Life Sciences 110 (2014) 53–60
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Mitochondrial dysfunction and oxidative damage in the brain of diet-induced obese rats but not in diet-resistant rats Weiwei Ma 1, Linhong Yuan 1, Huanling Yu, Yuandi Xi, Rong Xiao ⁎ School of Public Health, Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China
a r t i c l e
i n f o
Article history: Received 2 May 2014 Accepted 12 July 2014 Available online 21 July 2014 Keywords: High-fat diet Diet-induced obese rat Diet-resistant rats Oxidative damage Mitochondrial dysfunction
a b s t r a c t Aims: It has been suggested that obesity triggered by consuming a high-fat diet (HF) can account for oxidative damage and mitochondrial dysfunction. Thus, we aim to explore the oxidative stress and mitochondrial dysfunction detected in the brain of diet-induced obese (DIO) rats. Main methods: Sprague–Dawley (SD) rats were fed either a HF diet or a normal-fat (NF) diet for 10 weeks to obtain the control (CON), DIO and diet-resistant (DR) rats. D-Galactose was injected subcutaneously for 10 weeks to establish oxidative stress model (MOD) rats. Then, the levels of total antioxidant capacity (T-AOC), lipid peroxidation (LPO), malondialdehyde (MDA), both in plasma and brain tissue, and catalase (CAT) in plasma were measured using enzymic assay kits and the levels of ghrelin, neuropeptide Y (NPY) and leptin in both plasma and brain tissue were measured by using enzyme-linked immunosorbent assay (ELISA) kits. Mitochondrial reactive oxygen species (ROS) formation in brain tissues was detected with 2, 7-dichlorofluorescein diacetate (DCFH2-DA) dyeing. The mitochondrial membrane potential (MMP) was measured with tetrachloro-tetraethyl benzimidazol carbocyanine iodide (JC-1) by a flow cytometer. Key findings: HF diet leads to an obese or DR state characterized by increased or decreased adiposity. The HF diet increased brain LPO, which was accompanied by lower ghrelin levels in DIO rats compared with DR rats. In addition, the increased mitochondrial ROS and lower MMP were detected in DIO rat comparing with DR rats. Significance: The current results demonstrated that mitochondrial dysfunction and oxidative damage in the brains of DIO rats, induced by HF diets, might be measurable. © 2014 Elsevier Inc. All rights reserved.
Introduction Obesity has emerged as a public health problem associated with a number of diseases. Increased intake of high energy-density foods, such as those seen in high-fat (HF) diets, is the main reason for obesity. The availability of useful animal models, such as diet-induced obese (DIO) and diet-resistant (DR) rats, is crucial in the search for novel compounds for the treatment of obesity. Levin (Levin et al., 1983) first found that when Sprague–Dawley rats were fed a HF diet, some rats developed obesity while others remained lean. This phenomenon has also been confirmed in our laboratory (Li et al., 2011; Zhao et al., 2008). In recent years, increasing attention has been paid on the relationship between obesity and brain diseases (Shefer et al., 2013), such as Alzheimer's disease (AD). AD is characterized by increased betaamyloid deposition and neuronal dysfunction (Ding et al., 2013). HF
⁎ Corresponding author at: No. 10 Xitoutiao, You An Men, Beijing 100069, China. Tel./ fax: +86 010 83911512. E-mail address: [email protected] (R. Xiao). 1 Co-first author: contributed equally to this work.
http://dx.doi.org/10.1016/j.lfs.2014.07.018 0024-3205/© 2014 Elsevier Inc. All rights reserved.
diets were reported to increase brain expression of beta-amyloid associated with memory impairment (Thirumangalakudi et al., 2008; Knight et al., 2014; Pistell et al., 2010). Epidemiological studies also showed that people with higher body mass index (BMI) are at greater risk for developing AD than the subjects with normal BMI (Fitzpatrick et al., 2009; Whitmer et al., 2007). The linkage between obesity and cognitive impairment is also strongly supported by epidemiological cross-sectional and prospective studies (Yaffe et al., 2004). However, the neurobiological damage caused by obesity is poorly understood. It has been reported that obesity is associated with lower brain volumes in normal subjects (Ho pril et al., 2010), suggesting that the brain is involved in the pathogenesis of obesity. Obesity has been associated with structural abnormalities in the brain (Smucny et al., 2012). Animal studies also showed that high-energy diet can damage hippocampal structure and function (Davidson et al., 2009, 2012; Kanoski et al., 2010). Recent studies indicate that various mechanisms linking obesity to cognitive dysfunction have been postulated, including oxidative stress. A HF diet is correlated with increased oxidative stress. Brain function is sensitive to oxidative pathways, the expression of which is enhanced in the obese state (Rege et al., 2013). Obesity linked to mitochondrial
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dysfunction in neurons can cause impairment in central glucose sensing (Parton et al., 2007), which indicates that the mitochondria may play an important role in obesity. DIO mice differed from DR mice in a number of neurotransmitter systems, including decreased mRNA expression and increased NPY mRNA expression in the hypothalamic nucleus (Huang et al., 2003). However, the differences between the brain of DIO and DR rats have not been studied. Therefore, the aim of the study is to explore the oxidative stress and mitochondrial dysfunction in the brain of DIO and DR rats. The study will provide a base to uncover the relationship between obesity and brain oxidative damage by comparing the difference between DIO and DR rats. Materials and methods Animals and diets All experimental procedures were approved by the Animal Ethics Committee of Capital Medical University and conducted in compliance with the animal-use guidelines. Male 6-week-old Sprague–Dawley rats (n = 50; body weight 140– 160 g; SPF degree) were purchased from Academy of Military Medical Sciences (Beijing, China). All of the rats were housed in plastic boxes individually at 20–23 °C with available food and water. A 12:12 h light dark cycle with lights on at 8:00 am was maintained. Rats were fed with standard laboratory chow for the first week to adapt to the new environment. In the following experimental period, rats were given either a NF diet (345.3 kcal/100 g) or a HF diet (435.96 kcal/100 g) (Table 1). The HF and NF diet formulations (SPF degree), made from semisynthetic materials, were purchased form Academy of Military Medical Sciences (Beijing, China). Experimental protocol The experimental schedule and study groups are shown in Fig. 1. After the acclimation period, 10 rats were randomly assigned to receive a NF diet according to their body weights, and D -galactose (120 mg/kg·day) was injected subcutaneously through the back of the neck for 10 weeks to establish model (MOD) rats. Another 40 rats were placed on a HF diet for 2 weeks. Then, the 10 intermediate weight gainers were switched back to a NF diet and were designated as controls (CON). Another 30 rats were fed a HF diet continually, and 8 weeks later the upper tertile (n = 10) in body weight gained was designated as DIO, and the lower tertile (n = 10) in body weight gained was designated as diet-resistant (DR). Those in the middle tertile of body weight gained were removed from the experiment. The rats were anesthetized and the blood was collected from the heart. The brain, heart, liver, spleen, perirenal fat, epididymal fat and omental fat were all removed and weighed.
collected from the heart and immediately transferred into chilled polypropylene tubes for plasma preparation. These tubes were gently rocked several times immediately after blood collection to obtain an even mixture and to prevent coagulation. Blood samples were centrifuged at 3500 g for 15 min at 4 °C. Plasma was collected and stored at −80°C until the assay. Isolation of mitochondria of brain tissue Mitochondrial/cytoplasmatic protein extraction kits (Calbiochem, America) were used to isolate the mitochondria. Brain tissues were washed with ice-cold PBS and added to a 1 ml cytosol extraction buffer mix and incubated on ice for 10 min. We homogenized the tissues using an ice-cold Dounce tissue homogenizer. The tissues were maintained on ice during the homogenization procedure. In general, 30–50 passes with the grinder were used, as recommended. The homogenate was transferred to a 1.5 ml microcentrifuge tube and centrifuged at 700 g for 10 min at 4 °C. The supernatant was then transferred to a 1.5 ml microcentrifuge tube and centrifuged at 10,000 g for 30 min at 4 °C. The supernatant was finally transferred to a clean tube, and what remains is the cytosolic fraction and the precipitates are the mitochondria of brain tissues. Measurement of T-AOC, LPO, MDA and CAT in plasma The T-AOC, LPO, MDA and CAT level in plasma were measured using assay kits purchased from Nanjing Jiancheng Biotechnology Company (China). The activity of T-AOC, the LPO concentration, the MDA concentration and the CAT activity were calculated. Measurement of ghrelin, NPY and leptin in plasma and brain tissue Since all the rats were fasting for 12 h, the ghrelin, NPY and leptin level in plasma and brain tissue were measured using assay kits purchased from Ray Biotechnology Company (America). Sigma Plot software, which can perform four-parameter logistic regression models, was used to calculate the concentration of ghrelin, NPY and leptin. Measurement of mitochondrial ROS in brain tissues Mitochondrial reactive oxygen species (ROS) formation was detected with 2, 7-dichlorofluorescein diacetate (DCFH2-DA), a fluorescent probe, according to the instruction of ROS assay kit (Beyotime Institute of Biotechnology, China) with a slight change. The isolated mitochondria were incubated with 10 μM DCFH2-DA dissolved in none-serum DMEM at 37 °C for 20 min. The fluorescence was then measured at 488 nm excitation and 525 nm emission by a Cary Eclipse fluorescence spectrometer (Agilent Technologies, USA). Measurement of mitochondrial membrane potential
Preparation of plasma samples Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg per body weight), and blood samples were Table 1 Composition of the normal fat and high fat diets.a Diet component
Energy (NF diet, %)
Energy (HF diet, %)
Total crude protein Crude fat Crude carbohydrate
24 10.7 65.3
16 40.5 43.5
The HF diet is composed of 84 g NF diet and 16 g lard oil (added with 0.2 g cholesterol). NF, normal fat; HF, high fat. a In the diets, all micronutrients, proteins and fiber were balanced by energy according to the Chinese standard.
