Exogenous ketone supplements reduce anxiety-related behavior in Sprague-Dawley and Wistar Albino Glaxo/Rijswijk rats Ari, Csilla 1; Kovacs, Zsolt 2; Juhasz, Gabor 3; Murdun, Cem 1; Goldhagen, Craig R. 1; Koutnik, Andrew 1; Poff, Angela M. 1; Kesl, Shannon L. 1; D’Agostino, Dominic P. 1
1Department
of Molecular Pharmacology and Physiology, Hyperbaric Biomedical Research Laboratory, Morsani College of Medicine, University of South Florida, Tampa FL, USA; 2Department of Zoology, University of West Hungary, Savaria Campus, Szombathely, Hungary; 3Proteomics Laboratory, Eotvos Lorand University, Budapest, Hungary
Background Anxiety disorders are the most prevalent type of mental disorders, interfering with a person`s quality of life. Anxiety and depression are common problems affecting people with epilepsy, while the same brain regions involved in a significant proportion of patients with focal epilepsy, such as the amygdala and the hippocampus, also play a key role in the neurobiology of anxiety. Nutritional ketosis has been proven effective for seizure disorders and other neurological disorders, while anecdotal reports suggest that it can promote a reduction in anxiety. Nutritional ketosis is a state when the body utilizes ketones instead of glucose as the primary molecule of energy metabolism. It can be achieved by fasting, following a ketogenic diet (KD), which is a high-fat (70-85% of daily caloric intake) and low carbohydrate (5-10%) diet or by recently developed exogenous ketone supplements. The focus of this study was to determine the effects of ketone supplementation on anxiety.
Results
Response of SD rats to chronic feeding of exogenous ketone supplementation
Ketone supplementation reduced anxiety on EPM as measured by less entries to closed arms (subchronic SD+KS: SPD and WAG/Rij rats), more time spent in open arms (sub-chronic SD+KS: SPD and WAG/Rij rats; chronic SD+KS/KSMCT: SPD rats), more distance travelled in open arms (chronic SD+KS/KSMCT: SPD rats), and by delayed latency to entrance to closed arms (chronic SD+HKE/KS/KSMCT: SPD rats), when compared to control. The chronic and sub-chronic ketone supplements also caused significant elevation of blood βHB levels and changed blood glucose levels.
Figure1. A) Rats consuming SD+KS or SD+KSMCT supplements spent more time in open arms and less in closed arms, showing reduced anxiety; B) Rats consuming ketone supplements travelled more distance in open arms and less in closed arms, showing reduced anxiety. C) Rats consuming SD+KS or SD+HKE entered the closed arms later, showing reduced anxiety; D) Rats consuming either ketone supplements with ~25g/kg/day dose showed elevated blood ketone levels after 13 weeks. 1
Response of SD rats to 7 days oral administration of ketogenic diet or exogenous ketone supplementation
Response of Wag/Rij rats to 7 days oral administration of exogenous ketone supplementation
Figure 2. A) Less entries in center and closed arms by SD+KS group, compared to control. B) More time spent in open arms by SD+KE group and less time spent in center by KD, SD+KS, SD+KE groups, compared to control. C) Blood βHB levels were higher in all treatment groups after 24 hours and after 7 days as well, blood βHB level was higher in KD group at baseline, compared to control, blood βHB levels were higher in KD and SD+KE groups.
A
Methods 2 Anxiety level of a laboratory rat is tested on an elevated plus maze (EPM).
Summary
Figure 3. A) Less entries in closed arms by SD+KS group, compared to control. B) More time spent in open arms and less time spent in closed arms by SD+KS group, compared to control; C) Blood βHB levels were higher in both treatment groups after 7 days, compared to baseline.
3
A
A
B Time spent (p=0.017;0.021, respectively) and distance travelled (p=0.03; 0.02) in open arms were significantly more in BHB-S and BHB-S+MCT groups, while time spent (p=0.011; 0.005) and distance travelled (p=0.002) in closed arms were significantly less in these groups, compared to control (SD). Latency to first entrance to closed arms was significantly higher in HKE, BHB-S and BHB-S+MCT groups (p=0.037; 0.035; 0.015, respectively). Blood ketone levels were elevated in all ketone supplement treatment groups similar to levels reported. *
Less anxiety is detected by reduced fear from time spent in open arms.
B
B
In conclusion, based on the present study, we can conclude that chronic and sub-chronic administration of exogenous ketone supplementation may be an effective way to reduce anxiety. Achieving nutritional ketosis with exogenous ketone supplementation while maintaining a normal diet might be an alternative to the KD, or may further augment the therapeutic efficacy of the KD. Therefore, it is important to understand the long-term effects of these supplements fed chronically. These preliminary data show that chronic and subchronic feeding of ketone supplements not only elevated blood ketone levels, but also reduced anxiety-related behavior, which can be highly beneficial for patients managing diseases with nutritional ketosis. Since achieving nutritional ketosis requires strict dietary restrictions, compliance is a major difficulty in this treatment. The administration of exogenous ketone supplements that increase ketone levels in the blood without dietary restrictions may be an effective option to those willing to reach and stay at the state of nutritional ketosis. We propose that exogenous ketone supplementation (mainly KS and KSMCT) could provide an alternative method to reduce anxiety for healthy individuals, as well as those who need to maintain nutritional ketosis. Therefore, further studies are needed to determine the exact pathophysiological and molecular basis of ketone supplementation-induced changes in anxiety level.
C We tested exogenous ketone supplements fed chronically for 83 days and administered subchronically for 7 days by gavage, followed by assessment of anxiety measures on elevated plus maze (EPM) on 98 male SPD rats. The groups included standard diet (SD), ketogenic diet (KD) or SD + ketone supplementation. Lowdose ketone ester (LKE) (1,3-butanediolacetoacetate diester, ~10 g/kg/day, SD+LKE), high dose ketone ester (HKE) (~25 g/kg/day, SD+HKE), beta-hydroxybutyrate-mineral salt (βHB-S) (~25 g/kg/day, SD+KS), and BHB-S + medium chain triglyceride (MCT) (~25 g/kg/day, SD+KSMCT) were used as ketone supplementation. To extend our results on SPD rats, exogenous ketone supplements were also tested on 24 Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats for 7 days by gavage (SD+KE and SD+KS). Behavioral data collection was conducted manually by a blinded observer and with a video-tracking system (SMART V3.0 PLATFORM, Harvard Apparatus) at the end of treatments.
References D'Agostino, D., Pilla, R., Held, H., Landon, C., Puchowicz, M., Brunengraber, H., Ari, C., Arnold, P., and Dean, J. (2013) Therapeutic ketosis with ketone ester delays central nervous system oxygen toxicity seizures in rats. American Journal of Physiology. Regulatory, integrative and comparative physiology 304(10):R82936.doi:10.1152/ajpregu.00506.2012.
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Veech, R. (2004) The therapeutic implications of ketone bodies: the effects of ketone bodies in pathological conditions: ketosis, ketogenic diet, redox states, insulin resistance, and mitochondrial metabolism. Prostaglandins, leukotrienes, and essential fatty acids 70, 309-319.
Acknowledgements This work was supported by ONR Grant N000141310062 (DPD), the Natl. Devel. Agency of Hungary TIOP-1.3.1.-07/2-2F-2009-2008 (ZK), the Natl. Devel. Agency of Hungary TÁMOP 4.2.1./B-09/1/KMR-20100003, KTIA_NAP_13-2014-0023 (GJ). We thank Tamás Török for technical assistance.
Contact
Dominic D’Agostino, Ph.D.
[email protected]
