Glut4 storage vesicles without Glut4: transcriptional regulation of insulin-dependent vesicular traffic.
Two families of transcription factors that play a major role in the development of adipocytes are the CCAAT/enhancer-binding proteins (C/EBPs) and the peroxisome proliferator-activated receptors (PPARs), in particular PPAR gamma. Ectopic expression of either C/EBP alpha or PPAR gamma in NIH 3T3 fibroblasts results in the conversion of these cells to adipocyte-like cells replete with fat droplets. NIH 3T3 cells ectopically expressing C/EBP alpha (NIH-C/EBP alpha) differentiate into adipocytes and exhibit insulin-stimulated glucose uptake, whereas NIH 3T3 cells ectopically expressing PPAR gamma (NIH-PPAR gamma) differentiate but do not exhibit any insulin-stimulated glucose uptake, nor do they express any C/EBP alpha. The reason for the lack of insulin-responsive glucose uptake in the NIH-PPAR gamma cells is their virtual lack of the insulin-responsive glucose transporter, Glut4. The NIH-PPAR gamma cells express functionally active components of the insulin receptor-signaling pathway (the insulin receptor, IRS-1, phosphatidylinositol 3-kinase, and Akt2) at levels comparable to those in responsive cell lines. They also express components of the insulin-sensitive vesicular transport machinery, namely, VAMP2, syntaxin-4, and IRAP, the last of these being the other marker of insulin-regulated vesicular traffic along with Glut4. Interestingly, the NIH-PPAR gamma cells show normal insulin-dependent translocation of IRAP and form an insulin-responsive vesicular compartment as assessed by cell surface biotinylation and sucrose velocity gradient analysis, respectively. Moreover, expression of a Glut4-myc construct in the NIH-PPAR gamma cells results in its insulin-dependent translocation to the plasma membrane as assessed by immunofluorescence and Western blot analysis. Based on these data, we conclude that major role of C/EBP alpha in the context of the NIH-PPAR gamma cells is to regulate Glut4 expression. The differentiated cells possess a large insulin-sensitive vesicular compartment with negligible Glut4, and Glut4 translocation can be reconstituted on expression of this transporter.
Department of Biochemistry, Boston University School of Medicine, MA 02118, USA.
Danielle N
DN
Gross
Stephen R
SR
Farmer
Paul F
PF
Pilch
D017667:Adipocytes / Q000166:cytology / Q000502:physiology; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D010122:Cystinyl Aminopeptidase; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D003847:Deoxyglucose / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D051275:Glucose Transporter Type 4; D007328:Insulin / Q000378:metabolism*; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins; D041681:NIH 3T3 Cells; D021381:Protein Transport / Q000502:physiology; D011972:Receptor, Insulin / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D022763:CCAAT-Enhancer-Binding Protein-alpha; D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D018160:Receptors, Cytoplasmic and Nuclear; C495693:Slc2a4 protein, mouse; D014157:Transcription Factors; D003847:Deoxyglucose; D011972:Receptor, Insulin; D000626:Aminopeptidases; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase