Dose-Dependent Beneficial Effect of Ketone Supplement-Evoked Ketosis on Anxiety Level in Female WAG/Rij Rats: Sometimes Less Is More.

While one-third of the population can be affected by anxiety disorders during their lifetime, our knowledge of the pathophysiology of these disorders is far from complete. Previously, it has been demonstrated in male animals that exogenous ketone supplement-evoked ketosis can decrease anxiety levels in preclinical rodent models, such as Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. Thus, in this study, we investigated whether intragastric gavage of the exogenous ketone supplement KEMCT (mix of 1,3-butanediol-acetoacetate diester/ketone ester/KE and medium-chain triglyceride/MCT oil in 1:1 ratio) for 7 days can alter the anxiety levels of female WAG/Rij rats using the light-dark box (LDB) test. We demonstrated that a lower dose of KEMCT (3 g/kg/day) increased blood R-ßHB (R-ß-hydroxybutyrate) levels and significantly decreased anxiety levels (e.g., increased the time spent in the light compartment) in female WAG/Rij rats on the seventh day of administration. Although the higher KEMCT dose (5 g/kg/day) increased blood R-ßHB levels more effectively, compared with the lower KEMCT dose, anxiety levels did not improve significantly. We conclude that ketone supplementation might be an effective strategy to induce anxiolytic effects not only in male but also in female WAG/Rij rats. However, these results suggest that the optimal level may be moderately, not highly, elevated blood R-ßHB levels when the goal is to alleviate symptoms of anxiety. More studies are needed to understand the exact mechanism of action of ketone supplementation on anxiety levels and to investigate their use in other animal models and humans for the treatment of anxiety disorders and other mental health conditions.

A Glutamate Scavenging Protocol Combined with Deanna Protocol in SOD1-G93A Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive disease of neuronal degeneration in the motor cortex, brainstem, and spinal cord, resulting in impaired motor function and premature demise as a result of insufficient respiratory drive. ALS is associated with dysfunctions in neurons, neuroglia, muscle cells, energy metabolism, and glutamate balance. Currently, there is not a widely accepted, effective treatment for this condition. Prior work from our lab has demonstrated the efficacy of supplemental nutrition with the Deanna Protocol (DP). In the present study, we tested the effects of three different treatments in a mouse model of ALS. These treatments were the DP alone, a glutamate scavenging protocol (GSP) alone, and a combination of the two treatments. Outcome measures included body weight, food intake, behavioral assessments, neurological score, and lifespan. Compared to the control group, DP had a significantly slower decline in neurological score, strength, endurance, and coordination, with a trend toward increased lifespan despite a greater loss of weight. GSP had a significantly slower decline in neurological score, strength, endurance, and coordination, with a trend toward increased lifespan. DP+GSP had a significantly slower decline in neurological score with a trend toward increased lifespan, despite a greater loss of weight. While each of the treatment groups fared better than the control group, the combination of the DP+GSP was not better than either of the individual treatments. We conclude that the beneficial effects of the DP and the GSP in this ALS mouse model are distinct, and appear to offer no additional benefit when combined.

Ketone supplementation abolished isoflurane anesthesia-induced elevation in blood glucose level and increased recovery time from anesthesia in Wistar Albino Glaxo Rijswijk rats.

