ßOHB Protective Pathways in Aralar-KO Neurons and Brain: An Alternative to Ketogenic Diet.

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory component of the NADH malate-aspartate shuttle. AGC1 deficiency is a neuropediatric rare disease characterized by hypomyelination, hypotonia, developmental arrest, and epilepsy. We have investigated whether ß-hydroxybutyrate (ßOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in aralar-knock-out (KO) neurons and mice. We report that ßOHB efficiently recovers aralar-KO neurons from deficits in basal-stimulated and glutamate-stimulated respiration, effects requiring ßOHB entry into the neuron, and protects from glutamate excitotoxicity. Aralar-deficient mice were fed a KD to investigate its therapeutic potential early in development, but this approach was unfeasible. Therefore, aralar-KO pups were treated without distinction of gender with daily intraperitoneal injections of ßOHB during 5 d. This treatment resulted in a recovery of striatal markers of the dopaminergic system including dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio, and vesicular monoamine transporter 2 (VMAT2) protein. Regarding postnatal myelination, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) myelin proteins were markedly increased in the cortices of ßOHB-treated aralar-KO mice. Although brain Asp and NAA levels did not change by ßOHB administration, a 4-d ßOHB treatment to aralar-KO, but not to control, neurons led to a substantial increase in Asp (3-fold) and NAA (4-fold) levels. These results suggest that the lack of increase in brain Asp and NAA is possibly because of its active utilization by the aralar-KO brain and the likely involvement of neuronal NAA in postnatal myelination in these mice. The effectiveness of ßOHB as a therapeutic treatment in AGC1 deficiency deserves further investigation.SIGNIFICANCE STATEMENTAralar deficiency induces a fatal phenotype in humans and mice and is associated with impaired neurodevelopment, epilepsy, and hypomyelination. In neurons, highly expressing aralar, its deficiency causes a metabolic blockade hampering mitochondrial energetics and respiration. Here, we find that ßOHB, the main metabolic product in KD, recovers defective mitochondrial respiration bypassing the metabolic failure in aralar-deficient neurons. ßOHB oxidation in mitochondria boosts the synthesis of cytosolic aspartate (Asp) and NAA, which is impeded by aralar deficiency, presumably through citrate-malate shuttle. In aralar-knock-out (KO) mice, ßOHB recovers from the drastic drop in specific dopaminergic and myelin markers. The ßOHB-induced myelin synthesis occurring together with the marked increment in neuronal NAA synthesis supports the role of NAA as a lipid precursor during postnatal myelination.

Ketone Body, 3-Hydroxybutyrate: Minor Metabolite - Major Medical Manifestations.

Ketone bodies - 3-hydroxybutyrate (3-OHB), acetoacetate, and acetone - are ancient, evolutionarily preserved, small fuel substrates, which uniquely can substitute and alternate with glucose under conditions of fuel and food deficiency. Once canonized as a noxious, toxic pathogen leading to ketoacidosis in patients with diabetes, it is now becoming increasingly clear that 3-OHB possesses a large number of beneficial, life-preserving effects in the fields of clinical science and medicine. 3-OHB, the most prominent ketone body, binds to specific hydroxyl-carboxylic acid receptors and inhibits histone deacetylase enzymes, free fatty acid receptors, and the NOD-like receptor protein 3 inflammasome, tentatively inhibiting lipolysis, inflammation, oxidative stress, cancer growth, angiogenesis, and atherosclerosis, and perhaps contributing to the increased longevity associated with exercise and caloric restriction. Clinically ketone bodies/ketogenic diets have for a long time been used to reduce the incidence of seizures in epilepsy and may have a role in the treatment of other neurological diseases such as dementia. 3-OHB also acts to preserve muscle protein during systemic inflammation and is an important component of the metabolic defense against insulin-induced hypoglycemia. Most recently, a number of studies have reported that 3-OHB dramatically increases myocardial blood flow and cardiac output in control subjects and patients with heart failure. At the moment, scientific interest in ketone bodies, in particular 3-OHB, is in a hectic transit and, hopefully, future, much needed, controlled clinical studies will reveal and determine to which extent the diverse biological manifestations of 3-OHB should be introduced medically.

