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Attenuation of stress-induced weight loss with a ketogenic diet
Physiology & Behavior 212 (2019) 112654
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Attenuation of stress-induced weight loss with a ketogenic diet ⁎
T
Elizabeth Sahagun , Lauren M. Ward, Kimberly P. Kinzig Department of Psychological Sciences, Purdue University, West Lafayette, IN, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Ketogenic diet Chronic stress Hypothalamic-pituitary-adrenal axis Mood disorders Animal model
Ketogenic diets (KDs) are high-fat, low-carbohydrate diets that have been used therapeutically for decades, most notably for the treatment of epilepsy and diabetes. Recent data, however, suggest that KD may impart protective effects on mood disorders. The current experiments test the hypothesis that KDs can protect from stress-induced symptoms of mood disorders. To test this, we assessed behavioral and neuroendocrine effects of KD in male and female Long Evans rats. Animals experienced three weeks of chronic mild stress (CMS) while consuming KD or control chow (CH). Body weight and food intake data were recorded daily and behaviors were assayed after three weeks. Plasma beta-hydroxybutyrate (βHB), corticosterone (CORT) and interleukin-1 beta (IL-1β) were measured after behavioral testing, along with hypothalamic corticotropin-releasing hormone (CRH) and neuropeptide Y (NPY) mRNA expression. CMS induced weight loss in the CH groups, however the KD-fed rats were resistant to CMS-induced weight loss. Female rats fed KD were protected from CMS-induced reductions in plasma CORT and hypothalamic NPY expression. Collectively, these data suggest protective potential of KDs against chronic stress, particularly in females.
1. Introduction Lifestyle changes known to decrease stress and improve mental health are commonly suggested for individuals reporting symptoms of affective disorders [35]. Improved nutrition through dietary modifications, or implementing relatively undefined “healthy eating habits”, are suggested as beneficial to overall well-being. Even so, data supporting the health benefits of various dietary approaches have produced equivocal findings. Incomplete understanding of how dietary changes can impact mood and mental health can result in individuals turning to non-empirical sources of information, such as anecdotes from media sources making indirect untested claims. For example, the sustained popularity of ketogenic diets (KD) for improved health is largely fueled by lay reports in which there are widespread claims that severe restriction of carbohydrates can increase energy and mood. Empirical data demonstrate that KDs are effective in the treatment of epilepsy and diabetes, and are hypothesized to have other positive neuronal benefits. [28,30]. KD has been shown to protect from harmful diet-induced deficits in the brain such as impaired performance on cognitive tasks, decreased brain derived neurotropic factor (BDNF), as well as increased hippocampal inflammation and blood brain barrier permeability [10,14,15]. KDs can also protect from metabolic insult and peripheral immune challenges ([11]; Kimberly P. [18]; Kimberly P Kinzig, Scott, Hyun, Bi, & Moran, [19], [39]). More recently, data
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suggest that KD may be neuroprotective in response to the pathophysiology of mood disorders and adverse effects of chronic stress [6,7]. The neuroprotective effects of KD are suggested to rely on the induction of ketosis and elevation of beta-hydroxybutyrate (βHB). The elevation of βHB using a 80% lard-based diet has shown to protect from diet-induced cognitive deficits and blood brain barrier leakage [10,14,15]. βHB is produced by the liver and elevated in the blood stream in response to consuming a high-fat/low-carbohydrate diet, prolonged fasting, or exercising [23]. βHB enters the brain by active transport via monocarboxylate transporters [1]. The presence of βHB in the CNS has been demonstrated to decrease anxiety-related behavior and influence activity levels [3,27]. Mood disorders constitute neurological changes that result in emotional dysregulation and cognitive deficits. The core of some symptoms can also be the result of energy dysregulation driven by poor diet. Existing evidence shows that diets high in fat and sugar can cause depression-like symptoms, and epidemiological research shows that depression is often comorbid with metabolic disorders (Luppino, Floriana S., de Wit, Leonore M., Bouvy [22]). There is evidence suggesting KD can reverse the adverse metabolic effects of high-fat/ highsugar diet, however the effects on mental health are unclear. Collectively, data suggest that KD may be beneficial to both mental and metabolic health. Chronic stress increases one's susceptibility to develop a mood
Corresponding author. E-mail address: [email protected] (E. Sahagun).
https://doi.org/10.1016/j.physbeh.2019.112654 Received 28 March 2019; Received in revised form 29 July 2019; Accepted 15 August 2019 Available online 17 August 2019 0031-9384/ © 2019 Published by Elsevier Inc.
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animals were switched to their respective diets and given four days to habituate to their new diet. Then on days 5–26, stressors were presented once a day for various amounts of time in a pseudorandom manner to prevent habituation and predictability. After CMS, behavioral responses to the elevated plus maze (EPM), open field (OFT), and forced swim (FST) were evaluated on consecutive days. Experimental timeline is shown in Fig. 1. Rats were sacrificed approximately two hours after FST for plasma and hypothalamic tissue analyses.
disorder. Stressful stimuli activate the sympathetic nervous system, which leads to release of hormones that motivate a “fight or flight” response. Under normal conditions, this is an acute adaptive response of survival that is mediated by the hypothalamic-pituitary- adrenal (HPA) axis. The HPA axis has several feedback mechanisms to return the brain and body back to homeostasis once a stressful event is over. Chronic activation can lead to dysregulation of the HPA axis and becomes detrimental to homeostatic processes such as energy regulation [34], desensitization of neurotransmitter function in higher up brain regions [21], and immune function [38]. The goal of the following experiments was to elucidate the effects of KD on the development of anxiety- and depressive-like behaviors, analogous to symptoms of mood disorders, that result from exposure to chronic mild stress (CMS). Rats were given a KD with macronutrient ratios similar to a strict KD adopted by humans (80% fat, 15% protein, and 5% carbohydrate) (Kimberly P. [18]), and exposed to a series of mild stressors over the course of three weeks. We hypothesized that rats consuming KD during CMS will be resilient to deleterious effects typically induced by the model, as measured by behavioral and neuroendocrine outcomes.
2.3. Behavioral assessments Food intake was measured daily and final behavioral tests were done for three consecutive days following the end of CMS. All final testing was done in a room adjacent to the vivarium during the first half of the light cycle. Animals were given 10–15 min to habituate to the room before the start of each test. Test order was counterbalanced within each sex to minimize test time bias. 2.3.1. Elevated plus maze Anxiety-like behavior was assessed using the elevated plus maze (EPM). The EPM apparatus is 3 ft high, and has with four arms at 90degree angles, each 6 in. wide and 3 ft long. Two of the opposite side arms are enclosed and the other two are open. Animals were placed in the center of the EPM and behavior was video recorded from a ceiling mounted camera for 5 min. Rats were returned to home cages immediately after testing. The percent of time spent in open arms and in the center was scored manually by an observer blinded to the experimental conditions because AnyMaze tracking was not sensitive enough to track the head of Long Evans on the apparatus.
2. Materials methods 2.1. Animals and diets Adult (16–17 week old) male (N = 33) and female (N = 37) LongEvans rats were individually housed in suspended wire cages in humidity (55–65%) and temperature (20.5 ± 1 °C) controlled rooms that were maintained with a 12:12 h light/dark cycle unless stated in the stress schedule (Table 1). Males and females were housed in separate rooms with light/dark cycles staggered by 1 h to maintain consistency of stress and testing times between groups. Animals were weight matched and then assigned to either KD or chow (CH) diet. The KD (Research Diets D06040601, New Brunswick, NJ) consisted of 80% fat, 5% carbohydrates, and 15% protein by calories. This lard-based diet has been used to effectively impart therapeutic effects of elevated βHB ([15]; Kimberly P. [18]; Kimberly P Kinzig et al., 2005). The control diet, CH (Teklad 2018, Envigo, Indianapolis, IN), consisted of 18% fat, 58% carbohydrates, and 24% protein. Diet compositions are further described in Table 2. After four days of consuming the assigned diet, animals were randomly assigned to a stress (+) or non-stress (−) subgroup for the remainder of the experiment. There were four experimental groups per sex: CH-, CH+, KD-, KD+. Group n for males were 7, 9, 8, 9, and for females 9, 9, 9, 10, respectively. All animals had ad libitum access to food and water in the home cage. Animals were weighed daily and food intake was measured by weighting food container daily and subtracting food spillage. All procedures were approved by the Purdue Animal Care and Use Committee (PACUC).
2.3.2. Open field test The open field test (OFT) quantifies locomotor activity and anxietylike behavior. The apparatus is 4 ft by 4 ft, with a grid printed on the floor. The rat is placed in the center of the apparatus and video recorded for five minutes on a ceiling view mounted camera. Videos were later scored automatically using Any-maze behavior tracking software (Stoelting Co.) Total distance traveled, percent time spent in the center and time spent in the corners of the apparatus were quantified. 2.3.3. Forced swim test Depressive-like behaviors were assessed in rats by forced swim test (FST). The FST apparatus was a 22 × 17 × 25 inch glass container with enough water to prevent rats from stepping on the bottom of the apparatus. Water was maintained at 25–30 °C. Rats were monitored for the duration of the test and the test was stopped if rats did not maintain a swim or float behavior. The test was recorded for five minutes using a ceiling mounted camera. After five minutes, fur was immediately dried with a towel and rats were returned to the home cage. Any-maze behavior tracking software (Stoelting Co.) was used to score behaviors. An observer blinded to treatment conditions evaluated time spent immobile, latency to immobility, and time spent displaying escape behaviors.
