High-Fat Diets Impair Spatial Learning of Mice in the Y-Maze Paradigm: Ameliorative Potential of α-Lipoic Acid

Abstract

High-fat diets (HFDs) have been found to influence central nervous system development and to cause cognitive impairments in human epidemiologic studies, as well as in animal investigations. These adverse effects on learning and memory induced by an HFD have been associated with an impaired hippocampus, including hippocampal oxidative damage. Previously, we had found that α-lipoic acid (α-LA) could ameliorate the oxidative stress in non-neural organs (liver, jejunum, and spleen) induced by a 10-week HFD (21.2% fat) food regimen in mice. In this study, we investigated whether a 10-week HFD (21.2% fat) induced oxidative stress in the hippocampus or impaired spatial learning in mice and whether LA ameliorated these effects. The HFD was found to induce oxidative stress (a decrease in catalase activity, glutathione peroxidase activity, and total antioxidative capacity and an increase in malondialdehyde levels) in the mouse hippocampus. In addition, we found that the HFD impaired spatial recognition memory of mice in the Y-maze paradigm. Furthermore, the hippocampal oxidative stress and impaired spatial recognition memory of the mice were reduced in HFD diets supplemented with 0.1% LA. These findings suggest that LA, as a strong antioxidant, may help prevent HFD-induced learning impairments by ameliorating associated oxidative stress in the hippocampus.

Introduction

I n the past two decades, high fat diets (HFD) have become pandemic across the globe. In addition to being implicated as a cause of obesity, the increased intake of HFDs are known to lead to many health problems, such as insulin resistance, dyslipidemia, hypertension, and dementia.¹ Furthermore, several studies have suggested that HFDs influence the development of the central nervous system and cause cognitive impairment.^2

–5 For example, human epidemiologic studies demonstrated that chronic ingestion of HFDs are associated with cognitive impairment. Saturated and trans-unsaturated fats are identified as risk factors for Alzheimer's disease,^6,7 and ω6 and saturated fatty acids are associated with poorer performances on a variety of cognitive tasks.^8,9 Laboratory studies in animals have also shown that rats and mice fed HFDs display impaired cognitive function in various learning and memory tasks, such as the radial-arm maze, the stone T-maze, and variable interval delayed alternation tasks.^2,10
–12

It has been well established that the brain region responsible for learning and memory is the hippocampus¹³ and that a variety of learning tasks are dependent on hippocampal function.^14,15 It is interesting that the adverse effects of HFDs on learning and memory have been associated with an impaired hippocampus.^16
–18 Furthermore, the chronic consumption of HFDs increased oxidative stress and damage in the hippocampal structure.^14,15

Lipoic acid (LA) is an endogenously produced coenzyme that plays an essential role in α-ketoacid dehydrogenase reactions. Additionally, there is sufficient evidence available to support an antioxidant function for LA.^19
–21 LA quenches a number of oxygen free radical species in both the lipid and aqueous phase, chelates transition metals, and prevents membrane lipid peroxidation and protein damage via interactions with vitamin C and glutathione. LA also participates in the recycling of vitamin C and vitamin E, increases cellular levels of glutathione, and suppresses nonenzymatic glycation.²²

Previously, we had found that 10-week treatment of mice with an HFD induced considerable oxidative stress in the liver, jejunum, and spleen.^19
–21,23 In the present study, our objective was to examine whether the HFD induced oxidative damage in the hippocampus and impaired spatial learning in the Y-maze paradigm. We also examined whether the antioxidant capability of LA ameliorated hippocampal oxidative stress and any associated learning impairment induced by the HFD.

