Dominic D'Agostino

The ketone metabolite β-hydroxybutyrate blocks NLRP3 inflammasome–mediated inflammatory disease

The ketone bodies β-hydroxybutyrate (BHB) and acetoacetate (AcAc) support mammalian survival during states of energy deficit by serving as alternative sources of ATP. BHB levels are elevated by starvation, caloric restriction, high-intensity exercise, or the low-carbohydrate ketogenic diet. Prolonged fasting reduces inflammation; however, the impact that ketones and other alternative metabolic fuels produced during energy deficits have on the innate immune response is unknown. We report that BHB, but neither AcAc nor the structurally related short-chain fatty acids butyrate and acetate, suppresses activation of the NLRP3 inflammasome in response to urate crystals, ATP and lipotoxic fatty acids. BHB did not inhibit caspase-1 activation in response to pathogens that activate the NLR family, CARD domain containing 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome and did not affect non-canonical caspase-11, inflammasome activation. Mechanistically, BHB inhibits the NLRP3 inflammasome by preventing K+ efflux and reducing ASC oligomerization and speck formation. The inhibitory effects of BHB on NLRP3 are not dependent on chirality or starvation-regulated mechanisms like AMP-activated protein kinase (AMPK), reactive oxygen species (ROS), autophagy or glycolytic inhibition. BHB blocks the NLRP3 inflammasome without undergoing oxidation in the TCA cycle, and independently of uncoupling protein-2 (UCP2), sirtuin-2 (SIRT2), the G protein–coupled receptor GPR109A or hydrocaboxylic acid receptor 2 (HCAR2). BHB reduces NLRP3 inflammasome–mediated interleukin (IL)-1β and IL-18 production in human monocytes. In vivo, BHB or a ketogenic diet attenuates caspase-1 activation and IL-1β secretion in mouse models of NLRP3-mediated diseases such as Muckle–Wells syndrome, familial cold autoinflammatory syndrome and urate crystal–induced peritonitis. Our findings suggest that the anti-inflammatory effects of caloric restriction or ketogenic diets may be linked to BHB-mediated inhibition of the NLRP3 inflammasome.

Therapeutic ketosis with ketone ester delays central nervous system oxygen toxicity seizures in rats

Central nervous system oxygen toxicity (CNS-OT) seizures occur with little or no warning, and no effective mitigation strategy has been identified. Ketogenic diets (KD) elevate blood ketones and have successfully treated drug-resistant epilepsy. We hypothesized that a ketone ester given orally as R,S-1,3-butanediol acetoacetate diester (BD-AcAc(2)) would delay CNS-OT seizures in rats breathing hyperbaric oxygen (HBO(2)). Adult male rats (n = 60) were implanted with radiotelemetry units to measure electroencephalogram (EEG). One week postsurgery, rats were administered a single oral dose of BD-AcAc(2), 1,3-butanediol (BD), or water 30 min before being placed into a hyperbaric chamber and pressurized to 5 atmospheres absolute (ATA) O2. Latency to seizure (LS) was measured from the time maximum pressure was reached until the onset of increased EEG activity and tonic-clonic contractions. Blood was drawn at room pressure from an arterial catheter in an additional 18 animals that were administered the same compounds, and levels of glucose, pH, Po(2), Pco(2), β-hydroxybutyrate (BHB), acetoacetate (AcAc), and acetone were analyzed. BD-AcAc(2) caused a rapid (30 min) and sustained (>4 h) elevation of BHB (>3 mM) and AcAc (>3 mM), which exceeded values reported with a KD or starvation. BD-AcAc(2) increased LS by 574 ± 116% compared with control (water) and was due to the effect of AcAc and acetone but not BHB. BD produced ketosis in rats by elevating BHB (>5 mM), but AcAc and acetone remained low or undetectable. BD did not increase LS. In conclusion, acute oral administration of BD-AcAc(2) produced sustained ketosis and significantly delayed CNS-OT seizures by elevating AcAc and acetone.

