(W1230-04-24) Disruptions in Glucose and Amyloid-Beta Transport in Mouse Models Manifesting Metabolic Syndrome

Purpose: Studies in humans and diabetic mouse models have identified a correlation between high-fat diet (HFD) and Alzheimer’s disease (AD)-related pathologies, including insulin resistance [1], metabolic dysregulation, inflammation [2], accumulation of amyloid-beta (Aβ) in the brain and plasma [3], as well as cognitive impairment [4]. Glucose is transported from plasma to the brain via the blood-brain barrier (BBB). Moreover, the BBB also regulates Aβ levels in plasma and the brain. [5, 6]. Metabolic syndrome triggered by HFD is believed to disrupt these BBB functions. However, the underlying molecular mechanisms are poorly understood. Thus, we aimed to bridge these gaps by investigating how HFD impacts AD-related pathologies, explicitly focusing on the trafficking of Aβ peptides and glucose across the BBB.

Methods: To investigate the relationships and molecular mechanisms of HFD and BBB trafficking of Aβ peptides and glucose, we injected ¹²⁵I-Aβ40, ¹²⁵I-Aβ42, or ¹⁸F-FDG via the femoral vein to female wild-type (WT) mice fed either HFD or regular chow diet (RCD). The brain accumulation of these tracers was then monitored between 0-40 min by dynamic SPECT/CT or PET/CT imaging. The brain influx rate was estimated using the slope obtained from Gjedde-Patlak’s graphical analysis. Furthermore, cerebral microvessels were harvested from both RCD- and HFD-fed mice, and the expression of receptors/transporters was assessed using western blot analysis. To further investigate the effects of insulin resistance on cellular uptake of Aβ peptides and glucose, polarized human cerebral microvascular endothelial cells (hCMEC/D3) were stimulated with insulin with and without the exposure to insulin signaling inhibitors, specifically the MEK inhibitor trametinib (1 µM) or the AKT inhibitor MK2206 (12.5 µM). The cellular uptake of ¹²⁵I-Aβ40 and ¹²⁵I-Aβ42 (5 µCi/mL) was then measured using gamma counters, and the uptake of the glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) (440 µM), was measured using flow cytometry.

Results: The plasma pharmacokinetics of ¹²⁵I-Aβ peptides and ¹⁸F-FDG were not substantially differ between RCD- and HFD-fed mice. However, the plasma-to-brain influx rate of both ¹²⁵I-Aβ40 and ¹²⁵I-Aβ42 was significantly higher in HFD-fed mice compared to RCD-fed mice. Conversely, the plasma-to-brain influx rate of ¹⁸F-FDG was lower in HFD-fed mice. Western blot analysis revealed increased expression of the Aβ influx transporter RAGE and decreased expression of the glucose transporter GLUT1 in the cerebral microvessels harvested from HFD-fed mice compared to RCD-fed mice. Additionally, reduced levels of p-AKT and p-ERK were observed in HFD-fed mouse microvessels compared to those obtained from RCD mice. In BBB endothelial cell monolayers, inhibition of the insulin signaling pathway using either an AKT or MEK inhibitor increased the cellular uptake of ¹²⁵I-Aβ40 and ¹²⁵I-Aβ42 but reduced the uptake of 2-NBDG.

Conclusion: Our findings suggest that HFD-fed mice exhibited increased brain influx of Aβ and reduced influx of glucose. This was associated with altered expression of transporters and disrupted insulin signaling at the BBB. Furthermore, activation of AKT and ERK pathways was shown to regulate BBB trafficking of both Aβ and glucose. These pathways are disrupted in HFD-fed mice compared to RCD-fed mice.

References: 1. Hayden MR (2023) Overview and New Insights into the Metabolic Syndrome: Risk Factors and Emerging Variables in the Development of Type 2 Diabetes and Cerebrocardiovascular Disease. Medicina (Kaunas) 59
2. Walker JM, Dixit S, Saulsberry AC, May JM and Harrison FE (2017) Reversal of high fat diet-induced obesity improves glucose tolerance, inflammatory response, beta-amyloid accumulation and cognitive decline in the APP/PSEN1 mouse model of Alzheimer's disease. Neurobiol Dis 100:87-98.
3. Ramos-Rodriguez JJ, Ortiz-Barajas O, Gamero-Carrasco C, de la Rosa PR, Infante-Garcia C, Zopeque-Garcia N, Lechuga-Sancho AM and Garcia-Alloza M (2014) Prediabetes-induced vascular alterations exacerbate central pathology in APPswe/PS1dE9 mice. Psychoneuroendocrinology 48:123-135.
4. Bao J, Liang Z, Gong X, Yu J, Xiao Y, Liu W, Wang X, Wang JZ and Shu X (2022) High Fat Diet Mediates Amyloid-beta Cleaving Enzyme 1 Phosphorylation and SUMOylation, Enhancing Cognitive Impairment in APP/PS1 Mice. J Alzheimers Dis 85:863-876.
5. Cornford EM and Hyman S (2005) Localization of brain endothelial luminal and abluminal transporters with immunogold electron microscopy. NeuroRx 2:27-43.
6. Weiss N, Miller F, Cazaubon S and Couraud PO (2009) The blood-brain barrier in brain homeostasis and neurological diseases. Biochim Biophys Acta 1788:842-857.

Acknowledgements: This work was supported by the Minnesota Partnership for Biotechnology and Medical Genomics (MNP#15.31) and National Institutes of Health/National Institute of Neurological Disorders and Stroke (R01NS125437).

Figure 1. Higher plasma-to-brain uptake of 125I-Aβ peptides and lower plasma-to-brain influx rate of glucose in HFD-fed mice compared to RCD-fed mice. The Patlak plots of (A) 125I-Aβ40 and (B) 125I-Aβ42 in RCD- and HFD-fed mice. (C) The brain influx rates of 125I-Aβ40 and 125I-Aβ42 were estimated by the slope obtained from the Patlak plot (mean ± SD). Unpaired two-tailed student’s t-test (*p < 0.05, ***p < 0.001). (D) The Patlak plot of 18F-FDG in RCD- and HFD-fed mice. (E) The brain influx rate of 18F-FDG was estimated from the Patlak plot slope (mean ± SD). Paired two-tailed student’s t-test (**p < 0.01).

Fig 2. (A) Western blots representative blots are shown for Aβ influx transporter RAGE, glucose transporter (GLUT1), pAKT, tAKT, pERK, and tERK in cerebral microvessels harvested from RCD- and HFD-fed mice. (B-E) Bar chart of the quantification for (B) GLUT1/Vinculin, (C) RAGE/Vinculin, (D) pAKT/tAKT, and (E) pERK/tERK (mean ± SD). Unpaired two-tailed student’s t-test (*p < 0.05, **p < 0.01).

Fig 3. (A-C) Effects of MEK inhibitor (Trametinib) and AKT inhibitor (MK2206) on the expression of pAKT/tAKT and pERK/tAKT in BBB endothelial cell monolayers. One-way ANOVA (*p < 0.05; ****p < 0.0001, ns: not significant). (D-K) The Cellular uptake of (D&E) 125I-Aβ40, (F&G) 125I-Aβ42, and (H-K) 2-NBDG was assessed in hCMEC/D3 cell monolayers upon insulin stimulation following treatment with MEK inhibitor (Trametinib) and AKT inhibitor (MK2206) (mean ± SD). Unpaired two-tailed student t-test (*p < 0.05; ****p < 0.0001).