Regulation of Insulin-Responsive Aminopeptidase Expression and Targeting in the Insulin-Responsive Vesicle Compartment of Glucose Transporter Isoform 4-Deficient Cardiomyocytes

Abstract

In adipocytes and cardiac or skeletal muscle, glucose transporter isoform 4 (GLUT4) is targeted to insulin-responsive intracellular membrane vesicles (IRVs) that contain several membrane proteins, including insulin-responsive aminopeptidase (IRAP) that completely colocalizes with GLUT4 in basal and insulin-treated cells. Cardiac GLUT4 content is reduced by 65-85% in IRAP knockout mice, suggesting that IRAP may regulate the targeting or degradation of GLUT4. To determine whether GLUT4 is required for maintenance of IRAP content within IRVs, we studied the expression and cellular localization of IRAP and other GLUT4 vesicle-associated proteins, in hearts of mice with cardiac-specific deletion of GLUT4 (G4H-/-). In G4H-/- hearts, IRAP content was reduced by 60%, but the expression of other vesicle-associated proteins, namely cellugyrin, IGF-II/mannose-6-phosphate, and transferrin receptors, secretory carrier-associated membrane proteins and vesicle-associated membrane protein were unchanged. Using sucrose gradient centrifugation and cell surface biotinylation, we found that IRAP content in 50-80S vesicles where GLUT4 vesicles normally sediment was markedly depleted in G4H-/- hearts, and the remaining IRAP was found in the heavy membrane fraction. Although insulin caused a discernible increase in cell surface IRAP content of G4H-/- cardiomyocytes, cell surface IRAP remained 70% lower than insulin-stimulated controls. Immunoabsorption of intracellular vesicles with anticellugyrin antibodies revealed that IRAP content was reduced by 70% in both cellugyrin-positive and cellugyrin-negative vesicles. Endosomal recycling, as measured by transferrin receptor recycling was normal. Thus, GLUT4 and IRAP content of early endosome-derived sorting vesicles and of IRVs are coordinately regulated, and both proteins are required for maintenance of key constituents of these compartments in cardiac muscle cells in vivo.

Recycling of IRAP from the plasma membrane back to the insulin-responsive compartment requires the Q-SNARE syntaxin 6 but not the GGA clathrin adaptors

“…striated muscle and adipose tissue (Keller et al, 2002). In addition, the genetic ablation of GLUT4 altered the subcellular distribution and expression levels of the IRAP protein (Abel et al, 2004;Carvalho et al, 2004). These data suggest a functional relationship between IRAP and GLUT4; however, a molecular mechanism has not been elucidated.…”

The Inactivation of RabGAP Function of AS160 Promotes Lysosomal Degradation of GLUT4 and Causes Postprandial Hyperglycemia and Hyperinsulinemia

“…We are currently unable to distinguish these two possibilities, and further experimentation is required to address this question in the future. It is well known that the other protein residing in GSVs, IRAP, traffics together with GLUT4 in response to insulin (40), and its expression is decreased in various GLUT4-null cells (41,42). Interestingly, in contrast to GLUT4, IRAP levels were normal in tissues or primary adipocytes from the AS160 KO mice, suggesting that the degradation of these two proteins might be differentially regulated.…”

A Specific Dileucine Motif Is Required for the GGA-dependent Entry of Newly Synthesized Insulin-responsive Aminopeptidase into the Insulin-responsive Compartment

“…Other than GLUT4, the insulin-regulated aminopeptidase (IRAP) is the only known transmembrane protein that undergoes a dramatic redistribution to the cell surface in response to acute insulin stimulation. IRAP was identified as a major protein that colocalizes with GLUT4 in insulin-responsive storage vesicles (27,32,(41)(42)(43)(44)(45)(46)(47)(48). Although the physiological function of IRAP remains unclear (49), it has nevertheless been used successfully as a reporter molecule to analyze insulin-regulated intracellular trafficking (50 -53).…”