brain, regulating the function and structure of neurons, endothelial cells and glial cells.21 The long-chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) also modify neurotransmission, reduce neuroinflammation and promote neuronal survival and neurogenesis.21 The high intake of omega-6 fatty acids in the Western diet (ie, from vegetable oil) causes an imbalance of omega-3/6 fatty acids, resulting in lower than optimal levels of DHA and EPA.22 23 A high intake of omega-6 fatty acids has been suggested to adversely impact cognition by depleting DHA, the omega-3 PUFA most prevalent in the brain.24 In contrast, a low omega- 6/3 ratio has been shown to predict better cognitive function in healthy older people.25

Greater dietary intake of DHA (from consumption of fish) has been linked to better cognitive outcomes in older people26 and a 36% lower risk of AD across multiple observational studies.17 A dose–response meta-analysis showed that an increment of 100 g per week of fish intake was associated with an 11% lower risk of AD.17 In older people with mild memory complaints (n=437) 900 mg/day of DHA supplementation for 24 weeks improved episodic memory and learning on computerised measures.27 Of note, when compared with normative data, the improvements due to DHA supplementation on the verbal learning test approximated a 7-year improvement, compared with a 3.6-year improvement with placebo. Another study conducted in 50 older people with mild cognitive impairment found that both EPA rich (1.67 g EPA+0.16 g DHA/day) and DHA rich (1.55 g DHA+0.40 g EPA/day) supplements, taken daily for 6 months, improved depressive symptoms, compared with an omega-6 rich supplement.28 In this study, only the DHA-rich supplement improved a single cognitive domain as well as physical health. As not all studies have yielded positive results regarding omega-3 fatty acid supplementation on cognitive function,29–31 dose and target population may be crucial factors.

An emerging role for vitamin D in the maintenance of brain health and cognition is developing with reports that serum 25-hydroxyvitamin D (25(OH)D) levels are lower in those with impaired cognitive function and AD than healthy controls18 32 and low vitamin D levels increase AD risk 7 years later.33 Observational studies indicate that inadequate vitamin D intake is linked to poorer cognitive function34 and may increase the probability of cognitive impairment by as much as 30%.35 Relatively few RCTs have investigated the impact of vitamin D supplementation on cognition in older adults,35 although clinical trial registries indicate a number of studies are underway. In a recent 18-week study comparing high-dose vitamin D3 (4000 IU/day) to a low dose (400 IU/day) in 82 healthy adults, the high-dose supplementation improved visuospatial memory but not other cognitive domains.36 As even the low dose increased 25(OH)D concentrations above 75 nmol/L, this may have impacted the ability to detect cognitive changes dues to higher dose supplementation, as 75 nmol/L  is the suggested optimum value to exceed to reduce cognitive and brain dysfunction.37

Whether there is an additive effect of different nutrients with exercise in terms of benefits to brain health is an important question. Research in rodents has indicated that DHA can enhance the effects of exercise on BDNF-related synaptic plasticity,38 and vitamin D has the potential to contribute to these actions through the regulation of neurotrophins BDNF, glial cell line-derived neurotrophic factor, nerve growth factor and IGF-1.35 As a neuroprotective agent, vitamin D may target specific AD pathology, attenuating amyloid-beta (Aβ) accumulation by stimulating the phagocytosis of the Aβ peptide and inducing cytokines and macrophages to increase Aβ clearance.35 Exercise and DHA exert potent anti-inflammatory effects5 21 and vitamin D may further attenuate inflammation-induced degeneration of neurons by inhibiting the production of tumour necrosis factor-α, interleukin-6 and nitric oxide, preventing lipid peroxidation and reversing mitochondrial dysfunction under conditions of heightened oxidative stress.35

