Alpha Ketoglutarate (AKG)

GENERAL EFFECTS:

An important Krebs cycle intermediate and precursor for amino acids like glutamine and glutamate. Acts as a cofactor for enzymes involved in epigenetic regulation, ammonia detox, kidney health, bone health, hypoxia sensing etc (ref, ref, ref, ref).
Extended life span in worms, fruit flies and mice, and promoted healthy aging in mice (2020).
Calcium Alpha-Ketoglutarate Supplementation Reduced Epigenetic Age in Humans by 8.5 years (2021).

EFFECTS ON CANCER & COVID-19:

Supplementation mitigates metabolic disturbances in MPNs, reduces symptoms in mice such as splenomegaly and elevated platelets, and shows potential as a therapeutic agent for platelet hyperreactivity (2022).
Inhibits Akt-mediated activation in platelets and monocytes, reducing clot formation and inflammation, and alleviates COVID-19 effects in lungs (2021).
Improved the efficacy of anti-PD1 melanoma treatment through epigenetic modulation of PD-L1 (2023)
Elevated intracellular levels of AKG impinge (have a negative effect) on tumor progression (2020). Inhibited colon cancer growth in cell cultures (2020).

DEEPER DIVE:

Multiomic Profiling Reveals Metabolic Alterations Mediating Aberrant Platelet Activity and Inflammation in Myeloproliferative Neoplasms, 2022

α-KG is a key TCA cycle intermediate, which inhibits PI3K/AKT/mTOR pathway signaling via suppression of ATP synthase.
α-KG supplementation drastically reduced oxygen consumption and ATP generation in platelets from MPN patients.
Oral α-KG supplementation of Jak2 V617F mice decreased splenomegaly and reduced hematocrit, monocyte and platelet counts. Bone marrow samples revealed inhibition of heme metabolism, OXPHOS and mTOR signaling pathways.
α-KG inhibited platelet hyperreactivities in both human and mouse MPN samples. These findings highlight a prominent role for α-KG and mTOR signalling in aberrant MPN platelet activity and monocyte-driven inflammation.

α-KG is a key TCA cycle intermediate, which inhibits PI3K/AKT/mTOR pathway signaling via suppression of ATP synthase. α-KG supplementation drastically reduced oxygen consumption and ATP generation in platelets from MPN patients. We further investigated the effects of α-KG on MPN platelet activation. Ex vivo incubation of platelets from both MPN patients and Jak2 V617F knock-in mice with α-KG significantly reduced platelet surface P-selectin and integrin α2bβ3 activation. Additionally, α-KG inhibited the spreading and adhesion of platelets from Jak2 V617F knock-in mice to fibrinogen-coated surfaces. Platelet phosphoblots demonstrated significant downregulation of p-AKT and p-ERK after treatment with α-KG, suggesting the involvement of PI3K/AKT/mTOR and MAPK pathways in the inhibitory effects of α-KG on platelet activation. Thus, α-KG inhibited platelet hyperreactivities in both human and mouse MPN samples.

To test the therapeutic impact of α-KG on MPN disease features, we treated Jak2 V617F knock-in mice with α-KG for 6 weeks. Oral α-KG supplementation decreased splenomegaly and reduced elevated platelets and hematocrit. Additionally, monocytes were significantly decreased as early as 2 weeks after α-KG treatment in Jak2 V617F knock-in mice. Consistently, RNA-seq of bone marrow samples from α-KG treated mice revealed inhibition of heme metabolism, OXPHOS and mTOR signaling pathways. In ex vivo studies with MPN patient CD34+ cells, α-KG treatment for 10 days led to a decrease in CD41+ CD61+ cells, suggesting decreased megakaryocyte commitment. We further observed that α-KG incubation significantly decreased the secretion of proinflammatory cytokines from sorted CD14+ human monocytes. Mass cytometry analysis of whole blood from MPN patients demonstrated inhibition of MAPK pathway signaling after α-KG treatment. Taken together, these results suggest that α-KG supplementation may exert therapeutic effects through both direct inhibition of MPN platelet activity and via quenching of monocyte hyper-inflammation.

In summary, these studies reveal a previously unrecognized metabolic disorder in platelets from MPN patients and highlight a prominent role for α-KG and mTOR signalling in aberrant MPN platelet activity and monocyte-driven inflammation. These findings have potential relevance for novel therapeutic approaches for MPN patients.

α-Ketoglutarate Inhibits Thrombosis and Inflammation by Prolyl Hydroxylase-2 Mediated Inactivation of Phospho-Akt, 2021

Background: Phospho-Akt1 (pAkt1) undergoes prolyl hydroxylation at Pro125 and Pro313 by the prolyl hydroxylase-2 (PHD2) in a reaction decarboxylating α-ketoglutarate (αKG). We investigated whether the αKG supplementation could inhibit Akt-mediated activation of platelets and monocytes, in vitro as well as in vivo, by augmenting PHD2 activity.

Methods: We treated platelets or monocytes isolated from healthy individuals with αKG in presence of agonists in vitro and assessed the signalling molecules including pAkt1. We supplemented mice with dietary αKG and estimated the functional responses of platelets and monocytes ex vivo. Further, we investigated the impact of dietary αKG on inflammation and thrombosis in lungs of mice either treated with thrombosis-inducing agent carrageenan or infected with SARS-CoV-2.

