npg Axin/TNKS/KIF3A complex regulates GLUT4 translocation 1252 Figure 5 TNKS2 is required in insulin-stimulated glucose dis- posal. (A) Analysis of blood glucose concentrations in normal and mutant adult mice (12-week males). Mice (TNKS2+/+, n = 6; TNKS2−/−, n = 5) were fasted for 16 h, and re-fed for 1 h. Blood glucose levels in the fasted mice and re-fed mice were sepa- rately determined. (B) Blood glucose levels of young (7-week) males (TNKS2+/+, n = 7; TNKS2−/−, n = 8) were determined, showing a significant difference between the WT and mutant mice (*P < 0.05). (C) Glucose tolerance test. Animals were fasted for 16 h. D-Glucose (2 g/kg) was administrated into mice by intraperitoneal injection, and blood glucose concentrations were determined at the indicated times (12-week male mice; TNKS2+/+, n = 6, 24-27 g; TNKS2−/−, n = 5, 20-24 g; *P < 0.05, **P < 0.01). (D) WT (n = 13) and mutant mice (n = 12) were fed or fasted overnight, and their insulin levels were determined. (E) Insulin tolerance test. 1 U/kg human insulin was administered into 12-week mice (TNKS2+/+, n = 6, 24-27 g; TNKS2−/−, n = 5, 20-24 g) by intraperitoneal injection and blood glucose levels were determined at the indicated times. selves and Axin, increasing their ubiquitination and sub- sequent degradation [34], we next determined the extents of PARsylation and ubiquitination of TNKS and Axin in the presence or absence of insulin. 3T3-L1 adipocytes were serum-starved for 4 h, followed by insulin treatment in the presence of MG132 (Figure 6B). Axin and TNKS proteins were then immunoprecipitated and the effects of insulin on their PARsylation and ubiquitination were separately monitored by immunoblotting using anti-PAR and anti-ubiquitin (Ub) antibodies. The results showed that insulin treatment obviously reduced both PARsyla- tion and ubiquitination of TNKS and Axin, indicating that the TNKS PARsylase activity was suppressed upon insulin stimulation. We also checked whether GLUT4 is PARsylated or ubiquitinated with or without the treat- ment of insulin. The results show that GLUT4 is indeed PARsylated and ubiquitinated. However, the PARsylation and ubiquitination as well as the total GLUT4 protein levels are not affected by insulin treatment (Supplemen- tary information, Figure S8). We then tested whether levels of the Axin/TNKS/KI- F3A complex formation were changed with addition of insulin by carrying out co-IP of the endogenous complex proteins from 3T3-L1 adipocytes treated with or with- out insulin with the antibody against TNKS. Increased amounts of KIF3A and Axin were co-precipitated with TNKS in cells treated with insulin (Figure 6C). We also investigated Axin/TNKS/KIF3A complex formation in C2C12 cells through co-IP (Supplementary information, Figure S9), which showed that the complex formation is conserved between adipocytes and muscle cells. To support the notion that the Axin/TNKS/KIF3A complex is stabilized by insulin through inhibition of TNKS PARsylase activity, we synthesized the TNKS PARsylase inhibitor XAV939. As shown in Supplemen- tary information Figure S10A, XAV939 could promote the tripartite complex formation. Furthermore, XAV939 could increase insulin-stimulated glucose uptake by about 30% (Supplementary information, Figure S10B) and enhance insulin-simulated GLUT4 translocation to the PM (Supplementary information, Figure S10C). Together, these findings suggest that insulin stimulation can enhance the Axin/TNKS/KIF3A complex formation by suppressing TNKS-mediated PARsylation and sub- sequent ubiquitination/degradation of Axin and TNKS itself. Akt mediates insulin-stimulated Axin/TNKS/KIF3A com- plex stabilization To further analyze the direct involvement of insulin signaling in modulating the complex of Axin/TNKS/ KIF3A, we determined the effects of Akt inhibition on Cell Research | Vol 22 No 8 | August 2012