Ketogenic Diets Alter the Gut Microbiome Resulting in Decreased Intestinal Th17 Cells.

Very low-carbohydrate, high-fat ketogenic diets (KDs) induce a pronounced shift in metabolic fuel utilization that elevates circulating ketone bodies; however, the consequences of these compounds for host-microbiome interactions remain unknown. Here, we show that KDs alter the human and mouse gut microbiota in a manner distinct from high-fat diets (HFDs). Metagenomic and metabolomic analyses of stool samples from an 8-week inpatient study revealed marked shifts in gut microbial community structure and function during the KD. Gradient diet experiments in mice confirmed the unique impact of KDs relative to HFDs with a reproducible depletion of bifidobacteria. In vitro and in vivo experiments showed that ketone bodies selectively inhibited bifidobacterial growth. Finally, mono-colonizations and human microbiome transplantations into germ-free mice revealed that the KD-associated gut microbiota reduces the levels of intestinal pro-inflammatory Th17 cells. Together, these results highlight the importance of trans-kingdom chemical dialogs for mediating the host response to dietary interventions.

Ketogenic HMG-CoA lyase and its product ß-hydroxybutyrate promote pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDA) tumor cells are deprived of oxygen and nutrients and therefore must adapt their metabolism to ensure proliferation. In some physiological states, cells rely on ketone bodies to satisfy their metabolic needs, especially during nutrient stress. Here, we show that PDA cells can activate ketone body metabolism and that ß-hydroxybutyrate (ßOHB) is an alternative cell-intrinsic or systemic fuel that can promote PDA growth and progression. PDA cells activate enzymes required for ketogenesis, utilizing various nutrients as carbon sources for ketone body formation. By assessing metabolic gene expression from spontaneously arising PDA tumors in mice, we find HMG-CoA lyase (HMGCL), involved in ketogenesis, to be among the most deregulated metabolic enzymes in PDA compared to normal pancreas. In vitro depletion of HMGCL impedes migration, tumor cell invasiveness, and anchorage-independent tumor sphere compaction. Moreover, disrupting HMGCL drastically decreases PDA tumor growth in vivo, while ßOHB stimulates metastatic dissemination to the liver. These findings suggest that ßOHB increases PDA aggressiveness and identify HMGCL and ketogenesis as metabolic targets for limiting PDA progression.

Exercise-induced ß-hydroxybutyrate promotes Treg cell differentiation to ameliorate colitis in mice.

Increasing attention is being paid to the mechanistic investigation of exercise-associated chronic inflammatory disease improvement. Ulcerative colitis (UC) is one type of chronic inflammatory bowel disease with increasing incidence and prevalence worldwide. It is known that regular moderate aerobic exercise (RMAE) reduces the incidence or risk of UC, and attenuates disease progression in UC patients. However, the mechanisms of this RMAE's benefit are still under investigation. Here, we revealed that ß-hydroxybutyrate (ß-HB), a metabolite upon prolonged aerobic exercise, could contribute to RMAE preconditioning in retarding dextran sulfate sodium (DSS)-induced mouse colitis. When blocking ß-HB production, RMAE preconditioning-induced colitis amelioration was compromised, whereas supplementation of ß-HB significantly rescued impaired ß-HB production-associated defects. Meanwhile, we found that RMAE preconditioning significantly caused decreased colonic Th17/Treg ratio, which is considered to be important for colitis mitigation; and the downregulated Th17/Treg ratio was associated with ß-HB. We further demonstrated that ß-HB can directly promote the differentiation of Treg cell rather than inhibit Th17 cell generation. Furthermore, ß-HB increased forkhead box protein P3 (Foxp3) expression, the core transcriptional factor for Treg cell, by enhancing histone H3 acetylation in the promoter and conserved noncoding sequences of the Foxp3 locus. In addition, fatty acid oxidation, the key metabolic pathway required for Treg cell differentiation, was enhanced by ß-HB treatment. Lastly, administration of ß-HB without exercise significantly boosted colonic Treg cell and alleviated colitis in mice. Together, we unveiled a previously unappreciated role for exercise metabolite ß-HB in the promotion of Treg cell generation and RMAE preconditioning-associated colitis attenuation.

Impaired glucose utilization in the brain of patients with delirium following hip fracture.

