Search

Background Methotrexate (MTX) is widely used in chemotherapy but its associated hepatotoxicity is a major complication, limiting its use. This study evaluates possible therapeutic effect of oral alpha-ketoglutarate (AKG) supplementation against MTX-induced hepatotoxicity. Materials and methods HepG2 cells were used to evaluate in-vitro cyto-protection conferred by AKG against MTX induced cytotoxicity. For in-vivo animal study, rats were divided into three groups. Group-I served as control. Group-II animals were administered single intraperitoneal injection of MTX (20 mg/kg/body weight), while Group-III received MTX as in group-II followed by oral AKG (2 gm/kg body weight) for 5 days. 99mTc-Mebrofenin hepatobiliary study was performed under a gamma camera to determine real time functional status of rats’ livers. Multiple parameters concerning hepatic mebrofenin uptake and excretion, including Tpeak and T1/2 peak in control and treated animals were determined. Biochemical analysis of the liver homogenate in terms of hepatic enzyme activities in serum, antioxidant status, tissue factor activity, tissue collagen content and histological analysis of the liver tissue were also done. Results AKG supplementation significantly reversed MTX induced derangement in activities of serum liver enzymes [ALT and ALP (p = 0.003); AST (p = 0.005)], antioxidant status [LPO and GSH (p = 0.005); CAT (p = 0.004)], tissue factor activity (p = 0.005) and tissue collagen content (p = 0.005). Functional imaging confirmed that hepatic retention and fractional biliary excretion were significantly abnormal in MTX treated group (Tpeak: 234 s ± 40 s; T1/2 peak: 846sec ± 32sec) as compared to AKG supplemented group (Tpeak: 144 s ± 35sec; T1/2peak: 468sec ± 27sec). Hepatic extraction fraction (HEF) was 92.2 ± 1.8%, 48.7 ± 2.6% and 69.8 ± 4.3% in control, MTX and AKG supplemented rats respectively. Conclusion 99mTc-mebrofenin imaging strongly suggests therapeutic action of AKG in protecting liver damage by MTX in rats. Functional imaging parameters correlated well with biochemical and histopathological findings.

Alpha-ketoglutarate (α-KG) is a key metabolite and signaling molecule in cancer cells, but the low permeability of α-KG limits the study of α-KG mediated effects in vivo. Recently, cell-permeable monoester and diester α-KG derivatives have been synthesized for use in vivo, but many of these derivatives are not compatible for use in hyperpolarized carbon-13 nuclear magnetic resonance spectroscopy (HP-13C-MRS). HP-13C-MRS is a powerful technique that has been used to noninvasively trace labeled metabolites in real time. Here, we show that using diethyl-[1-13C]-α-KG as a probe in HP-13C-MRS allows for noninvasive tracing of α-KG metabolism in vivo.

BackgroundPhosphoinositide-3 kinase (PI3K)/AKT signaling participates in cellular proliferation, survival and tumorigenesis. The activation of AKT signaling promotes the cellular reprogramming including generation of induced pluripotent stem cells (iPSCs) and dedifferentiation of primordial germ cells (PGCs). Previous studies suggested that AKT promotes reprogramming by activating proliferation and glycolysis. Here we report a line of evidence that supports the notion that AKT signaling is involved in TET-mediated DNA demethylation during iPSC induction.MethodsAKT signaling was activated in mouse embryonic fibroblasts (MEFs) that were transduced with OCT4, SOX2 and KLF4. Multiomics analyses were conducted in this system to examine the effects of AKT activation on cells undergoing reprogramming.ResultsWe revealed that cells undergoing reprogramming with artificially activated AKT exhibit enhanced anabolic glucose metabolism and accordingly increased level of cytosolic α-ketoglutarate (αKG), which is an essential cofactor for the enzymatic activity of the 5-methylcytosine (5mC) dioxygenase TET. Additionally, the level of TET is upregulated. Consistent with the upregulation of αKG production and TET, we observed a genome-wide increase in 5-hydroxymethylcytosine (5hmC), which is an intermediate in DNA demethylation. Moreover, the DNA methylation level of ES-cell super-enhancers of pluripotency-related genes is significantly decreased, leading to the upregulation of associated genes. Finally, the transduction of TET and the administration of cell-permeable αKG to somatic cells synergistically enhance cell reprogramming by Yamanaka factors.ConclusionThese results suggest the possibility that the activation of AKT during somatic cell reprogramming promotes epigenetic reprogramming through the hyperactivation of TET at the transcriptional and catalytic levels.

Whole-cell systems offer many benefits for biochemical production, such as relatively easy enzyme control and higher tolerance toward harsh environments, than purified enzymes. These systems can be applied to many bioconversion reactions, but they sometimes require cofactor regeneration units to support reactions at high substrate concentrations. Here, we examined l-glutamate oxidase (GOX) from Streptomyces sp. X119-6, which produces α-ketoglutarate (α-KG) from l-glutamate, and catalase (KatE) from Escherichia coli, which removes hydrogen peroxide generated by GOX. After optimizing the expression vector, pH, strains, culture conditions, and isopropyl β-d-1-thiogalactopyranoside concentration, we compared their efficiency to that of a previously reported GOX from Streptomyces mobaraensis. Our results indicated that GOX from Streptomyces sp. X119-6 and KatE increased α-KG production by 2.76-fold. This GOX required high levels of α-KG as an amino donor to convert 5-aminovaleric acid to glutaric acid. Performing the reaction at pH 8 enabled us to avoid the exogenous addition of catalase, but severe substrate inhibition was observed, resulting in the production of 287 mM glutaric acid. This α-KG regeneration system has potential for improving production in various aminotransferase systems.

Age-related osteoporosis is characterized by the deterioration in bone volume and strength, partly due to the dysfunction of bone marrow mesenchymal stromal/stem cells (MSCs) during aging. Alpha-ketoglutarate (αKG) is an essential intermediate in the tricarboxylic acid (TCA) cycle. Studies have revealed that αKG extends the lifespan of worms and maintains the pluripotency of embryonic stem cells (ESCs). Here, we show that the administration of αKG increases the bone mass of aged mice, attenuates age-related bone loss, and accelerates bone regeneration of aged rodents. αKG ameliorates the senescence-associated (SA) phenotypes of bone marrow MSCs derived from aged mice, as well as promoting their proliferation, colony formation, migration, and osteogenic potential. Mechanistically, αKG decreases the accumulations of H3K9me3 and H3K27me3, and subsequently upregulates BMP signaling and Nanog expression. Collectively, our findings illuminate the role of αKG in rejuvenating MSCs and ameliorating age-related osteoporosis, with a promising therapeutic potential in age-related diseases., α-ketoglutarate is an intermediate of the Krebs Cycle that was recently reported to extend lifespan in C.Elegans. Here, the authors show that administration of α-ketoglutarate to mice reduces age-related bone loss, by ameliorating senescence of bone-marrow derived mesenchymal stem cells.

Anaplerosis and the associated mitochondrial metabolite transporters generate unique cytosolic metabolic signaling molecules that can regulate insulin release from pancreatic β-cells. It has been shown that mitochondrial metabolites, transported by the citrate carrier (CIC), dicarboxylate carrier (DIC), oxoglutarate carrier (OGC), and mitochondrial pyruvate carrier (MPC) play a vital role in the regulation of glucose-stimulated insulin secretion (GSIS). Metabolomic studies on static and biphasic insulin secretion, suggests that several anaplerotic derived metabolites, including α-ketoglutarate (αKG), are strongly associated with nutrient regulated insulin secretion. Support for a role of αKG in the regulation of insulin secretion comes from studies looking at αKG dependent enzymes, including hypoxia-inducible factor-prolyl hydroxylases (PHDs) in clonal β-cells, and rodent and human islets. This review will focus on the possible link between defective anaplerotic-derived αKG, PHDs, and the development of type 2 diabetes (T2D).

