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Melatonin prevents memory impairment induced by high-fat diet: Role of oxidative stress
Accepted Manuscript Title: Melatonin Prevents Memory Impairment Induced by High-fat Diet: Role of Oxidative StressMelatonin and high-fat diet −induced memory impairment–> Authors: Karem. H. Alzoubi, Fadia A. Mayyas, Rania Mahafzah, Onar F. Khabour PII: DOI: Reference:
S0166-4328(17)31072-0 http://dx.doi.org/10.1016/j.bbr.2017.08.047 BBR 11066
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
27-6-2017 12-8-2017 29-8-2017
Please cite this article as: Alzoubi Karem H, Mayyas Fadia A, Mahafzah Rania, Khabour Onar F.Melatonin Prevents Memory Impairment Induced by High-fat Diet: Role of Oxidative Stress.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2017.08.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Melatonin Prevents Memory Impairment Induced by High-fat Diet: Role of Oxidative Stress Running title: Melatonin and high-fat diet -induced memory impairment
Karem. H. Alzoubi1*, PhD, Fadia A. Mayyas1, PhD, Rania Mahafzah1, Msc, Onar F. Khabour2, PhD
1
Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan 2
Department of Applied Medical Laboratory Sciences, Faculty of Applied Medical Sciences, Jordan University of Science and Technology, Irbid, Jordan
*Corresponding
Author:
Karem Alzoubi, PhD Professor of Pharmacology Department of Clinical Pharmacy Faculty of Pharmacy Jordan University of Science and Technology Irbid, Jordan 22110 Tel.: +962-2-7201000 Ext.: (23525) Fax: +962-2-7201075 E-mail: [email protected]
Highlights
HFD induced memory impairment
This impairment was correlated with oxidative stress in the hippocampus
Melatonin prevented this impairment by protecting hippocampus antioxidant mechanisms
Abstract Consumption of high-fat diet (HFD) induces oxidative stress in the hippocampus that leads to memory impairment. Melatonin has antioxidant and neuroprotective effects. In this study, we hypothesized that chronic administration of melatonin can prevent memory impairment induced by consumption of HFD. Melatonin was administered to rats via oral gavage (100 mg/kg/day) for 4 weeks. HFD was also instituted for the same duration. Behavioral studies were conducted to test spatial memory using the radial arm water maze. Additionally, oxidative stress biomarkers were assessed in the hippocampus. Results showed that HFD impaired both short- and long- term memory (P<0.05), while melatonin treatment prevented such effects. Furthermore, melatonin prevented HFD-induced reduction in levels of GSH, and ratio of GSH/GSSG, and increase in GSSG in the hippocampus. Melatonin also prevented reduction in the catalase activity in hippocampus of animals on HFD. In conclusion, HFD induced memory impairment and melatonin prevented this impairment probably by preventing alteration of oxidative stress in the hippocampus.
Keywords Melatonin, High-fat diet, Memory, Hippocampus, Maze.
Introduction Consumption of high-fat diet (HFD) is common in most of world societies [31, 45, 85]. HFD is associated with increased risk of metabolic disorders such as diabetes, obesity and cardiovascular disease [19, 28, 50-52, 64]. Recent researches suggest that HFD can have profound effects on the brain and can lead to cognitive impairment [5, 6, 11, 12, 22, 42, 65, 76, 82]. Additionally, HFD contributes to decline in cognitive function during aging [18, 42, 56], stress [4, 5], sleep deprivation [10] and accelerates dementia in Alzheimer's disease [35, 82]. The mechanism of HFD in decreasing cognitive function is not fully understood. Several studies have reported that HFD increases oxidative stress [10, 11, 49, 51, 52, 90]. Excessive consumption of HFD increased the production of free radicals that causes lipid peroxidation and alters the structural components of blood brain barrier [10, 37]. Melatonin is a natural substance found in plants, animals and fungi [21, 71]. Melatonin is secreted from the pineal gland in a circadian fashion [27]. The concentration of melatonin increases nocturnally and decreases when exposed to light [69]. Melatonin is secreted into the blood, brain, tissues and cerebrospinal fluid [26]. Melatonin is an effective antioxidant because it can scavenge oxidative molecules such as superoxide anions and detoxify oxygen and nitrogen-based toxic reactants [3, 30, 70]. Furthermore, Melatonin enhances the glutathione antioxidant system [3, 60, 63, 74]. The protective effect of melatonin has been previously shown during chronic sleep deprivation-induced cognitive decline [15, 89]. In this study, we evaluated the protective effect of chronic melatonin treatment on chronic HFD-induced memory impairment using the radial arm water maze (RAWM). We also carried out molecular
