Ketone Body Β-hydroxybutyrate Research Articles

Rapid modulation of interscapular brown adipose tissue mitochondrial activity by ketosis induced by 1,3-butanediol administration to rats.

Some studies indicate that brown adipose tissue (BAT) represents a promising target in the fight against dysmetabolic diseases, with indications suggesting it as a potential target for the effects of ketone bodies. We investigate whether the elevation of plasma levels of the ketone body β-hydroxybutyrate, achieved through the invivo administration of its precursor 1,3-butanediol (BD) to rats, could impact interscapular BAT (iBAT) mitochondrial biochemistry and functionality. We examined the effects induced by BD within 3 h and after 2 weeks of treatment. A large-scale quantitative proteomics approach, coupling liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, and Western blot associated with functional studies by respirometry allowed us to evaluate the changes in iBAT mitochondrial protein expression and bioenergetics induced by BD. BD administration increased β-hydroxybutyrate plasma levels, which correlated with an enhancement in iBAT mitochondrial respiration rate, likely due to the activation of the respiratory chain and uncoupling protein-1. The proteomic analysis demonstrated that BD influenced the mitochondrial levels of specific subunits belonging to the five respiratory complexes, uncoupling protein-1, and proteins involved in propanoate metabolism. BD administration also induced lysine β-hydroxybutyrylation of mitochondrial proteins, including specific subunits of the respiratory chain complexes and uncoupling protein-1. Most of the BD-induced effects were observed within 3 h of its administration and persisted/increased after 2 weeks of treatment. In conclusion, by using BD to increase β-hydroxybutyrate levels, we provide evidence supporting the role of β-hydroxybutyrate as a signaling molecule capable of rapidly modulating BAT physiology by acting at the mitochondrial level.

The ketone body β-hydroxybutyrate ameliorates neurodevelopmental deficits in the GABAergic system of daf-18/PTEN Caenorhabditis elegans mutants.

A finely tuned balance between excitation and inhibition (E/I) is essential for proper brain function. Disruptions in the GABAergic system, which alter this equilibrium, are a common feature in various types of neurological disorders, including autism spectrum disorders (ASDs). Mutations in Phosphatase and Tensin Homolog (PTEN), the main negative regulator of the phosphatidylinositol 3-phosphate kinase/Akt pathway, are strongly associated with ASD. However, it is unclear whether PTEN deficiencies can differentially affect inhibitory and excitatory signaling. Using the Caenorhabditis elegans neuromuscular system, where both excitatory (cholinergic) and inhibitory (GABAergic) inputs regulate muscle activity, we found that daf-18/PTEN mutations impact GABAergic (but not cholinergic) neurodevelopment and function. This selective impact results in a deficiency in inhibitory signaling. The defects observed in the GABAergic system in daf-18/PTEN mutants are due to reduced activity of DAF-16/FOXO during development. Ketogenic diets (KGDs) have proven effective for disorders associated with E/I imbalances. However, the mechanisms underlying their action remain largely elusive. We found that a diet enriched with the ketone body β-hydroxybutyrate during early development induces DAF-16/FOXO activity, therefore improving GABAergic neurodevelopment and function in daf-18/PTEN mutants. Our study provides valuable insights into the link between PTEN mutations and neurodevelopmental defects and delves into the mechanisms underlying the potential therapeutic effects of KGDs.

The ketone body β-hydroxybutyrate ameliorates neurodevelopmental deficits in the GABAergic system of daf-18/PTEN Caenorhabditis elegans mutants

