Effects of anabolic agents on protein breakdown in L6 myoblasts.
Ontology highlight
ABSTRACT:
SUBMITTER: Ballard FJ
PROVIDER: S-EPMC1154211 | biostudies-other | 1983 Jan
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Insulin resistance and Type 2 diabetes are marked by an aberrant response in the insulin signaling network. The phosphoinositide-dependent serine/threonine kinase, Akt2, plays a key role in insulin signaling and glucose uptake, most notably within skeletal muscle. Protein-protein interaction regulates the functional consequence of Akt2 and in turn, Akt2's role in glucose uptake. However, only few insulin-responsive Akt2 interaction partners have been identified in skeletal muscle cells. In the present work, rat L6 myoblasts, a widely used insulin sensitive skeletal muscle cell line, were used to examine endogenous, insulin-stimulated Akt2 protein interaction partners. Akt2 co-immunoprecipitation was coupled with 1D-SDS-PAGE and fractions were analyzed by HPLC-ESI-MS/MS to reveal Akt2 protein-protein interactions. The pull-down assay displayed specificity for the Akt2 isoform; Akt1 and Akt3 unique peptides were not detected. A total of 49 were detected with a significantly increased (47) or decreased (2) association with Akt2 following insulin administration (n = 4; p<0.05). Multiple pathways were identified for the novel Akt2 interaction partners, such as the EIF2 and ubiquitination pathways. These data suggest that multiple new endogenous proteins may associate with Akt2 under basal as well as insulin-stimulated conditions, providing further insight into the insulin signaling network. Data are available via ProteomeXchange with identifier PXD002557.
| S-EPMC4605787 | biostudies-literature
Project description:Insulin resistance and Type 2 diabetes are marked by an aberrant response in the insulin signaling network. The phosphoinositide-dependent serine/threonine kinase, Akt2, plays a key role in insulin signaling and glucose uptake, most notably within skeletal muscle. Protein-protein interaction regulates the functional consequence of Akt2 and in turn, Akt2’s role in glucose uptake. However, only few insulin-responsive Akt2 interaction partners have been identified in skeletal muscle cells. In the present work, rat L6 myoblasts, a widely used insulin sensitive skeletal muscle cell line, were used to examine endogenous, insulin-stimulated Akt2 protein interaction partners. Akt2 co-immunoprecipitation was coupled with 1D-SDS-PAGE and fractions were analyzed by HPLC-ESI-MS/MS to reveal Akt2 protein-protein interactions. The pull-down assay displayed specificity for the Akt2 isoform; Akt1 and Akt3 unique peptides were not detected. A total of 330 proteins were significantly enriched as bonafide Akt2 protein interaction partners. Among them, 226 were novel for any cell types. Furthermore, 59 were detected with a significantly increased (57) or decreased (2) association with Akt2 following insulin administration (n=4; p<0.05). Multiple pathways were identified for the novel Akt2 interaction partners, such as the EIF2 and ubiquitination pathways. These data suggest that multiple new endogenous proteins may associate with Akt2 under basal as well as insulin-stimulated conditions, providing further insight into the insulin signaling network.
2015-09-30 | PXD002557 | Pride
Project description:Chronic metabolic acidosis plays a role in cachexia by enhancing total proteolysis in skeletal muscle. Glucocorticoid also triggers proteolysis and plays a permissive role in the effect of acidosis. The System A amino acid transporter SNAT2/SLC38A2 is ubiquitously expressed in mammalian cells including muscle, performing Na+-dependent active import of neutral amino acids, and is strongly inhibited by low pH. Exposure of rat skeletal muscle cell line L6-G8C5 to low pH rapidly inhibits SNAT2 transport activity and enhances total proteolysis rate. Pharmacological inhibition or silencing of SNAT2 also enhances proteolysis. This study tests the hypothesis that the glucocorticoid dexamethasone (DEX), like low pH, inhibits SNAT2 activity in L6-G8C5 myotubes, thus contributing to total proteolysis. Incubation with 500 nM DEX for 4 h reduced the System A amino acid transport rate to half the rate in control cultures. This inhibition depended on glucocorticoid receptor-mediated gene transcription, but SNAT2 mRNA levels were unaffected by DEX. In contrast, the SNAT2 protein assessed by immunoblotting was significantly depleted. The co-inhibitory effects of DEX and low pH on System A transport activity were additive in stimulating total proteolysis. In keeping with this mechanism, DEX's inhibitory effect on SNAT2 transport activity was significantly blunted by the proteasome inhibitor MG132. Proof of principle was achieved in similar experiments using recombinant expression of a GFP-tagged SNAT2 fusion protein in HEK293A cells. It is concluded that DEX acutely depletes the SNAT2 transporter protein, at least partly through proteasome-dependent degradation of this functionally important transporter.
