Iresjö Britt-Marie, Landin Andreas, Ohlsson Claes, Lundholm Kent
1Surgical Metabolic Research Lab, Department of Surgery, Institute of clinical sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
2Department of Surgery, Sahlgrenska University Hospital, Region Västra Götaland, Gothenburg, Sweden.
Genes Nutr. 2019 Nov 12;14:29. doi: 10.1186/s12263-019-0652-8. eCollection 2019.
Previous investigations have indicated upregulation of gene expression in cellular pathways related to the biosynthesis of steroids in response to amino acids (AA) in skeletal muscle cells. This suggests AA as modulators of de novo synthesis of sex steroids for muscle growth and improved functional capacity. The aim of the present study was to investigate if increased availability of amino acids induced biosynthesis of sex steroids in skeletal muscles.
Confluent L6 muscle cells were cultured in media with various AA concentrations (0.3 or 9 mM AA or 2.1 mM branched-chain (BCAA) only), following pre-culture in serum-free medium. Sex steroids were quantified by gas chromatography-tandem mass spectrometry (GC-MS/MS). Mevalonate (diphospho-) decarboxylase enzyme (MVD) was quantified by Western blot.
The experiments confirmed that estradiol and estrone increased in both L6 cell lysates and in conditioned media at the end of experiments on confluent cells, while progesterone or androgenic steroids were not detected in either cell lysates or culture media. Estradiol (+ 31 ± 3%) and estrone (+ 18 ± 4%) increased significantly in cells cultured at 9 mM AA ( < 0.001 vs. 0.3 mM AA, = 10). Similarly, MVD protein increased at 9 mM AA ( < 0.001 vs. 0.3 mM AA, = 17). An addition of BCAA alone to media increased MVD-protein levels to the same extent as all AA ( < 0.01 vs. 0.3 mM AA, = 3).
Female sex steroids and MVD enzyme production increased significantly in response to amino acid availability. The results indicate a role of amino acids as modulators of local muscle estrogen synthesis in muscle cells from rats at feeding.
先前的研究表明，骨骼肌细胞中与类固醇生物合成相关的细胞途径中的基因表达会因氨基酸（AA）而上调。这表明AA是肌肉生长和功能能力改善的性类固醇从头合成的调节剂。本研究的目的是调查氨基酸可用性增加是否会诱导骨骼肌中性类固醇的生物合成。
在无血清培养基中预培养后，将汇合的L6肌肉细胞培养在含有不同AA浓度（0.3或9 mM AA或仅2.1 mM支链氨基酸（BCAA））的培养基中。通过气相色谱-串联质谱法（GC-MS/MS）对性类固醇进行定量。通过蛋白质印迹法对甲羟戊酸（二磷酸）脱羧酶（MVD）进行定量。
实验证实，在汇合细胞实验结束时，L6细胞裂解物和条件培养基中的雌二醇和雌酮均增加，而在细胞裂解物或培养基中均未检测到孕酮或雄激素类固醇。在9 mM AA培养的细胞中，雌二醇（+31±3%）和雌酮（+18± 4%）显著增加（与0.3 mM AA相比，P<0.001，n = 10）。同样，在9 mM AA时MVD蛋白增加（与0.3 mM AA相比，P<0.001，n = 17）。仅在培养基中添加BCAA可使MVD蛋白水平增加到与所有AA相同的程度（与0.3 mM AA相比，P<0.01，n = 3）。
雌性性类固醇和MVD酶的产生因氨基酸可用性而显著增加。结果表明氨基酸在喂食大鼠的肌肉细胞中作为局部肌肉雌激素合成调节剂的作用。