A finely tuned balance between excitation and inhibition (E/I) is essential for proper brain function. Disruptions in the GABAergic system, which alter this equilibrium, are a common feature in various types of neurological disorders, including autism spectrum disorders (ASDs). Mutations in Phosphatase and Tensin Homolog (PTEN), the main negative regulator of the phosphatidylinositol 3-phosphate kinase/Akt pathway, are strongly associated with ASD. However, it is unclear whether PTEN deficiencies can differentially affect inhibitory and excitatory signaling. Using the Caenorhabditis elegans neuromuscular system, where both excitatory (cholinergic) and inhibitory (GABAergic) inputs regulate muscle activity, we found that daf-18/PTEN mutations impact GABAergic (but not cholinergic) neurodevelopment and function. This selective impact results in a deficiency in inhibitory signaling. The defects observed in the GABAergic system in daf-18/PTEN mutants are due to reduced activity of DAF-16/FOXO during development. Ketogenic diets (KGDs) have proven effective for disorders associated with E/I imbalances. However, the mechanisms underlying their action remain largely elusive. We found that a diet enriched with the ketone body β-hydroxybutyrate during early development induces DAF-16/FOXO activity, therefore improving GABAergic neurodevelopment and function in daf-18/PTEN mutants. Our study provides valuable insights into the link between PTEN mutations and neurodevelopmental defects and delves into the mechanisms underlying the potential therapeutic effects of KGDs.

The Ketone Body β-Hydroxybutyrate Mitigates Diabetic Kidney Disease through Metabolic Reprogramming

The Ketone Body β-Hydroxybutyrate Mitigates Diabetic Kidney Disease through Metabolic Reprogramming

Intestinal Ketogenesis and Permeability.

Consumption of a high-fat diet (HFD) has been suggested as a contributing factor behind increased intestinal permeability in obesity, leading to increased plasma levels of microbial endotoxins and, thereby, increased systemic inflammation. We and others have shown that HFD can induce jejunal expression of the ketogenic rate-limiting enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS). HMGCS is activated via the free fatty acid binding nuclear receptor PPAR-α, and it is a key enzyme in ketone body synthesis that was earlier believed to be expressed exclusively in the liver. The function of intestinal ketogenesis is unknown but has been described in suckling rats and mice pups, possibly in order to allow large molecules, such as immunoglobulins, to pass over the intestinal barrier. Therefore, we hypothesized that ketone bodies could regulate intestinal barrier function, e.g., via regulation of tight junction proteins. The primary aim was to compare the effects of HFD that can induce intestinal ketogenesis to an equicaloric carbohydrate diet on inflammatory responses, nutrition sensing, and intestinal permeability in human jejunal mucosa. Fifteen healthy volunteers receiving a 2-week HFD diet compared to a high-carbohydrate diet were compared. Blood samples and mixed meal tests were performed at the end of each dietary period to examine inflammation markers and postprandial endotoxemia. Jejunal biopsies were assessed for protein expression using Western blotting, immunohistochemistry, and morphometric characteristics of tight junctions by electron microscopy. Functional analyses of permeability and ketogenesis were performed in Caco-2 cells, mice, and human enteroids. Ussing chambers were used to analyze permeability. CRP and ALP values were within normal ranges and postprandial endotoxemia levels were low and did not differ between the two diets. The PPARα receptor was ketone body-dependently reduced after HFD. None of the tight junction proteins studied, nor the basal electrical parameters, were different between the two diets. However, the ketone body inhibitor hymeglusin increased resistance in mucosal biopsies. In addition, the tight junction protein claudin-3 was increased by ketone inhibition in human enteroids. The ketone body β-Hydroxybutyrate (βHB) did not, however, change the mucosal transition of the large-size molecular FD4-probe or LPS in Caco-2 and mouse experiments. We found that PPARα expression was inhibited by the ketone body βHB. As PPARα regulates HMGCS expression, the ketone bodies thus exert negative feedback signaling on their own production. Furthermore, ketone bodies were involved in the regulation of permeability on intestinal mucosal cells in vitro and ex vivo. We were not, however, able to reproduce these effects on intestinal permeability in vivo in humans when comparing two weeks of high-fat with high-carbohydrate diet in healthy volunteers. Further, neither the expression of inflammation markers nor the aggregate tight junction proteins were changed. Thus, it seems that not only HFD but also other factors are needed to permit increased intestinal permeability in vivo. This indicates that the healthy gut can adapt to extremes of macro-nutrients and increased levels of intestinally produced ketone bodies, at least during a shorter dietary challenge.

