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Multimodal imaging analyses of brain hippocampal formation reveals reduced Cu and lipid content and increased lactate content in non-insulin dependent diabetic mice Mark J. Hackett, Ashley Hollings, Maimuna Majimbi, Emily Brook, Blake Cochran, Corey Giles, Virginie Lam, Michael Nesbit, Kerry-Anne Rye, John C.L. Mamo, and Ryusuke Takechi ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00039 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 12, 2019
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ACS Chemical Neuroscience
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Multimodal imaging analyses of brain hippocampal formation reveals reduced Cu and lipid content
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and increased lactate content in non-insulin dependent diabetic mice.
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Mark J. Hackett1,2, Ashley Hollings1,2, Maimuna Majimbi1,3, Emily Brook1,3, Blake Cochran4, Corey Giles5,
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Virginie Lam1,6, Michael Nesbit1,6, Kerry-Anne Rye4, John C.L. Mamo1,6, Ryusuke Takechi1,6
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1Curtin Health innovation Research Institute, Faculty of Health Sciences, Curtin University, WA, Australia
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2Curtin
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of Science and Engineering, Curtin University, WA, Australia
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3School
Institute for Functional Molecules and Interfaces, School of Molecular and Life Science, Faculty
of Pharmacy and Biomedical Sciences, Faculty of Health Sciences, Curtin University, WA,
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Australia
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4School
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5Baker
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6School
of Medical Sciences, Faculty of Medicine, UNSW, NSW, Australia
Heart and Diabetes Institute, VIC, Australia of Public Health, Faculty of Health Sciences, Curtin University, WA, Australia
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Corresponding author: Associate Professor Ryusuke Takechi; GPO Box U1987, Perth, WA, 6164,
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Australia; Email:
[email protected]; Phone +61 8 9266 2607
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Abstract
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Non-insulin dependent diabetes mellitus (NIDDM) is reported to increase the risk of cognitive
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impairment and dementia. However, the underlying mechanisms are not fully understood. Whilst the
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brain homeostasis of metals and lipids are pivotal to maintaining energy metabolism and redox
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homeostasis for healthy brain function, no studies to date report hippocampal metal and biochemical
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changes in NIDDM. Therefore, we here utilized direct spectroscopic imaging to reveal the elemental
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distribution within the hippocampal subregions of an established murine model of NIDDM, db/db mice.
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In 26-week old insulin resistant db/db mice, X-ray fluorescent microscopy revealed that Cu content
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within the dentate gyrus and CA3 was significantly greater compared to the age-matched non-diabetic
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control mice. In addition, Fourier transform infrared (FTIR) spectroscopy analysis indicated significant
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elevation in the lactate abundance within corpus callosum (CC), dentate gyrus (DG), CA1, and CA3
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regions of diabetic db/db mice compared to the control, indicating altered energy metabolism. FTIR
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analysis also showed significant reduction of lipid methylene, and ester within CC of db/db mice.
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Furthermore, immunomicroscopy analyses demonstrated trending increase in glial fibrillary acidic
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protein expression and peri-vascular extravasation of IgG, indicating astrogliosis and blood-brain barrier
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dysfunction, respectively. These data suggest that astrogliosis-induced alterations in the supply of Cu,
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lipids and energy substrates may potentially be involved in the mechanisms of NIDDM-associated
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cognitive decline.
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Keywords: diabetes; brain elemental mapping; X-ray fluorescent microscopy; fourier transform infrared
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spectroscopy
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Introduction
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Non-insulin dependent diabetes mellitus (NIDDM) is an established risk factor for cognitive impairment
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and dementia including Alzheimer’s disease (2, 3). Cross-sectional studies reported that subjects with
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NIDDM have significantly lowered cognitive performance compared to age-matched healthy control
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subjects (4). In addition, magnetic resonance imaging (MRI) analyses of subjects with well-controlled
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NIDDM revealed marked reduction in cerebral white matter (5). Murine models of diabetic insulin
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resistance also demonstrated substantial neurodegeneration and cognitive decline (3). However, the
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mechanisms by which NIDDM induces neuronal atrophy and cognitive dysfunction have not been fully
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elucidated.
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Emerging evidence suggest that biochemical and elemental dyshomeostasis in the brain may facilitate
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oxidative stress through Fe and Cu redox cycling and neuroinflammation, which triggers or exacerbates
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neurodegeneration and cognitive decline. There is increasing interest in the field of neuroscience to
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research the roles of metal ions, which may influence neuronal function (6). Zn and Cu are reported to
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be involved in neuro-signalling (6-9), whilst a direct neuron-signalling role for Fe has not been proposed.
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The ions of Fe, Cu and Zn are demonstrated to influence and modulate neuronal function through other
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pathways, including free-radical driven redox changes (Fe and Cu), RNA binding, and modulation of
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neurotransmitter synthesis via acting is key enzyme co-factors (10). An accumulation of Fe and Cu ions
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in the brain is reported to catalyze free radical production via Fenton chemistry, mediating lipid
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oxidation and protein aggregation (10, 11). Indeed, altered abundance of redox-associated metals
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including Cu and Fe are commonly reported in the brains of neurodegenerative disorders such as
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Alzheimer’s disease (12). Furthermore, altered mitochondrial energy metabolism and lipid homeostasis,
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which is intrinsically linked to homeostasis of the ions of Fe, Cu, and Zn, is also demonstrated in the
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brains of Alzheimer’s disease (13). However, to date, no studies have investigated the elemental and
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metabolic homeostasis in the brains of NIDDM.
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The association between elemental homeostasis and biochemical markers of lipid homeostasis or
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metabolism is difficult to study with standard biochemical assays or histochemical analyses. We have
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previously applied a multimodal spectroscopic imaging approach to reveal association between
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biochemical and elemental alterations in the hippocampus of an accelerated ageing murine model,
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which build on previous work to characterise the baseline elemental composition of the rodent
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hippocampus (1, 14). Others have used a similar strategy to study the link of metal homeostasis or ionic
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disturbance with protein aggregation (15-19). Therefore, in order to identify potential mechanisms of
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cognitive decline and neurodegeneration in NIDDM, here we utilized the similar multimodal
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spectroscopic imaging techniques with synchrotron X-ray fluorescent microscopy (XFM) and Fourier
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transform infrared spectroscopy (FTIR) for the first time to analyse biochemical and elemental
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distribution within the hippocampal subregions at meso-/micro-spatial resolution in one of the most
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widely utilized pre-clinical NIDDM model.
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Results and Discussion
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The mean plasma concentration of glucose in db/db mice at 26 weeks of age was 26.652.06 mmol/L,
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which was significantly higher than the age-matched non-diabetic control mice (Table 1). Furthermore,
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plasma levels of insulin in db/db mice was over 10-fold higher than the control mice. Similarly, HOMA-
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IR index was significantly greater in db/db mice compared to the control, confirming that the db/db
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mice were severely diabetic.
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Table 1. Plasma measures
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Control db/db
glucose (mM) 13.5±0.94 26.65±2.06***
insulin (µg/L) 0.22±0.03 2.73±0.07****
HOMA-IR 3.34±0.53 80.03±5.84****
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*** p