Ketone supplementation decreases tumor cell viability and prolongs survival of mice with metastatic cancer

Cancer cells express an abnormal metabolism characterized by increased glucose consumption owing to genetic mutations and mitochondrial dysfunction. Previous studies indicate that unlike healthy tissues, cancer cells are unable to effectively use ketone bodies for energy. Furthermore, ketones inhibit the proliferation and viability of cultured tumor cells. As the Warburg effect is especially prominent in metastatic cells, we hypothesized that dietary ketone supplementation would inhibit metastatic cancer progression in vivo. Proliferation and viability were measured in the highly metastatic VM‐M3 cells cultured in the presence and absence of β‐hydroxybutyrate (βHB). Adult male inbred VM mice were implanted subcutaneously with firefly luciferase‐tagged syngeneic VM‐M3 cells. Mice were fed a standard diet supplemented with either 1,3‐butanediol (BD) or a ketone ester (KE), which are metabolized to the ketone bodies βHB and acetoacetate. Tumor growth was monitored by in vivo bioluminescent imaging. Survival time, tumor growth rate, blood glucose, blood βHB and body weight were measured throughout the survival study. Ketone supplementation decreased proliferation and viability of the VM‐M3 cells grown in vitro, even in the presence of high glucose. Dietary ketone supplementation with BD and KE prolonged survival in VM‐M3 mice with systemic metastatic cancer by 51 and 69%, respectively (p < 0.05). Ketone administration elicited anticancer effects in vitro and in vivo independent of glucose levels or calorie restriction. The use of supplemental ketone precursors as a cancer treatment should be further investigated in animal models to determine potential for future clinical use.

Acute hyperoxia increases lipid peroxidation and induces plasma membrane blebbing in human U87 glioblastoma cells

Atomic force microscopy (AFM), malondialdehyde (MDA) assays, and amperometric measurements of extracellular hydrogen peroxide (H(2)O(2)) were used to test the hypothesis that graded hyperoxia induces measurable nanoscopic changes in membrane ultrastructure and membrane lipid peroxidation (MLP) in cultured U87 human glioma cells. U87 cells were exposed to 0.20 atmospheres absolute (ATA) O(2), normobaric hyperoxia (0.95 ATA O(2)) or hyperbaric hyperoxia (HBO(2), 3.25 ATA O(2)) for 60 min. H(2)O(2) (0.2 or 2 mM; 60 min) was used as a positive control for MLP. Cells were fixed with 2% glutaraldehyde immediately after treatment and scanned with AFM in air or fluid. Surface topography revealed ultrastructural changes such as membrane blebbing in cells treated with hyperoxia and H(2)O(2). Average membrane roughness (R(a)) of individual cells from each group (n=35 to 45 cells/group) was quantified to assess ultrastructural changes from oxidative stress. The R(a) of the plasma membrane was 34+/-3, 57+/-3 and 63+/-5 nm in 0.20 ATA O(2), 0.95 ATA O(2) and HBO(2), respectively. R(a) was 56+/-7 and 138+/-14 nm in 0.2 and 2 mM H(2)O(2). Similarly, levels of MDA were significantly elevated in cultures treated with hyperoxia and H(2)O(2) and correlated with O(2)-induced membrane blebbing (r(2)=0.93). Coapplication of antioxidant, Trolox-C (150 microM), significantly reduced membrane R(a) and MDA levels during hyperoxia. Hyperoxia-induced H(2)O(2) production increased 189%+/-5% (0.95 ATA O(2)) and 236%+/-5% (4 ATA O(2)) above control (0.20 ATA O(2)). We conclude that MLP and membrane blebbing increase with increasing O(2) concentration. We hypothesize that membrane blebbing is an ultrastructural correlate of MLP resulting from hyperoxia. Furthermore, AFM is a powerful technique for resolving nanoscopic changes in the plasma membrane that result from oxidative damage.