The cognitive effects of omega-3 fatty acids combined with a more complex lifestyle intervention, incorporating exercise, have recently been examined in the large 3-year RCT Multidomain Alzheimer Prevention Trial (MAPT). This study examined the impact of omega-3 fatty acid supplementation (800 mg/day DHA +250 mg/day EPA) and a multidomain intervention (cognitive training, physical activity and nutrition advice and three preventative consultations), alone or in combination, compared with placebo on cognitive function in 1680 community-dwelling, non-demented adults aged 70+ years.39 The combination of omega-3 fatty acids and the multidomain intervention enhanced cognitive performance on a secondary measure of cognitive function and in subgroup analyses of those with an increased dementia risk score, but no benefits were found for the primary cognitive endpoints. The absence of an effect on the primary outcome for the multidomain intervention may have been influenced by the design of the exercise component that was limited to generic physical activity advice to be more active (eg, aim to walk 30 min per day) and did not include a structured programme designed to enhance physical function.

Muscle mass and strength and cognitive function

The ageing process targets multiple systems of the body, leaving older individuals vulnerable to deterioration of brain, vascular and musculoskeletal systems. A progressive loss of muscle mass, strength and function, termed as sarcopenia, occurs with ageing. After the age of 40–50 years, there is a progressive loss of muscle mass in the magnitude of 1%–2% per year.40 Sarcopenia is a growing public health concern, increasing the risk of low trauma fracture, disability, morbidity and reducing quality of life in older people.40

Low muscle strength has been linked to lower global cognitive performance41 and a higher risk of cognitive impairment.42 Greater frailty has been associated with increased cognitive decline in patients with AD.43 A recent investigation using data derived from the UK Biobank identified a significant relationship between grip strength and multiple cognitive domains in over 475 000 individuals from the general population.44 While these findings emphasise the connection between muscular strength and brain health, more research is needed to determine whether increasing muscle mass and strength can enhance cognitive function. Results from the Study of Mental and Resistance Training (SMART) trial showed that strength improvements due to 6 months of high intensity resistance training were associated with improvements to cognitive function in older people with mild cognitive impairment.45

Combining dietary protein supplementation with resistance training exercise has been proposed as a strategy to offset muscle decline in older people.46 A 16-week RCT conducted in older women found that increasing lean red meat intake, combined with twice weekly resistance training, increased lean tissue mass and muscle strength compared with resistance training coupled with a high carbohydrate diet.47 A recent meta-analysis has confirmed that dietary protein can augment the effects of resistance training exercise on muscle mass and strength, although the findings indicate that resistance training is the more potent stimulus for increasing muscle strength than protein intake.48 Protein dose may be an important consideration, with an RCT in men aged >70 years having demonstrated that providing double the recommended daily allowance (RDA) of protein (1.6 g protein/kg/day), compared with the RDA, for 10 weeks provided benefits to lean body mass and knee extension peak power.49 These findings are consistent with the premise that older adults need a higher intake of protein to increase muscle protein synthesis.46 With regards to the impact of combining exercise with protein on cognitive function, there is some evidence that the combination of 30 g daily protein supplementation and resistance training exercise for 6 months enhanced processing speed in frail and prefrail elderly.50 While a consistent benefit of protein supplementation for cognition has not been established, it has been proposed that amino acids such as tyrosine and tryptophan may be capable of modulating cognitive function.51

Objective

It is hypothesised that a multifaceted approach combining exercise with dietary modification involving omega-3 fatty acids, vitamin D and protein may offer cognitive benefits, relative to the control condition, as they will target complementary pathways related to neuroinflammation, neuroprotection and neuroplasticity.5 21 35

The primary aim of this study is to investigate the efficacy of a 6-month multifaceted intervention, incorporating an exercise programme with omega-3 fatty acid, vitamin D and protein supplementation on cognitive function in older people with subjective memory complaints. The secondary aims are to: (1) determine if any beneficial effects of this intervention on cognitive function are mediated through increases in muscle strength or mass, (2) explore the impact of the intervention on other aspects of psychological and physical health and (3) evaluate the longer term residual effects (6 months after cessation of the intervention) on cognition.

