Secretome of Primary Cultures of Human Adipose-derived Stem Cells: Modulation of Serpins by Adipogenesis

Sanjin Zvonic,Michael Lefevre,Gail Kilroy,Z Elizabeth Floyd,James P Delany,Indu Kheterpal,Amy Gravois,Ryan Dow,Angie White,Xiying Wu,Jeffrey M Gimble

doi:10.1074/mcp.m600217-mcp200

Abstract

Studies of adipogenic protein induction have led to a new appreciation of the role of adipose tissue as an endocrine organ. Adipocyte-derived "adipokines" such as adiponectin, leptin, and visceral adipose tissue-derived serine protease inhibitor (vaspin) exert hormone-like activities at the systemic level. In this study, we examined the secretome of primary cultures of human subcutaneous adipose-derived stem cells as an in vitro model of adipogenesis. Conditioned media obtained from four individual female donors after culture in uninduced or adipogenic induced conditions were compared by two-dimensional gel electrophoresis and tandem mass spectrometry. Over 80 individual protein features showing > or =2-fold relative differences were examined. Approximately 50% of the identified proteins have been described previously in the secretome of murine 3T3-L1 preadipocytes or in the interstitial fluid derived from human mammary gland adipose tissue. As reported by others, we found that the secretome included proteins such as actin and lactate dehydrogenase that do not display a leader sequence or transmembrane domain and are classified as "cytoplasmic" in origin. Moreover we detected a number of established adipokines such as adiponectin and plasminogen activator inhibitor 1. Of particular interest was the presence of multiple serine protease inhibitors (serpins). In addition to plasminogen activator inhibitor 1, these included pigment epithelium-derived factor (confirmed by Western immunoblot), placental thrombin inhibitor, pregnancy zone protein, and protease C1 inhibitor. These findings, together with the recent identification of vaspin, suggest that the serpin protein family warrants further proteomics investigation with respect to the etiology of obesity and type 2 diabetes.

Highlights

Studies of adipogenic protein induction have led to a new appreciation of the role of adipose tissue as an endocrine organ
Multiple proteomics tools have been used to characterize the secretome of preadipocyte cell lines, primary adipocytes, and adipose tissue (9 –12)
One- and two-dimensional gel electrophoresis-tandem mass spectrometry have been applied to murine 3T3-L1 preadipocytes undergoing adipogenesis

Summary

EXPERIMENTAL PROCEDURES

Liposuction Aspirate Cell Isolation and Culture—The procedures used are modifications of published methods (14 –20). The supernatant was removed, and the pelleted stromal vascular fraction was resuspended in Stromal Medium (DMEM/F-12 Ham’s, 10% fetal bovine serum, antibiotic/ antimycotic) and plated at a density of 0.156 ml of tissue digest/cm of surface area in T225 flasks using Stromal Medium for expansion and culture This initial passage of the primary cell culture is referred to as “Passage 0.”. Trypsin Digestion—Following electrophoresis, staining, scanning, spot detection, and match set preparation, proteins of interest were selected, and their standard spot numbers were entered into a “Cut List.” This Cut List was used by the automated spot cutter to select and excise the protein features in order of least to most abundant from one or more gels. Signal was detected using an ECL kit (Pierce) according to published procedures [23]

RESULTS

TABLE I Secretome proteins with secretory signal peptide

DISCUSSION

Findings

Human plasma

Serpin identification