Mitochondrial Branched-chain Aminotransferase Research Articles

Defective Muscle Ketone Body Oxidation Disrupts BCAA Catabolism by Altering Mitochondrial Branched-Chain Aminotransferase.

Ketone bodies are an endogenous fuel source generated primarily by the liver to provide alternative energy for extrahepatic tissues during prolonged fasting and exercise. Skeletal muscle is an important site of ketone body oxidation which occurs through a series of reactions requiring the enzyme succinyl-CoA:3-ketoacid-CoA transferase (SCOT/Oxct1). We have previously shown that deleting SCOT in the skeletal muscle protects against obesity-induced insulin resistance by increasing pyruvate dehydrogenase (PDH) activity, the rate-limiting enzyme of glucose oxidation. However, it remains unclear whether inhibiting muscle ketone body oxidation causes hypoglycemia and affects fuel metabolism in the absence of obesity. Here, we show that lean mice lacking skeletal muscle SCOT (SCOTSkM-/-) exhibited no overt phenotypic differences in glucose and fat metabolism from their human α-skeletal actin-Cre (HSACre) littermates. Of interest, we found that plasma and muscle branched-chain amino acid (BCAA) levels are elevated in SCOTSkM-/- lean mice compared to their HSACre littermates. Interestingly, this alteration in BCAA catabolism was only seen in SCOTSkM-/- mice under low-fat feeding and associated with decreased expression of mitochondrial branched-chain aminotransferases (BCATm/Bcat2), the first enzyme in BCAA catabolic pathway. Loss- and gain-of-function studies in C2C12 myotubes demonstrated that suppressing SCOT markedly diminished BCATm expression, whereas overexpressing SCOT resulted in an opposite effect without influencing BCAA oxidation enzymes. Further, SCOT overexpression in C2C12 myotubes significantly increased luciferase activity driven by a Bcat2 promoter construct. Together, our findings indicate that SCOT regulates the expression of the Bcat2 gene, which, through the abundance of its product BCATm, may influence circulating BCAA concentrations.

The emerging role of the branched chain aminotransferases, BCATc and BCATm, for anti-tumor T-cell immunity.

Challenges regarding successful immunotherapy are associated with the heterogeneity of tumors and the complex interactions within the surrounding tumor microenvironment (TME), particularly those between immune and tumor cells. Of interest, T cells receive a myriad of environmental signals to elicit differentiation to effector subtypes, which is accompanied by metabolic reprogramming needed to satisfy the high energy and biosynthetic demands of their activated state. However, T cells are subjected to immunosuppressive signals and areas of oxygen and nutrient depletion in the TME, which causes T-cell exhaustion and helps tumor cells escape immune detection. The cytosolic and mitochondrial branched chain amino transferases, BCATc and BCATm, respectively, are responsible for the first step of the branched chain amino acid (BCAA) degradation, of which, metabolites are shunted into various metabolic processes. In recent years, BCAT isoenzymes have been investigated for their role in a variety of cancers found throughout the body; however, a gap of knowledge exists regarding the role BCAT isoenzymes play within immune cells of the TME. The aim of this review is to summarize recent findings about BCAAs and their catabolism at the BCAT step during T-cell metabolic reprogramming and to discuss the BCAT putative role in the anti-tumor immunity of T cells. Not only does this review acknowledges gaps pertaining to BCAA metabolism in the TME but it also identifies the practical application of BCAA metabolism in T cells in response to cancer and spotlights a potential target for pharmacological intervention.

The mechanism of branched-chain amino acid transferases in different diseases: Research progress and future prospects.

It is well known that the enzyme catalyzes the first step of branched-chain amino acid (BCAA) catabolism is branched-chain amino transferase (BCAT), which is involved in the synthesis and degradation of leucine, isoleucine and valine. There are two main subtypes of human branched chain amino transferase (hBCAT), including cytoplasmic BCAT (BCAT1) and mitochondrial BCAT (BCAT2). In recent years, the role of BCAT in tumors has attracted the attention of scientists, and there have been continuous research reports that BCAT plays a role in the tumor, Alzheimer’s disease, myeloid leukaemia and other diseases. It plays a significant role in the growth and development of diseases, and new discoveries about this gene in some diseases are made every year. BCAT usually promotes cancer proliferation and invasion by activating the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathway and activating Wnt/β-catenin signal transduction. This article reviews the role and mechanism of BCAT in different diseases, as well as the recent biomedical research progress. This review aims to make a comprehensive summary of the role and mechanism of BCAT in different diseases and to provide new research ideas for the treatment, prognosis and prevention of certain diseases.