Findings: Octyl αKG supplementation to platelets promoted PHD2 activity through elevated intracellular αKG to succinate ratio, and reduced aggregation in vitro by suppressing pAkt1(Thr308). Augmented PHD2 activity was confirmed by increased hydroxylated-proline and 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 platelets and leukocytes ex vivo from mice supplemented with dietary αKG, unaccompanied by alteration in their number. Dietary αKG significantly reduced clot formation and leukocyte accumulation in various organs including lungs of mice treated with thrombosis-inducing agent carrageenan. Importantly, in SARS-CoV-2 infected hamsters, we observed a significant rescue effect of dietary αKG on inflamed lungs with significantly reduced leukocyte accumulation, clot formation and viral load alongside down-modulation of pAkt in the lung of the infected animals.

Interpretation: Our study suggests that dietary αKG supplementation prevents Akt-driven maladies such as thrombosis and inflammation and rescues pathology of COVID19-infected lungs.

Bodybuilding supplement promotes healthy aging and extends life span, at least in mice, 2020

The molecule grabbed attention as a possible antiaging treatment in 2014, when researchers reported AKG could extend life span by more than 50% in tiny Caenorhabditis elegans worms. That’s on par with a low-calorie diet, which has been shown to promote healthy aging, but is hard for most people to stick with. Other groups later showed life span improvements from AKG in fruit flies.

In the new study, Gordon Lithgow and Brian Kennedy of the Buck Institute for Research on Aging and colleagues turned to mammals. They gave groups of 18-month-old mice (about age 55 in human years) the equivalent of 2% of their daily chow as AKG until they died, or for up to 21 months. AKG levels in blood gradually drop with age, and the scientists’ aim was to restore levels to those seen in young animals.

Some differences jumped out within a few months: “They looked much blacker, shinier, and younger” than control mice, says Azar Asadi Shahmirzadi, a postdoc at the Buck Institute who did the experiments as a graduate student. In addition, the AKG-fed mice scored an average of more than 40% better on tests of “frailty,” as measured by 31 physiological attributes including hair color, hearing, walking gait, and grip strength. And female mice lived a median of 8% to 20% longer after AKG treatment began than control mice, the group reports today in Cell Metabolism.

The AKG-eating mice did not perform better on tests of heart function or treadmill endurance, however, and the tests did not include cognitive performance.

Probing the mechanism for these improvements, the researchers found that female mice receiving AKG produced higher levels of a molecule that tamps down on inflammation. Chronic inflammation can spur many diseases of aging such as cancer, heart disease, arthritis, and dementia.

4.5% median lifespan improvement in mice (ref):

An informed anecdote about epigenetic tests on a forum: "I’ve done a lot of testing – something like 7 or 8 methylation tests since 2017. And most things just don’t affect methylation age, even if they can be shown to benefit health in other ways. Even 2 years of 2-3mg/wk rapamycin had only a very slight reduction (a few months) on the methylation age of this ~40yo rat. AKG took about 6 months to work, but did reverse my methylation age by 5-6 years. GDF11 also appears to reverse methylation age quite substantially according to reports I have from friends that have tried it and tested."

Another user: “Six months after beginning regimen: C-reactive protein, albumin, lipoproteins, and other levels significantly better as measured by third party blood tests.”

Calcium Alpha-Ketoglutarate Supplementation Reduces Epigenetic Age in Humans, 2021

The company Ponce De Leon Health claims that a recent pilot study of calcium alpha-ketoglutarate supplementation results in an average reduction of 8.5 years of epigenetic age via the DNA methylation test offered by TrueMe Labs.

The best pace to start is with the 2019 paper on the effects of calcium alpha-ketoglutarate in mice, which is a reputable study authored by reputable researchers. Delivered late in life, this intervention reduced frailty to a meaningful degree, but with only a modest effect on life span. It did not reduce senescent cell burden, but did reduce inflammatory signaling - and chronic inflammation is an important aspect of degenerative aging.

The important point to consider here is that the TrueMe Labs assay is not a relabeling of any of the more established epigenetic clocks, those with significant research associated with their behavior. It is is its own beast, an independently developed test. It uses only 13 DNA methylation sites, and so it is very possible that it is much more sensitive to some interventions than others, in comparison to, say, the original Horvath clock, depending on which mechanisms influence those sites. Thus one cannot take any of the established research into the better studied clocks and use it to inform expectations as to how the TrueMe Labs assay will behave. 8.5 years might sound like a large effect size, but it is impossible to say whether or not that is the case.

Alpha-ketoglutarate (link)

AKG extends the lifespan of adult C. elegans by roughly 50% by inhibiting ATP synthase and the target of rapamycin (TOR) [1]. Essentially, what is happening here is that mitochondrial function is being somewhat suppressed, in particular the electron transport chain, and it is that partial suppression that is responsible for increased lifespans in C. elegans. AKG does not directly interact with TOR, though it does influence it, mainly via the inhibition of ATP synthase.

Finally, autophagy, which is activated by caloric restriction and also the direct inhibition of TOR, is increased significantly in C. elegans given additional AKG.

