High Dietary Advanced Glycation End Products Impair Mitochondrial and Cognitive Function

Abstract

Background: Advanced glycation end products (AGEs) are an important risk factor for the development of cognitive decline in aging and late-onset neurodegenerative diseases including Alzheimer's disease. However, whether and how dietary AGEs exacerbate cognitive impairment and brain mitochondrial dysfunction in the aging process remains largely unknown.

Objective: We investigated the direct effects of dietary AGEs on AGE adducts accumulation, mitochondrial function, and cognitive performance in mice.

Methods: Mice were fed the AGE+ diet or AGE- diet. We examined levels of AGE adducts in serum and cerebral cortexes by immunodetection and immunohistochemistry, determined levels of reactive oxygen species by biochemical analysis, detected enzyme activity associated with mitochondrial respiratory chain complexes I & IV and ATP levels, and assessed learning and memory ability by Morris Water Maze and nesting behavior.

Results: Levels of AGE adducts (MG-H1 and CEL) were robustly increased in the serum and brain of AGE+ diet fed mice compared to the AGE- group. Furthermore, greatly elevated levels of reactive oxygen species, decreased activities of mitochondrial respiratory chain complexes I & IV, reduced ATP levels, and impaired learning and memory were evident in AGE+ diet fed mice compared to the AGE- group.

Conclusion: These results indicate that dietary AGEs are important sources of AGE accumulation in vivo, resulting in mitochondrial dysfunction, impairment of energy metabolism, and subsequent cognitive impairment. Thus, reducing AGEs intake to lower accumulation of AGEs could hold therapeutic potential for the prevention and treatment of AGEs-induced mitochondrial dysfunction linked to cognitive decline.

Keywords: Advanced glycation end products; methylglyoxal; mitochondrial and cognitive dysfunction.

Fig. 1.

Characterization of MG-glycated BSA. A) Ultraviolet absorption spectra of native BSA (BSA) and BSA modified with 100 mM MG (MG-BSA) during 1–12 days of incubation. B) Increased % hyperchromicity of MG-BSA compared to day 4 BSA. C) Fluorescence emission spectra of BSA and MG-BSA. Excitation wavelength was 350 nm. D) % change in fluorescence intensity of MG-BSA compared to BSA. E) Far UV CD spectra of BSA and MG-BSA. F) % change in ellipticity (structural loss) of MG-BSA (*p < 0.01 versus day 4 MG-BSA). G) Thermal melting profile of BSA and MG-BSA. H) % decrease in melting temperature (°C) of MG-BSA versus BSA. Data presented as mean ± SEM, ^#p < 0.05, *p < 0.01 versus day 4 glycated sample or BSA group. All spectra/profile shown are the averages of three determinations.

Fig. 2.

Determination of AGE adducts in the AGE additive and AGE diet. A) Protein (2 μg) from BSA or MG-BSA samples following 12 days of incubation were subjected to SDS-PAGE. Protein bands were stained by Coomassie Blue (A, Lane 1 & 2). Glycation was detected by immunoblotting with antibody to MG-H1 (A, Lane 3 & 4) or CEL (A, Lane 5 & 6). Lane 1, 3, 5, BSA; lane 2, 4, 6, MG-BSA. Quantification of immunodot intensity of MG-H1 (B), and CEL (C) normalized to Ponceau staining intensity (as a protein loading control) in protein extracted from the samples of AGE− and AGE+ diet. Data are presented as mean ± SEM (n = 3), *p < 0.01 versus AGE− diet.

Fig. 3.

Measurement of levels of AGEs in serum from AGE diet fed mice. Quantification of immunodot intensity of MG-H1 (A) and CEL (B) normalized to Ponceau staining intensity in serum of AGE− and AGE+ diet fed mice. Ponceau staining of membranes of corresponding dot blots was used as a protein loading control (A & B, lower panel). ELISA for the measurement of AGE (C) levels in serum samples of AGE− and AGE+ fed mice. Data are presented as mean ± SEM (n = 5), *p < 0.01 versus AGE− fed mice as a control group.

Fig. 4.

Measurement of levels of AGEs in cerebral cortex from AGE diet fed mice. A–C) Quantification of immunodot intensity of MG-H1 (A), and CEL (B) normalized to Ponceau staining intensity in cerebral cortex of AGE− and AGE+ fed mice (A & B, lower panel). ELISA for measurement of AGE (C) levels in cerebral cortex of AGE− and AGE+ fed mice. D) Quantification of AGE immunofluorescence intensity in the cortex of the indicated group of mice using MetaMorph® Image Analysis Software. E) Representative AGEs staining images for AGEs (red) and nucleus (NeuN, neuronal marker, blue) under confocal microscopy. Scale bar = 25 μm. Data are presented as mean ± SEM (n = 3), *p < 0.01 verses AGE− fed mice.

Fig. 5.

Effect of AGE diet on ROS generation and mitochondrial function. Quantification of superoxide anion (A), hydrogen peroxide (H₂O₂) (B), hydroxyl radicals (C) in serum of AGE− and AGE+ fed mice. Representative in vitro EPR spectra measured in cortical brain homogenates (D, E) are shown. The peak height in the spectrum indicates levels of ROS. Quantification of EPR spectra in the indicated AGE− & AGE+ fed mice (D). *p < 0.01 compared to other groups of mice. Data are expressed as fold-increase relative to AGE− fed mice. N = 5 mice per group. Activities of complexes I (F) and complex IV (G) and ATP levels (H) were determined in the cortex of AGE− and AGE+ mice. Data presented as mean ± SEM (n = 5), *p < 0.01 versus AGE− control group.

Fig. 6.

Effect of AGE diet on learning and memory. Mice fed AGE− or AGE+ diet for 17 months starting at the age of 5 months were subjected to the Morris water maze test. A) Escape latency during MWM hidden platform task in AGE− and AGE+ fed mice. B) Time spent in the quadrant with the hidden platform. C) Mean number of crossings of the target during the probe test. (D) Average swim speed of mice in MWM. (NS, no significance between AGE− and AGE+ group). E) Representative traces during the probe test. Data are shown as mean ± SEM. *p < 0.01 versus AGE− fed mice (n = 10 mice per group). F, G) Evaluation of nest building performance. F) Representative nest photos showing the nest building behavior in the indicated mice. G) Nest score on the basis of nest building behavior. Nesting deficits indicated by incomplete nests and low nest score. Nesting ability indicated by complete nests, high nest score. *p < 0.01 compared with AGE− fed mouse nesting ability.