de Vasconcelos Diogo Antonio Alves, Giesbertz Pieter, Murata Gilson Masahiro, de Souza Diego Ribeiro, Fiamoncini Jarlei, Duque-Guimaraes Daniella, Leandro Carol Góis, Hirabara Sandro Massao, Daniel Hannelore, Curi Rui, Pithon-Curi Tania Cristina
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.
Post-graduate Program in Nutrition, Physical Activity and Phenotypic Plasticity, Federal University of Pernambuco, Vitoria de Santo Antao, Brazil,
Cell Physiol Biochem. 2019;53(1):200-214. doi: 10.33594/000000130.
BACKGROUND/AIMS: Skeletal mass loss is reported in several catabolic conditions and it has been associated with a reduced intracellular L-glutamine content. We investigated the association of intracellular L-glutamine concentration with the protein content in skeletal muscle cells.
We cultivated CC myotubes in the absence or presence of 2 (reference condition), 8 or 16 mM L-glutamine for 48 hours, and the variations in the contents of amino acids and proteins measured. We used an inhibitor of L-glutamine synthesis (L-methionine sulfoximine - MSO) to promote a further reduction in intracellular L-glutamine levels. Amino acids contents in cells and media were measured using LC-MS/MS. We measured changes in phosphorylated Akt, RP-S6, and 4E-BP1contents in the absence or presence of insulin by western blotting.
Reduced intracellular L-glutamine concentration was associated with decreased protein content and increased protein breakdown. Low intracellular glutamine levels were also associated with decreased p-Akt contents in the presence of insulin. A further decrease in intracellular L-glutamine caused by glutamine synthetase inhibitor reduced protein content and levels of amino acids generated from glutamine metabolism and increased bAib still further. Cells exposed to high medium glutamine levels did not have any change in protein content but exhibited increased contents of the amino acids derived from L-glutamine metabolism.
Intracellular L-glutamine levels per se play a role in the control of protein content in skeletal muscle myotubes.
背景/目的：在几种分解代谢状态下均有骨骼质量损失的报道，且这与细胞内L-谷氨酰胺含量降低有关。我们研究了细胞内L-谷氨酰胺浓度与骨骼肌细胞中蛋白质含量的关联。
我们在不存在或存在2（参照条件）、8或16 mM L-谷氨酰胺的情况下培养CC肌管48小时，并测量氨基酸和蛋白质含量的变化。我们使用L-谷氨酰胺合成抑制剂（L-蛋氨酸亚砜亚胺 - MSO）来促使细胞内L-谷氨酰胺水平进一步降低。使用液相色谱-串联质谱法测量细胞和培养基中的氨基酸含量。我们通过蛋白质印迹法测量在存在或不存在胰岛素的情况下磷酸化Akt、核糖体蛋白S6（RP-S6）和真核起始因子4E结合蛋白1（4E-BP1）含量的变化。
细胞内L-谷氨酰胺浓度降低与蛋白质含量减少和蛋白质分解增加有关。在存在胰岛素的情况下，低细胞内谷氨酰胺水平还与磷酸化Akt含量降低有关。谷氨酰胺合成酶抑制剂导致细胞内L-谷氨酰胺进一步减少，降低了蛋白质含量以及谷氨酰胺代谢产生的氨基酸水平，并进一步增加了β-丙氨酸异丁酯（bAib）。暴露于高培养基谷氨酰胺水平的细胞蛋白质含量没有任何变化，但源自L-谷氨酰胺代谢的氨基酸含量增加。
细胞内L-谷氨酰胺水平本身在骨骼肌肌管蛋白质含量的控制中发挥作用。