The Role of Microbiota in Depression - a brief review.

The microbiota-gut-brain axis is a bidirectional homeostatic route of communication between both of the organs direct via receptors of the CNS or via epigenetic mechanisms of divers metabolites e.g. SCFA, GABA, ß-hydroxybutyrate. Thus, a modulation of gut microbiota via nutrition, lifestyle etc. might be effective for emotional status and depressive disorders. The dietary composition has an influence on gut microbiota composition, microbial metabolite profile and the according consequences on emotional status and depression within a system biologic approach. There are changes in gut microbiota composition and gut microbial profile (butyrate, GABA, ß-hydroxybutyrate) effecting epigenetic regulation (histone acetylation, DNA methylation) and gene expression of receptors and mediators (SLC6A4, BDNF, GABA, GPRs) involved in depressive disorders.

CV Protection in the EMPA-REG OUTCOME Trial: A "Thrifty Substrate" Hypothesis.

The striking and unexpected relative risk reductions in cardiovascular (CV) mortality (38%), hospitalization for heart failure (35%), and death from any cause (32%) observed in the EMPA-REG OUTCOME trial using an inhibitor of sodium-glucose cotransporter 2 (SGLT2) in patients with type 2 diabetes and high CV risk have raised the possibility that mechanisms other than those observed in the trial-modest improvement in glycemic control, small decrease in body weight, and persistent reductions in blood pressure and uric acid level-may be at play. We hypothesize that under conditions of mild, persistent hyperketonemia, such as those that prevail during treatment with SGLT2 inhibitors, ß-hydroxybutyrate is freely taken up by the heart (among other organs) and oxidized in preference to fatty acids. This fuel selection improves the transduction of oxygen consumption into work efficiency at the mitochondrial level. In addition, the hemoconcentration that typically follows SGLT2 inhibition enhances oxygen release to the tissues, thereby establishing a powerful synergy with the metabolic substrate shift. These mechanisms would cooperate with other SGLT2 inhibition-induced changes (chiefly, enhanced diuresis and reduced blood pressure) to achieve the degree of cardioprotection revealed in the EMPA-REG OUTCOME trial. This hypothesis opens up new lines of investigation into the pathogenesis and treatment of diabetic and nondiabetic heart disease.

ß-Hydroxybutyric acid inhibits growth hormone-releasing hormone synthesis and secretion through the GPR109A/extracellular signal-regulated 1/2 signalling pathway in the hypothalamus.

ß-Hydroxybutyric acid (BHBA) has recently been shown to regulate hormone synthesis and secretion in the hypothalamus. However, little is known about the effects of BHBA-mediated hormone regulation or the detailed mechanisms by which BHBA regulates growth hormone-releasing hormone (GHRH) synthesis and secretion. In the present study, we examined the expression of the BHBA receptor GPR109A in primary hypothalamic cell cultures. We hypothesised that BHBA regulates GHRH via GPR109A and its downstream signals. Initial in vivo studies conducted in rats demonstrated that GHRH mRNA expression in the hypothalamus was strongly inversely correlated with BHBA levels in the cerebrospinal fluid during postnatal development (r = -0.89, P < 0.01). Furthermore, i.c.v. administration of BHBA acutely decreased GHRH mRNA expression in rats. Further in vitro studies revealed a decrease in GHRH synthesis and secretion in primary hypothalamic cells after treatment with BHBA; this effect was inhibited when hypothalamic cells were pretreated with pertussis toxin (PTX). BHBA had no effect on GHRH synthesis and secretion in GT1-7 cells, which do not exhibit cell surface expression of GPR109A. Furthermore, BHBA acutely decreased the transcription of the homeobox gene for Gsh-1 in the hypothalamus in both in vivo and in vitro, and this effect was also inhibited by PTX in vitro. In primary hypothalamic cells, BHBA activated the extracellular signal-regulated kinase (ERK)1/2, p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) kinases, as shown by western blot analysis. Moreover, inhibition of ERK1/2 with U0126 attenuated the BHBA-mediated reduction in Gsh-1 expression and GHRH synthesis and secretion. These results strongly suggest that BHBA directly regulates GHRH synthesis and secretion via the GPR109A/ERK1/2 MAPK pathway, and also that Gsh-1 is essential for this function.

