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JDR2016 3978428 PDF
JDR2016 3978428 PDF
Research Article
Brain Activation during Memory Encoding in Type 2 Diabetes
Mellitus: A Discordant Twin Pair Study
Copyright © 2016 Amanda G. Wood et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Type 2 diabetes mellitus increases the risk of dementia and neuronal dysfunction may occur years before perceptible cognitive
decline. We aimed to study the impact of type 2 diabetes on brain activation during memory encoding in middle-aged people,
controlling for age, sex, genes, and early-shared environment. Twenty-two twin pairs discordant for type 2 diabetes mellitus (mean
age 60.9 years) without neurological disease were recruited from the Australian Twin Registry (ATR) and underwent functional
magnetic resonance imaging (fMRI) during a memory encoding task, cognitive tests, and structural MRI. Type 2 diabetes was
associated with significantly reduced activation in left hemisphere temporoparietal regions including angular gyrus, supramarginal
gyrus, and middle temporal gyrus and significantly increased activation in bilateral posteriorly distributed regions. These findings
were present in the absence of within-pair differences in standard cognitive test scores, brain volumes, or vascular lesion load.
Differences in activation were more pronounced among monozygotic (MZ) pairs, with MZ individuals with diabetes also displaying
greater frontal activation. These results provide evidence for preclinical memory-related neuronal dysfunction in type 2 diabetes.
They support the search for modifiable later-life environmental factors or epigenetic mechanisms linking type 2 diabetes and
cognitive decline.
Genetics and early-shared environment play important drawings, or fixation, presented for three seconds each) were
roles in determining later-life brain structure [9, 10], cog- shown in the centre of the screen. For blocks of words,
nition [11], and fMRI activation [12]. Therefore, accounting participants were instructed to indicate via a button press
for confounding by genes and early-shared environment is whether the item was living or nonliving, to encourage
particularly important when isolating the effects of type 2 encoding via a semantic route, which improves encoding
diabetes on the earliest neural signs of impending cogni- [17]. Similarly, the objects depicted included animate and
tive decline, and this has not been achieved in the few inanimate items and participants indicated whether these
previous functional imaging studies in people with type 2 were living/nonliving via button press. There was an equal
diabetes. Studying cotwins who are discordant for type 2 number of living and nonliving items within each block,
diabetes provides powerful control for such confounders. presented in fixed random order. The resting block showed
Thus, we compared fMRI activation during implicit memory a fixation cross that varied in size. Participants indicated via
encoding, as well as cognitive function and brain structure button press whether the cross was large or small and were
within middle-aged twin pairs discordant for type 2 diabetes. given practice outside the scanner before the session to ensure
We hypothesized that diabetes-affected individuals would that they understood the task requirements. Each block lasted
have altered brain activation during a memory encoding 30 seconds, giving a total session length of ten minutes
paradigm, poorer cognitive function, and altered brain struc- and thirty seconds. The design of our experiment controlled
ture compared to their cotwins without diabetes. for unwanted effects of motor response, decision-making,
and visual stimulus. Participants were told that the task was
designed to examine brain activity for different stimuli types
2. Methods and were not advised to memorize the items. They were
not informed in advance of the later recognition memory
2.1. Sample. The sample was derived from the Australian
test. The incidental nature of the memory encoding phase
Twin Registry (ATR), a nation-wide volunteer agency funded
avoids directing the participant to use a particular strategy,
by the Australian National Health and Medical Research
which might account for interindividual differences in neural
Council (NHMRC). We invited participants (monozygotic
response. Thirty minutes after fMRI acquisition, participants
and dizygotic) with type 2 diabetes (aged ≥50 years) and
were presented with a surprise recognition memory task
their discordant pairs without diabetes from a cohort in
on a laptop computer with previously presented stimuli
which type 2 diabetes was confirmed with a high degree of
mixed with 35 previously unseen items (“foils”). In this
accuracy by a diabetologist [13]. Fasting finger-prick glucose
phase, participants were instructed to respond whether they
levels was used as an additional measure of phenotype
remembered seeing the stimulus during the scanning sessions
validation in our study, and fasting glucose <7.0 mmol/L
or whether it was new. Items correctly recognized in this
was used to confirm absence of diabetes in the cotwin [14];
test were assumed to reflect accurate encoding and were
HbA1c levels were not available. Zygosity was established
considered as outcomes in subsequent paired fMRI analyses.