2.2. Chronic mild stress (CMS) Stress groups underwent three weeks of CMS by exposure to daily stressors (Willner, [36]), which are described in Table 1. In short, Table 1 CMS details. Stressor
Description
Duration
Restraint Lights Flooding Cold Crowding Static Cage Tilt
Restrained in a clear PVC device with a perforated front at an unpredictable time during the light cycle. Moved to a neighboring housing room in novel cages, where the lights remained on during the dark cycle. 200 ml of room temp tap water added to bedding in novel cage during the light cycle. Moved to a novel cage and placed in a 4 degrees C chamber at an unpredictable time during the light cycle. Temporarily housed with 3–4 unfamiliar conspecifics also undergoing CMS during the light cycle. Static noise (80 dB) played in a neighboring housing room in the middle of the light cycle. Moved to a novel cage during different times of the day tilted 30–45 degrees in home room.
30 min 12 ha 16 ha 30 min 6 ha 6 ha 6 ha
Note: Animals had access to food and water in conditions lasting 6 or more hours. 2
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Table 2 Diet compositions (% kcal). Diet type
Carbs % kcal
Protein % kcal
Fat % kcal
Calorie Density
Product ID
Chow (CH) Ketogenic (KD)
58
24
18
3.1 kcal/g
5
15
80
6.1 kcal/g
Teklad (2018) Research Diets (D06040601)
ELISA Rat IL-1β /IL-1F2 immunoassay according to the manufacturer's instructions. In summary, Plasma samples were diluted 1:3 using the provided calibrator diluent and the assay standard was serially diluted with a dilution factor of 2 in 8 concentrations. 50 μl of assay diluent was added to rat IL-1β coated well plates, then 50 μl of sample or standard were loaded into each well plate then incubated at room temperature for 2 h. After the incubation period, wells were washed 5 times, loaded with 100 μl of conjugate, incubated again at room temperature for 2 h, then washed again five times. After second wash, 100 μl of substrate solution was added to each well, incubated for 30 min at room temperature, protected from light, then 100 μl of stop solution was added. Immediately following the addition of stop solution, 450 nm absorbance was measured in a microplate reader (Multiskan Ascent with Ascent software v2.6, ThermoFisher Scientific, Waltham, MA).
Fig. 1. Experimental Timeline. Rats were exposed to either ketogenic diet (KD) or chow (CH) diet for 4 days, then underwent three weeks of chronic mild stress (CMS). After CMS, animals were tested for three consecutive days on elevated plus maze (EPM), open field (OFT), and forced swim (FST). They were then sacrificed approximately two hours after FST for plasma and tissue analysis.
2.4. Terminal assessment Rats were sacrificed by pentobarbital (Beauthanasia, D, Merck Animal Health, Madison, NJ) overdose approximately two hours after FST test for collection of blood and removal of brains. Following rapid decapitation, 3 ml of trunk blood was collected in VACUETTE® K3EDTA blood collection tubes (Greiner Bio-One, Monroe, NC) and centrifuged for 15 min at 4 °C, and 2500 rpm. Plasma was isolated and aliquoted into three tubes and stored in a − 80 °C freezer for later analyses. Plasma analyses included circulating levels of corticosterone, βHB, and IL-1β. Brains were rapidly removed and rinsed with 1× phosphate buffered saline (PBS). The hypothalamus of each rat was micro-dissected on ice, flash frozen on dry ice, then stored at −80 °C for later analysis of CRH and NPY.
2.4.4. RNA extraction and cDNA synthesis Tissues were homogenized in 1 ml TriReagent (RT111, Molecular Research Center, Cincinnati, OH) then 50 µl bromanisole (BCP, Molecular Research Center, Cincinnati, OH) was used to extract RNA. RNA was quantified by spectrophotometer at 260 nm absorbance (Eppendorf BioPhotometer 22,331, Hauppauge, NY). First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA) was used to synthesize cDNA from 2 μg total RNA.
2.4.1. Corticosterone assay Plasma corticosterone (CORT) concentration was quantified in duplicates using an EIA kit according to manufacturer instructions (Enzo Life sciences, protocol ADI-900-097). Samples were diluted 1:40 using a steroid displacement reagent and standards were serially diluted 1:50. 100 μl of sample or standard were loaded on donkey anti-sheep IgG coated plates, followed by 50 μl of conjugate, then 50 μl of sheep antibody to rat CORT. Plates were then incubated at room temperature on a plate shaker for 2 h at 500 rpm, and then washed 3 times. After washing, 200 μl of pNpp substrate solution was added to each well, and then incubated at room temperature for one hour. 50 μl of stop solution was then added to every well, and optical density was immediately read at 405 nm absorbance using a microplate reader (Multiskan Ascent, Thermo Scientific).
2.4.5. CRH and NPY RT-qPCR Real-time quantitative PCR (qPCR) was performed with PowerUp SYBR Green Master Mix (Applied BioSystems, ThermoFisher Scientific, Waltham, MA) on an iCycler iQ + MyiQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). The primer sequences for β-actin (NM_031144), NPY(NM_012614), and CRH (NM_031019) were designed and/or validated using Primer-BLAST (National Center for Biotechnology Information, NIH), and the thermal profile for each primer was optimized to 90–110% efficiency. Primer sequences are listed in Table 3, as are sequence and thermal conditions. Target gene expression levels were normalized to β-actin by ΔΔCt method.
2.5. Statistical analyses
2.4.2. Beta-hydroxybutyrate assay Plasma βHB was measured in duplicate by β-Hydroxybutyrate LiquiColor® assay kit (Stanbio, Laboratory, Boerne, TX) using a microplate reader. First, 1 mM standard solution was diluted with deionized water into five concentrations: 1, 0.75, 0.5, 0.25, and 0.125 mM. 2.5 μl of sample or standard were loaded on a 96 well plate. Reagent 1 was incubated at 25 °C for 10 min then 90 μl were dispensed into each well. The absorbance of 505 nm was immediately taken as the baseline reading. After adding 15 μl of reagent 2 per well, the plate was incubated in 25 °C for 10 min and a second reading was taken. The difference between the second and the first measurements was used to quantify expression and the concentration of plasma samples were interpolated from the standard curve.
Data were analyzed using R Studio (version 1.1.464, R Studio, Inc. Boston, MA2018). Kruskal-Wallis test was first run to evaluate whether combined male and female data were normally distributed. χ2 values for sex comparisons are reported for each outcome measure. If data were parametric, then an analysis of variance (ANOVA) was run with three factors: stress, diet, and sex. If Kruskal-Wallis test indicates male and female groups are non-parametric, ANOVA was run with two factors, stress and diet, per sex. Time was also included as a repeated factor, when necessary. Follow-up Tukey's post hoc multiple comparison test are reported when significant. Combined male and female data are illustrated using GraphPad Prism software (v7.00, GraphPad software Inc., La Jolla, CA). Significance was set at 95% percent confidence intervals (p < .05) for all analyses, and effects of stress (#), diet (*), and sex (†) are reported. Mean ± standard error of the mean (SEM) are illustrated in figures (Fig. 1).
2.4.3. IL-1β assay Plasma IL-1β was quantified in duplicate with R & D Quantikine 3
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Table 3 Primer sequences and thermal profiles. Target gene
Primer sequences
NCBI sequence
Thermal profile
Efficiency
CRH
Forward: 5′ – CTC TCT GGA TCT CAC CTT CCA C– 3′ Reverse: 5′ - CTA AAT GCA GAA TCG TTT TGG C - 3′
NM_031019.1
100.4%
NPY
Forward: 5’- TAT CCC TGC TCG TGT GTT TG- 3’ Reverse: 5′- TGT CGC AGA GCG GAG TAG TA - 3′
NM_012614
β-actin
Forward: 5′ - ATT GGT GGC TCT ATC CTG GC - 3′ Reverse: 5′- AAA CGC AGC TCA GTA ACA GTC - 3′
NM_031144.3
95 °C/15 s 60 °C/15 s 72 °C/60s 95 °C/15 s 58 °C/15 s 72 °C/60s 95 °C/15 s 60 °C/15 s 72 °C/60s
97.6%
90.4%
3. Results 3.1. Beta-hydroxybutyrate levels Final βHB was measured to confirm animals on KD had elevated circulating βHB. Parametric analyses found male and female plasma βHB levels were normally distributed (Kruskal-Wallis test, χ2 = 2.143, p = .143), therefore, three way analyses were appropriate. Animals consuming KD were found to have significantly higher βHB than animals on CH (F(1,57) = 45.689, p ≤.001), as shown in Fig. 2. There were no significant effects of stress (F(1,57) = 0.035, p = .853), or sex (F(1,57) = 2.565, p = .115), and no significant interactions. Fig. 3. Average daily food intake. Food intake was not normally distributed between sexes (Kruskal-Wallis χ2 = 50.918, †: p < .001). There were no significant changes in male groups. Females undergoing CMS has lower food intake, particularly between CH+ and KD- groups (#: p = .042). Values are means +/− SEM; n = 7–10 per group.