Long-term elevation of ß-hydroxybutyrate in dairy cows through infusion: effects on feed intake, milk production, and metabolism.

Elevation of ketone bodies in dairy cows frequently occurs in early lactation, usually concomitantly with a lack of energy and glucose. The objective of this study was to induce an elevated plasma ß-hydroxybutyrate (BHBA) concentration over 48 h in mid-lactating dairy cows (i.e., during a period of positive energy balance and normal glucose plasma concentrations). Effects of BHBA infusion on feed intake, metabolism, and performance were investigated. Thirteen cows were randomly assigned to 1 of 2 infusion groups, including an intravenous infusion with Na-dl-ß-OH-butyrate (1.7 mol/L) to achieve a plasma concentration of 1.5 to 2.0 mmol/L of BHBA (HyperB; n=5), or an infusion of 0.9% saline solution (control; n=8). Blood was sampled before and hourly during the 48 h of infusion. In the liver, mRNA transcripts related to gluconeogenesis (pyruvate carboxylase, glucose 6-phosphatase, mitochondrial phosphoenolpyruvate carboxykinase), phosphofructokinase, pyruvate dehydrogenase complex, and fatty acid synthesis (acetyl-coenzyme A carboxylase, fatty acid synthase) were measured by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase and ubiquitin were used as housekeeping genes. Changes (difference between before and after 48-h infusion) during the infusion period were evaluated by ANOVA with treatment as fixed effect, and area under the curve of variables was calculated on the second day of experiment. The plasma BHBA concentration in HyperB cows was 1.74 ± 0.02 mmol/L (mean ± SE) compared with 0.59 ± 0.02 mmol/L for control cows. The change in feed intake, milk yield, and energy corrected milk did not differ between the 2 experimental groups. Infusion of BHBA reduced the plasma glucose concentration (3.47 ± 0.11 mmol/L) in HyperB compared with control cows (4.11 ± 0.08 mmol/L). Plasma glucagon concentration in HyperB was lower than the control group. All other variables measured in plasma were not affected by treatment. In the liver, changes in mRNA abundance for the selected genes were similar between 2 groups. Results demonstrate that intravenous infusion of BHBA decreased plasma glucose concentration in dairy cows, but this decrease could not be explained by alterations in insulin concentrations or key enzymes related to gluconeogenesis. Declined glucose concentration is likely functionally related to decreased plasma glucagon concentration.

Herd-level association of serum metabolites in the transition period with disease, milk production, and early lactation reproductive performance.