For assessment of changes in mitochondrial membrane potential (MMP), the brain mitochondria were incubated for 20 min with JC-1 (BD Bioscience, USA) at 37 °C. The fluorescence signals of cells were excited at 488 nm and emission was monitored at 525 nm wavelength in a flow cytometer, corresponding to the fluorescence peak of the monomer and that of the aggregate. The value of fluorescence intensity was analyzed by the FCS Express Version 3.0 software (De Novo Software, Canada). Statistical analysis Data are presented as means and their standard errors. All statistical analyses were performed using SPSS 13.0. All data were analyzed by a one-way ANOVA, followed by LSD test and Dunnett T3 test.
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Fig. 1. Experimental schedule and groups of the study. NF: normal fat; HF: high fat; DIO: diet-induced obesity; DR: diet resistant; CON: control; MOD: oxidative stress.
other rats (P b 0.05), which suggests that the body weight increase was related to diet composition and energy intake.
Results Body weights of DIO and DR rats
The body fat content of DIO and DR rats The body weight (g) of each rat was recorded weekly. A one-way ANOVA revealed significant differences in body weight between different groups. The initial body weight (0 weeks) of DIO rats were significantly higher than that in other groups (P b 0.05). After 10 weeks on a HF diet, a distinct phenotype of body weight was observed: DIO rats gained weight more rapidly than did DR and control rats (P b 0.05); DR rats gained less weight than DIO rats did despite consuming the same diet (P b 0.05). Consuming a HF diet significantly modified the weight gain in obese rats, and also led to obesity-resistance in low body weight rats (Table 2).
As shown in Fig. 2, the perirenal fat, epididymal fat, omental fat and body fat content of DIO rats were significantly higher than that of CON, MOD and DR rats (P b 0.05). Heavier visceral fat in DIO rats and lighter visceral fat in DR rats induced by a HF diet indicate the successful establishment of the DIO and DR rat models. The organ coefficient in DIO and DR rats The results from calculating the organ coefficient showed that DIO rats have lower brain and heart weight than CON, MOD and DR rats (P b 0.05). However, MOD rats had heavier spleens than DIO and DR rats (P b 0.05). In addition, DIO and DR rats had greater liver weight than that of CON rats (P b 0.05), but there was no significant difference when compared with MOD rats (Table 5).
Food and caloric intake of DIO and DR rats Food intake was measured every week. Food was weighed, and remaining food and spillage were collected and weighed at the end of each 7 day period (Table 3). Energy intake (kcal) was determined by multiplying the food weight (g) and energy density (kcal/g) of each diet together. At the end of the tenth week, the diet intake among the CON, MOD and DIO rats showed no significant differences, while DR rats consumed less than other rats (P b 0.05). The calories were also calculated by food intake (Table 4). At the end of the first week, CON rats consumed fewer calories than MOD, DIO and DR rats (P b 0.05), whereas DIO rats consumed more calories than DR rats (P b 0.05). After 10 weeks of a HF diet or a NF diet, the energy intake of DIO rats was higher than that of the CON, MOD and DR rats (P b 0.05). The results showed that the caloric intake of DIO rats was higher than that of
Oxidative parameters in plasma and brain tissue of DIO and DR rats The T-AOC levels in both plasma and brain tissue showed no significant differences among CON, MOD, DIO and DR rats, and neither did the CAT levels in the rats' plasma. Compared to CON and MOD rats, DIO and DR rats had higher levels of LPO in plasma, whereas DIO and MOD rats showed higher LPO levels in brain tissue than CON and DR rats (P b 0.05). The MDA levels in the rats of the four groups were similar to the LPO levels in both plasma and brain tissue, but there were no significant differences among groups (P N 0.05) (Table 6).
Table 2 Body weights of rats in different groups (mean ± SE, n = 10). Body weight (g)
CON
0W (g) 1W (g) 2W (g) 3W (g) 4W (g) 5W (g) 6W (g) 7W (g) 8W (g) 9W (g) 10W (g)
180.32 241.17 309.53 366.33 408.14 455.88 491.79 512.47 536.90 559.75 580.96
MOD ± ± ± ± ± ± ± ± ± ± ±
2.15c 2.04c 0.78c 5.39c 7.13c 7.04c 6.97c 10.01c 10.89c 11.72c 11.64c
181.46 239.43 309.86 369.01 412.18 458.62 493.77 517.64 546.08 567.51 588.89
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
DIO ± ± ± ± ± ± ± ± ± ± ±
2.08c 4.76c 4.07c 5.21c 6.80c 8.54c 11.60c 11.75c 12.87c 13.71c 14.48c
189.88 261.28 335.43 402.65 462.46 512.42 565.05 603.06 641.79 669.37 701.06
DR ± ± ± ± ± ± ± ± ± ± ±
3.90a,b 5.45a,b 6.48a,b 7.81a,b 9.27a,b 12.86 a,b 12.55a,b 13.27a,b 13.47a,b 14.04a,b 14.95a,b
172.45 233.46 295.37 345.35 394.42 432.19 470.76 501.15 525.90 539.10 556.17
± ± ± ± ± ± ± ± ± ± ±
2.69b,c 4.91c 3.56a,c 6.93a,b,c 8.03c 9.05c 9.59c 10.23c 9.99c 10.81c 12.11c
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Table 3 The diet intake of DIO and DR rats induced by high-fat (mean ± SE, n = 10). Diet intake (g)
CON
1W (g) 2W (g) 3W (g) 4W (g) 5W (g) 6W (g) 7W (g) 8W (g) 9W (g) 10W (g)
176.66 349.14 509.09 714.65 938.40 1147.39 1359.38 1578.60 1783.78 1991.75
MOD ± ± ± ± ± ± ± ± ± ±
6.24b 9.92b 11.18 20.48b 23.50 27.37 31.78 37.48 44.51 49.93
194.66 398.16 540.03 766.89 994.78 1211.67 1433.21 1657.90 1858.03 2077.99
DIO ± ± ± ± ± ± ± ± ± ±
7.78a 10.30ac 15.80 18.87a 23.78 27.94 32.75 38.59 59.31 54.89
182.41 360.84 510.94 732.36 950.60 1174.73 1391.87 1624.56 1865.36 2103.19
DR ± ± ± ± ± ± ± ± ± ±
5.25 17.09b 15.75 14.90 16.83 20.92 26.59 31.59 34.51 38.76
160.45 323.06 489.56 665.76 840.42 1020.79 1192.09 1371.70 1550.87 1719.23
± ± ± ± ± ± ± ± ± ±
2.65b,c 6.21a,b,c 8.54 12.547a,b,c 17.58a,b,c 20.55a,b,c 25.81a,b,c 28.98a,b,c 33.18a,b,c 36.36a,b,c
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
induced obese (DIO) and diet-resistant (DR) rat models are useful and crucial models in the search for treatment of obesity, and thus have been used to acquire information on obesity. To determine the factors closely related to obesity, it would be advisable to investigate differences between DIO and DR rats. In the present study, we investigated the hypothesis that obesity triggered by high-fat diets can account for oxidative damage in the brain and deficits of cognitive function. D-Galactose, result in oxidative stress and learning and memory impairment in rats, was injected subcutaneously for 10 weeks to be considered as model (MOD) rats. Another 40 rats were placed on a HF diet (Table 1), and 2 weeks later the 10 intermediate weight gainers, which were insensitive to HF diets, were switched back to the NF (Table 1) diet and were designated as controls (CON). Another 30 rats were fed with HF diet continually for 8 weeks. The diet treatment containing an elevated percentage (40%) of saturated fat caused a rapid increase of body weight (BW) and adiposity in rats and these rats were called DIO rats. In addition, according to the Levin's method (Levin et al., 1983), we also produced the DR rats though the same HF diet consuming as the DIO rats. It should be noted that HF diets, such as the diet used in our study, evoke milder obesity and obesity resistance. In this study, we also observed that the initial body weight (0 weeks) of DIO rats were significantly higher than that in CON, MOD and DR rats. These results indicate the rats which have heavier body weights at the beginning developed DIO rats, which also suggest that high fat diet is not the only factor contributing to obesity. The DIO rats used in our study underwent 10 weeks dietary treatment, which led to BW gain and enlargement of adipose pads in some rats, and lower BW and smaller adipose pads in the DR rats. Moreover, parameters such as food intake and energy intake in DIO rats was also altered, which is consistent with heavier BW and adiposity. DIO rats seem to eat more HF diets and consume more calories than DR rats,