Alterations in brain energy metabolism have long been proposed as one of several neurobiological processes contributing to delirium. This is supported by previous findings of altered CSF lactate and neuron-specific enolase concentrations and decreased glucose uptake on brain-PET in patients with delirium. Despite this, there are limited data on metabolic alterations found in CSF samples, and targeted metabolic profiling of CSF metabolites involved in energy metabolism has not been performed. The aim of the study was to investigate whether metabolites related to energy metabolism in the serum and CSF of patients with hip fracture are associated with delirium. The study cohort included 406 patients with a mean age of 81 years (standard deviation 10 years), acutely admitted to hospital for surgical repair of a hip fracture. Delirium was assessed daily until the fifth postoperative day. CSF was collected from all 406 participants at the onset of spinal anaesthesia, and serum samples were drawn concurrently from 213 participants. Glucose and lactate in CSF were measured using amperometry, whereas plasma glucose was measured in the clinical laboratory using enzymatic photometry. Serum and CSF concentrations of the branched-chain amino acids, 3-hydroxyisobutyric acid, acetoacetate and ß-hydroxybutyrate were measured using gas chromatography-tandem mass spectrometry (GC-MS/MS). In total, 224 (55%) patients developed delirium pre- or postoperatively. Ketone body concentrations (acetoacetate, ß-hydroxybutyrate) and branched-chain amino acids were significantly elevated in the CSF but not in serum among patients with delirium, despite no group differences in glucose concentrations. The level of 3-hydroxyisobutyric acid was significantly elevated in both CSF and serum. An elevation of CSF lactate during delirium was explained by age and comorbidity. Our data suggest that altered glucose utilization and a shift to ketone body metabolism occurs in the brain during delirium.

ß-hydroxybutyrate administered at reperfusion reduces infarct size and preserves cardiac function by improving mitochondrial function through autophagy in male mice.

Ischemia/reperfusion (I/R) injury after revascularization contributes ∼50% of infarct size and causes heart failure, for which no established clinical treatment exists. ß-hydroxybutyrate (ß-OHB), which serves as both an energy source and a signaling molecule, has recently been reported to be cardioprotective when administered immediately before I/R and continuously after reperfusion. This study aims to determine whether administering ß-OHB at the time of reperfusion with a single dose can alleviate I/R injury and, if so, to define the mechanisms involved. We found plasma ß-OHB levels were elevated during ischemia in STEMI patients, albeit not to myocardial protection level, and decreased after revascularization. In mice, compared with normal saline, ß-OHB administrated at reperfusion reduced infarct size (by 50%) and preserved cardiac function, as well as activated autophagy and preserved mtDNA levels in the border zone. Our treatment with one dose ß-OHB reached a level achievable with fasting and strenuous physical activity. In neonatal rat ventricular myocytes (NRVMs) subjected to I/R, ß-OHB at physiologic level reduced cell death, increased autophagy, preserved mitochondrial mass, function, and membrane potential, in addition to attenuating reactive oxygen species (ROS) levels. ATG7 knockdown/knockout abolished the protective effects of ß-OHB observed both in vitro and in vivo. Mechanistically, ß-OHB's cardioprotective effects were associated with inhibition of mTOR signaling. In conclusion, ß-OHB, when administered at reperfusion, reduces infarct size and maintains mitochondrial homeostasis by increasing autophagic flux (potentially through mTOR inhibition). Since ß-OHB has been safely tested in heart failure patients, it may be a viable therapeutic to reduce infarct size in STEMI patients.

Defining ketone supplementation: the evolving evidence for postexercise ketone supplementation to improve recovery and adaptation to exercise.

Over the last decade, there has been a growing interest in the use of ketone supplements to improve athletic performance. These ketone supplements transiently elevate the concentrations of the ketone bodies acetoacetate (AcAc) and d-ß-hydroxybutyrate (ßHB) in the circulation. Early studies showed that ketone bodies can improve energetic efficiency in striated muscle compared with glucose oxidation and induce a glycogen-sparing effect during exercise. As such, most research has focused on the potential of ketone supplementation to improve athletic performance via ingestion of ketones immediately before or during exercise. However, subsequent studies generally observed no performance improvement, and particularly not under conditions that are relevant for most athletes. However, more and more studies are reporting beneficial effects when ketones are ingested after exercise. As such, the real potential of ketone supplementation may rather be in their ability to enhance postexercise recovery and training adaptations. For instance, recent studies observed that postexercise ketone supplementation (PEKS) blunts the development of overtraining symptoms, and improves sleep, muscle anabolic signaling, circulating erythropoietin levels, and skeletal muscle angiogenesis. In this review, we provide an overview of the current state-of-the-art about the impact of PEKS on aspects of exercise recovery and training adaptation, which is not only relevant for athletes but also in multiple clinical conditions. In addition, we highlight the underlying mechanisms by which PEKS may improve exercise recovery and training adaptation. This includes epigenetic effects, signaling via receptors, modulation of neurotransmitters, energy metabolism, and oxidative and anti-inflammatory pathways.

