Leucine and branched-chain amino acid metabolism contribute to the growth of bone sarcomas by regulating AMPK and mTORC1 signaling.

Osteosarcoma and chondrosarcoma are sarcomas of the bone and the cartilage that are primarily treated by surgical intervention combined with high toxicity chemotherapy. In search of alternative metabolic approaches to address the challenges in treating bone sarcomas, we assessed the growth dependence of these cancers on leucine, one of the branched-chain amino acids (BCAAs), and BCAA metabolism. Tumor biopsies from bone sarcoma patients revealed differential expression of BCAA metabolic enzymes. The cytosolic branched-chain aminotransferase (BCATc) that is commonly overexpressed in cancer cells, was down-regulated in chondrosarcoma (SW1353) in contrast with osteosarcoma (143B) cells that expressed both BCATc and its mitochondrial isoform BCATm. Treating SW1353 cells with gabapentin, a selective inhibitor of BCATc, further revealed that these cells failed to respond to gabapentin. Application of the structural analog of leucine, N-acetyl-leucine amide (NALA) to disrupt leucine uptake, indicated that all bone sarcoma cells used leucine to support their energy metabolism and biosynthetic demands. This was evident from the increased activity of the energy sensor AMP-activated protein kinase (AMPK), down-regulation of complex 1 of the mammalian target of rapamycin (mTORC1), and reduced cell viability in response to NALA. The observed changes were most profound in the 143B cells, which appeared highly dependent on cytosolic and mitochondrial BCAA metabolism. This study thus demonstrates that bone sarcomas rely on leucine and BCAA metabolism for energy and growth; however, the differential expression of BCAA enzymes and the presence of other carbon sources may dictate how efficiently these cancer cells take advantage of BCAA metabolism.

Cytosolic branched chain aminotransferase (BCATc) regulates mTORC1 signaling and glycolytic metabolism in CD4+ T cells.

Here we show that expression of the cytosolic branched chain aminotransferase (BCATc) is triggered by the T cell receptor (TCR) of CD4(+) T cells. Induction of BCATc correlates with increased Leu transamination, whereas T cells from the BCATc(-/-) mouse exhibit lower Leu transamination and higher intracellular Leu concentrations than the cells from wild type (WT) mice. Induction of BCATc by TCR in WT cells is prevented by the calcineurin-nuclear factor of activated T cells (NFAT) inhibitor, cyclosporin A (CsA), suggesting that NFAT controls BCATc expression. Leu is a known activator of the mammalian target of rapamycin complex 1 (mTORC1). mTOR is emerging as a critical regulator of T cell activation, differentiation, and metabolism. Activated T cells from BCATc(-/-) mice show increased phosphorylation of mTORC1 downstream targets, S6 and 4EBP-1, indicating higher mTORC1 activation than in T cells from WT mice. Furthermore, T cells from BCATc(-/-) mice display higher rates of glycolysis, glycolytic capacity, and glycolytic reserve when compared with activated WT cells. These findings reveal BCATc as a novel regulator of T cell activation and metabolism and highlight the important role of Leu metabolism in T cells.

Targeted inhibition of branched-chain amino acid metabolism drives apoptosis of glioblastoma by facilitating ubiquitin degradation of Mfn2 and oxidative stress.

Glioblastoma is one of the most challenging malignancies with high aggressiveness and invasiveness and its development and progression of glioblastoma highly depends on branched-chain amino acid (BCAA) metabolism. The study aimed to investigate effects of inhibition of BCAA metabolism with cytosolic branched-chain amino acid transaminase (BCATc) Inhibitor 2 on glioblastoma, elucidate its underlying mechanisms, and explore therapeutic potential of targeting BCAA metabolism. The expression of BCATc was upregulated in glioblastoma and BCATc Inhibitor 2 precipitated apoptosis both in vivo and in vitro with the activation of Bax/Bcl2/Caspase-3/Caspase-9 axis. In addition, BCATc Inhibitor 2 promoted K63-linkage ubiquitination of mitofusin 2 (Mfn2), which subsequently caused lysosomal degradation of Mfn2, and then oxidative stress, mitochondrial fission and loss of mitochondrial membrane potential. Furthermore, BCATc Inhibitor 2 treatment resulted in metabolic reprogramming, and significant inhibition of expression of ATP5A, UQCRC2, SDHB and COX II, indicative of suppressed oxidative phosphorylation. Moreover, Mfn2 overexpression or scavenging mitochondria-originated reactive oxygen species (ROS) with mito-TEMPO ameliorated BCATc Inhibitor 2-induced oxidative stress, mitochondrial membrane potential disruption and mitochondrial fission, and abrogated the inhibitory effect of BCATc Inhibitor 2 on glioblastoma cells through PI3K/AKT/mTOR signaling. All of these findings indicate suppression of BCAA metabolism promotes glioblastoma cell apoptosis via disruption of Mfn2-mediated mitochondrial dynamics and inhibition of PI3K/AKT/mTOR pathway, and suggest that BCAA metabolism can be targeted for developing therapeutic agents to treat glioblastoma.

Adipose transplant for inborn errors of branched chain amino acid metabolism in mice.

Liver transplantation appears to be quite beneficial for treatment of maple syrup urine disease (MSUD, an inherited disorder of branched chain amino acid metabolism); however, there is a limited availability of donor livers worldwide and the first year costs of liver transplants are quite high. Recent studies have suggested that intact adipose tissue, already widely used in reconstructive surgery, may have an underappreciated high capacity for branched chain amino acid (BCAA) metabolism. Here we examined the potential for adipose tissue transplant to lower circulating BCAAs in two models of defective BCAA metabolism, BCATm and PP2Cm [branched chain keto acid dehydrogenase complex (BCKDC) phosphatase] knockout (KO) mice. After 1-2g fat transplant, BCATm and PP2Cm KO mice gained or maintained body weight 3weeks after surgery and consumed similar or more food/BCAAs the week before phlebotomy. Transplant of fat into the abdominal cavity led to a sterile inflammatory response and nonviable transplanted tissue. However when 1-2g of fat was transplanted subcutaneously into the back, either as small (0.1-0.3g) or finely minced pieces introduced with an 18-ga. needle, plasma BCAAs decreased compared to Sham operated mice. In two studies on BCATm KO mice and one study on PP2Cm KO mice, fat transplant led to 52-81% reductions in plasma BCAAs compared to baseline plasma BCAA concentrations of untreated WT type siblings. In PP2Cm KO mice, individual BCAAs in plasma were also significantly reduced by fat transplant, as were the alloisoleucine/Phe ratios. Therefore, subcutaneous fat transplantation may have merit as an adjunct to dietary treatment of MSUD. Additional studies are needed to further refine this approach.

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.

Leucine Metabolism in T Cell Activation: mTOR Signaling and Beyond.

In connection with the increasing interest in metabolic regulation of the immune response, this review discusses current advances in understanding the role of leucine and leucine metabolism in T lymphocyte (T cell) activation. T cell activation during the development of an immune response depends on metabolic reprogramming to ensure that sufficient nutrients and energy are taken up by the highly proliferating T cells. Leucine has been described as an important essential amino acid and a nutrient signal that activates complex 1 of the mammalian target of rapamycin (mTORC1), which is a critical regulator of T cell proliferation, differentiation, and function. The role of leucine in these processes is further discussed in relation to amino acid transporters, leucine-degrading enzymes, and other metabolites of leucine metabolism. A new model of T cell regulation by leucine is proposed and outlines a chain of events that leads to the activation of mTORC1 in T cells.