A high-sugar high-fat diet induced metabolic syndrome shows some symptoms of Alzheimer's disease in rats

Abstract

Objectives

Cases of sporadic Alzheimer's disease (SAD) are the predominant form of the age-related dementia. New evidence suggests that metabolic syndrome (MS), a metabolic disorder, is an initiating factor of some SAD cases. A high-sugar high-fat diet could cause MS, we aimed to investigate whether it could directly lead to SAD.

Measurements

The characteristic molecules of AD (hippocampus Aβ and Tau) were tested by using ELISA and western blotting to confirm the happening hallmarks of AD in brain. MS and inflammation related biochemical indicators were measured using immunological method. Proteins associated with the insulin resistance signal pathway (JNK, PI-3K, AKT, GSK-3β, GLUT3) were evaluated using western blotting method. The levels of reactive oxygen species (ROS) were measured by immunofluorescence method.

Results

Expressions of hippocampus Aβ, phosphorylation-Tau (p-Tau), inflammatory factors and p-JNK, Gsk-3βwere higher in the model rats than those in the control rats and expressions of p-PI3K, p-AKT and GLUT3 were reversed.

Conclusions

The MS model animals, which can induce the characteristics symptoms of AD, and therefore it may be preliminarily considered that the AD pertains to the MS-related diseases.

Introduction

Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system with the typical symptoms of progressive degradation of learning, memorization, and cognition functions. The characteristic pathology of AD is the formation of neurofibrillary tangles due to hyperphosphorylation of tau protein in the hippocampus of patients, along with the deposition of Aβ that forms senile plaques (1, 2, 3). Approximately 95% of AD cases are sporadic AD (SAD), which is an age-related disease that is mainly caused by environmental factors (4). However, the exact cause(s) and pathogenesis of the disease are still unknown.

Metabolic syndrome (MS) is associated with insulin resistance (IR) and defined by a clinical metabolic disorder syndrome characterized by abdominal (central) obesity, elevated blood pressure, fasting plasma glucose and serum triglycerides, and low high-density cholesterol (HDL) levels. IR is center position in many metabolic diseases, and is one of the many symptoms of MS (5). In recent years, researchers have come to appreciate the important role that chronic inflammation caused by macrophage infiltration plays in MS-related diseases. Interestingly, diabetes, cardiovascular diseases, and obesity are generally latent factors causing SAD (6, 7, 8, 9). AD patients were found to elevate levels of inflammatory molecules such as tumor necrosis factor-α(TNF-α) and interleukin (IL)-1βcompared to a control group[10]. And AD is also associated with the desensitization of insulin signaling in the brain (11, 12, 13). It seems that the impairment of insulin signaling in the brain may be closely related to the development of neurodegenerative disorders (11). Therefore, metabolic disorders maybe initiate SAD.

A shift in dietary habits worldwide has led to the overconsumption of sucrose-enriched foods and meat. To mimic this diet in laboratory rats, we have recently raised rats on a high-sugar high-fat diet for 1 year and found that this model captures many MS-related diseases such as obesity, nonalcoholic fatty liver disease (NAFLD), type-II diabetes, metabolic cardiomyopathy, and atherosclerosis (14). The common characteristics of these animal models are chronic, low-grade inflammatory disease, and metabolic disorder centering on IR, similar to other rodent models (15, 16, 17, 18), especially a high-fructose diet exacerbates memory deficits (18).

So, we further explore whether SAD can be mimicked in high-sugar high-fat raised rats that have MS-related diseases. To explore the correlation between SAD and MS, in rats, we measured various hallmarks of AD found in humans at different time points (6, 9, and 12 months).

Material and methods

Animals and MS model

Forty-eight Sprague-Dawley rats were randomly assigned to four groups. The control group included 24 rats that were fed with the normal diet, and the MS model groups included 24 rats and were divided into 3 groups with 8 rats per group. The rats of these 3 groups were fed with the high-sugar high-fat diet (70% normal fodder, plus 20% lard, 10% table sugar, 1% cholesterol, and 0.25% cholic acid) for 6 months (6M), 9 months (9M), or 12 months (12M). The total caloric intake of the rats in the control group was ~13.89 kJ/g whereas that in experimental group was ~68.40 kJ/g. Body weight and body length of the rats were measured monthly.