| S-EPMC7805547 | biostudies-literature
Project description:Methylglyoxal (MG), a highly reactive α-dicarbonyl metabolite of glucose degradation pathways, protein and fatty acid metabolism, plays an important role in the pathogenesis of diabetic complications. Hyperglycemia triggers enhanced production of MG and increased generation of advanced glycation endproducts (AGEs). In non-enzymatic reactions, MG reacts with arginine residues of proteins to form the AGEs argpyrimidine and hydroimidazolone. Glyoxalase 1 (GLO1), in combination with glyoxalase 2 and the co-factor glutathione constitute the glyoxalase system, which is responsible for the detoxification of MG. A GLO1 specific knock down results in accumulation of MG in targeted cells. The aim of this study was to investigate the effect of intracellularly accumulated MG on insulin signaling and on the translocation of the glucose transporter 4 (GLUT4). Therefore, L6 cells stably expressing a myc-tagged GLUT4 were examined. For the intracellular accumulation of MG, GLO1, the first enzyme of the glyoxalase pathway, was down regulated by siRNA knock down and cells were cultivated under hyperglycemic conditions (25 mM glucose) for 48 h. Here we show that GLO1 knock down augmented GLUT4 level on the cell surface of L6 myoblasts at least in part through reduction of GLUT4 internalization, resulting in increased glucose uptake. However, intracellular accumulation of MG had no effect on GLUT4 concentration or modification. The antioxidant and MG scavenger NAC prevented the MG-induced GLUT4 translocation. Tiron, which is also a well-known antioxidant, had no impact on MG-induced GLUT4 translocation.
| S-EPMC3662699 | biostudies-literature
Project description:Nutritional status, in particular overweight and obesity, as well as sedentarism and high-fat diet consumption, are important risk factors to develop chronic diseases, which have a higher impact on the elderly's health. Therefore, these nutritional problems have become a concern to human healthspan and longevity. The fatty acids obtained thru the diet or due to fatty acid synthesis during obesity accumulate within the body generating toxicity and cell death. Fat is not only stored in adipose tissue, but it can also be stored in skeletal muscle. Palmitic acid (PA) has been reported as one of the most important saturated free fatty acids; it is associated to chronic oxidative stress and increased mitochondrial ROS production causing cell death by apoptosis. In skeletal muscle, palmitate has been associated with various pathophysiological consequences, which lead to muscle deterioration during aging and obesity. Since molecules that modify redox state have been proven to prevent cellular damage by inducing a hormetic response, the aim of this study was to evaluate if tert-butylhydroquinone (tBHQ) could activate an antioxidant hormetic response that would be able to protect L6 myoblasts from palmitate toxic effect. Our results provide evidence that tBHQ is able to protect L6 myoblasts against the toxicity induced by sodium palmitate due to a synergistic activation of different signaling pathways such as Nrf2 and NF-?B.
| S-EPMC7246405 | biostudies-literature
Project description:Osteoporosis is a common skeletal disorder characterized by low bone mass, which leads to reduced bone strength and an increased risk of fractures. Anabolic agents have been shown to improve bone mass and decrease fracture risk in osteoporosis patients by directly stimulating osteoblasts to produce new bone. Currently, two anabolic agents are available in the USA: recombinantly produced teriparatide (TPTD), which is the fully active (1-34) amino active sequence of human parathyroid hormone (PTH), and abaloparatide (APTD), a synthetic analog of parathyroid hormone-related peptide (PTHrP). At present, both agents are approved only for treatment of patients with osteoporosis at high risk of fracture. Nonetheless, their anabolic properties have led to off-label application in additional settings which include spine fusion, osteonecrosis of the jaw, arthroplasty, and fracture healing. In this article, we summarize available scientific literature regarding the efficacy, effectiveness, and safety of TPTD in these off-label settings.
