Cachexia is a devastating muscle‐wasting condition found in many diseases, including cancer, chronic kidney disease and heart failure. Aside from both tumour burden and disease‐related malnutrition, the development of cachexia is associated with chemotherapy treatment. Branched‐ chain amino acids (BCAA: leucine, isoleucine and valine) are critical regulators of skeletal muscle protein anabolism due to their activation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1). However, BCAA supplementation/nutritional support does not fully reverse chemotherapy‐induced cachexia. Therefore, we investigated whether breakdown of BCAAs is affected by chemotherapy drugs. On day 4 of differentiation, L6 myotubes were treated with vehicle (1.4μL/mL DMSO) or a common chemotherapy drug cocktail, folfiri (a mixture of CPT‐11 (20μg/mL), leucovorin (10μg/mL), and 5‐fluorouracil (50μg/mL)) for 24‐48h. Myotubes treated with folfiri exhibited ~30% reductions in myotube diameter (p < 0.05, n=3) and ~50% reductions in abundance of myofibrillar proteins myosin heavy chain‐1 (MHC) and troponin (p < 0.05). Protein content of branched‐chain alpha‐ketoacid dehydrogenase complex (BCKD), the enzyme responsible for the irreversible decarboxylation of the BCAA ketoacids, was unchanged following folfiri treatment. However, the activity of this enzyme complex was significantly decreased (~20%) 24 and 48h following treatment with folfiri (p < 0.05). Branched‐chain alpha‐ketoacid dehydrogenase complex kinase (BDK), a negative regulator of BCKD, was increased 24h (~20%), but unchanged at 48h following folfiri treatment. Compared to vehicle, folfiri‐treated myotubes showed a non‐significant reduction (~30%) in phenylalanine incorporation into proteins. In line with studies showing a link between impaired BCAA catabolism and insulin resistance, our data suggest a link between chemotherapy‐induced muscle atrophy and altered BCAA catabolism.
High protein diets are sometimes prescribed in weight management interventions; however these diets and amino acids (AA) have been linked to insulin resistance. Much of the attention has been on the branched‐chain amino acid (BCAA) leucine because of its ability to activate the mammalian target of rapamycin complex 1 (mTORC1). This complex and its substrate (S6K1) are implicated in AA‐induced insulin resistance due to their inhibitory serine phosphorylation of insulin receptor substrate 1 (IRS1). Since AAs other than leucine are present in dietary proteins, we examined their roles. Valine suppressed insulin stimulated glucose uptake (Glu‐trans) in L6 myotubes by 47% (P<0.05) compared to a suppression of 22% observed with leucine. The effect was not due to a greater intracellular accumulation of valine. However, valine and leucine, but not isoleucine, increased phosphorylation of S6K1 (thr389) and IRS1 (ser612 (P<0.05)). This is consistent with a lack of effect of isoleucine on Glu‐trans. We also examined the effect of 2 metabolites of leucine, ketoisocaproic acid (KIC) and isovaleryl CoA. KIC, but not isovaleryl CoA, suppressed Glu‐trans by 25% (P<0.05) and increased S6K1 and IRS1 phosphorylation. Strikingly, AA other than the BCAA, especially arginine and glutamine, also suppressed Glu‐trans by 22‐47% (P<0.05). Inhibition of Glu‐trans was not always linked to mTORC1/S6K1 and IRS1 phosphorylation. We conclude that KIC may mediate the suppressive effects of leucine on insulin stimulated glucose transport, and that amino acids other than leucine can induce insulin resistance of glucose transport. Grant Funding Source: Supported by NSERC and Faculty of Health, York University
Objectives Branched-chain amino acids (BCAAs) are essential amino acids that are crucial for skeletal muscle anabolism. Thus, alterations in their levels are associated with muscle atrophic diseases such as cancer, chronic inflammatory and neurological disorders. Others have linked impairments in BCAA metabolism to the development of insulin resistance and its sequelae. Compared to the effects of theses amino acids, much less is known on how impairment in BCAA catabolism affects skeletal muscle. BCAA catabolism starts with the reversible transamination by the mitochondrial enzyme branched-chain aminotransferase 2 (BCAT2). This is followed by the irreversible carboxylation, catalyzed by branched-chain ketoacid dehydrogenase (BCKD) complex. We have shown that BCAT2 and BCKD are essential for the differentiation of skeletal myoblasts into myotubes. Here, we investigated the effect of depletion of BCAT2 or of E1a subunit of BCKD in differentiated myotubes. Methods On day 4 of differentiation, L6 myotubes were transfected with the following siRNA oligonucleotides: scrambled (control), BCAT2, or E1a subunit of BCKD. Results Forty-eight hours after transfection, compared to control or BCAT2 siRNA group, we observed improved myotube structure in BCKD-depleted cells. BCKD depletion augmented myofibrillar protein levels: myosin heavy chain (MHC, 2-fold) and tropomyosin (4-fold), P < 0.05, n = 3. To further analyze the increase in myofibrillar protein content, we examined signaling through mTORC1 (mechanistic target of rapamycin complex 1), a vital complex necessary for skeletal muscle anabolism. BCKD depletion increased the phosphorylation of mTORC1 upstream activator AKT (52%, P < 0.05, n = 3), and of mTORC1 downstream substrates by 25%-86%, consistent with the increase in myofibrillar proteins. Finally, in myotubes treated with the catabolic cytokine (tumor necrosis factor-a), BCKD depletion tended to increase the abundance of tropomyosin (a myofibrillar protein). Conclusions We showed that depletion of BCKD enhanced myofibrillar protein content and anabolic signaling. If these data are confirmed in vivo, development of dietary and other interventions that target BCKD abundance or functions may promote muscle protein anabolism in individuals with muscle wasting conditions. Funding Sources MHRC, NSERC York U.
The mammalian target of rapamycin complex 1/S6 ribosomal protein kinase 1 (mTORC1/S6K1) pathway is a critical regulator of mRNA translation and skeletal muscle mass. It does this in part by inhibiting the tumor suppressor protein, programmed cell death 4 (PDCD4). In C2C12 and L6 muscle cells, we showed that PDCD4 abundance was high on day 1 and then decreased as myoblasts differentiated into myotubes (p<0.05). siRNA‐mediated knockdown of S6K1 reversed the decrease in PDCD4 abundance and significantly decreased myosin heavy chain 1 (MHC 1) protein abundance, suggesting that PDCD4 regulation was vital for differentiation. Indeed, cells depleted of PDCD4 had reduced MHC abundance, showed delayed myoblast fusion and abnormal myotube formation. On days 3 and 4 of differentiation, myotubes depleted of PDCD4 showed 40–60% reductions in myotube protein synthesis. This study unravels a link between PDCD4 and muscle cell differentiation, and suggests that this mTORC1/S6K1 substrate may be of therapeutic significance for muscle recovery following injury or atrophy. Funded by NSERC.
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