Measurements of inflammation and metabolic related factors

At the various time points (6, 9, and 12 months), peripheral blood of rats was collected under pentobarbital anesthesia. Tissue around the kidney and uterus that represents visceral fat was removed. The rats were then executed and brains were removed. One-half of the brain tissue was fixed in 10% neutral formalin and embedded in paraffin, while the remaining half was stored at −80°C for reactive oxygen species (ROS) analysis and western blotting. Inflammation related indices such as levels of TNF-α, and IL-1β were tested by using immunological methods (reagents kits were purchased from R&D, California, USA). Metabolic related factors such as free fatty acids (FFA), triglycerides (TG), and fasting blood sugar (FPG) in blood, and FFA and diacylglycerol (DAG) in brain tissue were measured using the related biochemical reagents kits (BioSino Bio-technology and Science Inc., Beijing, China). IR index was calculated as follows: HOMA-IR = (FPG × FINS)/22.5, where FIN is fasting insulin.

Hematoxylin-eosin staining and immunofluorescence

To study pathological changes of the hippocampus, we performed hematoxylin-eosin (HE) staining of paraffinembedded brain sections (5-μm thickness) and observed hippocampal structure with light microscopy. For immunofluorescence analysis of brain tissues, frozen sections were incubated with a dihydroethidium probe (Sigma, St. Louis, MO) for 15 min at room temperature after being blocked with a blocking solution. The incubated sections were washed three times with phosphate buffered saline (PBS) and coverslipped with 50% glycerol, and then observed with fluorescence microscopy.

Enzyme-linked immunosorbent assay (ELISA) for Aβ

The Aβ concentration in brain tissue was analyzed with the rat Aβ ELISA kit (R&D Systems, California, USA) following the manufacturer's recommended protocol. Each sample was assayed in triplicate.

Western blotting

The homogenized frozen tissue samples were prepared with RIPA buffer (Cell Bioscience, London, United Kingdom) and the lysates were quantified using a BCA assay reagent kit (Pierce, Rockford, IL). Aliquots (20 μg each) were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes, and after transfer, each blot was stained with Ponceau to ensure even loading of protein across lanes. The membranes with blots were incubated with goat anti-rat primary antibody (Cell Signaling Technology, Inc., Beverly, MA) at 4°C overnight and were washed three times in PBST.The membranes were then incubated with alkaline phosphatase-conjugated secondary antibody for 2 h at room temperature and exposed to X-ray film after five washings in PBST. The images of the X-ray film were captured and were analyzed using ImageJ (NIH freeware).

Statistical analysis

All data are expressed as means ± SE. Comparisons among groups were made via one-way ANOVAs. T-test was used in comparison between two groups. The statistical calculations were performed using Statistica 6.1 software (StatSoft, Tulsa, OK). Differences were considered significant at P-values ≤ 0.05.

Results

Body weight and visceral fat weight

During the experiments, all rats were in good health. Compared with rats of the control group, body weight and visceral fat weights of experimental rats increased significantly (P < 0.05; Figure 1 A, B). We considered rats with Lee's index greater than 300 as obese. Lee's index provides a convenient method to qualify body type, and is calculated as: $\sqrt[3]{weight\times {10}^3/length(\text{cm})}$. Rats fed a high-sugar high-fat diet all had Lee's indices greater than 300 and was significantly higher than the control group (P < 0.05; Figure 1 C).

Figure 1.

Changes of body weight, visceral fat, and Lee's index

Metabolic markers in blood and brain tissue

The levels of FFA, TG, and FPG in the blood, as well as FFA and DAG in brain tissue of experimental rats were higher than those of the control group (Tables 1 and 2). The HOMA-IR value of experimental rats was dramatically increased compared with the HOMA-IR of control rats (Table 1). Highsugar high-fat diets can thus induce IR in rats.

Table 1.

The change of metabolic and inflammation related factors in the blood

	Cont.	6 months	9 months	12 months
TG (mmol/L)	0.08 ± 0.03	0.55 ± 0.15**	0.57 ± 0.18**	0.56 ± 0.14**
FFA (mmol/L)	418.9 ± 35.74	1164.3 ± 71.33**	1349.5 ± 53.15**	1369.2 ± 134.4**
FPG (mmol/L)	5.10 ± 0.63	11.71 ± 0.47*	13.78 ± 0.14**	14.66 ± 0.17**
HOMA-IR	1.68 ± 0.18	3.60 ± 0.20*	3.57 ± 0.184**	3.39 ± 0.23**
TNF-α (µg/ml)	0.98 ± 0.05	1.86 ± 0.04**	1.89 ± 0.11**	1.90 ± 0.08**
IL-1β (pg/mL)	163.37 ± 33.68	297.13 ± 40.67*	401.20 ± 34.71**	418.29 ± 22.25**

^*P < 0.05

^**P < 0.001

Table 2.

