Background: Insulin stimulates endoproteolytic cleavage of TUG proteins to promote glucose uptake in cultured adipocytes, but the role of this pathway in muscle is uncharacterized. Results: Transgenic mice with constitutive and unregulated TUG cleavage in muscle had increased whole-body and musclespecific glucose uptake during fasting. Conclusion: Insulin acts through TUG to control glucose uptake in muscle. Significance: Understanding insulin action will elucidate diabetes pathogenesis.

Insulin stimulates the exocytic translocation of specialized vesicles in adipocytes, which inserts GLUT4 glucose transporters into the plasma membrane to enhance glucose uptake. Previous results support a model in which TUG (ether containing a BX domain forLUT4) proteins trap these GLUT4 storage vesicles at the Golgi matrix and in which insulin triggers endoproteolytic cleavage of TUG to translocate GLUT4. Here, we identify the muscle splice form of Usp25 (Usp25m) as a protease required for insulin-stimulated TUG cleavage and GLUT4 translocation in adipocytes. Usp25m is expressed in adipocytes, binds TUG and GLUT4, dissociates from TUG-bound vesicles after insulin addition, and colocalizes with TUG and insulin-responsive cargoes in unstimulated cells. Previous results show that TUG proteolysis generates the ubiquitin-like protein, TUGUL (for biquitin-ike). We now show that TUGUL modifies the kinesin motor protein, KIF5B, and that TUG proteolysis is required to load GLUT4 onto these motors. Insulin stimulates TUG proteolytic processing independently of phosphatidylinositol 3-kinase. In nonadipocytes, TUG cleavage can be reconstituted by transfection of Usp25m, but not the related Usp25a isoform, together with other proteins present on GLUT4 vesicles. In rodents with diet-induced insulin resistance, TUG proteolysis and Usp25m protein abundance are reduced in adipose tissue. These effects occur soon after dietary manipulation, prior to the attenuation of insulin signaling to Akt. Together with previous data, these results support a model whereby insulin acts through Usp25m to mediate TUG cleavage, which liberates GLUT4 storage vesicles from the Golgi matrix and activates their microtubule-based movement to the plasma membrane. This TUG proteolytic pathway for insulin action is independent of Akt and is impaired by nutritional excess.

Background: Insulin stimulates glucose uptake by triggering TUG proteolysis, which liberates intracellular storage vesicles containing GLUT4. Results: TUG acetylation modulates its interaction with Golgi matrix proteins and enhances its function to trap GLUT4 storage vesicles within unstimulated cells. SIRT2 modulates TUG acetylation and controls insulin sensitivity in vivo. Conclusion: TUG acetylation promotes GLUT4 accumulation in insulin-responsive vesicles. Significance: Nutritional status modulates insulin-stimulated glucose uptake.

Insulin regulates glucose uptake through effects on the trafficking of the glucose transporter Glut4. To investigate the degree of overlap between Glut4 and the general endocytic pathways, the kinetics of trafficking of Glut4 and the receptors for transferrin (Tf) and ␣ 2 -macroglobulin (␣-2-M; LRP-1) were compared using quantitative flow cytometric assays. Insulin increased the exocytic rate constant (k ex ) for both Glut4 and Tf. However, the k ex of Glut4 was 5-15 times slower than Tf in both basal and insulin-stimulated cells. The endocytic rate constant (k en ) of Glut4 was also five times slower than Tf. Insulin did not affect the k en of either protein. In basal cells, the k en for ␣-2-M/ LRP-1 was similar to Glut4 but 5-fold slower than Tf. Insulin increased k en for ␣-2-M/LRP-1 by 30%. In contrast, the k ex for LRP-1 was five times faster than Glut4 in basal cells, and insulin did not increase this rate constant. Thus, although there is overlap in the protein machineries/compartments utilized, the differences in trafficking kinetics indicate that Glut4, the Tf receptor, and LRP-1 are differentially processed both within the cell and at the plasma membrane. It has been reported that insulin decreases the k en of Glut4 in adipocytes. However, the effect of exocytosis on the "internalization" assays was not considered. Because it is counterintuitive, the effect of exocytosis on these assays is often overlooked in endocytosis studies. Using mathematical modeling and simulation, we show that the reported decrease in Glut4 k en can be entirely accounted for by the well established increase in Glut4 k ex .

Mechanisms to coordinately regulate energy expenditure and glucose uptake into muscle and fat cells are not well described. Insulin stimulates glucose uptake in part by causing site-specific endoproteolytic cleavage of TUG, which mobilizes GLUT4 glucose transporters to the cell surface. Here, we show that the TUG C-terminal cleavage product enters the nucleus, binds the transcriptional regulators PGC-1a and PPARg, and increases oxidative metabolism and thermogenic protein expression. Muscle-specific genetic manipulation of this pathway impacts whole-body energy expenditure, independent of glucose uptake. The PPARg2 Pro12Ala polymorphism, which reduces diabetes risk, enhances TUG binding. The TUG cleavage product stabilizes PGC-1a and is itself susceptible to an Ate1 arginyltransferase -dependent degradation mechanism; binding of the TUG product confers Ate1-dependent stability upon PGC-1a. We conclude that TUG cleavage coordinates energy expenditure with glucose uptake, that this pathway may contribute to the thermic effect of food, and that its attenuation may be important in obesity..