Association between diabetes and cognitive function at baseline in the Brazilian Longitudinal Study of Adult Health (ELSA- Brasil)

Diabetes has been associated with cognitive changes and an increased risk of vascular dementia and Alzheimer’s disease, but it is unclear whether there are associations between diabetes and early alterations in cognitive performance. The present study consisted of a cross-section analysis of 14,444 participants aged 35–74 years and from a developing country at baseline in the Brazilian Longitudinal Study of Adult Health (ELSA–Brasil); these participants were recruited between 2008 and 2010. We investigated whether there was an association between diabetes and early changes in the cognitive performance of this Brazilian population. To assess cognitive domains, we used the word-list learning, word-list delayed recall and word recognition tests along. Phonemic verbal fluency tests included semantic phonemic test (animals) and a phonemic test (words beginning with the letter F). Executive functions associated with attention, concentration and psychomotor speed were evaluated using the Trail Making Test B. The exposure variable in the study was defined as diabetes. Multiple linear regression was used to estimate the association between diabetes and cognitive performance. The results were adjusted for age, sex, education, hypertension, coronary disease, depression, physical activity, smoking, alcohol consumption, and the cholesterol/HDL-C ratio. We found a significant association between diabetes and decreased memory, language and executive function (attention, concentration and psychomotor speed) performance in this population from a country with a distinct epidemiological profile, even after adjusting for the main intervening variables.


Material and Methods
Study population and ethics. The  . The purpose of the ELSA-Brasil study was to investigate the risk factors and incidence of chronic diseases, especially diabetes and cardiovascular diseases 15 . The data collection was performed via face-to-face interviews, anthropometric measures, arterial pressure and laboratorial tests in the six research centres 16 .
The baseline ELSA-Brasil cohort included 15,105 individuals aged between 35 and 74 years; 54.3% were women, 17.3% had diabetes, 35.4% had hypertension, 22.9% were obese, and 40.2% were overweight. In the present analysis, 184 participants were excluded due to a previous history of stroke; 330 were excluded due to their use of antiepileptic medications, neurotropic agents, or psychotropic agents that could interfere in the cognitive tests; and 108 were excluded due to a glycated haemoglobin (A1c) level below 4.0%. We excluded 39 patients with type 1 diabetes. The remaining final sample size was 14,444 individuals (Fig. 1).
All participants signed the free informed consent form from the ELSA-Brasil study. This study was approved by the Ethics and Research Committees of all institutions, including the Research Ethics Committee of the Federal University of Minas Gerais, Belo Horizonte (ETIC 186/06), and the National Research Ethics Committee. This study was approved by the National Commission of Ethics in Research (CONEP), and all methods were conducted according to the relevant guidelines and regulations of this cohort 16 . Diabetes (explanatory variable). Samples were collected after a 12-hour fast, stored in a freezer at −80 °C and sent to the certified central laboratory in São Paulo. An oral glucose tolerance test (OGTT) was administered to all participants without a known diabetes diagnosis [15]. Glycaemia was measured using the enzymatic colorimetric method (ADVIA 1200 Siemens Deerfield, IL, USA), and glycated haemoglobin A1c was measured using high-pressure chromatography (HPLC -Bio-Rad Laboratories, Hercules, CA, USA). Diabetes was defined as A1c ≥ 6.5% (48 mmol/mol), fasting glycaemia ≥ 126 mg/dL (7.0 mmol/L), or OGTT ≥ 200 mg/dL (11.1 mmol/L), according to the American Diabetes Association (ADA) criteria; by insulin and antidiabetic drug use; or by the self-reported medical diagnosis of diabetes.
covariates. We investigated and categorised sociodemographic variables such as age, sex and education.
Age was grouped as follows: 35 to 44, 45 to 54, 55 to 64 and ≥65 to 75 years old. Education was divided into four sub-groups: <8 years (incomplete fundamental school), eight years (complete fundamental school), 9-11 years (high school) and ≥12 years (university degree) 22 . Leisure time physical activity was assessed using the long modified version of the International Physical Activity Questionnaire (IPAQ) and was categorised as low, moderate and high 23 . Smoking status was categorised as never, former, and current smoker. Alcohol use was categorised as low (weekly consumption ≤175 g), moderate (176-350 g weekly consumption) and high (>350 g weekly consumption).
