Preconditioning with ß-hydroxybutyrate attenuates lung ischemia-reperfusion injury by suppressing alveolar macrophage pyroptosis through the SIRT1-FOXO3 signaling pathway.

The complex pathogenesis of lung ischemia-reperfusion injury (LIRI) was examined in a murine model, focusing on the role of pyroptosis and its exacerbation of lung injury. We specifically examined the levels and cellular localization of pyroptosis within the lung, which revealed alveolar macrophages as the primary site. The inhibition of pyroptosis by VX-765 reduced the severity of lung injury, underscoring its significant role in LIRI. Furthermore, the therapeutic potential of ß-hydroxybutyrate (ß-OHB) in ameliorating LIRI was examined. Modulation of ß-OHB levels was evaluated by ketone ester supplementation and 3-hydroxybutyrate dehydrogenase 1 (BDH-1) gene knockout, along with the manipulation of the SIRT1-FOXO3 signaling pathway using EX-527 and pCMV-SIRT1 plasmid transfection. This revealed that ß-OHB exerts lung-protective and anti-pyroptotic effects, which were mediated through the upregulation of SIRT1 and the enhancement of FOXO3 deacetylation, leading to decreased pyroptosis markers and lung injury. In addition, ß-OHB treatment of MH-S cells in vitro showed a concentration-dependent improvement in pyroptosis, linking its therapeutic benefits to specific cell mechanisms. Overall, this study highlights the significance of alveolar macrophage pyroptosis in the exacerbation of LIRI and indicates the potential of ß-OHB in mitigating injury by modulating the SIRT1-FOXO3 signaling pathway.

SGLT2 inhibitor promotes ketogenesis to improve MASH by suppressing CD8+ T cell activation.

During the progression of metabolic dysfunction-associated steatohepatitis (MASH), the accumulation of auto-aggressive CD8+ T cells significantly contributes to liver injury and inflammation. Empagliflozin (EMPA), a highly selective inhibitor of sodium-glucose co-transporter 2 (SGLT2), exhibits potential therapeutic benefits for liver steatosis; however, the underlying mechanism remains incompletely elucidated. Here, we found that EMPA significantly reduced the hepatic accumulation of auto-aggressive CD8+ T cells and lowered granzyme B levels in mice with MASH. Mechanistically, EMPA increased ß-hydroxybutyric acid by promoting the ketogenesis of CD8+ T cells via elevating 3-hydroxybutyrate dehydrogenase 1 (Bdh1) expression. The ß-hydroxybutyric acid subsequently inhibited interferon regulatory factor 4 (Irf4), which is crucial for CD8+ T cell activation. Furthermore, the ablation of Bdh1 in T cells aggravated the manifestation of MASH and hindered the therapeutic efficacy of EMPA. Moreover, a case-control study also showed that SGLT2 inhibitor treatment repressed CD8+ T cell infiltration and improved liver injury in patients with MASH. In summary, our study indicates that SGLT2 inhibitors can target CD8+ T cells and may be an effective strategy for treating MASH.

Differences in the Effect of Beta-Hydroxybutyrate on the Mitochondrial Biogenesis, Oxidative Stress and Inflammation Markers in Tissues from Young and Old Rats.

One of the therapeutic approaches to age-related diseases is modulation of body cell metabolism through certain diets or their pharmacological mimetics. The ketogenic diet significantly affects cell energy metabolism and functioning of mitochondria, which has been actively studied in various age-related pathologies. Here, we investigated the effect of the ketogenic diet mimetic beta-hydroxybutyrate (BHB) on the expression of genes regulating mitochondrial biogenesis (Ppargc1a, Nrf1, Tfam), quality control (Sqstm1), functioning of the antioxidant system (Nfe2l2, Gpx1, Gpx3, Srxn1, Txnrd2, Slc6a9, Slc7a11), and inflammatory response (Il1b, Tnf, Ptgs2, Gfap) in the brain, lungs, heart, liver, kidneys, and muscles of young and old rats. We also analyzed mitochondrial DNA (mtDNA) copy number, accumulation of mtDNA damage, and levels of oxidative stress based on the concentration of reduced glutathione and thiobarbituric acid-reactive substances (TBARS). In some organs, aging disrupted mitochondrial biogenesis and functioning of cell antioxidant system, which was accompanied by the increased oxidative stress and inflammation. Administration of BHB for 2 weeks had different effects on the organs of young and old rats. In particular, BHB upregulated expression of genes coding for proteins associated with the mitochondrial biogenesis and antioxidant system, especially in the liver and muscles of young (but not old) rats. At the same time, BHB contributed to the reduction of TBARS in the kidneys of old rats. Therefore, our study has shown that administration of ketone bodies significantly affected gene expression in organs, especially in young rats, by promoting mitochondrial biogenesis, improving the functioning of the antioxidant defense system, and partially reducing the level of oxidative stress. However, these changes were much less pronounced in old animals.