| S-EPMC5949085 | biostudies-literature
Project description:Studies with subconfluent day 2 cultures of rat myoblasts revealed that a cell surface 112 kDa protein could be phosphorylated by extracellular ATP. Analysis of the phosphorylated 112 kDa protein suggested the involvement of a serine protein kinase. The following evidence indicated the cell surface location of this protein kinase: (i) extracellular ATP was unable to penetrate the cell membrane under our experimental conditions; (ii) the phosphorylated protein profile of intact cells differed significantly from that of broken cells; (iii) the phosphorylation of the 112 kDa protein could be abolished by pretreatment of cells with very low concentrations of trypsin; (iv) the phosphorylated 112 kDa protein could be dephosphorylated by exogenously added alkaline phosphatase; (v) the phosphorylation of the 112 kDa protein was inhibited by exogenously added proteins; and (vi) exogenously added proteins could be phosphorylated by intact cells under similar experimental conditions. The phosphorylated 112 kDa protein was detected only when the reaction was carried out in the presence of Ca2+, Mg2+, and F- ions. Kinetic analysis that revealed that the Km value of the ecto-protein kinase for ATP was 0.04 microM, and the Vmax. value for phosphorylation of the 112 kDa protein was 1.67 x 10(-4) pmol/min per 10(5) cells. Data presented in the accompanying paper [Chen & Lo (1991) Biochem. J. 279, 475-482] show that there was a constant and adequate supply of ATP on the cell surface of rat myoblasts for efficient functioning of this protein kinase, and that mutants defective in either the ecto-protein kinase or the 112 kDa protein were also impaired in myogenic differentiation. This and other biochemical studies suggest that the ecto-protein kinase and the 112 kDa protein might play important roles in myogenic differentiation.
| S-EPMC1151628 | biostudies-other
Project description:Insulin at a concentration close to the physiological range (100 mu-units/ml) stimulated protein synthesis in L6 myoblasts by 17%. Pre-treatment with the phospholipase A2 inhibitors mepacrine or dexamethasone prevented this stimulation and decreased the release of prostaglandin F2 alpha, implicating the action of phospholipase A2 and the subsequent metabolism of arachidonic acid to prostaglandins in the stimulation of protein synthesis by physiological doses of insulin. Higher concentrations of insulin (500-1000 mu-units/ml) stimulated protein synthesis in the presence of mepacrine or dexamethasone, suggesting that an alternative pathway may become important in insulin action when phospholipase A2 is inhibited.
| S-EPMC1131793 | biostudies-other
Project description:MethodsCells were divided into 5 groups-control, high-fat, 10 nmol/L LR + 0.6 mmol/L palmitic acid (PA) (10LR), 100 nmol/L LR + 0.6 mmol/L PA (100LR), and 1000 nmol/L LR + 0.6 mmol/L PA (1000LR). CCK-8 method to detect cell viability, GPO-PAP enzymatic method to detect intracellular triglyceride content, and reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and western blotting methods to detect fatty acid translocase CD36 (FAT/CD36) and fatty acid binding protein 4 (FABP4) in L6 cells, glucose-regulated protein 78 (GRP78), glucose transporter 4 (GLUT4) expression at the mRNA and protein levels, respectively, were performed.ResultsWe found that after PA intervention for 24 h, the cell viability decreased significantly; the cell viability of the LR group was higher than that of the high-fat group (P < 0.01). After PA intervention, compared with those in the high-fat group, GRP-78, FAT/CD36, FABP4 mRNA ((4.36 ± 0.32 vs. 8.15 ± 0.35); (1.00 ± 0.04 vs. 2.46 ± 0.08); (2.88 ± 0.55 vs. 8.29 ± 0.52), P < 0.01) and protein ((3338.13 ± 333.15 vs. 4963.98 ± 277.29); (1978.85 ± 124.24 vs. 2676.07 ± 100.64); (3372.00 ± 219.84 vs. 6083.20 ± 284.70), both P < 0.01) expression decreased in the LR group. The expression levels of GLUT4 mRNA ((0.75 ± 0.04 vs. 0.34 ± 0.03), P < 0.01) and protein ((3443.71 ± 191.89 vs. 2137.79 ± 118.75), P < 0.01) increased.ConclusionTherefore, we conclude that LR can reverse PA-induced cell inactivation and lipid deposition, which may be related to the change in GRP-78, FAT/CD36, FABP4, GLUT4, and other factors.
| S-EPMC9812596 | biostudies-literature
Project description:Increasing numbers of dietary supplements with ecdysteroids are marketed as "natural anabolic agents". Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERβ mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class "S1.2 Other Anabolic Agents" of the list of prohibited substances of the World Anti-Doping Agency.
| S-EPMC4447764 | biostudies-literature