ABSTRACT
While adipocyte differentiation has been studied extensively in murine cultures, the lack of a readily available preadipocyte model has hindered equivalent studies in man. We describe methods for the isolation and culture of primary human stromal cells from surgical adipose tissue specimens. In vitro, the stromal cells rapidly differentiate in response to a combination of adipogenic agents. Among these, glucocorticoids and thiazolidinediones act together to induce the formation of lipid vacuoles within the cells. These morphologic changes accompany the increased expression of 2 characteristic adipocyte proteins, the cytoplasmic enzyme glycerol phosphate dehydrogenase (GPDH) and the secreted cytokine leptin. Likewise, stromal cell differentiation results in elevated mRNA levels for the fatty acid binding protein aP2 and the adipogenic regulatory transcription factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) in addition to leptin. The in vitro differentiated stromal cells exhibit a lipolytic response to beta-adrenergic agonists, comparable to that reported with primary human adipocytes. These studies demonstrate the validity of human adipose tissue-derived stromal cells as a reliable in vitro model for investigations of adipocyte metabolism in humans.
Subject(s)
Adipose Tissue/cytology , Glucocorticoids/pharmacology , Stromal Cells/drug effects , Thiazoles/pharmacology , Adipose Tissue/drug effects , Adipose Tissue/metabolism , Cell Differentiation/drug effects , Coloring Agents , Glycerolphosphate Dehydrogenase/metabolism , Humans , Immunoblotting , In Vitro Techniques , Leptin/biosynthesis , Lipectomy , Lipolysis/drug effects , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/metabolismABSTRACT
Intracellular calcium ([Ca(2+)](i)) modulates adipocyte lipid metabolism and inhibits the early stages of murine adipogenesis. Consequently, we evaluated effects of increasing [Ca(2+)](i) in early and late stages of human adipocyte differentiation. Increasing [Ca(2+)](i) with either thapsigargin or A23187 at 0-1 h of differentiation markedly suppressed differentiation, with a 40-70% decrease in triglyceride accumulation and glycerol-3 phosphate dehydrogenase (GPDH) activity (P < 0.005). However, a 1-h pulse of either agent at 47-48 h only modestly inhibited differentiation. Sustained, mild stimulation of Ca(2+) influx with either agouti protein or 10 mM KCl-induced depolarization during 0-48 h of differentiation inhibited triglyceride accumulation and GPDH activity by 20-70% (P < 0.05) and markedly suppressed peroxisome proliferator-activated receptor gamma (PPARgamma) expression. These effects were reversed by Ca(2+) channel antagonism. In contrast, Ca(2+) pulses late in differentiation (71-72 h or 48-72 h) markedly increased these markers of differentiation. Thus increasing [Ca(2+)](i) appears to exert a biphasic regulatory role in human adipocyte differentiation, inhibiting the early stages while promoting the late stage of differentiation and lipid filling.