Obese Skeletal Muscle-Expressed Interferon Regulatory Factor 4 Transcriptionally Regulates Mitochondrial Branched-Chain Aminotransferase Reprogramming Metabolome.

In addition to the significant role in physical activity, skeletal muscle also contributes to health through the storage and use of macronutrients associated with energy homeostasis. However, the mechanisms of regulating integrated metabolism in skeletal muscle are not well-defined. Here, we compared the skeletal muscle transcriptome from obese and lean control subjects in different species (human and mouse) and found that interferon regulatory factor 4 (IRF4), an inflammation-immune transcription factor, conservatively increased in obese subjects. Thus, we investigated whether IRF4 gain of function in the skeletal muscle predisposed to obesity and insulin resistance. Conversely, mice with specific IRF4 loss in skeletal muscle showed protection against the metabolic effects of high-fat diet, increased branched-chain amino acids (BCAA) level of serum and muscle, and reprogrammed metabolome in serum. Mechanistically, IRF4 could transcriptionally upregulate mitochondrial branched-chain aminotransferase (BCATm) expression; subsequently, the enhanced BCATm could counteract the effects caused by IRF4 deletion. Furthermore, we demonstrated that IRF4 ablation in skeletal muscle enhanced mitochondrial activity, BCAA, and fatty acid oxidation in a BCATm-dependent manner. Taken together, these studies, for the first time, established IRF4 as a novel metabolic driver of macronutrients via BCATm in skeletal muscle in terms of diet-induced obesity.

The Oncogene MYC Regulates the Branched Chain Amino Acid Metabolism and mTOR Signaling in Diffuse Large B Cell Lymphoma

Diffuse large B‐cell Lymphoma (DLBCL) is the most common type of aggressive non‐Hodgkin lymphoma (NHL) worldwide. While therapeutic strategies to combat DLBCL have advanced, these malignancies continue to be challenging to treat due to their heterogeneity and frequently recurring mutations. Aberrations in the oncogene MYC are commonly found in high grade DLBCL and are associated with poor responses to standard therapy.In search of downstream targets of MYC in DLBCL, we found MYC‐binding sites in the promoter regions of BCAT1, BCAT2, and BCKDHA. These genes encode enzymes in the metabolism of the branched chain amino acids (BCAAs), and they have been previously linked to cancer progression. BCAT1 and 2 encode the cytosolic (BCATc) and mitochondrial (BCATm) branched‐chain aminotransferases, while BCKDHA encodes the E1α subunit of the branched‐chain ketoacid dehydrogenase complex. We hypothesized that MYC activates these genes in DLBCL tissues leading to upregulation of BCAA metabolism and lymphoma growth.Human tissues collected from DLBCL patients (n=89) or controls (n=33) were used to analyze the gene expression of MYC, BCAT1, BCAT2 and BCKDHA followed by correlative analysis of the expression of these genes and the survival of DLBCL patients. Next, human DLBCL and B‐lymphoblast (control) cells were treated with the MYC inhibitor 10058‐F4 (0, 25, 50, 100 µM), the BCAA antagonist N‐acetyl leucine amide (NALA, 0, 10, 20, 40 mM) and the BCATc inhibitor gabapentin (0, 5, 10, 20 mM). 24 hours post treatment, the cell viability and cell proliferation, as well as the protein expression of BCATc, BCATm, and E1α, and the total and phosphorylation states of the ribosomal protein S6 (a downstream target of the mammalian target of rapamycin, mTOR) were determined.Results showed that the gene expression of BCAT1, BCAT2and BCKDHA were significantly elevated in tumor tissues from DLBCL patients compared to controls, and there was a positive correlation between their expression and the upregulation of MYC in DLBCL tissues. The high expression of MYC and BCAT2, but not that of BCAT1 and BCKDHA, correlated with poor overall cancer survival. Inhibiting the MYC function by 10058‐F4, the BCAA uptake by NALA, and the BCATc activity by gabapentin led to concentration dependent reductions in the cell viability and proliferation of the DLBCL cells. The protein expression of BCATm and E1α were reduced in the presence of 10058‐F4, which correlated with downregulation of mTOR signaling as measured by the deceased phosphorylation state of S6 in treated DLBCL cells. These results support the hypothesis that MYC is an upstream regulator of genes associated with BCAA metabolism. The results also demonstrate that DLBCL cells need MYC and BCAAs to upregulate mTOR signaling and to survive and may represent a new molecular mechanism via which MYC exerts its effects in DLBCL lymphoma.