Attenuation of brown adipocyte whitening in high-fat diet-induced obese rats: Effects of melatonin and ß-hydroxybutyrate on Cidea, Fsp27 and MT1 expression.

AIM: To investigate the effects of ß-hydroxybutyrate (BHB) and melatonin on brown adipose tissue (BAT) plasticity in rats fed a high-fat diet (HFD). METHODS: We employed a 7-week experimental design for a study on 30 male Sprague-Dawley rats divided into five groups: (1) a control-diet fed group; (2) a high-fat diet (HFD)-fed group; (3) a group that received an HFD and a BHB solution in their drinking water; (4) a group that received an HFD with 10 mg/kg/day melatonin in their drinking water; and (5) a group that received an HFD and were also treated with the combination of BHB and melatonin. Following the treatment period, biochemical indices, gene expression levels of key thermogenic markers (including uncoupling protein 1 [UCP1], PR domain containing 16 [PRDM16], Cidea, fat-specific protein 27 [Fsp27], and metallothionein 1 [MT1]), and stereological assessments of BAT were evaluated. RESULTS: Treatment with BHB and melatonin significantly boosted blood ketone levels, improved lipid profiles, and reduced weight gain from an HFD. It also downregulated genes linked to WAT, namely, Cidea and Fsp27, and upregulated key BAT markers, including UCP1, PRDM16 and peroxisome proliferator-activated receptor-gamma coactivator-1-alpha. Additionally, the co-treatment increased MT1 receptor expression and enhanced the structural density of BAT. CONCLUSION: The combined oral administration of BHB and melatonin successfully prevented the whitening of BAT in obese rats fed an HFD, indicating its potential as a therapeutic strategy for obesity-related BAT dysfunction. The synergistic effects of this treatment underscore the potential of a combined approach to address BAT dysfunction in obesity.

Ketone monoester attenuates oxygen desaturation during weighted ruck exercise under acute hypoxic exposure but does not impact cognitive performance.

Acute ingestion of exogenous ketone supplements in the form of a (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (R-BD R-ßHB) ketone monoester (KME) can attenuate declines in oxygen availability during hypoxic exposure and might impact cognitive performance at rest and in response to moderate-intensity exercise. In a single-blind randomized crossover design, 16 males performed assessments of cognitive performance before and during hypoxic exposure with moderate exercise [2 × 20 min weighted ruck (∼22 kg) at 3.2 km/h at 10% incline] in a normobaric altitude chamber (4572 m, 11.8% O2). The R-BD R-ßHB KME (573 mg/kg) or a calorie- and taste-matched placebo (∼50 g maltodextrin) were co-ingested with 40 g of dextrose before exposure to hypoxia. The R-ßHB concentrations were rapidly elevated and sustained (>3 mM; P < 0.001) by KME. The decline in oxygen saturation during hypoxic exposure was attenuated in KME conditions by 2.4%-4.2% (P < 0.05) compared with placebo. Outcomes of cognitive performance tasks, in the form of the Defense Automated Neurobehavioral Assessment (DANA) code substitution task, the Stroop color and word task, and a shooting simulation, did not differ between trials before and during hypoxic exposure. These data suggest that the acute exogenous ketosis induced by KME ingestion can attenuate declining blood oxygen saturation during acute hypoxic exposure both at rest and during moderate-intensity exercise, but this did not translate into differences in cognitive performance before or after exercise in the conditions investigated.

Ketone monoester attenuates declines in cognitive performance and oxygen saturation during acute severe hypoxic exposure under resting conditions.