To assess adiposity, we used anthropometric data and applied standardised techniques and equipment. Weight (kg) and height (m) were measured with the participant barefoot and wearing light clothes using a Toledo ™ scale (accuracy 100 g) and a stadiometer (accuracy 0.1 cm), respectively. Body mass index was also determined [weight (kg)/height (m 2 )]. Abdominal circumference (midpoint between the last rib and the iliac crest) was measured using an inelastic tape (cm), and the average of two measures was used in the analysis. The measures were confirmed by trained and certified study personnel according to the standards of the project 24 . Arterial hypertension was defined by a self-reported medical diagnosis of hypertension, use of anti-hypertensive drugs, or blood pressure ≥140/90 mmHg at the time of evaluation (sum of two measures). Coronary heart disease was defined as a history of myocardial revascularization and/or myocardial infarction. Depression was defined by the use of antidepressant drugs at the time of the interview.
Total cholesterol was determined by the enzymatic colorimetric method; high-density cholesterol (HDL-C) was determined by the homogeneous colorimetric method without precipitation, and triglycerides were determined by glycerol-phosphate peroxidase (ADVIA 1200 Siemens Deerfield, IL, USA). We used the total cholesterol/HDL-C ratio as a covariate 25 . Statistical analysis. The statistical analysis was accomplished using STATA ™ software (Stata Corporation, College Station, TX, USA), v. 14.0. The categorical variables were described as frequencies and percentages. The continuous variables were described by the means and standard deviations (SDs) or medians and interquartile ranges, according to the normality of data definition, which was tested in histograms, and coefficients of asymmetry (skewness) and kurtosis. In the univariate analysis, Pearson and Spearman's correlations were used for the continuous variables with normal and non-normal distributions, respectively, to assess the correlation between the potential factors and cognitive performance. To compare means and medians from the cognitive tests among the potential associated factors, we used Student's t-test, the Mann-Whitney test, ANOVA or the Kruskal-Wallis test. All the variables with p < 0.20 in the univariate analysis were pre-selected for inclusion in the multivariate analysis. The Trail Making Test B had a skewed distribution; therefore, we used the natural logarithmic transformation (log-transformed).
Multiple linear regression (forward) was applied for response variables memory test, verbal fluency test (animals and letter F), and log-transformed Trail Making Test B to estimate the association between cognitive performance and diabetes (yes/no). The data were adjusted for age (categorical), sex and education in model 1; for all model 1 variables plus hypertension, coronary disease, and depression in model 2; and for all model 2 variables plus physical activity, smoking, alcohol use, and total cholesterol/HDL-C ratio in model 3.

Results
There were 14,444 participants in this study; 54% were female, and the mean age was 52.0 years (±9.1). The education level was high; 52.8% of the participants had a university degree. The characteristics of the participants are depicted in Table 1: 76.7% were sedentary, 57.0% never smoked, 63.1% were overweight (BMI 25.0-29.9 kg/m 2 ), and 22.8% were obese (BMI ≥ 30.0 kg/m 2 ). In the univariate analysis, there was an association between a worse cognitive performance in all cognitive tests and the highest age group, decreased schooling and smoking. The variables with p < 0.2 in the univariate analysis were maintained in the multivariate analyses: hypertension, physical activity, alcohol use, coronary disease, and the total cholesterol/HDL-C ratio. Table 2 shows the result of the linear regression between the memory tests (learning, recall and word recognition tests) and diabetes. In the final model, we found that a lower cognitive performance was associated with diabetes. The results also showed better cognitive performance in women, and poor cognitive performance was associated with increased age, decreased schooling, smoking, moderate consumption of alcohol, and increased total cholesterol/HDL-C ratio. In Tables 3 and 4, we present the results of the linear regression analysis between verbal phonemic fluency (letter F), semantic phonemic test (animals) and diabetes. In this executive domain, the final model shows a worse cognitive performance with diabetes. Decreased cognitive performance was associated with increased age, among men, with decreased education, hypertension, depression, smoking and decreased physical activity levels.