Abstract 1807: T cell conditional knockout mouse model of the mitochondrial branched chain aminotransferase (BCATm) responds with delayed tumor growth to a lymphoma challenge

Abstract Enzymes in amino acid catabolism are emerging as attractive targets for cancer therapies. The mitochondrial branched-chain amino transferase (BCATm), an enzyme catalyzing the first step in leucine, valine, and isoleucine degradation, has been recently implicated in cancer growth but its role in T cell driven anti-cancer immunity has never been explored. To fulfill the gap in current scientific knowledge and address some of the challenges with lymphoma immunotherapies, we used the CD4-Cre-Lox recombination to create a conditional knockout mouse (T-BCATmKO) with CD4+ and CD8+ T cells deficient in the Bcat2 gene that encodes BCATm. Based on initial studies showing that a deletion of BCATm from T cells leads to increased glycolytic and oxygen metabolism and up-regulation of complex 1 of the mammalian target of rapamycin (mTORC1) signaling, we hypothesized that if BCATm is absent from T cells, this would stimulate T cell activation leading to improved T cell function in mice challenged with lymphoma. For this purpose, T-BCATmKO and T-BCATmfl/fl male mice (15-16 weeks old) were subcutaneously injected with 2.5x105 mouse lymphoma EG7-OVA cells. Tumor growth, animal weight, and food intake were recorded daily for a period of 20 days. Mice developed tumors around day 10 and this was not associated with weight loss or poor feeding of the mice as no changes in body weights or food intake between the different mouse groups were observed. Statistical analysis via one-way ANOVA revealed that T-BCATmKO mice had significantly delayed tumor growth compared to T-BCATmfl/fl mice (P= 0.05). In addition, CD4+ T cells isolated from T-BCATmKO mice released significantly more IFNγ after 48-72 hours of activation suggesting that CD4+ T cells from T-BCATmKO mice were more active than T cells from T-BCATmfl/fl mice. This study revealed that targeting BCATm in T cells may offer an advantage in mice to better fight lymphoma cancer and represents an attractive target for future immunotherapeutic anti-cancer approaches. Citation Format: Christie Adam, Alexander Martin, Rebekah Betar, Mercedes Foster, Michael Boyer, Elitsa Ananieva. T cell conditional knockout mouse model of the mitochondrial branched chain aminotransferase (BCATm) responds with delayed tumor growth to a lymphoma challenge [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1807.

Emerging role of the mitochondrial branched-chain aminotransferase (BCATm) as an immunosuppressive enzyme during CD4+ T cell activation

Abstract The mitochondrial branched-chain aminotransferase (BCATm) catalyzes the transamination of leucine, an amino acid that is essential for the upregulation of complex 1 of the mammalian target of rapamycin (mTORC1) during T cell activation. Because BCATm is responsible for the mitochondrial degradation of leucine, we hypothesized that BCATm limits leucine availability for mTORC1 signaling and subsequently suppresses T cell activation. To test the hypothesis, we isolated CD4+T cells from the global BCATmKO mouse and measured the rates of leucine transamination/oxidation, the activity of the mTORC1 downstream targets S6 and 4EBP-1, and the release of IFNγ after 24–72h of T cell activation. To elucidate the role of BCATm in T cells in vivo, we used the Cre-LoxP strategy to create a T cell specific deletion of BCATm in C57BL/6 mice. We completed the initial characterization of the mice, which included a validation of absence of BCATm in T cells by using qRT-PCR and Western blotting. Results demonstrated that BCATm-deficient T cells experienced significant reductions in the transamination and oxidation of leucine, which led to increased intracellular leucine concentrations. This correlated with an increased phosphorylation of S6, but not 4EBP-1, suggesting that BCATm regulates mTORC1 signaling in S6 dependent manner. The T cells released more IFNγ in the absence of BCATm, which was highest after 72h of T cell activation. Thus, BCATm appears to play an immunosuppressive role by reducing CD4+ T cell activation. The newly designed T cell conditional BCATmKO mice, which do not express BCATm in CD4+ and CD8+ T cells will be engaged in preclinical studies aiming at deciphering the therapeutic potential of BCATm in T cell-driven immunological responses.