Oxidative-related factors in both plasma and brain tissue of DIO and DR rats The level of leptin in the plasma of rats showed no significant differences among these four groups. However, compared with the CON group, the MOD, DIO and DR rats had significantly higher leptin levels in brain tissues (P b 0.05), and the MOD, DIO and DR groups has no significant differences between each other. The NPY levels in the plasma of DIO and DR rats were significantly lower than that of CON and MOD rats, whereas the NPY levels in the brain tissue of MOD, DIO and DR rats were significantly higher than that of CON rats (P b 0.05). The ghrelin levels in plasma of MOD and DIO rats were higher than that of CON rats, whereas DR rats had higher ghrelin levels in brain tissue than MOD and DIO rats (P b 0.05) (Table 7). ROS formation in mitochondrial in the brain of DIO and DR rats In Fig. 3, HF diets increased ROS formation in DIO animals compared with CON and DR rats. The MOD rats also showed significantly more ROS formation than CON rats, which indicated the contribution of D-galactose treatment to oxidative damage in the rat brain (P b 0.05). MMP of DIO and DR rats Fig. 4 shows the FSC, SSC and fluorescence intensity, and the ratio of P2/P3 in these four groups. The ratio of P2/P3 and fluorescence intensity in MOD and DIO rats were decreased compared with CON and DR rats (P b 0.05). Discussion Humans and other mammals show considerable inter-individual variation in susceptibility to weight gain in response to HF diets. Diet-
Table 4 The energy intake of DIO and DR rats induced by high-fat (mean ± SE, n = 10). Energy intake (kcal)
CON
1W (kcal) 2W (kcal) 3W (kcal) 4W (kcal) 5W (kcal) 6W (kcal) 7W (kcal) 8W (kcal) 9W (kcal) 10W (kcal)
610.00 1205.58 1757.89 2467.69 3240.30 3961.94 4693.94 5450.91 6159.39 6877.51
MOD ± ± ± ± ± ± ± ± ± ±
21.56b,c 34.27b,c 38.59b,c 70.71b,c 81.13b,c 94.50b,c 109.74b,c 129.41b,c 153.69b,c 172.40b,c
847.94 1734.39 2400.03 3183.38 3970.29 4719.21 5484.18 6260.04 6951.09 7710.61
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
DIO ± ± ± ± ± ± ± ± ± ±
33.89a 44.86a 59.26a 71.87a 88.92a 103.87a,c 120.10a,c 140.10a,c 210.51a,c 196.45a,c
794.58 1640.71 2268.61 3225.52 4170.43 5139.64 6081.61 7095.68 8137.64 9166.09
DR ± ± ± ± ± ± ± ± ± ±
22.85a 31.60a 59.79a 60.87a 74.96a 98.74a,b 127.80a,b 152.36a,b 167.50a,b 188.68a,b
698.92 1407.25 2132.52 2900.05 3660.87 4446.56 5192.74 5975.13 6755.59 7488.97
± ± ± ± ± ± ± ± ± ±
11.56a,b,c 27.04a,b,c 37.21a,b 54.66a,b,c 76.56a,b,c 89.52a,c 112.42a,c 126.22a,c 144.53a,c 158.36a,c
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Fig. 2. The body fat content in DIO and DR rats induced by high-fat diet (mean = SE: n = 10). Mean values were significantly different from those of the CON group: aP b 0.05; mean values were significantly different from those of the MOD group: cP b 0.05; mean values were significantly different from those of the MOD group: bP b 0.05; DIO: diet-induced obesity; DR: diet resistant; CON: control; MOD: oxidative stress. Body fat content = (perirenal fat + testicular fat + omental fat) body weight ∗ 100.
which indicate a high energy metabolism in DIO rats compared to DR rats, despite the same HF diet consuming. Brain structural integrity reflects underlying neuronal health. Neuron apoptosis, which is a common pathological phenomenon in nervous system diseases, has been reported to be linked strongly with lipid metabolism. Moreover, hippocampus-dependent learning mechanisms appear to contribute to the control of food intake (Davidson et al., 2009) and interfere with body weight regulation, perhaps controlling appetite and behavior. It has been reported that people who consume excess energy and fat develop AD at twice the rate compared to the general population, and obese people have smaller overall brain and hippocampal volumes (Pothos et al., 2009). Obesity induced by a HF diet can affect fetal brain development (Niculescu and Lupu, 2009). These data are consistent with our study on body weight and brain parameters in DIO rats. We found that HF diet-induced DIO rats have lower brain weight than that of DR and MOD rats. Moreover, we observed that the MOD rats had greater spleen weight than DIO and DR rats. It is well known that the spleen is an organ involved in the immune system, and spleen atrophy can prompt the body's immune function to decline, which contributes to changes in learning and memory ability (Haley et al., 2011). Therefore, the data of the present study indicated that HF diets might cause changes in rat organs, and the DIO rats appeared to have much more brain damage than DR rats. The oxidation of the brain can produce more byproducts than the other parts of body, and the brain is highly susceptible to oxidative damage (Christian, 2004). Furukawa (Furukawa et al., 2004) reported that the oxidative stress levels in plasma from obese mice were elevated compared to control mice. In the present study, we further detected Table 5 The organ coefficient in DIO and DR rats induced by high-fat diet (mean ± SE, n = 10). Group
Brain/weight
CON MOD DIO DR
0.28 0.28 0.23 0.28
± ± ± ±
0.0063c 0.0057c 0.0064a,b 0.0052c
Heart/weight 0.28 0.29 0.25 0.27
± ± ± ±
0.0077c 0.0064a 0.0063b,c 0.0029b,c
Spleen/weight
Liver/weight
0.15 0.16 0.13 0.14
2.58 2.77 2.89 2.95
± ± ± ±
0.0064 0.0072c 0.0054b 0.012b
± ± ± ±
0.072c 0.15 0.076a 0.074a
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
the biomarkers associated with oxidative damage in plasma and brain tissue. We detected a positive correlation between obesity and LPO levels and ROS in mitochondria. Elevated levels of LPO have been linked to injurious effects, such as loss of cell membrane potential (MMP). Excess ROS can make polyunsaturated fatty acids generate LPO, ruining the biological membrane and contributing to abnormal function. In 3T3-L1 adipocytes, increases in ROS occur with lipid accumulation. ROS may contribute to the development of obesity. Together with these reports, we speculate that consuming a HF diet induced the accumulation of adipose in rats and contributed to the increased LPO and ROS in plasma or in brain mitochondria. Additionally, the increased LPO in the brain and the accumulation of ROS in brain mitochondria, which is indicative of oxidative damage, were mainly detected in DIO and MOD rats, but not in DR rats. These data further prove that HF diets have a similar effect to D-galactose in inducing oxidative damage in the brain. A main finding of the current work is that 10 weeks of HF diet leads to oxidative stress in the brain tissue of DIO rats but not DR rats. However, we also observed the increased plasma LPO in DR rats, which was not consistent with the LPO level in the brain. HF intake may also result to the increased LPO level in DR rats. MMP can reflect the opening of the mitochondrial permeability transition pore (PTP) and the mitochondrial integrity. The PTP opening may result in ROS release from mitochondria, and then intracellular ROS levels may be increased. MMP is a good indicator of mitochondrial function and is used to study signaling mechanisms involved in oxidative stress. To evaluate associations between HF diet intervention and brain mitochondria, we investigate the MMP of rat brain. The results showed that the ratio of P2/P3 and fluorescence intensity in MOD and DIO rats were decreased compared to CON and DR rats. These data suggest the mitochondrial dysfunction in DIO and MOD rats, but not in the DR rats (Mantena et al., 2008; Zou et al., 2014). There is active apoptotic signaling in the hypothalamus of rodents placed on a high fat diet (De Souza et al., 2005). Hormonal alterations were associated with cognitive function in obesity, such as leptin, cholecystokinin (CCK), glucagon-like peptide (GLP-1) and brain derived neurotrophic factor (BDNF) (Nogueiras et al., 2009; Paz-Filho et al., 2008). DIO rats exhibit a different behavioral and neuronal response to hormones such as CCK than DR rats (Swartz et al., 2010). There is also the comparison of the NPY expression in DIO rats and DR rats (Huang et al., 2003).