BACKGROUND: It has been suggested that administration of exogenous ketone supplements (EKSs) not only increases blood ketone body levels but also decreases blood glucose level and modulates isoflurane-induced anesthesia in different rodents, such as Wistar Albino Glaxo Rijswijk (WAG/Rij) rats. Thus, we investigated whether administration of EKSs can modulate the isoflurane anesthesia-generated increase in blood glucose level and the time required to recover from isoflurane-induced anesthesia. METHODS: To investigate the effect of EKSs on isoflurane anesthesia-induced changes in blood glucose and R-ß-hydroxybutyrate (R-ßHB) level as well as recovery time from anesthesia, we used KEMCT (mix of ketone ester/KE and medium chain triglyceride/MCT oil in a 1:1 ratio) in WAG/Rij rats. First, to accustom the animals to the method, water gavage was carried out for 5 days (adaptation period). After adaptation period, rats of first group (group 1) were gavaged by water (3 g/kg), whereas, in the case of second group (group 2), the diet of animals was supplemented by KEMCT (3 g/kg, gavage) once per day for 7 days. One hour after the last gavage, isoflurane (3%) anesthesia was induced for 20 min (group 1 and group 2) and the time required for recovery from anesthesia was measured by using righting reflex. Subsequently, blood levels of both R-ßHB and glucose were also evaluated. Changes in blood glucose and R-ßHB levels were compared to control, which control glucose and R-ßHB levels were measured on the last day of the adaptation period (group 1 and group 2). Time required for recovery from isoflurane anesthesia, which was detected after 7th KEMCT gavage (group 2), was compared to recovery time measured after 7th water gavage (group 1). RESULTS: The KEMCT maintained the normal glucose level under isoflurane anesthesia-evoked circumstances preventing the glucose level elevating effect of isoflurane. Thus, we demonstrated that administration of KEMCT not only increased blood level of R-ßHB but also abolished the isoflurane anesthesia-generated increase in blood glucose level. Moreover, the time required for recovery from isoflurane-evoked anesthesia increased significantly in KEMCT treated animals. CONCLUSIONS: Putative influence of elevated blood ketone body level on isoflurane-evoked effects, such as modulation of blood glucose level and recovery time from anesthesia, should be considered by anesthesiologists.

Adenosine Receptors Modulate the Exogenous Ketogenic Supplement-Evoked Alleviating Effect on Lipopolysaccharide-Generated Increase in Absence Epileptic Activity in WAG/Rij Rats.

It has been previously demonstrated that KEKS food containing exogenous ketogenic supplement ketone salt (KS) and ketone ester (KE) decreased the lipopolysaccharide (LPS)-generated increase in SWD (spike-wave discharge) number in Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats, likely through ketosis. KEKS-supplemented food-generated ketosis may increase adenosine levels, and may thus modulate both neuroinflammatory processes and epileptic activity through adenosine receptors (such as A1Rs and A2ARs). To determine whether these adenosine receptors are able to modify the KEKS food-generated alleviating effect on LPS-evoked increases in SWD number, an antagonist of A1R DPCPX (1,3-dipropyl-8-cyclopentylxanthine; 0.2 mg/kg) with LPS (50 µg/kg) and an antagonist of A2AR SCH58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine; 0.5 mg/kg) with LPS were co-injected intraperitoneally (i.p.) on the ninth day of KEKS food administration, and their influence not only on the SWD number, but also on blood glucose, R-beta-hydroxybutyrate (R-ßHB) levels, and body weight were measured. We showed that inhibition of A1Rs abolished the alleviating effect of KEKS food on LPS-generated increases in the SWD number, whereas blocking A2ARs did not significantly modify the KEKS food-generated beneficial effect. Our results suggest that the neuromodulatory benefits of KEKS-supplemented food on absence epileptic activity are mediated primarily through A1R, not A2AR.

Beneficial Effects of Exogenous Ketogenic Supplements on Aging Processes and Age-Related Neurodegenerative Diseases.

Life expectancy of humans has increased continuously up to the present days, but their health status (healthspan) was not enhanced by similar extent. To decrease enormous medical, economical and psychological burden that arise from this discrepancy, improvement of healthspan is needed that leads to delaying both aging processes and development of age-related diseases, thereby extending lifespan. Thus, development of new therapeutic tools to alleviate aging processes and related diseases and to increase life expectancy is a topic of increasing interest. It is widely accepted that ketosis (increased blood ketone body levels, e.g., ß-hydroxybutyrate) can generate neuroprotective effects. Ketosis-evoked neuroprotective effects may lead to improvement in health status and delay both aging and the development of related diseases through improving mitochondrial function, antioxidant and anti-inflammatory effects, histone and non-histone acetylation, ß-hydroxybutyrylation of histones, modulation of neurotransmitter systems and RNA functions. Administration of exogenous ketogenic supplements was proven to be an effective method to induce and maintain a healthy state of nutritional ketosis. Consequently, exogenous ketogenic supplements, such as ketone salts and ketone esters, may mitigate aging processes, delay the onset of age-associated diseases and extend lifespan through ketosis. The aim of this review is to summarize the main hallmarks of aging processes and certain signaling pathways in association with (putative) beneficial influences of exogenous ketogenic supplements-evoked ketosis on lifespan, aging processes, the most common age-related neurodegenerative diseases (Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis), as well as impaired learning and memory functions.