Exogenous ketone supplements are a potential augmentation strategy for cognitive resilience during acute hypoxic exposure due to their capacity to attenuate the decline in oxygen (O2) availability, and by providing an alternative substrate for cerebral metabolism. Utilizing a single-blind randomized crossover design, 16 male military personnel (age, 25.3 ± 2.4 year, body mass, 86.2 ± 9.3 kg) performed tests of cognitive performance at rest in three environments: room air (baseline), normoxia (20 min; 0 m; 20.9% O2) and hypoxia (20 min; 6096 m, 9.7% O2) using a reduced O2 breathing device (ROBD). (R)-3-Hydroxybutyl (R)-3-hydroxybutyrate (R-BD R-ßHB) ketone monoester (KME; 650 mg/kg, split dose given at 30 min prior to each exposure) or taste-matched placebo (PLA) was ingested prior to normoxia and hypoxic exposure. Blood R-ßHB and glucose concentrations, cognitive performance and O2 saturation ( S p O 2 ${{S}_{{\mathrm{p}}{{{\mathrm{O}}}_{\mathrm{2}}}}}$ ) were collected throughout. KME ingestion increased blood R-ßHB concentration, which was rapid and sustained (>4 mM 30 min post; P < 0.001) and accompanied by lower blood glucose concentration (∼20 mg/dL; P < 0.01) compared to PLA. Declines in cognitive performance during hypoxic exposure, assessed as cognitive efficiency during a Defense Automated Neurobehavioral Assessment (DANA) code substitution task, were attenuated with KME leading to 6.8 (95% CL: 1.0, 12.6) more correct responses per minute compared to PLA (P = 0.018). The decline in S p O 2 ${{S}_{{\mathrm{p}}{{{\mathrm{O}}}_{\mathrm{2}}}}}$ during hypoxic exposure was attenuated (6.40% S p O 2 ${{S}_{{\mathrm{p}}{{{\mathrm{O}}}_{\mathrm{2}}}}}$ ; 95% CL: 0.04, 12.75; P = 0.049) in KME compared to PLA (KME, 76.8 ± 6.4% S p O 2 ${{S}_{{\mathrm{p}}{{{\mathrm{O}}}_{\mathrm{2}}}}}$ ; PLA, 70.4 ± 7.4% S p O 2 ${{S}_{{\mathrm{p}}{{{\mathrm{O}}}_{\mathrm{2}}}}}$ ). Acute ingestion of KME attenuated the decline in cognitive performance during acute severe hypoxic exposure, which coincided with attenuation of declines in O2 saturation.

Effects of betaine supplementation on dry matter intake, milk characteristics, plasma non-esterified fatty acids, and ß-hydroxybutyric acid in dairy cattle: a meta-analysis.

Betaine supplementation in dairy cattle has gained attention due to its potential benefits to production and health as a methyl donor, which can play a crucial role in the metabolism of dairy cows. The objective of the current meta-analysis was to quantify the effects of betaine supplementation on milk production, composition, ß-hydroxybutyric acid (BHBA), and non-esterified fatty acids (NEFA). A systematic literature search was carried out, all relevant studies were retrieved, and the meta-analysis was carried out. The mean difference (MD) for dry matter intake (DMI) using the random-effects model was 0.499 kg/d (P < 0.0001). The subgroup analysis indicated that supplementing betaine in heat-stressed cows increased DMI by 0.584 kg/d (P < 0.001), while in cows not exposed to heat stress, DMI was increased by 0.381 kg/d (P = 0.007). The energy-corrected milk (ECM) increased by 1.36 kg/d (P < 0.0001). The milk fat yield was significantly increased in betaine-supplemented cows (MD = 0.040 kg/d, 95% CI = 0.015 to 0.065). The milk protein yield (kg/d) (MD = 0.014, P = 0.138) was increased (MD = 0.035, P = 0.0005) by betaine supplementation. The lactose yield (kg/d) was also significantly higher (MD = 0.055, P = 0.020) in betaine-supplemented cows. The standardized mean difference (SMD) for NEFA (SMD = - 0.447, 95% CI = - 1.029 to 0.135, P = 0.114) and BHBA (SMD = - 0.130, 95% CI = - 0.491 to 0.234). In conclusion, the findings from this meta-analysis suggest that betaine supplementation positively influences DMI, ECM, milk fat yield, milk lactose yield, and milk protein yield. Subgroup analysis further indicated that the positive effects on DMI are greater in heat-stressed cows compared to those not exposed to heat stress. The analysis did not find significant effects on the levels of NEFA or BHBA, suggesting that betaine supplementation may not directly influence these metabolic parameters.

