Significant outcomes
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∙ Western-type diet (WD) might contribute to the production of Alzheimer-associated proteins.
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∙ Environmental enrichment may modify the diet-induced pathological effects associated with Alzheimer’s disease.
Limitations
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∙ As Alzheimer’s disease is a complex pathology, the metabolic characterisation of the animals, and the investigation of post-translational modifications and amyloid-β transporters would offer further information.
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∙ Further, protein deposition would be confirmed by utilising additional methods.
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∙ Moreover, including an additional control group in which environmental enrichment is applied without any dietary intervention would facilitate the evaluation of the environmental effects alone.
Introduction
The Western-type diet with principal components such as saturated fats and refined carbohydrates not only promotes excess energy intake and body weight gain, but also enhances the risk of dementia (Reference Whitmer, Gunderson, Barrett-Connor, Quesenberry and Yaffe1). The WD is associated with an elevated risk of Alzheimer’s disease (AD) and other milder forms of cognitive impairment (Reference Berrino2,Reference Morris, Evans, Bienias, Tangney and Wilson3). The WD prominently disturbes hippocampal learning and memory (Reference Kanoski and Davidson4,Reference Cordner and Tamashiro5). Obesity and Western-style high-fat diet (HFD) are associated with an increased risk of dementia and AD in humans (Reference Luchsinger, Tang, Shea and Mayeux6,Reference Profenno, Porsteinsson and Faraone7). In addition, the prevalence of AD is greater in countries where the intake of HFD/high-calorie diets is high, but lower in those countries with low-fat diet intakes (Reference Martin, Jameson, Allan and Lawrence8,Reference Panza, Solfrizzi and Colacicco9).
Many studies show that HFD increases tau expression and amyloid β-peptide (Aβ) levels in the brain of AD mice and leads to memory impairment (Reference Martin, Jameson, Allan and Lawrence8,Reference Vandal, White and Tremblay10,Reference Julien, Tremblay and Phivilay12). Moreover, a study indicates that the consumption of sucrose-sweetened water exacerbates memory impairment and increases insoluble Aβ levels and deposition in the brain (Reference Cao, Lu, Lewis and Li13). These data highlight the potential role of diet in the pathogenesis of AD.
Several authors have reported the mitigation of cognitive deficits by environmental enrichment (Reference Van Praag, Kempermann and Gage14–Reference Gergerlioglu, Oz, Demir, Nurullahoglu-Atalik and Yerlikaya19), and many studies suggested that enriched social, physical, and/or cognitive activities reduced the risk of AD development (Reference Wilson, Bennett and Bienias20–Reference Friedland, Fritsch and Smyth22). In a transgenic mouse model of familial AD, Hu et al. (Reference Hu, Xu, Pigino, Brady, Larson and Lazarov23) showed that EE reversed impaired neurogenesis, enhanced synaptic plasticity, and hence attenuated the neuropathology. In addition, many findings show that the exposure to EE was associated with a decrease in the Aβ levels in brain and decreased tau-hyperphosphorylation (Reference Beauquis, Pavía and Pomilio24,Reference Gerenu, Dobarro, Ramirez and Gil-Bea25). Maesako et al. (Reference Maesako, Uemura and Kubota26) demonstrated that EE ameliorated HFD-induced behavioural changes and AD pathology in amyloid precursor protein (APP) transgenic mice. Despite increasing studies, the mechanisms underlying the link between EE and diet-related cognitive dysfunction are not fully understood thus far.
The role of EE in the amyloid and tau metabolism of the brain is an important emerging research area. There is increasing evidence on the relationship between diet, EE, and amyloid-related mechanisms and tau pathology (Reference Beauquis, Pavía and Pomilio24,Reference Costa, Cracchiolo and Bachstetter27–Reference Patel, Gordon and Connor31). Studies on the effects of tau protein, APP, and Aβ on physiological senility, mild cognitive impairment (MCI), and AD suggest that these proteins lead to neurotoxicity and neurodegeneration mainly due to neurofibrillary tangle formation and amyloid plaque deposition and the loss of synapses and neurons (Reference Ballatore, Lee and Trojanowski32–Reference Kowall, McKee, Yankner and Beal36). APP is generated in large quantities in the neurons and degraded very rapidly (Reference Lee, Retamal and Cuitiño37). Aβ is derived from the proteolytic processing of APP by the action of β- and γ-secretases sequentially (Reference Masters, Simms, Weinman, Multhaup, McDonald and Beyreuther38). Neurofibrillary tangles contain aggregates of hyperphosphorylated tau protein, and are responsible for neurodegeneration (Reference Duyckaerts, Delatour and Potier39). The elevated deposition of Aβ and tau proteins is characteristically considered to be neuropathological in AD (Reference Bloom40,Reference Nisbet, Polanco, Ittner and Götz41).