Materials and Methods

HFD mouse models

Four-week-old KM male mice were purchased commercially and were housed in a light-controlled (12-h light cycle starting at 7 a.m.) and a temperature-regulated (24°C) space. For 1 week, the animals were allowed to acclimatize to their environment while consuming standard chow, before the dietary intervention was initiated. After this period, the following experiments lasted for an additional 10 weeks. Animals were randomly assigned to one of the following dietary groups (n=12 animals per group): Group I (Control) received a normal diet containing 4.89% fat, Group II (HFD) received an HFD containing 21.2% fat (including lard [17.5%] and cholesterol [0.5%]), and Group III (HFD+0.1% LA) was fed the HFD with a 0.1% LA supplementation (Table 1). This HFD has been found to cause significant oxidative damage and blood glucose and lipid metabolism disorders in KM mice.²⁴ All mice were allowed free access to the test diets and deionized water throughout the test period. After the 10-week period, all mice were evaluated in the Y-maze (see Behavioral apparatus and method). Following this, the mice were deprived of food for 12 h, then slightly anesthetized, and sacrificed by decapitation. The hippocampus was immediately dissected out, weighed, and analyzed for oxidative stress (see Determination of oxidative stress).

Table 1.

Percentage Compositions of Diets

Ingredient	Normal diet	HFD	HFD+LA
Corn meal	52.92	29.73	29.73
Soybean meal	25.00	28.00	28.00
Corn bran	6.50	9.20	9.20
Wheat flour	9.00	11.32	11.32
NaCl	0.20	0.20	0.20
CaHPO₄	1.15	1.25	1.25
Lard	2.80	18.00	18.00
Lysine	0.30	0.21	0.21
Methionine	0.21	0.22	0.22
Vitamins	0.02	0.02	0.02
Choline	0.10	0.10	0.10
Sucrose	0.10	0.10	0.10
Minerals	0.06	0.06	0.06
CaCO₃	1.65	1.60	1.60
LA	—	—	0.10

HFD, high-fat diet; LA, lipoic acid.

Behavioral apparatus and method

At the end of the feeding period, the acquisition of spatial recognition memory in all animals was evaluated in a Y-maze. The Y-maze was made of green painted timber and consisted of three identical arms. The angle of orientation between each arm was 120°, and the arm dimensions were 8 cm ×30 cm ×15 cm (width×length×height). The three arms were randomly designated: Start arm, where the mouse started to explore (always open); Novel arm, which was blocked during the first trial but opened during the second trial; and Other arm (always open). The maze was placed in a sound-attenuated room with dim illumination. The floor of the maze was covered with sawdust, which was mixed after each individual trial in order to eliminate olfactory stimuli. Visual cues were placed on the walls of the maze, and the observer was always in the same position at least 3 m from the maze. The Y-maze test consisted of two trials separated by an intertrial interval to assess spatial recognition memory. The first trial was 10 min in duration, and the mouse was allowed to explore only two arms (Start arm and Other arm) in the maze; the third arm (Novel arm) was blocked. After a 1-h intertrial interval, the second (retention) trial was conducted. During the retention trial, the mouse was placed back in the same starting arm of the Y-maze with free access to all three arms for 5 min. A 1-h intertrial interval was chosen because the recognition memory of mice in the Y-maze paradigm has been shown to fail after more than 4 h.²⁵ All trials were recorded with a ceiling-mounted CCD camera and analyzed later on a computer. The animal's behavior (locomotive speed, locomotive distance, and time spent in each arm) were analyzed, and the novelty (Novel arm) versus familiarity (Other arm) were compared during the retention trial. Data were expressed as percentage of total time.^26,27

Determination of oxidative stress

For each mouse, the hippocampus was removed and homogenized in 10:1 (vol/wt) ice-cold phosphate-buffered saline. A quantity of the homogenate was used to assess standard cellular markers of oxidative stress. The activities of catalase (CAT) and glutathione peroxidase (GSH-PX), total antioxidative capacity (T-AOC), and the concentration of malondialdehyde (MDA) from the hippocampal samples were measured spectrophotometrically with commercially available kits according to the manufacturers' instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Protein concentrations were determined according to the method of Lowry et al.²⁸

Statistical analysis

Statistical analysis was performed using SPSS (Chicago, IL, USA) software. All results were expressed as mean±SEM values. The results were analyzed by a one-way analysis of variance followed by a post hoc least significant difference (LSD) test. Statistical significance was set at P<.05.