The aim of this meta-analysis was to examine the effects of dietary betaine supplementation on dairy cows' dry matter intake, milk production, milk composition, non-esterified fatty acids, and ß-hydroxybutyric acid. The results indicated that the supplementation of betaine increased dry matter intake (+0.584 kg/d/cow), energy-corrected milk (+1.36 kg/d), milk fat yield (+ 0.040 kg/d), milk protein yield (+ 0.014 kg/d), and lactose yield (+ 0.055 kg/d). ß-Hydroxybutyric acid and plasma non-esterified fatty acids were not influenced by betaine supplementation in dairy cattle.

Caprylic Acid Inhibits High Mobility Group Box-1-Induced Mitochondrial Damage in Myocardial Tubes.

Myocardial damage significantly impacts the prognosis of patients with cancer; however, the mechanisms of myocardial damage induced by cancer and its treatment remain unknown. We previously reported that medium-chain fatty acids (MCFAs) improve cancer-induced myocardial damage but did not evaluate the differences in effect according to MCFA type. Therefore, this study investigated the role of inflammatory cytokines in cancer-induced myocardial damage and the effects of three types of MCFAs (caprylic acid [C8], capric acid [C10], and lauric acid [C12]). In a mouse model, the C8 diet showed a greater effect on improving myocardial damage compared with C10 and C12 diets. Myocardial tubes differentiated from H9C2 cardiomyoblasts demonstrated increased mitochondrial oxidative stress, decreased membrane potential and mitochondrial volume, and inhibited myocardial tube differentiation following treatment with high-mobility group box-1 (HMGB1) but not interleukin-6 and tumor necrosis factor-α cytokines. However, HMGB1 treatment combined with C8 improved HMGB1-induced mitochondrial damage, enhanced autophagy, and increased mitochondrial biogenesis and maturation. However, these effects were only partial when combined with beta-hydroxybutyrate, a C8 metabolite. Thus, HMGB1 may play an important role in cancer-related myocardial damage. C8 counteracts HMGB1's effects and improves cancer-related myocardial damage. Further clinical studies are required to investigate the effects of C8.

ß-Hydroxybutyrate alleviates brain aging through the MTA1 pathway in D-galactose injured mice.

Aging is an inevitable law of the process of life during which many physiological functions change. Brain aging is an important mechanism in the occurrence and development of degenerative diseases of the central nervous system. ß-Hydroxybutyrate (BHBA) is a water-soluble, endogenous small-molecule ketone that can cross the blood-brain barrier and induce neuroprotective effects. This study aimed to investigate the effects of BHBA on D-galactose (D-gal) induced aging in mice and its underlying mechanisms using in vitro and in vivo experiments. These results indicated that D-gal-induced senescence, oxidative stress, and inflammatory responses were inhibited by BHBA, and autophagy was promoted by BHBA. Mechanistically, we explored the role of metastasis-associated antigen-1 (MTA1) in D-gal-induced damaged in HT22 cells using small interfering RNA (siRNA). The results demonstrated that the expression of MTA1 was significantly increased by BHBA, which attenuated D-gal-induced aging, oxidative stress, and inflammatory responses, and promoted autophagy through the upregulation of MTA1. In conclusion, MTA1 may be a novel target for treating aging caused by neurological damage. BHBA improves brain aging by activating the MTA1 pathway.

BHBA attenuates endoplasmic reticulum stress-dependent neuroinflammation via the gut-brain axis in a mouse model of heat stress.