α-Ketoglutarate (αKG) is a metabolite of the tricarboxylic acid cycle, important for biomass synthesis and a precursor for biotechnological products like 1,4-butanediol. In the eukaryote Saccharomyces cerevisiae αKG is present in different compartments. Compartmentation and (intra-)cellular transport could interfere with heterologous product pathways, generate futile cycles and reduce product yields. Batch and chemostat cultivations at low pH (≤ 5) showed that αKG can be transported, catabolized and used for biomass synthesis. The uptake mechanism of αKG was further investigated under αKG limited chemostat conditions at different pH (3, 4, 5, and 6). At very low pH (3, 4) there is a fraction of undissociated αKG that could diffuse over the periplasmic membrane. At pH 5 this fraction is very low, and the observed growth and residual concentration requires a permease/facilitated uptake mechanism of the mono-dissociated form of αKG. Consumption of αKG under mixed substrate conditions was only observed for low glucose concentrations in chemostat cultivations, suggesting that the putative αKG transporter is repressed by glucose. Fully 13C-labeled αKG was introduced as a tracer during a glucose/αKG co-feeding chemostat to trace αKG transport and utilization. The measured 13C enrichments suggest the major part of the consumed 13C αKG was used for the synthesis of glutamate, and the remainder was transported into the mitochondria and fully oxidized. There was no enrichment observed in glycolytic intermediates, suggesting that there was no gluconeogenic activity under the co-feeding conditions. 13C based flux analysis suggests that the intracellular transport is bi-directional, i.e. there is a fast exchange between the cytosol and mitochondria. The model further estimates that most intracellular αKG (88%) was present in the cytosol. Using literature reported volume fractions, the mitochondria/cytosol concentration ratio was 1.33. Such ratio will not require energy investment for transport towards the mitochondria (based on thermodynamic driving forces calculated with literature pH values). Growth on αKG as sole carbon source was observed, suggesting that S. cerevisiae is not fully Krebs-negative. Using 13C tracing and modelling the intracellular use of αKG under co-feeding conditions showed a link with biomass synthesis, transport into the mitochondria and catabolism. For the engineering of strains that use cytosolic αKG as precursor, both observed sinks should be minimized to increase the putative yields.

Background Non-alcoholic liver disease is of increased concern and contributing to economic burdens not only in developing countries but in developed countries as well. Identifying the biomarker of early diagnosis and early intervention approaches for non-alcoholic liver disease is unmet and required further investigation. Although the alpha-ketoglutarate (α-KG) is recently proposed to be a potential biomarker in differentiating patients with obesity from those with non-alcoholic liver disease, how α-ketoglutatate is involved in the fatty liver progression is not clear. Methods A high-fat diet (HFD) feeding animal model, liver functional assays, and molecular approaches were adopted to clarify the impact of α-KG in fatty liver progression. Results In the current study, it was found that dietary α-KG would inhibit weight gain in male and female mice fed with a normal chew or HFD. HFD feeding caused fatty liver in male mice, but α-KG treatment could substantially inhibit hepatic steatosis progression. Biochemical studies revealed the possible linkage of α-KG protective functions to lipid metabolism. Further analysis identified the important role of peroxisome proliferator-activated receptors in beneficial α-KG-mediated effects on fatty liver progression. Conclusions The current study demonstrates the therapeutic potential of α-KG and how it may be used, via dietary supplementation, as a preventive intervention for non-alcoholic liver disease in obese patients.

A better understanding of the metabolic constraints of a tumor may lead to more effective anticancer treatments. Evidence has emerged in recent years shedding light on a crucial aspartate dependency of many tumor types. As a precursor for nucleotide synthesis, aspartate is indispensable for cell proliferation. Moreover, the malate–aspartate shuttle plays a key role in redox balance, and a deficit in aspartate can lead to oxidative stress. It is now recognized that aspartate biosynthesis is largely governed by mitochondrial metabolism, including respiration and glutaminolysis in cancer cells. Therefore, under conditions that suppress mitochondrial metabolism, including mutations, hypoxia, or chemical inhibitors, aspartate can become a limiting factor for tumor growth and cancer cell survival. Notably, aspartate availability has been associated with sensitivity or resistance to various therapeutics that are presently in the clinic or in clinical trials, arguing for a critical need for more effective aspartate-targeting approaches. In this review, we present current knowledge of the metabolic roles of aspartate in cancer cells and describe how cancer cells maintain aspartate levels under different metabolic states. We also highlight several promising aspartate level-modulating agents that are currently under investigation.

Coronaviruses belong to a well-known family of enveloped RNA viruses and are the causative agent of the common cold. Although the seasonal coronaviruses do not pose a threat to human life, three members of this family, i.e., SARS-CoV, MERS-CoV and recently, SARS-CoV2, may cause severe acute respiratory syndrome and lead to death. Unfortunately, COVID-19 has already caused more than 4.4 million deaths worldwide. Although much is better understood about the immunopathogenesis of the lung disease, important information about systemic disease is still missing, mainly concerning neurological parameters. In this context, we sought to evaluate immunometabolic changes using in vitro and in vivo models of hamsters infected with SARS-CoV-2. Here we show that, besides infecting hamster’s astrocytes, SARS-CoV-2 induces changes in protein expression and metabolic pathways involved in carbon metabolism, glycolysis, mitochondrial respiration, and synaptic transmission. Interestingly, many of the differentially expressed proteins are concurrent with proteins that correlate with neurological diseases, such as Parkinsons’s disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntington’s disease. Metabolic analysis by high resolution real-time respirometry evidenced hyperactivation of glycolysis and mitochondrial respiration. Further metabolomics analysis confirmed the consumption of many metabolites, including glucose, pyruvate, glutamine, and alpha ketoglutarate. Interestingly, we observed that glutamine was significantly reduced in infected cultures, and the blockade of mitochondrial glutaminolysis significantly reduced viral replication and pro-inflammatory response. SARS-CoV-2 was confirmed in vivo as hippocampus, cortex, and olfactory bulb of intranasally infected hamsters were positive for viral genome several days post-infection. Altogether, our data reveals important changes in overall protein expression, mostly of those related to carbon metabolism and energy generation, causing an imbalance in important metabolic molecules and neurotransmitters. This may suggest that some of the neurological features observed during COVID-19, as memory and cognitive impairment, may rely on altered energetic profile of brain cells, as well as an unbalanced glutamine/glutamate levels, whose importance for adequate brain function is unquestionable.

Introduction The tricarboxylic acid (TCA) cycle is a key metabolic pathway for driving the generation of mitochondrial energy in all oxidative organisms. Alpha-ketoglutarate (Alpha-KG), a precursor of glutamine, is known as a crucial intermediate of the TCA cycle and plays a pivotal role in multiple metabolic processes. As a precursor of glutamate and glutamine, AKG acts as an antioxidant agent as it directly reacts with hydrogen peroxide with formation of succinate, water, and carbon dioxide; meanwhile, it discharges plenty of ATP by oxidative decarboxylation. Several studies reported that Alpha-KG is a key participant in the detoxification of reactive oxygen species and acts as an integral part of the oxidative defense machinery. However, few studies have been reported on the efficacy of Alpha-KG in the maintenance of skin functions. This study demonstrated that Alpha-KG has beneficial effects on skin hydration and barrier function and that fermentation is an effective way to enhance the synthesis of Alpha-KG in yeast, which possesses mitochondria. Methods Evaluation of promoting effects on epidermal keratinocyte proliferation: Keratinocytes were incubated with a test sample, and the degree of proliferation was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Evaluation of promoting effects on mRNA expression of genes related to skin hydration and barrier function: Keratinocytes were incubated with a test sample, and gene expression levels of filaggrin (FLG), serine palmitoyltransferase (SPT), and involucrin (IVL) were analyzed by real-time RT-PCR. Analysis of Alpha-KG in rice fermented liquid: Alpha-KG in rice fermented liquid was quantitatively analyzed by capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS). Clinical study testing methods and VISIA testing: After 28 days of treatment use the cream with Alpha-KG and control sample without Alpha-KG, instrumentation measurements were adopted to assess skin wrinkles, texture, elasticity, and firmness, tested by the VISIA-CR. Results Immediately after using the sample for D0, the skin wrinkles were significantly reduced by 23.64%. After using the sample for D7, the average clinical score of outer corner wrinkles was significantly reduced by 15.23%, and nasolabial groove wrinkles were significantly reduced by 25.68%. After using the sample for D56, the mean clinical evaluation score of crow's feet decreased significantly by 25.42%; the average score of clinical evaluation of skin firmness increased significantly by 41.40%; the skin gloss increased significantly by 28.67%. Conclusion It was demonstrated that Alpha-KG is expected to promote skin hydration and barrier function by the activation of cell proliferation and the up-regulation of mRNA expression of genes related to the maintenance of epidermal function in in vitro tests. In order to develop a cosmetic ingredient including Alpha-KG, we focused on fermentation with yeast as an efficient production method of Alpha-KG and found that fermentation of rice with yeast efficiently produced Alpha-KG. Taken together, it is considered that rice fermented liquid containing Alpha-KG could be a promising ingredient for skin care products. Through the clinical testing, rice fermented liquid containing Alpha-KG could be a promising cosmetic ingredient for skin care products.