The change of metabolic and inflammation related factors in the brain

	Cont.	6 months	9 months	12 months
DG (mmol/L)	16.56 ± 3.04	38.12 ± 10.75	64.78 ± 9.82**	233.7 ± 45.42**
FFA (mmol/L)	75.15 ± 12.32	128.0 ± 14.47*	198.3 ± 44.39*	206.3 ± 46.44*
TNF-α (µg/mL)	2.98 ± 0.13	3.20 ± 0.20	4.31 ± 0.28*	4.45 ± 0.30*
IL-1β (pg/mL)	37.72 ± 4.11	59.07 ± 7.43*	104.12 ± 6.35*	118.33 ± 5.89*

^*P < 0.05

^**P < 0.001

Inflammation -related factors

We found that the production of the inflammatory factors TNF-α and IL-1β increased gradually and were significantly higher in experimental rats than in control rats (Table 1). Thus, experimental rats exhibit an elevated inflammatory status. Consistently, as in blood, inflammatory cytokines (TNF-α and IL-1β) in brain tissue also gradually increased (Table 2).

Pathological changes in brain tissue

When analyzing proteins characterizing AD, we found that experimental rats expressed high levels of tau and Aβ. Two phosphorylation sites of p-tau, S199 and S396, were expressed highest at 6 months following a high-sugar high-fat diet (Figure 2 A). The concentration of Aβ1-42 was significantly greater in experimental rats than in control rats (Figure 2 B).

Figure 2.

Expressions of proteins related to brain pathologies

Next, we found that rats fed with a high-sugar high-fat diet had inflamed brains cause by inflammatory cell infiltration (Figure 3 A). Moreover, we found that ROS accumulated gradually in the brain and peaked at 12 months following a high-sugar high-fat diet (Figure 3 B).

Figure 3.

Pathological changes and ROS production in brain tissue

Central insulin signaling pathway

To test whether rats fed a high-sugar high-fat develop a disrupted insulin signaling pathway, we examined the expression of various proteins in the insulin signaling pathway (Figure 4). We found that expression of phosphorylated c-Jun N-terminal kinase (p-JNK), which is upstream of insulin receptor substrate (IRS), is downregulated in experimental rats compared to control rats. However, expression of insulin signaling proteins p-P85, p-AKT, and glucose transporter 3 (GLUT3) were all downregulated in experimental rats. Expression of glycogen synthase kinase 3 (GSK-3β), which is downstream of AKT, is gradually increased in experimental rats compared to control rats. Therefore, a high-sugar high-fat diet disrupts insulin signaling in rats.

Figure 4.

Expressions of proteins related to insulin signaling

Discussion

By feeding rats for 1 year with a high-sugar high-fat diet, which could be initiated MS (14), can lead to chronic low-grade inflammation and IR (19). Importantly, these same rats also exhibited the pathological hallmarks of AD, strongly implicating metabolic disorders may be related to SAD.

Many studies have linked MS with disrupted cognitive functions, as well as with SAD. Hyperglycemia, dyslipidemia and hypercholesterolemia all contribute to the pathology of MS and are also independent risk factors for AD (20). Hyperglycemia can lead to oxidative stress and advanced glycation end products (AGEs) formation,which derived from the Maillard reaction of glucose and proteins and lipids. That can activate receptors for AGEs (RAGE) and can cause the pathological changes of AD (21). RAGE acts as a receptor for Aβ, and its expression level is increased in AD brains, morover recent study demonstrated that AGEs induces tau hyperphosphorylation and memory impairment through RAGE-mediated glycogen synthase kinase-3 activity (22). As one of the major physiological changes in MS is an elevated inflammatory status, it is currently considered that inflammation causes Aβ deposition, and is thus a key factor leading to neurodegeneration (23). In the present study, animals were fat and inflammatory factors were released by the adipose tissue, so we found elevated levels of inflammatory factors in the blood plasma and brain tissues at all time points, in line with previous findings of the well-characterized changes that occur in AD (24). We surmise that peripheral inflammatory factors enter the brain through the blood-brain barrier, which may then inflame the brain, and thus affect cognition. These inflammatory factors activate microglial cells to secrete more inflammatory factors, thereby triggering a vicious circle and amplifying brain inflammation. Moreover, activated microglial cells may generate a large amount of amyloid precursor protein (APP), which is the source of Aβ. Tamagno et al. (25) found that oxidative stress caused by Aβ can accelerate the abnormal cleavage of APP to generate more Aβ. Aβ is not only toxic to neurons, but activates astrocytes and microglial cells to release proinflammatory molecules, further damaging the brain. Moreover, brain inflammation not only promotes the deposition of Aβ, but also causes hyperphosphorylation of tau. Taken together, inflammatory molecules released by activated glial cells cause a positive feedback loop that continuously activates more cells to release inflammatory molecules, migrate to Aβ deposits, and damage neurons.

