ABSTRACT
Differentiating 3T3-L1 cells exhibit a dramatic increase in the rate of insulin-stimulated glucose transport during their conversion from proliferating fibroblasts to nonproliferating adipocytes. On day 3 of 3T3-L1 cell differentiation, basal glucose transport and cell surface transferrin binding are markedly diminished. This occurs concomitant with the formation of a distinct insulin-responsive vesicular pool of intracellular glucose transporter 1 (GLUT1) and transferrin receptors as assessed by sucrose velocity gradients. The intracellular distribution of the insulin-responsive aminopeptidase is first readily detectable on day 3, and its gradient profile and response to insulin at this time are identical to that of GLUT1. With further time of differentiation, GLUT4 is expressed and targeted to the same insulin-responsive vesicles as the other three proteins. Our data are consistent with the notion that a distinct insulin-sensitive vesicular cargo compartment forms early during fat call differentiation and its formation precedes GLUT4 expression. The development of this compartment may result from the differentiation-dependent inhibition of constitutive GLUT1 and transferrin receptor trafficking such that there is a large increase in, or the new formation of, a population of postendosomal, insulin-responsive vesicles.
Subject(s)
Adipocytes/cytology , Adipocytes/metabolism , Cell Compartmentation/physiology , Insulin/metabolism , Muscle Proteins , 3T3 Cells/cytology , 3T3 Cells/drug effects , 3T3 Cells/metabolism , Adipocytes/drug effects , Aminopeptidases/drug effects , Aminopeptidases/metabolism , Androstadienes/pharmacology , Animals , Antibodies/pharmacology , Biological Transport , Cell Compartmentation/drug effects , Cell Differentiation/physiology , Deoxyglucose/metabolism , Glucose/metabolism , Glucose Transporter Type 1 , Glucose Transporter Type 4 , Insulin/pharmacology , Insulin Antagonists/pharmacology , Mice , Monosaccharide Transport Proteins/immunology , Monosaccharide Transport Proteins/metabolism , Receptors, Transferrin/metabolism , Transferrin/metabolism , WortmanninABSTRACT
Adipocyte differentiation is regulated by at least two major transcription factors, CCAAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma). Expression of PPARgamma in fibroblasts converts them to fat-laden cells with an adipocyte-like morphology. Here, we investigate the ability of PPARgamma to confer insulin-sensitive glucose transport to a variety of murine fibroblast cell lines. When cultured in the presence of a PPARgamma ligand, Swiss-3T3 and BALB/c-3T3 cells ectopically expressing PPARgamma accumulate lipid droplets, express C/EBPalpha, aP2, insulin-responsive aminopeptidase, and glucose transporter isoform 4 (GLUT4), and exhibit highly insulin-responsive 2-deoxyglucose uptake. In contrast, PPARgamma-expressing NIH-3T3 cells, despite similar lipid accumulation, adipocyte morphology, and aP2 expression, do not express C/EBPalpha or GLUT4 and fail to acquire insulin sensitivity. In cells ectopically expressing PPARgamma, the development of insulin-responsive glucose uptake correlates with C/EBPalpha expression. Furthermore, ectopic expression of C/EBPalpha in NIH-3T3 cells converts them to the adipocyte phenotype and restores insulin-sensitive glucose uptake. We propose that the pathway(s) leading to fat accumulation and morphological changes are distinct from that leading to insulin-dependent glucose transport. Our results suggest that although PPARgamma is sufficient to trigger the adipogenic program, C/EBPalpha is required for establishment of insulin-sensitive glucose transport.
Subject(s)
DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Glucose/metabolism , Insulin/pharmacology , Muscle Proteins , Nuclear Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , 3T3 Cells , Adipocytes/cytology , Adipocytes/drug effects , Adipocytes/metabolism , Animals , Biological Transport/drug effects , CCAAT-Enhancer-Binding Proteins , Cell Differentiation/drug effects , Fibroblasts/metabolism , Glucose Transporter Type 4 , Mice , Mice, Inbred BALB C , Monosaccharide Transport Proteins/metabolismABSTRACT
Adipocyte differentiation is regulated by at least two families of transcription factors, CCAAT/enhancer binding proteins (C/EBPs) and peroxisome proliferator-activated receptors (PPARs). Induction of PPAR gamma gene transcription during the differentiation of preadipocytes into adipocytes in vitro occurs following an initial phase of cell proliferation and requires a direct involvement of C/EBP beta, C/EBP delta, and glucocorticoids. Ectopic expression of PPAR gamma in non-adipogenic, Swiss 3T3 fibroblasts promotes their conversion into adipocytes as indicated by the accumulation of lipid droplets and the induction of C/EBP alpha, aP2, insulin-responsive aminopeptidase (IRAP), and glucose transporter 4 (GLUT4) expression. These PPAR gamma-expressing Swiss cells also exhibit a high level of insulin-responsive glucose uptake that is comparable to that expressed in 3T3-L1 adipocytes. In contrast, PPAR gamma-expressing NIH-3T3 fibroblasts, despite similar lipid accumulation, adipocyte morphology, and aP2 expression, do not synthesize C/EBP alpha and fail to acquire insulin sensitivity. In Swiss 3T3 cells ectopically expressing PPAR gamma, the development of insulin-responsive glucose uptake correlates with C/EBP alpha expression. Furthermore, ectopic expression of C/EBP alpha in NIH-3T3 cells induces PPAR gamma expression and adipogenesis, but also restores insulin-sensitive glucose transport. These results suggest that although PPAR gamma is sufficient to trigger the adipogenic program, C/EBP alpha is required for establishment of insulin-sensitive glucose transport in adipocytes.