3.2. Daily caloric intake Combined caloric intake data were not normally distributed between sexes (Kruskal-Wallis χ2 = 50.918, p ≤.001). Because this failed to meet assumptions to analyze sex as an ANOVA factor, male and female data were analyzed separately with stress and diet as factors. Analysis of average daily food intake (Fig. 3) over the three weeks suggests there were no significant effects of stress (F(1,29) = 2.220, p = .147) or diet (F = (1,29)3.997, p = .055) within male groups however a significant effect of stress within females (F(1,33) = 6.035, p = .019). Tukey's multiple comparison tests suggest this was driven by CH+ females, which had significantly lower average food intake than KD- females (p = .042). No effects of diet (F(1,33) = 2.201, p = .147) were observed in females. 3.3. Body weight Baseline body weights were subtracted from final weight to calculate body weight gain from the start to the end of the experiment. Data were non-parametric (Kruskal-Wallis χ2 = 7.133, p = .007) so data were analyzed by stress and diet (Fig. 4). For males, there were significant effects of stress (F(1,29) = 10.676, p = .003) and diet (F
Fig. 4. Body weight change from start to end CMS. Body weight change was not normally distributed by sex (Kruskal-Wallis χ2 = 7.1334, †: p = .008). CH+ males gained less weight than CH– (#: p = .022), KD- (#: p < .001), and KD+ (#: p = .001). The same was observed within females. CH+ females gained less weight than CH– (*: p = .003), KD- (*: p = .001), and KD+ (*: p = .049). Values are means +/− SEM; n = 7–10 per group.
(1,29) = 19.712, p ≤.001). Tukey's post hoc test analysis found that these effects were driven by CH+ group. CH+ gained significantly less weight than CH– (p = .022), KD- (p ≤.001), and KD+ groups (p = .001). For females, there was only a significant effect of stress (F (1,33) = 9.281, p = .005) and not diet (F(1,33) = 3.329, p = .077). Tukey's post hoc analysis found that body weight gain differences were also driven by the CH+ group. CH+ females gained significantly less weight than CH– (p = .003), KD- (p = .001), and KD+ (p = .049). Collectively, CMS males and females consuming CH gained less weight than their respective non-stress CH consuming group. No differences were observed in KD consuming animals.
Fig. 2. Terminal plasma beta-hydroxybutyrate (βHB). βHB levels were normally distributed (Kruskal-Wallis test, χ2 = 2.143, p = .143). Animals consuming KD had significantly higher plasma βHB after three weeks of diet exposure than those consuming CH (*: p < .0001). Values are means +/− SEM; n = 6–10 per group. 4
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Fig. 5. Elevated Plus Maze (EPM) behavior. (A) Time spent in closed arms of apparatus was normally distributed between sexes (Kruskal-Wallis χ2 = 0.799, p = .371). There was a significant effect of stress, driven by CH+ females (#: p = .034). (B) Time spent in center of apparatus was normally distributed between sexes (Kruskal-Wallis χ2 = 0.779, p = .377). There was a significant interaction between stress and diet. KD+ groups spent less time in the center of the apparatus than CH+ groups (*: p = .049). (C) Time spent in open arms of apparatus was also normally distributed (Kruskal-Wallis χ2 = 1.615, p = .204). There was a significant effect of stress. Tukey's multiple comparison test suggests this was largely driven female stress groups (#: p = .02). Values are means +/− SEM; n = 7–10 per group.
all three behavior measures (center: Kruskal-Wallis χ2 = 3.198, p = .074, corner: Kruskal-Wallis χ2 = 1.059, p = .303, distance: Kruskal-Wallis χ2 = 0.287, p = .592). There were no significant effects of sex, stress, or diet on locomotor activity or anxiety-like behavior in OFT (Fig. 6A-C). There were also no interactions between the three factors.
3.4. Behavioral assessments 3.4.1. Elevated plus maze (EPM) Behaviors on the EPM were measured three weeks after CMS and analyzed for percent of time in closed, center, and open parts of the apparatus. Compiled sex data were normally distributed for all three measures (closed arms: Kruskal-Wallis χ2 = 0.799, p = .371, open arms: Kruskal-Wallis χ2 = 1.614, p = .204, center quadrant: KruskalWallis χ2 = 0.779, p = .377). There was a significant effect of stress on time spent in the closed arms of the apparatus (F(1,62) = 7.127, p = .01) (Fig. 5A). Tukey's multiple comparison test suggests this was largely driven female CH+ group, as they significantly less time spent in the closed arms than was spent by CH– females (p = .034). There were no significant effects of diet (F(1,62) = 1.203, p = .277) or sex (F(1,62) = 1.697, p = .197). There were no general treatment effects of stress, diet or sex on time spent in center of apparatus, however there was an interaction of stress and diet (F(1,62) = 4.043, p = .049) (Fig. 5B). Tukey's multiple comparison test suggests that stressed animals consuming KD spent less time in the center of the apparatus (p = .054). There was also a significant effect of stress on time spent in the open arms of the EPM (F(1,62) = 7.346, p = .009) (Fig. 5C). Tukey's multiple comparison test suggests this was largely driven by female stress groups (p = .02). There were no significant effects of diet (F (1,62) = 0.042, p = .839) or sex (F(1,62) = 3.597, p = .063).
3.4.3. Forced swim test (FST) FST behaviors were measured on third day of behavior testing. Sex data were not normally distrubuted across all FST measures (immobility: Kruskal-Wallis χ2 = 4.369, p = .037, latency to immobility: Kruskal-Wallis χ2 = 0.717, p = .397, escape: Kruskal-Wallis χ2 = 6.699, p = .009). Therefore, FST data are reported by two-way ANOVA per sex when they are not normally distributed and three way ANOVA when they are. There were no significant effects of stress (F(1,29) = 0.003, p = .956) or diet (F(1,29) = 0.064, p = .801) on time spent immobile in males or females (stress: (F(1,32) = 0.960, p = .335; diet (F (1,32) = 0.858, p = .361) (Fig. 7A). There were no signficant effects of stress (F(1,61) = 2.222, p = .141) or diet (F(1,61) = 0.000, p = .992) or sex (F(1,62) = 0.450, 0.505) on latency to immobility (Fig. 7B). There were no signficant effects of stress (F(1,29) = 0.327, p = .572) or diet (F(1,29) = 0.006, p = .937) on time spent escaping in males or females (stress: (F(1,32) = 0.030, p = .864; diet (F (1,32) = 0.462, p = .501) (Fig. 7C).
3.4.2. Open field test (OFT) OFT behaviors were measured on second day of behavior testing. Locomotor activity was measured as distance traveled in apparatus and percent of time spent in center or corner quadrants of apparatus as anxiety-like behavior. Compiled sex data were normally distributed for
3.5. Neuroendocrine assessment 3.5.1. Corticosterone (CORT) Terminal corticosterone (CORT) was analyzed using plasma samples 5
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Fig. 6. Open field test (OFT) behavior. (A) Distance traveled was normally distributed between sexes (Kruskal-Wallis χ2 = 0.287, p = .592). There were no significant effects of stress, diet, or sex. (B). Time spent in center was normally distributed (Kruskal-Wallis χ2 = 3.198, p = .074. There were no effects of stress, diet, or sex. (C) Time spent in corners was also normally distributed (Kruskal-Wallis χ2 = 1.059, p = .303). There were no significant effects of stress, diet, or sex. Values are means +/− SEM; n = 7–10 per group.
female groups, there was a significant effect of stress (F(1,30) = 4.291, p = .047), but not diet (F(1,30) = 0.857, p = .362). Tukey's post hoc analysis revealed the diet effect in females was driven by a significant difference between CH– and CH+ groups (p = .045). Changes in hypothalamic NPY expression were only observed in CH+ females. CRH expression was normally distributed (Kruskal- Wallis χ2 = 0.106, p = .744), therefore a three-way ANOVA was used, with stress, diet, and sex as factors (Fig. 10B). There were no significant effects of stress (F(1,59) = 0.665, p = .418), diet (F(1,59) = 0.344, p = .560), or sex (F(1,59) = 0.017, p = .897), on hypothalamic CRH expression and no significant interactions between the three factors.
collected approximately 2 h after forced swim test (FST). Data were not normally distributed across sexes (Kruskal-Wallis χ2 = 4.686, p = .03) so each sex was analyzed by two-way ANOVA by stress and diet (Fig. 8). There were no effects of stress (F(1,25) = 0.000, p = .985), or diet (F (1,25) = 0.072, p = .791) observed in male groups with no significant interaction. Significant effects of terminal CORT were observed in females. Although there were no main effects of stress (F(1,31) = 0.708, p = .406) or diet (F(1,31) = 0.297, 0.589), there was a significant interaction (F(1,31) = 7.806, p = .009). Tukey's multiple comparisons found that females that underwent CMS while consuming CH had lower CORT than CH female controls (p = .038).
4. Discussion
3.5.2. Interleukin-1β (IL-1β) Terminal plasma samples were used to analyze circulating levels of cytokine, IL-1β. Combined sex data were normally distributed (KruskalWallis χ2 = 3.554, p = .059) so three-way ANOVA was run with stress, diet, and sex as factors (Fig. 9). There were no effects of stress (F (1,62) = 0.003, p = .956), diet (F(1,62) = 0.095, p = .758), or sex (F (1,62) = 0.0489, p = .826). Notably, all values were toward the lower reliably detectable limit (9 ng) according to the kit manufacturer, suggesting minimal to no inflammation in any of the groups.