Acetoacetate and d- and l-ß-hydroxybutyrate have distinct effects on basal and insulin-stimulated glucose uptake in L6 skeletal muscle cells.

Ketone bodies (acetoacetate and ß-hydroxybutyrate) are oxidized in skeletal muscle mainly during fasting as an alternative source of energy to glucose. Previous studies suggest that there is a negative relationship between increased muscle ketolysis and muscle glucose metabolism in mice with obesity and/or type 2 diabetes. Therefore, we investigated the connection between increased ketone body exposure and muscle glucose metabolism by measuring the effect of a 3-h exposure to ketone bodies on glucose uptake in differentiated L6 myotubes. We showed that exposure to acetoacetate at a typical concentration (0.2 mM) resulted in increased basal glucose uptake in L6 myotubes, which was dependent on increased membrane glucose transporter type 4 (GLUT4) translocation. Basal and insulin-stimulated glucose uptake was also increased with a concentration of acetoacetate reflective of diabetic ketoacidosis or a ketogenic diet (1 mM). We found that ß-hydroxybutyrate had a variable effect on basal glucose uptake: a racemic mixture of the two ß-hydroxybutyrate enantiomers (d and l) appeared to decrease basal glucose uptake, while 3 mM d-ß-hydroxybutyrate alone increased basal glucose uptake. However, the effects of the ketone bodies individually were not observed when acetoacetate was present in combination with ß-hydroxybutyrate. These results provide insight that will help elucidate the effect of ketone bodies in the context of specific metabolic diseases and nutritional states (e.g., type 2 diabetes and ketogenic diets).NEW & NOTEWORTHY A limited number of studies investigate the effect of ketone bodies at concentrations reflective of both typical fasting and ketoacidosis. We tested a mix of physiologically relevant concentrations of ketone bodies, which allowed us to highlight the differential effects of d- and l-ß-hydroxybutyrate and acetoacetate on skeletal muscle cell glucose uptake. Our findings will assist in better understanding the mechanisms that contribute to muscle insulin resistance and provide guidance on recommendations regarding ketogenic diets.

Acute effects of a ketone monoester, whey protein, or their coingestion on mTOR trafficking and protein-protein colocalization in human skeletal muscle.

We recently demonstrated that acute oral ketone monoester intake induces a stimulation of postprandial myofibrillar protein synthesis rates comparable to that elicited following the ingestion of 10 g whey protein or their coingestion. The present investigation aimed to determine the acute effects of ingesting a ketone monoester, whey protein, or their coingestion on mechanistic target of rapamycin (mTOR)-related protein-protein colocalization and intracellular trafficking in human skeletal muscle. In a randomized, double-blind, parallel group design, 36 healthy recreationally active young males (age: 24.2 ± 4.1 yr) ingested either: 1) 0.36 g·kg-1 bodyweight of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 2) 10 g whey protein (PRO), or 3) the combination of both (KET + PRO). Muscle biopsies were obtained in the overnight postabsorptive state (basal conditions), and at 120 and 300 min in the postprandial period for immunofluorescence assessment of protein translocation and colocalization of mTOR-related signaling molecules. All treatments resulted in a significant (Interaction: P < 0.0001) decrease in tuberous sclerosis complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) colocalization at 120 min versus basal; however, the decrease was sustained at 300 min versus basal (P < 0.0001) only in KET + PRO. PRO and KET + PRO increased (Interaction: P < 0.0001) mTOR-Rheb colocalization at 120 min versus basal; however, KET + PRO resulted in a sustained increase in mTOR-Rheb colocalization at 300 min that was greater than KET and PRO. Treatment intake increased mTOR-wheat germ agglutinin (WGA) colocalization at 120 and 300 min (Time: P = 0.0031), suggesting translocation toward the fiber periphery. These findings demonstrate that ketone monoester intake can influence the spatial mechanisms involved in the regulation of mTORC1 in human skeletal muscle.NEW & NOTEWORTHY We explored the effects of a ketone monoester (KET), whey protein (PRO), or their coingestion (KET + PRO) on mTOR-related protein-protein colocalization and intracellular trafficking in human muscle. All treatments decreased TSC2-Rheb colocalization at 120 minutes; however, KET + PRO sustained the decrease at 300 min. Only PRO and KET + PRO increased mTOR-Rheb colocalization; however, the increase at 300 min was greater in KET + PRO. Treatment intake increased mTOR-WGA colocalization, suggesting translocation to the fiber periphery. Ketone bodies influence the spatial regulation of mTOR.