2014 C. elegans study [1] showed that AKG levels are elevated in starving worms but that AKG did not increase the lifespan of calorically restricted animals. This suggests that AKG is a key metabolite and player in the regulation of lifespan via starvation and caloric restriction. It also suggests that AKG is a molecular link between cellular energy generation and dietary restriction in the context of lifespan regulation. Finally, it means that AKG is a potential target for the delay of aging and the treatment of age-related diseases.

Supplementation with α-ketoglutarate improved the efficacy of anti-PD1 melanoma treatment through epigenetic modulation of PD-L1, 2023

Patients with advanced melanoma have shown an improved outlook after anti-PD1 therapy, but the low response rate restricts clinical benefit; therefore, enhancing anti-PD1 therapeutic efficacy remains a major challenge. Here, our findings showed a significantly increased abundance of α-KG in healthy controls, anti-PD1-sensitive melanoma-bearing mice, and anti-PD1-sensitive melanoma patients; moreover, supplementation with α-KG enhanced the efficacy of anti-PD1 immunotherapy and increased PD-L1 expression in melanoma tumors via STAT1/3. We also found that supplementation with α-KG significantly increased the activity of the methylcytosine dioxygenases TET2/3, which led to an increased 5-hydroxymethylcytosine (5-hmC) level in the PD-L1 promoter. As a consequence, STAT1/3 binding to the PD-L1 promoter was stabilized to upregulate PD-L1 expression. Importantly, single-cell sequencing of preclinical samples and analysis of clinical data revealed that TET2/3-STAT1/3-CD274 signaling was associated with sensitivity to anti-PD1 treatment in melanoma. Taken together, our results provide novel insight into α-KG’s function in anti-PD1 treatment of melanoma and suggest supplementation with α-KG as a novel promising strategy to improve the efficacy of anti-PD1 therapy.

Turning colon cancer cells around: Supplement inhibits cell growth, 2020

Eighty-percent of colon cancers stem from a genetic mutation of the protein adenomatous polyposis coli, or APC. While the majority of people with that mutation will develop polyps, only some of the polyps will become cancerous, a phenomenon that is not fully understood. The research team decided to investigate non-genetic factors that could propel the disease, focusing their inquiry on the role of the amino acid glutamine.

"Cancer cells consume a great amount of glutamine to proliferate," said Molecular Biology and Biochemistry Associate Professor Mei Kong. "But we found that depriving them of glutamine doesn't kill all the tumor cells. Some tumor cells are able to adapt and in fact, when their glutamine supply runs low, they turn into a more invasive form of cancer."

The researchers found that a drop in cellular levels of the metabolite alpha-ketoglutarate after glutamine starvation accompanied the transition from benign to cancerous cells. This finding led them to conduct further investigation into the metabolite's role. When they provided a modified version of alpha-ketoglutarate to animal models with APC mutations, the results were significant. Just 23% of those given the modified metabolite developed rectal bleeding, an indication of intestinal tumors, compared to 90% of the animal models who did not receive it. It also curbed tumor growth and protected against disease-associated conditions such as weight loss.

"Supplementation of the modified alpha-ketoglutarate inhibits a key cancer-development signaling pathway in colon cancer cells, turning them into more normal cells," said researcher Thai Q. Tran, the paper's first author. "What's also notable is that we administered it by mixing it into drinking water, so it was easy to take and it did not affect overall health."

α-Ketoglutarate attenuates Wnt signaling and drives differentiation in colorectal cancer, 2020

Genetic-driven deregulation of the Wnt pathway is crucial but not sufficient for colorectal cancer (CRC) tumorigenesis. Here, we show that environmental glutamine restriction further augments Wnt signaling in APC-mutant intestinal organoids to promote stemness, and leads to adenocarcinoma formation in vivo via decreasing intracellular α-ketoglutarate (αKG) levels. αKG supplementation is sufficient to rescue low-glutamine-induced stemness and Wnt hyperactivation. Mechanistically, we found that αKG promotes hypomethylation of DNA and histone H3K4me3, leading to an upregulation of differentiation-associated genes and downregulation of Wnt target genes, respectively. Using organoids derived from patients with CRC and several in vivo CRC tumor models, we show that αKG supplementation suppresses Wnt signaling and promotes cellular differentiation, thereby significantly restricting tumor growth and extending survival. Together, our results reveal how the metabolic microenvironment impacts Wnt signaling and identify αKG as a potent antineoplastic metabolite for potential differentiation therapy for patients with CRC.

Nitrogen Trapping as a Therapeutic Strategy in Tumors with Mitochondrial Dysfunction, 2020

Under conditions of inherent or induced mitochondrial dysfunction, cancer cells manifest overlapping metabolic phenotypes, suggesting that they may be targeted via a common approach. Here, we use multiple oxidative phosphorylation (OXPHOS)–competent and incompetent cancer cell pairs to demonstrate that treatment with α-ketoglutarate (aKG) esters elicits rapid death of OXPHOS-deficient cancer cells by elevating intracellular aKG concentrations, thereby sequestering nitrogen from aspartate through glutamic-oxaloacetic transaminase 1 (GOT1). Exhaustion of aspartate in these cells resulted in immediate depletion of adenylates, which plays a central role in mediating mTOR inactivation and inhibition of glycolysis. aKG esters also conferred cytotoxicity in a variety of cancer types if their cell respiration was obstructed by hypoxia or by chemical inhibition of the electron transport chain (ETC), both of which are known to increase aspartate and GOT1 dependencies. Furthermore, preclinical mouse studies suggested that cell-permeable aKG displays a good biosafety profile, eliminates aspartate only in OXPHOS-incompetent tumors, and prevents their growth and metastasis. This study reveals a novel cytotoxic mechanism for the metabolite aKG and identifies cell-permeable aKG, either by itself or in combination with ETC inhibitors, as a potential anticancer approach.