by the ATR using standard responses to questions shown
to have 95% accuracy for such a purpose [15]. Exclusion
criteria were a history of significant neurological disease 2.3. Cognitive Assessment. These tests were performed after
including stroke/TIA, seizures, dementia, Parkinson’s disease, fMRI and were selected for their sensitivity to early cognitive
and severe head trauma. This study was approved by the decline in dementia. We screened general intellectual abilities
Monash University Human Research Ethics Committee and with the National Adult Reading Test-Revised (NART-R)
written informed consent was obtained. [18]; basic attention processing skills with the Mental Control
and Digit Span subtests from the Wechsler Memory Scale
3 (WMS3) [19]; Memory with Hopkins Verbal Learning
2.2. MRI Acquisition. MRI scanning was performed on a
Test-Revised [20]; short (5 minute) recall for delayed Rey-
single Siemens Trio Tim 3-Tesla (3T) MRI at the Murdoch
Osterrieth Complex Figure [20]; Paired Associate Learning
Childrens Research Institute, Melbourne, Australia. Struc-
subtest from the Wechsler Memory Scale (WMS-1). In addi-
tural MRI (sMRI) sequences: these included high resolution
tion, we used the Cambridge Neuropsychological Automated
T1-weighted images (0.9 mm isotropic, TR =1800 ms, TE =
Test Battery (CANTAB) [21] to measure basic and choice
2.2 ms, Flip Angle = 9∘ , and FOV = 230) and T2/FLAIR
reaction time and visual Paired Associate Learning (VPAL), a
images (1 mm isotropic, TR = 6000 ms, TE = 405 ms, and
task which is highly sensitive to the early detection of AD [22].
FOV = 256); fMRI sequences: these included echoplanar
Paired 𝑡-tests were used to compare cognitive scores between
imaging (EPI) sequence (TR = 3000 ms, TE = 40 ms, slice
cotwins.
thickness = 3 mm no gap, FOV = 210, and N slices = 39)
with whole brain coverage using an incidental memory task,
based on a well-established mixed-design [16]. The person 2.4. Image Analyses. Preprocessing was conducted blind to
administering the task could not be completely blinded age, sex, diabetes status, clinical or cognitive measurements,
to diabetes status but was completely unaware of specific and zygosity.
study hypotheses. The fMRI paradigm was delivered via
Presentation software (Neurobehavioral Systems Inc.). The fMRI Analysis. We analyzed fMRI memory blood oxygen-
experiment comprised seven cycles of task and rest. For level dependent (BOLD) activation with FSL 4.1.4
each cycle, three blocks of ten stimuli (words, simple line (FMRIB Software Library, The University of Oxford) using
Journal of Diabetes Research 3
the standard analytic pipeline. Following brain extraction matter, and hippocampal volumes using in-house voxel-
with BET [23], linear registration was performed using counting algorithms. Paired comparison of global volumes
T1 images. EPI scans of each subject were motion and was followed by paired voxelwise comparison of regional
slice-timing corrected, normalized to the standard Montreal gray and white matter distribution, adopting a family-wise
Neurological Institute (MNI152) template in stereotaxic error (FWE) corrected 𝑝 < 0.05. In addition, restricted
coordinate space, and smoothed using a 5 mm isotropic paired region-of-interest (ROI) comparisons were made by
Gaussian kernel. After high-pass filtering (cut-off 100 s), applying the hippocampal mask derived from the Automated
data were convolved using Gamma function with temporal Anatomical Labelling AAL atlas [26].
