Putative Role of Adenosine A1 Receptors in Exogenous Ketone Supplements-Evoked Anti-Epileptic Effect.

Approximately 30% of patients with epilepsy are drug-refractory. There is an urgent need to elucidate the exact pathophysiology of different types of epilepsies and the mechanisms of action of both antiseizure medication and metabolic therapies to treat patients more effectively and safely. For example, it has been demonstrated that exogenous ketone supplement (EKS)-generated therapeutic ketosis, as a metabolic therapy, may decrease epileptic activity in both animal models and humans, but its exact mechanism of action is unknown. However, it was demonstrated that therapeutic ketosis, among others, can increase adenosine level, which may enhance activity of A1 adenosine receptors (A1Rs) in the brain. It has also been demonstrated previously that adenosine has anti-epileptic effect through A1Rs in different models of epilepsies. Thus, it is possible that (i) therapeutic ketosis generated by the administration of EKSs may exert its anti-epileptic effect through, among other mechanisms, increased adenosine level and A1R activity and that (ii) the enhanced activity of A1Rs may be a necessary anti-epileptic mechanism evoked by EKS administration-generated ketosis. Moreover, EKSs can evoke and maintain ketosis without severe side effects. These results also suggest that the therapeutic application of EKS-generated ketosis may be a promising opportunity to treat different types of epilepsies. In this literature review, we specifically focus on the putative role of A1Rs in the anti-epileptic effect of EKS-induced ketosis.

Exogenous Ketone Supplement Administration Abrogated Isoflurane-Anesthesia-Induced Increase in Blood Glucose Level in Female WAG/Rij Rats.

It has been demonstrated that isoflurane-induced anesthesia can increase the blood glucose level, leading to hyperglycemia and several adverse effects. The administration of a mix of ketone diester (KE) and medium-chain triglyceride (MCT) oil, named KEMCT, abolished the isoflurane-anesthesia-induced increase in blood glucose level and prolonged the recovery time from isoflurane anesthesia in a male preclinical rodent model, Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. While most preclinical studies use exclusively male animals, our previous study on blood glucose changes in response to KEMCT administration showed that the results can be sex-dependent. Thus, in this study, we investigated female WAG/Rij rats, whether KEMCT gavage (3 g/kg/day for 7 days) can change the isoflurane (3%)-anesthesia-induced increase in blood glucose level and the recovery time from isoflurane-evoked anesthesia using the righting reflex. Moreover, KEMCT-induced ketosis may enhance both the extracellular level of adenosine and the activity of adenosine A1 receptors (A1Rs). To obtain information on the putative A1R mechanism of action, the effects of an A1R antagonist, DPCPX (1,3-dipropyl-8-cyclopentylxanthine; intraperitoneal/i.p. 0.2 mg/kg), on KEMCT-generated influences were also investigated. Our results show that KEMCT supplementation abolished the isoflurane-anesthesia-induced increase in blood glucose level, and this was abrogated by the co-administration of DPCPX. Nevertheless, KEMCT gavage did not change the recovery time from isoflurane-induced anesthesia. We can conclude that intragastric gavage of exogenous ketone supplements (EKSs), such as KEMCT, can abolish the isoflurane-anesthesia-induced increase in blood glucose level in both sexes likely through A1Rs in WAG/Rij rats, while recovery time was not affected in females, unlike in males. These results suggest that the administration of EKSs as an adjuvant therapy may be effective in mitigating metabolic side effects of isoflurane, such as hyperglycemia, in both sexes.

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.

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.

Enhancement of Ketone Supplements-Evoked Effect on Absence Epileptic Activity by Co-Administration of Uridine in Wistar Albino Glaxo Rijswijk Rats.

