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1.
Physiol Rep ; 12(8): e16003, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38631892

ABSTRACT

Chemotherapy is a major contributor to cachexia, but studies often investigate male animals. Here, we investigated whether sex modifies the effects of chemotherapy on cachexia and BCAA metabolism. Ten-week-old CD2F1 male and female mice were treated with the chemotherapy drug cocktail folfiri (50 mg/kg 5-fluorouracil, 90 mg/kg leucovorin, and 24 mg/kg CPT11) (drug) or vehicle twice a week for 6 weeks. Insulin tolerance tests were conducted and BCAA levels and metabolism were measured in plasma and tissues. Drug treatment reduced body and skeletal muscle weights and anabolic signaling in both sexes, with females showing worsened outcomes (p < 0.05 for all). Drug treatment increased plasma BCAA only in males, but BCAA concentrations in the skeletal muscle of both sexes were decreased; this decrease was more profound in males (p = 0.0097). In addition, muscle expression of the BCAA transporter LAT1 was reduced; this reduction was more severe in females (p = 0.0264). In both sexes, the (inhibitory) phosphorylation of BCKD-E1αser293 was increased along with decreased BCKD activity. In the liver, drug treatment increased BCAA concentrations and LAT1 expression, but BCKD activity was suppressed in both sexes (p < 0.05 for all). Our results demonstrate that altered BCAA metabolism may contribute to chemotherapy-induced cachexia in a sex-dependent manner.


Subject(s)
Cachexia , Sex Characteristics , Mice , Female , Male , Animals , Cachexia/metabolism , Amino Acids, Branched-Chain/pharmacology , Liver/metabolism , Fluorouracil/pharmacology , Muscle, Skeletal/metabolism
2.
Am J Physiol Cell Physiol ; 326(3): C866-C879, 2024 Mar 01.
Article in English | MEDLINE | ID: mdl-38284122

ABSTRACT

Prevention/management of cachexia remains a critical issue in muscle wasting conditions. The branched-chain amino acids (BCAA) have anabolic properties in skeletal muscle, but their use in treating cachexia has minimal benefits. This may be related to altered BCAA metabolism consequent to the use of chemotherapy, a main cancer treatment. Since this topic is minimally studied, we investigated the effect of chemotherapy on BCAA concentrations, transporter expression, and their metabolism. L6 myotubes were treated with vehicle (1.4 µL/mL DMSO) or a chemotherapy drug cocktail, FOLFIRI [CPT-11 (20 µg/mL), leucovorin (10 µg/mL), and 5-fluorouracil (50 µg/mL)] for 24-48 h. Chemotherapy reduced myotube diameter (-43%), myofibrillar protein content (-50%), and phosphorylation of the mechanistic target of rapamycin complex 1 (mTORC1) substrate S6K1thr389 (-80%). Drug-treated myotubes exhibited decreased BCAA concentrations (-52%) and expression of their transporter, L-type amino acid transporter 1 (LAT1; -67%). BCAA transaminase BCAT2 level was increased, but there was a reduction in PP2CM (-54%), along with increased inhibitory phosphorylation of BCKD-E1αser293 (+98%), corresponding with decreased BCKD enzyme activity (-23%) in chemotherapy-treated myotubes. Decreases in BCAA concentrations were a later response, preceded by decreases in LAT1 and BCKD activity. Although supplementation with the BCAA restored myotube BCAA levels, it had minimal effects on preventing the loss of myofibrillar proteins. However, RNAi-mediated depletion of neural precursor cell-expressed developmentally downregulated gene 4 (NEdd4), the protein ligase responsible for ubiquitin-dependent degradation of LAT1, attenuated the effects of chemotherapy on BCAA concentrations, anabolic signaling, protein synthesis, and myofibrillar protein abundance. Thus, if our findings are validated in preclinical models, interventions regulating muscle amino acid transporters might represent a promising strategy to treat cachexia.NEW & NOTEWORTHY This is the first study to attenuate chemotherapy-induced myotube atrophy by manipulating a BCAA transporter. Our findings suggest that positive regulation of amino acid transporters may be a promising strategy to treat cachexia.


Subject(s)
Amino Acids, Branched-Chain , Cachexia , Humans , Amino Acids, Branched-Chain/metabolism , Muscle Fibers, Skeletal/metabolism , Amino Acid Transport Systems , Atrophy
4.
Front Physiol ; 12: 702826, 2021.
Article in English | MEDLINE | ID: mdl-34354601