Significance: These findings demonstrate that OXPHOS deficiency caused by either hypoxia or mutations, which can significantly increase cancer virulence, renders tumors sensitive to aKG esters by targeting their dependence upon GOT1 for aspartate synthesis.

The multifaceted contribution of α-ketoglutarate to tumor progression: An opportunity to exploit? 2020

Highlights

αKG is at the center of metabolic reactions essential for cells.
αKG regulates enzymes involved in hypoxic adaptation and epigenetic modifications.
Elevated intracellular levels of αKG impinge (have a negative effect) on tumor progression.
Increase of αKG levels may represent a possible anti-cancer strategy.

Abstract

The thriving field that constitutes cancer metabolism has unveiled some groundbreaking facts over the past two decades, at the heart of which is the TCA cycle and its intermediates. As such and besides its metabolic role, α-ketoglutarate was shown to withstand a wide range of physiological reactions from protection against oxidative stress, collagen and bone maintenance to development and immunity. Most importantly, it constitutes the rate-limiting substrate of 2-oxoglutarate-dependent dioxygenases family enzymes, which are involved in hypoxia sensing and in the shaping of cellular epigenetic landscape, two major drivers of oncogenic transformation. Based on literature reports, we hereby review the benefits of this metabolite as a possible novel adjuvant therapeutic opportunity to target tumor progression. This article is part of the special issue “Mitochondrial metabolic alterations in cancer cells and related therapeutic targets”.

Alpha-ketoglutarate (AKG) inhibits proliferation of colon adenocarcinoma cells in normoxic conditions, 2012

Background and objective. Alpha-ketoglutarate (AKG), a key intermediate in Krebs cycle, is an important biological compound involved in the formation of amino acids, nitrogen transport, and oxidation reactions. AKG is already commercially available as a dietary supplement and its supplementation with glutamine, arginine, or ornithine alpha-ketoglutarate has been recently considered to improve anticancer immune functions. It is well documented that AKG treatment of Hep3B hepatoma cells in hypoxia induced HIF-alpha (hypoxia-inducible factor) degradation and reduced vascular endothelial growth factor (VEGF) synthesis. Moreover, AKG showed potent antitumor effects in murine tumor xenograft model, inhibiting tumor growth, angiogenesis, and VEGF gene expression. However, the mechanisms of its anticancer activity in normoxia have not been examined so far. Results. Here, we report that in normoxia, AKG inhibited proliferation of colon adenocarcinoma cell lines: Caco-2, HT-29, and LS-180, representing different stages of colon carcinogenesis. Furthermore, AKG influenced the cell cycle, enhancing the expression of the inhibitors of cyclin-dependent kinases p21 Waf1/Cip1 and p27 Kip1. Moreover, expression of cyclin D1, required in G1/S transmission, was decreased, which accompanied with the significant increase in cell number in G1 phase. AKG affected also one the key cell cycle regulator, Rb, and reduced its activation status. Conclusion. In this study for the first time, the antiproliferative activity of AKG on colon adenocarcinoma Caco-2, HT-29, and LS-180 cells in normoxic conditions was revealed. Taking into consideration an anticancer activity both in hypoxic and normoxic conditions, AKG may be considered as a new potent chemopreventive agent.

α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis, 2018

Metastasis formation requires active energy production and is regulated at multiple levels by mitochondrial metabolism. The hyperactive metabolism of cancer cells supports their extreme adaptability and plasticity and facilitates resistance to common anticancer therapies. In spite the potential relevance of a metastasis metabolic control therapy, so far, limited experience is available in this direction. Here, we evaluated the effect of the recently described α-ketoglutarate dehydrogenase (KGDH) inhibitor, (S)-2-[(2,6-dichlorobenzoyl) amino] succinic acid (AA6), in an orthotopic mouse model of breast cancer 4T1 and in other human breast cancer cell lines. In all conditions, AA6 altered Krebs cycle causing intracellular α-ketoglutarate (α-KG) accumulation. Consequently, the activity of the α-KG-dependent epigenetic enzymes, including the DNA demethylation ten-eleven translocation translocation hydroxylases (TETs), was increased. In mice, AA6 injection reduced metastasis formation and increased 5hmC levels in primary tumours. Moreover, in vitro and in vivo treatment with AA6 determined an α-KG accumulation paralleled by an enhanced production of nitric oxide (NO). This epigenetically remodelled metabolic environment efficiently counteracted the initiating steps of tumour invasion inhibiting the epithelial-to-mesenchymal transition (EMT). Mechanistically, AA6 treatment could be linked to upregulation of the NO-sensitive anti-metastatic miRNA 200 family and down-modulation of EMT-associated transcription factor Zeb1 and its CtBP1 cofactor. This scenario led to a decrease of the matrix metalloproteinase 3 (MMP3) and to an impairment of 4T1 aggressiveness. Overall, our data suggest that AA6 determines an α-KG-dependent epigenetic regulation of the TET–miR200–Zeb1/CtBP1–MMP3 axis providing an anti-metastatic effect in a mouse model of breast cancer-associated metastasis.

