Proteome of Human Subcutaneous Adipose Tissue Stromal Vascular Fraction Cells versus Mature Adipocytes Based on DIGE

Abstract

Adipose tissue contains a heterogeneous population of mature adipocytes, endothelial cells, immune cells, pericytes, and pre-adipocytic stromal/stem cells. To date, the majority of proteomic analyses have focused on intact adipose tissue or isolated adipose stromal/stem cells in vitro. In this study, human subcutaneous adipose tissue from multiple depots (arm and abdomen) obtained from female donors was separated into populations of stromal vascular fraction cells and mature adipocytes. Out of 960 features detected by 2-D gel electrophoresis, a total of 200 features displayed a 2-fold up-or down-regulation relative to each cell population. The protein identity of 136 features was determined. Immunoblot analyses comparing SVF relative to adipocytes confirmed that carbonic anhydrase II was up-regulated in both adipose depots while catalase was up-regulated in the arm only. Bioinformatic analyses of the dataset determined that cytoskeletal, glycogenic, glycolytic, lipid metabolic, and oxidative stress related pathways were highly represented as differentially regulated between the mature adipocytes and stromal vascular fraction cells. These findings extend previous reports in the literature with respect to the adipose tissue proteome and the consequences of adipogenesis. The proteins identified may have value as biomarkers for monitoring the physiology and pathology of cell populations within subcutaneous adipose depots.

Comparison of fractionation strategies for offline two-dimensional liquid chromatography tandem mass spectrometry analysis of proteins from mouse adipose tissue

“…Investigations aimed at characterizing adipose tissue samples frequently employ LC- MS-based methods in combination with gel-based proteomics due to the high lipid content and low protein amount of the adipose tissue [6,7,10,12,14]. To overcome these challenges, in our previous investigations we identified a detergent-free protein extraction method directly compatible with subsequent 2D-LC-MS/MS analysis [16].…”

“…The aim of the current study was to apply a pH-based gradient separation and to compare its performance with the classical salt-based gradient separation for the protein analysis of entire mouse adipose tissue. The interaction between different WAT compartments, namely the mature adipocytes and the cells in the stromal vascular fractions (SVFs), is a critical component in the maintenance of their metabolic and endocrine function [14,40,48]. Thus, to gain a general overview of the mouse adipose tissue proteome, we investigated integral mouse WAT samples without preceding separation of the distinct tissue fractions.…”

“…After centrifugation at 800g (10 min), the lipid phase (i.e., organic phase) was removed and the protein disc and aqueous phase were kept for protein precipitation. Proteins were precipitated in at least 6 volumes of pure cold acetone (À20°C) overnight and then pelleted at 14 …”

“…Investigations aimed at uncovering modifications in WAT protein expression are of important clinical interest in the quest to identify new therapeutic targets or candidate biomarkers for these prevalent pathologies [4,5]. Scientific prominence of adipose tissue proteomics along with an increasing number of adipose tissue proteomics studies per year [6][7][8][9][10][11][12][13][14] was recently observed by Peinado and coworkers [15]. The main hurdles in the isolation and identification of adipose tissue proteins are its high lipid content combined with low protein amount and a high proteome complexity related to significant tissue vascularization and the presence of a multitude of functionally important cell types located in different tissue fractions [4,16,17].…”

Role of Adipose Secreted Factors and Kisspeptin in the Metabolic Control of Gonadotropin Secretion and Puberty

“…Recent proteomic studies of human and rat adipocytes have revealed the true scope of the adipose tissue secretome (Chen et al, 2005;Kheterpal et al, 2011;Lehr et al, 2012;Lim et al, 2008;Zhong et al, 2010). With refined and advanced proteomics techniques, these studies have revealed that many of the adipose tissue secreted factors identified at the gene level do indeed encode secreted proteins (Chen et al, 2005;Kheterpal et al, 2011;Lehr et al, 2012;Lim et al, 2008;Zhong et al, 2010).…”