Abstract MP125: Branched-chain Keto Acids, Not Branched-chain Amino Acids, Impairs Cardiac Insulin Sensitivity by Disrupting Insulin Signaling in the Mitochondria

Alterations in branched-chain amino acids (BCAA) oxidation have been linked to the development of cardiac insulin resistance and its negative impact on cardiac function. However, it is not clear if these detrimental effects are due to the accumulation of BCAAs or branched-chain keto acids (BCKAs). It is also unknown how impaired BCAAs oxidation mediates cardiac insulin resistance. To examine this, we specifically deleted mitochondrial branched-chain aminotransferase (BCATm) in the heart to selectively increase in BCAAs and decrease in BCKAs in the heart. BCATm -/- mice had normal cardiac function compared to their wildtype littermates (WT Cre+/+ ). However, there was a significant increase in insulin-stimulated cardiac glucose oxidation rates in BCATm -/- mice, independent of any changes in glucose uptake or glycolytic rates. This enhancement in cardiac insulin sensitivity was associated with an increase in the phosphorylation of Akt and activation of pyruvate dehydrogenase (PDH), the rate-limiting enzyme of glucose oxidation. To determine the impact of reversing these events, we examined the effects of increasing cardiac BCKAs on cardiac insulin sensitivity. We perfused isolated working mice hearts with high levels of BCKAs (α;-keto-isocaproate 80 μM, α;-keto-β;-methylvalorate 100μM, α;-keto-isovalorate 70 μM), levels that can be seen during diabetes and obesity. The BCKAs completely blunted insulin-stimulated glucose oxidation rates. We also found that BCKAs abolished insulin-stimulated mitochondrial translocation of Akt, an effect which was associated with PDH deactivation. We conclude that the accumulation of BCKAs, and not BCAAs, is a major contributor to cardiac insulin resistance via abrogating mitochondrial translocation of Akt.

A Loss of Function of the Mitochondrial Branched-Chain Aminotransferase (BCATm) Leads to Increased Glycolytic and Oxidative Metabolism in Activated CD4+ T Cells

Abstract Objectives T cells use the amino acid leucine to respond to their increased biosynthetic demands during activation. However, once inside T cells, leucine is subjected to degradation, which is initiated by the mitochondrial branched-chain aminotransferase (BCATm) that catalyzes the reversible transamination of leucine. We hypothesized that if BCATm is absent from T cells, this would provide more intracellular leucine to stimulate T cell metabolism. Methods To explore the dependence of T cells on BCATm function, we isolated CD4+ T cells from spleens of wild type (WT) and BCATm global knockout (KO) mice, and after cell activation with anti-CD3 and anti-CD28 for 24 h, we measured leucine transamination, glycolysis, mitochondrial respiration and ATP synthesis, the activity of the mammalian target of rapamycin (mTOR) pathway, and the release of IFN-γ. Results The global deletion of BCATm resulted in a 1.8-fold reduction in leucine transamination and a 1.2-fold increase in the intracellular leucine concentrations in activated CD4+ T cells from BCATmKO mice. These T cells demonstrated 4.0– and 5.0-fold increases in glycolysis and glycolytic capacity, along with 1.8– and 2-0-fold increases in the maximal respiration and spare respiratory capacity when compared to WT T cells after 24 h of activation. In addition, mTOR signaling was more active in BCATmKO T cells and their IFN-γ release was increased by 2.1-fold relative to WT T cells. Conclusions The results suggested that leucine catabolism at the BCATm step negatively affects T cell metabolism by limiting glycolytic intermediates for biosynthetic needs and mitochondrial respiration for energy. Thus, leucine catabolism is regarded as a metabolic checkpoint of T cells that may prove useful for therapeutic purposes. Funding Sources Des Moines University, (IOER-112-3705 to EAS), the National Institute of Health (DK 34,738 to SMH).

