Effects of Acerola (Malpighia emarginata DC.) Juice Intake on Brain Energy Metabolism of Mice Fed a Cafeteria Diet Daniela Dimer Leffa, Gislaine Tezza Rezin, Francine Daumann, Luiza M. Longaretti, Ana Luiza F. Dajori, Lara Mezari Gomes, et al. Molecular Neurobiology ISSN 0893-7648 Mol Neurobiol DOI 10.1007/s12035-016-9691-y
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Author's personal copy Mol Neurobiol DOI 10.1007/s12035-016-9691-y
Effects of Acerola (Malpighia emarginata DC.) Juice Intake on Brain Energy Metabolism of Mice Fed a Cafeteria Diet Daniela Dimer Leffa 1 & Gislaine Tezza Rezin 2 & Francine Daumann 1 & Luiza M. Longaretti 1 & Ana Luiza F. Dajori 1 & Lara Mezari Gomes 3 & Milena Carvalho Silva 3 & Emílio L. Streck 3 & Vanessa Moraes de Andrade 1
Received: 30 October 2015 / Accepted: 5 January 2016 # Springer Science+Business Media New York 2016
Abstract Obesity is a multifactorial disease that comes from an imbalance between food intake and energy expenditure. Moreover, studies have shown a relationship between mitochondrial dysfunction and obesity. In the present study, we investigated the effect of acerola juices (unripe, ripe, and industrial) and its main pharmacologically active components (vitamin C and rutin) on the activity of enzymes of energy metabolism in the brain of mice fed a palatable cafeteria diet. Two groups of male Swiss mice were fed on a standard diet (STA) or a cafeteria diet (CAF) for 13 weeks. Afterwards, the CAF-fed animals were divided into six subgroups, each of which received a different supplement for one further month (water, unripe, ripe or industrial acerola juices, vitamin C, or rutin) by gavage. Our results demonstrated that CAF diet inhibited the activity of citrate synthase in the prefrontal cortex, hippocampus, and hypothalamus. Moreover, CAF diet decreased the complex I activity in the hypothalamus, complex II in the prefrontal cortex, complex II–III in the hypothalamus, and complex IV in the posterior cortex and striatum.
* Daniela Dimer Leffa
[email protected]
1
Laboratory of Cellular and Molecular Biology, Graduate Program in Health Sciences, Health Sciences Unit, Universidade do Extremo Sul Catarinense (UNESC), Avenida Universitaria, 1105 Bloco S, Criciuma, SC 88806-100, Brazil
2
Laboratory of Clinical and Experimental Pathophysiology, Postgraduate Program in Health Sciences, Universidade do Sul de Santa Catarina, Av. José Acácio Moreira, 787, 88704-9000 Tubarão, SC, Brazil
3
Laboratory of Experimental Physiopathology, Postgraduate Program in Health Sciences, Universidade do Extremo Sul Catarinense, Av. Universitária, 1105, 88806-000 Criciúma, SC, Brazil
The activity of succinate dehydrogenase and creatine kinase was not altered by the CAF diet. However, unripe acerola juice reversed the inhibition of the citrate synthase activity in the prefrontal cortex and hypothalamus. Ripe acerola juice reversed the inhibition of citrate synthase in the hypothalamus. The industrial acerola juice reversed the inhibition of complex I activity in the hypothalamus. The other changes were not reversed by any of the tested substances. In conclusion, we suggest that alterations in energy metabolism caused by obesity can be partially reversed by ripe, unripe, and industrial acerola juice. Keywords Acerola . Obesity . Krebs cycle . Respiratory mitochondrial chain . Energy metabolism
Introduction Overweight and obesity are defined as an abnormal or excessive fat accumulation that may impair health. The main cause of obesity and overweight is an increased intake of energydense, high-fat foods and physical inactivity. Furthermore, overweight and obesity are major risk factors for cardiovascular diseases, diabetes, musculoskeletal disorders, and some cancers. The worldwide prevalence of obesity more than doubled since 1980, and the World Health Organization (WHO) estimated that 39 % of the world’s adult population was overweight, and about 13 % were obese in 2014 [1]. Although the effects of obesity are initially observed in peripheral organs and systems, some studies have shown that obesity can cause abnormalities in the hypothalamus [2–7]. Furthermore, the presence of oxidative stress [8–15] and mitochondrial dysfunction [16–18] have been demonstrated in obesity.
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In addition, during the electron transfer in the respiratory chain, free radicals are produced, which may damage the mitochondrial respiratory chain [19]. The brain is particularly vulnerable to the production of reactive oxygen species (ROS) because it metabolizes 20 % of the total body oxygen and has a limited antioxidant capacity [20, 21]. Oxidative stress arises from an imbalance between oxidant and antioxidant compounds, which generates excessive reactive species or overwhelms the body’s capacity to remove them [21–23]. This process leads to the oxidation of biomolecules with consequent loss of its biological functions and/or homeostatic imbalances, which cause oxidative damage to cells and tissues [22, 23]. The aerobic organisms are integrated antioxidant systems that encompass enzymatic and non-enzymatic antioxidants. The function of these antioxidants is to neutralize and block the harmful effects of ROS. The non-enzymatic antioxidants include some vitamins (vitamins C and E), carotenoids, flavonoids, among others [23, 24]. The importance of the antioxidants to human health is becoming increasingly apparent, as is the case with obesity, which is associated with a decrease in the antioxidant capacity. Studies have shown beneficial effects of antioxidant supplementation in the diet of obesity-related diseases [25]. Further research should be done to investigate the possible corrective or therapeutic effects of antioxidants on this disease. In this context, several natural antioxidants have been investigated to evaluate their potential therapeutic effect in conditions associated with oxidative stress [26]. Malpighia emarginata DC. is a fruit popularly known as Bacerola^ or BAntilles cherry^ [27]. Acerola has a high amount of vitamin C (695–4827 mg/100 g), carotenoids, and flavonoids, which provide important nutritional value and make this fruit a good source of antioxidants [27, 28]. The purpose of the present study was to investigate the effect of acerola juices (unripe, ripe, and industrial) and its main pharmacologically active components (vitamin C and rutin) on the activity of enzymes of Krebs cycle (citrate synthase and succinate dehydrogenase), on the activity of complexes (complex I, II, II–III, and IV) of the mitochondrial respiratory chain, and on the activity of creatine kinase enzyme in the brain of mice fed a palatable cafeteria diet.
Materials and Methods Acerola Fruit Samples Unripe and ripe acerola (M. emarginata DC.) were purchased from Nutrilite Farm (Ceará, Brazil), whereas industrial acerola juice was obtained from Da Fruta® (Pernambuco, Brazil). Frozen ripe or unripe acerola were received from the manufacturer in packages of 1.5 kg. They were stored at −20 °C in
packages of 50 g until the experiments took place. Prior to the experiments, the fruits were thawed and processed in a food juicer, and juice doses were administered to the animals by gavage. Industrial acerola juice was stored at −4 °C. L-ascorbic acid (CAS Number 50-81-7) and rutin hydrates (CAS Number 207671-50-9) were purchased from Sigma-Aldrich (USA). In order to obtain the desired final doses, L-ascorbic acid and rutin were dissolved in distilled water immediately prior to each experiment. All experiments were carried out in minimal, indirect light. Animals In the present study, 42 healthy male Swiss albino mice (average body weight, 25 ± 0.5 g; age, 5–6 weeks) were obtained from the Animal Center of the Universidade do Extremo Sul Catarinense (UNESC, Brazil). All procedures involving animals and their care were carried out in accordance with national and international laws and guidelines on the use of animals in biomedical research. Moreover, the local ethics committee for animal use (CEUA—UNESC; Register No. 130 /2011) approved the experimental procedures. Mice were randomized by weight and housed in polycarbonate cages with steel wire tops (six animals per cage). Cages were kept at standard room temperature (22 ± 2 °C) and humidity (55 ± 10 %) at 12-h alternating light-dark cycles. Meticulous efforts were made to ensure minimal suffering and reduce external sources of stress, pain, and discomfort to the animals. Only a minimum number of animals necessary to produce reliable scientific data was used. Experimental Design All the animals were allowed to acclimatize in their new environment for 1 week prior to the beginning of the experiments. The mice were subsequently divided into a control group and a cafeteria-diet group. The control group consisted of six animals fed on a standard diet (STA), whereas the cafeteria-diet group (CAF) was composed of 36 animals. Both groups were kept on their respective diet for 13 weeks. After this period, the CAF-fed animals were divided into six subgroups (six animals per group), which were subjected to a treatment with 0.1 mL/10 g/day of the following dietary supplements: (1) water, (2) unripe acerola juice, (3) ripe acerola juice, (4) industrial acerola juice, (5) 1 mg/kg/day of vitamin C, or (6) 200 mg/kg/day of rutin. The schematic diagram of acerola corrective treatment is presented in Fig. 1. Doses of vitamin C and rutin were administered according to Franke et al. [29] and La Casa et al. [30], respectively. Vitamin C and rutin were chosen to treat the animals because of their greater presence in acerola juices evaluated by previous HPLC [31]. After 1 month of treatment with the dietary supplements, the
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Curitiba, PR, Brazil), the STA diet provides 2.93 kcal/g (see Table 2). Food supplies of both STA and CAF diets were renewed daily in the animal cages. The animals receiving the CAF diet had also access to STA diet and water (weekly menus are outlined in Table 1). The animals receiving the CAF diet developed obesity according to the feed efficiency parameter (body weight gain/energy intake, p < 0.001) and presented glucose intolerance (p < 0.05) when compared to the STA diet. These data are shown in our previously published work [31]. Furthermore, we also showed that the CAF group (obese animals) presented a significant increase in their adiposity indices, triacylglycerol levels, and body weight when compared with STA diet (control group, p < 0.05) [33]. Tissue and Homogenate Preparation Fig. 1 Schematic diagram of acerola corrective treatments
animals were killed by decapitation and brain samples were collected for biochemical analysis.
Experimental Diets A palatable high-calorie CAF diet was devised in order to resemble modern patterns of human food consumption that leads to obesity in lean animals [32]. The CAF diet used in this study was adapted from a CAF diet previously described by Shafat et al. [32]. Food in this CAF diet included chocolate crackers, wafers, marshmallows, mortadella, hot dog sausages, cheese and bacon chips, Doritos® chips, peanut candy, and calf’s foot jelly. Finally, soft drinks such as the popular guarana and cola were included as well (see Table 1). According to calculations based on the package information provided by the manufacturers, the CAF diet provides 4.12 kcal/g on average. On the other hand, according to the information provided by the manufacturer (Nuvilab CR-1, NUVITAL®, Table 1 Weekly menu of animals fed a cafeteria diet
Posterior cortex, prefrontal cortex, striatum, hippocampus, and hypothalamus were homogenized (1:10, w/v) in SETH buffer, pH 7.4 (250 mM sucrose, 2 mM ethylenediaminetetraacetic acid, 10 mM Trizma base, 50 IU/mL heparin). The homogenates were centrifuged at 750 g for 10 min at 4 °C, and the supernatants were kept at −70 °C until they were used to determine enzyme activity. The maximal period between homogenate preparation and enzyme analysis was always 5 days. Protein content was determined by the method described by Lowry et al. [34] using bovine serum albumin as a standard. Activities of Enzymes of Krebs Cycle Citrate synthase activity was assayed according to the method described by Srere [35]. The reaction mixture contained 100 mM Tris, pH 8.0, 0.1 mM acetyl CoA, 0.1 mM 5,50-dithiobis-(2-nitrobenzoic acid), 0.1 % Triton X-100, and 2–4 μg supernatant protein, and it was initiated with 0.2 mM oxaloacetate and monitored at 412 nm for 3 min at 25 °C. Succinate dehydrogenase activity was determined according to the method described by Fischer et al. [36], by following the