Male and female rats that experienced chronic mild stress (CMS) while consuming a chow diet (CH+) showed some phenotypic characteristics of affective disorders, such as decreased body weight. Differences in time spent in the open arms of the EPM, plasma CORT, and hypothalamic NPY expression were also observed, but only in female groups. Additionally, our data did not demonstrate any behavioral or neuroendocrine differences in either KD+ or KD- condition. This suggests that KD animals were protected from stress-induced changes in body weight, CORT, and NPY. Collectively, these data suggest KDs have some therapeutic potential in normalizing physiological changes related to energy expenditure, particularly in females. Increased time spent in the open arms of EPM and decreased CORT in females is consistent with chronic stress experiments from other groups, and indicative of anxiety-like behavior [25]. The FST served as an acute stressor, and females that undergo acute stress have been shown to have higher CORT than those that have undergone chronic stress [20]. The direction of behavioral changes is opposite of what is
3.5.3. Neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) expression Hypothalamic tissue was collected and micro dissected approximately two hours after final behavior test. NPY expression was not normally distributed across sexes (Kruskal-Wallis χ2 = 3.926, p= 0.048) so each sex was analyzed by two-way ANOVA (Fig. 10A). Within male groups, there were no significant effects of stress (F (1,29) = 0.148, p = .703), or diet (F(1,29) = 0.727, p = .401). Within 6
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Fig. 7. Forced swim test (FST) behavior. (A) Time spent immobile. Sex distributions were significantly different (Kruskal-Wallis χ2 = 4.46, †: p = .034). There were no significant effects within male or female groups of stress or diet. (B) Latency to immobility. Sex distributions were not significantly different (Kruskal-Wallis χ2 = 0.717, p = .397). There were no significant effects of stress, diet, or sex observed. (C) Time spent escaping. Sex distributions were significantly different (Kruskal-Wallis χ2 = 6.699, †: p = .01). There were no significant effects within male or female groups of stress or diet. Values are means +/− SEM; n = 7–10 per group.
Fig. 9. Final plasma IL-1β. Combine sex data were normally distributed (Kruskal-Wallis χ2 = 3.553, p = .059). There were no significant effects of CMS exposure or exposure to KD in either males or females. Values are means +/− SEM; n = 7–10.
Fig. 8. Terminal plasma corticosterone (CORT). Data were not normally distributed across sexes (Kruskal-Wallis χ2 = 4.686, †: p = .03). Females undergoing stress while consuming chow (CH+) had lower CORT than control females (CH-) (#: p = .038) and there were no changes observed between male groups. Values are means +/− SEM; n = 6–10 per group.
lack of change in time spent floating in the FST, no changes in time in the center of OFT, no changes in plasma CORT and IL-1β, or changes in hypothalamic CRH [16]. This could be attributed to a number of issues such as external environmental factors, stressors being too mild, habituation to stress, and even the sex of experimenters ([32,37,36]). Because of these additional factors that can influence the paradigm, criticism for this particular model for its issues of reproducibility across labs has been growing [2,9,26,29]. Lack of changes in CRH and IL-1β could also be due to how plasma and tissue were processed. Animals were sacrificed approximately 2 h (+/− 15 min) after FST, when animals were expected to return to a basal level. The high variability suggests there were some differences in some animals, and it is possible that repeated blood collections at different time points post-FST would have revealed differences in stress responses in our experiments. It is known, for example, that males that
typically observed in males. The dampened response in females may be related to differences in adaptive response to chronic stress [8]. Females exhibit hypoactivity in the HPA axis in response to chronic stress, whereas males typically exhibit hyperactivity. This sexual dimorphism has been attributed to the multifaceted roles of estrogens and reproductive cycles. This phenomenon is still not fully understood, albeit its critical importance in understanding how and why there are sex differences in not only the development of mood disorders, but also metabolism [4,24]. Estrous cycle was not measured in this experiment as typical methods, such as vaginal swabs, are stressful and an extra stress variable to females could have influenced the data interpretation of general sex differences. Some of our data were inconsistent with what has been reported by others using CMS models. Inconsistencies in stress responses include a 7
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protective effects on metabolic and cognitive health ([14]; K P Kinzig & Taylor, [17]; Kimberly P. [18]) The goal of this experiment was to elucidate the protective effects of KDs to the development of stress-induced mood disorders in a preclinical model. The model chosen is reportedly influenced by numerous factors that are difficult to replicate between labs, thus limiting the findings in this study. Although the range of depressive and anxiety-like behaviors that are analogous to those observed in mood disorders were not reached with this model, we did find that KD somewhat increased resiliency to stress-induced changes observed in groups consuming a normal chow diet. Perhaps a different pre-clinical model, such as chronic psychosocial stress would further test our hypothesis, however, sex differences in these models are challenging to interpret as males and females respond to different psychosocial stressors [13]. Further research is warranted not only on the effects of KD on mental health, but also on what other aspects of high fat/high carbohydrate diets play roles in mood disorders. Acknowledgements We would like to express our appreciation to Dr. Julia Chester for assisting with corticosterone assays, and Melissa McCurley for technical assistance with animal husbandry, behavioral testing, and tissue collection. Funding provided by Purdue University Department of Psychological Sciences. References Fig. 10. Hypothalamic neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) mRNA expression. Cycle thresholds (Ct) normalized to ß-actin, and ΔΔ Ct was normalized to chow control condition (CH-). (A) Male and female groups had different NPY expression distributions (Kruskal-Wallis χ2 = 3.927, †: p = .048), therefore sexes were analyzed separately. There were no significant differences within males. Within female analyses found CH+ group had lower NPY expression than CH– (#: p = .045). (B) Male and female groups had normal CRH distributions (Kruskal- Wallis χ2 = 0.106, p = .744). There were no significant effects of stress, diet, or stress on CRH expression in the hypothalamus. Values are means +/− SEM; n = 7–10 per group.
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experience CMS have higher basal levels, but respond differently at 15, 30, and 60 min post- acute stress [5]. Pre-behavior testing samples would clarify basal levels, however, the stress of blood collection could introduce an acute stress episode before testing and influence behavior testing. Additionally, NPY expression levels were more variable in males under experimental conditions in this experiment than is typically reported. This greater variability may also be attributed to the timing of tissue collection, given that CORT, CRH, and NPY mechanisms are interrelated. Other high fat diet studies, such as those using a ‘Western diet’ (WD) that is high in both fat and carbohydrate, demonstrate that caloriedense diets high in fat that are not ketogenic disrupt HPA activity. This has been related to disordered feeding behavior and induction of a depressive-like phenotype [12,31,33,40]. Our current data demonstrate that minimizing the carbohydrate level of a very high fat diet does not produce the negative effects of WD, and do not increase susceptibility to the development of symptoms consistent with affective disorders. Our data show that KD can play a role in preventing stress-induced decreases in body weight which is indicative of symptoms of anxiety and depression identified in the DSM-V. This could be attributed to the elevation of BHB observed in KD-consuming animals. Our experiments demonstrate no depressive- or anxiety-like behavior, body weight, food intake, or hypothalamic dysregulation when animals consumed a diet with 80% fat. This suggests that the effects of high fat diets (containing 42–60% fat) are non-linear and that it is likely that other elements of these diets, such as simple carbohydrates, alone or in combination with high fat induce deleterious effects on mental health. This is consistent with previous experiments from our lab suggesting that KDs may impart 8
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Attenuation of stress-induced weight loss with a ketogenic diet ⁎
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Elizabeth Sahagun , Lauren M. Ward, Kimberly P. Kinzig Department of Psychological Sciences, Purdue University, West Lafayette, IN, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Ketogenic diet Chronic stress Hypothalamic-pituitary-adrenal axis Mood disorders Animal model
Ketogenic diets (KDs) are high-fat, low-carbohydrate diets that have been used therapeutically for decades, most notably for the treatment of epilepsy and diabetes. Recent data, however, suggest that KD may impart protective effects on mood disorders. The current experiments test the hypothesis that KDs can protect from stress-induced symptoms of mood disorders. To test this, we assessed behavioral and neuroendocrine effects of KD in male and female Long Evans rats. Animals experienced three weeks of chronic mild stress (CMS) while consuming KD or control chow (CH). Body weight and food intake data were recorded daily and behaviors were assayed after three weeks. Plasma beta-hydroxybutyrate (βHB), corticosterone (CORT) and interleukin-1 beta (IL-1β) were measured after behavioral testing, along with hypothalamic corticotropin-releasing hormone (CRH) and neuropeptide Y (NPY) mRNA expression. CMS induced weight loss in the CH groups, however the KD-fed rats were resistant to CMS-induced weight loss. Female rats fed KD were protected from CMS-induced reductions in plasma CORT and hypothalamic NPY expression. Collectively, these data suggest protective potential of KDs against chronic stress, particularly in females.