The effects of Ketone Body β-hydroxybutyrate on eNOS Levels and VCAM-1 Expression in Wistar Rats Exposed to Cigarette Smoke

The effects of Ketone Body β-hydroxybutyrate on eNOS Levels and VCAM-1 Expression in Wistar Rats Exposed to Cigarette Smoke

Histone β-hydroxybutyrylation-mediated Chromatin Remodeling Upregulates β-oxidation of Fatty Acids and Attenuates Metabolic Syndrome

Center for Hypertension and Precision Medicine, Department of Physiology and Pharmacology, University of Toledo, Toledo, OH Introduction: Metabolic syndrome (MetS) is a cluster of metabolic dysregulations including high blood pressure (BP), dyslipidemia, central obesity, hyperglycemia, and insulin resistance. Regular exercise is one of the lifestyle modifications known to prevent MetS by the generation of ketone bodies. Precisely how ketone bodies operate to lower MetS is largely unknown. Previously, we demonstrated that the ketone body, β-hydroxybutyrate, is a potent anti-hypertensive metabolite. β-hydroxybutyrate has been recently recognized to epigenetically modify histones by β-hydroxybutyrylation. Using assay for transposase-accessible chromatin with sequencing and chromatin immunoprecipitation assays, we found that histone β-hydroxybutyrylation causes chromatin remodeling and exposes regions for transcription of genes for β-oxidation of fatty acids. Therefore, we hypothesized that β-hydroxybutyrate alleviates MetS via histone β-hydroxybutyrylation-mediated transcriptional upregulation of β-oxidation of fatty acids. To test this hypothesis, we examined the epigenetic effect of increasing systemic β-hydroxybutyrate in a genetic rat model of MetS inbred in our laboratory, the low-capacity runner (LCR/Tol) rats. Methods: Randomly selected two groups of six months old female LCR/Tol rats (n=5/group) were surgically implanted with radiotelemetry transmitters for BP measurement. The control group was given water, whereas the experimental group was given water containing 20% v/v of 1,3-butanediol (1,3-BD), which is a precursor of β-hydroxybutyrate for 10 weeks. Following euthanasia, sera and tissues were harvested. Histones were isolated and subjected to western blotting for quantitation of β-hydroxybutyrylation. Results: Compared to the control group, rats given 1,3-BD had a significantly increased circulating β-hydroxybutyrate (mean 1.98 mM vs 0.71 mM, p<0.05), lower body weight (mean 231.6 gm vs 248.4 gm, p<0.05), lower fasting glucose (mean 79.60 mg/dl vs 100.8 mg/dl, p<0.05) and lower 24-hour average systolic BP (116±2 mmHg vs. 120±2 mmHg, p<0.05). Interestingly, histones isolated from the experimental group with higher circulating β-hydroxybutyrate also displayed a marked increase in β-hydroxybutyrylation (mean 0.82 vs 0.22, p<0.01). Further, the target transcriptional genes of histone β-hydroxybutyrylation, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) and cytochrome P450 family 2 subfamily d polypeptide 4 (Cyp2d4) were both significantly upregulated in the experimental group (p<0.01). However, markers of liver injury, alanine transaminase, alkaline phosphatase, aspartate transaminase, and total bile acid levels, were comparable between the two groups, indicating no liver damage. Conclusion: This is the first study to demonstrate that enhanced systemic β-hydroxybutyrate attenuates MetS by histone β-hydroxybutyrylation-mediated chromatin remodeling to upregulate the transcription of Hmgcs2 and Cyp2d4 to promote β-oxidation of fatty acids. Further, our results demonstrate that for individuals with MetS who may be unable to exercise, utilizing 1,3-BD could serve as an alternate source to combat the MetS ameliorating benefits of the ketone body β-hydroxybutyrate. Funding Sources: Authors acknowledge the National Institutes of Health for funding support to Bina Joe (R01HL1430820). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The ketone body β-Hydroxybutyrate as a fuel source of chondrosarcoma cells