Crystal structure of an oxidized mutant of human mitochondrial branched-chain aminotransferase.

This study presents the crystal structure of a thiol variant of the human mitochondrial branched-chain aminotransferase protein. Human branched-chain aminotransferase (hBCAT) catalyzes the transamination of the branched-chain amino acids leucine, valine and isoleucine and α-ketoglutarate to their respective α-keto acids and glutamate. hBCAT activity is regulated by a CXXC center located approximately 10 Å from the active site. This redox-active center facilitates recycling between the reduced and oxidized states, representing hBCAT in its active and inactive forms, respectively. Site-directed mutagenesis of the redox sensor (Cys315) results in a significant loss of activity, with no loss of activity reported on the mutation of the resolving cysteine (Cys318), which allows the reversible formation of a disulfide bond between Cys315 and Cys318. The crystal structure of the oxidized form of the C318A variant was used to better understand the contributions of the individual cysteines and their oxidation states. The structure reveals the modified CXXC center in a conformation similar to that in the oxidized wild type, supporting the notion that its regulatory mechanism depends on switching the Cys315 side chain between active and inactive conformations. Moreover, the structure reveals conformational differences in the N-terminal and inter-domain region that may correlate with the inactivated state of the CXXC center.

Natural and Artificial Sweeteners and High Fat Diet Modify Differential Taste Receptors, Insulin, and TLR4-Mediated Inflammatory Pathways in Adipose Tissues of Rats.

It is difficult to know if the cause for obesity is the type of sweetener, high fat (HF) content, or the combination of sweetener and fat. The purpose of the present work was to study different types of sweeteners; in particular, steviol glycosides (SG), glucose, fructose, sucrose, brown sugar, honey, SG + sucrose (SV), and sucralose on the functionality of the adipocyte. Male Wistar rats were fed for four months with different sweeteners or sweetener with HF added. Taste receptors T1R2 and T1R3 were differentially expressed in the tongue and intestine by sweeteners and HF. The combination of fat and sweetener showed an additive effect on circulating levels of GIP and GLP-1 except for honey, SG, and brown sugar. In adipose tissue, sucrose and sucralose stimulated TLR4, and c-Jun N-terminal (JNK). The combination of HF with sweeteners increased NFκB, with the exception of SG and honey. Honey kept the insulin signaling pathway active and the smallest adipocytes in white (WAT) and brown (BAT) adipose tissue and the highest expression of adiponectin, PPARγ, and UCP-1 in BAT. The addition of HF reduced mitochondrial branched-chain amino transferase (BCAT2) branched-chain keto acid dehydrogenase E1 (BCKDH) and increased branched chain amino acids (BCAA) levels by sucrose and sucralose. Our data suggests that the consumption of particular honey maintained functional adipocytes despite the consumption of a HF diet.

Mice deficient in the mitochondrial branched-chain aminotransferase (BCATm) respond with delayed tumour growth to a challenge with EL-4 lymphoma

BackgroundThe mitochondrial branched-chain aminotransferase (BCATm) is a recently discovered cancer marker with a poorly defined role in tumour progression.MethodsTo understand how a loss of function of BCATm affects cancer, the global knockout mouse BCATmKO was challenged with EL-4 lymphoma under different diet compositions with varying amounts of branched-chain amino acids (BCAAs). Next, the growth and metabolism of EL-4 cells were studied in the presence of different leucine concentrations in the growth medium.ResultsBCATmKO mice experienced delayed tumour growth when fed standard rodent chow or a normal BCAA diet. Tumour suppression correlated with 37.6- and 18.9-fold increases in plasma and tumour BCAAs, 37.5% and 30.4% decreases in tumour glutamine and alanine, and a 3.5-fold increase in the phosphorylation of tumour AMPK in BCATmKO mice on standard rodent chow. Similar results were obtained with a normal but not with a choice BCAA diet.ConclusionsGlobal deletion of BCATm caused a dramatic build-up of BCAAs, which could not be utilised for energy or amino acid synthesis, ultimately delaying the growth of lymphoma tumours. Furthermore, physiological, but not high, leucine concentrations promoted the growth of EL-4 cells. BCATm and BCAA metabolism were identified as attractive targets for anti-lymphoma therapy.