Increasing evidence indicates the involvement of myocardial oxidative injury and mitochondrial dysfunction in the pathophysiology of heart failure (HF). Alpha-ketoglutarate (AKG) is an intermediate metabolite of the tricarboxylic acid (TCA) cycle that participates in different cellular metabolic and regulatory pathways. The circulating concentration of AKG was found to decrease with ageing and is elevated after acute exercise and resistance exercise and in HF. Recent studies in experimental models have shown that dietary AKG reduces reactive oxygen species (ROS) production and systemic inflammatory cytokine levels, regulates metabolism, extends lifespan and delays the occurrence of age-related decline. However, the effects of AKG on HF remain unclear. In the present study, we explored the effects of AKG on left ventricular (LV) systolic function, the myocardial ROS content and mitophagy in mice with transverse aortic constriction (TAC). AKG supplementation inhibited pressure overload-induced myocardial hypertrophy and fibrosis and improved cardiac systolic dysfunction; in vitro, AKG decreased the Ang II-induced upregulation of β-MHC and ANP, reduced ROS production and cardiomyocyte apoptosis, and repaired Ang II-mediated injury to the mitochondrial membrane potential (MMP). These benefits of AKG in the TAC mice may have been obtained by enhanced mitophagy, which cleared damaged mitochondria. In summary, our study suggests that AKG improves myocardial hypertrophy remodelling, fibrosis and LV systolic dysfunction in the pressure-overloaded heart by promoting mitophagy to clear damaged mitochondria and reduce ROS production; thus, AKG may have therapeutic potential for HF., Graphical abstract Image 1, Highlights • AKG attenuated pressure overload-induced myocardial fibrosis and hypertrophy remodelling. • AKG improved cardiac function and left ventricular strain in TAC mice. • AKG increased myocardial mitophagy to clear damaged mitochondria and rescued Ang II-induced MMP impairment in TAC mice. • AKG reduced intracellular ROS and myocardial apoptosis in the pressure overloaded heart.

BACKGROUND Gliomas are the most common brain cancer and survival is poor, with 11–15 months for high-grade glioblastoma patients, despite treatment. Gliomas are hypoxic tumours, which increases with tumour grade. Under hypoxia, the transcription factor hypoxia inducible factor-1 (HIF) accumulates and upregulates expression of genes involved in tumour development and progression. HIF-1 levels and activity are controlled by HIF hydroxylases which target HIF-1α for degradation and prevent co-activation. HIF hydroxylases are part of the 2-oxoglutarate (2-OG)-dependent dioxygenase enzyme family, that require 2-OG and oxygen as substrates and ascorbate and iron as co-factors. The role of ascorbate in regulating the hypoxic pathway in cancer is of interest, with previous research showing reduced HIF pathway activity with increasing tumour ascorbate levels. Brain tissue has one of the highest ascorbate levels in the body, and is one of the last to become depleted under deficiency, indicating an important role for ascorbate in this tissue. One previous study has analysed ascorbate levels in 11 human glioblastoma patients, and showed lower ascorbate in tumour tissue compared to normal brain tissue. There have been no studies investigating the relationship between ascorbate levels and the hypoxic pathway in human glioma tissues. MATERIAL AND METHODS Human glioma tissues (n = 39), obtained from the Cancer Society Tissue Bank Christchurch (ethics approval H19/163), were processed for ascorbate and hypoxic pathway proteins (HIF-1α, CA-IX, BNIP3, HKII, GLUT1 and VEGF). Ascorbate levels were quantified by HPLC-ED, and proteins were measured by Western blotting and ELISA. Spearman’s correlations were used to identify relationships between ascorbate and HIF pathway proteins. RESULTS Of the samples, 64% were GBM. Ascorbate was significantly lower in GBM compared to low-grade gliomas (p = 0.04). VEGF was significantly higher in GBM compared to astrocytomas (p = 0.01). Increased tumour ascorbate was associated with lower VEGF and CA-IX proteins. HIF-1α and BNIP3 protein were positively associated, and VEGF was positively associated with HKII and CA-IX. VEGF inversely associated with BNIP3, and CA-IX inversely associated with HKII. The hypoxic pathway score (calculated from protein levels of members of the hypoxic pathway) was reduced in tumours with higher ascorbate but this did not reach significance (p = 0.2). CONCLUSION This is the first study to show that ascorbate levels were reduced in high-grade gliomas compared to low-grade. Some members of the hypoxic pathway were associated with ascorbate levels. The overall hypoxic pathway score did not significantly correlate with ascorbate and increased numbers of samples are required to confirm any associations. Other variables, such as IDH-1 mutation status of the tumours may affect the correlation and will be analysed next.

Metabolites control epigenetic mechanisms, and conversly, cell metabolism is regulated at the epigenetic level in response to changes in the cellular environment. In recent years, this metabolo-epigenetic control of gene expression has been implicated in the regulation of multiple stages of embryonic development. The developmental potency of stem cells and their embryonic counterparts is directly determined by metabolic rewiring. Here, we review the current knowledge on the interplay between epigenetics and metabolism in the specific context of early germ cell development. We explore the implications of metabolic rewiring in primordial germ cells in light of their epigenetic remodeling during cell fate determination. Finally, we discuss the relevance of concerted metabolic and epigenetic regulation of primordial germ cells in the context of mammalian transgenerational epigenetic inheritance. Summary sentence The interplay between metabolism and epigenetics participates in the development of primordial germ cells and may play a role in transgenerational epigenetic inheritance.