Exogenous Ketone Supplements Improved Motor Performance in Preclinical Rodent Models.

Nutritional ketosis has been proven effective for neurometabolic conditions and disorders linked to metabolic dysregulation. While inducing nutritional ketosis, ketogenic diet (KD) can improve motor performance in the context of certain disease states, but it is unknown whether exogenous ketone supplements-alternatives to KDs-may have similar effects. Therefore, we investigated the effect of ketone supplements on motor performance, using accelerating rotarod test and on postexercise blood glucose and R-beta-hydroxybutyrate (R-ßHB) levels in rodent models with and without pathology. The effect of KD, butanediol (BD), ketone-ester (KE), ketone-salt (KS), and their combination (KE + KS: KEKS) or mixtures with medium chain triglyceride (MCT) (KE + MCT: KEMCT; KS + MCT: KSMCT) was tested in Sprague-Dawley (SPD) and WAG/Rij (WR) rats and in GLUT-1 Deficiency Syndrome (G1D) mice. Motor performance was enhanced by KEMCT acutely, KE and KS subchronically in SPD rats, by KEKS and KEMCT groups in WR rats, and by KE chronically in G1D mice. We demonstrated that exogenous ketone supplementation improved motor performance to various degrees in rodent models, while effectively elevated R-ßHB and in some cases offsets postexercise blood glucose elevations. Our results suggest that improvement of motor performance varies depending on the strain of rodents, specific ketone formulation, age, and exposure frequency.

Exogenous Ketones Lower Blood Glucose Level in Rested and Exercised Rodent Models.

Diseases involving inflammation and oxidative stress can be exacerbated by high blood glucose levels. Due to tight metabolic regulation, safely reducing blood glucose can prove difficult. The ketogenic diet (KD) reduces absolute glucose and insulin, while increasing fatty acid oxidation, ketogenesis, and circulating levels of ß-hydroxybutyrate (ßHB), acetoacetate (AcAc), and acetone. Compliance to KD can be difficult, so alternative therapies that help reduce glucose levels are needed. Exogenous ketones provide an alternative method to elevate blood ketone levels without strict dietary requirements. In this study, we tested the changes in blood glucose and ketone (ßHB) levels in response to acute, sub-chronic, and chronic administration of various ketogenic compounds in either a post-exercise or rested state. WAG/Rij (WR) rats, a rodent model of human absence epilepsy, GLUT1 deficiency syndrome mice (GLUT1D), and wild type Sprague Dawley rats (SPD) were assessed. Non-pathological animals were also assessed across different age ranges. Experimental groups included KD, standard diet (SD) supplemented with water (Control, C) or with exogenous ketones: 1, 3-butanediol (BD), ßHB mineral salt (KS), KS with medium chain triglyceride/MCT (KSMCT), BD acetoacetate diester (KE), KE with MCT (KEMCT), and KE with KS (KEKS). In rested WR rats, the KE, KS, KSMCT groups had lower blood glucose level after 1 h of treatment, and in KE and KSMCT groups after 24 h. After exercise, the KE, KSMCT, KEKS, and KEMCT groups had lowered glucose levels after 1 h, and in the KEKS and KEMCT groups after 7 days, compared to control. In GLUT1D mice without exercise, only KE resulted in significantly lower glucose levels at week 2 and week 6 during a 10 weeks long chronic feeding study. In 4-month and 1-year-old SPD rats in the post-exercise trials, blood glucose was significantly lower in KD and KE, and in KEMCT groups, respectively. After seven days, the KSMCT group had the most significantly reduced blood glucose levels, compared to control. These results indicate that exogenous ketones were efficacious in reducing blood glucose levels within and outside the context of exercise in various rodent models of different ages, with and without pathology.

