AIMS: The overarching aim of our study was to establish whether and how glucose availability and AMPK affect expression of Na+,K+-ATPase (NKA) subunits in cultured skeletal muscle cells. Specific aims were: (1) to determine if glucose affects expression of NKA subunits in isoform-specific manner; (2) to uncover transcription factors involved in regulation of NKA subunit expression by glucose; (3) to dissect the role of AMPK activation in regulation of NKA subunit expression.
HYPOTHESES: We tested three hypotheses: (H1) Glucose regulates expression of NKA subunits in isoform specific-manner in skeletal muscle cells. (H2) Glucose-sensitive transcription factors modulate expression of NKA subunits in skeletal muscle cells. (H3) AMPK regulates the abundance of NKA subunits in skeletal muscle cells.
METHODS: Experiments were performed on standard models used for skeletal muscle research in vitro: rat L6 skeletal muscle cell line and primary human skeletal muscle cells (HSMCs). Expression of NKA subunits was estimated using quantitative polymerase chain reaction (qPCR) and Western Blot. Gene silencing was used to assess the role of AMPK in regulation of NKA.
RESULTS: Ad H1: To investigate the effects of glucose on NKA expression and protein abundance, we performed experiments in rat L6 cells and HSMCs under different glucose concentrations (0 mM, 5.5 mM and 25 mM). In L6 cells, glucose deprivation led to an increase in protein levels of NKAα1 and NKAα2, and the trend of mRNA expression was consistent with the results of protein expression of NKAα1. Conversely, the mRNA expression of NKAα2 was downregulated in glucose-free media. In HSMCs, NKAα1 protein levels increased under high glucose, while NKAα2 remained unchanged. Ad H2: Treating rat L6 myotubes with glucose concentration of 5.5 mM resulted in upregulation of mRNA of Sp1 and ZEB1 compared to glucose deprivation and high glucose concentration. On the other hand, Mondo A and ATF1 were downregulated after treatment with a glucose concentration of 25 mM. ATF1 was also downregulated at a glucose concentration of 5.5 mM. In addition, glucose deprivation downregulated mRNA expression of REST at both 5.5 mM and 25 mM glucose concentrations. In HSMCs, the only significant difference mRNA expression in response to different glucose concentrations was observed for ChREBP, which was upregulated under glucose-free conditions. Ad H3: L6 myotubes were treated with AICAR and A-769662 for 24 hours. AMPK activators decreased the protein abundance of NKAα1 and NKAα2, consistent with mRNA expression. We also detected decreased mRNA expression of FXYD1 after treatment with AMPK activators. To investigate the involvement of AMPK in NKA regulation, knock-down of AMPKα1/α2 was performed in L6 myotubes, and the protein abundance of NKAα1 was increased at a glucose concentration of 5.5 mM. Furthermore, knock-down of AMPKα1/α2 resulted in decreased protein abundance of NKAα2 under glucose deprivation. In HSMCs, at a glucose concentration of 5.5 mM, mRNA expression of NKAα1 was upregulated in AMPKα1/α2 knockdown cells, along with downregulation of mRNA expression of NKAβ1 and NKAβ2.
CONCLUSIONS: (1) Glucose affects the expression of NKA subunits in L6 cells and HSMCs in an isoform-specific manner, supporting our first hypothesis. (2) Our study indirectly suggests that mRNA expression of some transcription factors (Sp1, ZEB1, ATF1 and REST v L6 cells and ChREBP in HSMCs) is glucose-responsive and may play a role in influencing the expression of NKA subunits in skeletal muscle cells. We can neither confirm nor reject our second hypothesis. (3) In L6 cells, AMPK regulates protein and mRNA levels of NKA subunits in an isoform-dependent manner. Furthermore, we indicate that AMPK is a negative regulator of FXYD1 in L6 cells. Overall, our results support the third hypothesis.
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