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Table 6 The T-AOC, CAT, LPO and MDA levels both in the plasma (mean ± SE, n = 10) and brain tissue (mean ± SE, n = 8) of DIO and DR rats induced by high-fat diet. Group
T-AOC
MDA
Plasma (U/ml) CON MOD DIO DR
3.89 3.86 4.93 4.50
± ± ± ±
0.34 0.34 0.56 0.36
LPO
Brain tissue (U/mg prot)
Plasma (nmol/ml)
Brain tissue (nmol/mg prot)
Plasma (nmol/ml)
3.98 0.50 0.83 2.30
5.82 5.70 7.04 6.14
6.87 7.45 7.41 7.04
5.01 5.00 6.76 7.03
± ± ± ±
1.60 0.29 0.19 1.38
± ± ± ±
0.42 0.52 0.40 0.23
± ± ± ±
0.68 1.54 0.51 0.77
± ± ± ±
0.52c 0.64c 0.50a,b 0.50a,b
Brain tissue (nmol/mg prot) 7.45 10.54 10.09 5.84
± ± ± ±
0.59b,c 0.98a 0.89a 0.43b,c
CAT (U/ml)
1.65 1.83 2.63 2.60
± ± ± ±
0.22 0.41 0.34 0.46
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress; T-AOC, total antioxidant capacity; CAT, catalase; LPO, lipid peroxide; MDA, malonaldehyde. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
Table 7 The leptin, NPY, ghrelin levels in both plasma and brain tissue of DIO and DR rats induced by high-fat diet (n = 6–8). Group
Leptin
NPY
Plasma (pg/ml) CON MOD DIO DR
1432.57 655.63 1210.87 1013.80
Tissue (pg/mg prot) ± ± ± ±
494.09 95.16 349.74 441.80
3864.30 6006.80 5603.43 6219.27
± ± ± ±
641.59b,c 629.31a 407.16a 428.04a
Ghrelin
Plasma (pg/ml) 428.17 400.11 206.30 120.84
± ± ± ±
Tissue (pg/mg prot) 77.18c 88.78c 44.63,ab 45.53a,b
605.31 867.43 877.74 966.32
± ± ± ±
92.66b,c 82.72a 58.54a 44.99a
Plasma (pg/ml) 45.12 98.20 158.46 91.73
± ± ± ±
Tissue (pg/mg prot) 5.58b,c 11.87a 23.28a 12.86
17.33 7.30 11.86 62.99
± ± ± ±
4.76 3.69 3.37 13.08b,c
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress; NPY, neuropeptide Y. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
Fig. 3. A Forward scatter (FS) and side scatter (SS) of brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry. B and C Fluorescence intensity of the brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry (mean = SE: n = 6). Mean values were significantly different from those of the CON group: aP b 0.05; mean values were significantly different from those of the MOD group: cP b ().05; mean values were significantly different from those of the MOD group: bP b 0.05; DIO: diet-induced obesity; DR: diet resistant; CON: control; MOD: oxidative stress.
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Fig. 4. A Forward scatter (FS) and side scatter (SS) of brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry. B Fluorescence intensity of the brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry. C The ratio of green and red of brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry. D Fluorescence intensity, the ratio of green and red, the forward scatter (FS) and side scatter (SS) of brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry (mean = SE, n = 6). Mean values were significantly different from those of the CON group: aP b 0.05; mean values were significantly different from those of the MOD group: cP b 0.05; mean values were significantly different from those of the MOD group: bP b 0.05; DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress.
Leptin replacement changes brain structure and improves cognitive function in a boy with leptin deficiency (Paz-Filho et al., 2008). Leptin might be associated with oxidative stress in patients with impaired glucose tolerance or diabetes (Nakanishi et al., 2005). Bouloumıe et al. (1999) reported that leptin led to ROS generation in cultured human umbilical vein endothelial cells. However, in our study, we did not observe a significant difference in leptin levels among DIO, DR and MOD rats (Bruijnzeel et al., 2013). Ghrelin was the first orexigenic protein to be identified. The physiological role of ghrelin is to stimulate growth hormone release, food intake, and gastric motility in both humans and rodents (Luttikhold et al., 2013; Gasco et al., 2010). Decreased plasma ghrelin levels may have a protective role against the development of obesity. The regulatory role of ghrelin in intracellular energy balance, autophagy and anti-
oxidative actions under cardiac hypoxic conditions has been described (Kishimoto et al., 2012) and shown in MES23.5 cells (Liu et al., 2010). Accordingly, in the current study, we wondered if ghrelin plays a role in obesity induced by a HF diet. We found that DR rats showed higher ghrelin levels in brain tissue than DIO rats did, which indicates that ghrelin is related to oxidative stress in the brain of DIO rats, but not DR rats, and is related to the HF diet. NPY is widely distributed in the central nervous system that has been associated with the modulation of several functions including food intake, learning, memory and neuroprotection (dos Santos et al., 2013). It was found that central administration of NPY prevents spatial memory deficits in mice and this response is mediated by prevention of oxidative stress. In our study, the NPY levels in the plasma of DIO and DR rats were higher than that of CON and MOD rats, whereas the NPY level
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in brain tissue of MOD, DIO and DR rats was significantly higher than that of CON rats. We did not find a NPY difference between DIO rats and DR rats in either plasma or brain tissue. Conclusion Obesity caused by a HF diet was accompanied by an increase of food intake, energy intake and enlarged fat pads in DIO rats. The HF diet also induced obesity resistance in experimental animals. Moreover, the HF diet-induced DIO animals exhibited oxidative damage, demonstrated by the elevated formation of ROS and mitochondrial dysfunction, in both plasma and brain tissues, but these effects were not seen in DR rats. The oxidative stress state appears in DIO rats, but not in DR rats, and contributed to the NPY, ghrelin and leptin changes. However, the current study did not explore molecular mechanisms to elucidate the relationship between HF diet-induced obesity and brain oxidative damage. Nevertheless, our work hints at an association of HF dietinduced obesity and brain oxidative damage, and our work provides basic data for a diet pattern-based strategy for preventing obesity. Conflict of interest There is no conflict of interest to report in this study.
Acknowledgments This work was supported by the grants from National Natural Science Foundation of China (No. 81102122). Ma Weiwei and Yuan Linhong contributed to the implementation of the experiment and data analysis; Xi Yuandi and Yu Huanling performed the animal experiment and data analysis; and Xiao Rong designed the study. References Bouloumıe A, Marumo T, Lafontan M, Busse R. Leptin induces oxidative stress in human endothelial cells. FASEB J 1999;13:1231–8. Bruijnzeel AW, Qi X, Corrie LW. Anorexic effects of intra-VTA leptin are similar in low-fat and high-fat-fed rats but attenuated in a subgroup of high-fat-fed obese rats. Pharmacol Biochem Behav 2013;103(3):573–81. Christian B. Alzheimer's disease and oxidative stress. Brain Res 2004;1000:32. Davidson TL, Chan K, Jarrard LE, Kanoski SE, Clegg DJ, Benoit SC. Contributions of the hippocampus and medial prefrontal cortex to energy and body weight regulation. Hippocampus 2009;19(3):235–52. Davidson TL, Monnot A, Neal AU, Martin AA, Horton JJ, Zheng W. The effects of a highenergy diet on hippocampal-dependent discrimination performance and blood– brain barrier integrity differ for diet-induced obese and diet-resistant rats. Physiol Behav 2012;107(1):26–33. De Souza CT, Araujo EP, Bordin S, Ashimine R, Zollner RL, Boschero AC, et al. Consumption of a fat-rich diet activates a proinflammatory response and induces insulin resistance in the hypothalamus. Endocrinology 2005;146:4192–9. Ding J, Yu HL, Ma WW, Xi YD, Zhao X, Yuan HL, et al. Soy isoflavone attenuates brain mitochondrial oxidative stress induced by beta-amyloid peptides 1–42 injection in lateral cerebral ventricle. J Neurosci Res 2013;91:562–7. dos Santos VV, Santos DB, Lach G, Rodrigues AL, Farina M, De Lima TC, et al. Neuropeptide Y (NPY) prevents depressive-like behavior, spatial memory deficits and oxidative stress following amyloid-β (Aβ1–40) administration in mice. Brain Res 2013;244: 107–15. Fitzpatrick AL, Kuller LH, Lopez OL, Diehr P, O'Meara ES, Longstreth Jr WT, et al. Midlife and late-life obesity and the risk of dementia: cardiovascular health study. Arch Neurol 2009;66:336–42. Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 2004;114:1752–61. Gasco V, Beccuti G, Marotta F, Benso A, Granata R, Broglio F, et al. Endocrine and metabolic actions of ghrelin. Endocr Dev 2010;17:86–95.