Results

Body weight

During the treatment period, all animals were in the growth state. There was no significant difference between the two treatment groups (HFD and HFD+LA) in daily food intake; however, both treatment groups exhibited significantly lower food intake compared with the control. Nonetheless, there was a significant difference in weight gain across the three groups (Table 2).

Table 2.

Body Weight

	Control	HFD	HFD+LA
0 week	22.31±0.51	—	—
1 week	24.34±0.24	24.12±0.63	23.98±0.45
2 weeks	26.30±0.34	25.18±1.22	24.89±0.69
4 weeks	27.65±0.45	27.28±1.26	26.42±0.71
6 weeks	29.88±1.46	31.67±1.60	30.67±1.03
8 weeks	35.89±1.38^a	38.65±2.11^b	35.13±1.05^a
10 weeks	40.45±1.64^a	42.34±2.21^b	40.92±1.15^a

Data are mean±SD values for 12 animals.

Means with different superscript letters within a row are significantly different (P<.05).

HFD+LA, HFD plus 0.1% LA.

α-LA attenuated spatial recognition memory deficits induced by HFD

The effects of the HFD on the spatial recognition memory of mice are depicted in Figure 1. The percentage of time spent visiting the novel arm was significantly higher than the time spent exploring the other arm for mice fed a normal diet (F _2,35=22.38, P<.001; by LSD, novel vs. start P<.001, novel vs. other P<.001) and an HFD with an LA supplement (F _2,35=27.94, P<.001; by LSD, novel vs. start P<.001, novel vs. other P<.001). However, the percentage of time spent exploring the novel arm by HFD fed mice was not significantly different from the time spent visiting the other arm (F _2,35=1.63, P=1.64; by LSD, novel vs. start P=.24, novel vs. other P=.08). As shown in Figure 2, an arm preference (depicted as the percentage of total arm visits) was found for normal diet mice (F _2,35=40.78, P<.001; by LSD, novel vs. start P<.001, novel vs. other P<.001) and HFDA+LA mice (F _2,35=31.30, P<.001; by LSD, novel vs. start P<.001, novel vs. other P<.001). However, no arm preference was found in mice fed HFD only (F _2,35=1.46, P=.25; by LSD, novel vs. start P=.84, novel vs. other P=.12). The distances traveled and the speed of movement by the mice were not statistically significant different among the three groups (F _2,35=0.99, P=.38) (Fig. 3). These results indicated that an HFD or LA did not affect locomotor ability in the mice. This suggested that the HFD impaired the spatial recognition memory in the mice and that the LA administration attenuated this memory impairment.

FIG. 1.

Percentage of time spent visiting the novel arm, the start arm, and the other arm in the Y-maze spatial learning recognition task. Treatment groups were fed with a normal diet, HFD, or HFD+LA for 10 consecutive weeks. An HFD impaired the spatial learning of the mice. In contrast, this impairment was ameliorated by supplementing the HFD with LA. Data are mean±SE values (n=12). *P<.05.

FIG. 2.

Percentage of times the mice visited the novel arm, the start arm, or the other arm in the Y-maze spatial learning recognition task. Treatment groups were fed with a normal diet, HFD, or HFD+LA for 10 consecutive weeks. An arm preference in the percentage of times visiting the novel arm visit was found in mice fed a normal diet and HFD+LA. However, no arm preference was found in mice fed HFD only. Data are mean±SE values (n=12). *P<.05.

FIG. 3.

Distance traveled by mice in the Y-maze. Treatment groups were fed with a normal diet, HFD, or HFD+LA for 10 consecutive weeks. The total distance traveled was not affected by either the HFD or HFD+LA. Data are mean±SE values (n=12).