Chondrosarcoma (CS) is a malignant bone tumor arising from cartilage-producing cells. The conventional subtype of CS typically develops within a dense cartilaginous matrix, creating an environment deficient in oxygen and nutrients, necessitating metabolic adaptation to ensure proliferation under stress conditions. Although ketone bodies (KBs) are oxidized by extrahepatic tissue cells such as the heart and brain, specific cancer cells, including CS cells, can undergo ketolysis. In this study, we found that KBs catabolism is activated in CS cells under nutrition-deprivation conditions. Interestingly, cytosolic β-hydroxybutyrate dehydrogenase 2 (BDH2), rather than mitochondrial BDH1, is expressed in these cells, indicating a specific metabolic adaptation for ketolysis in this bone tumor. The addition of the KB, β-Hydroxybutyrate (β-HB) in serum-starved CS cells re-induced the expression of BDH2, along with the key ketolytic enzyme 3-oxoacid CoA-transferase 1 (OXCT1) and monocarboxylate transporter-1 (MCT1). Additionally, internal β-HB production was quantified in supplied and starved cells, suggesting that CS cells are also capable of ketogenesis alongside ketolysis. These findings unveil a novel metabolic adaptation wherein nutrition-deprived CS cells utilize KBs for energy supply and proliferation.

Tracking the therapeutic efficacy of a ketone mono ester and β-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 β-hydroxybutyrate inhibits NLRP3 demonstrating its anti-inflammatory properties. Additionally, BD-AcAc 2 is ketone mono ester that increases β-hydroxybutyrate blood levels. It has the potential to address the constraints associated with exogenous β-hydroxybutyrate as a therapeutic agent, including issues related to stability and short duration of action. However, the effects of β-hydroxybutyrate and BD-AcAc 2 on colitis have not been fully investigated. This study found that while both exogenous β-hydroxybutyrate and BD-AcAc 2 produced the same levels of plasma β-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 β-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.

Abstract 3022: GPR109A activation limits cell growth by inducing p53 and its targets in human colon cancer cells

Abstract Chemical carcinogens or xenobiotics present in the diet play a significant role in colon cancer (CC) formation. p53, a tumor suppressor and multi-functional protein, provides an important barrier against the initiation and maintenance of CC. p53 mutation or inactivation is observed in about 60% of CCs and about 90% of all cancers. Therefore, therapeutic avenues that will rejuvenate wild-type p53 or target mutant p53 should have a significant impact on the prevention and treatment of CC. GPR109A, a G-protein coupled receptor for the FDA-approved lipid-lowering drug Niacin (NA), bacterial metabolite Butyrate (BTR), and ketone body β-hydroxybutyrate (BHB), functions as a novel tumor suppressor in the colon by providing the molecular link between colonic bacteria and p53 function. GPR109A activation limits cAMP by inhibiting adenylate cyclase (AC), which induces RAS and its downstream growth-promoting signaling. cAMP also enhances the binding of MDM2 to p53 and induces the proteasomal degradation of p53. p53 is an NAD+-dependent molecule and deficiency of NA, the precursor of NAD+, impairs p53 function. We tested the role of GPR109A and its agonists (NA, BTR, and BHB) in normal immortalized colon epithelial cell lines (CCD33 and CCD81) and various CC lines such as HCT116-p53+/+, HCT116-p53−/−, DLD1 (p53 point mutation), VACO4A (p53 insertion mutation), and SW498 (p53 deletion) and measured cell proliferation, cell cycle, apoptosis, and analyzed the expression of GPR109A and p53 (total, phosphorylated, and acetylated), MDM2, p21, Bax, and Puma in the presence and absence of GPR109109 agonists. We found that GPR109A expression is significantly reduced in CC cell lines, especially in p53 mutant cell lines, when compared to normal immortalized colonic epithelial cell lines. Treatment of CC cell lines with GPR109A agonists-induced apoptosis in wild-type p53 expressing CC by activating p53 and its targets. However, some lesser extent in p53 mutant or p53 null cell lines. Lentiviral-mediated GPR109A expression in CC cell lines itself induced apoptosis in both wild-type and mutant p53 expressing CC cell lines and GPR109A agonists treatment induced further. Interestingly, GPR109A agonists BTR and BHB significantly increased apoptosis in p53 mutant and p53 null CC cell lines in a p53-independent manner. Overall, our results provide evidence that GPR109A activation can be used as an effective therapeutic strategy for the treatment of CC. Citation Format: Muthusamy Thangaraju, Nanditi N. Thangaraju, Lauren Gilstrap, Madison Chimenti, Tulshi Patel, Sathish Sivaprakasam, Nikhil Patel, Santhakumar Manicassamy, Puttur D. Prasad, Vadivel Ganapathy. GPR109A activation limits cell growth by inducing p53 and its targets in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3022.

