The adipose compartment houses the largest energy reserves of the body in humans in the form of triacylglycerols and plays an essential role in maintaining the energy balance1
. It is now well recognized that adipogenesis can occur throughout the lifetime of humans and the capacity to increase the adipocyte number is retained in adulthood2
. Thus, identification of key players in the process of adipogenesis is important especially in regard to manipulating them for therapeutic purposes. Over the last several years, adipocytes have also been shown to possess regulatory functions in processes such as satiety and reproduction through the secretion of a variety of hormones, cytokines, growth factors and other bioactive substances, conceptualized as adipocytokines3
. These include adiponectin, leptin, plasminogen activated inhibitor 1 (PAI-1), and other secreted factors4
. Another important class of proteins that play a vital role in adipogenesis is transcription factors such as peroxisome proliferator activated receptor-γ5,6
and the CCAAT-enhancer binding protein-α7
, which have been characterized in detail during adipocyte differentiation. Thus, we sought to identify additional molecules involved in adipogenesis by analyzing nuclear and secreted compartments during the differentiation of adipocytes.
Cell lines and primary cultures are useful cell models for elucidation of molecular and developmental pathways leading to adipogenesis. 3T3-L1 preadipocytes are one of the commonly used model systems for studying the process of adipocyte differentiation. DNA microarray studies have been carried out to investigate specific cellular programs in the regulation of gene expression during differentiation of 3T3-L1 preadipocytes8
. They found that that expression levels of 1259 transcripts changed 3-fold or more during differentiation of 3T3-L1 preadipocytes to adipocyte. In a similar study, Burton et al.9
reported 636 transcripts to be upregulated at least 2 fold and 380 transcripts downregulated in adipocytes as compared to preadipocytes. However, since adipogenesis is a dynamic and actively regulated process, a temporal analysis of differential protein expression during adipogenesis would be crucial for understanding adipocyte biology. We have previously identified factors that are secreted by adipocytes using liquid chromatography tandem mass spectrometry (LC-MS/MS)10
. In that study, which was not designed to be quantitative, the differential expression of a small subset of molecules was confirmed by RT-PCR and Northern blot analysis. More recently, using 1D gel based LC-MS/MS, another non-quantitative proteomic analysis was reported in which various subcellular fractions from adipocytes were characterized11
Thus far, there is no study describing a quantitative analysis of the dynamics of the adipocyte proteome during differentiation. Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and robust in vivo labeling method that can be applied to studies analyzing cells at multiple time points such as during cellular differentiation. Traditionally, SILAC has been used for analyzing two or three different states. Here, we describe the development and application of a five-plex SILAC experiment using four different heavy stable isotopic forms of arginine for the analysis of nuclear and secreted proteins of adipocytes at 5 different time points during adipogenesis. We identified 882 nuclear/secreted proteins using SILAC labeling followed by 2D-LC-MS/MS. Almost half of the identified proteins from each compartment could be quantitated enabling the identification of potential regulators and markers at different states of adipocyte differentiation.