Therefore, diabetes was associated with a decreased cognitive performance in the memory tests (learning, recall and word recognition tests) (β −0.53 [−10.77; −0.29] adjusted R 2 0.21). The verbal fluency test (letter F) performance was decreased among patients with diabetes (β −0.50 [−0.69; −0.31] adjusted R 2 0.18) and semantic phonemic test (animals) also was decreased among patients with diabetes (β −0.27[−0.50; −0,04] adjusted R 2 0.21), although the magnitude of the association decreased slightly after all adjustments. Finally, an association was found between diabetes and the Trail Making Test B (e β 1.03 [1.02; 1.06] adjusted R 2 0.30), after adjustments (Table 5). Therefore, in the final model, decreased cognitive performance was demonstrated in men, older individuals, patients with low education levels, hypertensive individuals and those who smoke.

Discussion
At baseline in this large Brazilian cohort with relatively young participants (52.0 ± 9.1 years old), an association between cognitive performance in the domains of memory (learning, recall and word recognition tests), phonemic verbal fluency tests (letter F and animals), trail making and diabetes has been shown. This association occurred independently of education and other traditional risk factors (such as lipid levels), comorbidities (such as hypertension), and health-related behaviours (such as smoking). Previous cross-sectional and longitudinal studies have focused on the substantial epidemiological evidence suggesting that diabetes is associated with cognitive impairment 5,7 . What is unclear is whether there is a specific pattern for impaired function in terms of the affected cognitive domains 26 . Many researchers have described an association between diabetes and cognitive performance in studies in Asia, North America and Europe 2,10,25,27,28 . However, the majority of studies in developed countries have been conducted in cohorts of patients over 60 years old and focused on cognitive diagnoses of conditions such as mild cognitive impairment and dementia 2,4,10,28 .
The influence of diabetes on cognitive performance among young and middle-aged adults, such as the participants in the ELSA-Brasil cohort at baseline, is not well understood, especially in middle-and low-income countries. Our findings show associations between diabetes and cognitive performance in Brazilian individuals, where demographic and nutritional changes are increasing the prevalence of metabolic risk factors and threatening to accelerate the incidence of diabetes and neurodegenerative diseases 29 .
At the present study, diabetes had a significant impact on the performance of memory, phonemic verbal fluency and Trail B tests. In a prospective Dutch study, cognitive performance was measured twice over a five-year interval in 2,613 individuals aged 43-70 years at baseline (1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002). They evaluated changes in cognitive performance of individuals with previous or incident diabetes and compared global cognitive performance and specific domains of cognitive function (memory, speed, and cognitive flexibility) among individuals with and without type 2 diabetes. The results showed a decline in global cognitive function in individuals with diabetes that was 2.6 times greater than that in subjects without the disease. Interestingly, the magnitude of cognitive decline in most of the different domains was intermediate in subjects with incident diabetes in comparison with individuals without diabetes or individuals with diabetes that was prevalent at the study baseline. The results of this study seem to indicate that diabetes affects different domains of cognitive functioning at different stages of the disease process 30 .