enzymatic assays to determine some molecular targets for the studied effect of HFD and /or melatonin. Methods and Materials Animals and Housing Conditions Adult male Wister rats weighting 160-200 g, were used in this study. The animals were housed in plastic cages (5/cage) and were kept on a 12:12-h light–dark cycle (lights on at 7 AM), under hygienic condition and at 24º C with free access to food and water. All animal experiments were done during the daylight and were approved by Animal Care and Use Committee (ACUC) at Jordan University of Science and Technology, Irbid, Jordan. Animal Groups and Diets The rats were randomly divided into four groups (n= 12-16): Control, Melatonin, high-fat diet (HFD) and HFD with melatonin (Melatonin/HFD). HFD and Melatonin/HFD groups were fed high-fat diet for 4 weeks. HFD contained (gm %): total fat (25%) including 11% unsaturated fats from butter and soybean oil, carbohydrates (44%) from starch, protein (18%) from casein, and 13% fibers, ash and other ingredients. The remaining groups were fed conventional diet containing (g%): 5% total fat including 2% unsaturated fats, 62% carbohydrates, 20% proteins, 13% fibers, and ash and other ingredients . In both diets, casein was the main source of proteins, butter and soybean oil were the main source of fats, and starch was the main source of carbohydrates. Both diets contained similar amount of standard vitamins and mineral mix with all essential nutrients [6, 10, 11, 51]. Food was provided ad libitum for the duration of the experiments.
Melatonin Administration Melatonin was dissolved in distilled water and dimethylsulfoxide (DMSO) (each 100 mg of melatonin dissolved in 0.5 ml water and 0.5 ml DMSO). Melatonin (100 mg/kg) was given by oral gavage to Melatonin and Melatonin/HFD groups [15]. The Control and HFD groups received vehicle treatment. HFD and/or melatonin treatment were started on the same day and continued for 4 weeks and throughout the day of behavioral test. Behavioral Test The RAWM was used to test spatial learning and memory among all groups of animals [1, 7, 8, 24, 38]. This model details were previously described [7, 16, 55, 58]. Hippocampus dissection Rats were killed after 4 week of HFD and/or melatonin treatment [4, 39]. In brief, rats were killed by decapitation using guillotine and the brain was removed immediately from the skull and placed on a filter paper soaked in normal saline over a glass plate filled with crushed ice. The hippocampus was isolated, kept in Eppendorf tubes and preserved in liquid nitrogen, then at -80 C until analysis. Calorimetric Assays The hippocampus tissues were homogenized as described previously [7]. Total protein concentrations were estimated using commercially available kit BioRAD procedure (Hercules, CA, USA). The oxidized glutathione (GSH) and reduced glutathione (GSSG) were assayed according to manufacturer's instructions (Glutathione Assay Kit, Sigma-Aldrich Crop, MI, USA). Activity of superoxide dismutase (SOD) was measured in the hippocampus using SOD assay kit (Sigma-
Aldrich Crop, MI, USA). Activity of catalase was determined in the hippocampal homogenate using Catalase Assay Kit (Cayman Chem. Com. Ann Arbor, MI, USA). Thiobarbituric acid reactive substance (TBARS) levels in hippocampus were measured using TBARS Assay Kit (Cayman Chem. Com. Ann Arbor, MI, USA). All procedures were performed according to kits’ instructions and the absorbance was measured using an automated reader (Bio-tek Instruments, Highland Park, Winooski, USA). Statistical Analysis All statistics were carried out using the GraphPad Prism (4.0) computer program (LA Jolle, CA). Comparisons of the number of errors for RAWM experiments (learning phase) were made using two-way AVOVA; followed by the Bonferroni posttest. Time (repeated measures factor) and treatment (between-subjects factor) groups were the independent variables. Comparisons of memory tests and immunoassays results were made using one-way AVOVA; followed by the Bonferroni posttest. P<0.05 was considered significant. All values are represented as mean ± standard error of the mean (SEM).
Result Melatonin protects against HFD-induced impairment of short- and long- term memory At the beginning of training, the rats made high number of errors and with time the errors decreased (P<0.05 within each experimental group) with no significant difference among experimental groups in each of the learning trials (treatment: F(3, 647) = 2.75, P > 0.05; time: F(11, 647) = 12.41, P < 0.05; interaction: F(33, 647) = 0.34, P > 0.05; Fig. 1).