Study design

This is a 12-month, community-based, parallel group, randomised, double-blind, placebo-controlled trial involving a 6-month supervised and structured intervention, followed by an additional 6-month follow-up to evaluate any long-term residual effects. A total of 148 individuals with subjective memory impairment (SMI) aged 60–85 years will be randomised to: (1) a supervised multimodal exercise programme involving progressive resistance training and aerobic exercise, combined with omega-3 fatty acid, vitamin D and protein supplementation or (2) a stretching/flexibility exercise programme and placebo supplements. During the 6-month postintervention follow-up, participants will no longer be provided with the exercise programmes or supplements but will be encouraged to continue to exercise and follow a healthy diet. This study protocol is reported in accordance with the Standard Protocol Items: Recommendations for Interventional Trials statement,52 and results will be reported in line with the Consolidated Standards of Reporting Trials (CONSORT) statement53 and the CONSORT statement for non-pharmacological interventions.54 The study was prospectively registered on the Australian New Zealand Clinical Trials Registry on 10 November  2016.

Participants and screening

Participants will be included into the study based on the following criteria: (1) presence of subjective memory complaints, defined as a positive response to the question ‘do you feel like your memory is becoming worse?’ and a score >17 on the Montreal Cognitive Assessment (MoCA), (2) community dwelling and aged 60–85 years, (3) fluent in written and spoken English and (4) with normal or corrected to normal vision and hearing.

To minimise extraneous causes of cognitive impairment, participants will be excluded based on the following: (1) diagnosis of AD or other dementia and Parkinson’s disease; (2) history of brain damage including significant head trauma, stroke or serious neurological disorder, including loss of consciousness >24 hours; (3) clinical diagnosis of psychiatric disorder likely to affect cognition, including current clinical diagnosis of depression, commencement of anti-anxiety or antidepressant medication within the past 6 months, score of >5 on the short Geriatric Depression Scale; (4) or alcohol or drug dependency within the last 2 years. Exclusion criteria relevant to the study intervention, supplement and/or exercise programme, will include: (5) existing diagnosed gastrointestinal disorders likely to impact absorption of fatty acids (eg, coeliac disease); (6) known allergy to fish or any other component in the dietary supplement; (7) type two diabetes; (8) body mass index >40; (9) health conditions that contraindicate exercise or insufficient mobility to exercise; (10) participating in progressive resistance training >1 week or more >150 min per week of moderate intensity physical activity; (11) current smokers; (12) oily fish consumption (≥1/week); (13) daily use of omega-3 rich supplement >500 mg or vitamin D supplements >500 IU within the past 12 weeks; (14) use of cholinesterase inhibitors, benzodiazepines, antipsychotics or high-dose antidepressants (judged by medical advisor), use of warfarin or high dose blood thinners, resistant hypertension defined as the use of more than three antihypertensive agents and medications contraindicated for use with omega-3 fatty acids, vitamin D or protein. Any uncertainty or discrepancies about participants’ clinical history will be resolved by consulting with a physician familiar with the study (investigator SM).

Participants will be recruited from within the local community in Melbourne (Australia) and surrounding areas via newspaper advertisements, community presentations, social media and community notice boards. Participants will be provided with the exercise programme free of charge as an incentive to participate in the trial.

Randomisation

Participants will be randomly allocated to one of the two parallel groups using a simple randomisation computer generated random sampling set, with an allocation ratio of 1:1. Randomisation will be stratified for gender and age, separated into three categories of 60–69.9 years, 70–79.9 and 80–85.9 years. Randomisation will be conducted by personnel not otherwise involved in the trial. Participant group allocation will be provided in an opaque envelope to the researcher involved in assigning participants to the intervention, on completion of the baseline appointment. The researcher who assigns participants to the intervention will distribute the study treatment and provide information about the assigned exercise programme. This researcher will no longer be blinded from this point.