Heme oxygenase is necessary for the excitatory response of cultured neonatal rat rostral ventrolateral medulla neurons to hypoxia

Heme oxygenase has been linked to the oxygen-sensing function of the carotid body, pulmonary vasculature, cerebral vasculature, and airway smooth muscle. We have shown previously that the cardiorespiratory regions of the rostral ventrolateral medulla are excited by local hypoxia and that heme oxygenase-2 (HO-2) is expressed in the hypoxia-chemosensitive regions of the rostral ventrolateral medulla (RVLM), the respiratory pre-Bötzinger complex, and C1 sympathoexcitatory region. To determine whether heme oxygenase is necessary for the hypoxic-excitation of dissociated RVLM neurons (P1) cultured on confluent medullary astrocytes (P5), we examined their electrophysiological responses to hypoxia (NaCN and low Po(2)) using the whole-cell perforated patch clamp technique before and after blocking heme oxygenase with tin protoporphyrin-IX (SnPP-IX). Following the electrophysiological recording, immunocytochemistry was performed on the recorded neuron to correlate the electrophysiological response to hypoxia with the expression of HO-2. We found that the responses to NaCN and hypoxia were similar. RVLM neurons responded to NaCN and low Po(2) with either depolarization or hyperpolarization and SnPP-IX blocked the depolarization response of hypoxia-excited neurons to both NaCN and low Po(2) but had no effect on the hyperpolarization response of hypoxia-depressed neurons. Consistent with this observation, HO-2 expression was present only in the hypoxia-excited neurons. We conclude that RVLM neurons are excited by hypoxia via a heme oxygenase-dependent mechanism.

Ketone ester supplementation attenuates seizure activity, and improves behavior and hippocampal synaptic plasticity in an Angelman syndrome mouse model

Angelman syndrome (AS) is a rare genetic and neurological disorder presenting with seizures, developmental delay, ataxia, and lack of speech. Previous studies have indicated that oxidative stress-dependent metabolic dysfunction may underlie the phenotypic deficits reported in the AS mouse model. While the ketogenic diet (KD) has been used to protect against oxidative stress and has successfully treated refractory epilepsy in AS case studies, issues arise due to its strict adherence requirements, in addition to selective eating habits and weight issues reported in patients with AS. We hypothesized that ketone ester supplementation would mimic the KD as an anticonvulsant and improve the behavioral and synaptic plasticity deficits in vivo. AS mice were supplemented R,S-1,3-butanediol acetoacetate diester (KE) ad libitum for eight weeks. KE administration improved motor coordination, learning and memory, and synaptic plasticity in AS mice. The KE was also anticonvulsant and altered brain amino acid metabolism in AS treated animals. Our findings suggest that KE supplementation produces sustained ketosis and ameliorates many phenotypes in the AS mouse model, and should be investigated further for future clinical use.

Hyperbaric Environment: Oxygen and Cellular Damage versus Protection

The elevation of tissue pO2 induced by hyperbaric oxygen (HBO) is a physiological stimulus that elicits a variety of cellular responses. These effects are largely mediated by, or in response to, an increase in the production of reactive oxygen and nitrogen species (RONS). The major consequences of elevated RONS include increased oxidative stress and enhanced antioxidant capacity, and modulation of redox-sensitive cell signaling pathways. Interestingly, these phenomena underlie both the therapeutic and potentially toxic effects of HBO. Emerging evidence indicates that supporting mitochondrial health is a potential method of enhancing the therapeutic efficacy of, and preventing oxygen toxicity during, HBO. This review will focus on the cellular consequences of HBO, and explore how these processes mediate a delicate balance of cellular protection versus damage.

Commentary: Ketone Diester Ingestion Impairs Time Trial Performance in Professional Cyclists