Previous analysis of this Brazilian cohort has found that education plays a greater role than age in performance on cognitive tests 31 . High education levels were the strongest predictor of maintained cognitive function according to different authors 32,33 . In this study, the impact of education on cognitive performance remained higher than the impact of diabetes, age, and other characteristics.  www.nature.com/scientificreports www.nature.com/scientificreports/ In the final model relating diabetes and memory tests (learning, recall and word recognition tests), phonemic verbal fluency tests (letter F and animals) and the Trail Making Test B, women presented with better cognitive performance than men, some resemblance to the results found in a German study with middle-aged participants 34 . We found an association between diabetes and cognitive performance, mainly in the domains of memory, attention, concentration, psychomotor speed and executive function. A meta-analysis examining the nature and magnitude of cognitive deficits in individuals with type 2 diabetes was performed to determine the magnitude of the Cohen effect (d) on the cognitive dysfunction of individuals with or without diabetes. The Cohen effect is characterised by differences in the standardised means between the experimental and comparison groups, divided by the standard deviation. The effect size "d" is characterised as small (0.2-0.3), medium (0.4-0.7) and large (≥0.8) 35  To date, insulin has been considered to have neuromodulatory effects that promote the plasticity of synapses 37 . The impairment of insulin signalling, the presence of chronic inflammation and hyperglycaemia, the accumulation of advanced glycation end-products (AGEs) and increases in oxidative stress play an essential role in the pathogenesis of both diabetes and Alzheimer's disease 38,39 . Inflammatory cytokines produced by macrophages and adipocytes may cross the blood-brain barrier and activate stress kinases, inducing insulin resistance. Moreover, amyloid β protein oligomers can indirectly contribute to brain insulin resistance through the microglial induction of kinases. Neuronal insulin resistance impairs synaptic function and contributes to the neurodegenerative process 40 . In a sample of 998 non-diabetic participants from a subanalysis of the ELSA-Brasil study, an association between a cluster of selected inflammatory biomarkers and poor cognitive scores was also demonstrated in middle-aged women 41 . Micro-and macrovascular disease and unstable metabolic control in individuals with diabetes, including severe hypoglycaemic events, are also critical for cognitive changes 1,12 .
Furthermore, the loss of brain volume has been described in patients with diabetes, particularly in regions of the hippocampus, thalamus, and cerebellar area 42 . Meta-analysis identified reduced resting-state brain activity in all brain regions in individuals with type 2 diabetes 43   www.nature.com/scientificreports www.nature.com/scientificreports/ be one of the main complications of diabetes, according to the current recommendations for good practice in the care of individuals with diabetes, it has become important to perform cognitive screening or cognitive assessment of these individuals 13,44,45 .
The strengths of this study include the large sample, methodological rigor in data collection, centralised analysis of the laboratory tests and quality assurance control. The use of this brief battery of neuropsychological tests was standardised and validated for the Brazilian population, thus increasing the reliability of the results 17,46 . In addition, a large set of covariables was evaluated, which allowed adjustment for a wide range of possible confounding factors. However, this study has some limitations. By its cross-sectional design, one cannot infer any causal relationship between cognitive performance and the associated variables. The investigation of the same variables in the follow-up of this cohort may add additional enlightening information. The ELSA-Brasil cohort has a much higher percentage of people with high education levels than the Brazil population. However, it is comparable to the cohorts of other international studies 47 . Moreover, we were unable to evaluate all cognitive tests for each domain, as is the case with large longitudinal studies of adult health in the world. Unfortunately, this comprehensive assessment is logistically impossible in the vast majority of cases.
Although Brazil has a distinct epidemiological profile, the observed associations between diabetes, cognitive performance and metabolic risk factors are similar to those observed in cohorts in developed countries 48,49 . Preventive strategies may be more effective to avoid the worsening of cognition functions in these high-risk individuals 50 . Our data demonstrate that even a brief cognition evaluation is important in assessing the impact of diabetes on the mental health of this population, which may be of great relevance to many similar low-and middle-income countries. The ageing of our population will generate sharp increases in the number of older adults living with diabetes and, possibly, comorbid cognitive impairment. It is estimated, therefore, that the impaired treatment adherence and diabetes self-care represent a major challenge for future health systems around the world, particularly for those with fewer resources 50 .
In conclusion, we found a significant association between diabetes and cognitive function test performance in this relatively young and highly educated Brazilian population.

Data availability
The datasets generated during the current study are available from the corresponding author upon reasonable request.  Model 3 -Model 2 plus physical activity, smoking, alcohol use, total cholesterol / HDL-C. *p < 0.05; **p < 0.01; ***p < 0.001 Final models included variables with p < 0.05.