The present study aimed to investigate the effects of EE on amyloid and tau pathology in high-fat- and high-sucrose-fed rats.
Material and methods
Animals, housing conditions, and diets
40 adult male (8–12 weeks old) Wistar albino rats were selected for this study from Experimental Medicine Research and Application Center of Necmettin Erbakan University (Konya, Turkey). Ethical consent was received from the local ethical committee for animal studies. The animals were distributed into two main groups according to the type of housing condition: enriched environment (EE, n=16) and standard housing condition (n=24). The groups were further divided into five subgroups that received standard diet (SD), high-fat diet (HFD), or high-sucrose diet (HSD). The five subgroups were as follows: Group I (StNE), SD – in normal environment (n=8); Group II (FaNE), HFD – in normal environment (n=8); Group III (SuNE), HSD – in normal environment (n=8); Group IV (FaEE), HFD – in EE (n=8); Group V (SuEE), HSD – in EE (n=8).
Animals were provided with HFD or HSD for 4 weeks. The caloric value of food intake was determined on the basis of 35% of total calorie intake for the HFD and 100% of total carbohydrate-derived calorie intake for the HSD (Table 1).
Compositions and energy loads of the diets
* Starch was used in the standard diet while it was substituted with sucrose in the high-sucrose diet.
† Vegetable oil was used in the standard and high-sucrose diets, whereas in the high-fat diet tallow was used.
The EE protocol that has been used was similar to that described by Zhang et al. (Reference Zhang, Zhang, Sun, Zhu, Liu and Yang42). Environmental enrichment was provided by using specially designed polypropylene cages (90×75×45 cm) with a wire-mesh roof. The roof could be removed to replace the bedding material and provide access to the rats. The animals in the enrichment groups were housed in these cages (eight rats in each cage) that contained objects such as toys, tunnels of different shapes, running wheels, and stairs and platforms made from wood or metal. The design of EE was changed twice a week for each cage. All rats were given ad libitum access to food and water. The housing facility was light- and climate-controlled (12 : 12 light–dark cycle, 22±2°C temperature, and 50±5% humidity).
Measurement of Aβ(1–40), Aβ(1–42), APP, and Tau by enzyme-linked immunosorbent assay (ELISA)
On completion of the experiment, the rats were anaesthetised by a high dose of ketamine (60 mg/kg)–xylazine (10 mg/kg) anaesthesia via a single intraperitoneal injection and sacrificed via exsanguination. The brains were removed from the cranium, and the hippocampus were dissected and rapidly frozen in liquid hydrogen for biochemical analysis. Samples were stored at −80°C until further analysis could be performed. The hippocampal tissue was homogenised in ice-cold buffer containing 0.25 M sucrose, 10 mM Tris-HCl, and 0.25 mM phenylmethylsulfonyl fluoride at pH 7.4. Homogenates were centrifuged (+4°C) at 3000 rpm for 10 min, and the supernatant was collected. The levels of Aβ(1–40), Aβ(1–42), APP, and tau were measured using specific ELISA kits that are validated for use in rats (Hangzhou Eastbiopharm Co., Ltd., Hangzhou, China) according to the manufacturer’s instructions. Amino acids targeted by the antibodies were as follows: Aβ(1–42): amino acid residues 33–42, Aβ(1–40): amino acid residues 31–40, APP: target unknown, and tau protein: amino acid residues 201–241.
Statistical analysis
Data were analysed by using one-way analyses of variance (one-way ANOVA) with post hoc Dunnett’s test. The statistical significance threshold was considered to be p<0.05. All the data were expressed as mean±SEM.