Both uridine and exogenous ketone supplements decreased the number of spike-wave discharges (SWDs) in a rat model of human absence epilepsy Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. It has been suggested that alleviating influence of both uridine and ketone supplements on absence epileptic activity may be modulated by A1 type adenosine receptors (A1Rs). The first aim was to determine whether intraperitoneal (i.p.) administration of a specific A1R antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 0.2 mg/kg) and a selective adenosine A2A receptor antagonist (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine) (SCH 58261; 0.5 mg/kg) have a modulatory influence on i.p. 1000 mg/kg uridine-evoked effects on SWD number in WAG/Rij rats. The second aim was to assess efficacy of a sub-effective dose of uridine (i.p. 250 mg/kg) combined with beta-hydroxybutyrate salt + medium chain triglyceride (KSMCT; 2.5 g/kg, gavage) on absence epilepsy. DPCPX completely abolished the i.p. 1000 mg/kg uridine-evoked alleviating effect on SWD number whereas SCH 58261 was ineffective, confirming the A1R mechanism. Moreover, the sub-effective dose of uridine markedly enhanced the effect of KSMCT (2.5 g/kg, gavage) on absence epileptic activity. These results demonstrate the anti-epilepsy benefits of co-administrating uridine and exogenous ketone supplements as a means to treat absence epilepsy.

Prior ingestion of a ketone monoester supplement reduces postprandial glycemic responses in young healthy-weight individuals.

The main objective of this study was to determine whether acute ingestion of a ketone monoester (KME) supplement impacted mixed-meal tolerance test (MMTT) glucose area under the curve (AUC). Nineteen healthy young volunteers (10 males/9 females; age, 24.7 ± 4.9 years; body mass index, 22.7 ± 2.4 kg/m2) participated in a double-blind, placebo-controlled crossover study. Following overnight fasting (≥10 h), participants consumed 0.45 mL/kg of a KME supplement or taste-matched placebo followed by an MMTT 15 min later. Blood samples were collected every 15-30 min over 2.5 h. KME supplementation acutely raised ß-hydroxybutyrate AUC (590%, P < 0.0001, d = 2.4) and resulted in decreases in blood glucose AUC (-9.4%, P = 0.03, d = 0.56) and nonesterified fatty acid (NEFA) AUC (-27.3%, P = 0.023, d = 0.68) compared with placebo. No differences were found for plasma insulin AUC (P = 0.70) or gastric emptying estimated by co-ingested acetaminophen AUC (P = 0.96) between ketone and placebo. Overall, results indicate that KME supplementation attenuates postprandial glycemic and NEFA responses when taken 15 min prior to a mixed meal in young healthy individuals. Future studies are warranted to investigate whether KME supplementation may benefit individuals with impaired glycemic control. Novelty: Acute ketone monoester supplementation 15 min prior to a mixed meal decreased postprandial glucose and NEFA levels without significantly impacting postprandial insulin or estimates of gastric emptying. Glucose- and NEFA-lowering effects of ketone monoester supplementation are apparently not mediated by changes in insulin release or gastric emptying.

Evaluation of the safety and tolerability of a nutritional Formulation in patients with ANgelman Syndrome (FANS): study protocol for a randomized controlled trial.

BACKGROUND: Ketogenic and low-glycemic-index diets are effective in treating drug-resistant seizures in children with Angelman syndrome. Cognition, mobility, sleep, and gastrointestinal health are intrinsically linked to seizure activity and overall quality of life. Ketogenic and low-glycemic diets restrict carbohydrate consumption and stabilize blood glucose levels. The ketogenic diet induces ketosis, a metabolic state where ketone bodies are preferentially used for fuel. The use of exogenous ketones in promoting ketosis in Angelman syndrome has not been previously studied. The study formulation evaluated herein contains the exogenous ketone beta-hydroxybutyrate to rapidly shift the body towards ketosis, resulting in enhanced metabolic efficiency. METHODS/DESIGN: This is a 16-week, randomized, double-blind, placebo-controlled, crossover study to assess the safety and tolerability of a nutritional formula containing exogenous ketones. It also examines the potential for exogenous ketones to improve the patient's nutritional status which can impact the physiologic, symptomatic, and health outcome liabilities of living with Angelman syndrome. DISCUSSION: This manuscript outlines the rationale for a study designed to be the first to provide data on nutritional approaches for patients with Angelman syndrome using exogenous ketones. TRIAL REGISTRATION: ClinicalTrials.gov, ID: NCT03644693. Registered on 23 August 2018. Last updated on 23 August 2018.