BACKGROUND: Heat stress (HS) commonly occurs as a severe pathological response when the body's sensible temperature exceeds its thermoregulatory capacity, leading to the development of chronic brain inflammation, known as neuroinflammation. Emerging evidence suggests that HS leads to the disruption of the gut microbiota, whereas abnormalities in the gut microbiota have been demonstrated to affect neuroinflammation. However, the mechanisms underlying the effects of HS on neuroinflammation are poorly studied. Meanwhile, effective interventions have been unclear. ß-Hydroxybutyric acid (BHBA) has been found to have neuroprotective and anti-inflammatory properties in previous studies. This study aims to explore the modulatory effects of BHBA on neuroinflammation induced by HS and elucidate the underlying molecular mechanisms. METHODS: An in vivo and in vitro model of HS was constructed under the precondition of BHBA pretreatment. The modulatory effects of BHBA on HS-induced neuroinflammation were explored and the underlying molecular mechanisms were elucidated by flow cytometry, WB, qPCR, immunofluorescence staining, DCFH-DA fluorescent probe assay, and 16S rRNA gene sequencing of colonic contents. RESULTS: Heat stress was found to cause gut microbiota disruption in HS mouse models, and TM7 and [Previotella] spp. may be the best potential biomarkers for assessing the occurrence of HS. Fecal microbiota transplantation associated with BHBA effectively reversed the disruption of gut microbiota in HS mice. Moreover, BHBA may inhibit microglia hyperactivation, suppress neuroinflammation (TNF-α, IL-1ß, and IL-6), and reduce the expression of cortical endoplasmic reticulum stress (ERS) markers (GRP78 and CHOP) mainly through its modulatory effects on the gut microbiota (TM7, Lactobacillus spp., Ruminalococcus spp., and Prevotella spp.). In vitro experiments revealed that BHBA (1 mM) raised the expression of the ERS marker GRP78, enhanced cellular activity, and increased the generation of reactive oxygen species (ROS) and anti-inflammatory cytokines (IL-10), while also inhibiting HS-induced apoptosis, ROS production, and excessive release of inflammatory cytokines (TNF-α and IL-1ß) in mouse BV2 cells. CONCLUSION: ß-Hydroxybutyric acid may be an effective agent for preventing neuroinflammation in HS mice, possibly due to its ability to inhibit ERS and subsequent microglia neuroinflammation via the gut-brain axis. These findings lay the groundwork for future research and development of BHBA as a preventive drug for HS and provide fresh insights into techniques for treating neurological illnesses by modifying the gut microbiota.

ß-Hydroxybutyrate and melatonin suppress maladaptive UPR, excessive autophagy and pyroptosis in Aß 1-42 and LPS-Induced SH-SY5Y cells.

BACKGROUND: Alzheimer's disease is a neurological disease characterized by the build-up of amyloid beta peptide (Aß) and lipopolysaccharide (LPS), which causes synapse dysfunction, cell death, and neuro-inflammation. A maladaptive unfolded protein response (UPR), excessive autophagy, and pyroptosis aggravate the disease. Melatonin (MEL) and hydroxybutyrate (BHB) have both shown promise in terms of decreasing Aß pathology. The goal of this study was to see how BHB and MEL affected the UPR, autophagy, and pyroptosis pathways in Aß1-42 and LPS-induced SH-SY5Y cells. MATERIALS AND METHODS: Human neuroblastoma SH-SY5Y cells were treated with BHB, MEL, or a combination of the two after being exposed to A ß1-42 and LPS. Cell viability was determined using the MTT test, and gene expression levels of UPR (ATF6, PERK, and CHOP), autophagy (Beclin-1, LC3II, P62, and Atg5), and pyroptosis-related markers (NLRP3, TXNIP, IL-1ß, and NFκB1) were determined using quantitative Real-Time PCR (qRT-PCR). For statistical analysis, one-way ANOVA was employed, followed by Tukey's post hoc test. RESULTS: BHB and MEL significantly increased SH-SY5Y cell viability in the presence of A ß1-42 and LPS. Both compounds inhibited the expression of maladaptive UPR and autophagy-related genes, as well as inflammatory and pyroptotic markers caused by Aß1-42 and LPS-induced SH-SY5Y cells. CONCLUSION: BHB and MEL rescue neurons in A ß1-42 and LPS-induced SH-SY5Y cells by reducing maladaptive UPR, excessive autophagy, and pyroptosis. More research is needed to fully comprehend the processes behind their beneficial effects and to discover their practical applications in the treatment of neurodegenerative disorders.

Lentinan Ameliorates ß-Hydroxybutyrate-Induced Lipid Metabolism Disorder in Bovine Hepatocytes by Upregulating the Expression of Acetyl-coenzyme A Acetyltransferase 2.