The generation of peroxynitrite (ONOO−) is associated with several diseases, including atherosclerosis, hypertension, neurodegeneration, cancer, inflammation, and sepsis. Alpha-ketoglutarate (αKG) is a known potential highly antioxidative agent for radical oxidative species such as peroxides. The question arises as to whether αKG is also a potential scavenger of ONOO− and a potential protector against ONOO−-mediated nitration of proteins. NMR studies of 1 mM αKG in 100 mM phosphate-buffered saline at pH 7.4 and pH 6.0 were carried out in the presence or absence of a final concentration of 2 mM ONOO−. An ONOO−–luminol-induced chemiluminescence reaction was used to measure the scavenging function of several concentrations of αKG, quantification of αKG was performed via spectrophotometric enzymatic assay of αKG in the absence or presence of 0, 1, or 2 mM ONOO−. The nitration of tyrosine residues on proteins was measured on ONOO−-treated bovine serum albumin (BSA) in the presence or absence of 0–24 mM αKG by an ELISA technique using a specific anti-IgG against nitro-tyrosine. The addition of ONOO− to αKG led to the formation of succinic acid and nitrite at pH 7.0, but not at pH 6.0, as αKG was stable against ONOO−. The absorbance of enzymatically estimated αKG at the time point of 30 min was significantly lower in favour of ONOO− (1 mM: 0.21 ± 0.03, 2 mM: 0.12 ± 0.05 vs. 0 mM: 0.32 ± 0.02, p &lt, 0.001). The luminol technique showed an inverse logarithmic correlation of the ONOO− and αKG concentrations (y = −2 × 105 ln(x) + 1 × 106, r2 = 0.99). The usage of 4 mM αKG showed a significant reduction by nearly half in the chemiluminescence signal (284,456 ± 29,293 cps, p &lt, 0.001) compared to the control (474,401 ± 18,259), for 20 and 200 mM αKG, there were further reductions to 163,546 ± 26,196 cps (p &lt, 0.001) and 12,658 ± 1928 cps (p &lt, 0.001). Nitrated tyrosine residues were estimated using the ELISA technique. A negative linear correlation was obtained by estimating nitrated tyrosine residues in the presence of αKG (r2 = 0.94): a reduction by half of nitrated tyrosine was estimated using 12 mM αKG compared to the control (326.1 ± 39.6 nmol vs. 844.5 ± 128.4 nmol, 0.001).

Isocitrate dehydrogenase 1 (IDH1) mutations that generate the oncometabolite 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG) have been identified in many types of tumors and are an important prognostic factor in gliomas. 2-hydroxyglutarate production can be determined by hyperpolarized carbon-13 magnetic resonance spectroscopy (HP-(13)C-MRS) using [1-(13)C]-α- KG as a probe, but peak contamination from naturally-occurring [5-(13)C]-α-KG overlaps with the [1-(13)C]-2-HG peak. Via a newly developed oxidative-Stetter reaction, [1-(13)C-5-(12)C]-α-KG was synthesized. α-KG metabolism was measured via HP-(13)C-MRS using [1-(13)C-5-(12)C]-α-KG as a probe. [1-(13)C-5-(12)C]-α-KG was synthesized in high yields, and successfully eliminated the signal from C5 of α-KG in the HP-(13)C-MRS spectra. In HCT116 IDH1 R132H cells, [1-(13)C-5-(12)C]-α-KG allowed for unimpeded detection of [1-(13)C]-2-HG. (12)C-enrichment represents a novel method to circumvent spectral overlap, and [1-(13)C-5-(12)C]-α-KG shows promise as a probe to study IDH1 mutant tumors and α-KG metabolism.

As an intermediate substance of the tricarboxylic acid cycle and a precursor substance of glutamic acid synthesis, the effect of alpha-ketoglutarate on growth and protein synthesis has been extensively studied. However, its prevention and treatment of pathogenic bacteria and its mechanism have not yet been noticed. To evaluate the effects of alpha-ketoglutarate on intestinal antioxidant capacity and immune response of Songpu mirror carp, a total of 360 fish with an average initial weight of 6.54 ± 0.08 g were fed diets containing alpha-ketoglutarate with 1% for 8 weeks. At the end of the feeding trial, the fish were challenged with Aeromonas hydrophila for 2 weeks. The results indicated that alpha-ketoglutarate supplementation significantly increased the survival rate of carp after infection with Aeromonas hydrophila (P < 0.05), and the contents of immune digestion enzymes including lysozyme, alkaline phosphatase and the concentration of complement C4 were markedly enhanced after alpha-ketoglutarate supplementation (P < 0.05). Also, appropriate alpha-ketoglutarate increased the activities of total antioxidant capacity and catalase and prevented the up-regulation in the mRNA expression levels of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1β, interleukin-6, and interleukin-8 (P < 0.05). Furthermore, the mRNA expression levels of toll-like receptor 4 (TLR4), and nuclear factor kappa-B (NF-κB) were strikingly increased after infection with Aeromonas hydrophila (P < 0.05), while the TLR4 was strikingly decreased with alpha-ketoglutarate supplementation (P < 0.05). Moreover, the mRNA expression levels of tight junctions including claudin-1, claudin-3, claudin-7, claudin-11 and myosin light chain kinases (MLCK) were upregulated after alpha-ketoglutarate supplementation (P < 0.05). In summary, the appropriate alpha-ketoglutarate supplementation could increase survival rate, strengthen the intestinal enzyme immunosuppressive activities, antioxidant capacities and alleviate the intestinal inflammation, thereby promoting the intestinal immune responses and barrier functions of Songpu mirror carp via activating TLR4/MyD88/NF-κB and MLCK signaling pathways after infection with Aeromonas hydrophila.

Phosphorylation of Akt (pAkt) regulates multiple physiological and pathological processes including thrombosis and inflammation. In an approach to inhibit the pathological signalling of pAkt by prolyl-hydroxylase-2 (PHD2) we employed α-ketoglutarate (αKG), a cofactor of PHD2. Octyl-αKG supplementation to platelets promoted PHD2 activity through elevated intracellular αKG:succinate ratio and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline alongside enhanced binding of PHD2 to pAkt in αKG-treated platelets. Contrastingly, inhibitors of PHD2 significantly increased pAkt1 in platelets. Octyl-αKG followed similar mechanism in monocytes to inhibit cytokine secretion in vitro. Our data also describe a suppressed pAkt1 and reduced activation of platelet and leukocyte obtained from mice supplemented with dietary-αKG, unaccompanied by alteration in their counts. Dietary-αKG significantly reduced clot formation and leukocyte accumulation in various organs including lung of mice treated with thrombosis-inducing agent carrageenan. Importantly, we observed a significant rescue effect of dietary-αKG on inflamed lung of SARS-CoV-2 infected hamsters. αKG significantly reduced leukocyte accumulation, clot formation and viral load alongside downmodulation of pAkt in lung of the infected animals. Therefore, our study suggests a safe implementation of dietary-αKG in prevention of Akt-driven anomalies including thrombosis and inflammation, highlighting a better pulmonary management in COVID-19.

OBJECTIVES: Milk and dairy products are a major component of dietary intake, with milk consumption estimated at 6 billion people globally. Adults are reported to consume two or more portions of dairy daily. Cow's milk is composed of a variety of micro and macro nutrients, which is impacted by a range of factors, including lactation stage, and feeding system (indoor vs. pasture). Application of Metabolomics provides in depth analysis of the metabolite composition of milk. The objective was to examine the impact of lactation on bovine milk metabolite levels. METHODS: Metabolomic analysis of bovine milk powder samples across lactation (N = 18), was performed using (1)H-NMR. Metabolites were quantified using Chenomx software (7.7). One-way repeated measures of analysis variance (ANOVA) with Tukeys post hoc test, was preformed on NMR data using SPSS (version 24.0), to assess the impact of lactation stages. P-values were corrected for multiple comparisons using Benjamini and Hochberg false discovery rate (FDR) procedure. Multivariate statistical analysis of the (1)H-NMR data was carried out using SIMCA 13 (SIMCA Version 13.0.3.0 Umetrics, AB). RESULTS: 42 metabolites were identified and quantified in the (1)H-NMR spectra. Statistical analysis revealed that 17 metabolites were significantly different across lactation stages (FDR

In this issue of Cell Metabolism, Asadi Shahmirzadi et al. (2020) demonstrate that late-onset dietary supplementation with calcium alpha-ketoglutarate results in increased survival, compressed morbidity, and reduced frailty in mice. The study provides further evidence for critical links between metabolism, inflammation, and aging.