At both short- and long- term memory tests done 30 min and 5 hrs after the end of the learning trials, the errors committed by the HFD group were significantly higher than the errors in the Control, Melatonin, and Melatonin/HFD groups (short-term memory test: F(3, 81) = 117.8, P < 0.05; long-term memory test: F(3, 78) = 42.5, P < 0.05; Fig. 2). No significant difference was found in the number of errors among Control, Melatonin, and Melatonin/HFD groups (P>0.05; Fig. 2).
Melatonin prevents HFD-induced elevation hippocampus oxidative stress The HFD resulted in a significant reduction in GSH levels as compared to Control, Melatonin and Melatonin/HFD groups (F(3,
50)
= 10.6, P<0.05; Fig. 3A). It also
resulted in a significant elevation in GSSG levels (F(3, 50) = 15.6, P<0.05; Fig. 3B) and a reduction in GSH/GSSG ratio (F(3, 50) = 43.6, P<0.05; Fig 3C) compared to Control, Melatonin and Melatonin/HFD groups. Thus, HFD effects on GSH, GSSG and GSH/GSSG ratio were prevented by melatonin administration. As for SOD, no difference was observed in its levels among all experimental groups (F(3, 49) = 0.3, p>0.05, Fig. 4A). HFD significantly decreased catalase activity as compared to Control group (F(3, 50) = 7.1, P<0.05; Fig. 4B). On the other hand, catalase activity in Melatonin and Melatonin/HFD groups was similar to Control group, indicating that melatonin prevented HFD-induced reduction in hippocampal catalase activity. Levels of TBARS were elevated in the HFD group compared to Control, Melatonin and Melatonin/HFD groups (F(3,
50)
= 5.6, P<0.05; Fig. 5). Thus, administration of
melatonin with HFD prevented the rise in the levels of TBARS induced by HFD.
Discussion The principal finding of the current study is that melatonin prevents short- and longterm memory impairment induced by HFD. In correlation, melatonin prevented levels of major oxidative stress biomarkers in the hippocampus such as GSH, and GSSG levels, and GSH/GSSG ratio, catalase enzyme induced by HFD. Currently, there is growing interest in clarifying the roles of life style and dietary habits in neural health. Several studies have shown that diet rich in saturated fat and refined sugar can decrease cognitive functions [5, 6, 18, 22, 23, 53, 67]. HFD aggravates cognitive function impairment during other conditions/diseases such as aging [42, 77], chronic stress [4, 5], sleep deprivation [9], and can accelerate the course of dementia in Alzheimer’s disease [34, 36, 82]. In accordance, current work showed that HFD consumption impairs to short- and long- term memories. HFD was shown to induce increased production of free radicals that leads to cell damage [10, 11, 25, 72, 90]. HFD leads to an imbalance between reactive oxygen species and antioxidant enzyme/species [10, 11, 32, 48, 72]. It has been reported that HFD increases hippocampal oxidative stress via decreasing the levels of GSH and increasing the levels of GSSG, leading to reduced GSH/GSSG ratio [10, 11]. This occurs alongside, a reduced activity of major antioxidative enzymes such as catalase [10, 11]. These results support the current study findings that showed that HFD caused significant changes in hippocampal oxidative stress biomarkers. The activity of antioxidant enzyme, catalase, was reduced by HFD. In addition, the GSH/GSSG ratio was reduced, decreasing the scavenging effect of glutathione in the hippocampus. The reduction in the antioxidant defense mechanisms increases oxidative stress in the hippocampus and provides a reasonable explanation for memory deficits accompanying HFD. In fact, oxidative stress is associated with
cognitive functions impairment in several other health conditions such as Alzheimer’s disease [20, 41, 44, 46, 47], sleep deprivation [13, 14], and aging [57]. Lipid peroxidation is a major consequence associated with oxidative stress. For instance, in diabetic mice fed high-cholesterol diet, TBARS, was significantly elevated in the frontal cortex and the hippocampus, which resulted in apoptosis of nerve cells, and impairment in learning and memory [87]. In addition, diet high in fat was shown to impair hippocampal neurogenesis through elevation of lipid peroxidation [66]. Others studies showed that high fat diet was associated with an increase in TBARS levels in rats’ cerebellum and cerebral cortex [29]. Recently, we have reported that HFD induced elevation in hippocampal TBARS levels [9, 10]. Results of this study confirm this finding, and further shows the elevation in hippocampal TBARS induced by HFD.