Delaying latency to hyperbaric oxygen-induced CNS oxygen toxicity seizures by combinations of exogenous ketone supplements.

Central nervous system oxygen toxicity (CNS-OT) manifests as tonic-clonic seizures and is a limitation of hyperbaric oxygen therapy (HBOT), as well as of recreational and technical diving associated with elevated partial pressure of oxygen. A previous study showed that ketone ester (1,3-butanediol acetoacetate diester, KE) administration delayed latency to seizures (LS) in 3-month-old Sprague-Dawley (SD) rats. This study explores the effect of exogenous ketone supplements in additional dosages and formulations on CNS-OT seizures in 18 months old SD rats, an age group correlating to human middle age. Ketogenic agents were given orally 60 min prior to exposure to hyperbaric oxygen and included control (water), KE (10 g/kg), KE/2 (KE 5 g/kg + water 5 g/kg), KE + medium-chain triglycerides (KE 5 g/kg + MCT 5 g/kg), and ketone salt (Na+ /K+ ßHB, KS) + MCT (KS 5 g/kg + MCT 5 g/kg). Rats were exposed to 100% oxygen at 5 atmospheres absolute (ATA). Upon seizure presentation (tonic-clonic movements) experiments were immediately terminated and blood was tested for glucose and D-beta-hydroxybutyrate (D-ßHB) levels. While blood D-ßHB levels were significantly elevated post-dive in all treatment groups, LS was significantly delayed only in KE (P = 0.0003), KE/2 (P = 0.023), and KE + MCT (P = 0.028) groups. In these groups, the severity of seizures appeared to be reduced, although these changes were significant only in KE-treated animals (P = 0.015). Acetoacetate (AcAc) levels were also significantly elevated in KE-treated animals. The LS in 18-month-old rats was delayed by 179% in KE, 219% in KE + MCT, and 55% in KE/2 groups, while only by 29% in KS + MCT. In conclusion, KE supplementation given alone and in combination with MCT elevated both ßHB and AcAc, and delayed CNS-OT seizures.

Exogenous Ketone Supplements Reduce Anxiety-Related Behavior in Sprague-Dawley and Wistar Albino Glaxo/Rijswijk Rats.

Nutritional ketosis has been proven effective for seizure disorders and other neurological disorders. The focus of this study was to determine the effects of ketone supplementation on anxiety-related behavior in Sprague-Dawley (SPD) and Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. We tested exogenous ketone supplements added to food and fed chronically for 83 days in SPD rats and administered sub-chronically for 7 days in both rat models by daily intragastric gavage bolus followed by assessment of anxiety measures on elevated plus maze (EPM). The groups included standard diet (SD) or SD + ketone supplementation. Low-dose ketone ester (LKE; 1,3-butanediol-acetoacetate diester, ~10 g/kg/day, LKE), high dose ketone ester (HKE; ~25 g/kg/day, HKE), beta-hydroxybutyrate-mineral salt (ßHB-S; ~25 g/kg/day, KS) and ßHB-S + medium chain triglyceride (MCT; ~25 g/kg/day, KSMCT) were used as ketone supplementation for chronic administration. To extend our results, exogenous ketone supplements were also tested sub-chronically on SPD rats (KE, KS and KSMCT; 5 g/kg/day) and on WAG/Rij rats (KE, KS and KSMCT; 2.5 g/kg/day). At the end of treatments behavioral data collection was conducted manually by a blinded observer and with a video-tracking system, after which blood ßHB and glucose levels were measured. Ketone supplementation reduced anxiety on EPM as measured by less entries to closed arms (sub-chronic KE and KS: SPD rats and KSMCT: WAG/Rij rats), more time spent in open arms (sub-chronic KE: SPD and KSMCT: WAG/Rij rats; chronic KSMCT: SPD rats), more distance traveled in open arms (chronic KS and KSMCT: SPD rats) and by delayed latency to entrance to closed arms (chronic KSMCT: SPD rats), when compared to control. Our data indicates that chronic and sub-chronic ketone supplementation not only elevated blood ßHB levels in both animal models, but reduced anxiety-related behavior. We conclude that ketone supplementation may represent a promising anxiolytic strategy through a novel means of inducing nutritional ketosis.