We observed that brain tissue from the rats fed a high-sugar high-fat diet were insulin resistant. Proinflammatory factors entering the brain may activate JNK signaling, and JNK phosphorylates IRS1-Ser but not IRS1-Tyr; this may induce IR (26). IR may also be caused by the release of proinflammatory factors from macrophages (27). Moreover, FFA is related to the IR, it can affect the insulin signal pathway, glucose uptake and usage in peripheral tissue and central. Insulin signaling plays many vital roles in the brain, especially protection from apoptosis (28, 29). We found that p-P85 and p-AKT in the brain tissues of the experimental rats gradually decreased with time, strongly implicating developing IR. In addition, reduced activation of P85 increases apoptosis. GLUT-3 is the most important GLUT in mature neurons and plays a considerable role in the glucose metabolism of the brain (30). Interesting, before the manifestation of the clinical symptoms of AD, glucose metabolism in cognitive related brain areas is reduced followed by Aβ deposition. This indicates that glucose metabolic disruption occurs before encephalatrophy and cognitive decline. Like p-P85 and p-AKT, we found that GLUT3 is reduced in high-sugar high-fat fed rats, suggesting that neurons are starved for glucose, compromising their essential energy source. Furthermore, GSK-3β is located downstream of the insulin signal, and is an important kinase that phosphorylates tau. After killing pancreatic cells with chronic streptozotocin treatment, tau protein in the mouse cerebral cortex and hippocampus becomes phosphorylated at multiple sites (31), and IR in the brain reduces the activation of p-AKT, activates GSK-3β, and phosphorylates tau. In our present study, IR likely leads to overactivation of GSK-3β, as well as tau phosphorylation, another hallmark of AD.

Another feature of AD is oxidative stress in general, and the accumulation of ROS in particular. On the one hand, ROS attack neuronal DNA, and energy consumption of the cells is increased and apoptosis of mitochondria is accelerated, thereby inducing the release of acute-phase proteins and inflammatory factors (32). On the other hand, ROS adversely affect mitochondrial energy metabolism and cause more damage to neurons (33). In addition to a ROS-inflammation link, IR is also associated with systematic oxidative stress (34). Long-term exposure of fat cells to ROS may inactivate P85 and AKT, affect fat formation by means of insulin stimulation, decrease glucose intake and the translocation of GLUT (35). Oxidative stress was also induced by FFA generated under lipolytic action. Regardless of the mechanism, oxidative stress, IR and inflammation promote each other, common in MS-related diseases.

In conclusion, after feeding rats a high-sugar high-fat diet for a year, rats became obese, showed MS and histopathological characteristics of AD. Importantly, chronic inflammation and IR were central features, which negatively influenced many pathways. This pathway, which leads to a SAD-like pathology, suggests SAD was may be MS related disease, provides an novel strategy for the prevention of SAD.

Ethical Standards: This study was initiated following approval from the ethics committee of Shanxi medical University, and informed consent was obtained.

Acknowledgments: This study was supported by the the natural science foundation of China. (Grant No. 81302220) and Scientific and technological project of Shanxi Province. (Grant No.2014021040-3, No. 2014021037-1). The Corresponding Authors have the right to grant on behalf of all authors and does grant on behalf of all authors. The manuscript has not been published elsewhere and has not been submitted simultaneously for publication elsewhere. All study participants provided informed consent, and the study design was approved by the appropriate ethics review boards. All the authors have approved the manuscript and agree with submission to your esteemed journal. There are no conflicts of interest to declare. An exclusive license (or non exclusive for government employees) on a worldwide basis to the Elsevier to permit this article (if accepted) to be published in The journal of nutrition,health & aging and sublicenses such use and exploit all subsidiary rights, as set out in our license.

Conflict of interest: The authors declare no conflicts of interest.