Melatonin is a powerful antioxidant substance [40, 70]. A number of studies have shown that melatonin has a significant and positive effect on the cognitive functions [15, 61, 81]. Melatonin administration for 4 weeks significantly restored the impairment of cognitive function induced by sleep deprivation [15]. In this study, we showed that administration of melatonin prevents short- and long-term memory impairment induced by HFD. Previous studies have documented the protective effect of melatonin on learning and memory deficits induced by a number of pathological or physiological conditions such as Alzheimer’s disease [33, 88], aging [2], diabetes [75], and head trauma [62]. Additionally, melatonin was shown to prevents cognitive deficits in memory due to administration/ingestion of chemicals and medications such as aromatic thinner solvents [17], pesticides [54], D-galactose [2] and dexamethasone [80].
The mechanisms by which melatonin prevents HFD-induced memory impairment is not fully understood. Melatonin reduces oxidative stress by scavenging oxidative molecules such as superoxide anions and detoxifying oxygen and nitrogen-based toxic reactant [70]. In this study, melatonin treatment for 4 weeks showed a protective effect against hippocampus oxidative stress. Melatonin prevented changes in GSH, GSSG, GSH/GSSG ratio catalase, and TBARS produced by HFD consumption. Thus, it is likely that melatonin prevents memory impairment through its effect on antioxidant molecules and enzyme activities in the hippocampus of HFD fed rats. In fact, melatonin has the ability to neutralize free radicals [68] and to prevent tissue damage associated with oxidative stress. This can be achieved utilizing different mechanisms such as scavenging the free radicals [73, 78, 79], increasing mRNA levels and activities of several important antioxidant enzymes [84], and preventing free radical formation at the mitochondrial level by reducing the leakage of electrons from the electron transport chain [43]. Thus, current results highlight the importance of using antioxidants such as melatonin in relieving some of the deleterious effects of HFD on individual’s health. In this study, melatonin was applied at the same time as HFD induced changes in memory functions and oxidative stress. Future studies are recommended to test the therapeutic effect of melatonin administration after HFD induces its impairment of memory and oxidative stress. Being a key hormone in the regulation of the circadian rhythm, unwanted side effects such as changes in sleep pattern could be expected when using melatonin as a therapy for reducing/preventing oxidative stress in certain disease conditions [83]. Such side effects in turn could have negative effects on cognitive functioning as well [86]. However, this scenario seems to be not the one manifesting in the current study as
melatonin successfully prevented memory functions impairment and oxidative stress elevation induced by HFD. Multiple previous studies have reported impairment of memory formation along with intact learning/acquisition processes [7, 16, 58, 59]. Our previous work showed that HFD for 3 months duration to impair both learning and short- and long- term memories [5, 6]. However, institution of HFD for durations of 6 weeks affected only short- and long- term memory, whereas learning processes remain intact [10, 11]. Results of the current study where HFD was applied for 4 weeks are in accordance with these previous works showing impaired short- and longterm memory with unaffected learning phase. The exact mechanism for such time-dependent effect for HFD on learning processes is not fully understood. Prolonged oxidative stress could be a factor; yet, more work is warranted toward this end.
In conclusion, melatonin prevents short-term memory and long-term memory impairments induced by HFD, probably via preventing oxidative stress in the hippocampus.
Acknowledgment: Financial Support was via grant number: 219/2016, from Deanship of Research at the Jordan University of Science and Technology to KA
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Legends Fig. 1 Animal learning performance in the radial arm water maze. Comparison of control, high-fat diet (HFD), melatonin 100 mg/kg/day and Melatonin/HFD groups for 4 weeks. Each animal was trained for six consecutive trials separated by 5 min rest, then another six consecutive trials (the learning phase). Similar learning performance was observed among all groups. Data are expressed as mean ± SEM (n = 15 per group).
Fig. 2 Animals’ performance in the RAWM during (A) short-term and (B) long-term memory tests. In both tests, the HFD group committed more errors compared to control, Melatonin, and Melatonin/HFD groups, indicating that melatonin treatment prevented HFD-induced short- and long- term memory impairment. Each column represent the mean ± SEM of 15 rats. * indicates significant difference from all other groups (P<0.05).
Fig. 3 HFD reduced hippocampus (A) GSH levels, (B) increased GSSG levels, and (C) the ratio GSH/GSSG. Adminstration of melatonin prevented these alterations in GSH, GSSG and GSH/GSSG ratio induced by HFD. Each bar represent the mean ± SEM of 15 rats. * indicates significant difference (P<0.05) from control group.
Fig. 4 Activity of SOD and catalase in control, Melatonin, and Melatonin/HFD groups. (A) No change was observed in the activity of SOD among experimental groups. (B) Activity of catalase was reduced in the HFD group as compared to other experimental groups. Thus, melatonin administration prents HFD-induced reduction in catalase activity. Each bar represent mean ± SEM of 15 rats. * indicates significant difference (P<0.05) from control group.
