Figure 2 Axin, TNKS and KIF3A are co-localized and highly enriched in the perinuclear region. Differentiated 3T3-L1 adipo- cytes (d 8) were incubated with or without 200 nM insulin for 30 min after starvation in low-glucose DMEM for 4 h. (A) Cellular Axin, KIF3A and TNKS are co-localized in the perinuclear re- gion. The nuclei were indicated by DAPI staining (gray). Images were acquired with a confocal laser-scanning microscope. After superimposition of images, white color indicates co-localization of Axin (red), KIF3A (green), and TNKS (blue). Arrow heads point to the PM. (B) Axin and KIF3A are partially co-localized with the trans Golgi network marker TGN38. (C) TNKS, KIF3A and Axin have co-localization with GLUT4. Scale bars represent 15 µm. Axin/TNKS/KIF3A complex is required for insulin-stimu- lated glucose uptake We then assessed whether the Axin/TNKS/KIF3A www.cell-research.com | Cell Research Hui-Ling Guo et al . npg 1249 complex affects glucose uptake by knocking down the expression of Axin, TNKS1/2 or KIF3A individually in 3T3-L1 adipocytes by siRNA-expressing adenoviruses. As can be seen from Figure 4A-4C and Supplementary information, Figure S3, depletion of Axin, KIF3A or TNKS2 attenuated insulin-stimulated 2-deoxyglucose uptake. Re-introduction of each protein by adenoviruses expressing human Axin, TNKS2 and KIF3A restored the insulin-stimulated glucose uptake. Double knock- down of TNKS1 and TNKS2 further decreased insulin- stimulated glucose uptake than single knockdown (Figure 4D, Supplementary information, Figure S3C), indicating that TNKS1 and TNKS2 have possibly overlapping func- tions in the regulation of glucose uptake in adipocytes. Furthermore, we knocked down TNKS2 and KIF3A together and found that the effect on glucose uptake was similar to that of KIF3A single knockdown (Figure 4E), suggesting that TNKS and KIF3A work in conjunction to regulate insulin-stimulated glucose uptake. We next determined the physiological consequence of TNKS regulation of GLUT4 translocation. We mea- sured the blood glucose levels of adult (12-week-old) wildtype and TNKS2−/− mice at normally fed, fasted and re-fed states (Figure 5A), and found that the normally fed and fasted TNKS2−/− mice had similar blood glucose levels compared to WT mice. However, the blood glu- cose levels were significantly higher in the re-fed (fed after fasting) TNKS2−/− mice (9.06 ± 0.33 mM) compared to wildtype littermates (7.68 ± 0.26 mM, P = 0.043). The food consumption between wildtype and TNKS2−/− mice showed no significant difference (Supplementary information, Figure S5). Intriguingly, we observed in- creased blood glucose levels in normally fed 7-week- old TNKS2−/− mice (8.33 ± 0.19 mM) compared to WT mice of the same age (7.2 ± 0.29 mM, P = 0.012) (Figure 5B). Moreover, glucose tolerance test (GTT) showed that adult TNKS2−/− mice had higher levels of blood glucose following administration of an exogenous load of glucose compared with that of WT mice (Figure 5C), suggest- ing a decreased glucose disposal in the TNKS2-deficient mice. Insulin secretion was similar between the WT and TNKS2−/− mice, at either fasted or fed state (Figure 5D). In fact, in the GTT test, due to elevated blood glucose levels in TNKS2−/− mice, their secreted insulin level was proportionally increased (Supplementary information, Figure S6), indicating that the decreased glucose disposal observed in TNKS2−/− mice during the GTT assay is not caused by an abnormal insulin secretion. Following in- jection of excessive amounts of insulin for the insulin tolerance test (ITT), TNKS2−/− mice showed higher blood glucose levels compared to WT mice (Figure 5E). These results collectively indicate that TNKS2 plays an impor-