Ketone bodies promote epididymal white adipose expansion to alleviate liver steatosis in response to a ketogenic diet

Liver can sense the nutrient status and send signals to other organs to regulate overall metabolic homoeostasis. Herein, we demonstrate that ketone bodies act as signals released from the liver that specifically determine the distribution of excess lipid in epididymal white adipose tissue (eWAT) when exposed to a ketogenic diet (KD). An acute KD can immediately result in excess lipid deposition in the liver. Subsequently, the liver sends the ketone body β-hydroxybutyrate (BHB) to regulate white adipose expansion, including adipogenesis and lipogenesis, to alleviate hepatic lipid accumulation. When ketone bodies are depleted by deleting 3-Hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) gene in liver, the enhanced lipid deposition in eWAT but not in inguinal white adipose tissue (iWAT) is preferentially blocked, while lipid accumulation in liver is not alleviated. Mechanistically, ketone body BHB can significantly decrease lysine acetylation of peroxisome proliferator-activated receptor gamma (PPARγ) in eWAT, causing enhanced activity of PPARγ, the key adipogenic transcription factor. These observations suggest that the liver senses metabolic stress first and sends a corresponding signal, that is, ketone body BHB, to specifically promote eWAT expansion to adapt to metabolic challenges.

The ketone body β-hydroxybutyrate shifts microglial metabolism and suppresses amyloid-β oligomer-induced inflammation in human microglia.

Fatty acids are metabolized by β-oxidation within the "mitochondrial ketogenic pathway" (MKP) to generate β-hydroxybutyrate (BHB), a ketone body. BHB can be generated by most cells but largely by hepatocytes following exercise, fasting, or ketogenic diet consumption. BHB has been shown to modulate systemic and brain inflammation; however, its direct effects on microglia have been little studied. We investigated the impact of BHB on Aβ oligomer (AβO)-stimulated human iPS-derived microglia (hiMG), a model relevant to the pathogenesis of Alzheimer's disease (AD). HiMG responded to AβO with proinflammatory activation, which was mitigated by BHB at physiological concentrations of 0.1-2 mM. AβO stimulated glycolytic transcripts, suppressed genes in the β-oxidation pathway, and induced over-expression of AD-relevant p46Shc, an endogenous inhibitor of thiolase, actions that are expected to suppress MKP. AβO also triggered mitochondrial Ca2+ increase, mitochondrial reactive oxygen species production, and activation of the mitochondrial permeability transition pore. BHB potently ameliorated all the above mitochondrial changes and rectified the MKP, resulting in reduced inflammasome activation and recovery of the phagocytotic function impaired by AβO. These results indicate that microglia MKP can be induced to modulate microglia immunometabolism, and that BHB can remedy "keto-deficiency" resulting from MKP suppression and shift microglia away from proinflammatory mitochondrial metabolism. These effects of BHB may contribute to the beneficial effects of ketogenic diet intervention in aged mice and in human subjects with mild AD.