Results
We quantified the changes in the levels of Aβ(1–40), Aβ(1–42), APP, and tau by ELISA in the hippocampus, and the one-way ANOVA test revealed a statistical significance in the mentioned parameters (p=0.01, <0.01, <0.01, and <0.01, respectively). The measurements of Aβ(1–40), Aβ(1–42), and tau levels showed no significant effect (p>0.05) of HFD despite the observed increases in all the parameters, whereas HFD intake had led to an appreciable increase in the level of APP comparison with the controls, as shown in Fig. 1c (p=0.03). Moreover, there was a significant increase in the production of Aβ(1–40), Aβ(1–42), APP, and tau in response to high sucrose feeding (p=0.04, <0.01, <0.01, =0.02, respectively) (Figs 1a–1d). In addition, we performed the analysis of Aβ(1–40), Aβ(1–42), APP, and tau levels in the hippocampus of rats that were maintained under standard housing conditions or exposed to an EE. The environmental enrichment decreased the levels of Aβ(1–40), Aβ(1–42), APP, and tau to those of the controls in high-fat-fed and high-sucrose-fed rats (p>0.05). The initial and final weights of the animals and the change in the body weights were not statistically significant (see Fig. 2). This observation shows that EE may rescue diet-induced amyloid and tau pathology by reducing these parameters (see Fig. 1a–1d).
The effect of high-fat (HF), high-sucrose (HS) diet and enriched environment (EE) on (a) Amyloid-β (Aβ)(1–40), (b) Aβ(1–42), (c) Amyloid precursor protein, and (d) Tau protein. The sample size (n) was 8 in each group. *Statistical significance (p<0.05 vs. Control. One-way ANOVA and post hoc Dunnett’s test).
The weight gain of the animals over the study. CON, control; EE, enriched environment; HFD, high-fat diet; HSD, high-sucrose diet.
Discussion
In early studies, it has been reported that mice exposed to EE showed reductions in cerebral Aβ levels and amyloid deposits compared with animals in standard housing conditions (Reference Lazarov, Robinson and Tang43,Reference Mainardi, Di Garbo, Caleo, Berardi, Sale and Maffei44). Moreover, there is substantial evidence that WD is associated with AD-related neuropathology (Reference Ghribi, Larsen, Schrag and Herman28,Reference Hooijmans, Rutters and Dederen29). Therefore, intense investigations have been made lately to deepen causal mechanistic links underlying this association, particularly in animal models. It is indicated that HFD, regardless of the genotype and peripheral metabolic status, increases tau expression in the brain (Reference Takalo, Haapasalo and Martiskainen11). In addition, it is found that high sucrose intake is related to an increase the production and deposition of Aβ and exacerbation in tau phosphorylation (Reference Orr, Salinas, Buffenstein and Oddo45). However, there is little information about how EE affect diet-induced changes in AD neuropathology. Therefore, in this study, we have examined the effects of EE on diet-related changes in amyloid proteins and tau. We found that HFD is associated with an increase in the levels of APP and HSD, thereby resulting in a significant increase in the levels of Aβ(1–40), Aβ(1–42), APP, and tau in the hippocampus. In addition, our results demonstrate that EE can ameliorate HFD- and HSD-induced Aβ(1–40), Aβ(1–42), APP, and tau deposition in the hippocampus. To the best of our knowledge, this study is the first to demonstrate that EE reduces the WD-related accumulation of Aβ(1–40), Aβ(1–42), APP, and tau protein in the brain.