Divergent Induction of Branched-Chain Aminotransferases and Phosphorylation of Branched Chain Keto-Acid Dehydrogenase Is a Potential Mechanism Coupling Branched-Chain Keto-Acid-Mediated-Astrocyte Activation to Branched-Chain Amino Acid Depletion-Mediated Cognitive Deficit after Traumatic Brain Injury.

Deficient branched-chain amino acids (BCAAs) are implicated in cognitive dysfunction after traumatic brain injury (TBI). The mechanism remains unknown. BCAAs are catabolized by neuron-specific cytosolic and astrocyte-specific mitochondrial branched-chain aminotransferases (BCATc, BCATm) to generate glutamate and branched-chain keto-acids (BCKAs) that are metabolized by the mitochondrial branched-chain keto-acid dehydrogenase (BCKD) whose activity is regulated by its phosphorylation state. BCKD phosphorylation by BCKD kinase (BCKDK) inactivates BCKD and cause neurocognitive dysfunction, whereas dephosphorylation by specific phosphatase restores BCKD activity. Real-time polymerase chain reaction showed rapidly and significantly decreased BCATc messenger RNA (mRNA) levels, but significantly increased BCATm mRNA level post-CCI (controlled cortical impact). BCKD and BCKDK mRNA decreased significantly immediately after CCI-induced TBI (CCI) in the rat. Phosphorylated BCKD proteins (pBCKD) increased significantly in the ipsilateral-CCI hemisphere. Immunohistochemistry revealed significantly increased pBCKD proteins in ipsilateral astrocytes post-CCI. BCKD protein expression is higher in primarily cultured cortical neurons than in astrocytes, whereas pBCKD protein level is higher in astrocytes than in cortical neurons. Transforming growth factor beta treatment (10 μg/mL for 48 h) significantly increased pBCKD protein expression in astrocytes, whereas glutamate treatment (25 μM for 24 h) significantly decreased pBCKD protein in neurons. Because increased pBCKD would lead to increased BCKA accumulation, BCKA-mediated astrocyte activation, cell death, and cognitive dysfunction as found in maple syrup urine disease; thus, TBI may potentially induce cognitive deficit through diverting BCAA from glutamate production in neurons to BCKA production in astrocytes through the pBCKD-dependent mechanism.

Branched-chain amino acid metabolism in cancer.

Purpose of reviewThe current review aims to provide an update on the recent biomedical interest in oncogenic branched-chain amino acid (BCAA) metabolism, and discusses the advantages of using BCAAs and expression of BCAA-related enzymes in the treatment and diagnosis of cancers.Recent findingsAn accumulating body of evidence demonstrates that BCAAs are essential nutrients for cancer growth and are used by tumors in various biosynthetic pathways and as a source of energy. In addition, BCAA metabolic enzymes, such as the cytosolic branched-chain aminotransferase 1 (BCAT1) and mitochondrial branched-chain aminotransferase 2, have emerged as useful prognostic cancer markers. BCAT1 expression commonly correlates with more aggressive cancer growth and progression, and has attracted substantial scientific attention in the past few years. These studies have found the consequences of BCAT1 disruption to be heterogeneous; not all cancers share the same requirements for BCAA metabolites and the function of BCAT1 appears to vary between cancer types.SummaryBoth oncogenic mutations and cancer tissue-of-origin influence BCAA metabolism and expression of BCAA-associated metabolic enzymes. These new discoveries need to be taken into consideration during the development of new cancer therapies that target BCAA metabolism.

Liver BCATm transgenic mouse model reveals the important role of the liver in maintaining BCAA homeostasis

Unlike other amino acids, the branched-chain amino acids (BCAAs) largely bypass first-pass liver degradation due to a lack of hepatocyte expression of the mitochondrial branched-chain aminotransferase (BCATm). This sets up interorgan shuttling of BCAAs and liver–skeletal muscle cooperation in BCAA catabolism. To explore whether complete liver catabolism of BCAAs may impact BCAA shuttling in peripheral tissues, the BCATm gene was stably introduced into mouse liver. Two transgenic mouse lines with low and high hepatocyte expression of the BCATm transgene (LivTg-LE and LivTg-HE) were created and used to measure liver and plasma amino acid concentrations and determine whether the first two BCAA enzymatic steps in liver, skeletal muscle, heart and kidney were impacted. Expression of the hepatic BCATm transgene lowered the concentrations of hepatic BCAAs while enhancing the concentrations of some nonessential amino acids. Extrahepatic BCAA metabolic enzymes and plasma amino acids were largely unaffected, and no growth rate or body composition differences were observed in the transgenic animals as compared to wild-type mice. Feeding the transgenic animals a high-fat diet did not reverse the effect of the BCATm transgene on the hepatic BCAA catabolism, nor did the high-fat diet cause elevation in plasma BCAAs. However, the high-fat-diet-fed BCATm transgenic animals experienced attenuation in the mammalian target of rapamycin (mTOR) pathway in the liver and had impaired blood glucose tolerance. These results suggest that complete liver BCAA metabolism influences the regulation of glucose utilization during diet-induced obesity.

