Endocrinol Metab.  2014 Sep;29(3):363-370. 10.3803/EnM.2014.29.3.363.

Vav3, a GEF for RhoA, Plays a Critical Role under High Glucose Conditions

• Sha J
• Na J
• Lee JO
• Kim N
• Lee SK
• Kim JH
• Moon JW
• Kim SJ
• Lee HJ
• Choi JI
• Park SH
• Kim HS

• KMID: 2169474
• DOI: http://doi.org/10.3803/EnM.2014.29.3.363

Abstract

BACKGROUND
The role of small GTPase molecules is poorly understood under high glucose conditions.
METHODS
We analyzed the expression pattern of Vav3 in skeletal muscle C2C12 cells under high glucose culture condition with reverse transcription-polymerase chain reaction and Western blot analysis. We also measured glucose uptake using isotope-labelled glucose.
RESULTS
We showed that expression of Vav3 (a guanine nucleotide exchange factor for RhoA) increased. mRNA and protein levels in skeletal muscle C2C12 cells under high glucose conditions. The AMP-activated protein kinase (AMPK) activator AMPK agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR) suppressed high glucose-induced Vav3 induction. In addition, exposure of cells to high glucose concentration increased the phosphorylation of PAK-1, a molecule downstream of RhoA. The phosphorylation of paxillin, a downstream molecule of PAK-1, was also increased by exposure to high glucose. Phosphorylation of these molecules was not observed in the presence of AICAR, indicating that AMPK is involved in the RhoA signal pathway under high glucose conditions. Knock down of Vav3 enhances metformin-mediated glucose uptake. Inhibition of AMPK blocked the increases of Vav3 knock down-induced glucose uptake. Metformin-mediated Glut4 translocation was also increased by Vav3 knock-down, suggesting that Vav3 is involved in metformin-mediated glucose uptake.
CONCLUSION
These results demonstrate that Vav3 is involved in the process of metformin-mediated glucose regulation.

Keyword

AMP-activated protein kinases; Diabetes; High glucose; Metformin; Vav3

MeSH Terms

AMP-Activated Protein Kinases
Blotting, Western
Glucose*
GTP Phosphohydrolases
Guanine Nucleotide Exchange Factors
Metformin
Muscle, Skeletal
Paxillin
Phosphorylation
RNA, Messenger
Signal Transduction
AMP-Activated Protein Kinases
GTP Phosphohydrolases
Glucose
Guanine Nucleotide Exchange Factors
Metformin
Paxillin
RNA, Messenger

Figure

• 

  Fig. 1 (A) Total RNA was prepared for these cells after high glucose culture, and reverse transcription-polymerase chain reaction (RT-PCR) was conducted using specific Vav3 primers. PCR products were then run on 1% agarose gels and visualized under ultraviolet light. β-Actin mRNA was employed as a positive control. (B) C2C12 cells were stimulated at the indicated times with high glucose concentrations. The cell lysates (25 µg) were analyzed via Western blotting for anti-Vav3 antibody. Blotting with anti-β-actin antibody was used as a protein loading control. The results shown are from three independent experiments.

• 

  Fig. 2 (A) Total RNA was cultured under high glucose culture conditions in the presence or absence of 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR) treatment, and reverse transcription-polymerase chain reaction (RT-PCR) was conducted using specific Vav3 primers. PCR products were then run on 1% agarose gels and visualized under ultraviolet light. β-Actin mRNA was employed as a positive control. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMP-activated protein kinase (AMPK) agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). The cell lysates (25 µg) were analyzed via Western blotting for anti-Vav3 antibody. Blotting with anti-β-actin antibody was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPK antibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. (D) C2C12 cells were cultured under high glucose conditions in the presence or absence of metformin. The cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-AMPKantibody. Blotting with anti-AMPK antibody was conducted as a protein loading control. The results shown are from three independent experiments.

• 

  Fig. 3 (A) C2C12 cells were cultured under high glucose conditions in the presence or absence of AMPK agonist 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR). Total cell lysates (25 µg) were analyzed via Western blotting for anti-phospho-PAK and anti-PAK antibodies. (B) C2C12 cells were cultured under high glucose conditions in the presence or absence of AICAR. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-paxillin and anti-paxillin antibodies was conducted as a protein loading control. (C) C2C12 cells were cultured under high glucose conditions in the presence of metformin. The cell lysates (25 µg) were analyzed via Western blotting. Blotting with anti-phospho-PAK and anti-phospho-paxillin antibodies was performed. Blotting with anti-PAK and anti-paxillin antibodies was conducted as a protein loading control. The results shown are from three independent experiments.

• 

  Fig. 4 (A) C2C12 cells were transiently transfected with 50 nM siRNA Vav3 for 2 days. Total cell lysates (25 µg) were analyzed via Western blotting for anti-Vav3 and anti-β-actin antibodies. (B) C2C12 cells were allowed to differentiate for 7 days. Cells were transiently transfected with Vav3 siRNA for 48 hours and then treated with metformin for 16 hours either in the presence or absence of compound C. Glucose uptake was measured using 2-deoxy-[3H]-d-glucose. The results shown are from three independent experiments. (C) Myoblasts stably expressing L6-GLUT4myc were differentiated and were transiently transfected with Vav3 siRNA and maintained for 48 hours, then incubated with metformin for 1 hour. Cell surface expression of GLUT4myc was detected using an antibody-coupled colorimetric absorbance assay. Results are displayed as the mean±SEM from three experiments. aP<0.05 for the basal values; bP<0.05 for the metformin-treated condition; cP<0.05 for the metformin plus Vav3 siRNA; dP<0.05 compared with metformin-treated condition.

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