Wednesday, November 7, 2007

Elucidating The Transcriptional Regulatory Network Underlying 3T3-L1 Adipocyte Differentiation And Hypertrophic Enlargement

Colby Moya1, Hai Shi2, Kyongbum Lee2, and Arul Jayaraman3. (1) Chemical Engineering, Texas A&M University, College Station, TX 77843-3122, (2) Chemical and Biological Engineering, Tufts University, 4 Colby Street, Room 142, Medford, MA 02155, U.S.A., (3) Department of Chemical Engineering, Texas A&M University, College Station, TX 77843-3122

Our overall objective is to develop complementary experimental-mathematical models for elucidating the interactions between key regulatory and signaling molecules underlying white adipocyte differentiation and enlargement. Adiposity occurs through both increases in fat cell number and cell size. The increase in cell number involves the recruitment, proliferation, and differentiation of locally derived precursor cells (preadipocytes) into mature adipocytes. Terminally differentiated adipocytes generally do not undergo further mitotic division; however, genetic or environmental conditions can bring about significant enlargement through progressive lipid loading. Numerous molecular studies, including microarray experiments and proteomic analyses, have established that several key transcription factors such as PPAR-γ and C/EBP-α act as master switches for a complex cascade of gene expression events to convert preadipocytes into adipocytes. Pharmacological agents in the form of synthetic ligands targeting these transcription factors have yielded mixed results; for example, significant inhibition of PPAR-γ in vivo, while reducing adipogenesis, also increases insulin resistance, one of the chief complications of type-2 diabetes mellitus (T2DM). Therefore, the ability to control hypertrophy, without significantly altering the extent of adipogenesis, is crucial for the development of therapeutic approaches to combat obesity and related disorders. We hypothesize that adipogenesis and hypertrophic enlargement are differentially regulated through the concerted actions of a network of transcription factors and signaling molecules. Differential regulation can be due to differences in the molecules involved and/or timing of the induction/deactivation of these molecules. To this end, we are characterizing the molecular regulatory network associated with the two major adiposity modalities differentiation-driven adipogenesis per se and lipid loading-driven enlargement and hypertrophic growth - and demonstrate that interfering with this network during enlargement will attenuate hypertrophic expansion. We have developed green fluorescence protein (GFP)-based reporter plasmids for profiling the activity of different transcriptional regulators (e.g., C/EBPα, PPARγ, SREBP1c) that are involved in the regulation of adipocyte differentiation. These reporter plasmids consists of 4 copies of the DNA binding sequence (response element) for the different transcription factors, inserted upstream of a minimal (CMVmin) promoter. No expression from this promoter is observed in the absence of binding to the response element sequence. A destabilized (2 h half-life) variant of the egfp gene was inserted downstream of the minimal promoter as the reporter gene. Reporter plasmids (e.g., C/EBPα) have been stably integrated into 3T3-L1 preadipocytes by electroporation. Our data shows that the expression of EGFP (and hence, the activation of C/EBPα) correlates with the extent of differentiation (i.e., fluorescence is observed only in areas where differentiated adipocytes are present) and is significantly more than that observed with undifferentiated controls. These results are consistent with the established role of C/EBPα as a master regulator of adipocyte differentiation. Prospectively, better understanding of the differential dynamics could lead to a comprehensive pharmacological treatment regimen that targets different regulatory molecules in the network at different times.