Effects of exogenous ketone supplementation on blood ketone, glucose, triglyceride, and lipoprotein levels in Sprague-Dawley rats.

BACKGROUND: Nutritional ketosis induced by the ketogenic diet (KD) has therapeutic applications for many disease states. We hypothesized that oral administration of exogenous ketone supplements could produce sustained nutritional ketosis (>0.5 mM) without carbohydrate restriction. METHODS: We tested the effects of 28-day administration of five ketone supplements on blood glucose, ketones, and lipids in male Sprague-Dawley rats. The supplements included: 1,3-butanediol (BD), a sodium/potassium ß-hydroxybutyrate (ßHB) mineral salt (BMS), medium chain triglyceride oil (MCT), BMS + MCT 1:1 mixture, and 1,3 butanediol acetoacetate diester (KE). Rats received a daily 5-10 g/kg dose of their respective ketone supplement via intragastric gavage during treatment. Weekly whole blood samples were taken for analysis of glucose and ßHB at baseline and, 0.5, 1, 4, 8, and 12 h post-gavage, or until ßHB returned to baseline. At 28 days, triglycerides, total cholesterol and high-density lipoprotein (HDL) were measured. RESULTS: Exogenous ketone supplementation caused a rapid and sustained elevation of ßHB, reduction of glucose, and little change to lipid biomarkers compared to control animals. CONCLUSIONS: This study demonstrates the efficacy and tolerability of oral exogenous ketone supplementation in inducing nutritional ketosis independent of dietary restriction.

Metabolic therapy with Deanna Protocol supplementation delays disease progression and extends survival in amyotrophic lateral sclerosis (ALS) mouse model.

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig's disease, is a neurodegenerative disorder of motor neurons causing progressive muscle weakness, paralysis, and eventual death from respiratory failure. There is currently no cure or effective treatment for ALS. Besides motor neuron degeneration, ALS is associated with impaired energy metabolism, which is pathophysiologically linked to mitochondrial dysfunction and glutamate excitotoxicity. The Deanna Protocol (DP) is a metabolic therapy that has been reported to alleviate symptoms in patients with ALS. In this study we hypothesized that alternative fuels in the form of TCA cycle intermediates, specifically arginine-alpha-ketoglutarate (AAKG), the main ingredient of the DP, and the ketogenic diet (KD), would increase motor function and survival in a mouse model of ALS (SOD1-G93A). ALS mice were fed standard rodent diet (SD), KD, or either diets containing a metabolic therapy of the primary ingredients of the DP consisting of AAKG, gamma-aminobutyric acid, Coenzyme Q10, and medium chain triglyceride high in caprylic triglyceride. Assessment of ALS-like pathology was performed using a pre-defined criteria for neurological score, accelerated rotarod test, paw grip endurance test, and grip strength test. Blood glucose, blood beta-hydroxybutyrate, and body weight were also monitored. SD+DP-fed mice exhibited improved neurological score from age 116 to 136 days compared to control mice. KD-fed mice exhibited better motor performance on all motor function tests at 15 and 16 weeks of age compared to controls. SD+DP and KD+DP therapies significantly extended survival time of SOD1-G93A mice by 7.5% (p = 0.001) and 4.2% (p = 0.006), respectively. Sixty-three percent of mice in the KD+DP and 72.7% of the SD+DP group lived past 125 days, while only 9% of the control animals survived past that point. Targeting energy metabolism with metabolic therapy produces a therapeutic effect in ALS mice which may prolong survival and quality of life in ALS patients.