The therapeutic potential of ketones in cardiometabolic disease: impact on heart and skeletal muscle.

ß-Hydroxybutyrate (ßOHB) is the major ketone in the body, and it is recognized as a metabolic energy source and an important signaling molecule. While ketone oxidation is essential in the brain during prolonged fasting/starvation, other organs such as skeletal muscle and the heart also use ketones as metabolic substrates. Additionally, ßOHB-mediated molecular signaling events occur in heart and skeletal muscle cells, and via metabolism and/or signaling, ketones may contribute to optimal skeletal muscle health and cardiac function. Of importance, when the use of ketones for ATP production and/or as signaling molecules becomes disturbed in the presence of underlying obesity, type 2 diabetes, and/or cardiovascular diseases, these changes may contribute to cardiometabolic disease. As a result of these disturbances in cardiometabolic disease, multiple approaches have been used to elevate circulating ketones with the goal of optimizing either ketone metabolism or ketone-mediated signaling. These approaches have produced significant improvements in heart and skeletal muscle during cardiometabolic disease with a wide range of benefits that include improved metabolism, weight loss, better glycemic control, improved cardiac and vascular function, as well as reduced inflammation and oxidative stress. Herein, we present the evidence that indicates that ketone therapy could be used as an approach to help treat cardiometabolic diseases by targeting cardiac and skeletal muscles.

Ketone bodies in cell physiology and cancer.

Ketogenic diets (KDs), fasting, or prolonged physical activity elevate serum ketone bodies (KBs) levels, providing an alternative fuel source for the brain and other organs. However, KBs play pleiotropic roles that go beyond their role in energy production. KBs can act as signaling metabolites, influence gene expression, proteins' posttranslational modifications (PTMs), inflammation, and oxidative stress. Here, we explore the impact of KBs on mammalian cell physiology, including aging and tissue regeneration. We also concentrate on KBs and cancer, given the extensive evidence that dietary approaches inducing ketosis, including fasting-mimicking diets (FMDs) and KDs, can prevent cancer and affect tumor progression.

A combination of ß-hydroxybutyrate and citrate ameliorates disease progression in a rat model of polycystic kidney disease.

Our research has shown that interventions producing a state of ketosis are highly effective in rat, mouse, and cat models of polycystic kidney disease (PKD), preventing and partially reversing cyst growth and disease progression. The ketone ß-hydroxybutyrate (BHB) appears to underlie this effect. In addition, we have demonstrated that naturally formed microcrystals within kidney tubules trigger a renoprotective response that facilitates tubular obstruction clearance in healthy animals but, alternatively, leads to cyst formation in PKD. The administration of citrate prevents microcrystal formation and slows PKD progression. Juvenile Cy/+ rats, a nonorthologous PKD model, were supplemented from 3 to 8 wk of age with water containing titrated BHB, citrate, or in combination to find minimal effective and optimal dosages, respectively. Adult rats were given a reduced BHB/citrate combination or equimolar control K/NaCl salts from 8 to 12 wk of age. In addition, adult rats were placed in metabolic cages following BHB, citrate, and BHB/citrate administration to determine the impact on mineral, creatinine, and citrate excretion. BHB or citrate alone effectively ameliorates disease progression in juvenile rats, decreasing markers of cystic disease and, in combination, producing a synergistic effect. BHB/citrate leads to partial disease regression in adult rats with established cystic disease, inhibiting cyst formation and kidney injury. BHB/citrate confers benefits via multiple mechanisms, increases creatinine and citrate excretion, and normalizes mineral excretion. BHB and citrate are widely available and generally recognized as safe compounds and, in combination, exhibit high promise for supporting kidney health in polycystic kidney disease.NEW & NOTEWORTHY Combining ß-hydroxybutyrate (BHB) and citrate effectively slows and prevents cyst formation and expansion in young Cy/+ rats using less BHB and citrate than when used alone, demonstrating synergy. In adult rats, the combination causes a partial reversal of existing disease, reducing cyst number and cystic area, preserving glomerular health, and decreasing markers of kidney injury. Our results suggest a safe and feasible strategy for supporting kidney health in polycystic kidney disease (PKD) using a combination of BHB and citrate.