Days of week
Food
Monday
Mortadella, marshmallow, cheese chips, chocolate wafer, chow Nuvilab®, water, and Guaraná soft drink Chocolate crackers, Doritos®, chow Nuvilab®, sausage hot dog, water, and Guaraná soft drink Paçoca peanuts, chow Nuvilab®, mortadella, cheese chips, water, and Guaraná soft drink Mortadella, chocolate wafer, calf’s foot jelly, Doritos®, chow Nuvilab®, water, and cola soft drink Chocolate crackers, chow Nuvilab®, sausage hot dog, bacon chips, marshmallow, water, and cola soft drink
Tuesday Wednesday Thursday Friday, Saturday, and Sunday
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Macronutrient composition of the diets
Composition Standard diet (2.96 Kcal/g) Cafeteria diet (4.12 Kcal/g) g/100 g
Kcal/100 g
g/100 g
Kcal/100 g
Protein 22.00 Carbohydrate 43.00
88.00 172.00
7.67 41.97
30.68 167.88
Saturated fat
36.00
23.76
213.84
4.00
decrease in absorbance, due to the reduction of 2,6-di-chloroindophenol (2,6-DCIP) at 600 nm, with 700 nm as a reference wavelength (ε = 19.1/mM/cm), in the presence of phenazine methosulfate (PMS). The reaction mixture, consisting of 40 mM potassium phosphate, pH 7.4, 16 mM succinate, and 8 μM 2,6-DCIP, was preincubated with 40–80 μg homogenate protein at 30 °C, for 20 min. Subsequently, 4 mM sodium azide, 7 μM rotenone, and 40 μM 2,6-DCIP were added, and the reaction was initiated by the addition of 1 mM PMS, and monitored for 5 min. Activities of Mitochondrial Respiratory Chain Enzymes Complex I activity was evaluated according to Cassina and Radi [37] by the determination of the rate of nicotinamide adenine dinucleotide (NADH)-dependent ferricyanide reduction at λ = 420 nm. Complex II activity was determined using the method described by Fischer et al. [36], measured by following the decrease in absorbance due to the reduction of 2,6DCIP at λ = 600 nm. Complex II–III activity was determined using the method described by Fischer et al. [36], measured by cytochrome c reduction, using succinate as substrate at λ = 550 nm. Complex IV activity was assayed according to the method described by Rustin et al. [38], by following the decrease in absorbance due to the oxidation of previously reduced cytochrome c at λ = 550 nm. The activity of complexes of the mitochondrial respiratory chain were expressed as nanomoles per minute times milligram protein. Activity of Creatine Kinase Enzyme Creatine kinase activity was measured in brain homogenates pretreated with 0.625 mM n-dodecyl-β-D-maltoside. The reaction mixture consisted of 60 mM Tris–HCl, pH 7.5, containing 7 mM phosphocreatine, 9 mM MgSO4, and approximately 0.4–1.2 μg protein, in a final volume of 100 μL. After 15 min of preincubation at 37 °C, the reaction was started by the addition of 3.2 mmol of ADP. The reaction was stopped after 10 min by the addition of 1 μmol of phydroxymercuribenzoic acid. The creatine formed was estimated according to the colorimetric method of Hughes [39]. The color was developed by the addition of 100 μL 2 % αnaphtol and 100 μL 0.05 % diacetyl in a final volume of 1 mL
and read spectrophotometrically after 20 min at 540 nm. Results were expressed as nanomoles per minute times milligram protein. Statistical Analysis Statistical analyses were carried out using the SPSS 19.0 software package. The results were expressed as mean values ± standard deviation. The normality of variables was evaluated using the Kolmogorov–Smirnov test. Statistical analyses for the STA group and CAF diet groups were carried out using one-way analysis of variance (ANOVA). When ANOVA showed significant differences (p < 0.05), post hoc analysis was performed using Tukey’s test. The critical level for rejection of the null hypothesis was considered to be at p > 0.05.