ABSTRACT

Branched-chain amino acids (BCAAs) are critical for skeletal muscle and whole-body anabolism and energy homeostasis. They also serve as signaling molecules, for example, being able to activate mammalian/mechanistic target of rapamycin complex 1 (mTORC1). This has implication for macronutrient metabolism. However, elevated circulating levels of BCAAs and of their ketoacids as well as impaired catabolism of these amino acids (AAs) are implicated in the development of insulin resistance and its sequelae, including type 2 diabetes, cardiovascular disease, and of some cancers, although other studies indicate supplements of these AAs may help in the management of some chronic diseases. Here, we first reviewed the catabolism of these AAs especially in skeletal muscle as this tissue contributes the most to whole body disposal of the BCAA. We then reviewed emerging mechanisms of control of enzymes involved in regulating BCAA catabolism. Such mechanisms include regulation of their abundance by microRNA and by post translational modifications such as phosphorylation, acetylation, and ubiquitination. We also reviewed implications of impaired metabolism of BCAA for muscle and whole-body metabolism. We comment on outstanding questions in the regulation of catabolism of these AAs, including regulation of the abundance and post-transcriptional/post-translational modification of enzymes that regulate BCAA catabolism, as well the impact of circadian rhythm, age and mTORC1 on these enzymes. Answers to such questions may facilitate emergence of treatment/management options that can help patients suffering from chronic diseases linked to impaired metabolism of the BCAAs.

5.
Physiol Rep ; 9(13): e14927, 2021 07.
Article in English | MEDLINE | ID: mdl-34197700

ABSTRACT

Cachexia, a condition prevalent in many chronically ill patients, is characterized by weight loss, fatigue, and decreases in muscle mass and function. Cachexia is associated with tumor burden and disease-related malnutrition, but other studies implicate chemotherapy as being causative. We investigated the effects of a chemotherapy drug cocktail on myofibrillar protein abundance and synthesis, anabolic signaling mechanisms, and substrate availability. On day 4 of differentiation, L6 myotubes were treated with vehicle (1.4 µl/ml DMSO) or a chemotherapy drug cocktail (a mixture of cisplatin [20 µg/ml], leucovorin [10 µg/ml], and 5-fluorouracil [5-FLU; 50 µg/ml]) for 24-72 h. Compared to myotubes treated with vehicle, those treated with the drug cocktail showed 50%-80% reductions in the abundance of myofibrillar proteins, including myosin heavy chain-1, troponin, and tropomyosin (p < 0.05). Cells treated with only a mixture of cisplatin and 5-FLU had identical reductions in myofibrillar protein abundance. Myotubes treated with the drug cocktail also showed >50% reductions in the phosphorylation of AKTSer473 and of mTORC1 substrates ribosomal protein S6Ser235/236 , its kinase S6K1Thr389 and eukaryotic translation initiation factor 4E-binding protein 1 (all p < 0.05). Drug treatment impaired peptide chain initiation in myofibrillar protein fractions and insulin-stimulated glucose uptake (p = 0.06) but increased the expression of autophagy markers beclin-1 and microtubule-associated proteins 1A/1B light chain 3B (p < 0.05), and of apoptotic marker, cleaved caspase 3 (p < 0.05). Drug treatment reduced the expression of mitochondrial markers cytochrome oxidase and succinate dehydrogenase (p < 0.05). The observed profound negative effects of this chemotherapy drug cocktail on myotubes underlie a need for approaches that can reduce the negative effects of these drugs on muscle metabolism.


Subject(s)
Muscle Fibers, Skeletal/drug effects , Muscle Proteins/drug effects , Animals , Blotting, Western , Cachexia/chemically induced , Cells, Cultured , Cisplatin/administration & dosage , Cisplatin/pharmacology , Drug Therapy, Combination , Fluorouracil/administration & dosage , Fluorouracil/pharmacology , Leucovorin/administration & dosage , Leucovorin/pharmacology , Muscle Fibers, Skeletal/chemistry , Muscle Fibers, Skeletal/ultrastructure , Muscle Proteins/analysis , Muscle Proteins/physiology , Myosin Heavy Chains/analysis , Rats , Tropomyosin/analysis , Troponin/analysis
6.
Physiol Rep ; 9(1): e14673, 2021 01.
Article in English | MEDLINE | ID: mdl-33400857

ABSTRACT

Branched-chain amino acids (BCAAs) are regulators of protein metabolism. However, elevated levels of BCAAs and their metabolites are linked to insulin resistance. We previously demonstrated that the leucine metabolite, α-ketoisocaproate (KIC), inhibited insulin-stimulated glucose transport in myotubes. Like KIC, inflammatory factors are implicated in the development of insulin resistance. Here, we analyzed the effect of KIC and inflammatory factors (homocysteine [50 µM], TNF-α [10 ng/ml], and interleukin 6 (IL-6) [10 ng/ml]) on myotubes. Although KIC suppressed insulin-stimulated glucose transport, addition of the inflammatory factors did not worsen this effect. Depletion of branched-chain aminotransferase 2, the enzyme that catalyzes the conversion of leucine into KIC, abrogated the effect of KIC and the inflammatory factors. The effect of insulin on AKTS473 and S6K1T389 phosphorylation was not modified by treatments. There were no treatment effects on glycogen synthase phosphorylation. Depletion of E1α subunit of branched-chain α-keto acid dehydrogenase, the enzyme that catalyzes the oxidative decarboxylation of KIC, suppressed insulin-stimulated glucose transport, especially in cells incubated in KIC. Thus, defects in BCAA catabolism are contributory to insulin resistance of glucose transport in myotubes, especially in the presence of KIC. Interventions that increase BCAA catabolism may promote muscle glucose utilization and improve insulin resistance and its sequelae.