1. Introduction Lifestyle changes known to decrease stress and improve mental health are commonly suggested for individuals reporting symptoms of affective disorders [35]. Improved nutrition through dietary modifications, or implementing relatively undefined “healthy eating habits”, are suggested as beneficial to overall well-being. Even so, data supporting the health benefits of various dietary approaches have produced equivocal findings. Incomplete understanding of how dietary changes can impact mood and mental health can result in individuals turning to non-empirical sources of information, such as anecdotes from media sources making indirect untested claims. For example, the sustained popularity of ketogenic diets (KD) for improved health is largely fueled by lay reports in which there are widespread claims that severe restriction of carbohydrates can increase energy and mood. Empirical data demonstrate that KDs are effective in the treatment of epilepsy and diabetes, and are hypothesized to have other positive neuronal benefits. [28,30]. KD has been shown to protect from harmful diet-induced deficits in the brain such as impaired performance on cognitive tasks, decreased brain derived neurotropic factor (BDNF), as well as increased hippocampal inflammation and blood brain barrier permeability [10,14,15]. KDs can also protect from metabolic insult and peripheral immune challenges ([11]; Kimberly P. [18]; Kimberly P Kinzig, Scott, Hyun, Bi, & Moran, [19], [39]). More recently, data
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suggest that KD may be neuroprotective in response to the pathophysiology of mood disorders and adverse effects of chronic stress [6,7]. The neuroprotective effects of KD are suggested to rely on the induction of ketosis and elevation of beta-hydroxybutyrate (βHB). The elevation of βHB using a 80% lard-based diet has shown to protect from diet-induced cognitive deficits and blood brain barrier leakage [10,14,15]. βHB is produced by the liver and elevated in the blood stream in response to consuming a high-fat/low-carbohydrate diet, prolonged fasting, or exercising [23]. βHB enters the brain by active transport via monocarboxylate transporters [1]. The presence of βHB in the CNS has been demonstrated to decrease anxiety-related behavior and influence activity levels [3,27]. Mood disorders constitute neurological changes that result in emotional dysregulation and cognitive deficits. The core of some symptoms can also be the result of energy dysregulation driven by poor diet. Existing evidence shows that diets high in fat and sugar can cause depression-like symptoms, and epidemiological research shows that depression is often comorbid with metabolic disorders (Luppino, Floriana S., de Wit, Leonore M., Bouvy [22]). There is evidence suggesting KD can reverse the adverse metabolic effects of high-fat/ highsugar diet, however the effects on mental health are unclear. Collectively, data suggest that KD may be beneficial to both mental and metabolic health. Chronic stress increases one's susceptibility to develop a mood
Corresponding author. E-mail address: [email protected] (E. Sahagun).
https://doi.org/10.1016/j.physbeh.2019.112654 Received 28 March 2019; Received in revised form 29 July 2019; Accepted 15 August 2019 Available online 17 August 2019 0031-9384/ © 2019 Published by Elsevier Inc.
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animals were switched to their respective diets and given four days to habituate to their new diet. Then on days 5–26, stressors were presented once a day for various amounts of time in a pseudorandom manner to prevent habituation and predictability. After CMS, behavioral responses to the elevated plus maze (EPM), open field (OFT), and forced swim (FST) were evaluated on consecutive days. Experimental timeline is shown in Fig. 1. Rats were sacrificed approximately two hours after FST for plasma and hypothalamic tissue analyses.
disorder. Stressful stimuli activate the sympathetic nervous system, which leads to release of hormones that motivate a “fight or flight” response. Under normal conditions, this is an acute adaptive response of survival that is mediated by the hypothalamic-pituitary- adrenal (HPA) axis. The HPA axis has several feedback mechanisms to return the brain and body back to homeostasis once a stressful event is over. Chronic activation can lead to dysregulation of the HPA axis and becomes detrimental to homeostatic processes such as energy regulation [34], desensitization of neurotransmitter function in higher up brain regions [21], and immune function [38]. The goal of the following experiments was to elucidate the effects of KD on the development of anxiety- and depressive-like behaviors, analogous to symptoms of mood disorders, that result from exposure to chronic mild stress (CMS). Rats were given a KD with macronutrient ratios similar to a strict KD adopted by humans (80% fat, 15% protein, and 5% carbohydrate) (Kimberly P. [18]), and exposed to a series of mild stressors over the course of three weeks. We hypothesized that rats consuming KD during CMS will be resilient to deleterious effects typically induced by the model, as measured by behavioral and neuroendocrine outcomes.
2.3. Behavioral assessments Food intake was measured daily and final behavioral tests were done for three consecutive days following the end of CMS. All final testing was done in a room adjacent to the vivarium during the first half of the light cycle. Animals were given 10–15 min to habituate to the room before the start of each test. Test order was counterbalanced within each sex to minimize test time bias. 2.3.1. Elevated plus maze Anxiety-like behavior was assessed using the elevated plus maze (EPM). The EPM apparatus is 3 ft high, and has with four arms at 90degree angles, each 6 in. wide and 3 ft long. Two of the opposite side arms are enclosed and the other two are open. Animals were placed in the center of the EPM and behavior was video recorded from a ceiling mounted camera for 5 min. Rats were returned to home cages immediately after testing. The percent of time spent in open arms and in the center was scored manually by an observer blinded to the experimental conditions because AnyMaze tracking was not sensitive enough to track the head of Long Evans on the apparatus.
2. Materials methods 2.1. Animals and diets Adult (16–17 week old) male (N = 33) and female (N = 37) LongEvans rats were individually housed in suspended wire cages in humidity (55–65%) and temperature (20.5 ± 1 °C) controlled rooms that were maintained with a 12:12 h light/dark cycle unless stated in the stress schedule (Table 1). Males and females were housed in separate rooms with light/dark cycles staggered by 1 h to maintain consistency of stress and testing times between groups. Animals were weight matched and then assigned to either KD or chow (CH) diet. The KD (Research Diets D06040601, New Brunswick, NJ) consisted of 80% fat, 5% carbohydrates, and 15% protein by calories. This lard-based diet has been used to effectively impart therapeutic effects of elevated βHB ([15]; Kimberly P. [18]; Kimberly P Kinzig et al., 2005). The control diet, CH (Teklad 2018, Envigo, Indianapolis, IN), consisted of 18% fat, 58% carbohydrates, and 24% protein. Diet compositions are further described in Table 2. After four days of consuming the assigned diet, animals were randomly assigned to a stress (+) or non-stress (−) subgroup for the remainder of the experiment. There were four experimental groups per sex: CH-, CH+, KD-, KD+. Group n for males were 7, 9, 8, 9, and for females 9, 9, 9, 10, respectively. All animals had ad libitum access to food and water in the home cage. Animals were weighed daily and food intake was measured by weighting food container daily and subtracting food spillage. All procedures were approved by the Purdue Animal Care and Use Committee (PACUC).
2.3.2. Open field test The open field test (OFT) quantifies locomotor activity and anxietylike behavior. The apparatus is 4 ft by 4 ft, with a grid printed on the floor. The rat is placed in the center of the apparatus and video recorded for five minutes on a ceiling view mounted camera. Videos were later scored automatically using Any-maze behavior tracking software (Stoelting Co.) Total distance traveled, percent time spent in the center and time spent in the corners of the apparatus were quantified. 2.3.3. Forced swim test Depressive-like behaviors were assessed in rats by forced swim test (FST). The FST apparatus was a 22 × 17 × 25 inch glass container with enough water to prevent rats from stepping on the bottom of the apparatus. Water was maintained at 25–30 °C. Rats were monitored for the duration of the test and the test was stopped if rats did not maintain a swim or float behavior. The test was recorded for five minutes using a ceiling mounted camera. After five minutes, fur was immediately dried with a towel and rats were returned to the home cage. Any-maze behavior tracking software (Stoelting Co.) was used to score behaviors. An observer blinded to treatment conditions evaluated time spent immobile, latency to immobility, and time spent displaying escape behaviors.
2.2. Chronic mild stress (CMS) Stress groups underwent three weeks of CMS by exposure to daily stressors (Willner, [36]), which are described in Table 1. In short, Table 1 CMS details. Stressor
Description
Duration
Restraint Lights Flooding Cold Crowding Static Cage Tilt
Restrained in a clear PVC device with a perforated front at an unpredictable time during the light cycle. Moved to a neighboring housing room in novel cages, where the lights remained on during the dark cycle. 200 ml of room temp tap water added to bedding in novel cage during the light cycle. Moved to a novel cage and placed in a 4 degrees C chamber at an unpredictable time during the light cycle. Temporarily housed with 3–4 unfamiliar conspecifics also undergoing CMS during the light cycle. Static noise (80 dB) played in a neighboring housing room in the middle of the light cycle. Moved to a novel cage during different times of the day tilted 30–45 degrees in home room.
30 min 12 ha 16 ha 30 min 6 ha 6 ha 6 ha
Note: Animals had access to food and water in conditions lasting 6 or more hours. 2
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Table 2 Diet compositions (% kcal). Diet type
Carbs % kcal
Protein % kcal
Fat % kcal
Calorie Density
Product ID
Chow (CH) Ketogenic (KD)
58
24
18
3.1 kcal/g
5
15
80
6.1 kcal/g
Teklad (2018) Research Diets (D06040601)
ELISA Rat IL-1β /IL-1F2 immunoassay according to the manufacturer's instructions. In summary, Plasma samples were diluted 1:3 using the provided calibrator diluent and the assay standard was serially diluted with a dilution factor of 2 in 8 concentrations. 50 μl of assay diluent was added to rat IL-1β coated well plates, then 50 μl of sample or standard were loaded into each well plate then incubated at room temperature for 2 h. After the incubation period, wells were washed 5 times, loaded with 100 μl of conjugate, incubated again at room temperature for 2 h, then washed again five times. After second wash, 100 μl of substrate solution was added to each well, incubated for 30 min at room temperature, protected from light, then 100 μl of stop solution was added. Immediately following the addition of stop solution, 450 nm absorbance was measured in a microplate reader (Multiskan Ascent with Ascent software v2.6, ThermoFisher Scientific, Waltham, MA).