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Mitochondrial dysfunction and oxidative damage in the brain of diet-induced obese rats but not in diet-resistant rats Weiwei Ma 1, Linhong Yuan 1, Huanling Yu, Yuandi Xi, Rong Xiao ⁎ School of Public Health, Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China
a r t i c l e
i n f o
Article history: Received 2 May 2014 Accepted 12 July 2014 Available online 21 July 2014 Keywords: High-fat diet Diet-induced obese rat Diet-resistant rats Oxidative damage Mitochondrial dysfunction
a b s t r a c t Aims: It has been suggested that obesity triggered by consuming a high-fat diet (HF) can account for oxidative damage and mitochondrial dysfunction. Thus, we aim to explore the oxidative stress and mitochondrial dysfunction detected in the brain of diet-induced obese (DIO) rats. Main methods: Sprague–Dawley (SD) rats were fed either a HF diet or a normal-fat (NF) diet for 10 weeks to obtain the control (CON), DIO and diet-resistant (DR) rats. D-Galactose was injected subcutaneously for 10 weeks to establish oxidative stress model (MOD) rats. Then, the levels of total antioxidant capacity (T-AOC), lipid peroxidation (LPO), malondialdehyde (MDA), both in plasma and brain tissue, and catalase (CAT) in plasma were measured using enzymic assay kits and the levels of ghrelin, neuropeptide Y (NPY) and leptin in both plasma and brain tissue were measured by using enzyme-linked immunosorbent assay (ELISA) kits. Mitochondrial reactive oxygen species (ROS) formation in brain tissues was detected with 2, 7-dichlorofluorescein diacetate (DCFH2-DA) dyeing. The mitochondrial membrane potential (MMP) was measured with tetrachloro-tetraethyl benzimidazol carbocyanine iodide (JC-1) by a flow cytometer. Key findings: HF diet leads to an obese or DR state characterized by increased or decreased adiposity. The HF diet increased brain LPO, which was accompanied by lower ghrelin levels in DIO rats compared with DR rats. In addition, the increased mitochondrial ROS and lower MMP were detected in DIO rat comparing with DR rats. Significance: The current results demonstrated that mitochondrial dysfunction and oxidative damage in the brains of DIO rats, induced by HF diets, might be measurable. © 2014 Elsevier Inc. All rights reserved.
Introduction Obesity has emerged as a public health problem associated with a number of diseases. Increased intake of high energy-density foods, such as those seen in high-fat (HF) diets, is the main reason for obesity. The availability of useful animal models, such as diet-induced obese (DIO) and diet-resistant (DR) rats, is crucial in the search for novel compounds for the treatment of obesity. Levin (Levin et al., 1983) first found that when Sprague–Dawley rats were fed a HF diet, some rats developed obesity while others remained lean. This phenomenon has also been confirmed in our laboratory (Li et al., 2011; Zhao et al., 2008). In recent years, increasing attention has been paid on the relationship between obesity and brain diseases (Shefer et al., 2013), such as Alzheimer's disease (AD). AD is characterized by increased betaamyloid deposition and neuronal dysfunction (Ding et al., 2013). HF
⁎ Corresponding author at: No. 10 Xitoutiao, You An Men, Beijing 100069, China. Tel./ fax: +86 010 83911512. E-mail address: [email protected] (R. Xiao). 1 Co-first author: contributed equally to this work.
http://dx.doi.org/10.1016/j.lfs.2014.07.018 0024-3205/© 2014 Elsevier Inc. All rights reserved.
diets were reported to increase brain expression of beta-amyloid associated with memory impairment (Thirumangalakudi et al., 2008; Knight et al., 2014; Pistell et al., 2010). Epidemiological studies also showed that people with higher body mass index (BMI) are at greater risk for developing AD than the subjects with normal BMI (Fitzpatrick et al., 2009; Whitmer et al., 2007). The linkage between obesity and cognitive impairment is also strongly supported by epidemiological cross-sectional and prospective studies (Yaffe et al., 2004). However, the neurobiological damage caused by obesity is poorly understood. It has been reported that obesity is associated with lower brain volumes in normal subjects (Ho pril et al., 2010), suggesting that the brain is involved in the pathogenesis of obesity. Obesity has been associated with structural abnormalities in the brain (Smucny et al., 2012). Animal studies also showed that high-energy diet can damage hippocampal structure and function (Davidson et al., 2009, 2012; Kanoski et al., 2010). Recent studies indicate that various mechanisms linking obesity to cognitive dysfunction have been postulated, including oxidative stress. A HF diet is correlated with increased oxidative stress. Brain function is sensitive to oxidative pathways, the expression of which is enhanced in the obese state (Rege et al., 2013). Obesity linked to mitochondrial
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dysfunction in neurons can cause impairment in central glucose sensing (Parton et al., 2007), which indicates that the mitochondria may play an important role in obesity. DIO mice differed from DR mice in a number of neurotransmitter systems, including decreased mRNA expression and increased NPY mRNA expression in the hypothalamic nucleus (Huang et al., 2003). However, the differences between the brain of DIO and DR rats have not been studied. Therefore, the aim of the study is to explore the oxidative stress and mitochondrial dysfunction in the brain of DIO and DR rats. The study will provide a base to uncover the relationship between obesity and brain oxidative damage by comparing the difference between DIO and DR rats. Materials and methods Animals and diets All experimental procedures were approved by the Animal Ethics Committee of Capital Medical University and conducted in compliance with the animal-use guidelines. Male 6-week-old Sprague–Dawley rats (n = 50; body weight 140– 160 g; SPF degree) were purchased from Academy of Military Medical Sciences (Beijing, China). All of the rats were housed in plastic boxes individually at 20–23 °C with available food and water. A 12:12 h light dark cycle with lights on at 8:00 am was maintained. Rats were fed with standard laboratory chow for the first week to adapt to the new environment. In the following experimental period, rats were given either a NF diet (345.3 kcal/100 g) or a HF diet (435.96 kcal/100 g) (Table 1). The HF and NF diet formulations (SPF degree), made from semisynthetic materials, were purchased form Academy of Military Medical Sciences (Beijing, China). Experimental protocol The experimental schedule and study groups are shown in Fig. 1. After the acclimation period, 10 rats were randomly assigned to receive a NF diet according to their body weights, and D -galactose (120 mg/kg·day) was injected subcutaneously through the back of the neck for 10 weeks to establish model (MOD) rats. Another 40 rats were placed on a HF diet for 2 weeks. Then, the 10 intermediate weight gainers were switched back to a NF diet and were designated as controls (CON). Another 30 rats were fed a HF diet continually, and 8 weeks later the upper tertile (n = 10) in body weight gained was designated as DIO, and the lower tertile (n = 10) in body weight gained was designated as diet-resistant (DR). Those in the middle tertile of body weight gained were removed from the experiment. The rats were anesthetized and the blood was collected from the heart. The brain, heart, liver, spleen, perirenal fat, epididymal fat and omental fat were all removed and weighed.