Excessive protein levels in diets result in incomplete digestion of nitrogenous nutrients that are excreted from the body, causing environment pollution. Alpha-ketoglutarate (AKG) has been reported to decrease dietary protein levels, promote intestinal health in piglets and reduce environmental pollution. However, the underlying mechanisms of AKG are largely unknown. The objective of this study was to determine the effects of low-protein diet supplementation of AKG on the growth performance, nitrogen metabolism, relative expression of amino acid transporter genes and mTOR signalling pathway of skeletal muscle in piglets. Forty-eight piglets with an initial weight of 11.53 ± 0.04 kg were randomly divided into four groups. Each group had four replicates, and each replicate had three pigs. A low-protein (LP) diet (crude protein was 14.96%) served as the control without AKG, while 0.5%, 1.0% and 1.5% AKG were added to the LP diet for the other experimental groups. The trial period lasted for 28 days. Compared with the LP group, the LP + 1.0%A and LP + 1.5%A groups increased the growth performance (p < .05);increased the mRNA levels of amino acid transporters in the duodenum, anterior jejunum and posterior jejunum (p < .05); and reduced faecal nitrogen and urine nitrogen emissions (p < .05). They also showed greater mRNA levels and phosphorylated protein levels for S6 kinase beta (S6K) (p < .05), mammalian target of rapamycin (mTOR) (p < .05) and 4E-binding protein 1 (4EBP1) (p < .05) in skeletal muscle. An LP diet supplemented with AKG activated the mTOR signalling and promoted the ability of the small intestine to absorb protein, thereby increasing protein deposition. Taken together, an LP diet supplemented with AKG provides a theoretical basis for the promotion and application of AKG in piglet production.

The anthracycline anticancer drug doxorubicin is an effective and frequently used chemotherapeutic agent for various malignancies but it causes acute ventricular dysfunction, and also induces cardiomyopathy and heart failure. One of the mechanisms of cardiotoxicity of doxorubicin is oxidative stress, which stimulates myocardial remodeling. Matrix metalloproteinases MMP2 and MMP9 play a key role in this process. Despite extensive research, the expression and activity of these enzymes in the doxorubicin-damaged heart and the effect of antioxidants on these indicators have not been sufficiently studied. The aim of this work was to study the possible cardioprotective effect of the antioxidant drugs corvitin and alpha-ketoglutarate in rats with doxorubicin-induced cardiomyopathy. Cardiomyopathy in rats was induced by intraperitoneal administration of doxorubicin at the dose of 2.5 mg/kg body weight weekly for 28 days. Animals were divided into four groups: group 1 (control) received saline injections (2.5 mL/kg); group 2 – injections of doxorubicin, 3 – corvitin (5 mg/kg) 60 minutes before doxorubicin administration, 4 – doxorubicin and 1% solution of alpha-ketoglutarate in drinking water ad libitum. Heart weight and shape indexes, the ratio of muscle to connective tissues, and heart histology were examined 7 days after the end of drug administration. Activity of MMP2 and MMP9, their intracellular distribution in myocardial tissues were evaluated by gelatin-zymography and immunohistochemistry. It was found that doxorubicin cardiomyopathy in rats was accompanied by a decrease in heart weight index, adaptive change of heart shape from ellipsoid to globular, increase of connective tissue content. Administration of doxorubicin results in profound lesion of the cardiomyocytes of the atria and ventricle, manifested by excessive cytoplasmic expression of MMP2 and MMP9 and an increase their activity in the heart. Antioxidants corvitin and alpha-ketoglutarate have insufficient regenerative effect on mass and shape indexes of heart however, exhibit potent cardioprotective effect by regulation of expression and activity of MMP2 and MMP9.

The aim of the study was to investigate if dietary alpha-ketoglutarate (AKG) supplementation may improve the performance of lactating sows and their suckling piglets. After farrowing, 24 lactating sows (Large White × Landrace) with similar body weight (BW) were assigned to the control and AKG groups based on parity, and their lactation diets were supplemented with 0.00 or 0.25% AKG, respectively. It was found that supplementing the diet of lactating sows with 0.25% AKG enhanced growth performance of the suckling piglets from d 7 to d 21 of the lactation period, improved villus height of ileum and tended (p = 0.085) to increase mean volumetric bone mineral density of femur in the weanling piglets. In the lactating sows, dietary supplementation of AKG decreased plasma urea level on d 14 of lactation, decreased plasma calcium (Ca) concentrations from d 7 to d 21 of lactation and increased lactose and Ca levels in ordinary milk. Thus, it was proposed that AKG supplementation stimulates the capacity for lactose synthesis and Ca uptake in the mammary gland, thereby altering the composition of the ordinary milk which might be associated with the enhanced performance of piglets during the suckling period. These findings could lead to a better application of AKG in lactating nutrition, and therefore, promoting pork production.

α-Ketoglutarate (α-KG) is an intermediary metabolite in the tricarboxylic acid (TCA) cycle and functions to inhibit ATPase and maintain the pluripotency of embryonic stem cells (ESCs); however, little is known regarding the effects of α-KG on the development of preimplantation embryos. Herein, we report that α-KG (150 μM) treatment significantly promoted the blastocyst rate, the number of inner cell mass (ICM) cells and foetal growth after embryo transfer. Mechanistic studies revealed two important pathways involved in the α-KG effects on embryo development. First, α-KG modulates mitochondria function by inducing relatively low ATP production without modification of mitochondrial copy number. The relatively low energy metabolism preserves the pluripotency and competence of the ICM. Second, α-KG modifies epigenetics in embryos cultured in vitro by affecting the activity of the DNA demethylation enzyme TET and the DNA methylation gene Dnmt3a to increase the ratio of 5hmC/5mC ratio. Elevation of the 5hmC/5mC ratio not only promotes the pluripotency of the ICM but also leads to a methylation level in an in vitro embryo close to that in an in vivo embryo. All these functions of α-KG collectively contribute to an increase in the number of ICM cells, leading to greater adaptation of cultured embryos to in vitro conditions and promoting foetal growth after embryo transfer. Our findings provide basic knowledge regarding the mechanisms by which α-KG affects embryo development and cell differentiation.

Acute myeloid leukemia (AML) is symbolized by an increase in the number of myeloid cells in the bone marrow and an arrest in their maturation, frequently resulting in hematopoietic insufficiency (granulocytopenia, thrombocytopenia, or anemia) with or without leukocytosis either by a predominance of immature forms or a loss of normal hematopoiesis. IDH2 gene encodes for isocitrate dehydrogenase enzyme which is involved in the TCA cycle domino effect and converts isocitrate to alpha-ketoglutarate. In the U.S, the annual incidence of AML progressively increases with age to a peak of 12.6 per 100,000 adults of 65 years or older. Mutations in isocitrate dehydrogenase 2 (arginine 132) have been demonstrated to be recurrent gene alterations in acute myeloid leukemia (AML) by forming 2-Hydroxy alpha ketoglutarate which, instead of participating in TCA cycle, accumulates to form AML. The current study approaches by molecular docking and virtual screening to elucidate inhibitor with superior affinity against IDH2 and achieve a pharmacological profile. To obtain the best established drug Molegro Virtual Docker algorithm was executed. The compound AG-221 (Pub CID 71299339) having the high affinity score was subjected to similarity search to retrieve the drugs with similar properties. The virtual screened compound SCHEMBL16391748 (PubChem CID-117816179) shows high affinity for the protein. Comparative study and ADMET study for both the above compounds resulted in equivalent chemical properties. Virtual screened compound SCHEMBL16391748 (PubChem CID-117816179) shows the lowest re-rank score. These drugs are identified as high potential IDH2 inhibitors and can halt AML when validated through further In vitro screening.

Alpha-ketoglutarate (AKG) is a key metabolite of the tricarboxylic acid (TCA) cycle, an essential process influencing the mitochondrial oxidative respiration rate. Recent studies have shown that dietary AKG reduces mTOR pathway activation by inhibiting ATP synthase, thereby extending the lifespan of nematodes. Although AKG also extends lifespan in fruit flies, the antiaging mechanisms of AKG in these organisms remain unclear. In the present study, we explored changes in gene expression associated with the extension of Drosophila lifespan mediated by dietary AKG. Supplementation of the flies' diets with 5 μM AKG extended their lifespan but reduced their reproductive performance. Dietary AKG also enhanced vertical climbing ability, but did not protect against oxidative stress or increase tolerance to starvation. AKG-reared flies were resistant to heat stress and demonstrated higher expression of heat shock protein genes (Hsp22 and Hsp70) than control flies. In addition, AKG significantly upregulated mRNA expression of cry, FoxO, HNF4, p300, Sirt1 and AMPKα, and downregulated expression of HDAC4, PI3K, TORC, PGC, and SREBP. The metabolic effects of AKG supplementation included a reduction in the ATP/ADP ratio and increased autophagy. Collectively, these observations indicate that AKG extends Drosophila lifespan by activating AMPK signaling and inhibiting the mTOR pathway.