Fig. 1. Experimental Timeline. Rats were exposed to either ketogenic diet (KD) or chow (CH) diet for 4 days, then underwent three weeks of chronic mild stress (CMS). After CMS, animals were tested for three consecutive days on elevated plus maze (EPM), open field (OFT), and forced swim (FST). They were then sacrificed approximately two hours after FST for plasma and tissue analysis.
2.4. Terminal assessment Rats were sacrificed by pentobarbital (Beauthanasia, D, Merck Animal Health, Madison, NJ) overdose approximately two hours after FST test for collection of blood and removal of brains. Following rapid decapitation, 3 ml of trunk blood was collected in VACUETTE® K3EDTA blood collection tubes (Greiner Bio-One, Monroe, NC) and centrifuged for 15 min at 4 °C, and 2500 rpm. Plasma was isolated and aliquoted into three tubes and stored in a − 80 °C freezer for later analyses. Plasma analyses included circulating levels of corticosterone, βHB, and IL-1β. Brains were rapidly removed and rinsed with 1× phosphate buffered saline (PBS). The hypothalamus of each rat was micro-dissected on ice, flash frozen on dry ice, then stored at −80 °C for later analysis of CRH and NPY.
2.4.4. RNA extraction and cDNA synthesis Tissues were homogenized in 1 ml TriReagent (RT111, Molecular Research Center, Cincinnati, OH) then 50 µl bromanisole (BCP, Molecular Research Center, Cincinnati, OH) was used to extract RNA. RNA was quantified by spectrophotometer at 260 nm absorbance (Eppendorf BioPhotometer 22,331, Hauppauge, NY). First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA) was used to synthesize cDNA from 2 μg total RNA.
2.4.1. Corticosterone assay Plasma corticosterone (CORT) concentration was quantified in duplicates using an EIA kit according to manufacturer instructions (Enzo Life sciences, protocol ADI-900-097). Samples were diluted 1:40 using a steroid displacement reagent and standards were serially diluted 1:50. 100 μl of sample or standard were loaded on donkey anti-sheep IgG coated plates, followed by 50 μl of conjugate, then 50 μl of sheep antibody to rat CORT. Plates were then incubated at room temperature on a plate shaker for 2 h at 500 rpm, and then washed 3 times. After washing, 200 μl of pNpp substrate solution was added to each well, and then incubated at room temperature for one hour. 50 μl of stop solution was then added to every well, and optical density was immediately read at 405 nm absorbance using a microplate reader (Multiskan Ascent, Thermo Scientific).
2.4.5. CRH and NPY RT-qPCR Real-time quantitative PCR (qPCR) was performed with PowerUp SYBR Green Master Mix (Applied BioSystems, ThermoFisher Scientific, Waltham, MA) on an iCycler iQ + MyiQ Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). The primer sequences for β-actin (NM_031144), NPY(NM_012614), and CRH (NM_031019) were designed and/or validated using Primer-BLAST (National Center for Biotechnology Information, NIH), and the thermal profile for each primer was optimized to 90–110% efficiency. Primer sequences are listed in Table 3, as are sequence and thermal conditions. Target gene expression levels were normalized to β-actin by ΔΔCt method.
2.5. Statistical analyses
2.4.2. Beta-hydroxybutyrate assay Plasma βHB was measured in duplicate by β-Hydroxybutyrate LiquiColor® assay kit (Stanbio, Laboratory, Boerne, TX) using a microplate reader. First, 1 mM standard solution was diluted with deionized water into five concentrations: 1, 0.75, 0.5, 0.25, and 0.125 mM. 2.5 μl of sample or standard were loaded on a 96 well plate. Reagent 1 was incubated at 25 °C for 10 min then 90 μl were dispensed into each well. The absorbance of 505 nm was immediately taken as the baseline reading. After adding 15 μl of reagent 2 per well, the plate was incubated in 25 °C for 10 min and a second reading was taken. The difference between the second and the first measurements was used to quantify expression and the concentration of plasma samples were interpolated from the standard curve.
Data were analyzed using R Studio (version 1.1.464, R Studio, Inc. Boston, MA2018). Kruskal-Wallis test was first run to evaluate whether combined male and female data were normally distributed. χ2 values for sex comparisons are reported for each outcome measure. If data were parametric, then an analysis of variance (ANOVA) was run with three factors: stress, diet, and sex. If Kruskal-Wallis test indicates male and female groups are non-parametric, ANOVA was run with two factors, stress and diet, per sex. Time was also included as a repeated factor, when necessary. Follow-up Tukey's post hoc multiple comparison test are reported when significant. Combined male and female data are illustrated using GraphPad Prism software (v7.00, GraphPad software Inc., La Jolla, CA). Significance was set at 95% percent confidence intervals (p < .05) for all analyses, and effects of stress (#), diet (*), and sex (†) are reported. Mean ± standard error of the mean (SEM) are illustrated in figures (Fig. 1).
2.4.3. IL-1β assay Plasma IL-1β was quantified in duplicate with R & D Quantikine 3
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Table 3 Primer sequences and thermal profiles. Target gene
Primer sequences
NCBI sequence
Thermal profile
Efficiency
CRH
Forward: 5′ – CTC TCT GGA TCT CAC CTT CCA C– 3′ Reverse: 5′ - CTA AAT GCA GAA TCG TTT TGG C - 3′
NM_031019.1
100.4%
NPY
Forward: 5’- TAT CCC TGC TCG TGT GTT TG- 3’ Reverse: 5′- TGT CGC AGA GCG GAG TAG TA - 3′
NM_012614
β-actin
Forward: 5′ - ATT GGT GGC TCT ATC CTG GC - 3′ Reverse: 5′- AAA CGC AGC TCA GTA ACA GTC - 3′
NM_031144.3
95 °C/15 s 60 °C/15 s 72 °C/60s 95 °C/15 s 58 °C/15 s 72 °C/60s 95 °C/15 s 60 °C/15 s 72 °C/60s
97.6%
90.4%
3. Results 3.1. Beta-hydroxybutyrate levels Final βHB was measured to confirm animals on KD had elevated circulating βHB. Parametric analyses found male and female plasma βHB levels were normally distributed (Kruskal-Wallis test, χ2 = 2.143, p = .143), therefore, three way analyses were appropriate. Animals consuming KD were found to have significantly higher βHB than animals on CH (F(1,57) = 45.689, p ≤.001), as shown in Fig. 2. There were no significant effects of stress (F(1,57) = 0.035, p = .853), or sex (F(1,57) = 2.565, p = .115), and no significant interactions. Fig. 3. Average daily food intake. Food intake was not normally distributed between sexes (Kruskal-Wallis χ2 = 50.918, †: p < .001). There were no significant changes in male groups. Females undergoing CMS has lower food intake, particularly between CH+ and KD- groups (#: p = .042). Values are means +/− SEM; n = 7–10 per group.
3.2. Daily caloric intake Combined caloric intake data were not normally distributed between sexes (Kruskal-Wallis χ2 = 50.918, p ≤.001). Because this failed to meet assumptions to analyze sex as an ANOVA factor, male and female data were analyzed separately with stress and diet as factors. Analysis of average daily food intake (Fig. 3) over the three weeks suggests there were no significant effects of stress (F(1,29) = 2.220, p = .147) or diet (F = (1,29)3.997, p = .055) within male groups however a significant effect of stress within females (F(1,33) = 6.035, p = .019). Tukey's multiple comparison tests suggest this was driven by CH+ females, which had significantly lower average food intake than KD- females (p = .042). No effects of diet (F(1,33) = 2.201, p = .147) were observed in females. 3.3. Body weight Baseline body weights were subtracted from final weight to calculate body weight gain from the start to the end of the experiment. Data were non-parametric (Kruskal-Wallis χ2 = 7.133, p = .007) so data were analyzed by stress and diet (Fig. 4). For males, there were significant effects of stress (F(1,29) = 10.676, p = .003) and diet (F
Fig. 4. Body weight change from start to end CMS. Body weight change was not normally distributed by sex (Kruskal-Wallis χ2 = 7.1334, †: p = .008). CH+ males gained less weight than CH– (#: p = .022), KD- (#: p < .001), and KD+ (#: p = .001). The same was observed within females. CH+ females gained less weight than CH– (*: p = .003), KD- (*: p = .001), and KD+ (*: p = .049). Values are means +/− SEM; n = 7–10 per group.
(1,29) = 19.712, p ≤.001). Tukey's post hoc test analysis found that these effects were driven by CH+ group. CH+ gained significantly less weight than CH– (p = .022), KD- (p ≤.001), and KD+ groups (p = .001). For females, there was only a significant effect of stress (F (1,33) = 9.281, p = .005) and not diet (F(1,33) = 3.329, p = .077). Tukey's post hoc analysis found that body weight gain differences were also driven by the CH+ group. CH+ females gained significantly less weight than CH– (p = .003), KD- (p = .001), and KD+ (p = .049). Collectively, CMS males and females consuming CH gained less weight than their respective non-stress CH consuming group. No differences were observed in KD consuming animals.