collected from the heart and immediately transferred into chilled polypropylene tubes for plasma preparation. These tubes were gently rocked several times immediately after blood collection to obtain an even mixture and to prevent coagulation. Blood samples were centrifuged at 3500 g for 15 min at 4 °C. Plasma was collected and stored at −80°C until the assay. Isolation of mitochondria of brain tissue Mitochondrial/cytoplasmatic protein extraction kits (Calbiochem, America) were used to isolate the mitochondria. Brain tissues were washed with ice-cold PBS and added to a 1 ml cytosol extraction buffer mix and incubated on ice for 10 min. We homogenized the tissues using an ice-cold Dounce tissue homogenizer. The tissues were maintained on ice during the homogenization procedure. In general, 30–50 passes with the grinder were used, as recommended. The homogenate was transferred to a 1.5 ml microcentrifuge tube and centrifuged at 700 g for 10 min at 4 °C. The supernatant was then transferred to a 1.5 ml microcentrifuge tube and centrifuged at 10,000 g for 30 min at 4 °C. The supernatant was finally transferred to a clean tube, and what remains is the cytosolic fraction and the precipitates are the mitochondria of brain tissues. Measurement of T-AOC, LPO, MDA and CAT in plasma The T-AOC, LPO, MDA and CAT level in plasma were measured using assay kits purchased from Nanjing Jiancheng Biotechnology Company (China). The activity of T-AOC, the LPO concentration, the MDA concentration and the CAT activity were calculated. Measurement of ghrelin, NPY and leptin in plasma and brain tissue Since all the rats were fasting for 12 h, the ghrelin, NPY and leptin level in plasma and brain tissue were measured using assay kits purchased from Ray Biotechnology Company (America). Sigma Plot software, which can perform four-parameter logistic regression models, was used to calculate the concentration of ghrelin, NPY and leptin. Measurement of mitochondrial ROS in brain tissues Mitochondrial reactive oxygen species (ROS) formation was detected with 2, 7-dichlorofluorescein diacetate (DCFH2-DA), a fluorescent probe, according to the instruction of ROS assay kit (Beyotime Institute of Biotechnology, China) with a slight change. The isolated mitochondria were incubated with 10 μM DCFH2-DA dissolved in none-serum DMEM at 37 °C for 20 min. The fluorescence was then measured at 488 nm excitation and 525 nm emission by a Cary Eclipse fluorescence spectrometer (Agilent Technologies, USA). Measurement of mitochondrial membrane potential
Preparation of plasma samples Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg per body weight), and blood samples were Table 1 Composition of the normal fat and high fat diets.a Diet component
Energy (NF diet, %)
Energy (HF diet, %)
Total crude protein Crude fat Crude carbohydrate
24 10.7 65.3
16 40.5 43.5
The HF diet is composed of 84 g NF diet and 16 g lard oil (added with 0.2 g cholesterol). NF, normal fat; HF, high fat. a In the diets, all micronutrients, proteins and fiber were balanced by energy according to the Chinese standard.
For assessment of changes in mitochondrial membrane potential (MMP), the brain mitochondria were incubated for 20 min with JC-1 (BD Bioscience, USA) at 37 °C. The fluorescence signals of cells were excited at 488 nm and emission was monitored at 525 nm wavelength in a flow cytometer, corresponding to the fluorescence peak of the monomer and that of the aggregate. The value of fluorescence intensity was analyzed by the FCS Express Version 3.0 software (De Novo Software, Canada). Statistical analysis Data are presented as means and their standard errors. All statistical analyses were performed using SPSS 13.0. All data were analyzed by a one-way ANOVA, followed by LSD test and Dunnett T3 test.
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Fig. 1. Experimental schedule and groups of the study. NF: normal fat; HF: high fat; DIO: diet-induced obesity; DR: diet resistant; CON: control; MOD: oxidative stress.
other rats (P b 0.05), which suggests that the body weight increase was related to diet composition and energy intake.
Results Body weights of DIO and DR rats
The body fat content of DIO and DR rats The body weight (g) of each rat was recorded weekly. A one-way ANOVA revealed significant differences in body weight between different groups. The initial body weight (0 weeks) of DIO rats were significantly higher than that in other groups (P b 0.05). After 10 weeks on a HF diet, a distinct phenotype of body weight was observed: DIO rats gained weight more rapidly than did DR and control rats (P b 0.05); DR rats gained less weight than DIO rats did despite consuming the same diet (P b 0.05). Consuming a HF diet significantly modified the weight gain in obese rats, and also led to obesity-resistance in low body weight rats (Table 2).
As shown in Fig. 2, the perirenal fat, epididymal fat, omental fat and body fat content of DIO rats were significantly higher than that of CON, MOD and DR rats (P b 0.05). Heavier visceral fat in DIO rats and lighter visceral fat in DR rats induced by a HF diet indicate the successful establishment of the DIO and DR rat models. The organ coefficient in DIO and DR rats The results from calculating the organ coefficient showed that DIO rats have lower brain and heart weight than CON, MOD and DR rats (P b 0.05). However, MOD rats had heavier spleens than DIO and DR rats (P b 0.05). In addition, DIO and DR rats had greater liver weight than that of CON rats (P b 0.05), but there was no significant difference when compared with MOD rats (Table 5).
Food and caloric intake of DIO and DR rats Food intake was measured every week. Food was weighed, and remaining food and spillage were collected and weighed at the end of each 7 day period (Table 3). Energy intake (kcal) was determined by multiplying the food weight (g) and energy density (kcal/g) of each diet together. At the end of the tenth week, the diet intake among the CON, MOD and DIO rats showed no significant differences, while DR rats consumed less than other rats (P b 0.05). The calories were also calculated by food intake (Table 4). At the end of the first week, CON rats consumed fewer calories than MOD, DIO and DR rats (P b 0.05), whereas DIO rats consumed more calories than DR rats (P b 0.05). After 10 weeks of a HF diet or a NF diet, the energy intake of DIO rats was higher than that of the CON, MOD and DR rats (P b 0.05). The results showed that the caloric intake of DIO rats was higher than that of
Oxidative parameters in plasma and brain tissue of DIO and DR rats The T-AOC levels in both plasma and brain tissue showed no significant differences among CON, MOD, DIO and DR rats, and neither did the CAT levels in the rats' plasma. Compared to CON and MOD rats, DIO and DR rats had higher levels of LPO in plasma, whereas DIO and MOD rats showed higher LPO levels in brain tissue than CON and DR rats (P b 0.05). The MDA levels in the rats of the four groups were similar to the LPO levels in both plasma and brain tissue, but there were no significant differences among groups (P N 0.05) (Table 6).