Pre-treatment with ß-hydroxybutyrate mitigates cisplatin-induced acute kidney injury.

ß-Hydroxybutyrate (ß-HB), the primary circulating ketone body, plays a dual role as both a metabolic fuel and an endogenous signaling molecule, offering diverse systemic benefits. Recent studies have highlighted the renoprotective effects of exogenous ß-HB therapy in various animal models of kidney disease. In this investigation, our goal was to assess whether pre-treatment with exogenous ß-HB could alleviate kidney damage in a mouse model of cisplatin-induced acute kidney injury (AKI). Prior to cisplatin administration, intraperitoneal administration of ß-HB was carried out, and the groups were classified into four: Sham, ß-HB, cisplatin, and ß-HB + cisplatin. The tubular damage score and serum creatinine levels were significantly lower in the ß-HB + cisplatin group compared to the cisplatin group. Furthermore, the expression of phosphorylated NF-κB, inflammatory cytokines, and the quantity of F4/80-positive macrophages in the ß-HB + cisplatin group were reduced compared to those in the cisplatin group. Additionally, oxidative stress markers for DNA, protein, and lipid in the ß-HB + cisplatin group were markedly diminished compared to those in the cisplatin group. The number of TUNEL-positive and cleaved caspase 3-positive tubular cells in the ß-HB + cisplatin group was lower than in the cisplatin group. Pre-treating with exogenous ß-HB effectively mitigated kidney damage by suppressing inflammation, oxidative stress, and tubular apoptosis in cisplatin-induced AKI. Therefore, exogenous ß-HB as a pre-treatment emerges as a promising and novel strategy for preventing cisplatin-induced AKI.

Protein acylations induced by a ketogenic diet demonstrate diverse patterns depending on organs and differ between histones and global proteins.

An essential ketone body, ß-hydroxybutyrate (BOHB), plays various roles in physiological regulations via protein acylations such as lysine acetylation and ß-hydroxybutyrylation. Here, to understand how BOHB systemically regulates acylations from an overarching perspective, we administered a ketogenic diet to mice to increase BOHB concentration and examined acylations. We found that global acetylation and ß-hydroxybutyrylation dramatically increase in various organs except for the brains, where the increase was much smaller than in the other organs. Interestingly, we observe no increase in histone acetylation in the organs where significant global protein acetylation occurs despite a substantial rise in histone ß-hydroxybutyrylation. Finally, we compared the transcriptome data of the mice's liver after the ketogenic diet to the public databases, showing that upregulated genes are enriched in those related to histone ß-hydroxybutyrylation in starvation. Our data indicate that a ketogenic diet induces diverse patterns of acylations depending on organs and protein localizations, suggesting that different mechanisms regulate acylations and that the ketogenic diet is associated with starvation in terms of protein modifications.

Ketogenic diet ameliorates high-fat diet-induced insulin resistance in mouse skeletal muscle by alleviating endoplasmic reticulum stress.

OBJECTIVE: Ketogenic diets (KD) have been shown to alleviate insulin resistance (IR) by exerting anti-lipogenic and insulin sensitizing effects in the liver through a variety of pathways. The present study sought to investigate whether a ketogenic diet also improves insulin sensitization in skeletal muscle cells through alleviating endoplasmic reticulum stress. METHODS: High-fat diet-induced IR mice were allowed to a 2-week ketogenic diet. Insulin resistance and glucose tolerance were evaluated through GTT, ITT, and HOMA-IR. The C2C12 myoblasts exposed to palmitic acid were used to evaluate the insulin sensitization effects of ß-hydroxybutyric acid (ß-OHB). Molecular mechanisms concerning ER stress signaling activation and glucose uptake were assessed. RESULTS: The AKT/GSK3ß pathway was inhibited, ER stress signaling associated with IRE1, PERK, and BIP was activated, and the number of Glut4 proteins translocated to membrane decreased in the muscle of HFD mice. However, all these changes were reversed after 2 weeks of feeding on a ketogenic diet. Consistently in C2C12 myoblasts, the AKT/GSK3ß pathway was inhibited by palmitic acid (PA) treatment. The endoplasmic reticulum stress-related proteins, IRE1, and BIP were increased, and the number of Glut4 proteins on the cell membrane decreased. However, ß-OHB treatment alleviated ER stress and improved the glucose uptake of C2C12 cells. CONCLUSION: Our data reveal that KD ameliorated HFD-induced insulin resistance in skeletal muscle, which was partially mediated by inhibiting endoplasmic reticulum stress. The insulin sensitization effect of ß-OHB is associated with up regulation of AKT/GSK3ß pathway and the increase in the number of Glut4 proteins on the cell membrane.

