Identification of Glucose Transporter 4 Knockdown-dependent Transcriptional Activation Element on the Retinol Binding Protein 4 Gene Promoter and Requirement of the 20 S Proteasome Subunit for Transcriptional Activity

Abstract

Retinol binding protein 4 (RBP4) is the transport protein that carries retinol in blood. RBP4 was described recently as a new adipokine that reduced insulin sensitivity. Mice lacking glucose transporter 4 (GLUT4) in adipocytes have enhanced Rbp4 gene expression; however, the molecular mechanism is unknown. We found a G4KA (GLUT4 knockdown-dependent transcriptional activation) element located ϳ1.3 kb upstream of the Rbp4 promoter. Mutations within the G4KA sequence significantly reduced expression of the Rbp4 promoter-reporter construct in G4KD-L1 (GLUT4 knockdown 3T3-L1) adipocyte cells. In a yeast one-hybrid screen of a G4KD-L1 cell cDNA library, using the G4KA element as bait, we identified subunits of the 20 S proteasome, PSMB1 and PSMA4, as binding partners. In chromatin immunoprecipitation assays, both subunits bound to the G4KA element; however, only PSMB1 was tightly bound in the GLUT4 knockdown model. PSMB1 RNA interference, but not PSMA4, significantly inhibited Rbp4 transcription. Nuclear transportation of PSMB1 was increased in G4KD-L1 cells. These results provide evidence for an exclusive proteasome subunit-related mechanism for transcriptional activation of RBP4 within a GLUT4 knockdown model. Retinol binding protein 4 (RBP4)2 is a protein with the specific function of transporting retinol in blood. In addition, RBP4 serves to solubilize lipid-soluble retinol in aqueous serum, protecting it from oxidative destruction and maintaining it at a constant concentration in circulation (1). RBP4 is synthesized in the liver and adipocytes and is secreted into the circulation, carrying one molecule of retinol per molecule of RBP4. RBP4 circulates attached to the serum thyroid binding protein transthyretin in a 1:1 molar ratio (1). The retinol-RBP4-transthyretin complex breaks up as retinol is released from RBP4 at the target tissue. Retinol then enters the target cell through a supposed cell surface RBP4 receptor (2). Thus, RBP4 has crucial roles in retinol concentration, transport, and uptake by target cells (3).A major cause of type 2 diabetes is impaired insulin action in adipose tissue, skeletal muscle, and liver. Even in the absence of diabetes, insulin resistance is a major risk factor in cardiovascular disease and early mortality (4). Resistance to insulin-stimulated glucose transport in adipose tissue and skeletal muscle is one of the earliest defects detected in an insulin-resistant status (5). Transport of glucose by glucose transporter 4 (GLUT4) is the rate-limiting step in glucose use by muscle and adipose tissue (5). With the development of insulin resistance, GLUT4 expression is selectively down-regulated in adipose tissue but not in skeletal muscle (5, 6). Down-regulation of GLUT4 expression in adipose tissue is an almost universal factor in diseases with insulin resistant status, including obesity, type 2 diabetes, and metabolic syndrome (5).Genetic knock-out of GLUT4 in adipocytes of mice (7) is associated with increased serum levels of RBP4 (8). Injection of RBP4 or transgenic overexpression...

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