Ketosis in dairy cows is often accompanied by the dysregulation of lipid homeostasis in the liver. Acetyl-coenzyme A acetyltransferase 2 (ACAT2) is specifically expressed in the liver and is important for regulating lipid homeostasis in ketotic cows. Lentinan (LNT) has a wide range of pharmacological activities, and this study investigates the protective effects of LNT on ß-hydroxybutyrate (BHBA)-induced lipid metabolism disorder in bovine hepatocytes (BHECs) and elucidates the underlying mechanisms. BHECs were first pretreated with LNT to investigate the effect of LNT on BHBA-induced lipid metabolism disorder in BHECs. ACAT2 was then silenced or overexpressed to investigate whether this mediated the protective action of LNT against BHBA-induced lipid metabolism disorder in BHECs. Finally, BHECs were treated with LNT after silencing ACAT2 to investigate the interaction between LNT and ACAT2. LNT pretreatment effectively enhanced the synthesis and absorption of cholesterol, inhibited the synthesis of triglycerides, increased the expression of ACAT2, and elevated the contents of very low-density lipoprotein and low-density lipoprotein cholesterol, thereby ameliorating BHBA-induced lipid metabolism disorder in BHECs. The overexpression of ACAT2 achieved a comparable effect to LNT pretreatment, whereas the silencing of ACAT2 aggravated the effect of BHBA on inducing disorder in lipid metabolism in BHECs. Moreover, the protective effect of LNT against lipid metabolism disorder in BHBA-induced BHECs was abrogated upon silencing of ACAT2. Thus, LNT, as a natural protective agent, can enhance the regulatory capacity of BHECs in maintaining lipid homeostasis by upregulating ACAT2 expression, thereby ameliorating the BHBA-induced lipid metabolism disorder.

Beta-Hydroxybutyrate Promotes Basal Insulin Secretion While Decreasing Glucagon Secretion in Mouse and Human Islets.

Dietary carbohydrates raise blood glucose levels, and limiting carbohydrate intake improves glycemia in patients with type 2 diabetes. Low carbohydrate intake (< 25 g) allows the body to utilize fat as its primary fuel. As a consequence of increased fatty acid oxidation, the liver produces ketones to serve as an alternative energy source. ß-Hydroxybutyrate (ßHB) is the most abundant ketone. While ßHB has a wide range of functions outside of the pancreas, its direct effects on islet cell function remain understudied. We examined human islet secretory response to acute racemic ßHB treatment and observed increased insulin secretion at a low glucose concentration of 3 mM. Because ßHB is a chiral molecule, existing as both R and S forms, we further studied insulin and glucagon secretion following acute treatment with individual ßHB enantiomers in human and C57BL/6J mouse islets. We found that acute treatment with R-ßHB increased insulin secretion and decreased glucagon secretion at physiological glucose concentrations in both human and mouse islets. Proteomic analysis of human islets treated with R-ßHB over 72 hours showed altered abundance of proteins that may promote islet cell health and survival. Collectively, our data show that physiological concentrations of ßHB influence hormone secretion and signaling within pancreatic islets.

Dapagliflozin improves skeletal muscle insulin sensitivity through SIRT1 activation induced by nutrient deprivation state.

Lipid peroxidation and mitochondrial damage impair insulin sensitivity in skeletal muscle. Sirtuin-1 (SIRT1) protects mitochondria and activates under energy restriction. Dapagliflozin (Dapa) is an antihyperglycaemic agent that belongs to the sodium-glucose cotransporter-2 (SGLT2) inhibitors. Evidence shows that Dapa can induce nutrient deprivation effects, providing additional metabolic benefits. This study investigates whether Dapa can trigger nutrient deprivation to activate SIRT1 and enhance insulin sensitivity in skeletal muscle. We treated diet-induced obese (DIO) mice with Dapa and measured metabolic parameters, lipid accumulation, oxidative stress, mitochondrial function, and glucose utilization in skeletal muscle. ß-hydroxybutyric acid (ß-HB) was intervened in C2C12 myotubes. The role of SIRT1 was verified by RNA interference. We found that Dapa treatment induced nutrient deprivation state and reduced lipid deposition and oxidative stress, improved mitochondrial function and glucose tolerance in skeletal muscle. The same positive effects were observed after ß-HB intervening for C2C12 myotubes, and the promoting effects on glucose utilization were diminished by SIRT1 RNA interference. Thus, Dapa promotes a nutrient deprivation state and enhances skeletal muscle insulin sensitivity via SIRT1 activation. In this study, we identified a novel hypoglycemic mechanism of Dapa and the potential mechanistic targets.

ß-Hydroxybutyrate Improves the Redox Status, Cytokine Production and Phagocytic Potency of Glucose-Deprived HMC3 Human Microglia-like Cells.