Subject(s)
Amino Acids, Branched-Chain/pharmacology , Glucose/metabolism , Inflammation Mediators/pharmacology , Insulin/pharmacology , Keto Acids/pharmacology , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)/genetics , 3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)/metabolism , Animals , Biological Transport , Cells, Cultured , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Muscle Fibers, Skeletal/drug effects , Muscle, Skeletal/drug effects , Phosphorylation , Rats , Transaminases/genetics , Transaminases/metabolism
7.
Physiol Rep ; 7(23): e14299, 2019 12.
Article in English | MEDLINE | ID: mdl-31833233

ABSTRACT

Much is known about the positive effects of branched-chain amino acids (BCAA) in regulating muscle protein metabolism. Comparatively much less is known about the effects of these amino acids and their metabolites in regulating myotube formation. Using cultured myoblasts, we showed that although leucine is required for myotube formation, this requirement is easily met by α-ketoisocaproic acid, the ketoacid of leucine. We then demonstrated increases in the expression of the first two enzymes in the catabolism of the three BCAA, branched-chain amino transferase (BCAT2) and branched-chain α-ketoacid dehydrogenase (BCKD), with ~3× increase in BCKD protein expression (p < .05) during differentiation. Furthermore, depletion of BCAT2 abolished myoblast differentiation, as indicated by reduction in the levels of myosin heavy chain-1, troponin and myogenin. Supplementation of incubation medium with branched-chain α-ketoacids or related metabolites derivable from BCAT2 functions did not rescue the defects. However, co-depletion of BCKD kinase partially rescued the defects. Collectively, our data indicate a requirement for BCAA catabolism during myotube formation and that this requirement for BCAT2 likely goes beyond the need for this enzyme to generate the α-ketoacids of the BCAA.


Subject(s)
Cell Differentiation , Mitochondrial Proteins/metabolism , Muscle Fibers, Skeletal/metabolism , Myoblasts/metabolism , Transaminases/metabolism , Animals , Cell Line , Mitochondrial Proteins/deficiency , Mitochondrial Proteins/genetics , Muscle Fibers, Skeletal/cytology , Myoblasts/cytology , Myogenin/genetics , Myogenin/metabolism , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Rats , Transaminases/deficiency , Transaminases/genetics , Troponin/genetics , Troponin/metabolism
8.
Int J Biochem Cell Biol ; 117: 105638, 2019 12.
Article in English | MEDLINE | ID: mdl-31678320

ABSTRACT

Homeostatic regulation of energy and metabolic status requires that anabolic and catabolic signaling pathways be precisely regulated and coordinated. Mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is a mega protein complex that promotes energy-consuming anabolic processes of protein and nucleic acid synthesis as well lipogenesis in times of energy and nutrient abundance. However, it is best characterized as the regulator of steps leading to protein synthesis. The ubiquitin-proteasome proteolytic system (UPS) is a major intracellular proteolytic system whose activity is increased during periods of nutrient scarcity and in muscle wasting conditions such as cachexia. Recent studies have examined the impact of mTORC1 on levels and functions of the 26S proteasome, the mega protease complex of the UPS. Here we first briefly review current understanding of the regulation of mTORC1, the UPS, and the 26S proteasome complex. We then review evidence of the effect of each complex on the abundance and functions of the other. Given the fact that drugs that inhibit either complex are either in clinical trials or are approved for treatment of cancer, a muscle wasting condition, we identify studying the effect of combinatory mTORC1-proteasome inhibition on skeletal muscle mass and health as a critical area requiring investigation.


Subject(s)
Amino Acids/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Proteasome Endopeptidase Complex/metabolism , Animals , Humans
9.
Physiol Rep ; 5(17)2017 Sep.
Article in English | MEDLINE | ID: mdl-28899909