derivative and analyzed using a general linear model. We
used event-related analyses to compare encoding-related Cerebrovascular Lesions. All images were reviewed by two
activation responses for individual stimuli (drawings and stroke experts (Thanh Phan, Velandai Srikanth) to iden-
words) that were correctly recognized versus activation tify brain infarcts defined as a hypointense lesion with a
during the baseline condition (cross-hairs) for each hyperintense rim on FLAIR measuring greater than 3 mm.
participant. Group-level analysis across the whole brain WML were manually segmented on FLAIR images by a single
was first performed to characterize the pattern of activation trained expert using established in-house methods with high
during memory encoding in the T2DM affected and test-retest reliability (intraclass correlation coefficient, ICC
unaffected twins using 𝑧 > 2.3 and cluster family-wise error 0.98; 95% CI; 0.97–0.99) [27].
(FWE) corrected 𝑝 < 0.05. Where stated, we controlled for
relevant health-related variables (e.g., waist circumference) 2.5. Other Measurements and Analyses. Standardized, struc-
by entering data in FSL during higher-level group analyses. tured questionnaires were used to record educational qual-
The study hypotheses were tested by first examining ifications, self-reported medical history including hyperten-
differences in activation across the whole brain, within sion, ischemic heart disease, stroke, hyperlipidemia, smok-
twin pairs by paired-𝑡 test of the contrast images. We also ing, alcohol consumption, and prior head injury, medication
used mesial temporal region-of-interest analysis to examine use, a measure of current mood with the Geriatric Depression
BOLD activation within the hippocampi based on an in- Scale (GDS) [28], and age of onset of diabetes. Resting arm
house hippocampal mask generated by the average of manual blood pressure, waist and hip circumferences, height, and
segmentation of the hippocampi in the 22 pairs, as well weight were obtained using standardized protocols.
as hippocampal and parahippocampal masks derived from
an automated anatomic labelling scheme. Standard analyses
using the general linear model were first conducted in the 3. Results
whole sample and then repeated after stratifying for zygosity
and controlling for sex (except among MZ), history of Sample characteristics are provided in Table 1. There were
hypertension, and waist circumference by including them as 22 discordant twin pairs with mean age of 60.9 years (SD
variables of no interest. We additionally explored the rela- 6.7), of whom 8 pairs were monozygotic (MZ, mean 60.6
tionship of duration of diabetes and waist circumference on years; SD 7.5) and 14 pairs were dizygotic (DZ, mean 61.5
regional activation among those with type 2 diabetes alone, years; SD 5.6). Overall duration of diabetes in affected
controlling for age, sex, and hypertension. This regression individuals was 10.9 years (SD 9.9). In paired comparisons,
analysis was performed on a voxelwise basis, across the whole those with type 2 diabetes had higher fasting blood glucose
brain, corrected for multiple comparisons (𝑍 > 2.3, corrected levels, waist circumference (hence weight), and self-reported
(cluster) 𝑝 = 0.05). A final, exploratory, conjunction analysis frequency of hypertension and were more likely to be on
using the minimum statistic [23] was used to examine cholesterol and blood pressure lowering medication than
whether activation differences between discordant pairs were their cotwin (all 𝑝 < 0.05). Among those with diabetes,
similar for monozygotic and dizygotic twins. most (20/22, 91%) were on oral antidiabetes medication
and few (4/22, 18%) were on insulin. Five (23%) people
sMRI Analysis. An optimized voxel-based morphometry reported sensory symptoms in their lower extremities, and
(VBM) method was implemented in statistical parametric four (18%) reported visual problems; all four cases were tested
mapping software version 8 (SPM 8, Functional Imaging prior to scanning and confirmed as being able to view the
Laboratories, Institute of Neurology, London) [24]. Auto- in-scanner stimuli satisfactorily. Individuals with diabetes
mated segmentation of brain from nonbrain structures was among the MZ group had greater mean waist circumference
conducted followed by coregistration of the brains of all (114.3 cm versus 102.8 cm; 𝑝 = 0.009) than their counterparts
participants to the MNI152 template. VBM detects changes among the DZ group but showed similar mean fasting
in the regional concentration of gray matter after correcting glucose (7.4 mmol/L versus 6.7 mmol/L; 𝑝 = 0.28), levels
for global differences in brain shape. The optimized method of all other clinical/demographic variables, and duration of
includes recursive segmentation and spatial normalization diabetes (data not shown). No participant reported other
and a modulation step to allow comparison of tissue vol- serious neurological disorders and all were independently
umes. For region-of-interest analysis, hippocampal bound- living in the community.