Blinding

Both the active supplement and the placebo will be packaged in non-transparent sachets. All researchers involved in outcome assessments will be blinded to participant group allocation, with the exception of the 6-month dual-energy X-ray absorptiometry (DXA) scan, which may be conducted by an unblinded researcher due to limited qualified personnel. All DXA analyses will be carried out by a blinded researcher. During the intervention period, only unblinded researchers will be in contact with participants enrolled in the trial. In order to maintain a double-blind study design, participants will not be informed that all individuals assigned to the multimodal exercise programme will also receive the supplement drink containing omega-3, vitamin D and protein, nor that all participants assigned to the stretching/flexibility condition will also receive the placebo. The purpose of concealment will be explained when all participants have completed the trial. Exercise trainers delivering the exercise programme will not be informed that participants in the multimodal exercise programme are receiving the active supplements or those in the stretching group are receiving the placebo. All data analysis will be completed by personnel who are blinded to the intervention assignment. Participants will be unblinded after the 12-month assessment.

The codes will only be broken in an emergency, such as a severe adverse event that requires knowledge of the treatment being taken in order to manage a participant’s condition. The principal investigator and ethics committee will be informed within 24 hours of the code-break envelope being opened.

Intervention

Compliance

Compliance to the exercise programmes will be assessed using attendance records and for the multimodal group, monthly exercise cards collected from the exercise trainers. Each exercise card contains information regarding sessions attended, exercises completed, set, repetitions, time and training intensity. A compliance calendar showing each month will be provided to assess supplement compliance. Participants will mark off each day the supplement is consumed and record any reasons for variations (ie, missed supplement or taken later in the day). Participants who are non-compliant with the intervention will still be requested to attend all follow-up appointments.

Adverse events

Any adverse events potentially or confirmed to be associated, with the exercise programme will be recorded on the exercise cards provided to the exercise trainers. The supplement calendar will be used to record adverse events potentially due to the supplement. Any health-related unfavourable or unintended medical occurrence (eg, sign, symptom, syndrome and illness), deemed to be relevant to the study, that develops or worsens during the trial will be classed as an adverse event. All adverse events will be assessed for seriousness, severity, causality and expected outcome by the researchers. There will be no formal data monitoring committee, due to the size of the study and the single site; however, all data will be reviewed by the research team at regular intervals throughout the study.

Sample size calculation

To date, no studies have investigated the cognitive effects of the specific intervention combination examined in the current study. For this reason, the anticipated effect size is largely based on findings derived from the exercise literature. Exercise exerts an average 0.5 SD effect size (equating to a medium effect size) on executive function.5 A recent meta-analysis has indicated that multimodal exercise interventions of at least 4 weeks’ duration have a positive effect on cognition in the order of a 0.33 effect size, in people over the age of 50 years.10 Based on these figures, we anticipate at least a medium effect size (f=0.25). Allowing for 15% attrition rate, a sample of 148 participants, with 74 individuals assigned to each study arm, will provide a power level of 0.8 at the 5% probability level.

Procedure

Participants will attend a total of four visits to Deakin University in Melbourne to undergo study assessments (see figure 1). All participants will be requested to abstain from alcohol for 24 hours prior to study visits. No caffeine is to be consumed 2 hours prior to study visits, and participants will consume the same food and drink at the baseline (V1) and 6-month (V2) visits. Baseline and 6-month visits will be held at the approximate same time of day (within 2 hours).

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Figure 1

Flow diagram: participant recruitment and screening flow chart.

• Prescreening: all participants will undergo telephone screening.

• Screening (V0): potential participants who meet the prescreening study criteria will attend a face-to-face session with a researcher to provide written informed consent and undergo screening for inclusion/exclusion criteria. Eligible individuals will undergo familiarisation of core study measures and will be provided with a physical activity monitor and pathology collection requests to complete blood collection prior to V1. Participants requiring medical approval must obtain this prior to baseline.

• Baseline (V1): to be conducted within 4 weeks of the V0 screening visit. During V1, cognitive assessments will be carried out first, followed by cardiovascular measures, anthropometry, DXA, functional measures and questionnaires. On completion of V1, participants will be allocated to either the intervention group or control group and provided with the supplement and details of the relevant gym programme. Participants are requested not to commence additional omega-3 or vitamin supplementation during the trial or to take part in more than one additional structured exercise class per week.