In multiple animal studies, supplementing alpha-ketoglutarate reduced blood supply to tumors, resulting in tumor death [39].

In cell-based studies, alpha-ketoglutarate prevented the multiplication of cancerous cells [40, 41].

Cell-Permeating α-Ketoglutarate Derivatives Alleviate Pseudohypoxia in Succinate Dehydrogenase-Deficient Cells

Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are components of the tricarboxylic acid (TCA) cycle and tumor suppressors. Loss of SDH or FH induces pseudohypoxia, a major tumor-supporting event, which is the activation of hypoxia-inducible factor (HIF) under normoxia. In SDH- or FH-deficient cells, HIF activation is due to HIF1α stabilization by succinate or fumarate, respectively, either of which, when in excess, inhibits HIFα prolyl hydroxylase (PHD). To reactivate PHD, we focused on its substrate, α-ketoglutarate. We designed and synthesized cell-permeating α-ketoglutarate derivatives, which build up rapidly and preferentially in cells with a dysfunctional TCA cycle. This study shows that succinate- or fumarate-mediated inhibition of PHD is competitive and is reversed by pharmacologically elevating intracellular α-ketoglutarate. Introduction of α-ketoglutarate derivatives restores normal PHD activity and HIF1α levels to SDH-suppressed cells, indicating new therapy possibilities for the cancers associated with TCA cycle dysfunction.

Anti-Tumour Activity of Exogenous AKG: In Vitro and In Vivo Studies (link)

One of the very important elements of response to hypoxia and HIF-1 activation is transcription of genes playing a key role in angiogenesis. This process is crucial for the development of solid tumours. The growing tumour tissue needs high amounts of oxygen and nutrients that are supplied by diffusion from the nearby blood vessels in the initial stage of tumour development. As the tumour develops and increases its size, the cells of the nearby blood vessels start running out of oxygen, which activates HIF-1 and initiates the process of neoangiogenesis. HIF-1 in tumour cells activates the transcription of genes for pro-angiogenic factors such as vascular endothelial growth factor (VEGF), PDGF-B (platelet-derived growth factor, type B), hepatocyte growth factor, epidermal growth factor, angiopoietin-2, or placental growth factor (Sacewicz et al. 2009). Matsumoto et al. (2006, 2009) showed that exogenous AKG exhibited anti-tumour activity by reducing the level of the HIF-1α subunit and inhibition of angiogenesis in hypoxic conditions. In their study, AKG inhibited the expression of the HIF-1α subunit and the ability to connect HIF-1 protein subunits, decreased the activity of the Vegf gene promoter, and, consequently, inhibited VEGF and erythropoietin production in the Hep3B cell line. Furthermore, AKG inhibited tube formation in an in vitro angiogenesis model. Those anti-angiogenic effects of AKG were confirmed in another in vitro study, carried out using the Lewis lung carcinoma (LLC) cell line (Matsumoto et al. 2009). Additionally, AKG administered alone showed anti-tumour activity and enhanced the activity of a chemotherapeutic agent (5-fluorouracil, 5-FU) in vivo. In the mouse dorsal air sac assay, AKG reduced the amount of newly formed blood vessels caused by administration of the cancer cell line (LLC). Also intraperitoneal administration of AKG alone or its combination with 5-FU to mice with transplanted tumours significantly inhibited tumour growth and angiogenesis in tumour tissue (Matsumoto et al. 2009) which suggests the clinical usefulness of this molecule. In turn, the results of the experiments carried out by Brière et al. (2005) and MacKenzie et al. (2007) suggest a possibility of application of AKG in the treatment of neoplastic diseases in which Krebs cycle enzyme defects cause pseudohypoxia and activation of HIF-1 in normoxia. Brière et al. (2005) have shown that exogenous AKG prevented translocation of HIF-1 into the nucleus of fibroblasts with a mutation in the gene for SDHA (succinate dehydrogenase subunit A). In another study, the MacKenzie et al. (2007) have shown that the competitive inhibition of PHDs by succinate or fumarate may be reversed by increasing the cellular level of AKG. However, native AKG does not easily penetrate into cells; therefore, to increase the effectiveness of its function, tests were carried out using cell-permeating AKG derivatives, i.e. AKG esters with increased hydrophobicity—octyl-AKG and 1-trifluoromethyl benzyl-AKG (converted by cytoplasmic esterases to AKG). These derivatives restored the normal activity of PHDs, thereby decreasing the level of HIF-1α in SDH-deficient cells (MacKenzie et al. 2007). Other in vitro studies (Tennant et al. 2009) have shown that AKG esters (1-trifluoromethyl benzyl-AKG, TaAKG) can restore the activity of PHDs under hypoxic conditions, which has far-reaching effects on tumour cells. Restoration of the PHD activity in such conditions not only resulted in destabilisation of HIF-1α, but also induced functional changes in cells—reversal of the hypoxia-induced increased glycolysis process and cell death as its consequence. In addition, it has been shown that AKG derivatives (TaAKG and ETAKG—5-ethyl,4-1-trifluoromethylbenzyl AKG) may also function well in vivo. TaAKG penetrated several layers of cells in spheroids derived from cell line HCT116 (human colon carcinoma) and destabilised their HIF-1α subunit. Moreover, after oral administration of ETAKG to a mouse xenograft tumour model, increased levels of AKG within the tumour tissues were observed, as well as decreased levels of HIF-1α, and reduced glucose metabolism. The results of the studies mentioned above suggest that AKG in the form of a diester can reactivate PHDs and destabilise HIF-1α in vivo, thereby reducing the expression of genes targeted by this factor (Tennant et al. 2009). However, it has recently been shown that not all types of AKG esters that penetrate the cell membrane have the same activity on HIF-1. In the studies of Hou et al. (2014), membrane permeable ester dimethyl AKG (DAKG), which is the precursor of AKG, temporarily stabilized HIF-1α by inhibition of PHD2. During the long-term impact of DAKG on cells under normoxia conditions, an increase in the level of HIF-1α and expression of its target genes was observed, which indicates that DAKG, in contrast to AKG, promotes the state of pseudohypoxia. The authors speculate that at a high availability of nutrients, DAKG may have been quickly converted to succinate or fumarate, which inhibited the activity of PHD2. On the other hand, the activity of PHD2 might have been inhibited by increasing intracellular levels of ROS in pseudohypoxic conditions induced by DAKG.