The objective was to identify herd-level indicators expressed as a proportion of sampled animals with increased nonesterified fatty acids (NEFA) or ß-hydroxybutyric acid (BHBA), or decreased calcium in wk -1 and wk +1 relative to calving that were associated with herd-level incidence of retained placenta, metritis and displaced abomasum, milk production, and probability of pregnancy at the first artificial insemination (AI). Fifty-five Holstein freestall dairy herds in the United States and Canada were visited weekly. Blood was collected from 2,365 cows around parturition, and serum concentrations of NEFA, BHBA, and calcium were determined. Different cow-level metabolite thresholds associated with detrimental health or productivity in previous studies were used to classify animals into high- and low-risk metabolite concentration groups. For wk -1 and wk +1 relative to calving, a herd-level threshold was determined as the proportion of sampled animals in the high-risk metabolite concentration groups with the strongest association with increased incidence of disease, milk loss, or decreased pregnancy at the first AI. The odds of displaced abomasum after calving were higher in herds that had ≥ 25% of the animals with BHBA ≥ 1,400 µmol/L in wk +1 [odds ratio (OR)=2.1; 95% confidence interval (CI)=1.0-4.2)] or ≥ 35% of the animals with calcium ≤ 2.1 mmol/L in wk +1 (OR=2.4; CI=1.3-4.3). Herd-level thresholds of ≥15% of the cows with BHBA ≥ 800 µmol/L in wk -1 and ≥ 15% of the cows with calcium ≤2.1mmol/L in wk +1 were associated with milk loss (±SE) of 4.4±1.7 and 3.8 ± 1.4 kg/d per cow, respectively. When only multiparous cows were considered, herds with ≥30% of the multiparous cows with NEFA ≥0.5 mEq/L in wk -1 were associated with a 3.0 ± 1.5 kg/d per cow milk loss. The odds of pregnancy at first AI were lower in herds that had ≥ 5% of the cows with calcium ≤ 2.1 mmol/L in wk -1 (OR=0.7; CI=0.5-1.0), or ≥ 30% of the cows with NEFA ≥ 1.0 mEq/L (OR=0.6; CI=0.4-0.9) or ≥ 25% of the cows with calcium ≤2.1 mmol/L in wk +1 (OR=0.7; CI=0.5-0.9). When only multiparous cows were considered, the odds of pregnancy at first AI were lower in herds that had ≥50% of multiparous cows with NEFA ≥0.5 mEq/L in wk -1 (OR=0.5; CI=0.2-0.9). In conclusion, several herd-level thresholds for the proportion of cows with increased NEFA or BHBA, or decreased calcium in the week before and after calving were associated with higher risk of displaced abomasum, milk loss at the first Dairy Herd Improvement Association test, and decreased pregnancy at first AI. The association found between precalving BHBA and milk production is promising due to the availability of several cow-side tests for measuring BHBA. Some of the herd-level associations differed from the previously described cow-level associations, suggesting the potential of interpreting periparturient metabolic challenges at the herd level, where changes in diet and management are generally implemented.

Protein and fat mobilization and associations with serum ß-hydroxybutyrate concentrations in dairy cows.

The objective of this study was to obtain information on variation between dairy cows in muscle and fat tissue mobilization around parturition and to study the association between protein and fat mobilization and serum ß-hydroxybutyrate (BHBA) concentrations (hyperketonemia) in this period. Thirty-four cows kept under similar conditions at a university dairy farm (no experimental treatments) were monitored from 4 wk before until 8 wk after calving. Mobilization of muscle protein was investigated by analysis of plasma 3-methylhistidine concentrations (3-MH, analyzed by a recently developed HPLC tandem mass spectrometry method) and ultrasound measurements of longissimus muscle thickness. Mobilization of fat tissue was monitored by serum nonesterified fatty acid (NEFA) concentrations and ultrasound measurements of backfat thickness. Large variation was observed between cows in onset and duration of periparturient protein and fat mobilization. Plasma 3-MH concentrations and muscle thickness profiles indicated that protein mobilization started, on average, before parturition and continued until approximately wk 4 of lactation. Serum NEFA concentrations and backfat thickness profiles showed that fat mobilization occurred from parturition until the end of the study. Thus, muscle protein mobilization occurred in advance of fat mobilization in most cows from this study. We hypothesized that this might be due to a prepartum amino acid deficiency in the absence of negative energy balance. The incidence of hyperketonemia in this study was 16/34 = 47%. With the exception of 3 cows defined as having severe hyperketonemia, cows with lower 3-MH concentrations had higher serum BHBA concentrations. A possible explanation for this observation might be that higher mobilization of protein around calving might restrict ketone body production due to the higher availability of glucogenic precursors in the period of most severe negative energy balance and highest fat mobilization. The validity of this hypothesis needs to be confirmed, but data from this study indicate that further research on the role of protein mobilization in the etiology of hyperketonemia in dairy cows is needed.

Effects of ß-hydroxybutyrate and different calcium and potassium concentrations on the membrane potential and motility of abomasal smooth muscle cells in cattle.