Acute hyperketonaemia alters T-cell-related cytokine gene expression within stimulated peripheral blood mononuclear cells following prolonged exercise.

PURPOSE: We investigated the effect of the racemic ß-hydroxybutyrate precursor, R,S-1,3-butanediol (BD), on T-cell-related cytokine gene expression within stimulated peripheral blood mononuclear cells (PBMC) following prolonged, strenuous exercise. METHODS: A repeated-measures, randomised, crossover study was conducted in nine healthy, trained male cyclists (age, 26.7 ± 5.2 years; VO2peak, 63.9 ± 2.5 mL kg-1 min-1). Participants ingested 0.35 g kg-1 of BD or placebo 30 min before and 60 min during 85 min of steady-state (SS) exercise, which preceded a ~ 30 min time-trial (TT) (7 kJ kg-1). Blood samples were collected at pre-supplement, pre-exercise, post-SS, post-TT and 1-h post-TT. Whole blood cultures were stimulated with Staphylococcal enterotoxin B (SEB) for 24 h to determine T-cell-related interleukin (IL)-4, IL-10 and interferon (IFN)-γ mRNA expression within isolated PBMCs in vitro. RESULTS: Serum cortisol, total circulating leukocyte and lymphocyte, and T-cell subset concentrations were similar between trials during exercise and recovery (all p > 0.05). BD ingestion increased T-cell-related IFN-γ mRNA expression compared with placebo throughout exercise and recovery (p = 0.011); however, IL-4 and IL-10 mRNA expression and the IFN-γ/IL-4 mRNA expression ratio were unaltered (all p > 0.05). CONCLUSION: Acute hyperketonaemia appears to transiently amplify the initiation of the pro-inflammatory T-cell-related IFN-γ response to an immune challenge in vitro during and following prolonged, strenuous exercise; suggesting enhanced type-1 T-cell immunity at the gene level.

A ketone monoester drink reduces the glycemic response to an oral glucose challenge in individuals with obesity: a randomized trial.

BACKGROUND: Exogenous ketones make it possible to reach a state of ketosis that may improve metabolic control in humans. OBJECTIVES: The main objective of this study was to determine whether the ingestion of a ketone monoester (KE) drink before a 2-h oral-glucose-tolerance test (OGTT) would lower blood glucose concentrations. Secondary objectives were to determine the impact of KE on nonesterified fatty acid (NEFA) concentration and glucoregulatory hormones. METHODS: We conducted a randomized controlled crossover experiment in 15 individuals with obesity (mean ± SD age: 47 ± 10 y; BMI: 34 ± 5 kg/m2). After an overnight fast, participants consumed a KE drink [(R)-3-hydroxybutyl (R)-3-hydroxybutyrate; 0.45 mL/kg body weight] or taste-matched control drink 30 min before completing a 75-g OGTT. Participants and study personnel performing laboratory analyses were blinded to each condition. RESULTS: The KE increased d-ß-hydroxybutyrate to a maximum of ∼3.4 mM (P < 0.001) during the OGTT. Compared with the control drink, KE reduced glucose (-11%, P = 0.002), NEFA (-21%, P = 0.009), and glucagon-like peptide 1 (-31%, P = 0.001) areas under the curve (AUCs), whereas glucagon AUC increased (+11%, P = 0.030). No differences in triglyceride, C-peptide, and insulin AUCs were observed after the KE drink. Mean arterial blood pressure decreased and heart rate increased after the KE drink (both P < 0.01). CONCLUSIONS: A KE drink consumed before an OGTT lowered glucose and NEFA AUCs with no increase in circulating insulin. Our results suggest that a single drink of KE may acutely improve metabolic control in individuals with obesity. Future research is warranted to examine whether KE could be used safely to have longer-term effects on metabolic control. This trial was registered at clinicaltrials.gov as NCT03461068.

Anxiolytic Effect of Exogenous Ketone Supplementation Is Abolished by Adenosine A1 Receptor Inhibition in Wistar Albino Glaxo/Rijswijk Rats.