Empagliflozin induces the transcriptional program for nutrient homeostasis in skeletal muscle in normal mice

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) improve heart failure (HF) outcomes across a range of patient characteristics. A hypothesis that SGLT2i induce metabolic change similar to fasting has recently been proposed to explain their profound clinical benefits. However, it remains unclear whether SGLT2i primarily induce this change in physiological settings. Here, we demonstrate that empagliflozin administration under ad libitum feeding did not cause weight loss but did increase transcripts of the key nutrient sensors, AMP-activated protein kinase and nicotinamide phosphoribosyltransferase, and the master regulator of mitochondrial gene expression, PGC-1α, in quadriceps muscle in healthy mice. Expression of these genes correlated with that of PPARα and PPARδ target genes related to mitochondrial metabolism and oxidative stress response, and also correlated with serum ketone body β-hydroxybutyrate. These results were not observed in the heart. Collectively, this study revealed that empagliflozin activates transcriptional programs critical for sensing and adaptation to nutrient availability intrinsic to skeletal muscle rather than the heart even in normocaloric condition. As activation of PGC-1α is sufficient for metabolic switch from fatigable, glycolytic metabolism toward fatigue-resistant, oxidative mechanism in skeletal muscle myofibers, our findings may partly explain the improvement of exercise tolerance in patients with HF receiving empagliflozin.

Short-term fasting of mice elevates circulating fibroblast growth factor 23 (FGF23).

Phosphate and vitamin D homeostasis are controlled by fibroblast growth factor 23 (FGF23) from bone suppressing renal phosphate transport and enhancing 24-hydroxylase (Cyp24a1), thereby inactivating 1,25(OH)2 D3 . Serum FGF23 is correlated with outcomes in several diseases. Fasting stimulates the production of ketone bodies. We hypothesized that fasting can induce FGF23 synthesis through the production of ketone bodies. UMR106 cells and isolated neonatal rat ventricular myocytes (NRVM) were treated with ketone body β-hydroxybutyrate. Mice were fasted overnight, fed ad libitum, or treated with β-hydroxybutyrate. Proteins and further blood parameters were determined by enzyme-linked immunoassay (ELISA), western blotting, immunohistochemistry, fluorometric or colorimetric methods, and gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). β-Hydroxybutyrate stimulated FGF23 production in UMR106 cells in a nuclear factor kappa-light-chain enhancer of activated B-cells (NFκB)-dependent manner, and in NRVMs. Compared to fed animals, fasted mice exhibited higher β-hydroxybutyrate and FGF23 serum levels (based on assays either detecting C-terminal or intact, biologically active FGF23 only), cardiac, pancreatic, and thymic Fgf23 and renal Cyp24a1 expression, and lower 1,25(OH)2 D3 serum concentration as well as renal Slc34a1 and αKlotho (Kl) expression. In contrast, Fgf23 expression in bone and serum phosphate, calcium, plasma parathyroid hormone (PTH) concentration, and renal Cyp27b1 expression were not significantly affected by fasting. Short-term fasting increased FGF23 production, as did administration of β-hydroxybutyrate, effects possibly of clinical relevance in view of the increasing use of FGF23 as a surrogate parameter in clinical monitoring of diseases. The fasting state of patients might therefore affect FGF23 tests.

Biased allosteric activation of ketone body receptor HCAR2 suppresses inflammation

Hydroxycarboxylic acid receptor 2 (HCAR2), modulated by endogenous ketone body β-hydroxybutyrate and exogenous niacin, is a promising therapeutic target for inflammation-related diseases. HCAR2 mediates distinct pathophysiological events by activating Gi/o protein or β-arrestin effectors. Here, we characterize compound 9n as a Gi-biased allosteric modulator (BAM) of HCAR2 and exhibit anti-inflammatory efficacy in RAW264.7 macrophages via a specific HCAR2-Gi pathway. Furthermore, four structures of HCAR2-Gi complex bound to orthosteric agonists (niacin or monomethyl fumarate), compound 9n, and niacin together with compound 9n simultaneously reveal a common orthosteric site and a unique allosteric site. Combined with functional studies, we decipher the action framework of biased allosteric modulation of compound 9n on the orthosteric site. Moreover, co-administration of compound 9n with orthosteric agonists could enhance anti-inflammatory effects in the mouse model of colitis. Together, our study provides insight to understand the molecular pharmacology of the BAM and facilitates exploring the therapeutic potential of the BAM with orthosteric drugs.