aries were identified according to previously defined and val-
idated anatomical landmarks and manually segmented by a 3.1. Cognitive Function. There were no significant within-
single expert [25]. We derived whole brain gray matter, white pair differences in predicted full scale intelligence quotient
4 Journal of Diabetes Research
(NART-R FSIQ) or any other cognitive test either in the in response to the task as well as a typical pattern of
whole sample or within zygosity subgroups (Table 2). There suprathreshold activation. As expected, BOLD activation for
were no significant within-pair differences in the number correctly encoded items during the incidental memory task
of correctly recognized (i.e., encoded) items administered was observed in the mesial temporal region as well as a
during the fMRI scanning session. distributed network of brain regions (Figure 1: 𝑍 > 2.3 and
corrected cluster level threshold of 𝑝 = 0.05).
3.2. fMRI BOLD Activation. To confirm that the paradigm
performed in the expected manner, we conducted a group 3.2.1. Within-Pair Comparisons among Discordant Twins.
analysis across the entire sample of twins without diabetes Reduced activation during encoding was found in type
(i.e., ND). The analysis was performed only in ND because 2 diabetes in the following regions (Figure 2 (blue areas)
this group should demonstrate normal BOLD signal change and Table 3): (a) all pairs: left hemisphere temporoparietal
Journal of Diabetes Research 5
5
R L
5
x = 69 y = 32 z = 49
2.3
5
x = 48 y = 37 z = 63
2.3
Table 3: Regions (and 𝑥, 𝑦, and 𝑧 coordinates) associated with Table 4: Regions associated with increased activation in type 2
reduced activation in type 2 diabetes. diabetes.
such as carriers of apolipoprotein E4 (ApoE4) [33] or the recent study has demonstrated differences in histone deacety-
presenilin 1 mutation [34]. Such increases in activation may lase expression in brain between people with and without
also reflect a pathological loss of inhibition preceding deacti- T2D, and these differences correlate with altered expression
vation [35] or purely be a physiological response to decreased of synaptic proteins and consequent synaptic dysfunction
activation in other regions not necessarily due to neuronal [40].