We read with interest the recent article by Leckey et al., reporting that consumption of a ketone diester drink impaired 31 km cycling time trial performance (Leckey et al., 2017). Exogenous ketones are new to the field of sports science, with only six athlete studies published to date (Cox et al., 2016; Holdsworth et al., 2017; O’Malley et al., 2017; Rodger et al., 2017; Vandoorne et al., 2017; Evans et al., 2018). Therefore, this article is a welcomed contribution to the discussion surrounding their optimal use. Having worked with novel ketone compounds in animals, sedentary individuals, and athletes, we wish to offer some reflections on this study, which may assist with future investigations of exogenous ketones in athletics. We suggest that fully understanding the human pharmacokinetics of novel ketone compounds will permit optimal dosing. Reaching blood ketone concentrations >2mM is likely a key mediator of any potential ergogenic effect (Cox et al., 2016; Egan and D’Agostino, 2016; Evans et al., 2017). Blood ketone kinetics after identical doses of a beta-hydroxybutyrate (BHB) monoester or acetoacetate (AcAc) diester are unlikely to be comparable with respect to BHB and AcAc. These esters deliver a different number of “ketone equivalents.” The BHB monoester delivers two BHB equivalents. The AcAc diester delivers two AcAc equivalents and one racemic BHB equivalent. Even if ketone equivalents were matched, different compounds have distinct pharmacokinetics. For example, ketone levels after the BHB monoester differed to those after matched quantities of racemic BHB salts (Stubbs et al., 2017). Therefore, future studies should ensure that the dose chosen delivers sufficient blood ketone concentrations. There are metabolic differences between the ketone molecules delivered by exogenous ketones. This can impact their physiological and neurological effects. Optical isoforms of BHB are not equivalent: D-BHB is released by the liver, whereas L-BHB is an intracellular metabolite. It appears that L-BHB less readily undergoes oxidative metabolism (Webber and Edmond, 1977; Desrochers et al., 1992); therefore elevating L-BHB may not improve athletic performance. Furthermore, there

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 (βHB), 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 βHB levels were significantly increased after administration of KSMCT, while DPCPX did not change the KSMCT induced increase in blood βHB 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.

Understanding the behavior of manta rays: answer to a critique

Animal consciousness, especially fish consciousness, is a debated field. Very few studies focus on it, therefore critique and debate in regards to our presented results (Ari and D’Agostino 2016) was expected. There are currently several potential roles suggested for cephalic fin movements and body turns of manta rays. As the significance of these behaviors in regards to the existence of self-awareness is uncertain, these areas of research remain to be explored further. It is undeniably questionable whether Gallup’s mirror self-recognition test is the most appropriate test to demonstrate self-awareness in manta rays or other fish species, but since this is what scientists used in previous studies, this test represents the best-known comparator for exploring the question of self-awareness in manta rays. As technological advances allow us to examine more closely the biology of teleosts and chondrichthyans, science is uncovering social, cognitive and emotional capacities that rival those of virtually any other animal (Brown 2014; Bshary et al. 2002; Balcombe 2016). In light of these developments, we call for a more open-minded, less dismissive view of this diverse, successful and highly evolved group of vertebrates. The most important point of Stewart et al.’s critique that we would like to address first is their assumption that our paper’s conclusion is that manta rays are self-aware; this is based on misinterpretation of our results, and an inflation of our interpretation. We would like to point out that our paper provided evidence for behavior that is a prerequisite of self-awareness, but we did not state anywhere in our paper that it demonstrates self-awareness. The definition of the word ‘prerequisite’ is: a thing that is required as a prior condition for something else to happen, but it does not equal proof. In addition, those who are familiar with behavioral tests on animal consciousness, especially Gallup’s mirror test (Gallup 1970), know that without completing the mark test it is impossible to state that an animal is self-aware. We did not present any data on the mark test, therefore we could not demonstrate self-awareness based on our presented results, and we cannot be responsible for others misinterpreting our results. Even our title is a question, and our discussion describes qualitatively and quantitatively two manta rays’ behavioral responses in the presence of a mirror. The discussion was in light of similar studies using previous protocols and definitions during similar studies on different species. Although in the discussion we speculate that self-directed behavior possibly shows a capacity for self-awareness, based on previous studies on other species where self-directed behavior in response to a mirror has been taken as evidence of self-recognition, we also caution readers that to confirm this possibility the mark test would need to be completed and more animals tested. We also mentioned in the discussion that our findings should be interpreted with caution because of the small sample size and because the Electronic supplementary material The online version of this article (doi:10.1007/s10164-016-0497-1) contains supplementary material, which is available to authorized users.