Global Energy Metabolism Deficit in Alzheimer Disease Brain

BackgroundThe understanding of Alzheimer’s disease (AD) has been dominated by the amyloid hypothesis. However, therapies targeting beta-amyloid have largely failed, generating interest in other potential pathogenic factors including energy metabolism.ObjectivesTo interrogate canonical energy metabolism pathways from human prefrontal cortical tissue samples obtained from necropsy comparing AD and control.Design, Setting, and ParticipantsPostmortem pre-frontal cortical tissue from 10 subjects histologically diagnosed with AD and 10 control (CTRL) subjects was subjected to untargeted metabolomics to interrogate energy metabolism pathways. The samples were matched by age, sex, and postmortem interval.Metabolite MeasurementsUntargeted metabolomics analyses were via Metabolon®.ResultsGlucose-derived energy metabolites in the glycolytic and pentose phosphate pathway and the ketone body β-hydroxybutyrate were uniformly decreased in AD brain vs. CTRL brain.ConclusionThis pilot study aimed to identify energy metabolism abnormalities using untargeted brain metabolomics in two independent subject cohorts. Our study revealed a pattern of global energy deficit in AD brain, supporting a growing body of evidence of deficient energy metabolism in AD.

Modulation of the autophagy-lysosomal pathway and endoplasmic reticulum stress by ketone bodies in experimental models of stroke.

Ischemic stroke is a leading cause of disability worldwide. There is no simple treatment to alleviate ischemic brain injury, as thrombolytic therapy is applicable within a narrow time window. During the last years, the ketogenic diet (KD) and the exogenous administration of the ketone body β-hydroxybutyrate (BHB) have been proposed as therapeutic tools for acute neurological disorders and both can reduce ischemic brain injury. However, the mechanisms involved are not completely clear. We have previously shown that the D enantiomer of BHB stimulates the autophagic flux in cultured neurons exposed to glucose deprivation (GD) and in the brain of hypoglycemic rats. Here, we have investigated the effect of the systemic administration of D-BHB, followed by its continuous infusion after middle cerebral artery occlusion (MCAO), on the autophagy-lysosomal pathway and the activation of the unfolded protein response (UPR). Results show for the first time that the protective effect of BHB against MCAO injury is enantiomer selective as only D-BHB, the physiologic enantiomer of BHB, significantly reduced brain injury. D-BHB treatment prevented the cleavage of the lysosomal membrane protein LAMP2 and stimulated the autophagic flux in the ischemic core and the penumbra. In addition, D-BHB notably reduced the activation of the PERK/eIF2α/ATF4 pathway of the UPR and inhibited IRE1α phosphorylation. L-BHB showed no significant effect relative to ischemic animals. In cortical cultures under GD, D-BHB prevented LAMP2 cleavage and decreased lysosomal number. It also abated the activation of the PERK/eIF2α/ATF4 pathway, partially sustained protein synthesis, and reduced pIRE1α. In contrast, L-BHB showed no significant effects. Results suggest that protection elicited by D-BHB treatment post-ischemia prevents lysosomal rupture allowing functional autophagy, preventing the loss of proteostasis and UPR activation.

βHB inhibits glucose-induced GLP-1 secretion in GLUTag and human jejunal enteroids.