Cyanide is an important industrial pollutant, major occupational hazard, and a potential chemical warfare agent. Its intentional or accidental exposure to humans is a big clinical problem because of its rapid mode of action. Certain plant origin foods also contain substantial amount of cyanide and cause chronic toxicity. This study explores the protective efficacy of co-treatment of alpha-ketoglutarate (AKG) and an antioxidant N-acetyl cysteine (NAC) against toxicity of subchronically exposed cyanide in rats. We explore the effect of AKG + NAC co-treatment on oxidative stress, inflammation, and histological changes induced due to long-term sublethal cyanide exposure. Cyanide induces oxidative stress by inhibiting metalloenzymes (catalase and superoxide dismutase) causing increase in lipid peroxidation (malondialdehyde) and decrease in reduced glutathione (GSH). It also increases the activity of cyclo-oxygenase enzymes causing oxidative stress-mediated inflammation in the brain. Cyanide exposure also causes degenerative changes in the brain as shown in histology. It also causes pathology in liver and kidney. AKG is known to form cyanohydrins with cyanide reducing the free cyanide levels, and its combination with NAC showed overall improvement in by reducing the oxidative stress and subsequent neuroinflammation. Their combination was also found to improve the histological outcome of vital tissues. AKG, an over-the-counter sport medicine, and the antioxidant NAC per se did not show any detrimental effects in any tested parameter. Hence, oral treatment with AKG and NAC can be beneficial for the treatment of chronic cyanide poisoning.

Alcohol is the most abused psychoactive substance and known hepatotoxicant. Present study elucidates possible therapeutic effect of oral alpha-ketoglutarate (AKG) supplementation against alcohol in...

Breast cancer susceptibility gene 1 (BRCA1) has been implicated in modulating metabolism via transcriptional regulation. However, direct metabolic targets of BRCA1 and the underlying regulatory mechanisms are still unknown. Here, we identified several metabolic genes, including the gene which encodes glutamate‐oxaloacetate transaminase 2 (GOT2), a key enzyme for aspartate biosynthesis, which are repressed by BRCA1. We report that BRCA1 forms a co‐repressor complex with ZBRK1 that coordinately represses GOT 2 expression via a ZBRK1 recognition element in the promoter of GOT2. Impairment of this complex results in upregulation of GOT2, which in turn increases aspartate and alpha ketoglutarate production, leading to rapid cell proliferation of breast cancer cells. Importantly, we found that GOT2 can serve as an independent prognostic factor for overall survival and disease‐free survival of patients with breast cancer, especially triple‐negative breast cancer. Interestingly, we also demonstrated that GOT2 overexpression sensitized breast cancer cells to methotrexate, suggesting a promising precision therapeutic strategy for breast cancer treatment. In summary, our findings reveal that BRCA1 modulates aspartate biosynthesis through transcriptional repression of GOT2, and provides a biological basis for treatment choices in breast cancer.

In recent years, Drosophila melanogaster has emerged as a model for studies on aluminum toxicity. The current study aimed to disclose the mechanisms of aluminum toxicity in D. melanogaster at larval and adult stages and examined the potential protective effects of dietary alpha-ketoglutarate (AKG). Flies were reared on food containing 10 mM AlCl3, 10 mM AKG or both additives. Rearing on an AlCl3-containing diet induced behavioral defects, and decreased fecundity and long-term survival of female flies. The addition of dietary AKG did not ameliorate locomotor and taste behavior defects or the higher sensitivity to oxidative stress, but improved heat stress resistance, egg-laying capability and survival of females treated with AlCl3. Metabolic effects of AlCl3 exposure on flies included an imbalance of metal content, decreased glucose levels, increased free iron and storage triacylglyceride (TAG) levels, mitochondria dysfunction, and the development of oxidative stress. Dietary AKG did not prevent AlCl3 effects on glucose and TAG, but improved metal homeostasis, inhibited the increase in free Fe and restored the functional activity of iron-containing enzymes such as aconitase. In addition, AKG decreased the intensity of oxidative stress seen in AlCl3-reared adult flies, probably due to inhibition of iron mobilization. The results show that AKG is not a full antidote against Al toxicity but is able to relieve multiple metabolic effects of high aluminum. Furthermore, the modulating ability of AKG can clearly be helpful in exploring the molecular mechanisms of Al toxicity.

Ageing is a complex physiological process characterized by a gradual decline in cell, tissue and organ function. Consequently, ageing is a major risk factor for cardiovascular disease, diabetes and neurodegenerative disorders. Altered nervous system function can lead to behavioural or psychological disorders. Recent research has shown that alpha ketoglutarate (AKG) is a key intermediate in the Krebs cycle that extends the lifespan of adult animal organisms. The objective of this study was to assess the effect of AKG on the behaviour of laboratory mice in an open field test. The Na-AKG form was found to enhance perceptual-motor exploration in the mice (expressed as the number of rearings), and at the same time to suppress anxiety-related behaviour.

The aim of the study was to examine whether a rat model of liver cirrhosis induced by carbon tetrachloride (CCl4) is a suitable model of muscle wasting and alterations in amino acid metabolism in cirrhotic humans. Rats were treated by intragastric gavage of CCl4 or vehicle for 45 days. Blood plasma and different muscle types-tibialis anterior (mostly white fibres), soleus (red muscle) and extensor digitorum longus (white muscle) - were analysed at the end of the study. Characteristic biomarkers of impaired hepatic function were found in the plasma of cirrhotic animals. The weights and protein contents of all muscles of CCl4-treated animals were lower when compared with controls. Increased concentrations of glutamine (GLN) and aromatic amino acids (phenylalanine and tyrosine) and decreased concentrations of branched-chain amino acids (BCAA), glutamate (GLU), alanine and aspartate were found in plasma and muscles. In the soleus muscle, GLN increased more and GLU and BCAA decreased less than in the extensor digitorum and tibialis muscles. Increased chymotrypsin-like activity (indicating enhanced proteolysis) and decreased α-ketoglutarate and ATP levels were found in muscles of cirrhotic animals. ATP concentration also decreased in blood plasma. It is concluded that a rat model of CCl4-induced cirrhosis is a valid model for the investigation of hepatic cachexia that exhibits alterations in line with a theory of role of ammonia in pathogenesis of BCAA depletion, citric cycle and mitochondria dysfunction, and muscle wasting in cirrhotic subjects. The findings indicate more effective ammonia detoxification to GLN in red than in white muscles.