Brain glucose deprivation is a component of the pathophysiology of ischemia, glucose transporter1 (GLUT1) deficiency, neurological disorders and occurs transiently in diabetes. Microglia, the neuroimmune cells must function effectively to offer immune defence and debris removal in low-energy settings. Brain glucose deprivation may compromise microglial functions further escalating the disease pathology and deteriorating the overall mental health. In the current study, HMC3 human microglia-like cells were cultured in vitro and exposed to glucose deprivation to investigate the effects of glucose deprivation on phenotypic state, redox status, secretion of cytokines and phagocytic capabilities of HMC3 cells. However, HMC3 cells were able to proliferate in the absence of glucose but showed signs of redox imbalance and mitochondrial dysfunction, as demonstrated by decreased MTT reduction and Mito Tracker™ staining of cells, along with a concomitant reduction in NOX2 protein, superoxide, and nitrite levels. Reduced levels of secreted TNF and IL-1ß were the signs of compromised cytokine secretion by glucose-deprived HMC3 microglia-like cells. Moreover, glucose-deprived HMC3 cells also showed reduced phagocytic activity as assessed by fluorescently labelled latex beads-based functional phagocytosis assay. ß-hydroxybutyrate (BHB) supplementation restored the redox status, mitochondrial health, cytokine secretion, and phagocytic activity of glucose-deprived HMC3 microglia-like cells. Overall, impaired brain glucose metabolism may hinder microglia's capacity to release diffusible immune factors and perform phagocytosis. This could escalate the mental health issues in neurological diseases where brain glucose metabolism is compromised. Moreover, nutritional ketosis or exogenous ketone supplementation such as BHB may be utilized as a potential metabolic therapies for these conditions.

Simplification of Dietary Treatment in Pharmacoresistant Epilepsy: Impact of C8 and C10 Fatty Acids on Sirtuins of Neuronal Cells In Vitro.

Pharmacotherapy is the therapeutic mainstay in epilepsy; however, in about 30% of patients, epileptic seizures are drug-resistant. A ketogenic diet (KD) is an alternative therapeutic option. The mechanisms underlying the anti-seizure effect of a KD are not fully understood. Epileptic seizures lead to an increased energy demand of neurons. An improvement in energy provisions may have a protective effect. C8 and C10 fatty acids have been previously shown to activate mitochondrial function in vitro. This could involve sirtuins (SIRTs) as regulatory elements of energy metabolism. The aim of the present study was to investigate whether ß-hydroxybutyrate (ßHB), C8 fatty acids, C10 fatty acids, or a combination of C8 and C10 (250/250 µM) fatty acids, which all increase under a KD, could up-regulate SIRT1, -3, -4, and -5 in HT22 hippocampal murine neurons in vitro. Cells were incubated for 1 week in the presence of these metabolites. The sirtuins were measured at the enzyme (fluorometrically), protein (Western blot), and gene expression (PCR) levels. In hippocampal cells, the C8, C10, and C8 and C10 incubations led to increases in the sirtuin levels, which were not inferior to a ßHB incubation as the 'gold standard'. This may indicate that both C8 and C10 fatty acids are important for the antiepileptic effect of a KD. A KD may be replaced by nutritional supplements of C8 and C10 fatty acids, which could facilitate the diet.

ß-hydroxybutyrate resensitizes colorectal cancer cells to oxaliplatin by suppressing H3K79 methylation in vitro and in vivo.

BACKGROUND: Ketone ß-hydroxybutyrate (BHB) has been reported to prevent tumor cell proliferation and improve drug resistance. However, the effectiveness of BHB in oxaliplatin (Oxa)-resistant colorectal cancer (CRC) and the underlying mechanism still require further proof. METHODS: CRC-Oxa-resistant strains were established by increasing concentrations of CRC cells to Oxa. CRC-Oxa cell proliferation, apoptosis, invasion, migration, and epithelial-mesenchymal transition (EMT) were checked following BHB intervention in vitro. The subcutaneous and metastasis models were established to assess the effects of BHB on the growth and metastasis of CRC-Oxa in vivo. Eight Oxa responders and seven nonresponders with CRC were enrolled in the study. Then, the serum BHB level and H3K79me, H3K27ac, H3K14ac, and H3K9me levels in tissues were detected. DOT1L (H3K79me methyltransferase) gene knockdown or GNE-049 (H3K27ac inhibitor) use was applied to analyze further whether BHB reversed CRC-Oxa resistance via H3K79 demethylation and/or H3K27 deacetylation in vivo and in vitro. RESULTS: Following BHB intervention based on Oxa, the proliferation, migration, invasion, and EMT of CRC-Oxa cells and the growth and metastasis of transplanted tumors in mice were suppressed. Clinical analysis revealed that the differential change in BHB level was associated with drug resistance and was decreased in drug-resistant patient serum. The H3K79me, H3K27ac, and H3K14ac expressions in CRC were negatively correlated with BHB. Furthermore, results indicated that H3K79me inhibition may lead to BHB target deletion, resulting in its inability to function. CONCLUSIONS: ß-hydroxybutyrate resensitized CRC cells to Oxa by suppressing H3K79 methylation in vitro and in vivo.