Ingestion of nutrients stimulates incretin secretion from enteroendocrine cells (EECs) of the epithelial layer of the gut. Glucagon-like peptide-1 (GLP-1) is one of these incretins that stimulate postprandial insulin release and signal satiety to the brain. Understanding the regulation of incretin secretion might open up new therapeutic options for obesity and type-2 diabetes mellitus. To investigate the inhibitory effect of the ketone body β-hydroxybutyrate (βHB) on glucose-induced GLP-1 secretion from EECs, in vitro cultures of murine GLUTag cells and differentiated human jejunal enteroid monolayers were stimulated with glucose to induce GLP-1 secretion. The effect of βHB on GLP-1 secretion was studied using ELISA and ECLIA methods. GLUTag cells stimulated with glucose and βHB were analysed using global proteomics focusing on cellular signalling pathways and the results were verified by Western blot. Results demonstrated βHB had a significant inhibitory effect on glucose-induced GLP-1 secretion at a dose of 100 mM in GLUTag cells. In differentiated human jejunal enteroid monolayers, glucose-induced secretion of GLP-1 was inhibited at a much lower dose of 10 mM βHB. The addition of βHB to GLUTag cells resulted in decreased phosphorylation of kinase AKT and transcription factor STAT3 and also influenced the expressions of signalling molecule IRS-2, kinase DGKε and receptor FFAR3. In conclusion, βHB displays an inhibitory effect on glucose-induced GLP-1 secretion in vitro in GLUTag cells and in differentiated human jejunal enteroid monolayers. This effect may be mediated through multiple downstream mediators of G-protein coupled receptor activation, such as PI3K signalling.

The Ketogenic Diet in Colorectal Cancer: A Means to an End.

Some diets, such as high lipid and high glucose diets, are known to increase the risk of colorectal cancer. On the other hand, little is known about diets that prevent colonic carcinogenesis. The ketogenic diet, which is characterized by high fat and very low carbohydrate content, is one such diet. The ketogenic diet decreases the amount of available glucose for tumors and shifts to the production of ketone bodies as an alternative energy source for healthy cells. Cancer cells are unable to use the ketone bodies for energy thus depriving them of the energy needed for progression and survival. Many studies reported the beneficial effects of the ketogenic diet in several types of cancers. Recently, the ketone body β-hydroxybutyrate has been found to possess anti-tumor potential in colorectal cancer. Despite its beneficial effects, the ketogenic diet also has some drawbacks, some of which are related to gastrointestinal disorders and weight loss. Thus, studies are being directed at this time towards finding alternatives to following a strict ketogenic diet and supplementing patients with the ketone bodies responsible for its beneficial effects in the hope of overcoming some potential setbacks. This article discusses the mechanism by which a ketogenic diet influences growth and proliferation of tumor cells, it sheds the light on the most recent trials regarding its use as an adjunctive measure to chemotherapy in patients with metastatic colorectal cancer, and it explains the limitations of its usage in metastatic patients and the promising role of exogenous ketone supplementation in this setting.

Effects of a ketogenic diet on reproductive and metabolic phenotypes in mice with polycystic ovary syndrome†.

Polycystic ovary syndrome (PCOS) is one of the most common female reproductive and metabolic disorders. The ketogenic diet (KD) is a diet high in fat and low in carbohydrate. The beneficial effects of KD intervention have been demonstrated in obese women with PCOS. The underlying mechanisms, however, remain unknown. The aim of the present study was to investigate the effects of a KD on both reproductive and metabolic phenotypes of dehydroepiandrosterone (DHEA)-induced PCOS mice. Female C57BL/6 mice were divided into three groups, designated Control, DHEA, and DHEA+KD groups. Mice of both Control and DHEA groups were fed the control diet, whereas DHEA+KD mice were fed a KD with 89%(kcal) fat for 1or 3weeks after PCOS mouse model was completed. At the end of the experiment, both reproductive and metabolic characteristics were assessed. Our data show that KD treatment significantly increased blood ketone levels, reduced body weight and random and fasting blood glucose levels in DHEA+KD mice compared with DHEA mice. Glucose tolerance, however, was impaired in DHEA+KD mice. Ovarian functions were improved in some DHEAmice after KD feeding, especially in mice treated with KD for 3weeks. In addition, inflammation and cell apoptosis were inhibited in the ovaries of DHEA+KD mice. Results from in vitro experiments showed that the main ketone body β-hydroxybutyrate reduced inflammation and cell apoptosis in DHEA-treated KGN cells. These findings support the therapeutic effects of KD and reveal a possible mechanism by which KD improves ovarian functions in PCOS mice.