Results Results of this study showed that the activity of enzyme of Krebs cycle citrate synthase was inhibited in the prefrontal cortex (p < 0.001), hippocampus (p < 0.005), and hypothalamus (p < 0.005) of mice fed a CAF diet (CAF + vehicle) when compared to the control group (STA + vehicle). Regarding effects of the acerola juices (unripe, ripe, and industrial), as well as its synthetic constituents (vitamin C and rutin) in the brain of mice fed a cafeteria diet, unripe juice reversed the inhibition of the citrate synthase activity in the prefrontal cortex (p < 0.001) and in the hypothalamus (p < 0.038), and ripe juice reversed the inhibition in the hypothalamus (p < 0.001). Our results also showed that the activity of citrate synthase was inhibited in the posterior cortex (p < 0.026) in the CAF + ripe group, inhibited in the posterior cortex (p < 0.036), prefrontal cortex (p < 0.005), and hypothalamus (p < 0.001) in the CAF + vitamin C group, as well was inhibited in the prefrontal cortex (p < 0.001), striatum (p < 0.001), and hypothalamus (p < 0.001) in the CAF + rutin group when compared to the STA + vehicle group. Moreover, the results showed that the citrate synthase activity was inhibited in the hippocampus (p < 0.001) in the CAF + industrial group when compared to the STA + vehicle group, and in the CAF + rutin group when compared to the control group (p < 0.001) and CAF diet (p < 0.017) (Fig. 2). The succinate dehydrogenase activity in the brain of mice was not altered by CAF diet. Our results also showed that the succinate dehydrogenase activity was increased in the posterior cortex in the CAF + unripe (p < 0.001), CAF + industrial (p < 0.001), and CAF + vitamin C (p < 0.001) groups and that it was inhibited in the hippocampus (p < 0.003) in the CAF + vitamin C group and in the prefrontal cortex (p < 0.008), striatum (p < 0.012), and hippocampus (p < 0.001) in the CAF + rutin group, when compared to the STA + vehicle group. Compared to the CAF + vehicle group, the succinate dehydrogenase
Author's personal copy Mol Neurobiol Fig. 2 Citrate synthase activity in the brain of mice fed STA + vehicle, CAF + vehicle, CAF + unripe, CAF + ripe, CAF + industrial, CAF + vitamin C, and CAF + rutin. *p < 0.05 versus STA + vehicle; #p < 0.05 versus CAF + vehicle (one-way ANOVA followed by Tukey’s test)
activity was also increased in the posterior cortex in the CAF + unripe (p < 0.001), CAF + industrial (p < 0.001) and CAF + vitamin C (p < 0.004) groups and was inhibited in the striatum (p < 0.026) in the CAF + rutin group (Fig. 3). Regarding enzyme activities of the mitochondrial respiratory chain, the results showed a significant decrease in the activity of complex I in the hypothalamus (p < 0.015) of mice fed a CAF diet (CAF + vehicle) when compared to the control group (STA + vehicle). The treatment with industrial juice was able to reverse the inhibition of complex I observed in the hypothalamus (p < 0.046) in relation to the CAF + vehicle group. The results also showed that the activity of complex I was increased in the hippocampus (p < 0.003) in the CAF + unripe group and was inhibited in the hypothalamus (p < 0.001) in the CAF + vitamin C group when compared to the STA + vehicle group (Fig. 4). Complex II activity was inhibited in the prefrontal cortex (p < 0.003) of mice fed a CAF diet (CAF + vehicle) when compared to the control group (STA + vehicle). None of the treatments were able to reverse this inhibition. Our results also showed that the complex II activity was increased in the Fig. 3 Succinate dehydrogenase activity in the brain of mice fed STA + vehicle, CAF + vehicle, CAF + unripe, CAF + ripe, CAF + industrial, CAF + vitamin C, and CAF + rutin. *p < 0.05 versus STA + vehicle; #p < 0.05 versus CAF + vehicle (one-way ANOVA followed by Tukey’s test)