Fig. 5 Levels of TBARS in the hippocampus. HFD increased TBARS levels. This increase was prevented by melatonin administration. Each bar represent mean ± SEM of 15 rats. * indicate significant difference (P<0.05) from control group.
S0166-4328(17)31072-0 http://dx.doi.org/10.1016/j.bbr.2017.08.047 BBR 11066
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Behavioural Brain Research
Received date: Revised date: Accepted date:
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Please cite this article as: Alzoubi Karem H, Mayyas Fadia A, Mahafzah Rania, Khabour Onar F.Melatonin Prevents Memory Impairment Induced by High-fat Diet: Role of Oxidative Stress.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2017.08.047 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Melatonin Prevents Memory Impairment Induced by High-fat Diet: Role of Oxidative Stress Running title: Melatonin and high-fat diet -induced memory impairment
Karem. H. Alzoubi1*, PhD, Fadia A. Mayyas1, PhD, Rania Mahafzah1, Msc, Onar F. Khabour2, PhD
1
Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan 2
Department of Applied Medical Laboratory Sciences, Faculty of Applied Medical Sciences, Jordan University of Science and Technology, Irbid, Jordan
*Corresponding
Author:
Karem Alzoubi, PhD Professor of Pharmacology Department of Clinical Pharmacy Faculty of Pharmacy Jordan University of Science and Technology Irbid, Jordan 22110 Tel.: +962-2-7201000 Ext.: (23525) Fax: +962-2-7201075 E-mail: [email protected]
Highlights
HFD induced memory impairment
This impairment was correlated with oxidative stress in the hippocampus
Melatonin prevented this impairment by protecting hippocampus antioxidant mechanisms
Abstract Consumption of high-fat diet (HFD) induces oxidative stress in the hippocampus that leads to memory impairment. Melatonin has antioxidant and neuroprotective effects. In this study, we hypothesized that chronic administration of melatonin can prevent memory impairment induced by consumption of HFD. Melatonin was administered to rats via oral gavage (100 mg/kg/day) for 4 weeks. HFD was also instituted for the same duration. Behavioral studies were conducted to test spatial memory using the radial arm water maze. Additionally, oxidative stress biomarkers were assessed in the hippocampus. Results showed that HFD impaired both short- and long- term memory (P<0.05), while melatonin treatment prevented such effects. Furthermore, melatonin prevented HFD-induced reduction in levels of GSH, and ratio of GSH/GSSG, and increase in GSSG in the hippocampus. Melatonin also prevented reduction in the catalase activity in hippocampus of animals on HFD. In conclusion, HFD induced memory impairment and melatonin prevented this impairment probably by preventing alteration of oxidative stress in the hippocampus.
Keywords Melatonin, High-fat diet, Memory, Hippocampus, Maze.
Introduction Consumption of high-fat diet (HFD) is common in most of world societies [31, 45, 85]. HFD is associated with increased risk of metabolic disorders such as diabetes, obesity and cardiovascular disease [19, 28, 50-52, 64]. Recent researches suggest that HFD can have profound effects on the brain and can lead to cognitive impairment [5, 6, 11, 12, 22, 42, 65, 76, 82]. Additionally, HFD contributes to decline in cognitive function during aging [18, 42, 56], stress [4, 5], sleep deprivation [10] and accelerates dementia in Alzheimer's disease [35, 82]. The mechanism of HFD in decreasing cognitive function is not fully understood. Several studies have reported that HFD increases oxidative stress [10, 11, 49, 51, 52, 90]. Excessive consumption of HFD increased the production of free radicals that causes lipid peroxidation and alters the structural components of blood brain barrier [10, 37]. Melatonin is a natural substance found in plants, animals and fungi [21, 71]. Melatonin is secreted from the pineal gland in a circadian fashion [27]. The concentration of melatonin increases nocturnally and decreases when exposed to light [69]. Melatonin is secreted into the blood, brain, tissues and cerebrospinal fluid [26]. Melatonin is an effective antioxidant because it can scavenge oxidative molecules such as superoxide anions and detoxify oxygen and nitrogen-based toxic reactants [3, 30, 70]. Furthermore, Melatonin enhances the glutathione antioxidant system [3, 60, 63, 74]. The protective effect of melatonin has been previously shown during chronic sleep deprivation-induced cognitive decline [15, 89]. In this study, we evaluated the protective effect of chronic melatonin treatment on chronic HFD-induced memory impairment using the radial arm water maze (RAWM). We also carried out molecular
enzymatic assays to determine some molecular targets for the studied effect of HFD and /or melatonin. Methods and Materials Animals and Housing Conditions Adult male Wister rats weighting 160-200 g, were used in this study. The animals were housed in plastic cages (5/cage) and were kept on a 12:12-h light–dark cycle (lights on at 7 AM), under hygienic condition and at 24º C with free access to food and water. All animal experiments were done during the daylight and were approved by Animal Care and Use Committee (ACUC) at Jordan University of Science and Technology, Irbid, Jordan. Animal Groups and Diets The rats were randomly divided into four groups (n= 12-16): Control, Melatonin, high-fat diet (HFD) and HFD with melatonin (Melatonin/HFD). HFD and Melatonin/HFD groups were fed high-fat diet for 4 weeks. HFD contained (gm %): total fat (25%) including 11% unsaturated fats from butter and soybean oil, carbohydrates (44%) from starch, protein (18%) from casein, and 13% fibers, ash and other ingredients. The remaining groups were fed conventional diet containing (g%): 5% total fat including 2% unsaturated fats, 62% carbohydrates, 20% proteins, 13% fibers, and ash and other ingredients . In both diets, casein was the main source of proteins, butter and soybean oil were the main source of fats, and starch was the main source of carbohydrates. Both diets contained similar amount of standard vitamins and mineral mix with all essential nutrients [6, 10, 11, 51]. Food was provided ad libitum for the duration of the experiments.