Fig. 2. Terminal plasma beta-hydroxybutyrate (βHB). βHB levels were normally distributed (Kruskal-Wallis test, χ2 = 2.143, p = .143). Animals consuming KD had significantly higher plasma βHB after three weeks of diet exposure than those consuming CH (*: p < .0001). Values are means +/− SEM; n = 6–10 per group. 4
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Fig. 5. Elevated Plus Maze (EPM) behavior. (A) Time spent in closed arms of apparatus was normally distributed between sexes (Kruskal-Wallis χ2 = 0.799, p = .371). There was a significant effect of stress, driven by CH+ females (#: p = .034). (B) Time spent in center of apparatus was normally distributed between sexes (Kruskal-Wallis χ2 = 0.779, p = .377). There was a significant interaction between stress and diet. KD+ groups spent less time in the center of the apparatus than CH+ groups (*: p = .049). (C) Time spent in open arms of apparatus was also normally distributed (Kruskal-Wallis χ2 = 1.615, p = .204). There was a significant effect of stress. Tukey's multiple comparison test suggests this was largely driven female stress groups (#: p = .02). Values are means +/− SEM; n = 7–10 per group.
all three behavior measures (center: Kruskal-Wallis χ2 = 3.198, p = .074, corner: Kruskal-Wallis χ2 = 1.059, p = .303, distance: Kruskal-Wallis χ2 = 0.287, p = .592). There were no significant effects of sex, stress, or diet on locomotor activity or anxiety-like behavior in OFT (Fig. 6A-C). There were also no interactions between the three factors.
3.4. Behavioral assessments 3.4.1. Elevated plus maze (EPM) Behaviors on the EPM were measured three weeks after CMS and analyzed for percent of time in closed, center, and open parts of the apparatus. Compiled sex data were normally distributed for all three measures (closed arms: Kruskal-Wallis χ2 = 0.799, p = .371, open arms: Kruskal-Wallis χ2 = 1.614, p = .204, center quadrant: KruskalWallis χ2 = 0.779, p = .377). There was a significant effect of stress on time spent in the closed arms of the apparatus (F(1,62) = 7.127, p = .01) (Fig. 5A). Tukey's multiple comparison test suggests this was largely driven female CH+ group, as they significantly less time spent in the closed arms than was spent by CH– females (p = .034). There were no significant effects of diet (F(1,62) = 1.203, p = .277) or sex (F(1,62) = 1.697, p = .197). There were no general treatment effects of stress, diet or sex on time spent in center of apparatus, however there was an interaction of stress and diet (F(1,62) = 4.043, p = .049) (Fig. 5B). Tukey's multiple comparison test suggests that stressed animals consuming KD spent less time in the center of the apparatus (p = .054). There was also a significant effect of stress on time spent in the open arms of the EPM (F(1,62) = 7.346, p = .009) (Fig. 5C). Tukey's multiple comparison test suggests this was largely driven by female stress groups (p = .02). There were no significant effects of diet (F (1,62) = 0.042, p = .839) or sex (F(1,62) = 3.597, p = .063).
3.4.3. Forced swim test (FST) FST behaviors were measured on third day of behavior testing. Sex data were not normally distrubuted across all FST measures (immobility: Kruskal-Wallis χ2 = 4.369, p = .037, latency to immobility: Kruskal-Wallis χ2 = 0.717, p = .397, escape: Kruskal-Wallis χ2 = 6.699, p = .009). Therefore, FST data are reported by two-way ANOVA per sex when they are not normally distributed and three way ANOVA when they are. There were no significant effects of stress (F(1,29) = 0.003, p = .956) or diet (F(1,29) = 0.064, p = .801) on time spent immobile in males or females (stress: (F(1,32) = 0.960, p = .335; diet (F (1,32) = 0.858, p = .361) (Fig. 7A). There were no signficant effects of stress (F(1,61) = 2.222, p = .141) or diet (F(1,61) = 0.000, p = .992) or sex (F(1,62) = 0.450, 0.505) on latency to immobility (Fig. 7B). There were no signficant effects of stress (F(1,29) = 0.327, p = .572) or diet (F(1,29) = 0.006, p = .937) on time spent escaping in males or females (stress: (F(1,32) = 0.030, p = .864; diet (F (1,32) = 0.462, p = .501) (Fig. 7C).
3.4.2. Open field test (OFT) OFT behaviors were measured on second day of behavior testing. Locomotor activity was measured as distance traveled in apparatus and percent of time spent in center or corner quadrants of apparatus as anxiety-like behavior. Compiled sex data were normally distributed for
3.5. Neuroendocrine assessment 3.5.1. Corticosterone (CORT) Terminal corticosterone (CORT) was analyzed using plasma samples 5
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Fig. 6. Open field test (OFT) behavior. (A) Distance traveled was normally distributed between sexes (Kruskal-Wallis χ2 = 0.287, p = .592). There were no significant effects of stress, diet, or sex. (B). Time spent in center was normally distributed (Kruskal-Wallis χ2 = 3.198, p = .074. There were no effects of stress, diet, or sex. (C) Time spent in corners was also normally distributed (Kruskal-Wallis χ2 = 1.059, p = .303). There were no significant effects of stress, diet, or sex. Values are means +/− SEM; n = 7–10 per group.
female groups, there was a significant effect of stress (F(1,30) = 4.291, p = .047), but not diet (F(1,30) = 0.857, p = .362). Tukey's post hoc analysis revealed the diet effect in females was driven by a significant difference between CH– and CH+ groups (p = .045). Changes in hypothalamic NPY expression were only observed in CH+ females. CRH expression was normally distributed (Kruskal- Wallis χ2 = 0.106, p = .744), therefore a three-way ANOVA was used, with stress, diet, and sex as factors (Fig. 10B). There were no significant effects of stress (F(1,59) = 0.665, p = .418), diet (F(1,59) = 0.344, p = .560), or sex (F(1,59) = 0.017, p = .897), on hypothalamic CRH expression and no significant interactions between the three factors.
collected approximately 2 h after forced swim test (FST). Data were not normally distributed across sexes (Kruskal-Wallis χ2 = 4.686, p = .03) so each sex was analyzed by two-way ANOVA by stress and diet (Fig. 8). There were no effects of stress (F(1,25) = 0.000, p = .985), or diet (F (1,25) = 0.072, p = .791) observed in male groups with no significant interaction. Significant effects of terminal CORT were observed in females. Although there were no main effects of stress (F(1,31) = 0.708, p = .406) or diet (F(1,31) = 0.297, 0.589), there was a significant interaction (F(1,31) = 7.806, p = .009). Tukey's multiple comparisons found that females that underwent CMS while consuming CH had lower CORT than CH female controls (p = .038).
4. Discussion
3.5.2. Interleukin-1β (IL-1β) Terminal plasma samples were used to analyze circulating levels of cytokine, IL-1β. Combined sex data were normally distributed (KruskalWallis χ2 = 3.554, p = .059) so three-way ANOVA was run with stress, diet, and sex as factors (Fig. 9). There were no effects of stress (F (1,62) = 0.003, p = .956), diet (F(1,62) = 0.095, p = .758), or sex (F (1,62) = 0.0489, p = .826). Notably, all values were toward the lower reliably detectable limit (9 ng) according to the kit manufacturer, suggesting minimal to no inflammation in any of the groups.
Male and female rats that experienced chronic mild stress (CMS) while consuming a chow diet (CH+) showed some phenotypic characteristics of affective disorders, such as decreased body weight. Differences in time spent in the open arms of the EPM, plasma CORT, and hypothalamic NPY expression were also observed, but only in female groups. Additionally, our data did not demonstrate any behavioral or neuroendocrine differences in either KD+ or KD- condition. This suggests that KD animals were protected from stress-induced changes in body weight, CORT, and NPY. Collectively, these data suggest KDs have some therapeutic potential in normalizing physiological changes related to energy expenditure, particularly in females. Increased time spent in the open arms of EPM and decreased CORT in females is consistent with chronic stress experiments from other groups, and indicative of anxiety-like behavior [25]. The FST served as an acute stressor, and females that undergo acute stress have been shown to have higher CORT than those that have undergone chronic stress [20]. The direction of behavioral changes is opposite of what is
3.5.3. Neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) expression Hypothalamic tissue was collected and micro dissected approximately two hours after final behavior test. NPY expression was not normally distributed across sexes (Kruskal-Wallis χ2 = 3.926, p= 0.048) so each sex was analyzed by two-way ANOVA (Fig. 10A). Within male groups, there were no significant effects of stress (F (1,29) = 0.148, p = .703), or diet (F(1,29) = 0.727, p = .401). Within 6
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Fig. 7. Forced swim test (FST) behavior. (A) Time spent immobile. Sex distributions were significantly different (Kruskal-Wallis χ2 = 4.46, †: p = .034). There were no significant effects within male or female groups of stress or diet. (B) Latency to immobility. Sex distributions were not significantly different (Kruskal-Wallis χ2 = 0.717, p = .397). There were no significant effects of stress, diet, or sex observed. (C) Time spent escaping. Sex distributions were significantly different (Kruskal-Wallis χ2 = 6.699, †: p = .01). There were no significant effects within male or female groups of stress or diet. Values are means +/− SEM; n = 7–10 per group.
Fig. 9. Final plasma IL-1β. Combine sex data were normally distributed (Kruskal-Wallis χ2 = 3.553, p = .059). There were no significant effects of CMS exposure or exposure to KD in either males or females. Values are means +/− SEM; n = 7–10.
