Oxidation of branched-chain amino acids (BCAAs) is tightly regulated in mammals. We review here the distribution and regulation of whole-body BCAA oxidation. Phosphorylation and dephosphorylation of the rate-limiting enzyme, branched-chain α-ketoacid dehydrogenase complex directly regulates BCAA oxidation, and various other indirect mechanisms of regulation also exist. Most tissues throughout the body are capable of BCAA oxidation, and the flux of oxidative BCAA disposal in each tissue is influenced by three key factors: 1. tissue-specific preference for BCAA oxidation relative to other fuels, 2. the overall oxidative activity of mitochondria within a tissue, and 3. total tissue mass. Perturbations in BCAA oxidation have been implicated in many disease contexts, underscoring the importance of BCAA homeostasis in overall health.

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.

The mammalian or mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) is a ubiquitously expressed multimeric protein kinase complex that integrates nutrient and growth factor signals for the co-ordinated regulation of cellular metabolism and cell growth. Herein, we demonstrate that suppressing the cellular activity of glycogen synthase kinase-3 (GSK3), by use of pharmacological inhibitors or shRNA-mediated gene silencing, results in substantial reduction in amino acid (AA)-regulated mTORC1-directed signalling, as assessed by phosphorylation of multiple downstream mTORC1 targets. We show that GSK3 regulates mTORC1 activity through its ability to phosphorylate the mTOR-associated scaffold protein raptor (regulatory-associated protein of mTOR) on Ser 859 . We further demonstrate that either GSK3 inhibition or expression of a S859A mutated raptor leads to reduced interaction between mTOR and raptor and under these circumstances, irrespective of AA availability, there is a consequential loss in phosphorylation of mTOR substrates, such as p70S6K1 (ribosomal S6 kinase 1) and uncoordinated-51-like kinase (ULK1), which results in increased autophagic flux and reduced cellular proliferation.

In the present study, the BCAAs (branched-chain amino acids) leucine and valine caused a significant suppression in the loss of body weight in mice bearing a cachexia-inducing tumour (MAC16), producing a significant increase in skeletal muscle wet weight, through an increase in protein synthesis and a decrease in degradation. Leucine attenuated the increased phosphorylation of PKR (double-stranded-RNA-dependent protein kinase) and eIF2α (eukaryotic initiation factor 2α) in skeletal muscle of mice bearing the MAC16 tumour, due to an increased expression of PP1 (protein phosphatase 1). Weight loss in mice bearing the MAC16 tumour was associated with an increased amount of eIF4E bound to its binding protein 4E-BP1 (eIF4E-binding protein 1), and a progressive decrease in the active eIF4G–eIF4E complex due to hypophosphorylation of 4E-BP1. This may be due to a reduction in the phosphorylation of mTOR (mammalian target of rapamycin), which may also be responsible for the decreased phosphorylation of p70 S6k (70 kDa ribosomal S6 kinase). There was also a 5-fold increase in the phosphorylation of eEF2 (eukaryotic elongation factor 2), which would also decrease protein synthesis through a decrease in translation elongation. Treatment with leucine increased phosphorylation of mTOR and p70 S6k , caused hyperphosphorylation of 4E-BP1, reduced the amount of 4E-BP1 associated with eIF4E and caused an increase in the eIF4G–eIF4E complex, together with a reduction in phosphorylation of eEF2. These changes would be expected to increase protein synthesis, whereas a reduction in the activation of PKR would be expected to attenuate the increased protein degradation.