Determination of the safety of Halomonas sp. KM-1-derived d-ß-hydroxybutyric acid and its fermentation-derived impurities in mice and Japanese adults.

The use of halophilic bacteria in industrial chemical and food production has received great interest because of the unique properties of these bacteria; however, their safety remains under investigation. Halomonas sp. KM-1 intracellularly stores poly-D-ß-hydroxybutyric acid under aerobic conditions and successively secretes D-ß-hydroxybutyric acid (D-BHB) under microaerobic conditions. Therefore, we tested the safety of Halomonas sp. KM-1-derived D-BHB and the impurities generated during D-BHB manufacturing at a 100-fold increased concentration in acute tests using mice and daily intake of 16.0 g D-BHB in Japanese adults for 12 weeks. In the mice test, there were no abnormalities in the body weights or health of mice fed the purified D-BHB or its impurities. In the Japanese adult test, blood parameters and body condition showed no medically problematic fluctuations. These findings indicate that Halomonas sp. KM-1 is safe and can be used for commercial production of D-BHB and its derivatives.

Beta-Hydroxybutyrate Mitigates Sensorimotor and Cognitive Impairments in a Photothrombosis-Induced Ischemic Stroke in Mice.

The consequences of stroke include cognitive deficits and sensorimotor disturbances, which are largely related to mitochondrial impairments in the brain. In this work, we have shown that the mimetic of the ketogenic diet beta-hydroxybutyrate (ßHB) can improve neurological brain function in stroke. At 3 weeks after photothrombotic stroke, mice receiving ßHB with drinking water before and after surgery recovered faster in terms of sensorimotor functions assessed by the string test and static rods and cognitive functions assessed by the Morris water maze. At the same time, the ßHB-treated mice had lower expression of some markers of astrocyte activation and inflammation (Gfap, Il-1b, Tnf). We hypothesize that long-term administration of ßHB promotes the activation of the nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathway, which leads to increased expression of antioxidant genes targeting mitochondria and genes involved in signaling pathways necessary for the maintenance of synaptic plasticity. ßHB partially maintained mitochondrial DNA (mtDNA) integrity during the first days after photothrombosis. However, in the following three weeks, the number of mtDNA damages increased in all experimental groups, which coincided with a decrease in Ogg1 expression, which plays an important role in mtDNA repair. Thus, we can assume that ßHB is not only an important metabolite that provides additional energy to brain tissue during recovery from stroke under conditions of mitochondrial damage but also an important signaling molecule that supports neuronal plasticity and reduces neuroinflammation.

Epithelial-mesenchymal transition-related signaling pathways in gastric Cancer cells distinctively respond to long-term experimental ketosis.

BACKGROUND: The interrelationship between cellular metabolism and the epithelial-to-mesenchymal transition (EMT) process has made it an interesting topic to investigate the adjuvant effect of therapeutic diets in the treatment of cancers. However, the findings are controversial. In this study, the effects of glucose limitation along and with the addition of beta-hydroxybutyrate (bHB) were examined on the expression of specific genes and proteins of EMT, Wnt, Hedgehog, and Hippo signaling pathways, and also on cellular behavior of gastric cancer stem-like (MKN-45) and non-stem-like (KATO III) cells. METHODS AND RESULTS: The expression levels of chosen genes and proteins studied in cancer cells gradually adopted a low-glucose condition of one-fourth, along and with the addition of bHB, and compared to the unconditioned control cells. The long-term switching of the metabolic fuels successfully altered the expression profiles and behaviors of both gastric cancer cells. However, the results for some changes were the opposite. Glucose limitation along and with the addition of bHB reduced the CD44+ population in MKN-45 cells. In KATO III cells, glucose restriction increased the CD44+ population. Glucose deprivation alleviated EMT-related signaling pathways in MKN-45 cells but stimulated EMT in KATO III cells. Interestingly, bHB enrichment reduced the beneficial effect of glucose starvation in MKN-45 cells, but also alleviated the adverse effects of glucose restriction in KATO III cells. CONCLUSIONS: The findings of this research clearly showed that some controversial results in clinical trials for ketogenic diet in cancer patients stemmed from the different signaling responses of various cells to the metabolic changes in a heterogeneous cancer mass.