The ketogenic diet and hyperbaric oxygen therapy prolong survival in mice with systemic metastatic cancer.

INTRODUCTION: Abnormal cancer metabolism creates a glycolytic-dependency which can be exploited by lowering glucose availability to the tumor. The ketogenic diet (KD) is a low carbohydrate, high fat diet which decreases blood glucose and elevates blood ketones and has been shown to slow cancer progression in animals and humans. Abnormal tumor vasculature creates hypoxic pockets which promote cancer progression and further increase the glycolytic-dependency of cancers. Hyperbaric oxygen therapy (HBO2T) saturates tumors with oxygen, reversing the cancer promoting effects of tumor hypoxia. Since these non-toxic therapies exploit overlapping metabolic deficiencies of cancer, we tested their combined effects on cancer progression in a natural model of metastatic disease. METHODS: We used the firefly luciferase-tagged VM-M3 mouse model of metastatic cancer to compare tumor progression and survival in mice fed standard or KD ad libitum with or without HBO2T (2.5 ATM absolute, 90 min, 3x/week). Tumor growth was monitored by in vivo bioluminescent imaging. RESULTS: KD alone significantly decreased blood glucose, slowed tumor growth, and increased mean survival time by 56.7% in mice with systemic metastatic cancer. While HBO2T alone did not influence cancer progression, combining the KD with HBO2T elicited a significant decrease in blood glucose, tumor growth rate, and 77.9% increase in mean survival time compared to controls. CONCLUSIONS: KD and HBO2T produce significant anti-cancer effects when combined in a natural model of systemic metastatic cancer. Our evidence suggests that these therapies should be further investigated as potential non-toxic treatments or adjuvant therapies to standard care for patients with systemic metastatic disease.

Evolutionary changes of astroglia in Elasmobranchii comparing to amniotes: a study based on three immunohistochemical markers (GFAP, S-100, and glutamine synthetase).

This paper supplements former studies on elasmobranch species with an immunohistochemical investigation into glutamine synthetase and S-100 protein, in addition to GFAP, and extends its scope to the representatives of almost every group of Elasmobranchii: squalomorph sharks, galeomorph sharks, skates (Rajiformes) and rays (Torpediniformes and Myliobatifomes). More glial elements were labeled by S-100 protein, and even more so by using glutamine synthetase immunostaining than by GFAP: more astrocytes (mainly non-perivascular ones) were detected in the telencephalon of sharks, skates and rays. Only the markers S-100 and glutamine synthetase, but not GFAP, characterized the Bergmann-glia of skates and rays and astrocyte-like non-ependymal cells in Squalus acanthias. Another squalomorph shark species, Pristiophorus cirratus, however, had GFAP immunopositive astrocytes. Of all the species studied, the greatest number of GFAP positive astrocytes could be observed in Mobula japanica (order Myliobatiformes), in each major brain part. According to anatomical location, perivascular glia comprised varied types, including even a location in Mobula, which can also be found in mammals. Remnants of radial glia were found in confined areas of skates, less so in rays. In the rhombencephalon and in the spinal cord modified ependymoglia predominated in every group. In conclusion, there was no meaningful difference between the astroglial architectures of squalomorph and galeomorph sharks. The difference in the astroglial structure between sharks and batoids, however, was confined to the telencephalon and mesencephalon, and did not take place in the rhombencephalon, the latter structure being quite similar in all the species studied. The appearance of astrocytes in the relatively thin-walled shark telencephalon, however, indicates that the brain thickening promoted the preponderance of astrocytes rather than their appearance itself. Although the evolutionary changes of astroglia had some similarities in Elasmobranchii and Amniota, there was one meaningful difference: in Elasmobranchii astrocytes did not prevail in conservative brain regions as they did in the progressive brain regions.