Melatonin Administration Melatonin was dissolved in distilled water and dimethylsulfoxide (DMSO) (each 100 mg of melatonin dissolved in 0.5 ml water and 0.5 ml DMSO). Melatonin (100 mg/kg) was given by oral gavage to Melatonin and Melatonin/HFD groups [15]. The Control and HFD groups received vehicle treatment. HFD and/or melatonin treatment were started on the same day and continued for 4 weeks and throughout the day of behavioral test. Behavioral Test The RAWM was used to test spatial learning and memory among all groups of animals [1, 7, 8, 24, 38]. This model details were previously described [7, 16, 55, 58]. Hippocampus dissection Rats were killed after 4 week of HFD and/or melatonin treatment [4, 39]. In brief, rats were killed by decapitation using guillotine and the brain was removed immediately from the skull and placed on a filter paper soaked in normal saline over a glass plate filled with crushed ice. The hippocampus was isolated, kept in Eppendorf tubes and preserved in liquid nitrogen, then at -80 C until analysis. Calorimetric Assays The hippocampus tissues were homogenized as described previously [7]. Total protein concentrations were estimated using commercially available kit BioRAD procedure (Hercules, CA, USA). The oxidized glutathione (GSH) and reduced glutathione (GSSG) were assayed according to manufacturer's instructions (Glutathione Assay Kit, Sigma-Aldrich Crop, MI, USA). Activity of superoxide dismutase (SOD) was measured in the hippocampus using SOD assay kit (Sigma-
Aldrich Crop, MI, USA). Activity of catalase was determined in the hippocampal homogenate using Catalase Assay Kit (Cayman Chem. Com. Ann Arbor, MI, USA). Thiobarbituric acid reactive substance (TBARS) levels in hippocampus were measured using TBARS Assay Kit (Cayman Chem. Com. Ann Arbor, MI, USA). All procedures were performed according to kits’ instructions and the absorbance was measured using an automated reader (Bio-tek Instruments, Highland Park, Winooski, USA). Statistical Analysis All statistics were carried out using the GraphPad Prism (4.0) computer program (LA Jolle, CA). Comparisons of the number of errors for RAWM experiments (learning phase) were made using two-way AVOVA; followed by the Bonferroni posttest. Time (repeated measures factor) and treatment (between-subjects factor) groups were the independent variables. Comparisons of memory tests and immunoassays results were made using one-way AVOVA; followed by the Bonferroni posttest. P<0.05 was considered significant. All values are represented as mean ± standard error of the mean (SEM).
Result Melatonin protects against HFD-induced impairment of short- and long- term memory At the beginning of training, the rats made high number of errors and with time the errors decreased (P<0.05 within each experimental group) with no significant difference among experimental groups in each of the learning trials (treatment: F(3, 647) = 2.75, P > 0.05; time: F(11, 647) = 12.41, P < 0.05; interaction: F(33, 647) = 0.34, P > 0.05; Fig. 1).
At both short- and long- term memory tests done 30 min and 5 hrs after the end of the learning trials, the errors committed by the HFD group were significantly higher than the errors in the Control, Melatonin, and Melatonin/HFD groups (short-term memory test: F(3, 81) = 117.8, P < 0.05; long-term memory test: F(3, 78) = 42.5, P < 0.05; Fig. 2). No significant difference was found in the number of errors among Control, Melatonin, and Melatonin/HFD groups (P>0.05; Fig. 2).