Table 2 Body weights of rats in different groups (mean ± SE, n = 10). Body weight (g)
CON
0W (g) 1W (g) 2W (g) 3W (g) 4W (g) 5W (g) 6W (g) 7W (g) 8W (g) 9W (g) 10W (g)
180.32 241.17 309.53 366.33 408.14 455.88 491.79 512.47 536.90 559.75 580.96
MOD ± ± ± ± ± ± ± ± ± ± ±
2.15c 2.04c 0.78c 5.39c 7.13c 7.04c 6.97c 10.01c 10.89c 11.72c 11.64c
181.46 239.43 309.86 369.01 412.18 458.62 493.77 517.64 546.08 567.51 588.89
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
DIO ± ± ± ± ± ± ± ± ± ± ±
2.08c 4.76c 4.07c 5.21c 6.80c 8.54c 11.60c 11.75c 12.87c 13.71c 14.48c
189.88 261.28 335.43 402.65 462.46 512.42 565.05 603.06 641.79 669.37 701.06
DR ± ± ± ± ± ± ± ± ± ± ±
3.90a,b 5.45a,b 6.48a,b 7.81a,b 9.27a,b 12.86 a,b 12.55a,b 13.27a,b 13.47a,b 14.04a,b 14.95a,b
172.45 233.46 295.37 345.35 394.42 432.19 470.76 501.15 525.90 539.10 556.17
± ± ± ± ± ± ± ± ± ± ±
2.69b,c 4.91c 3.56a,c 6.93a,b,c 8.03c 9.05c 9.59c 10.23c 9.99c 10.81c 12.11c
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Table 3 The diet intake of DIO and DR rats induced by high-fat (mean ± SE, n = 10). Diet intake (g)
CON
1W (g) 2W (g) 3W (g) 4W (g) 5W (g) 6W (g) 7W (g) 8W (g) 9W (g) 10W (g)
176.66 349.14 509.09 714.65 938.40 1147.39 1359.38 1578.60 1783.78 1991.75
MOD ± ± ± ± ± ± ± ± ± ±
6.24b 9.92b 11.18 20.48b 23.50 27.37 31.78 37.48 44.51 49.93
194.66 398.16 540.03 766.89 994.78 1211.67 1433.21 1657.90 1858.03 2077.99
DIO ± ± ± ± ± ± ± ± ± ±
7.78a 10.30ac 15.80 18.87a 23.78 27.94 32.75 38.59 59.31 54.89
182.41 360.84 510.94 732.36 950.60 1174.73 1391.87 1624.56 1865.36 2103.19
DR ± ± ± ± ± ± ± ± ± ±
5.25 17.09b 15.75 14.90 16.83 20.92 26.59 31.59 34.51 38.76
160.45 323.06 489.56 665.76 840.42 1020.79 1192.09 1371.70 1550.87 1719.23
± ± ± ± ± ± ± ± ± ±
2.65b,c 6.21a,b,c 8.54 12.547a,b,c 17.58a,b,c 20.55a,b,c 25.81a,b,c 28.98a,b,c 33.18a,b,c 36.36a,b,c
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
induced obese (DIO) and diet-resistant (DR) rat models are useful and crucial models in the search for treatment of obesity, and thus have been used to acquire information on obesity. To determine the factors closely related to obesity, it would be advisable to investigate differences between DIO and DR rats. In the present study, we investigated the hypothesis that obesity triggered by high-fat diets can account for oxidative damage in the brain and deficits of cognitive function. D-Galactose, result in oxidative stress and learning and memory impairment in rats, was injected subcutaneously for 10 weeks to be considered as model (MOD) rats. Another 40 rats were placed on a HF diet (Table 1), and 2 weeks later the 10 intermediate weight gainers, which were insensitive to HF diets, were switched back to the NF (Table 1) diet and were designated as controls (CON). Another 30 rats were fed with HF diet continually for 8 weeks. The diet treatment containing an elevated percentage (40%) of saturated fat caused a rapid increase of body weight (BW) and adiposity in rats and these rats were called DIO rats. In addition, according to the Levin's method (Levin et al., 1983), we also produced the DR rats though the same HF diet consuming as the DIO rats. It should be noted that HF diets, such as the diet used in our study, evoke milder obesity and obesity resistance. In this study, we also observed that the initial body weight (0 weeks) of DIO rats were significantly higher than that in CON, MOD and DR rats. These results indicate the rats which have heavier body weights at the beginning developed DIO rats, which also suggest that high fat diet is not the only factor contributing to obesity. The DIO rats used in our study underwent 10 weeks dietary treatment, which led to BW gain and enlargement of adipose pads in some rats, and lower BW and smaller adipose pads in the DR rats. Moreover, parameters such as food intake and energy intake in DIO rats was also altered, which is consistent with heavier BW and adiposity. DIO rats seem to eat more HF diets and consume more calories than DR rats,
Oxidative-related factors in both plasma and brain tissue of DIO and DR rats The level of leptin in the plasma of rats showed no significant differences among these four groups. However, compared with the CON group, the MOD, DIO and DR rats had significantly higher leptin levels in brain tissues (P b 0.05), and the MOD, DIO and DR groups has no significant differences between each other. The NPY levels in the plasma of DIO and DR rats were significantly lower than that of CON and MOD rats, whereas the NPY levels in the brain tissue of MOD, DIO and DR rats were significantly higher than that of CON rats (P b 0.05). The ghrelin levels in plasma of MOD and DIO rats were higher than that of CON rats, whereas DR rats had higher ghrelin levels in brain tissue than MOD and DIO rats (P b 0.05) (Table 7). ROS formation in mitochondrial in the brain of DIO and DR rats In Fig. 3, HF diets increased ROS formation in DIO animals compared with CON and DR rats. The MOD rats also showed significantly more ROS formation than CON rats, which indicated the contribution of D-galactose treatment to oxidative damage in the rat brain (P b 0.05). MMP of DIO and DR rats Fig. 4 shows the FSC, SSC and fluorescence intensity, and the ratio of P2/P3 in these four groups. The ratio of P2/P3 and fluorescence intensity in MOD and DIO rats were decreased compared with CON and DR rats (P b 0.05). Discussion Humans and other mammals show considerable inter-individual variation in susceptibility to weight gain in response to HF diets. Diet-
Table 4 The energy intake of DIO and DR rats induced by high-fat (mean ± SE, n = 10). Energy intake (kcal)
CON
1W (kcal) 2W (kcal) 3W (kcal) 4W (kcal) 5W (kcal) 6W (kcal) 7W (kcal) 8W (kcal) 9W (kcal) 10W (kcal)
610.00 1205.58 1757.89 2467.69 3240.30 3961.94 4693.94 5450.91 6159.39 6877.51
MOD ± ± ± ± ± ± ± ± ± ±
21.56b,c 34.27b,c 38.59b,c 70.71b,c 81.13b,c 94.50b,c 109.74b,c 129.41b,c 153.69b,c 172.40b,c
847.94 1734.39 2400.03 3183.38 3970.29 4719.21 5484.18 6260.04 6951.09 7710.61
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
DIO ± ± ± ± ± ± ± ± ± ±
33.89a 44.86a 59.26a 71.87a 88.92a 103.87a,c 120.10a,c 140.10a,c 210.51a,c 196.45a,c
794.58 1640.71 2268.61 3225.52 4170.43 5139.64 6081.61 7095.68 8137.64 9166.09
DR ± ± ± ± ± ± ± ± ± ±
22.85a 31.60a 59.79a 60.87a 74.96a 98.74a,b 127.80a,b 152.36a,b 167.50a,b 188.68a,b
698.92 1407.25 2132.52 2900.05 3660.87 4446.56 5192.74 5975.13 6755.59 7488.97
± ± ± ± ± ± ± ± ± ±
11.56a,b,c 27.04a,b,c 37.21a,b 54.66a,b,c 76.56a,b,c 89.52a,c 112.42a,c 126.22a,c 144.53a,c 158.36a,c
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Fig. 2. The body fat content in DIO and DR rats induced by high-fat diet (mean = SE: n = 10). Mean values were significantly different from those of the CON group: aP b 0.05; mean values were significantly different from those of the MOD group: cP b 0.05; mean values were significantly different from those of the MOD group: bP b 0.05; DIO: diet-induced obesity; DR: diet resistant; CON: control; MOD: oxidative stress. Body fat content = (perirenal fat + testicular fat + omental fat) body weight ∗ 100.
which indicate a high energy metabolism in DIO rats compared to DR rats, despite the same HF diet consuming. Brain structural integrity reflects underlying neuronal health. Neuron apoptosis, which is a common pathological phenomenon in nervous system diseases, has been reported to be linked strongly with lipid metabolism. Moreover, hippocampus-dependent learning mechanisms appear to contribute to the control of food intake (Davidson et al., 2009) and interfere with body weight regulation, perhaps controlling appetite and behavior. It has been reported that people who consume excess energy and fat develop AD at twice the rate compared to the general population, and obese people have smaller overall brain and hippocampal volumes (Pothos et al., 2009). Obesity induced by a HF diet can affect fetal brain development (Niculescu and Lupu, 2009). These data are consistent with our study on body weight and brain parameters in DIO rats. We found that HF diet-induced DIO rats have lower brain weight than that of DR and MOD rats. Moreover, we observed that the MOD rats had greater spleen weight than DIO and DR rats. It is well known that the spleen is an organ involved in the immune system, and spleen atrophy can prompt the body's immune function to decline, which contributes to changes in learning and memory ability (Haley et al., 2011). Therefore, the data of the present study indicated that HF diets might cause changes in rat organs, and the DIO rats appeared to have much more brain damage than DR rats. The oxidation of the brain can produce more byproducts than the other parts of body, and the brain is highly susceptible to oxidative damage (Christian, 2004). Furukawa (Furukawa et al., 2004) reported that the oxidative stress levels in plasma from obese mice were elevated compared to control mice. In the present study, we further detected Table 5 The organ coefficient in DIO and DR rats induced by high-fat diet (mean ± SE, n = 10). Group
Brain/weight
CON MOD DIO DR
0.28 0.28 0.23 0.28
± ± ± ±
0.0063c 0.0057c 0.0064a,b 0.0052c
Heart/weight 0.28 0.29 0.25 0.27
± ± ± ±
0.0077c 0.0064a 0.0063b,c 0.0029b,c
Spleen/weight
Liver/weight
0.15 0.16 0.13 0.14
2.58 2.77 2.89 2.95
± ± ± ±
0.0064 0.0072c 0.0054b 0.012b
± ± ± ±
0.072c 0.15 0.076a 0.074a
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
the biomarkers associated with oxidative damage in plasma and brain tissue. We detected a positive correlation between obesity and LPO levels and ROS in mitochondria. Elevated levels of LPO have been linked to injurious effects, such as loss of cell membrane potential (MMP). Excess ROS can make polyunsaturated fatty acids generate LPO, ruining the biological membrane and contributing to abnormal function. In 3T3-L1 adipocytes, increases in ROS occur with lipid accumulation. ROS may contribute to the development of obesity. Together with these reports, we speculate that consuming a HF diet induced the accumulation of adipose in rats and contributed to the increased LPO and ROS in plasma or in brain mitochondria. Additionally, the increased LPO in the brain and the accumulation of ROS in brain mitochondria, which is indicative of oxidative damage, were mainly detected in DIO and MOD rats, but not in DR rats. These data further prove that HF diets have a similar effect to D-galactose in inducing oxidative damage in the brain. A main finding of the current work is that 10 weeks of HF diet leads to oxidative stress in the brain tissue of DIO rats but not DR rats. However, we also observed the increased plasma LPO in DR rats, which was not consistent with the LPO level in the brain. HF intake may also result to the increased LPO level in DR rats. MMP can reflect the opening of the mitochondrial permeability transition pore (PTP) and the mitochondrial integrity. The PTP opening may result in ROS release from mitochondria, and then intracellular ROS levels may be increased. MMP is a good indicator of mitochondrial function and is used to study signaling mechanisms involved in oxidative stress. To evaluate associations between HF diet intervention and brain mitochondria, we investigate the MMP of rat brain. The results showed that the ratio of P2/P3 and fluorescence intensity in MOD and DIO rats were decreased compared to CON and DR rats. These data suggest the mitochondrial dysfunction in DIO and MOD rats, but not in the DR rats (Mantena et al., 2008; Zou et al., 2014). There is active apoptotic signaling in the hypothalamus of rodents placed on a high fat diet (De Souza et al., 2005). Hormonal alterations were associated with cognitive function in obesity, such as leptin, cholecystokinin (CCK), glucagon-like peptide (GLP-1) and brain derived neurotrophic factor (BDNF) (Nogueiras et al., 2009; Paz-Filho et al., 2008). DIO rats exhibit a different behavioral and neuronal response to hormones such as CCK than DR rats (Swartz et al., 2010). There is also the comparison of the NPY expression in DIO rats and DR rats (Huang et al., 2003).