Metformin inhibits Branched Chain Amino Acid (BCAA) derived ketoacidosis and promotes metabolic homeostasis in MSUD.

Maple Syrup Urine Disease (MSUD) is an inherited disorder caused by the dysfunction in the branched chain keto-acid dehydrogenase (BCKDH) enzyme. This leads to buildup of branched-chain keto-acids (BCKA) and branched-chain amino acids (BCAA) in body fluids (e.g. keto-isocaproic acid from the BCAA leucine), leading to numerous clinical features including a less understood skeletal muscle dysfunction in patients. KIC is an inhibitor of mitochondrial function at disease relevant concentrations. A murine model of intermediate MSUD (iMSUD) shows significant skeletal muscle dysfunction as by judged decreased muscle fiber diameter. MSUD is an orphan disease with a need for novel drug interventions. Here using a 96-well plate (liquid chromatography- mass spectrometry (LC-MS) based drug-screening platform we show that Metformin, a widely used anti-diabetic drug, reduces levels of KIC in patient-derived fibroblasts by 20–50%. This Metformin-mediated effect was conserved in vivo; Metformin-treatment significantly reduced levels of KIC in the muscle (by 69%) and serum (by 56%) isolated from iMSUD mice, and restored levels of mitochondrial metabolites (e.g. AMP and other TCA). The drug also decreased the expression of mitochondrial branched chain amino transferase (BCAT) which produces KIC in skeletal muscle. This suggests that Metformin can restore skeletal muscle homeostasis in MSUD by decreasing mitochondrial KIC production.

Leucine metabolism is a promising new approach to study the interplay between T cells and cancer cells

Abstract Cancer and T cells undergo similar metabolic reprogramming during proliferation to support increased biosynthetic and energy demands. They both rely on high rates of glycolysis while retaining mitochondrial respiration, a phenomenon known as the Warburg effect. In the tumor microenvironment, tumor cells can escape immune surveillance by competing with T cells for common metabolites and redirecting nutrients for their own advantage. To gain insight into the relationship between T cells and cancer cells, we studied the effect of leucine metabolism on T cells and their cancerous counterpart, EL-4 lymphoma cells. Our previous results showed that a loss of expression of leucine degrading enzymes (cytosolic or mitochondrial branched chain aminotransferases, BCATc and BCATm, respectively) increased intracellular leucine concentrations and stimulated glycolytic rate in activated T cells. These results correlated with activation of complex 1 of the mammalian target of rapamycin (mTORC1). Continuation of this study with EL-4 lymphoma cells, grown in leucine free media or treated with the leucine antagonist, n-acetyl-leucine amide, revealed a decrease in glycolysis, glycolytic capacity, and glycolytic reserve while mTORC1 was only moderately impacted. The oxygen consumption rate was stimulated in T cells lacking BCATc and/or BCATm but was decreased in the absence of leucine in EL-4 cells. The results suggest that T cells and their cancerous counterpart use leucine in a similar manner and that leucine is essential for the regulation of glucose and oxygen metabolism in either cell type. Future studies targeting leucine metabolism in the tumor microenvironment will aim to improve the effectiveness of T cells to fight cancer cells.

Structurally Diverse Mitochondrial Branched Chain Aminotransferase (BCATm) Leads with Varying Binding Modes Identified by Fragment Screening

Inhibitors of mitochondrial branched chain aminotransferase (BCATm), identified using fragment screening, are described. This was carried out using a combination of STD-NMR, thermal melt (Tm), and biochemical assays to identify compounds that bound to BCATm, which were subsequently progressed to X-ray crystallography, where a number of exemplars showed significant diversity in their binding modes. The hits identified were supplemented by searching and screening of additional analogues, which enabled the gathering of further X-ray data where the original hits had not produced liganded structures. The fragment hits were optimized using structure-based design, with some transfer of information between series, which enabled the identification of ligand efficient lead molecules with micromolar levels of inhibition, cellular activity, and good solubility.