There are debatable data about the linkage between diet and amyloid pathology, with reports of increased (Reference Julien, Tremblay and Phivilay12,Reference Ho, Qin and Pompl46) and reduced (Reference Howland, Trusko and Savage47) amyloid accumulation in transgenic mice fed with a high-cholesterol diet. In our study, the HFD-related increase observed in Aβ(1–40) and Aβ(1–42) levels was not statistically significant. This might be related to the use of different types of animal models, diet content, and duration of diets in other studies. Moreover, we found that HFD increased the APP level, and this change was likely to appear regardless of the accumulation of Aβ(1–40), Aβ(1–42), and tau protein in the hippocampus. It would be expected that an increase in the APP level might be concomitantly observed with an increase in the Aβ levels on the basis of the understanding that large amounts of APP are continuously metabolised to Aβ in the brain (Reference O’Brien and Wong48). However, there are growing data about multiple alternate pathways of APP processing, some of which may or may not lead to the generation of Aβ. APP, β-, and γ-secretases are the principal players involved in Aβ production. On the cell surface, APP can be directly proteolyzed by α-secretase and then γ-secretase and this process does not generate Aβ (Reference O’Brien and Wong48). It is unclear how the access of these enzymes to APP is regulated. Ehehalt et al. (Reference Ehehalt, Keller, Haass, Thiele and Simons49) suggested the access of α-and β-secretases to APP and therefore, Aβ generation might be determined by the dynamic interactions of APP with lipid rafts. Thirumangalakudi et al. (Reference Thirumangalakudi, Prakasam and Zhang50) suggested that dietary cholesterol plays a role in the induction of APP processing, potentially contributing to AD-like dementia. They reported that increased APP-processing activity could be due to cholesterol’s direct effect on the activities of the APP-processing enzymes. Therefore, we believe that our results about amyloid pathology may be a consequence of APP processing.
Moreover, in our study, no significant change was observed in the level of tau protein associated with high fat intake. In support, there are studies reporting no effect on amyloid and tau pathology in AD mice fed a HFD. Phivilay et al. (Reference Phivilay, Julien and Tremblay51) indicated that very high trans-fatty acid intake modifies the fatty acid profile of the brain without significantly affecting the markers of AD pathology such as Aβ, tau protein, and synaptic protein content. Knight et al. (Reference Knight, Martins, Gümüsgöz, Allan and Lawrence52) demonstrated that a HFD increased the onset and severity of memory deficits in transgenic mice, but the effects of a HFD on cognition were independent of the effect on AD neuropathology as no difference was observed in the deposition of Aβ and tau protein. However, several studies using transgenic mouse models of AD indicated that HFD could cause increased tau phosphorylation (Reference Julien, Tremblay and Bendjelloul53,Reference Ma, Yang and Rosario54) and tau expression (Reference Takalo, Haapasalo and Martiskainen11). The reason for this discrepancy was unclear, but it is likely related to the different methodologies used in the investigations. Thus, the mechanisms responsible for the effect of a HFD on cognition related to amyloid and tau pathology remain unknown and further investigations are needed in this field.
Growing evidence shows that certain eating habits such as sugar- and fat-enriched diet increase the risk of AD development (Reference Biessels, Staekenborg, Brunner, Brayne and Scheltens55). Or et al. (Reference Orr, Salinas, Buffenstein and Oddo45) conducted a study using an animal model of AD and showed that high sucrose intake induces obesity with changes in central and peripheral insulin signalling. They reported that these prediabetic changes were associated with an increase in the production and deposition of Aβ and high sucrose ingestion exacerbated tau phosphorylation. Similarly, it is shown that the induction of experimental diabetes with a drug led to an increase in the Aβ and tau protein levels in a rabbit animal model (Reference Bitel, Kasinathan, Kaswala, Klein and Frederikse56). Moreover, it is suggested that, under diabetic conditions, impairments in certain insulin resistance-responsive cellular signalling pathways contribute to AD-related neuropathology and cognitive deterioration (Reference Li, Zhang and Sima57,Reference Craft58). Gasparini et al. (Reference Gasparini, Gouras and Wang59) suggested a mechanism for this finding, and it was found that insulin stimulates APP/Aβ trafficking from the trans-Golgi network, a major cellular site for Aβ generation, to the plasma membrane and suggested that insulin itself may significantly promote Aβ accumulation. Showing good agreement with these findings, we found a significant increase in Aβ(1–40), Aβ(1–42), APP, and tau levels induced by HSD in animals. Therefore, our study suggested that poor dietary lifestyle might be a risk for the development of AD and cognitive impairment through amyloid- and tau-dependent pathogenic brain mechanisms.