Fig. 8. Terminal plasma corticosterone (CORT). Data were not normally distributed across sexes (Kruskal-Wallis χ2 = 4.686, †: p = .03). Females undergoing stress while consuming chow (CH+) had lower CORT than control females (CH-) (#: p = .038) and there were no changes observed between male groups. Values are means +/− SEM; n = 6–10 per group.
lack of change in time spent floating in the FST, no changes in time in the center of OFT, no changes in plasma CORT and IL-1β, or changes in hypothalamic CRH [16]. This could be attributed to a number of issues such as external environmental factors, stressors being too mild, habituation to stress, and even the sex of experimenters ([32,37,36]). Because of these additional factors that can influence the paradigm, criticism for this particular model for its issues of reproducibility across labs has been growing [2,9,26,29]. Lack of changes in CRH and IL-1β could also be due to how plasma and tissue were processed. Animals were sacrificed approximately 2 h (+/− 15 min) after FST, when animals were expected to return to a basal level. The high variability suggests there were some differences in some animals, and it is possible that repeated blood collections at different time points post-FST would have revealed differences in stress responses in our experiments. It is known, for example, that males that
typically observed in males. The dampened response in females may be related to differences in adaptive response to chronic stress [8]. Females exhibit hypoactivity in the HPA axis in response to chronic stress, whereas males typically exhibit hyperactivity. This sexual dimorphism has been attributed to the multifaceted roles of estrogens and reproductive cycles. This phenomenon is still not fully understood, albeit its critical importance in understanding how and why there are sex differences in not only the development of mood disorders, but also metabolism [4,24]. Estrous cycle was not measured in this experiment as typical methods, such as vaginal swabs, are stressful and an extra stress variable to females could have influenced the data interpretation of general sex differences. Some of our data were inconsistent with what has been reported by others using CMS models. Inconsistencies in stress responses include a 7
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protective effects on metabolic and cognitive health ([14]; K P Kinzig & Taylor, [17]; Kimberly P. [18]) The goal of this experiment was to elucidate the protective effects of KDs to the development of stress-induced mood disorders in a preclinical model. The model chosen is reportedly influenced by numerous factors that are difficult to replicate between labs, thus limiting the findings in this study. Although the range of depressive and anxiety-like behaviors that are analogous to those observed in mood disorders were not reached with this model, we did find that KD somewhat increased resiliency to stress-induced changes observed in groups consuming a normal chow diet. Perhaps a different pre-clinical model, such as chronic psychosocial stress would further test our hypothesis, however, sex differences in these models are challenging to interpret as males and females respond to different psychosocial stressors [13]. Further research is warranted not only on the effects of KD on mental health, but also on what other aspects of high fat/high carbohydrate diets play roles in mood disorders. Acknowledgements We would like to express our appreciation to Dr. Julia Chester for assisting with corticosterone assays, and Melissa McCurley for technical assistance with animal husbandry, behavioral testing, and tissue collection. Funding provided by Purdue University Department of Psychological Sciences. References Fig. 10. Hypothalamic neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) mRNA expression. Cycle thresholds (Ct) normalized to ß-actin, and ΔΔ Ct was normalized to chow control condition (CH-). (A) Male and female groups had different NPY expression distributions (Kruskal-Wallis χ2 = 3.927, †: p = .048), therefore sexes were analyzed separately. There were no significant differences within males. Within female analyses found CH+ group had lower NPY expression than CH– (#: p = .045). (B) Male and female groups had normal CRH distributions (Kruskal- Wallis χ2 = 0.106, p = .744). There were no significant effects of stress, diet, or stress on CRH expression in the hypothalamus. Values are means +/− SEM; n = 7–10 per group.
[1] L.B. Achanta, C.D. Rae, Beta-hydroxybutyrate in the brain: one molecule, multiple mechanisms, Neurochem. Res. 42 (1) (2016) 1–15, https://doi.org/10.1007/ s11064-016-2099-2. [2] S. Antoniuk, M. Bijata, E. Ponimaskin, J. Wlodarczyk, Chronic unpredictable mild stress for modeling depression in rodents: meta-analysis of model reliability, Neurosci. Biobehav. Rev. 99 (December 2018) (2019) 101–116, https://doi.org/10. 1016/j.neubiorev.2018.12.002. [3] C. Ari, Z. Kovács, G. Juhasz, C. Murdun, C.R. Goldhagen, A.M. Koutnik, ... D.P. D'Agostino, Exogenous ketone supplements reduce anxiety-related behavior in Sprague-Dawley and Wistar Albino Glaxo/Rijswijk rats, Front. Mol. Neurosci. 9 (December) (2016) 1–10, https://doi.org/10.3389/fnmol.2016.00137. [4] S.L. Baker, A.C. Kentner, A.T.M. Konkle, L. Santa-Maria Barbagallo, C. Bielajew, Behavioral and physiological effects of chronic mild stress in female rats, Physiol. Behav. 87 (2) (2006) 314–322, https://doi.org/10.1016/j.physbeh.2005.10.019. [5] C. Bielajew, A.T. Konkle, Z. Merali, The effects of chronic mild stress on male Sprague–Dawley and long Evans rats: I. biochemical and physiological analyses, Behav. Brain Res. 136 (2) (2002) 583–592, https://doi.org/10.1016/S01664328(02)00222-X. [6] E. Brietzke, R.B. Mansur, M. Subramaniapillai, V. Balanzá-martínez, M. Vinberg, A. González-pinto, ... R.S. Mcintyre, Neuroscience and biobehavioral reviews ketogenic diet as a metabolic therapy for mood disorders : evidence and developments, Neurosci. Biobehav. Rev. 94 (July) (2018) 11–16, https://doi.org/10.1016/ j.neubiorev.2018.07.020. [7] M.L. Brownlow, S.H. Jung, R.J. Moore, N. Bechmann, R. Jankord, Nutritional ketosis affects metabolism and behavior in Sprague-Dawley rats in both control and chronic stress environments, Front. Mol. Neurosci. 10 (May) (2017) 1–17, https:// doi.org/10.3389/fnmol.2017.00129. [8] S.C. Cook, C.L. Wellman, Chronic stress alters dendritic morphology in rat medial prefrontal cortex, J. Neurobiol. 60 (2) (2004) 236–248, https://doi.org/10.1002/ neu.20025. [9] J.F. Cryan, A. Markou, I. Lucki, Assessing antidepressant activity in rodents: recent developments and future needs, Trends Pharmacol. Sci. 23 (5) (2002) 238–245, https://doi.org/10.1016/S0165-6147(02)02017-5. [10] T.L. Davidson, S.L. Hargrave, S.E. Swithers, C.H. Sample, X. Fu, K.P. Kinzig, W. Zheng, Inter-relationships among diet, obesity and hippocampal-dependent cognitive function, Neuroscience 253 (2013) 110–122, https://doi.org/10.1016/j. neuroscience.2013.08.044. [11] N. Dupuis, N. Curatolo, J.F. Benoist, S. Auvin, Ketogenic diet exhibits anti-inflammatory properties, Epilepsia 56 (7) (2015) e95–e98, https://doi.org/10.1111/ epi.13038. [12] S. Dutheil, K.T. Ota, E.S. Wohleb, K. Rasmussen, R.S. Duman, High fat diet induced anxiety and anhedonia: impact on brain homeostasis and inflammation, Neuropsychopharmacology 41 (October) (2015) 1–30, https://doi.org/10.1038/ npp.2015.357. [13] E. Fuchs, Defeat is a major stressor in males while social instability is stressful mainly in females: Towards the development of a social stress model in female rats, Brain. Res. Bull. 50 (1) (1999) 33–39. [14] S.L. Hargrave, T.L. Davidson, T.J. Lee, K.P. Kinzig, Brain and behavioral perturbations in rats following Western diet access, Appetite 93 (2015) 35–43, https://
experience CMS have higher basal levels, but respond differently at 15, 30, and 60 min post- acute stress [5]. Pre-behavior testing samples would clarify basal levels, however, the stress of blood collection could introduce an acute stress episode before testing and influence behavior testing. Additionally, NPY expression levels were more variable in males under experimental conditions in this experiment than is typically reported. This greater variability may also be attributed to the timing of tissue collection, given that CORT, CRH, and NPY mechanisms are interrelated. Other high fat diet studies, such as those using a ‘Western diet’ (WD) that is high in both fat and carbohydrate, demonstrate that caloriedense diets high in fat that are not ketogenic disrupt HPA activity. This has been related to disordered feeding behavior and induction of a depressive-like phenotype [12,31,33,40]. Our current data demonstrate that minimizing the carbohydrate level of a very high fat diet does not produce the negative effects of WD, and do not increase susceptibility to the development of symptoms consistent with affective disorders. Our data show that KD can play a role in preventing stress-induced decreases in body weight which is indicative of symptoms of anxiety and depression identified in the DSM-V. This could be attributed to the elevation of BHB observed in KD-consuming animals. Our experiments demonstrate no depressive- or anxiety-like behavior, body weight, food intake, or hypothalamic dysregulation when animals consumed a diet with 80% fat. This suggests that the effects of high fat diets (containing 42–60% fat) are non-linear and that it is likely that other elements of these diets, such as simple carbohydrates, alone or in combination with high fat induce deleterious effects on mental health. This is consistent with previous experiments from our lab suggesting that KDs may impart 8
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