α-LA attenuated hippocampal oxidative stress markers induced by HFD

The levels of CAT, GSH-PX, T-AOC, and MDA were evaluated as markers of oxidative stress in the mouse hippocampus. The experimental mice that were fed an HFD for 10 weeks were found to have undergone oxidative stress in the hippocampus. This was indicated by a decline in CAT and GSH-PX activities and in the T-AOC in the hippocampus from mice fed an HFD compared with controls. Furthermore, the level of MDA, a parameter of lipid oxidative damage, was significantly increased in HFD-fed mice compared with control mice. Moreover, the decline in CAT (F _2,35=95.2, P<.001), GSH-PX (F _2,35=278.2, P<.001), and T-AOC (F _2,35=45.1, P<.001) and the increase in MDA (F _2,35=55.2, P<.001) associated with the HFD were ameliorated in mice supplemented with LA (Fig. 4). All told, these results suggest that a HFD induces an imbalance between intracellular oxidants and antioxidants. Consequently, this imbalance lead to a level of oxidative stress in the hippocampus that may have caused oxidative damage to other biomolecules such as proteins and nucleic acids in addition to lipids.

FIG. 4.

Antioxidant status in the hippocampus of mice fed with a normal diet, an HFD, or HFD+LA: catalase (CAT), glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), and malondialdehyde (MDA). Mice were fed for 10 consecutive weeks. The HFD impaired the antioxidant status in the hippocampus of mice. In contrast, this impairment was ameliorated by supplementing the HFD with LA. Data are mean±SE values (n=12). *P<.05.

Discussion

In general, HFDs lead to significant increases in weight, glucose intolerance, insulin resistance and ultimately to obesity.^11,29 Furthermore, insulin resistance, which is usually associated with obesity, is believed to be a major underlying factor implicated in learning impairments.^3,18 This belief is supported by experimental evidence from HFD animal models. For example, mice fed HFDs have been found to have impaired spatial learning memory in the radial-arm maze paradigm.¹¹ This study presented here further explored the effect of HFDs on special learning in mice using the Y-maze paradigm. It is notable that the HFD administered here was also found to impair spatial learning, in support of the same causation. However, the molecular foundations for the connection between HFD and memory impairments are not fully established. Therefore, it is also important to note that this study found that LA was able to prevent the memory impairment caused by the HFD.

The present study focused on the hippocampus as it is well established to be important to learning and memory processes. Furthermore, several studies have implicated a functional role of hippocampal neurons in memory formation.¹³ Specifically, HFDs in animal studies have been found to damage hippocampal structure and function,¹⁴ and it was confirmed here that damage to the hippocampus caused by HFD is associated with oxidative stress.

The mechanism of oxidative stress in the hippocampus can be best explained by the oxygen paradox. That is, although oxygen is essential for aerobic life, excessive amounts of its free radical metabolic by-products are toxic. These free radicals, being highly unstable molecules with unpaired electrons, have considerable potential to reduce organic molecules and cause damage to cellular proteins, lipids, and nucleic acids.³⁰ The hippocampus is considered particularly vulnerable to oxidative damage for its comparatively high oxygen utilization, generation of excess free radical by-products, and its modest antioxidant defenses.¹⁷

However, it is not explicitly known that the oxidative damage associated with HFDs is the cause of the memory impairments witnessed in HFD animal models or in obese people with cognitive deficits. Therefore, the HFD administered here was also supplemented with LA, which has strong antioxidant properties and is known to quench a number of free radical species.²³ The findings that LA administration prevented the build-up of oxidative stress markers in the hippocampus and attenuated spatial recognition memory deficits induced by a HFD suggest that there is a direct relationship between the two. These results, together with other studies, further suggest that LA, being a strong antioxidant, may be beneficial to ameliorate cognitive damage induced by HFDs.^19
–21

In summary, we postulated that an HFD would induce oxidative damage in the hippocampus, thereby damaging hippocampal structure and function and leading to memory impairments. We then evaluated hippocampal learning deficits in the Y-maze with mice fed a normal diet, an HFD, or an HFD supplemented with LA. The results presented here confirmed that HFDs negatively affect hippocampal learning and memory. More important is that they suggested that these impairments can be ameliorated by LA supplementation.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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