Altered Expression of Human Mitochondrial Branched Chain Aminotransferase in Dementia with Lewy Bodies and Vascular Dementia

Cytosolic and mitochondrial human branched chain aminotransferase (hBCATc and hBCATm, respectively) play an integral role in brain glutamate metabolism. Regional increased levels of hBCATc in the CA1 and CA4 region of Alzheimer’s disease (AD) brain together with increased levels of hBCATm in frontal and temporal cortex of AD brains, suggest a role for these proteins in glutamate excitotoxicity. Glutamate toxicity is a key pathogenic feature of several neurological disorders including epilepsy associated dementia, AD, vascular dementia (VaD) and dementia with Lewy bodies (DLB). To further understand if these increases are specific to AD, the expression profiles of hBCATc and hBCATm were examined in other forms of dementia including DLB and VaD. Similar to AD, levels of hBCATm were significantly increased in the frontal and temporal cortex of VaD cases and in frontal cortex of DLB cases compared to controls, however there were no observed differences in hBCATc between groups in these areas. Moreover, multiple forms of hBCATm were observed that were particular to the disease state relative to matched controls. Real-time PCR revealed similar expression of hBCATm mRNA in frontal and temporal cortex for all cohort comparisons, whereas hBCATc mRNA expression was significantly increased in VaD cases compared to controls. Collectively our results suggest that hBCATm protein expression is significantly increased in the brains of DLB and VaD cases, similar to those reported in AD brain. These findings indicate a more global response to altered glutamate metabolism and suggest common metabolic responses that might reflect shared neurodegenerative mechanisms across several forms of dementia.

Maternal Factors Are Associated with the Expression of Placental Genes Involved in Amino Acid Metabolism and Transport.

IntroductionMaternal environment and lifestyle factors may modify placental function to match the mother’s capacity to support the demands of fetal growth. Much remains to be understood about maternal influences on placental metabolic and amino acid transporter gene expression. We investigated the influences of maternal lifestyle and body composition (e.g. fat and muscle content) on a selection of metabolic and amino acid transporter genes and their associations with fetal growth.MethodsRNA was extracted from 102 term Southampton Women’s Survey placental samples. Expression of nine metabolic, seven exchange, eight accumulative and three facilitated transporter genes was analyzed using quantitative real-time PCR.ResultsIncreased placental LAT2 (p = 0.01), y + LAT2 (p = 0.03), aspartate aminotransferase 2 (p = 0.02) and decreased aspartate aminotransferase 1 (p = 0.04) mRNA expression associated with pre-pregnancy maternal smoking. Placental mRNA expression of TAT1 (p = 0.01), ASCT1 (p = 0.03), mitochondrial branched chain aminotransferase (p = 0.02) and glutamine synthetase (p = 0.05) was positively associated with maternal strenuous exercise. Increased glutamine synthetase mRNA expression (r = 0.20, p = 0.05) associated with higher maternal diet quality (prudent dietary pattern) pre-pregnancy. Lower LAT4 (r = -0.25, p = 0.05) and aspartate aminotransferase 2 mRNA expression (r = -0.28, p = 0.01) associated with higher early pregnancy diet quality. Lower placental ASCT1 mRNA expression associated with measures of increased maternal fat mass, including pre-pregnancy BMI (r = -0.26, p = 0.01). Lower placental mRNA expression of alanine aminotransferase 2 associated with greater neonatal adiposity, for example neonatal subscapular skinfold thickness (r = -0.33, p = 0.001).ConclusionA number of maternal influences have been linked with outcomes in childhood, independently of neonatal size; our finding of associations between placental expression of transporter and metabolic genes and maternal smoking, physical activity and diet raises the possibility that their effects are mediated in part through alterations in placental function. The observed changes in placental gene expression in relation to modifiable maternal factors are important as they could form part of interventions aimed at maintaining a healthy lifestyle for the mother and for optimal fetal development.