Additionally, in vitro studies have shown that exogenous AKG may affect the level of DNA methylation (Letouzé et al. 2013). In contrast to genetic mutations, DNA methylation is a reversible process, which creates a possibility of introduction of new drugs for the treatment of certain tumours (Rodríguez-Paredes and Esteller 2011). Cancer cells are often characterised by a decrease in total DNA methylation and by hypermethylation of promoter CpG islands, which results in transcriptional silencing of tumour suppressor genes. This phenotype, which is characterised by simultaneous multiple gene hypermethylation, occurs, for example, in glioma, in which it is the result of mutations in IDH1/IDH2 genes and the inhibiting activity of 2HG on enzymes belonging to the KDM and TET groups. Also in paraganglioma, mutations in the SDH genes determine such a phenotype (Letouzé et al. 2013). In the studies of Letouzé et al. (2013), SDH-deficient chromaffin cells displayed an increased 5-mC/5-hmC ratio and histone methylation, while the addition of AKG to the culture medium reversed the accumulation of 5mC in vitro and consequently changed this phenotype. These results suggest that exogenous AKG can restore the TETs enzyme activity inhibited by succinate accumulated in cells and restore the cellular normal phenotype.

Given the contradictory results obtained in studies using various esters of AKG, currently, it seems to be safer to use AKG in its native form in cancer therapy. This is supported by the fact that recent studies have shown an antiproliferative effect of AKG on three cancer cell lines: Caco-2, HT-29, and LS-180, representing different stages of the development of colon adenocarcinoma (Rzeski et al. 2012). AKG interfered in the cell cycle of the tumour cells by increasing the expression of cyclin-dependent kinase (CDK) inhibitors p21 Waf/CIP1 and p27 Kip1. Affecting the cell cycle by influencing the proteins involved in its regulation (cyclins) is a very promising feature of AKG as a potential anticancer agent. Each stage of the cell cycle is supported by specific cyclins forming complexes with CDKs, which are involved in phosphorylation of certain proteins. This facilitates maintenance of the cycle, and consequently leads to cell division. The above-mentioned CDK inhibitors subsequently inhibit DNA replication and are responsible for cell cycle arrest resulting in the absence of cell division (Meeran and Katiyar 2008; Xiong et al. 1993). The influence of AKG on the cell cycle of tumour cells is not limited to increasing the expression of the CDK inhibitors. It also decreased the protein level of cyclin D1 and inhibited phosphorylation of the key regulator of the cell cycle, i.e., the Rb protein, which resulted in arresting a large number of cells in the G1 phase, preventing them from entering the division phase (Rzeski et al. 2012).

It should be noted that AKG in combination with 5-hydroxymethylfurfural, prepared in the form of an infusion solution, are currently being investigated for the treatment of patients with non-small-cell lung carcinoma, not responding to any conventional therapy. This combination, named KARAL®, is currently in phase II clinical study and shows great promises as cancer treatment (Donnarumma et al. 2013).