Tracking the therapeutic efficacy of a ketone mono ester and ß-hydroxybutyrate for ulcerative colitis in rats: New perspectives.

Ulcerative colitis (UC) is an inflammatory condition that affects the colon's lining and increases the risk of colon cancer. Despite ongoing research, there is no identified cure for UC. The recognition of NLRP3 inflammasome activation in the pathogenesis of UC has gained widespread acceptance. Notably, the ketone body ß-hydroxybutyrate inhibits NLRP3 demonstrating its anti-inflammatory properties. Additionally, BD-AcAc 2 is ketone mono ester that increases ß-hydroxybutyrate blood levels. It has the potential to address the constraints associated with exogenous ß-hydroxybutyrate as a therapeutic agent, including issues related to stability and short duration of action. However, the effects of ß-hydroxybutyrate and BD-AcAc 2 on colitis have not been fully investigated. This study found that while both exogenous ß-hydroxybutyrate and BD-AcAc 2 produced the same levels of plasma ß-hydroxybutyrate, BD-AcAc 2 demonstrated superior effectiveness in mitigating dextran sodium sulfate-induced UC in rats. The mechanism of action involves modulating the NF-κB signaling, inhibiting the NLRP3 inflammasome, regulating antioxidant capacity, controlling tight junction protein expression and a potential to inhibit apoptosis and pyroptosis. Certainly, BD-AcAc 2's anti-inflammatory effects require more than just increasing plasma ß-hydroxybutyrate levels and other factors contribute to its efficacy. Local ketone concentrations in the gastrointestinal tract, as well as the combined effect of specific ketone bodies, are likely to have contributed to the stronger protective effect observed with ketone mono ester ingestion in our experiment. As a result, further investigations are necessary to fully understand the mechanisms of BD-AcAc 2 and optimize its use.

ß-hydroxybutyrate inhibits malignant phenotypes of prostate cancer cells through ß-hydroxybutyrylation of indoleacetamide-N-methyltransferase.

BACKGROUND: Prostate cancer (PCa) is one of the most prevalent cancers in men and is associated with high mortality and disability rates. ß-hydroxybutyrate (BHB), a ketone body, has received increasing attention for its role in cancer. However, its role in PCa remains unclear. This study aimed to explore the mechanism and feasibility of BHB as a treatment alternative for PCa. METHODS: Colony formation assay, flow cytometry, western blot assay, and transwell assays were performed to determine the effect of BHB on the proliferation and metastasis of PCa cells. Tumor sphere formation and aldehyde dehydrogenase assays were used to identify the impact of BHB or indoleacetamide-N-methyltransferase (INMT) on the stemness of PCa cells. N6-methyladenosine (m6A)-meRIP real-time reverse transcription polymerase chain reaction and dual luciferase assays were conducted to confirm INMT upregulation via the METTL3-m6A pathway. Co-IP assay was used to detect the epigenetic modification of INMT by BHB-mediated ß-hydroxybutyrylation (kbhb) and screen enzymes that regulate INMT kbhb. Mouse xenograft experiments demonstrated the antitumor effects of BHB in vivo. RESULTS: BHB can inhibit the proliferation, migration, and invasion of PCa cells by suppressing their stemness. Mechanistically, INMT, whose expression is upregulated by the METTL3-m6A pathway, was demonstrated to be an oncogenic gene that promotes the stem-like characteristics of PCa cells. BHB can suppress the malignant phenotypes of PCa by kbhb of INMT, which in turn inhibits INMT expression. CONCLUSIONS: Our findings indicate a role of BHB in PCa metabolic therapy, thereby suggesting an epigenetic therapeutic strategy to target INMT in aggressive PCa. TRIAL REGISTRATION: Not applicable.

