The role of small GTPase molecules is poorly understood under high glucose conditions.

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.

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.

These results demonstrate that Vav3 is involved in the process of metformin-mediated glucose regulation.

The tubby protein has a motif that might be relevant for its action in the insulin signaling pathway. Previous studies have indicated that tubby undergoes phosphorylation on tyrosine residues in response to several stimuli and is known to localize in the nucleus as well as in the plasma membrane. However, the relationship between phosphorylation and nuclear translocation is not well understood. Here, we report that insulin directly phosphorylates tubby, which translocates into the nucleus.

The effects of insulin on Tubby were performed with Western blot. The immunoprecipitation and confocal microscopy were performed to prove phosphorylation and nuclear translocation.

Mutation study reveals that tyrosine residue 464 of tubby gene (Tub) is a phosphorylation site activated by insulin. In addition, major portions of tubby protein in the plasma membrane are translocated into the nucleus after insulin treatment. Tyrosine kinase inhibitor pretreatment blocked insulin-induced tubby translocation, suggesting that phosphorylation is important for nuclear translocation. Moreover, mutant tyrosine residue 464 did not translocate into the nucleus in respond to insulin. These findings demonstrate that insulin phosphorylates tyrosine residue 464 of Tub, and this event is important for insulin-induced tubby nuclear translocation.

Insulin phosphorylates tyrosine residue 464 of Tub and translocates tubby into the nuclei of HIRcB cells.