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Regulatory mechanisms of SNAT2, an amino acid transporter, in L6 rat skeletal muscle cells by insulin, osmotic shock and amino acid deprivation

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Abstract

Several studies have demonstrated that the activity of system A is upregulated by insulin, osmotic shock and amino acid deprivation. However, the mechanisms are not clear. We carried out studies using L6 rat skeletal muscle cells to clarify the mechanisms of upregulation of system A activity by insulin, osmotic shock and amino acid deprivation. The upregulation was found to be due to an increase in V max, not K m. Chloroquine and wortmannin inhibited the upregulation induced by insulin stimulation and amino acid deprivation but not that induced by osmotic shock. On the other hand, cycloheximide and actinomycin D inhibited the upregulation by each stimulation. Moreover, PD98059 and SP600125 inhibited only amino acid deprivation-induced upregulation and SB202190 inhibited only insulin-induced upregulation. Our findings indicate that the mechanisms of upregulation of system A activity by insulin, osmotic shock and amino acid deprivation are different in L6 cells. Western blot and RT-PCR analysis showed an increase in system A at the protein and mRNA levels with each stimulation.

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Abbreviations

SNAT2:

Sodium-coupled neutral amino acid transporter 2

Me-AIB:

α-Methylaminoisobutyric acid

MAPK:

Mitogen-activated protein kinase

ERK:

Extracellular regulated kinase

JNK:

Jun N-terminal kinase

MDCK:

Madin–Darby canine kidney

CHO:

Chinese hamster ovary

PI3K:

Phosphatidylinositol 3-kinase

MEM:

Minimum essential medium Eagle

FBS:

Fetal bovine serum

SDS:

Sodium dodecyl sulfate

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

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Acknowledgements

This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Culture and Sports of Japan.

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Authors and Affiliations

  1. Department of Pharmacy, Hokkaido University Hospital, Sapporo, Hokkaido, Japan

    Hitoshi Kashiwagi, Kojiro Yamazaki, Yoh Takekuma & Mitsuru Sugawara

  2. Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, USA

    Vadivel Ganapathy

Authors
  1. Hitoshi Kashiwagi
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  2. Kojiro Yamazaki
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  3. Yoh Takekuma
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  4. Vadivel Ganapathy
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  5. Mitsuru Sugawara
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Correspondence to Mitsuru Sugawara.

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Kashiwagi, H., Yamazaki, K., Takekuma, Y. et al. Regulatory mechanisms of SNAT2, an amino acid transporter, in L6 rat skeletal muscle cells by insulin, osmotic shock and amino acid deprivation. Amino Acids 36, 219–230 (2009). https://doi.org/10.1007/s00726-008-0050-9

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