• Intervention period: an unblinded study member will remain in contact with participants via telephone and face-to-face visits at the gyms and will collect exercise cards, including adverse event logs and supplement compliance logs. Possible adverse events, gym non-attendance and supplement non-compliance will be followed up by telephone by an unblinded researcher.

• 6 month follow-up (V2): participants will repeat physical activity monitoring and blood collection within the week prior to V2. All measures from baseline will be repeated along with the MaCA.

• 12-month follow-up (V3): due to funding constraints, only cognitive assessments including the Cogstate, Trail Making Test and MoCA and study questionnaires will be completed at V3.

Primary outcome

Change in global cognitive function derived from the CogState computerised battery and the Trail Making Test after 6 months.

Secondary outcomes

Changes in cognitive function for the six individual measures and the composite scores of working memory/learning and attention/psychomotor function assessed using the CogState battery, the Trail Making Test, the MoCA after 6 months and 12 months, and changes in mood, health-related quality of life, strength, physical function, body composition, cardiovascular health and sleep quality after 6 months. A list of outcomes and time points are shown in table 2.

Screening measures

The self-administered 15-item Short Form Geriatric Depression Scale (GDS)59 will be used to screen for the presence of depression at V0. Scores greater than 5 potentially indicate depression and those who score >5 will not be eligible for the study. The interviewer administered MoCA60 will be used to screen for the possible presence of dementia. The MoCA is scored out of 30 and assesses short-term memory, visuospatial ability, executive function, verbal fluency, attention and working memory and orientation to time and place and is summed to create a global measure of cognition. Participants require a score >17 to be eligible for the study. The MoCA has been shown to have 90% sensitivity and 87% specificity for detecting MCI (n=94) and 100% and 87%, respectively, for detecting early AD.60 The MOCA will be used for screening purposes at V0 and repeated as a secondary outcome after 6 months (V2) and 12 months (V3).

Primary outcome measures

Secondary outcome measures

Demographic variables and potential covariates

Demographic and lifestyle information will be collected on participant’s ethnic and educational background, employment history/status, medical history, history of falls and fractures, family history of osteoporosis, current medication and dietary supplement use, alcohol intake, weekly television viewing and sitting time and sun exposure habits.

Statistical analysis

Data will be analysed using SPSS V.25 and STATA V.15 applying standard statistical thresholds (p<0.05), corrected for multiple comparisons where appropriate. There will be no interim data analysis. All data will be checked for normality prior to analysis. Data will be analysed based on an intention-to-treat approach using linear mixed models with random effects. Changes in the primary outcomes (CogState global cognitive function and Trail making test of executive function), and all secondary outcomes will be expressed as the absolute or percentage difference for the change between the intervention group and control group after 6 months or 12 months, as appropriate. For the cognitive outcomes, the results will be analysed adjusted for age, gender and years of education (model 1) as well as baseline MoCA score and ApoE status (model 2). Per-protocol analysis will be used to determine the effectiveness of the intervention among participants who are considered to be compliant with the intervention. Participants who attended 67% (two-thirds) of exercise sessions and consumed 80% of the allocated supplement will be classified as adherent for the purpose of completer analysis. All data will be presented as mean±SD or 95% CIs.

Ethical approval, data management and dissemination plan

The current protocol is based on version 4, April 2017. The first participants were enrolled into the study on 4 April 2017. In line with the ethical approval for this study, all data will be de-identified and stored in a locked filing cabinet or on a secure data drive at Deakin University. Only members of the research team will have access to the data. Only study investigators will have access to the final data set. Any changes to the trial protocol will be reported to Deakin University Human Research Ethics Committee and updated on the clinical trial registry. On completion of the trial, participants will receive a report outlining major study findings. They may also request pathology results. Study investigators will disseminate group results to the public in the form of journal articles, conference presentations, seminars and community presentations.

Patient and public involvement

Participants were not explicitly involved in the development of the trial. Feedback will be sought from all participants at the end of the study to assess burden of intervention and to help develop future trials. Participants will receive individual result summaries of key measures and a summary of the overall study findings.