Background Pancreatic cancer is a malignant tumor and ranks the sixth in incidence among cancers. Circular RNA (circRNA) has been reported to regulate the progression of pancreatic cancer. However, the effects of circ-membrane bound O-acyltransferase domain containing 2 (circ-MBOAT2) on regulating pancreatic cancer process were unclear. Methods The expression levels of circ-MBOAT2, microRNA-433-3p (miR-433-3p) and glutamic-oxaloacetic transaminase 1 (GOT1) mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). GOT1 protein expression was determined by western blot analysis. Cell proliferation was illustrated by 3-(4,5)-dimethylthiahiazo (−z-y1)-3,5-di-phenytetrazoliumromide (MTT) and cell colony formation assay. Cell apoptosis was demonstrated by flow cytometry analysis. Cell invasion and migration were investigated by transwell invasion and wound-healing assays. Glutamine catabolism was explained by detecting glutamine consumption, alpha ketoglutarate (α-KG) production and glutamate production. In vivo assay was performed to illustrate the impacts of circ-MBOAT2 silencing on tumor formation in vivo. The binding relationship between miR-433-3p and circ-MBOAT2 or GOT1 was predicted by circinteractome or starbase online databases, and identified by dual-luciferase reporter assay. Results Circ-MBOAT2 and GOT1 expression were significantly upregulated, while miR-433-3p expression was downregulated in pancreatic cancer tissues and cells compared with normal pancreatic tissues or cells. Circ-MBOAT2 silencing repressed cell proliferation, migration, invasion and glutamine catabolism, whereas promoted cell apoptosis in pancreatic cancer. Additionally, circ-MBOAT2 acted as a sponge of miR-433-3p, which was found to be associate with GOT1. MiR-433-3p inhibitors hindered circ-MBOAT2 silencing-mediated impacts on pancreatic cancer progression and glutamine catabolism. Furthermore, circ-MBOAT2 silencing repressed tumor formation in vivo. Conclusion Circ-MBOAT2 modulated tumor development and glutamine catabolism by miR-433-3p/GOT1 axis in pancreatic cancer. This finding suggests that circ-MBOAT2 may be a therapeutic target for pancreatic cancer.

The current study aimed to assess the effects of dietary alpha-ketoglutarate (AKG) supplementation to laying hens on the fatty acid (FA) profile and cholesterol levels of the egg yolk at the end of production cycle. The experiment was performed on forty-eight Bovans Brown laying hens randomly assigned to either a control group (CONT) or a group supplemented with AKG. The CONT group was fed the basal diet, and the AKG group was fed the basal diet plus 1.0% AKG from the 31st until the 60th week of age, when FA profile, fat and cholesterol content of the egg yolks were determined. No significant changes in the cholesterol and total fat content of the egg yolks were observed. However, there were positive (the decrease in n-6 FA and the increase in MUFA), and negative (decrease in PUFA and n-3 FA, increase in TI and n-6/n-3 ratio) changes in FA profile following AKG supplementation. In conclusion, it was shown that dietary AKG after a 30-week long supplementation influence FA profile in egg yolk and its nutritional value.

IDH-wildtype glioma and IDH-mutant glioma have different genetical and metabolic background although their histological appearances are similar. The aim of the study is to reveal the difference in metabolites between IDH-wildtype glioma and IDH-mutant glioma, and to find the effective treatment targeting cancer metabolism according to the status of IDH in gliomas. Two artificial cell lines made from normal human astrocyte were used: NHAE6E7hTERTRas (IDH-wildtype) and NHAE6E7hTERTIDHmut (IDH-mutant). Capillary electrophoresis time-of-flight-mass spectrometry (CE-TOFMS) revealed that the amount of asparagine was lower in NHAE6E7hTERTRas cells compared with NHAE6E7hTERTIDHmut cells. L-asparaginase, which converts asparagine into aspartate, was more effective in the former cells than the latter cells. L-asparaginase induced autophagy and inhibition of autophagy by 3-MA suppressed L-asparaginase-induced antitumor effect. Adding asparagine into the culture medium rescued the antitumor effect of L-asparaginase. L-asparaginase increased the expression of asparagine synthetase (ASNS) and genetical or pharmacological inhibition of ASNS enhanced the antitumor effect of L-asparaginase. CE-TOFMS showed the lower amount of glutamine, glutamate and 2-oxoglutarate in NHAE6E7hTERTIDHmut cells than NHAE6E7hTERTRas cells. GLUD1 inhibitor inhibited proliferation by inducing higher ROS level and apoptosis in NHAE6E7hTERTIDHmut cells than NHAE6E7hTERTRas cells. ROS inhibitor, NAC suppressed GLUD1 inhibitor-induced ROS, apoptosis, and cytotoxicity in NHAE6E7hTERTIDHmut cells. Exogeneous dimethyl 2-oxoglutarate rescued the cytotoxicity induced by GLUD1 inhibitor. L-asparaginase and GLUD1 inhibitor will be new therapeutic option for IDH-wildtype glioma and IDH-mutant glioma, respectively.

Nephropathic cystinosis is an autosomal recessive monogenic kidney disorder characterized by lysosomal accumulation of cystine throughout the body, causing organ damage, particularly the kidneys. This is due to pathogenic mutations in the CTNS gene, which encodes for the lysosomal cystine transporter cystinosin, transporting cystine from the lysosome into the cytoplasm. Patients with nephropathic cystinosis usually develop symptoms of renal Fanconi syndrome, and if not treated, progress to end stage renal disease within the first 12 years of life. The mainstay for cystinosis treatment is life-long therapy with cysteamine, a cystine depleting agent. Although cysteamine efficiently lowers lysosomal cystine levels and improves some clinical outcomes, it does not reverse the established renal Fanconi syndrome and cannot prevent the loss in renal function. This suggests involvement of complex pathophysiological processes in disease progression that may not be directly related to cystine overload. Therefore, this thesis aimed to investigate the disease pathophysiology and to find new potential therapeutic interventions that could replace or compliment cysteamine therapy for patients with cystinosis.

Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the β-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

The metabolic changes controlling the stepwise differentiation of hematopoietic stem and progenitor cells (HSPCs) to mature erythrocytes are poorly understood. Here, we show that HSPC development to an erythroid-committed proerythroblast results in augmented glutaminolysis, generating alpha-ketoglutarate (αKG) and driving mitochondrial oxidative phosphorylation (OXPHOS). However, sequential late-stage erythropoiesis is dependent on decreasing αKG-driven OXPHOS, and we find that isocitrate dehydrogenase 1 (IDH1) plays a central role in this process. IDH1 downregulation augments mitochondrial oxidation of αKG and inhibits reticulocyte generation. Furthermore, IDH1 knockdown results in the generation of multinucleated erythroblasts, a morphological abnormality characteristic of myelodysplastic syndrome and congenital dyserythropoietic anemia. We identify vitamin C homeostasis as a critical regulator of ineffective erythropoiesis; oxidized ascorbate increases mitochondrial superoxide and significantly exacerbates the abnormal erythroblast phenotype of IDH1-downregulated progenitors, whereas vitamin C, scavenging reactive oxygen species (ROS) and reprogramming mitochondrial metabolism, rescues erythropoiesis. Thus, an IDH1-vitamin C crosstalk controls terminal steps of human erythroid differentiation.

Scope As a natural compound in foods, alpha-ketoglutarate (aKG) is one of the key metabolites maintaining energy homeostasis. This study examines the beneficial effects of dietary aKG against the development of experimental colitis and further explores the underlying molecular mechanisms. Methods and results Eight-week-old male C57BL/6 mice receive drinking water with or without 1% aKG for 4 weeks. At week 3, colitis is induced by 2.5% dextran sulfate sodium (DSS) for 7 days followed by 7 days recovery. Dietary aKG supplementation decreases DSS-induced body weight loss, gross bleeding, fecal consistency score, and disease activity index. In agreement, aKG supplementation restores DSS-associated colon shortening, ameliorated mucosal damage, and macrophage infiltration into colonic tissue, which are associated with suppressed gut inflammation and Wnt signaling, and improved epithelial structure. Consistently, aKG supplementation enhances M1 to M2 macrophage polarization and strengthens intestinal barrier function. Additionally, aKG supplementation elevates colonic aKG levels while decreasing 2-hydroxyglutarate levels, which increases oxidative instead of glycolytic metabolism. Conclusion aKG supplementation protects against epithelial damage and ameliorates DSS-induced colitis, which are associated with suppressed inflammation, Wnt signaling pathway, and glycolysis. Intake of foods enriched with aKG or aKG supplementation can be an alternative approach for the prevention or treatment of colitis that are common in Western societies.