Improved therapy for clear cell renal cell carcinoma: beta-hydroxybutyrate and quercetin target hypoxia-induced angiogenesis and multidrug resistance.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is a form of kidney cancer characterized by dysregulated angiogenesis and multidrug resistance. Hypoxia-induced tumor progression plays a crucial role in ccRCC pathogenesis. Beta-hydroxybutyrate (BHB) and quercetin (QCT) have shown potential in targeting angiogenesis and drug resistance in various cancer types. This study investigates the combined effects of BHB and QCT in hypoxia-induced Caki-1 cells. METHODS: Caki-1 cells were subjected to normoxic and hypoxic conditions and treated with BHB, QCT, or a combination of both. Cell-viability was assessed using the MTT assay, and mRNA expression levels of key angiogenesis-related genes (HIF-1α/2α, VEGF, Ang-1, Ang-2, and MDR4) were quantified through real-time PCR during 24 and 48 h. RESULTS: BHB and QCT treatments, either alone or in combination, significantly reduced cell-viability in Caki-1 cells (p < 0.05). Moreover, the combined therapy demonstrated a potential effect in downregulating the expression of angiogenesis-related genes and MDR4 in hypoxia-induced cells, with a marked reduction in HIF-1α/2α, VEGF, Ang-1, and MDR4 expression (p < 0.05). The expression of Ang-2 increases significantly in presence of BHB combined QCT treatment. CONCLUSION: This study highlights the promising potential of a combination therapy involving BHB and QCT in mitigating angiogenesis and MDR4 expression in hypoxia-induced ccRCC cells. These findings support further investigation into the underlying mechanisms and warrant clinical studies to evaluate the therapeutic value of this combined treatment for ccRCC patients. This research provides new insights into addressing the challenges posed by angiogenesis and drug resistance in ccRCC.

Targeting apoptosis and unfolded protein response: the impact of ß-hydroxybutyrate in clear cell renal cell carcinoma under glucose-deprived conditions.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) plays a significant role in the mortality associated with kidney cancer. Targeting biological processes that inhibit cancer growth opens up new treatment possibilities. The unfolded protein response (UPR) and apoptosis have crucial roles in RCC progression. This study investigates the impact of ß-hydroxybutyrate (BHB) on ccRCC cells under glucose deprivation resembling as a ketogenic diet. METHOD: Caki-1 ccRCC cells were exposed to decreasing glucose concentrations alone or in combination with 10 or 25 mM BHB during 48 and 72 h. Cell viability was determined using MTT assay. The mRNA expression level of apoptosis-and UPR-related markers (Bcl-2, Bax, caspase 3, XBP1s, BIP, CHOP, ATF4, and ATF6) were assayed by qRT-PCR. RESULTS: Cell viability experiments demonstrated that combining different doses of BHB with decreasing glucose levels initially improved cell viability after 48 h. Nevertheless, this trend reversed after 72 h, with higher impacts disclosed at 25 mM BHB. Apoptosis was induced in BHB-treated cells as caspase-3 and Bax were increased and Bcl-2 was downregulated. BHB supplementation reduced UPR-related gene expression (XBP1s, BIP, CHOP, ATF4, and ATF6), revealing a possible mechanism by which BHB affects cell survival. CONCLUSION: This research emphasizes the dual effect of BHB, initially suppressing cell- survival under glucose deprivation but eventually triggering apoptosis and suppressing UPR signaling. These data highlight the intricate connection between metabolic reprogramming and cellular stress response in ccRCC. Further research is recommended to explore the potential of BHB as a therapeutic strategy for managing ccRCC.

Metabolic Clusters of Early-Lactating Dairy Cows Based on Blood ß-hydroxybutyrate Trajectories and Predicted from Milk Compounds.