ABSTRACT

The mechanistic (mammalian) target of rapamycin complex 1 (mTORC1) signaling is vital for optimal muscle mass and function. Although the significance of mTORC1 in stimulating muscle growth is unequivocal, evidence in support of its role during muscle regeneration is less clear. Here, we showed that the abundance (protein and mRNA) of the mTORC1/S6K1 substrate, programmed cell death protein 4 (PDCD4), is upregulated at the onset of differentiation of L6 and C2C12 cells. The increase in PDCD4 was not associated with any changes in S6K1 activation, but the abundance of beta transducing repeat-containing protein (ß-TrCP), the ubiquitin ligase that targets PDCD4 for degradation, increased. Myoblasts lacking PDCD4 showed impaired myotube formation and had markedly low levels of MHC-1. Analysis of poly (ADP-ribose) Polymerase (PARP), caspase 7 and caspase 3 indicated reduced apoptosis in PDCD4-deficient cells. Our data demonstrate a role for PDCD4 in muscle cell formation and suggest that interventions that target this protein may hold promise for managing conditions associated with impaired myotube formation.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Cell Differentiation , Muscle Fibers, Skeletal/metabolism , Animals , Apoptosis , Apoptosis Regulatory Proteins/genetics , Cell Line , Mechanistic Target of Rapamycin Complex 1/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Muscle Fibers, Skeletal/cytology , Rats , Ribosomal Protein S6 Kinases/genetics , Ribosomal Protein S6 Kinases/metabolism , Up-Regulation , beta-Transducin Repeat-Containing Proteins/genetics , beta-Transducin Repeat-Containing Proteins/metabolism
10.
Am J Physiol Cell Physiol ; 311(3): C518-27, 2016 09 01.
Article in English | MEDLINE | ID: mdl-27488662

ABSTRACT

Although leucine has many positive effects on metabolism in multiple tissues, elevated levels of this amino acid and the other branched-chain amino acids (BCAAs) and their metabolites are implicated in obesity and insulin resistance. While some controversies exist about the direct effect of leucine on insulin action in skeletal muscle, little is known about the direct effect of BCAA metabolites. Here, we first showed that the inhibitory effect of leucine on insulin-stimulated glucose transport in L6 myotubes was dampened when other amino acids were present, due in part to a 140% stimulation of basal glucose transport (P < 0.05). Importantly, we also showed that α-ketoisocaproic acid (KIC), an obligatory metabolite of leucine, stimulated mTORC1 signaling but suppressed insulin-stimulated glucose transport (-34%, P < 0.05) in an mTORC1-dependent manner. The effect of KIC on insulin-stimulated glucose transport was abrogated in cells depleted of branched-chain aminotransferase 2 (BCAT2), the enzyme that catalyzes the reversible transamination of KIC to leucine. We conclude that although KIC can modulate muscle glucose metabolism, this effect is likely a result of its transamination back to leucine. Therefore, limiting the availability of leucine, rather than those of its metabolites, to skeletal muscle may be more critical in the management of insulin resistance and its sequelae.


Subject(s)
Biological Transport/physiology , Glucose/metabolism , Insulin/metabolism , Keto Acids/metabolism , Leucine/metabolism , Membrane Transport Proteins/metabolism , Muscle, Skeletal/metabolism , Amino Acids/metabolism , Animals , Cells, Cultured , Insulin Resistance/physiology , Mechanistic Target of Rapamycin Complex 1 , Mitochondrial Proteins , Monocarboxylic Acid Transporters , Multiprotein Complexes/metabolism , Muscle Fibers, Skeletal/metabolism , Rats , TOR Serine-Threonine Kinases/metabolism , Transaminases/metabolism
11.
Eur J Nutr ; 54(5): 751-9, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25120109

ABSTRACT

PURPOSE: Protein metabolism is altered in obesity, accompanied by elevated plasma amino acids (AA). Previously, we showed that exercise delayed progression to type 2 diabetes in obese ZDF rats with maintenance of ß cell function and reduction in hyperglucocorticoidemia. We hypothesized that exercise would correct the abnormalities we found in circulating AA and other indices of skeletal muscle protein metabolism. METHODS: Male obese prediabetic ZDF rats (7-10/group) were exercised (swimming) 1 h/day, 5 days/week from ages 6-19 weeks, and compared with age-matched obese sedentary and lean ZDF rats. RESULTS: Food intake and weight gain were unaffected. Protein metabolism was altered in obese rats as evidenced by increased plasma concentrations of essential AA, and increased muscle phosphorylation (ph) of Akt(ser473) (187%), mTOR(ser2448) (140%), eIF4E-binding protein 1 (4E-BP1) (111%), and decreased formation of 4E-BP1*eIF4E complex (75%, 0.01 ≤ p ≤ 0.05 for all measures) in obese relative to lean rats. Exercise attenuated the increase in plasma essential AA concentrations and muscle Akt and mTOR phosphorylation. Exercise did not modify phosphorylation of S6K1, S6, and 4E-BP1, nor the formation of 4E-BP1*eIF4E complex, mRNA levels of ubiquitin or the ubiquitin ligase MAFbx. Positive correlations were observed between ph-Akt and fed circulating branched-chain AA (r = 0.56, p = 0.008), postprandial glucose (r = 0.42, p = 0.04) and glucose AUC during an IPGTT (r = 0.44, p = 0.03). CONCLUSION: Swimming exercise-induced attenuation of hyperglycemia in ZDF rats is independent of changes in body weight and could result in part from modulation of muscle AKT activation acting via alterations of systemic AA metabolism.