Nitrogen Trapping as a Therapeutic Strategy in Tumors with Mitochondrial Dysfunction

Dietary alpha-ketoglutarate promotes beige adipogenesis and prevents obesity in middle-aged mice

Aging usually involves the progressive development of certain illnesses, including diabetes and obesity. Due to incapacity to form new white adipocytes, adipose expansion in aged mice primarily depends on adipocyte hypertrophy, which induces metabolic dysfunction. On the other hand, brown adipose tissue burns fatty acids, preventing ectopic lipid accumulation and metabolic diseases. However, the capacity of brown/beige adipogenesis declines inevitably during the aging process. Previously, we reported that DNA demethylation in the Prdm16 promoter is required for beige adipogenesis. DNA methylation is mediated by ten–eleven family proteins (TET) using alpha-ketoglutarate (AKG) as a cofactor. Here, we demonstrated that the circulatory AKG concentration was reduced in middle-aged mice (10-month-old) compared with young mice (2-month-old). Through AKG administration replenishing the AKG pool, aged mice were associated with the lower body weight gain and fat mass, and improved glucose tolerance after challenged with high-fat diet (HFD). These metabolic changes are accompanied by increased expression of brown adipose genes and proteins in inguinal adipose tissue. Cold-induced brown/beige adipogenesis was impeded in HFD mice, whereas AKG rescued the impairment of beige adipocyte functionality in middle-aged mice. Besides, AKG administration up-regulated Prdm16 expression, which was correlated with an increase of DNA demethylation in the Prdm16 promoter. In summary, AKG supplementation promotes beige adipogenesis and alleviates HFD-induced obesity in middle-aged mice, which is associated with enhanced DNA demethylation of the Prdm16 gene.

Alpha-ketoglutarate (AKG) lowers body weight and affects intestinal innate immunity through influencing intestinal microbiota

Alpha-ketoglutarate (AKG), a precursor of glutamate and a critical intermediate in the tricarboxylic acid cycle, shows beneficial effects on intestinal function. However, the influence of AKG on the intestinal innate immune system and intestinal microbiota is unknown. This study explores the effect of oral AKG administration in drinking water (10 g/L) on intestinal innate immunity and intestinal microbiota in a mouse model. Mouse water intake, feed intake and body weight were recorded throughout the entire experiment. The ileum was collected for detecting the expression of intestinal proinflammatory cytokines and innate immune factors by Real-time Polymerase Chain Reaction. Additionally, the ileal luminal contents and feces were collected for 16S rDNA sequencing to analyze the microbial composition. The intestinal microbiota in mice was disrupted with an antibiotic cocktail. The results revealed that AKG supplementation lowered body weight, promoted ileal expression of mammalian defensins of the alpha subfamily (such as cryptdins-1, cryptdins-4, and cryptdins-5) while influencing the intestinal microbial composition (i.e., lowering the Firmicutes to Bacteroidetes ratio). In the antibiotic-treated mouse model, AKG supplementation failed to affect mouse body weight and inhibited the expression of cryptdins-1 and cryptdins-5 in the ileum. We concluded that AKG might affect body weight and intestinal innate immunity through influencing intestinal microbiota.

The Health Benefits of Alpha-Ketoglutaric Acid (link)

The natural form is known to enhance athletic performance and metabolism

Chronic Kidney Disease

In a 2017 study published in the journal PLoS One, researchers identified and followed 1,483 people with advanced CKD who used an alpha-ketoglutaric acid supplement called Ketosteril. The average duration of the follow-up was 1.57 years.

Compared to a matched set of individuals who didn't take the supplement, those who did were less likely to require long-term dialysis. The benefits extended only to those who took more than 5.5 tablets per day, indicating the effects were dose-dependent.

Alpha-Ketoglutarate (link)

Possibly Effective for

Long-term kidney disease (chronic kidney disease or CKD). Taking calcium alpha-ketoglutarate seems to improve results of certain lab tests used to monitor the effectiveness of hemodialysis in patients receiving this treatment.

Tissue damage caused when there is limited blood flow and then blood flow is restored (ischemia-reperfusion injury). Administering alpha-ketoglutarate intravenously (by IV) seems to reduce blood supply problems during heart surgery.

The Influence of AKG on Bone Tissue (link)

In recent years, numerous papers (Dobrowolski et al. 2008; Filip et al. 2007; Filip 2007; Harrison et al. 2004; Radzki et al. 2012; Tatara et al. 2005, 2006, 2007) have been published to suggest that AKG may have an anabolic effect on bone tissue. Many in vivo studies (Harrison et al. 2004; Tatara et al. 2006, 2007) have demonstrated that supplementation of AKG or its derivatives during the animal growth has positive effects on the development of skeleton by improving the mechanical properties of skeletal bone. Moreover, other in vivo studies have shown that AKG supplementation prevented the development of osteopenia in female ovariectomized rats (Radzki et al. 2012), in rats after gastrectomy (Dobrowolski et al. 2008) or in model of osteopenia induced by denervation in turkeys (Tatara et al. 2005). In a study of menopausal women, it was also observed that administration of AKG (with Ca) inhibited bone resorption and reduced the effects of osteopenia. In women treated with AKG sodium salt, after 24 weeks of treatment, a significant decrease (about 37 %) in the level of CTX in the bloodstream was observed as well as higher bone density of the lumbar spine in comparison with the control group (receiving only CaCO3) (Filip et al. 2007). The results of above studies suggest that AKG not only can inhibit bone resorption, but can also induce reconstruction of bone tissue in the states of osteopenia and osteoporosis.

Although the positive influence of AKG on bone mineral density and strength is well documented in many in vivo studies (Dobrowolski et al. 2008; Filip et al. 2007; Harrison et al. 2004; Radzki et al. 2012; Tatara et al. 2005, 2006, 2007), its mechanism has not been elucidated so far. It is believed that AKG can contribute to an increase in the body pool of amino acids necessary for synthesis of type I collagen (proline and hydroxyproline) and thus have a positive effect on bone quality (Harrison and Pierzynowski 2008; Majamaa et al. 1987; Petersen et al. 2003).

