A novel 3 T 3 - L 1 preadipocyte variant that expresses PPAR g 2 and RXR a but does not undergo differentiation

This report describes a novel adipocyte-like cell line termed 3T3-L1/RB1 that was derived from preadipocyte cell line, 3T3-L1. The 3T3-L1/RB1 cells continued to divide after reaching confluence, formed foci, and constitutively expressed a low level of adipose fatty acid binding protein (A-FABP) mRNA. However, 3T3L-1/RB cells did not undergo terminal differentiation as indicated by the failure of insulin and thiazolidendiones to induce the expression of A-FABP, lipoprotein lipase, and fatty acid synthase. We hypothesized that the 3T3-L1/RB1 variant did not respond to differentiation stimuli because it did not express either peroxisomal proliferator activated receptor g 2 (PPAR g 2) or its heterodimer partner, retinoid X receptor a (RXR a ). Surprisingly, Western blots revealed that 3T3-L1/ RB1 cells contained both PPAR g 2 and RXR a proteins at levels equal to or greater than that of the parent cell line. However, gel retardation assays using the adipose response element from A-FABP and nuclear protein extracts from 3T3-L1/RB1 cells treated with insulin or pioglitazone revealed that nuclear protein extracts from 3T3-L1/RB1 cells had very little ability to bind the PPAR g 2 recognition sequence of the A-FABP gene. These data suggest that the 3T3-L1/RB1 variant contains a mutation that may prevent ligand activation of PPAR g 2, and the subsequent conversion of 3T3-L1/RB1 cells to mature fat cells.— Baillie, R. A., X. Sha, P. Thuillier, and S. D. Clarke. A novel 3T3-L1 preadipocyte variant that expresses PPAR g 2 and RXR a but does not undergo differentiation. J. Lipid Res. 1998. 39: 2048– 2053. Supplementary key words fatty acid binding protein • adipocytes • differentiation • 3T3-L1 cells • PPAR The conversion of preadipocytes to mature fat cells is a coordinated response to a collection of external stimuli including signals from cAMP, glucocorticoids, IGF-1, and arachidonate (1–6). These signals initiate a cascade of transcriptional changes which lead to the down-regulation of differentiation suppressors (7), and the up-regulation of differentiation inducers (1). Two transcription factors that are essential to terminal fat cell differentiation and the expression of fat specific genes are PPAR g 2 and RXR a (8–11). PPAR g 2 is a ligand-activated transcription factor that stimulates the transcription of fat specific genes (e.g., A-FABP) by forming a heterodimer with RXR a and subsequently binding to its DNA recognition site, DR-1 (direct repeat, one base pair spacer) (4, 10). The endogenous ligand activator for PPAR g 2 may be the prostanoid 15deoxy-d12,14-prostaglandin J2 (4). However, the ligand domain of PPAR g 2 can bind a number of different activating compounds including fibrates, ETYA, and the novel antidiabetic drugs, thiazolidenediones (12, 13). If PPAR g 2, RXR a , or the appropriate PPAR g 2 ligand are not present, then the expression of fat specific genes and terminal fat cell differentiation does not occur (6, 11). As an example, the ectopic expression of PPAR g 2 in 3T3 fibroblasts did not initiate fat cell differentiation until a ligand activator for PPAR g 2 was introduced (14). Similarly, 3T3-L1 preadipocytes do not express A-FABP and terminally differentiate until RXR a expression has been induced by glucocorticoids (9, 11). Thus, it is generally accepted that the events of terminal fat cell differentiation and the expression of fat specific genes such as A-FABP require the presence of PPAR g 2, a PPAR g 2 ligand activator, and RXR a . In this report we describe the isolation and characterization of a monoclonal cell line termed 3T3-L1/RB1 that spontaneously arose from the 3T3-L1 adipocyte cell line. Because the 3T3-L1/RB1 variant does not withdraw from the cell cycle and does not undergo fat cell differentiation in response to insulin and the PPAR g 2 activator pioglitazone, we have examined the hypothesis that the variant line does not express either PPAR g 2 or its heterodimer partner RXR a , and/or that the variant does not produce the ligand activator that is essential for terminal fat cell differentiation and the induction of the fat specific gene, A-FABP. Abbreviations: PPAR g 2, peroxisomal proliferator-activated receptor g ; RXR a , retinoid X receptor a ; RXR g , retinoid X receptor g ; AFABP, adipose fatty acid binding protein; IGF-1, insulin-like growth factor 1; PREF-1, preadipocyte factor 1; CUP, C/EBP undifferentiated protein; DR-1, direct repeat 1; C/EBP a , ccaat enhancer binding protein; GAPDH, glycceraldehyde phosphate dehydrogenase; ETYA, eicosatetraynoic acid; LPL, lipoprotein lipase. 1 To whom correspondence should be addressed. at P E N N S T A T E U N IV E R S IT Y , on F ebuary 0, 2013 w w w .j.org D ow nladed fom