Subject(s)
Amino Acids/blood , Hyperglycemia/prevention & control , Muscle, Skeletal/metabolism , Physical Conditioning, Animal , Weight Gain , Amino Acids/metabolism , Animals , Carrier Proteins/blood , Carrier Proteins/genetics , Intracellular Signaling Peptides and Proteins , Male , Obesity/metabolism , Obesity/therapy , Phosphoproteins/blood , Phosphoproteins/genetics , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Ribosomal Protein S6 Kinases/metabolism , TOR Serine-Threonine Kinases/metabolism
12.
Physiol Rep ; 2(7)2014 Jul 01.
Article in English | MEDLINE | ID: mdl-24997070

ABSTRACT

High-protein diets (HPDs) promote weight loss but other studies implicate these diets and their constituent amino acids (AAs) in insulin resistance. We hypothesized that AA-induced insulin resistance is a temporal and reversible metabolic event. L6 myotubes were serum deprived for 4 h and then incubated in AA and/or insulin (100 nmol/L). Another group of cells was incubated overnight in AA + insulin, starved again, and then reincubated with AA and insulin. Mammalian (mechanistic) target of rapamycin complex 1 (mTORC1) signaling and glucose uptake were then measured. Healthy or insulin-resistant rats were gavaged with leucine (0.48 g/kg) and insulin sensitivity was examined. In myotubes, incubation with AA and insulin significantly (P < 0.05) increased the phosphorylation of the mTORC1 substrate ribosomal protein S6 kinase 1 (S6K1, T389) and of insulin receptor substrate 1 (IRS-1, serine residues), but suppressed insulin-stimulated glucose uptake by 40% (P < 0.01). These modifications were mTORC1-dependent and were reversible. In vivo, leucine gavage reversibly increased S6K1 phosphorylation and IRS-1 serine phosphorylation 5- to 12-fold in skeletal muscle and impaired insulin tolerance of glucose (P < 0.05) in lean rats. In insulin-resistant rats, the impairment of whole blood glucose and AA metabolism induced by leucine gavage (0.001 < P < 0.05) was more severe than that observed in lean rats; however, the impairment was reversible within 24 h of treatment. If these data are confirmed in long-term studies, it would imply that the use of leucine/HPD in treating metabolic diseases is unlikely to have lasting negative effects on insulin sensitivity.

13.
BMC Cell Biol ; 15: 2, 2014 Jan 09.
Article in English | MEDLINE | ID: mdl-24405715

ABSTRACT

BACKGROUND: Reduced muscle mass is a hallmark of metabolic diseases like diabetes and cancer. The mammalian (mechanistic) target of rapamycin complex 1/S6 kinase 1 (mTORC1/S6K1) pathway is critical to the regulation of muscle protein synthesis and mass but its mechanism of action is not completely understood. RESULTS: Using L6 myotubes, we characterized the regulation of programmed cell death 4 (PDCD4), a recently described substrate of S6K1. The abundance, but not Ser67 phosphorylation, of PDCD4 was sensitive to amino acid and serum deprivation: values in starved cells were 4.5X of control (P < 0.001). Refeeding had opposite effects. Growth factors, compared to amino acids, appeared more critical in regulating PDCD4 abundance. Furthermore, inhibition of mTORC1 or the proteasome prevented the refeeding-associated decrease in PDCD4 abundance. Amino acid and serum deprivation significantly increased PDCD4 binding to eIF4A (P < 0.05); this was reversed during refeeding. PDCD4 depletion by RNA interference had no significant effect on phenylalanine incorporation into myotube mixed proteins in control cells but further suppressed (30%) this measure in nutrient-deprived cells (P < 0.0005). This was not observed in myoblasts. In starved myotubes, PDCD4 depletion further reduced the association of eIF4G with eIF4E. CONCLUSION: Our data suggest that in myotubes, PDCD4 abundance is sensitive to nutritional manipulation in an mTORC1 and proteasome depended manner. Furthermore, the role of PDCD4 in regulating protein synthesis appears dependent on the developmental state of the cell.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Muscle Fibers, Skeletal/metabolism , Myoblasts/metabolism , Animals , Apoptosis Regulatory Proteins/antagonists & inhibitors , Apoptosis Regulatory Proteins/genetics , Cells, Cultured , Culture Media/pharmacology , Eukaryotic Initiation Factor-4A/metabolism , Eukaryotic Initiation Factor-4E/metabolism , Eukaryotic Initiation Factor-4G/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/metabolism , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/drug effects , Myoblasts/cytology , Myoblasts/drug effects , Phenylalanine/metabolism , Phosphorylation , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , RNA Interference , Rats , TOR Serine-Threonine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/metabolism
14.
Appl Physiol Nutr Metab ; 37(3): 395-406, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22509811