posterior cortex in the CAF + vitamin C group when compared to both the STA + vehicle (p < 0.002) and CAF + vehicle (p < 0.003) groups and was inhibited in the prefrontal cortex in the CAF + unripe (p < 0.002), CAF + ripe (p < 0.028), and CAF + vitamin C (p < 0.005) groups when compared to the STA + vehicle group (Fig. 5). Complex II–III activity was inhibited in the hypothalamus (p < 0.017) of mice fed a CAF diet (CAF + vehicle) when compared to the control group (STA + vehicle). None of the treatments were able to reverse this inhibition. The results also showed that complex II–III activity was increased in the posterior cortex (p < 0.024) in the CAF + unripe group and in the hippocampus (p < 0.009) in the CAF + industrial group when compared to the STA + vehicle group (Fig. 6). Complex IV activity was inhibited in the posterior cortex (p < 0.029) and in the striatum (p < 0.001) of mice fed a CAF diet (CAF + vehicle) when compared to the control group (STA + vehicle). None of the treatments were able to reverse this inhibition. Complex IV activity was also inhibited in the posterior cortex in the CAF + ripe (p < 0.039) and CAF + vitamin C (p < 0.039) groups; as well, it was inhibited in the
Author's personal copy Mol Neurobiol Fig. 4 Complex I activity in the brain of mice fed STA + vehicle, CAF + vehicle, CAF + unripe, CAF + ripe, CAF + industrial, CAF + vitamin C, and CAF + rutin. *p < 0.05 versus STA + vehicle; #p < 0.05 versus CAF + vehicle (one-way ANOVA followed by Tukey’s test)
striatum in the CAF + unripe (p < 0.001), CAF + ripe (p < 0.001), CAF + vitamin C (p < 0.001), and CAF + rutin (p < 0.001) groups when compared to the STA + vehicle group. The results also showed that complex IV activity was increased in the hypothalamus in the CAF + rutin group as compared to both the STA + vehicle (p < 0.001) and CAF + vehicle (p < 0.001) groups (Fig. 7). Creatine kinase activity was increased in the posterior cortex (p < 0.037) in the CAF + rutin group and in the striatum (p < 0.005) in the CAF + unripe group when compared to the CAF + vehicle group. Moreover, creatine kinase activity was inhibited in the prefrontal cortex (p < 0.037) in the CAF + ripe group when compared to the CAF + vehicle group (Fig. 8).
Discussion Recently, the brain was identified as an insulin-sensitive organ regulating food intake [40]. Fatty acid sensitive neurons located in hypothalamus, hippocampus, or striatum are able to detect daily variations of plasma fatty acid levels. Thus, these neurons play a role to regulate energy balance by controlling Fig. 5 Complex II activity in the brain of mice fed STA + vehicle, CAF + vehicle, CAF + unripe, CAF + ripe, CAF + industrial, CAF + vitamin C, and CAF + rutin. *p < 0.05 versus STA + vehicle; #p < 0.05 versus CAF + vehicle (one-way ANOVA followed by Tukey’s test)
food intake, insulin secretion, or hepatic glucose production [41]. However, obesogenic diets are known to induce changes in synaptic plasticity in the prefrontal cortex and hippocampus leading to learning and memory impairments [42]. Moreover, diet-induced obesity appears to cause peripheral insulin resistance, increased brain oxidative stress, hippocampal synaptic dysfunction, brain mitochondrial dysfunction, and brain insulin resistance, which together can lead to cognitive impairment [43]. Therefore, the deregulation of this brain lipid sensing may be an early event leading to further dysfunction of energy balance leading to obesity and type 2 diabetes [41]. Despite this, therapeutic interventions targeting cafeteria diet-induced brain mitochondrial dysfunction are lacking. In this study, we discussed the possible beneficial effects of the different treatments with acerola juices or active compounds of acerola fruit on brain energy metabolism in mice fed a CAF diet that developed obesity. Regarding the main results of Krebs cycle enzymes, citrate synthase activity was inhibited by CAF diet in the prefrontal cortex, hippocampus, and hypothalamus. The unripe juice reversed the inhibition in the prefrontal cortex and in the hypothalamus, and the ripe juice reversed the inhibition in the hypothalamus.