The left displacement of the abomasum (LDA) is a common disease in periparturient dairy cows. Plasma ß-hydroxybutyrate (BHBA) levels above the reference range are regarded as risk factors for the occurrence of LDA. Additionally, hypokalemia and hypocalcemia have been observed in LDA cows. The aim of the present study was to characterize the membrane potential and the slow waves in abomasal smooth muscle cells by a microelectrode technique and to determine possible effects of BHBA and of various calcium and potassium concentrations on the membrane potential. Subsequently, the results obtained by the microelectrode technique were combined with in vitro motility experiments of abomasal smooth muscles. Strips of the abomasal circular smooth muscles were prepared and incubated in different buffer solutions. For the microelectrode technique, healthy bulls and cows that underwent surgery for LDA were sampled. These measurements showed a frequency of the slow waves between 3.5 and 10.9 per minute (for amplitudes ≥ 3mV) and between 0.6 and 4.5 per minute (for amplitudes ≥ 5mV). The frequency of contractions (1.8 to 3.1 per minute) were in the same order as the frequency of the slow waves with amplitudes ≥ 5 mV. Blocking potassium conductance with barium chloride induced a depolarization of the basal membrane potential (from -43±2.9 to -37±4.1mV; mean ± standard error of the mean) without affecting the frequency or the height of the slow waves. The reduction in the potassium concentration from 5.4 to 2 mmol/L resulted in a nominal decrease in the activity of contractions (from 22.2 to 18.6 mN/min). The subsequent addition of 1 mmol of KCl/L induced a nominal increase in contraction activity (from 18.6 to 25.7 mN/min). An effect of BHBA (5 mmol/L) could not be demonstrated, neither on the electric nor on the motility parameters. A simulated hypocalcemia (1.2 mmol/L total, 0.9 mmol/L ionized Ca) did not change slow waves and motility. In conclusion, changes in membrane potential ≥ 5 mV correlated with contractions of abomasal muscles. Hypokalemic conditions may reduce abomasal contraction activity via an effect on the membrane potential. An exclusive increase in BHBA or a slight hypocalcemia (0.9 mmol/L ionized Ca) had no effects on the motility of healthy abomasal smooth muscle cells. A prolonged or severe exposition of muscles to a combination of low Ca and high BHBA concentrations might nevertheless be able to affect abomasal motility.

Effect of postcalving serum nonesterified fatty acids concentration on the functionality of bovine immune cells.

The periparturient period is marked by metabolic, hormonal, and immunological changes, which have an effect on the incidence of infectious and metabolic diseases. In a previous study, a slower increase in milk production was induced by milking cows once daily during the first week of lactation, leading to an improvement in levels of several metabolites, including nonesterified fatty acids (NEFA) and ß-hydroxybutyrate (BHBA). The aim was to determine the influence of serum collected on d 2, 5, and 61 postpartum from cows milked once or twice daily on immune cell functions and to determine which of the constituents were responsible for these effects. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes were collected from healthy midlactation cows and their immune functions (i.e., proliferation and interferon-γ production and chemotaxis, phagocytosis, and oxidative burst, respectively), were evaluated in presence of serum, NEFA, and BHBA. Proliferation of PBMC was greater with d-61 (65.1±1.6%) serum than with d-2 (37.3±2.4%) or d-5 (48.4±1.6%) serum and greater with d-2 and -5 serum from cows milked once (42.2±3.7 and 54.0±2.5) compared with cows milked twice daily (32.4±3.0 and 42.9±2.1). Proliferation was inversely correlated with the concentration of NEFA and BHBA in the serum (r=-0.86). Adding NEFA to d-61 serum to reach the level present in d-5 serum decreased proliferation to the level observed with d-5 serum. No effect of BHBA addition was observed. The release of interferon-γ by PBMC was lower in d-5 serum (766±63 pg/mL) than in d-61 serum (1,187±90 pg/mL) and by NEFA. Milking frequency did not affect chemotaxis, phagocytosis, or oxidative burst of polymorphonuclear leukocytes. Phagocytosis decreased over time in serum from d 2 to 61. Similarly, oxidative burst was greater with d-5 serum (12.7×10(8) ± 1.6×10(8) relative light units) than with d-61 serum (9.0×10(8) ± 1.6×10(8) relative light units). The NEFA had a negative effect on oxidative burst, but BHBA did not. In conclusion, several immune cell functions appear affected by the NEFA concentration. Therefore, strategies that prevent increases in blood NEFA during the transition period may limit postpartum immunosuppression.

