title: Standard isolation of primary adipose cells from mouse epididymal fat pads induces inflammatory mediators and down-regulates adipocyte genes.
abstract: Isolation and subsequent in vitro culture of primary adipose cells are associated with down-regulation of GLUT4 mRNA and simultaneous induction of GLUT1 gene expression. Progressive loss of insulin-responsive GLUT4 contributes to the decrease in insulin-mediated glucose uptake in these cells when cultured in vitro. The mechanisms underlying these alterations are unknown. Here, we report that the standard procedure for isolating primary adipose cells from mouse adipose tissue triggers induction of many genes encoding inflammatory mediators including TNF-alpha, interleukin (IL)-1 alpha, IL-6, multiple chemokines, cell adhesion molecules, acute-phase proteins, type I IL-1 receptor, and multiple transcription factors implicated in the cellular inflammatory response. Secretion of TNF-alpha protein was also significantly induced during the 2-h collagenase digestion of adipose tissue. Isolated primary adipose cells exhibit dramatic changes in expression of multiple mRNAs that are characteristic of TNF-alpha-treated 3T3-L1 adipocytes including down-regulation of many genes important for insulin action and triglyceride synthesis. Addition of TNF-alpha to primary adipose cells in culture did not change the kinetics or the extent of the repression of adipose cell-abundant genes. Moreover, TNF-alpha-neutralizing antibody failed to block the changes in gene transcription in isolated primary adipose cells. Also, the standard isolation procedure induced the expression of NF-kappa B family members and their target genes in primary adipose cells prepared from TNF-alpha-/- mice to the same extent as in cells isolated from wild-type mice and resulted in almost identical changes in global gene expression when these cells were cultured in vitro. Thus, these data suggest that the standard isolation procedure-triggered reprogramming of gene expression in primary adipose cells that results in decreased insulin sensitivity does not require TNF-alpha, at least in this in vitro model system, but may be dependent on other inflammatory cytokines produced by these cells.
authors.affiliation: Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Ruan
authors.forename: Mary Jane
authors.initials: MJ
authors.lastname: Zarnowski
authors.forename: Samuel W
authors.initials: SW
authors.lastname: Cushman
authors.forename: Harvey F
authors.initials: HF
authors.lastname: Lodish
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology*; D000818:Animals; D002448:Cell Adhesion; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D000078404:Epidermal Cells; D005947:Glucose / Q000493:pharmacokinetics; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D066298:In Vitro Techniques; D007249:Inflammation; D007328:Insulin / Q000378:metabolism; D007375:Interleukin-1 / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D016328:NF-kappa B / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000737:chemistry; D018075:RNA, Complementary / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D018925:Chemokines; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D007375:Interleukin-1; D015850:Interleukin-6; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D016328:NF-kappa B; D009841:Oligonucleotides; D018075:RNA, Complementary; D012333:RNA, Messenger; C495678:Slc2a1 protein, mouse; C495693:Slc2a4 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D017364:Collagenases; D005947:Glucose