Author's personal copy Mol Neurobiol Fig. 6 Complex II–III activity in the brain of mice fed STA + vehicle, CAF + vehicle, CAF + unripe, CAF + ripe, CAF + industrial, CAF + vitamin C, and CAF + rutin. *p < 0.05 versus STA + vehicle; #p < 0.05 versus CAF + vehicle (one-way ANOVA followed by Tukey’s test)
In the mitochondrial respiratory chain, the main results showed that complex I activity was inhibited by CAF diet in the hypothalamus, and the treatment with industrial juice was able to reverse this inhibition. It is possible that the efficacy of acerola juices will be related to its chemical composition. In our previously published study [31], the high-performance liquid chromatography analyses have confirmed high concentrations of vitamin C in unripe, followed by ripe and industrial juice. Rutin was present in high concentrations in ripe juice, followed by industrial and unripe juice. These findings are similar to those found by Nunes [44]. In addition, additional synergistic effects between the individual phytochemical components in acerola have to be considered. In the same study [31], the 2, 2-diphenyl-1-picryl-hydrazylhydrate (DPPH) free radical assay quantified the antioxidant properties of these juices (unripe, ripe, and industrial) and revealed higher antioxidant potentials compared to pure vitamin C and rutin. Besides, complex II activity was inhibited by CAF diet in the prefrontal cortex, and none of the treatments reversed this inhibition. Complex II–III activity was inhibited in the hypothalamus by CAF diet, and none of the treatments reversed Fig. 7 Complex IV activity in the brain of mice fed STA + vehicle, CAF + vehicle, CAF + unripe, CAF + ripe, CAF + industrial, CAF + vitamin C, and CAF + rutin. *p < 0.05 versus STA + vehicle; #p < 0.05 versus CAF + vehicle (one-way ANOVA followed by Tukey’s test)
this inhibition. Complex IV activity was inhibited in the posterior cortex and in the striatum by CAF diet, and none of the treatments reversed the inhibitions. Although, the acerola juices were statistically similar to CAF diet group, the inhibition not reversed by CAF diet, the results showed an increase in the respiratory chain complex activity in acerola juices compared to CAF group. Overconsumption of saturated fats and high-sugar diets are related to the emergence of the obesity epidemic. Apart from an increase in the energy storage as fat, these dietary changes are accompanied by increased mitochondrial oxidation of macronutrients, leading to excessive production of ROS, and hence oxidative stress, because studies have shown that obesity is related to this phenomenon [8–10, 45]. In our previously publicated study [46], we found that the CAF diet increased protein and lipid oxidative damage in the brain compared with animals fed with STA diet. Moreover, after CAF mice received diet supplements (acerola juices, vitamin C, or rutin) for 1 month, unripe and ripe acerola juices and rutin led to a decrease of the diet-induced lipid and protein oxidative damage in the brain, showing the antioxidant beneficial effects present in this fruit.