ABSTRACT

The mass and integrity of skeletal muscle is vital to whole-body substrate metabolism and health. Indeed, defects in muscle metabolism and functions underlie or exacerbate diseases like diabetes, rheumatoid arthritis, and cancer. Physical activity and nutrition are the 2 most important environmental factors that can affect muscle health. At the molecular level, the mammalian target of rapamycin complex 1 (mTORC1) is a critical signalling complex that regulates muscle mass. In response to nutrition and resistance exercise, increased muscle mass and activation of mTORC1 occur in parallel. In this review, we summarize recent findings on mTORC1 and its regulation in skeletal muscle in response to resistance exercise, alone or in combination with intake of protein or amino acids. Because increased activity of the complex is implicated in the development of muscle insulin resistance, obesity, and some cancers (e.g., ovarian, breast), drugs that target mTORC1 are being developed or are in clinical trials. However, various cancers are associated with extensive muscle wasting, due in part to tumour burden and malnutrition. This muscle wasting may also be a side effect of anticancer drugs. Because loss of muscle mass is associated not only with metabolic abnormalities but also dose limiting toxicity, we review the possible implications for skeletal muscle of long-term inhibition of mTORC1, especially in muscle wasting conditions.


Subject(s)
Muscle Proteins/biosynthesis , Muscle, Skeletal/metabolism , Muscular Atrophy/metabolism , Proteins/metabolism , Amino Acids/metabolism , Animals , Body Weights and Measures , Dietary Proteins/metabolism , Humans , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes , Muscle Proteins/metabolism , Rats , Resistance Training/methods , Signal Transduction , TOR Serine-Threonine Kinases
15.
Am J Physiol Endocrinol Metab ; 300(6): E986-92, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21406616

ABSTRACT

Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P < 0.05); refeeding reversed these changes (P < 0.05). Consistent with these effects being regulated by S6K1, activation of this kinase was suppressed by FD (-91%, P < 0.05) but was increased by refeeding. Gavaging rats subjected to FD with a mixture of amino acids partially restored muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P < 0.0001). Thus feeding stimulates fractional protein synthesis in skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.


Subject(s)
Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/physiology , Eating/physiology , Fasting/physiology , Muscle Proteins/biosynthesis , Muscle, Skeletal/metabolism , RNA, Messenger/genetics , Amino Acids/pharmacology , Amino Acids, Branched-Chain/metabolism , Animals , Cell Count , Cell Line , Insulin/blood , Male , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/cytology , Myoblasts/metabolism , Phenylalanine/metabolism , RNA Interference/physiology , Rats , Rats, Sprague-Dawley , Transcription Factors/biosynthesis , Transcription Factors/genetics
16.
PLoS One ; 5(11): e14032, 2010 Nov 17.
Article in English | MEDLINE | ID: mdl-21103335

ABSTRACT

This present study investigated the temporal effects of type 1 diabetes mellitus (T1DM) on adolescent skeletal muscle growth, morphology and contractile properties using a 90% partial pancreatecomy (Px) model of the disease. Four week-old male Sprague-Dawley rats were randomly assigned to Px (n = 25) or Sham (n = 24) surgery groups and euthanized at 4 or 8 weeks following an in situ assessment of muscle force production. Compared to Shams, Px were hyperglycemic (>15 mM) and displayed attenuated body mass gains by days 2 and 4, respectively (both P<0.05). Absolute maximal force production of the gastrocnemius plantaris soleus complex (GPS) was 30% and 50% lower in Px vs. Shams at 4 and 8 weeks, respectively (P<0.01). GP mass was 35% lower in Px vs Shams at 4 weeks (1.24±0.06 g vs. 1.93±0.03 g, P<0.05) and 45% lower at 8 weeks (1.57±0.12 vs. 2.80±0.06, P<0.05). GP fiber area was 15-20% lower in Px vs. Shams at 4 weeks in all fiber types. At 8 weeks, GP type I and II fiber areas were ∼25% and 40% less, respectively, in Px vs. Shams (group by fiber type interactions, P<0.05). Phosphorylation states of 4E-BP1 and S6K1 following leucine gavage increased 2.0- and 3.5-fold, respectively, in Shams but not in Px. Px rats also had impaired rates of muscle protein synthesis in the basal state and in response to gavage. Taken together, these data indicate that exposure of growing skeletal muscle to uncontrolled T1DM significantly impairs muscle growth and function largely as a result of impaired protein synthesis in type II fibers.