Lifelong multilineage hematopoiesis critically depends on rare hematopoietic stem cells (HSCs) that reside in the hypoxic bone marrow microenvironment. Although the role of the canonical oxygen sensor hypoxia-inducible factor prolyl hydroxylase has been investigated extensively in hematopoiesis, the functional significance of other members of the 2-oxoglutarate (2-OG)-dependent protein hydroxylase family of enzymes remains poorly defined in HSC biology and multilineage hematopoiesis. Here, by using hematopoietic-specific conditional gene deletion, we reveal that the 2-OG–dependent protein hydroxylase JMJD6 is essential for short- and long-term maintenance of the HSC pool and multilineage hematopoiesis. Additionally, upon hematopoietic injury, Jmjd6-deficient HSCs display a striking failure to expand and regenerate the hematopoietic system. Moreover, HSCs lacking Jmjd6 lose multilineage reconstitution potential and self-renewal capacity upon serial transplantation. At the molecular level, we found that JMJD6 functions to repress multiple processes whose downregulation is essential for HSC integrity, including mitochondrial oxidative phosphorylation (OXPHOS), protein synthesis, p53 stabilization, cell cycle checkpoint progression, and mTORC1 signaling. Indeed, Jmjd6-deficient primitive hematopoietic cells display elevated basal and maximal mitochondrial respiration rates and increased reactive oxygen species (ROS), prerequisites for HSC failure. Notably, an antioxidant, N-acetyl-l-cysteine, rescued HSC and lymphoid progenitor cell depletion, indicating a causal impact of OXPHOS-mediated ROS generation upon Jmjd6 deletion. Thus, JMJD6 promotes HSC maintenance and multilineage differentiation potential by suppressing fundamental pathways whose activation is detrimental for HSC function.

Osteosarcoma (OS) is the most common type of primary bone tumor. Currently, there are limited treatment options for metastatic OS. Alpha-ketoglutarate (AKG), i.e., a multifunctional intermediate of the Krebs cycle, is one of the central metabolic regulators of tumor fate and plays an important role in cancerogenesis and tumor progression. There is growing evidence suggesting that AKG may represent a novel adjuvant therapeutic opportunity in anti-cancer therapy. The present study was intended to check whether supplementation of Saos-2 and HOS osteosarcoma cell lines (harboring a TP53 mutation) with exogenous AKG exerted an anti-cancer effect. The results revealed that AKG inhibited the proliferation of both OS cell lines in a concentration-dependent manner. As evidenced by flow cytometry, AKG blocked cell cycle progression at the G1 stage in both cell lines, which was accompanied by a decreased level of cyclin D1 in HOS and increased expression of p21Waf1/Cip1 protein in Saos-2 cells (evaluated with the ELISA method). Moreover, AKG induced apoptotic cell death and caspase-3 activation in both OS cell lines (determined by cytometric analysis). Both the immunoblotting and cytometric analysis revealed that the AKG-induced apoptosis proceeded predominantly through activation of an intrinsic caspase 9-dependent apoptotic pathway and an increased Bax/Bcl-2 ratio. The apoptotic process in the AKG-treated cells was mediated via c-Jun N-terminal protein kinase (JNK) activation, as the specific inhibitor of this kinase partially rescued the cells from apoptotic death. In addition, the AKG treatment led to reduced activation of extracellular signal-regulated kinase (ERK1/2) and significant inhibition of cell migration and invasion in vitro concomitantly with decreased production of pro-metastatic transforming growth factor &beta, (TGF-&beta, ) and pro-angiogenic vascular endothelial growth factor (VEGF) in both OS cell lines suggesting the anti-metastatic potential of this compound. In conclusion, we showed the anti-osteosarcoma potential of AKG and provided a rationale for a further study of the possible application of AKG in OS therapy.

The aim of the experiment was to assess the effect of dietary alpha-ketoglutarate (AKG) supplementation on performance, serum hormonal indices, duodenum and jejunum histomorphometry, meat quality characteristics, bone quality traits and cartilage degradation in laying hens with a mature skeletal system. Forty-eight 30 week-old Bovans Brown laying hens were randomly assigned to a control group or the group fed the basal diet plus 1.0% AKG. The experimental trial lasted 30 weeks. The supplementation of AKG increases blood serum content of leptin, ghrelin, bone alkaline phosphatate and receptor activator of nuclear factor kappa-&Beta, ligand, while osteoprotegerin and osteocalcin decrease. While dietary AKG was given to laying hens negatively influenced villus length, crypt depth, villus/crypt ratio and absorptive surface area in duodenum and jejunum, these changes have no effect on feed intake, weight gain, nor laying performance. In breast muscles, no significant changes in skeletal muscle fatty acid composition were observed, however, a higher shear force and decreased cholesterol content following AKG supplementation were noted, showing the improvement of muscle quality. While dietary AKG supplementation did not affect the general geometric and mechanical properties of the tibia, it increased collagen synthesis and enhanced immature collagen content. In medullary bone, an increase of bone volume fraction, trabecular thickness, fractal dimension and decrease of trabecular space were observed in AKG supplemented group. The trabeculae in bone metaphysis were also significantly thicker after AKG supplementation. AKG promoted fibrillogenesis in articular cartilage, as indicated by increased cartilage oligomeric matrix protein immunoexpression. By improving the structure and maintaining the proper bone turnover rate of highly reactive and metabolically active medullar and trabecular bones AKG showed its anti-osteoporotic action in laying hens.

Skeletal muscle weight loss is accompanied by small fiber size and low protein content. Alpha-ketoglutarate (AKG) participates in protein and nitrogen metabolism. The effect of AKG on skeletal muscle hypertrophy has not yet been tested, and its underlying mechanism is yet to be determined. In this study, we demonstrated that AKG (2%) increased the gastrocnemius muscle weight and fiber diameter in mice. Our in vitro study also confirmed that AKG dose increased protein synthesis in C2C12 myotubes, which could be effectively blocked by the antagonists of Akt and mTOR. The effects of AKG on skeletal muscle protein synthesis were independent of glutamate, its metabolite. We tested the expression of GPR91 and GPR99. The result demonstrated that C2C12 cells expressed GPR91, which could be upregulated by AKG. GPR91 knockdown abolished the effect of AKG on protein synthesis but failed to inhibit protein degradation. These findings demonstrated that AKG promoted skeletal muscle hypertrophy via Akt/mTOR signaling pathway. In addition, GPR91 might be partially attributed to AKG-induced skeletal muscle protein synthesis.

Obesity has become a serious problem in public health worldwide, causing numerous metabolic diseases. Once the differentiation to mature adipocytes is disrupted, adipocyte hypertrophy and ectopic lipid accumulation leads to the inflammation in adipose tissue and systemic metabolic disorders. Intracellular metabolic state is known to change during cell differentiation and it affects the cell fate or the differentiation through epigenetic mechanism. Although the mechanism of preadipocyte differentiation has been well established, it is unknown how metabolic state changes and how it affects the differentiation in predipocyte differentiation. Nicotinamide adenine dinucleotide (NAD+) plays crucial roles in energy metabolism as a coenzyme in multiple redox reactions in major catabolic pathways and as a substrate of sirtuins or poly(ADP-ribose)polymerases. NAD+ is mainly synthesized from salvage pathway mediated by two enzymes, Nampt and Nmnat. The manipulation to NAD+ metabolism causes metabolic change in each tissue and changes in systemic metabolism. However, the role of NAD+ and Nampt in adipocyte differentiation remains unknown. In this study, we employed liquid chromatography-mass spectrometry (LC-MS)- and gas chromatography-mass spectrometry (GC-MS)-based targeted metabolomics to elucidate the metabolic reprogramming events that occur during 3T3-L1 preadipocyte differentiation. We found that the tricarboxylic acid (TCA) cycle was enhanced, which correlated with upregulated NAD+ synthesis. Additionally, increased alpha-ketoglutarate (αKG) contributed to histone H3K9 demethylation in the promoter region of PPARγ, leading to its transcriptional activation. Thus, we concluded that NAD+-centered metabolic reprogramming is necessary for the differentiation of 3T3-L1 preadipocytes.