Author's personal copy Mol Neurobiol Fig. 8 Creatine kinase activity in the brain of mice fed STA + vehicle, CAF + vehicle, CAF + unripe, CAF + ripe, CAF + industrial, CAF + vitamin C, and CAF + rutin. *p < 0.05 versus STA + vehicle; #p < 0.05 versus CAF + vehicle (one-way ANOVA followed by Tukey’s test)
In this context, the consumption of fruits and vegetables rich in antioxidants can protect the body against oxidative damage, helping to deal with these metabolic consequences of westernized diet rich in high-energy-dense foods [24]. Furthermore, food is a powerful tool for the regulation of glucose metabolism, and it has been reported that foods rich in antioxidants may have protective effects [24, 45], given that the balance between oxidants and antioxidants is essential for cell viability and, consequently, for the organs to function properly [23]. Energy overload (in the form of carbohydrates, lipids, or proteins) can result in the increased production of ROS and may lead to an oxidative stress state [47]. Oxidative stress is an important event that has been related to the pathogenesis of diseases affecting the central nervous system. The brain is highly sensitive to ROS levels because of their high oxygen consumption, high iron concentration and lipid contents (especially polyunsaturated fatty acids), and low activity of enzymatic antioxidant defenses [48]. Reactive oxygen species inhibit the mitochondrial respiratory chain, resulting in the generation of more reactive species, forming a cyclic phenomenon [49]. Defects in energy production (such as oxidative phosphorylation) sometimes lead to a wide spectrum of problems, contributing to a variety of physiological abnormalities. Energy availability disorders do not cause brain malformations but do contribute to the onset of moderate neurological problems [50]. A study by Leffa et al. [31] has shown that CAF diet increased feed efficiency but also induced glucose intolerance and DNA damage, which was established by comet assays and micronucleus tests. Dias and colleagues [33] have demonstrated that the CAF water group (control obese) showed a significant increase in their adiposity indices and triacylglycerol levels, in addition to a reduced IL-10/TNF-α ratio in the adipose tissue, compared with the control lean group. However, the mechanisms by which fat accumulation leads to such a dysregulation of adipocytokines have not been clearly elucidated yet. A study by Furukawa et al. [9] has suggested that adipose tissue is the major source of the elevated plasma ROS levels.
Therefore, in accumulated fat of obesity, elevated ROS levels appear to upregulate mRNA expression of NADPH oxidase, establishing a vicious cycle that augments oxidative stress in blood. Thus, we assume that a transient increase of intracellular ROS is important for the insulin signaling pathway, while excessive and long-term exposure to ROS reduces insulin sensitivity and impairs glucose and lipid metabolism. In this way, we suggest that the decrease in activity of energy metabolism occurs by a decrease in glucose uptake by the cells, and in some situations, acerola juices have reversed this inhibition due to their antioxidant action. Acerola juice is a complex mixture containing high levels of L-ascorbic acid, carotenoids, and polyphenols, including quercetin and rutin [51]. These bioactive ingredients are able to reduce oxidative stress levels and the intake of acerola juice can, therefore, induce antigenotoxic and antimutagenic effects [44, 52]. Apart from the antigenotoxic and antimutagenic potential of vitamin C and rutin, which is most likely a result of the ability to quench free radicals [53], rutin has also antiinflammatory, antiallergic, antiviral, and anticarcinogenic properties [54]. Vitamin C is considered to be one of the most potent antioxidants contained in fruits and vegetables, allegedly contributing to the protection against cardiovascular diseases and cancer [51]. Vitamin C can also inhibit or activate the oxidation of certain flavonoids such as quercetin and rutin. Rutin has the ability to scavenge, e.g., hydroxyl radicals are increased at lower and inhibited at higher concentrations of vitamin C [55]. High concentrations of reactive species observed in the brains of obese rats led to a depletion of protective physiological moieties, such as the activity of glutathione peroxidase or superoxide dismutase [56]. Leffa and colleagues [57] have suggested that the simultaneous intake of acerola juice, vitamin C, or rutin, in association with a hypercaloric and hyperlipidic diet, provides a change in the mineral composition of organisms, which plays an important role in the antioxidant defenses of the body, that may help to reduce the metabolism of the fat tissue or even
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reduce the oxidative stress. Leffa and colleagues [31] have demonstrated that food supplementation with acerola juice led to a decreased DNA damage. Dias and colleagues [33] have suggested that acerola juice reduces low-grade inflammation and ameliorates obesity-associated defects in the lipolytic processes. Based on the above studies, we believe that acerola juice helps to reduce oxidative stress in obese subjects, and partially reverses the inhibition caused in energy metabolism. This study concluded that obesity might cause changes in the enzymes of energy metabolism due to oxidative stress, leading to a cyclical phenomenon, whereas the acerola juices reverses this damage due to its antioxidant effect. Acknowledgments We thank UNESC, CAPES, and CNPq for the financial support. Compliance with Ethical Standards Conflict of Interest The authors declare that they have no competing interests.
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