Subject(s)
Diabetes Mellitus, Type 1/physiopathology , Muscle, Skeletal/physiopathology , Muscular Diseases/physiopathology , Pancreatectomy/methods , Adolescent , Analgesics/pharmacology , Animals , Diabetes Mellitus, Type 1/complications , Disease Models, Animal , Humans , Ketamine/pharmacology , Male , Muscle Fatigue/drug effects , Muscle Fatigue/physiology , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/physiology , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscular Diseases/etiology , Random Allocation , Rats , Rats, Sprague-Dawley , Signal Transduction , Succinate Dehydrogenase/metabolism , TOR Serine-Threonine Kinases/metabolism , Ubiquitination , Xylazine/pharmacology
17.
Pediatr Res ; 67(3): 268-73, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19952868

ABSTRACT

Malnutrition and cytokine-induced catabolism are pervasive in children with inflammatory bowel diseases (IBD), however, the benefits of aggressive nutrition support or of probiotics on nutrient and functional deficiencies and growth remain unclear. Piglets with dextran sulfate (DS)-induced colitis consuming a 50% macronutrient restricted diet (C-MR) were compared with those receiving probiotics (C-MRP) or adequate nutrition (C-WN) and with healthy well-nourished controls (REF). C-WN versus REF had reduced growth (-34% chest circumference and -22% snout-to-rump length gain) and a tendency toward lesser weight gain, but no differences in skeletal muscle protein fractional synthesis rates (FSR) or initiation of translation via the mTOR pathway were observed. Compared with C-WN, the C-MR and C-MRP piglets had lower weight gain, growth, and skeletal muscle FSR, and lower phosphorylated p70S6K1 with higher eIF4E*4E-BP1, indicative of reduced initiation of protein translation. Finally, plasma leucine concentrations were positively correlated with weight and phosphorylated p70S6K1, whereas negatively correlated with eIF4E*4E-BP1. In conclusion, reductions in weight gain, growth, protein turnover, skeletal muscle FSR, and initiation of protein translation with moderate macronutrient restriction in colitis are not ameliorated by probiotic supplementation. However, maintaining adequate nutrient intake during colitis preserves whole body protein metabolism, but growth remains compromised.


Subject(s)
Colitis/therapy , Enteral Nutrition , Growth Disorders/prevention & control , Muscle Proteins/biosynthesis , Muscle, Skeletal/metabolism , Probiotics/administration & dosage , Animal Nutritional Physiological Phenomena , Animals , Animals, Newborn , Blood Glucose/metabolism , Body Size , Colitis/chemically induced , Colitis/metabolism , Colitis/physiopathology , Dextran Sulfate , Disease Models, Animal , Eukaryotic Initiation Factor-4E/metabolism , Growth Disorders/etiology , Growth Disorders/metabolism , Growth Disorders/physiopathology , Hydrocortisone/blood , Insulin/blood , Intracellular Signaling Peptides and Proteins/metabolism , Leucine/blood , Nutritional Status , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Swine , TOR Serine-Threonine Kinases , Ubiquitination , Weight Gain
18.
Am J Physiol Endocrinol Metab ; 296(1): E105-13, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18957614

ABSTRACT

Since maximum anabolism occurs postprandially, we developed a simulated fed state with clamped hyperinsulinemia, physiological hyperglycemia, and hyperaminoacidemia (Hyper-3) and explored muscle cellular mechanisms. Whole body [1-(13)C]leucine and [3-(3)H]glucose kinetics in healthy men were compared between hyperinsulinemic, euglycemic, isoaminoacidemic (Hyper-1, n = 10) and Hyper-3 (n = 9) clamps. In Hyper-3 vs. Hyper-1, nonoxidative leucine R(d) [rate of disappearance (synthesis)] was stimulated more (45 +/- 4 vs. 24 +/- 4 micromol/min, P < 0.01) and endogenous R(a) [rate of appearance (breakdown)] was inhibited similarly; hence net balance increased more (86 +/- 6 vs. 49 +/- 2 micromol/min, P < 0.001). Glucose R(d) was similar; thus Hyper-3 metabolic clearance rate (331 +/- 23 vs. 557 +/- 41 ml/min, P < 0.0005) and R(d)/insulin (M, 0.65 +/- 0.10 vs. 1.25 +/- 0.10 mg.min(-1).pmol(-1).l, P < 0.001) were less, despite higher insulin (798 +/- 74 vs. 450 +/- 24 pmol/l, P < 0.005). In vastus lateralis muscle biopsies, phosphorylation of Akt (P = 0.025), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70(S6K1); P = 0.008), S6 (P = 0.049), and 4E-binding protein 1 (4E-BP1; P = 0.001) increased. With decreased eukaryotic initiation factor-4E (eIF4E).4E-BP1 complex (P = 0.01), these are consistent with increased mTOR complex 1 (mTORC1) signaling and translation initiation of protein synthesis. Although mRNA expression of ubiquitin, MAFbx 1, and MuRF-1 was unchanged, total ubiquitinated proteins decreased 20% (P < 0.01), consistent with proteolysis suppression. The Hyper-3 clamp increases whole body protein synthesis, net anabolism, and muscle protein translation initiation pathways and decreases protein ubiquitination. The main contribution of hyperaminoacidemia is stimulation of synthesis rather than inhibition of proteolysis, and it attenuates the expected increment of glucose disposal.