The molecular link between diet and cognitive functions has been proposed in an increasing number of studies (Reference Kanoski and Davidson4,Reference Greenwood and Winocur60). In particular, WD has been linked to an increase in the incidence of AD and cognitive dysfunction (Reference Berrino2,Reference Pasinetti and Eberstein61,Reference Eskelinen, Ngandu and Helkala62). However, an effective intervention that adequately attenuates or prevents the existing cognitive impairment or its progression has not been found thus far. In the light of the collecting data that shows EE (Reference Maesako, Uemura and Iwata63,Reference Fischer64) provide the groundwork for the improvement of cognitive performance, we chose a paradigm of EE to search whether it is effective or not for diminishing diet-related neuropathology of cognitive impairment. EE provides physical and intellectual stimulation (Reference Krech, Rosenzweig and Bennett65). In animal studies, investigating the impact of EE on the pathophysiology of AD has revealed uncertain results. Jankowsky et al. (Reference Jankowsky, Melnikova and Fadale18) found that transgenic mice housed under enriched conditions developed a greater amyloid plaque burden, contained more aggregated Aβ, and accumulated a higher concentration of total Aβ than their standard-housed counterparts. However, mostly, studies have reported the decreased risk for cognitive impairment with EE. Yuede et al. (Reference Yuede, Zimmerman and Dong66) suggested that EE, particularly exercise, partly reduced brain Aβ in transgenic mice by increasing brain peptidases and also stimulated synaptic processes and growth factors. Herring et al. (Reference Herring, Lewejohann and Panzer30) have demonstrated that EE could reduce the Aβ plaque burden and the extent of cerebral amyloid angiopathy. In a previous study, Maesako et al. (Reference Maesako, Uemura and Kubota26) investigated the role of EE on HFD-induced Aβ deposition and memory deficit in APP transgenic mice and found that EE reversed HFD-related memory impairment and increased Aβ oligomers as well as Aβ deposition. To elucidate the effect of EE on HFD/HSD-induced amyloid or tau pathology, we first investigated how HFD/HSD affected the levels of Aβ(1–40), Aβ(1–42), APP, and tau in rats. Showing consistency with the previous study results, in the setting of the present study, the brain disturbances of the HFD/HSD+EE rats were found to have clearly reduced in comparison with that of the HFD/HSD+SD rats. However, EE failed to reduce the level of pathological proteins in rats fed with a regular diet. Recent findings have suggested that EE decreased adiposity, increased energy expenditure, and caused resistance to obesity (Reference Cao, Choi and Liu67). This might be a possible mechanism underlying the effect of EE on diet-related AD neuropathology in our study.
The present study has been conducted by using conventionally bred 8–12-week-old Wistar albino rats that were fed with HSD and HFD. There are no natural mouse models of AD (Reference Windisch68,Reference Elder, Gama Sosa and De Gasperi69), which is an age-related disease in human beings; thus, the age of the rats used in this study does not seem to play a crucial role. Moreover, we aimed to investigate the effect of special diet not on the clinical and behavioural symptoms of AD, but on AD-related parameters that play an important role in neuropathology. As we know, there is no investigational model which can reflect the human organism and how disease develop within it. Therefore, in our study, we focused on the effect of diet on Alzheimer-related neuropathology.
In summary, these data have important implications for discovering how WD may potentially contribute to brain dysfunction and the development of neurodegenerative disorders such as Alzheimer’s disease. Our investigation demonstrates the possibility of diet contributing to the neuropathology of cognitive decline. However, the underlying mechanism of fat/sucrose-induced changes in the pathological proteins in brain is still unclear and should be further verified in future studies. Moreover, this study provides evidence that the use of EE conditions reduces diet-related biochemical disturbances and leads to a healthier brain in rats. Overall, these findings emphasise the importance of the use of physical and intellectual stimulation in reducing the risk of cognitive impairment.
Acknowledgements
The authors would like to thank Selcuk University Neuroscience Research Center.
Authors’ Contributions: contribution by respective authors: Y.S. and H.S.G., study concept, design and manuscript preparation; N.A. and A.K., acquisition of data; E.A.D., data interpretation; F.H.Y and E.N.A., biochemical analyses; and M.O., revision of the manuscript.
Financial Support
The authors thank the Selcuk University Scientific Research Projects Coordination Unit for the financial support (Project No.: 15401033).
Conflicts of Interest
None.
Ethical standards
The authors assert that all the procedures contributing to this work conform to the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals.