Anxiety disorders are one of the most common mental health problems worldwide, but the exact pathophysiology remains largely unknown. It has been demonstrated previously that administration of exogenous ketone supplement KSMCT (ketone salt/KS + medium chain triglyceride/MCT oil) by intragastric gavage for 7 days decreased the anxiety level in genetically absence epileptic Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. To investigate the potential role of the adenosinergic system in the pathomechanism of anxiety we tested whether the inhibition of adenosine A1 receptors (A1Rs) influence the anxiolytic effect of the exogenous ketone supplement. As A1Rs may mediate such an effect, in the present study we used a specific A1R antagonist, DPCPX (1,3-dipropyl-8-cyclopentylxanthine) to test whether it modulates the anxiolytic effect of sub-chronically (7 days) applied KSMCT in the previously tested animal model by using elevated plus maze (EPM) test. We administered KSMCT (2.5 g/kg/day) alone by intragastric gavage and in combination with intraperitoneally (i.p.) injected of DPCPX in two doses (lower: 0.15 mg/kg, higher: 0.25 mg/kg). Control groups represented i.p saline and water gavage with or without i.p. DPCPX administration (2.5 g/kg/day). After treatments, the level of blood glucose and beta-hydroxybutyrate (ßHB), as well as body weight were recorded. KSMCT alone significantly increased the time spent in the open arms and decreased the time spent in the closed arms, supporting our previous results. Injection of lower dose of DPCPX decreased, while higher dose of DPCPX abolished the effect of KSMCT administration on EPM. Blood ßHB levels were significantly increased after administration of KSMCT, while DPCPX did not change the KSMCT induced increase in blood ßHB levels. These results demonstrate that A1R inhibition modified (decreased) the anti-anxiety effect of KSMCT administration implying that the adenosinergic system, likely via A1Rs, may modulate the exogenous ketone supplement induced anxiolytic influence.

The use of nutritional supplements to induce ketosis and reduce symptoms associated with keto-induction: a narrative review.

BACKGROUND: Adaptation to a ketogenic diet (keto-induction) can cause unpleasant symptoms, and this can reduce tolerability of the diet. Several methods have been suggested as useful for encouraging entry into nutritional ketosis (NK) and reducing symptoms of keto-induction. This paper reviews the scientific literature on the effects of these methods on time-to-NK and on symptoms during the keto-induction phase. METHODS: PubMed, Science Direct, CINAHL, MEDLINE, Alt Health Watch, Food Science Source and EBSCO Psychology and Behavioural Sciences Collection electronic databases were searched online. Various purported ketogenic supplements were searched along with the terms "ketogenic diet", "ketogenic", "ketosis" and ketonaemia (/ ketonemia). Additionally, author names and reference lists were used for further search of the selected papers for related references. RESULTS: Evidence, from one mouse study, suggests that leucine doesn't significantly increase beta-hydroxybutyrate (BOHB) but the addition of leucine to a ketogenic diet in humans, while increasing the protein-to-fat ratio of the diet, doesn't reduce ketosis. Animal studies indicate that the short chain fatty acids acetic acid and butyric acid, increase ketone body concentrations. However, only one study has been performed in humans. This demonstrated that butyric acid is more ketogenic than either leucine or an 8-chain monoglyceride. Medium-chain triglycerides (MCTs) increase BOHB in a linear, dose-dependent manner, and promote both ketonaemia and ketogenesis. Exogenous ketones promote ketonaemia but may inhibit ketogenesis. CONCLUSIONS: There is a clear ketogenic effect of supplemental MCTs; however, it is unclear whether they independently improve time to NK and reduce symptoms of keto-induction. There is limited research on the potential for other supplements to improve time to NK and reduce symptoms of keto-induction. Few studies have specifically evaluated symptoms and adverse effects of a ketogenic diet during the induction phase. Those that have typically were not designed to evaluate these variables as primary outcomes, and thus, more research is required to elucidate the role that supplementation might play in encouraging ketogenesis, improve time to NK, and reduce symptoms associated with keto-induction.

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.