loss [36]. There are limitations to our study that may be addressed
An interesting observation in our study was the greater in future work. The current analysis is cross-sectional, and
BOLD activation among MZ individuals with diabetes com- hence causal links cannot be proven. BOLD activation also
pared with their cotwin than that observed in DZ pairs, in does not clearly separate the effects of changes in blood flow
the presence of complete control for genetic variation and from actual neuronal metabolism but to counter this we
strong control for early-shared environment. Since frontal excluded those with stroke, and there was no within-pair
activation during a cognitive task has high heritability [12], difference in the volume of white matter lesions. The effect
it is conceivable that the presence of type 2 diabetes interacts of cerebrovascular lesions may however become more potent
with as yet undefined genotypes to influence activation in the with advancing age and severity of type 2 diabetes and a lon-
frontal cortex. These results raise the interesting possibility gitudinal and/or older cohort would help answer these ques-
that adult age environmental, lifestyle, or epigenetic factors tions. Further research using additional and concurrent func-
may influence diabetes-related neuronal loss/dysfunction. tional images methodologies (e.g., Arterial Spin Labelling,
Central obesity, which is closely related to adverse lifestyle, hypercapnia experiments, and neuronal metabolism using
was greater among MZ individuals with diabetes in our study FDG-PET) may assist in further disentangling neurovascular
and is known to be associated with a greater risk of dementia mechanisms underlying altered cortical function in type 2
[37] and brain atrophy [38], and its proinflammatory or diabetes. Furthermore, cerebrovascular disease caused by
metabolic effects on the brain may conceivably explain the diabetes may itself influence the BOLD response during
stronger findings among MZ diabetics. However these results cognitive tasks. For example, prescan blood glucose may
did not survive correction for multiple testing, and therefore influence neuronal metabolism as measured by positron
any interpretation is speculative. The regions of increased emission tomography (FDG-PET). However, its impact on
activation are similar to those found in other studies in BOLD-fMRI activation is less clear. Hypoglycemia-related
which compensation for early cognitive decline is mooted. changes in activation occur only at very low glucose levels
Indeed, there was also evidence of reduced activation in the (2.5 mmol) [41], and higher levels (9.6–18.6 mmol/L) had a
diabetic twin in functionally relevant areas (e.g., left middle negligible effect on BOLD activation during a visual stimulus
temporal gyrus). The reduction of temporal lobe activation [42]. The physiological range of blood glucose at screening
and concomitant increased activation in putative compen- in our study (4.6–10 mmol/L) and the lack of hypoglycemic
satory networks supports speculation that cognitive change symptoms in our subjects prior to scan suggest a low likeli-
experienced by older people with diabetes may be caused by hood for our results to be influenced by prescan glucose, but
neurodegeneration. An exploratory uncorrected conjunction we cannot definitively exclude this. We did not have sensitive
analysis showed overlap in activation patterns in MZ and DZ imaging measures of white matter microstructural integrity
twin pairs compared to their ND cotwins in these regions. or beta-amyloid binding or genetic markers of dementia risk
This suggests that this pattern of decreased and increased such as apolipoprotein epsilon 4 (ApoE4), and these have
activation is specifically related to the neural consequences the potential to provide greater mechanistic information and
of diabetes and therefore may represent a neural biomarker potentially link AD pathology to the observed differences.
of incipient cognitive decline. We emphasise that this analysis In summary, these results present evidence for dys-
requires replication in larger samples. functional neuronal network activity during episodic mem-
In addition to the common areas of activation or deac- ory encoding in type 2 diabetes. The strong control for
tivation in MZ and DZ twins within the broad network genes, age, sex, and early-shared environment in our study
of brain regions associated with memory encoding, there highlights a need to identify potentially modifiable later-
were a number of regions of activation that differed between life environmental/lifestyle-related or epigenetic factors that
MZ and DZ twins. We cannot exclude a role for glucose promote or modify the adverse effect of diabetes on neuronal
in the MZ-DZ differences even though blood glucose was health.
the same between MZ and DZ diabetics, because we did
not have data for HbA1c, a marker of longer-term glucose-
control. There are potentially several other nongenetic factors Disclosure
of interest that we were unable to measure that could also
The funding sources had no role in design and conduct of the
explain our findings. Apart from white matter lesions, we did
study; collection, management, analysis, and interpretation of
not have any objective measures of cerebral microvascular
the data; and preparation, review, or approval of the paper;
structure and function, aortic stiffening, physical activity,
and decision to submit the paper for publication.
and dietary patterns. There is emerging understanding of the
role of epigenetic modification of neuronal gene expression
in the ageing brain and consequent impairment of neu- Competing Interests
ronal function [39], and it would be of interest to study
whether type 2 diabetes is associated with such changes. A The authors declare that they have no competing interests.
Journal of Diabetes Research 9