Melatonin prevents HFD-induced elevation hippocampus oxidative stress The HFD resulted in a significant reduction in GSH levels as compared to Control, Melatonin and Melatonin/HFD groups (F(3,
50)
= 10.6, P<0.05; Fig. 3A). It also
resulted in a significant elevation in GSSG levels (F(3, 50) = 15.6, P<0.05; Fig. 3B) and a reduction in GSH/GSSG ratio (F(3, 50) = 43.6, P<0.05; Fig 3C) compared to Control, Melatonin and Melatonin/HFD groups. Thus, HFD effects on GSH, GSSG and GSH/GSSG ratio were prevented by melatonin administration. As for SOD, no difference was observed in its levels among all experimental groups (F(3, 49) = 0.3, p>0.05, Fig. 4A). HFD significantly decreased catalase activity as compared to Control group (F(3, 50) = 7.1, P<0.05; Fig. 4B). On the other hand, catalase activity in Melatonin and Melatonin/HFD groups was similar to Control group, indicating that melatonin prevented HFD-induced reduction in hippocampal catalase activity. Levels of TBARS were elevated in the HFD group compared to Control, Melatonin and Melatonin/HFD groups (F(3,
50)
= 5.6, P<0.05; Fig. 5). Thus, administration of
melatonin with HFD prevented the rise in the levels of TBARS induced by HFD.
Discussion The principal finding of the current study is that melatonin prevents short- and longterm memory impairment induced by HFD. In correlation, melatonin prevented levels of major oxidative stress biomarkers in the hippocampus such as GSH, and GSSG levels, and GSH/GSSG ratio, catalase enzyme induced by HFD. Currently, there is growing interest in clarifying the roles of life style and dietary habits in neural health. Several studies have shown that diet rich in saturated fat and refined sugar can decrease cognitive functions [5, 6, 18, 22, 23, 53, 67]. HFD aggravates cognitive function impairment during other conditions/diseases such as aging [42, 77], chronic stress [4, 5], sleep deprivation [9], and can accelerate the course of dementia in Alzheimer’s disease [34, 36, 82]. In accordance, current work showed that HFD consumption impairs to short- and long- term memories. HFD was shown to induce increased production of free radicals that leads to cell damage [10, 11, 25, 72, 90]. HFD leads to an imbalance between reactive oxygen species and antioxidant enzyme/species [10, 11, 32, 48, 72]. It has been reported that HFD increases hippocampal oxidative stress via decreasing the levels of GSH and increasing the levels of GSSG, leading to reduced GSH/GSSG ratio [10, 11]. This occurs alongside, a reduced activity of major antioxidative enzymes such as catalase [10, 11]. These results support the current study findings that showed that HFD caused significant changes in hippocampal oxidative stress biomarkers. The activity of antioxidant enzyme, catalase, was reduced by HFD. In addition, the GSH/GSSG ratio was reduced, decreasing the scavenging effect of glutathione in the hippocampus. The reduction in the antioxidant defense mechanisms increases oxidative stress in the hippocampus and provides a reasonable explanation for memory deficits accompanying HFD. In fact, oxidative stress is associated with
cognitive functions impairment in several other health conditions such as Alzheimer’s disease [20, 41, 44, 46, 47], sleep deprivation [13, 14], and aging [57]. Lipid peroxidation is a major consequence associated with oxidative stress. For instance, in diabetic mice fed high-cholesterol diet, TBARS, was significantly elevated in the frontal cortex and the hippocampus, which resulted in apoptosis of nerve cells, and impairment in learning and memory [87]. In addition, diet high in fat was shown to impair hippocampal neurogenesis through elevation of lipid peroxidation [66]. Others studies showed that high fat diet was associated with an increase in TBARS levels in rats’ cerebellum and cerebral cortex [29]. Recently, we have reported that HFD induced elevation in hippocampal TBARS levels [9, 10]. Results of this study confirm this finding, and further shows the elevation in hippocampal TBARS induced by HFD.
Melatonin is a powerful antioxidant substance [40, 70]. A number of studies have shown that melatonin has a significant and positive effect on the cognitive functions [15, 61, 81]. Melatonin administration for 4 weeks significantly restored the impairment of cognitive function induced by sleep deprivation [15]. In this study, we showed that administration of melatonin prevents short- and long-term memory impairment induced by HFD. Previous studies have documented the protective effect of melatonin on learning and memory deficits induced by a number of pathological or physiological conditions such as Alzheimer’s disease [33, 88], aging [2], diabetes [75], and head trauma [62]. Additionally, melatonin was shown to prevents cognitive deficits in memory due to administration/ingestion of chemicals and medications such as aromatic thinner solvents [17], pesticides [54], D-galactose [2] and dexamethasone [80].