Polycythaemia in infants of diabetic mothers: ß-hydroxybutyrate stimulates erythropoietic activity.

This study tested whether elevated maternal ß-hydroxybutyrate (ß-OHB) levels contribute to polycythaemia in infants of diabetic mothers. Pregnant diabetic women (n = 27) and non-diabetic controls (n = 20) and their singleton infants were included. Maternal glycosylated haemoglobin and ß-OHB levels were studied at 34-36 weeks' gestation; levels were significantly higher in mothers with diabetes than in controls. Birth weights and cord blood levels of insulin and fetal haemoglobin were significantly higher in infants from diabetic mothers compared with control infants, as were haematocrit levels in venous blood samples taken from each infant at 4 h following delivery. Cord blood erythropoietin levels were similar in both groups. There was a positive strong correlation between maternal ß-OHB levels and polycythaemia in newborn infants, indicating that ß-OHB could activate erythropoiesis independently from intrauterine hyperinsulinaemia and/or erythropoietin levels, and may be important in the pathogenesis of polycythaemia in infants born to diabetic mothers.

Antiepileptic effects of endogenous beta-hydroxybutyrate in suckling infant rats.

Physiological ketosis is a hallmark of metabolism in suckling infants. However, little is known on the impact of physiological ketosis on brain excitability. We addressed this question in suckling rats in vivo. 16-channel extracellular field potential recordings were performed from somatosensory barrel cortex at postnatal days 5-9 non-anaesthetized rat pups. Seizures were induced by the volatile convulsant agent flurothyl. One hour after blockade of physiological ketogenesis using combined administration of beta-oxidation inhibitors mercaptoacetate, insulin and glucose to prevent hypoglycemia, we found no significant change in the flurothyl-induced electrographic seizures. However, build-up of seizures during two repetitive flurothyl applications was strongly aggravated in the animals with blocked ketogenesis. The effect of ketogenesis inhibitors was reversed by exogenous beta-hydroxybutyrate. Diazepam exerted anticonvulsive action both under physiological ketosis and after blockade of ketogenesis, and bumetanide had no significant anticonvulsive effects in both conditions. Thus, physiological ketosis reduces excitability in the immature brain and elimination of physiological ketosis results in elimination of this anticonvulsant effect. Our study raises concern that the changes in diet, and pharmacological manipulations such as glucose infusion, and pathologies such as hyperinsulinism which break natural ketosis, may be a potential risk factor for epileptogenesis in nursing infants.

GABA depolarizes immature neocortical neurons in the presence of the ketone body ß-hydroxybutyrate.

A large body of evidence suggests that the neurotransmitter GABA undergoes a developmental switch from being predominantly depolarizing-excitatory to predominantly hyperpolarizing-inhibitory. Recently published data, however, point to the possibility that the presumed depolarizing mode of GABA action during early development might represent an artifact due to an insufficient energy supply of the in vitro preparations used. Specifically, addition of the ketone body dl-ß-hydroxybutyrate (ßHB) to the extracellular medium was shown to prevent GABA from exerting excitatory effects. Applying a complementary set of minimally invasive optical and electrophysiological techniques in brain slices from neonatal mice, we investigated the effects of ßHB on GABA actions in immature cells of the upper cortical plate. Fluorescence imaging revealed that GABA-mediated somatic [Ca(2+)] transients, that required activation of GABA(A) receptors and voltage-gated Ca(2+) channels, remained unaffected by ßHB. Cell-attached current-clamp recordings showed that, in the presence of ßHB, GABA still induced a membrane potential depolarization. To estimate membrane potential changes quantitatively, we used cell-attached recordings of voltage-gated potassium currents and demonstrated that the GABA-mediated depolarization was independent of supplementation of the extracellular solution with ßHB. We conclude that, in vitro, GABA depolarizes immature cells of the upper cortical plate in the presence of the ketone body ßHB. Our data thereby support the general concept of an excitatory-to-inhibitory switch of GABA action during early development.