High-yielding dairy cows encounter metabolic challenges in early lactation. Typically, ß-hydroxybutyrate (BHB), measured at a specific time point is employed to diagnose the metabolic status of cows based on a predetermined threshold. However, in early lactation, BHB is highly dynamic, and there is high interindividual variability in its time profile. This could limit the effectiveness of the single measurement and threshold-based diagnosis probably contributing to the disparities in reports linking metabolic status with productive and reproductive outcomes. This research delves into the examination of the trajectories of BHB to unveil inter-cow variations and identify latent metabolic groups. We compiled a data set from 2 observational studies involving a total of 195 lactations from multiparous Holstein Friesian cows. The data set encompasses measurements of BHB, NEFA, and insulin from blood samples collected at 3, 6, 9, and 21 d in milk (DIM), along with weekly determinations of milk composition and fatty acids (FA) proportions in milk fat. In both experiments, milk yield (MY) and feed intake were recorded daily during the first month of lactation. We explored interindividual and intraindividual variations in metabolic responses using the trajectories of blood BHB and evaluated the presence of distinct metabolic groups based on such variations. For this purpose, we employed the growth mixture model (GMM), a trajectory clustering technique. Our findings unveil novel insights into the diverse metabolic responses among cows, encompassing both trajectory patterns and the magnitude of blood BHB concentrations. Specifically, we identified 3 latent metabolic groups: the "QuiBHB" cluster (≈10%) exhibited a higher initial BHB concentration than other clusters, peaking on d 9 (average maximum BHB of 2.4 mM) and then declining by d 21; the "SloBHB" cluster (≈23%) started with a lower BHB concentration, gradually increasing until d 9, and at the highest BHB concentration at d 21 (1.6 mM serum BHB at the end of the experimental period); and the "LoBHB" cluster (≈67%) began with the lowest serum BHB concentration (serum BHB <0.75 mM), remaining relatively stable throughout the sampling period. Notably, the 3 metabolic groups exhibited significant physiological disparities, evident in blood NEFA and insulin concentrations. The QuiBHB and SloBHB cows exhibited higher NEFA and lower insulin concentrations as compared with the LoBHB cows. Interestingly, these metabolic differences extended to MY and DMI during the first month of lactation. The elevated BHB concentrations observed in QuiBHB cows were linked with lower DMI and MY as compared with SloBHB and LoBHB cows. Accordingly, these animals were considered metabolically impaired. Conversely, SloBHB cows displayed higher MY along with increased DMI, and thus the elevated BHB might be indicative of an adaptive response for these cows. The QuiBHB cows also displayed higher proportions of unsaturated FA (UFA), monounsaturated FA (MUFA), and total C18:1 FA in milk during the first week of lactation. Prediction of the QuiBHB cows using these FA and test day variables resulted in moderate predictive accuracy (ROCAUC > 0.7). Given the limited sample size for the development of prediction models, and the variation in DIM among samples in the same week, the result is indicative of the predictive potential of the model and room for model optimization. In summary, distinct metabolic groups of cows could be identified based on the trajectories of blood BHB in early lactation.

ß-Hydroxybutyrate Protects Against Cisplatin-Induced Renal Damage via Regulating Ferroptosis.

Cisplatin is a particularly potent antineoplastic drug. However, its usefulness is restricted due to the induction of nephrotoxicity. More recent research has indicated that ß-hydroxybutyrate (ß-HB) protects against acute or chronic organ damage as an efficient healing agent. Nonetheless, the therapeutic mechanisms of ß-HB in acute kidney damage caused by chemotherapeutic drugs remain unclear. Our study developed a model of cisplatin-induced acute kidney injury (AKI), which involved the administration of a ketogenic diet or ß-HB. We analyzed blood urea nitrogen (BUN) and creatinine (Cr) levels in serum, and used western blotting and immunohistochemical staining to assess ferroptosis and the calcium/calmodulin-dependent kinase kinase 2 (Camkk2)/AMPK pathway. The mitochondrial morphology and function were examined. Additionally, we conducted in vivo and in vitro experiments using selective Camkk2 inhibitor or activator to investigate the protective mechanism of ß-HB on cisplatin-induced AKI. Exogenous or endogenous ß-HB effectively alleviated cisplatin-induced abnormally elevated levels of BUN and Cr and renal tubular necrosis in vivo. Additionally, ß-HB reduced ferroptosis biomarkers and increased the levels of anti-ferroptosis biomarkers in the kidney. ß-HB also improved mitochondrial morphology and function. Moreover, ß-HB significantly attenuated cisplatin-induced cell ferroptosis and damage in vitro. Furthermore, western blotting and immunohistochemical staining indicated that ß-HB may prevent kidney injury by regulating the Camkk2-AMPK pathway. The use of the Camkk2 inhibitor or activator verified the involvement of Camkk2 in the renal protection by ß-HB. This study provided evidence of the protective effects of ß-HB against cisplatin-induced nephrotoxicity and identified inhibited ferroptosis and Camkk2 as potential molecular mechanisms.

ß-HB protects against cisplatin-induced renal damage both in vivo and in vitro.Moreover, ß-HB is effective in attenuating cisplatin-induced lipid peroxidation and ferroptosis.The regulation of energy metabolism, as well as the treatment involving ß-HB, is associated with Camkk2.