Alpha-Ketoglutarate: Physiological Functions and Applications (link)

Alpha-ketoglutarate (AKG) is a key molecule in the Krebs cycle determining the overall rate of the citric acid cycle of the organism. It is a nitrogen scavenger and a source of glutamate and glutamine that stimulates protein synthesis and inhibits protein degradation in muscles. AKG as a precursor of glutamate and glutamine is a central metabolic fuel for cells of the gastrointestinal tract as well. AKG can decrease protein catabolism and increase protein synthesis to enhance bone tissue formation in the skeletal muscles and can be used in clinical applications.

In the cellular metabolism, AKG provides an important source of glutamine and glutamate that stimulates protein synthesis, inhibits protein degradation in muscle, and constitutes an important metabolic fuel for cells of the gastrointestinal tract (Hixt and Müller, 1996; Jones et al., 1999)

Alpha-Ketoglutarate: Physiological Functions and Applications - NCBI, 2016

Longevity

Studies released linked α-ketoglutarate with increased lifespan in nematode worms [7] and increased healthspan/lifespan in mice.[8][9][10]

Shahmirzadi, Azar Asadi; Edgar, Daniel; Liao, Chen-Yu (2020-09-01). "Alpha-Ketoglutarate, an Endogenous Metabolite, Extends Lifespan and Compresses Morbidity in Aging Mice".

Highlights

A CaAKG-supplemented diet extends lifespan of middle-aged female mice
AKG supplementation extends healthspan of both female and male mice
AKG compresses morbidity. Reduction in frailty is more dramatic than lifespan extension
AKG reduces chronic inflammation and induces IL-10 in T cells of female mice

Summary

Metabolism and aging are tightly connected. Alpha-ketoglutarate is a key metabolite in the tricarboxylic acid (TCA) cycle, and its levels change upon fasting, exercise, and aging. Here, we investigate the effect of alpha-ketoglutarate (delivered in the form of a calcium salt, CaAKG) on healthspan and lifespan in C57BL/6 mice. To probe the relationship between healthspan and lifespan extension in mammals, we performed a series of longitudinal, clinically relevant measurements. We find that CaAKG promotes a longer, healthier life associated with a decrease in levels of systemic inflammatory cytokines. We propose that induction of IL-10 by dietary AKG suppresses chronic inflammation, leading to health benefits. By simultaneously reducing frailty and enhancing longevity, AKG, at least in the murine model, results in a compression of morbidity.

A Summary of Alpha-ketoglutarate (link)

Studies of interest

There is evidence that AKG can influence aging, and a number of studies suggest that this is the case. A 2014 study showed that AKG extends the lifespan of adult C. elegans by roughly 50% by inhibiting ATP synthase and the target of rapamycin (TOR) [1].

During this study, it was found that AKG not only increased lifespan but also delayed certain age-related phenotypes, such as the loss of rapid coordinated body movement commonly seen in aged C. elegans. In order to understand how AKG influences aging, we will describe the mechanism by which AKG inhibits ATP synthase and TOR to extend lifespan in C. elegans and likely other species as well.

The researchers found that ATP synthase subunit β is a binding protein of AKG. They discovered that AKG inhibits ATP synthase, which leads to a reduction of available ATP, decreased oxygen consumption, and an increase of autophagy in the cells of both C. elegans and mammals.

The direct binding of ATP-2 by AKG, the associated inhibition of enzymes, the reduction of ATP levels, reduction of oxygen consumption, and increased lifespan were almost the same as when ATP synthase 2 (ATP-2) is directly, genetically knocked out. From these findings, the researchers concluded that AKG likely increases lifespan by targeting ATP-2.

Essentially, what is happening here is that mitochondrial function is being somewhat suppressed, in particular the electron transport chain, and it is that partial suppression that is responsible for increased lifespans in C. elegans.

The key is to reduce mitochondrial function just enough without going too far and it becoming detrimental. So, the old saying “live fast, die young” is absolutely correct, only in this case, the worms are living slow and dying old thanks to ATP suppression.

Finally, autophagy, which is activated by caloric restriction and also the direct inhibition of TOR, is increased significantly in C. elegans given additional AKG. This means that AKG and TOR inhibition are increasing lifespan either via the same pathway or through independent/parallel pathways and mechanisms that ultimately converge on the same downstream target.

Further support for this has been shown in studies with starving yeast and bacteria [7] and in humans post-exercise [8], in which AKG levels are shown to be elevated. This increase is believed to be a starvation response, in this case anaplerotic gluconeogenesis, which activates glutamate-associated transaminases in the liver to generate carbon derived from amino acid catabolism.

This is consistent with the findings of the 2014 C. elegans study [1], which showed that AKG levels are elevated in starving worms but that AKG did not increase the lifespan of calorically restricted animals. This suggests that AKG is a key metabolite and player in the regulation of lifespan via starvation and caloric restriction. It also suggests that AKG is a molecular link between cellular energy generation and dietary restriction in the context of lifespan regulation. Finally, it means that AKG is a potential target for the delay of aging and the treatment of age-related diseases.

My Longevity Journey

Alpha Ketoglutarate (AKG)

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