The mechanisms by which melatonin prevents HFD-induced memory impairment is not fully understood. Melatonin reduces oxidative stress by scavenging oxidative molecules such as superoxide anions and detoxifying oxygen and nitrogen-based toxic reactant [70]. In this study, melatonin treatment for 4 weeks showed a protective effect against hippocampus oxidative stress. Melatonin prevented changes in GSH, GSSG, GSH/GSSG ratio catalase, and TBARS produced by HFD consumption. Thus, it is likely that melatonin prevents memory impairment through its effect on antioxidant molecules and enzyme activities in the hippocampus of HFD fed rats. In fact, melatonin has the ability to neutralize free radicals [68] and to prevent tissue damage associated with oxidative stress. This can be achieved utilizing different mechanisms such as scavenging the free radicals [73, 78, 79], increasing mRNA levels and activities of several important antioxidant enzymes [84], and preventing free radical formation at the mitochondrial level by reducing the leakage of electrons from the electron transport chain [43]. Thus, current results highlight the importance of using antioxidants such as melatonin in relieving some of the deleterious effects of HFD on individual’s health. In this study, melatonin was applied at the same time as HFD induced changes in memory functions and oxidative stress. Future studies are recommended to test the therapeutic effect of melatonin administration after HFD induces its impairment of memory and oxidative stress. Being a key hormone in the regulation of the circadian rhythm, unwanted side effects such as changes in sleep pattern could be expected when using melatonin as a therapy for reducing/preventing oxidative stress in certain disease conditions [83]. Such side effects in turn could have negative effects on cognitive functioning as well [86]. However, this scenario seems to be not the one manifesting in the current study as
melatonin successfully prevented memory functions impairment and oxidative stress elevation induced by HFD. Multiple previous studies have reported impairment of memory formation along with intact learning/acquisition processes [7, 16, 58, 59]. Our previous work showed that HFD for 3 months duration to impair both learning and short- and long- term memories [5, 6]. However, institution of HFD for durations of 6 weeks affected only short- and long- term memory, whereas learning processes remain intact [10, 11]. Results of the current study where HFD was applied for 4 weeks are in accordance with these previous works showing impaired short- and longterm memory with unaffected learning phase. The exact mechanism for such time-dependent effect for HFD on learning processes is not fully understood. Prolonged oxidative stress could be a factor; yet, more work is warranted toward this end.
In conclusion, melatonin prevents short-term memory and long-term memory impairments induced by HFD, probably via preventing oxidative stress in the hippocampus.
Acknowledgment: Financial Support was via grant number: 219/2016, from Deanship of Research at the Jordan University of Science and Technology to KA
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Legends Fig. 1 Animal learning performance in the radial arm water maze. Comparison of control, high-fat diet (HFD), melatonin 100 mg/kg/day and Melatonin/HFD groups for 4 weeks. Each animal was trained for six consecutive trials separated by 5 min rest, then another six consecutive trials (the learning phase). Similar learning performance was observed among all groups. Data are expressed as mean ± SEM (n = 15 per group).
Fig. 2 Animals’ performance in the RAWM during (A) short-term and (B) long-term memory tests. In both tests, the HFD group committed more errors compared to control, Melatonin, and Melatonin/HFD groups, indicating that melatonin treatment prevented HFD-induced short- and long- term memory impairment. Each column represent the mean ± SEM of 15 rats. * indicates significant difference from all other groups (P<0.05).
Fig. 3 HFD reduced hippocampus (A) GSH levels, (B) increased GSSG levels, and (C) the ratio GSH/GSSG. Adminstration of melatonin prevented these alterations in GSH, GSSG and GSH/GSSG ratio induced by HFD. Each bar represent the mean ± SEM of 15 rats. * indicates significant difference (P<0.05) from control group.
Fig. 4 Activity of SOD and catalase in control, Melatonin, and Melatonin/HFD groups. (A) No change was observed in the activity of SOD among experimental groups. (B) Activity of catalase was reduced in the HFD group as compared to other experimental groups. Thus, melatonin administration prents HFD-induced reduction in catalase activity. Each bar represent mean ± SEM of 15 rats. * indicates significant difference (P<0.05) from control group.
Fig. 5 Levels of TBARS in the hippocampus. HFD increased TBARS levels. This increase was prevented by melatonin administration. Each bar represent mean ± SEM of 15 rats. * indicate significant difference (P<0.05) from control group.