Subject(s)
Glucose/metabolism , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adult , Amino Acids/administration & dosage , Amino Acids/blood , Biopsy , Cell Cycle Proteins , Glucose/pharmacokinetics , Glucose Clamp Technique/methods , Humans , Leucine/administration & dosage , Leucine/blood , Leucine/pharmacokinetics , Male , Muscle Proteins/biosynthesis , Muscle Proteins/genetics , Phosphoproteins/metabolism , Postprandial Period , Protein Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Ribosomal Protein S6 Kinases/metabolism , SKP Cullin F-Box Protein Ligases/biosynthesis , SKP Cullin F-Box Protein Ligases/genetics , TOR Serine-Threonine Kinases , Tripartite Motif Proteins , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/genetics
19.
Mol Cell Biol ; 29(2): 547-58, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19015242

ABSTRACT

p27(Kip1) is a cyclin-dependent kinase inhibitor that regulates the G(1)/S transition. Increased degradation of p27(Kip1) is associated with cellular transformation. Previous work demonstrated that the ubiquitin ligases KPC1/KPC2 and SCF(Skp2) ubiquitinate p27(Kip1) in G(1) and early S, respectively. The regulation of these ligases remains unclear. We report here that the USP19 deubiquitinating enzyme interacts with and stabilizes KPC1, thereby modulating p27(Kip1) levels and cell proliferation. Cells depleted of USP19 by RNA interference exhibited an inhibition of cell proliferation, progressing more slowly from G(0)/G1 to S phase, and accumulated p27(Kip1). This increase in p27(Kip1) was associated with normal levels of Skp2 but reduced levels of KPC1. The overexpression of KPC1 or the use of p27(-/-) cells inhibited significantly the growth defect observed upon USP19 depletion. KPC1 was ubiquitinated in vivo and stabilized by proteasome inhibitors and by overexpression of USP19, and it also coimmunoprecipitated with USP19. Our results identify USP19 as the first deubiquitinating enzyme that regulates the stability of a cyclin-dependent kinase inhibitor and demonstrate that progression through G(1) to S phase is, like the metaphase-anaphase transition, controlled in a hierarchical, multilayered fashion.


Subject(s)
Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Ubiquitin Thiolesterase/metabolism , Ubiquitin-Protein Ligases/metabolism , Ubiquitin/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Line , Immunoblotting , Mice , RNA Interference , Rats , S-Phase Kinase-Associated Proteins/metabolism , Ubiquitin Thiolesterase/genetics , Ubiquitin-Protein Ligase Complexes , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/genetics
20.
Am J Physiol Endocrinol Metab ; 288(4): E693-700, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15562254

ABSTRACT

Ubiquitin-dependent proteolysis is activated in skeletal muscle atrophying in response to various catabolic stimuli. Previous studies have demonstrated activation of ubiquitin conjugation. Because ubiquitination can also be regulated by deubiquitinating enzymes, we used degenerate oligonucleotides derived from conserved sequences in the ubiquitin-specific protease (UBP) family of deubiquitinating enzymes in RT-PCR with skeletal muscle RNA to amplify putative deubiquitinating enzymes. We identified USP19, a 150-kDa deubiquitinating enzyme that is widely expressed in various tissues including skeletal muscle. Expression of USP19 mRNA increased by approximately 30-200% in rat skeletal muscle atrophying in response to fasting, streptozotocin-induced diabetes, dexamethasone treatment, and cancer. Increased mRNA levels during fasting returned to normal with refeeding, but 1 day later than the normalization of rates of proteolysis and coincided instead with recovery of muscle mass. Indeed, in all catabolic treatments, USP19 mRNA was inversely correlated with muscle mass and provided an index of muscle mass that may be useful in many pathological conditions, using small human muscle biopsies. The increased expression of this deubiquitinating enzyme under conditions of increased proteolysis suggests that it may play a role in regeneration of free ubiquitin either coincident with or after proteasome-mediated degradation of substrates. USP19 may also be involved in posttranslational processing of polyubiquitin produced de novo in response to induction of the polyubiquitin genes seen under these conditions. Deubiquitinating enzymes thus appear involved in muscle wasting and implicate a widening web of regulation of genes in the ubiquitin system in this process.


Subject(s)
Endopeptidases/metabolism , Muscle, Skeletal/enzymology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , Diabetes Mellitus, Experimental/enzymology , Endopeptidases/biosynthesis , Endopeptidases/genetics , Food Deprivation/physiology , Gene Expression Regulation, Enzymologic , Male , Molecular Sequence Data , Muscular Atrophy/enzymology , Neoplasms, Experimental/enzymology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Ubiquitin-Specific Proteases
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