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Table 6 The T-AOC, CAT, LPO and MDA levels both in the plasma (mean ± SE, n = 10) and brain tissue (mean ± SE, n = 8) of DIO and DR rats induced by high-fat diet. Group
T-AOC
MDA
Plasma (U/ml) CON MOD DIO DR
3.89 3.86 4.93 4.50
± ± ± ±
0.34 0.34 0.56 0.36
LPO
Brain tissue (U/mg prot)
Plasma (nmol/ml)
Brain tissue (nmol/mg prot)
Plasma (nmol/ml)
3.98 0.50 0.83 2.30
5.82 5.70 7.04 6.14
6.87 7.45 7.41 7.04
5.01 5.00 6.76 7.03
± ± ± ±
1.60 0.29 0.19 1.38
± ± ± ±
0.42 0.52 0.40 0.23
± ± ± ±
0.68 1.54 0.51 0.77
± ± ± ±
0.52c 0.64c 0.50a,b 0.50a,b
Brain tissue (nmol/mg prot) 7.45 10.54 10.09 5.84
± ± ± ±
0.59b,c 0.98a 0.89a 0.43b,c
CAT (U/ml)
1.65 1.83 2.63 2.60
± ± ± ±
0.22 0.41 0.34 0.46
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress; T-AOC, total antioxidant capacity; CAT, catalase; LPO, lipid peroxide; MDA, malonaldehyde. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
Table 7 The leptin, NPY, ghrelin levels in both plasma and brain tissue of DIO and DR rats induced by high-fat diet (n = 6–8). Group
Leptin
NPY
Plasma (pg/ml) CON MOD DIO DR
1432.57 655.63 1210.87 1013.80
Tissue (pg/mg prot) ± ± ± ±
494.09 95.16 349.74 441.80
3864.30 6006.80 5603.43 6219.27
± ± ± ±
641.59b,c 629.31a 407.16a 428.04a
Ghrelin
Plasma (pg/ml) 428.17 400.11 206.30 120.84
± ± ± ±
Tissue (pg/mg prot) 77.18c 88.78c 44.63,ab 45.53a,b
605.31 867.43 877.74 966.32
± ± ± ±
92.66b,c 82.72a 58.54a 44.99a
Plasma (pg/ml) 45.12 98.20 158.46 91.73
± ± ± ±
Tissue (pg/mg prot) 5.58b,c 11.87a 23.28a 12.86
17.33 7.30 11.86 62.99
± ± ± ±
4.76 3.69 3.37 13.08b,c
DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress; NPY, neuropeptide Y. a P b 0.05, mean values were significantly different from those of the CON group. b P b 0.05, mean values were significantly different from those of the MOD group. c P b 0.05, mean values were significantly different from those of the DIO group.
Fig. 3. A Forward scatter (FS) and side scatter (SS) of brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry. B and C Fluorescence intensity of the brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry (mean = SE: n = 6). Mean values were significantly different from those of the CON group: aP b 0.05; mean values were significantly different from those of the MOD group: cP b ().05; mean values were significantly different from those of the MOD group: bP b 0.05; DIO: diet-induced obesity; DR: diet resistant; CON: control; MOD: oxidative stress.
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Fig. 4. A Forward scatter (FS) and side scatter (SS) of brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry. B Fluorescence intensity of the brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry. C The ratio of green and red of brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry. D Fluorescence intensity, the ratio of green and red, the forward scatter (FS) and side scatter (SS) of brain mitochondrial in diet-induced DIO and DR rats by using flow cytometry (mean = SE, n = 6). Mean values were significantly different from those of the CON group: aP b 0.05; mean values were significantly different from those of the MOD group: cP b 0.05; mean values were significantly different from those of the MOD group: bP b 0.05; DIO, diet-induced obesity; DR, diet resistant; CON, control; MOD, oxidative stress.
Leptin replacement changes brain structure and improves cognitive function in a boy with leptin deficiency (Paz-Filho et al., 2008). Leptin might be associated with oxidative stress in patients with impaired glucose tolerance or diabetes (Nakanishi et al., 2005). Bouloumıe et al. (1999) reported that leptin led to ROS generation in cultured human umbilical vein endothelial cells. However, in our study, we did not observe a significant difference in leptin levels among DIO, DR and MOD rats (Bruijnzeel et al., 2013). Ghrelin was the first orexigenic protein to be identified. The physiological role of ghrelin is to stimulate growth hormone release, food intake, and gastric motility in both humans and rodents (Luttikhold et al., 2013; Gasco et al., 2010). Decreased plasma ghrelin levels may have a protective role against the development of obesity. The regulatory role of ghrelin in intracellular energy balance, autophagy and anti-
oxidative actions under cardiac hypoxic conditions has been described (Kishimoto et al., 2012) and shown in MES23.5 cells (Liu et al., 2010). Accordingly, in the current study, we wondered if ghrelin plays a role in obesity induced by a HF diet. We found that DR rats showed higher ghrelin levels in brain tissue than DIO rats did, which indicates that ghrelin is related to oxidative stress in the brain of DIO rats, but not DR rats, and is related to the HF diet. NPY is widely distributed in the central nervous system that has been associated with the modulation of several functions including food intake, learning, memory and neuroprotection (dos Santos et al., 2013). It was found that central administration of NPY prevents spatial memory deficits in mice and this response is mediated by prevention of oxidative stress. In our study, the NPY levels in the plasma of DIO and DR rats were higher than that of CON and MOD rats, whereas the NPY level
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in brain tissue of MOD, DIO and DR rats was significantly higher than that of CON rats. We did not find a NPY difference between DIO rats and DR rats in either plasma or brain tissue. Conclusion Obesity caused by a HF diet was accompanied by an increase of food intake, energy intake and enlarged fat pads in DIO rats. The HF diet also induced obesity resistance in experimental animals. Moreover, the HF diet-induced DIO animals exhibited oxidative damage, demonstrated by the elevated formation of ROS and mitochondrial dysfunction, in both plasma and brain tissues, but these effects were not seen in DR rats. The oxidative stress state appears in DIO rats, but not in DR rats, and contributed to the NPY, ghrelin and leptin changes. However, the current study did not explore molecular mechanisms to elucidate the relationship between HF diet-induced obesity and brain oxidative damage. Nevertheless, our work hints at an association of HF dietinduced obesity and brain oxidative damage, and our work provides basic data for a diet pattern-based strategy for preventing obesity. Conflict of interest There is no conflict of interest to report in this study.
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