Coordinated Regulation of Vasopressin Inactivation and Glucose Uptake by Action of TUG Protein in Muscle

Abstract

In adipose and muscle cells, insulin stimulates the exocytic translocation of vesicles containing GLUT4, a glucose transporter, and insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase. A substrate of IRAP is vasopressin, which controls water homeostasis. The physiological importance of IRAP translocation to inactivate vasopressin remains uncertain. We previously showed that in skeletal muscle, insulin stimulates proteolytic processing of the GLUT4 retention protein, TUG, to promote GLUT4 translocation and glucose uptake. Here we show that TUG proteolysis also controls IRAP targeting and regulates vasopressin action in vivo. Transgenic mice with constitutive TUG proteolysis in muscle consumed much more water than wild-type control mice. The transgenic mice lost more body weight during water restriction, and the abundance of renal AQP2 water channels was reduced, implying that vasopressin activity is decreased. To compensate for accelerated vasopressin degradation, vasopressin secretion was increased, as assessed by the cosecreted protein copeptin. IRAP abundance was increased in T-tubule fractions of fasting transgenic mice, when compared with controls. Recombinant IRAP bound to TUG, and this interaction was mapped to a short peptide in IRAP that was previously shown to be critical for GLUT4 intracellular retention. In cultured 3T3-L1 adipocytes, IRAP was present in TUG-bound membranes and was released by insulin stimulation. Together with previous results, these data support a model in which TUG controls vesicle translocation by interacting with IRAP as well as GLUT4. Furthermore, the effect of IRAP to reduce vasopressin activity is a physiologically important consequence of vesicle translocation, which is coordinated with the stimulation of glucose uptake.

Keywords: aminopeptidase; glucose transporter type 4 (GLUT4); insulin; membrane trafficking; skeletal muscle; translocation; vasopressin.

FIGURE 1.

Reduced vasopressin action and increased copeptin in mTUG^UBX-Cter mice. A and B, water intake was measured in 12-week-old control (WT) and mTUG^UBX-Cter TG (TG) mice and is plotted per body weight (A) and per mouse (B). C, WT and TG mice were treated without food for 18 h, allowed to recover, and then treated without food and water for 18 h. The weight decrease attributable to water removal is plotted as a percentage of initial body weight. D, mice were sacrificed after removal of food and water, and kidney lysates were immunoblotted to detect the vasopressin-responsive protein AQP2 and, as a control, α-tubulin. The relative abundance of AQP2 is plotted in WT and TG mice. E, plasma concentrations of copeptin, a product of the vasopressin prohormone, were measured in fasted WT and TG mice with free access to water. Data are presented as mean ± S.E.; n = 6–12 in each group.

FIGURE 2.

TUG binds IRAP and controls its targeting. A and B, T-tubule membrane fractions were prepared from quadriceps muscles of fasted mTUG^UBX-Cter TG and WT control mice. Immunoblots were performed to detect IRAP and insulin receptor β-chain (IRβ), as indicated. B, densitometry was done on immunoblots from A, and the relative abundance of IRAP in T-tubule fractions was plotted. C, GST fusion proteins containing the indicated regions of the IRAP cytosolic N terminus were used to purify TUG from lysates of transfected 293 cells. TUG was detected by immunoblotting (WB), and GST proteins were stained using GelCode Blue. D, a biotinylated peptide containing IRAP residues 55–84 was used to purify TUG from lysates of 293 cells transfected with full-length TUG (residues 1–550) or with a truncated form (residues 165–550). Purified proteins and lysates were immunoblotted as indicated. E, TUG-bound vesicles were purified from basal and insulin-stimulated 3T3-L1 adipocytes expressing biotin-tagged TUG, using immobilized streptavidin beads. Control precipitations were done using biotin-saturated streptavidin. Eluted proteins were immunoblotted to detect IRAP, GLUT4, and TUG as indicated. F, replicates of the experiment shown in E were analyzed using densitometry of the immunoblots, and data were quantified and plotted. Data are presented as mean ± S.E.; n = 3. G, cell surface proteins were biotinylated in control 3T3-L1 adipocytes, in cells containing an shRNA to deplete TUG (TUG KD), and in cells containing both the shRNA and the shRNA-resistant TUG (rescue). Cells were treated with insulin as indicated. Surface-exposed proteins were purified and immunoblotted to detect IRAP and, as a control, IRβ. The ratio of surface IRAP to surface IRβ band intensity is indicated below each lane.