Myoung-Sool Do, et al. hypertension, and dyslipidemia increases the likelihood of type II diabetes and cardiovascular disease.5 TNF-α regulates key components of fat metabo- lism, with a net effect of preventing fat deposition through inhibition of lipogenesis, increased lipolysis, and facilitation of adipocyte death by apoptosis.6 TNF-α has been suggested as a media- tor of insulin resistance in obesity,13 although controversy surrounds the exact nature of this proposed role. The effects of TNF-α and other proinflammatory cytokines on feeding, energy balance, and neuroendocrine and immune func- tion during infection and inflammation are well- recognized.7 It is likely that more inflammatory adipokines will be discovered. Most studies on adipocyte- secreted molecules have been conducted, using 3T3-L1 cells originating from mice, but recently Simpson-Golabi-Behmel Syndrome (SGBS) human preadipocytes have been introduced. These cells can be induced to differentiate into adipocytes in primary culture, enabling human adipocyte- secreted molecules to be studied.8 In this study, we describe the utilization of MWG human inflammatory microarrays to study the inflammatory gene expression profile of human SGBS adipocytes. We also studied the inflammatory gene expression profile of SGBS cells following TNF-α treatment. MATERIALS AND METHODS SGBS cell culture Cells were obtained from Dr. M. Wabitsch (Univ. of Ulm, Ulm, Germany),8 and these cells were derived from the stromal cell fraction of subcutaneous adipose tissue of an infant with SGBS. The cells were grown in serum-containing DMEM/F12 medium (Invitrogen, Paisley, UK), supplemented with 33 mM biotin, 17 mM pantothenate, and antibiotics. To induce adipocyte differentiation, cells were washed with phosphate buffered saline (PBS) buffer and cultured in serum-free medium (DMEM/F12), supplemented with 10 μg/mL of transferrin, 10 nM insulin, 200 pM triiodothyronine, 1 μM cortisol, 2 μM BRL Yonsei Med J Vol. 47, No. 5, 2006 49653, 500 μM 1-methyl-3-isobutylxanthine (IBMX), and 0.25 M dexamethasone for four days. After four days, the medium was replaced with serum- free medium (DMEM/F12), supplemented with 10 μg/mL of transferrin, 10 nM insulin, 200 pM triiodothyronine, and 1 μM cortisol, and then changed twice per week. One day before sample treatment, the medium was changed to serum-free medium, supplemented with only transferrin, pantothenate, and biotin, and, the next day, cells were treated with 50 ng of TNF-α for 24 h. All materials except DMEM/F12 were from Sigma (Poole, UK). Oil Red O staining Cells were fixed with 10% fresh formaldehyde (Sigma, Poole, UK). They were rinsed in PBS (Biowhittaker Molecular Application, Walkerville, MD, USA), incubated with filtered Oil Red O stock solution for at least 1 h, gently washed twice with water, stained with 1 mL of Harris' Haematoxylin (Brainwood lab., E. Sussex, UK), and finally washed with warm distilled water. cRNA preparation and probe preparation RNA extraction and cDNA synthesis Total RNA was extracted, using a standard Trizol RNA isolation protocol (Life Technologies, Grand Island, NY, USA) and quantified specto- photometrically. To each 2 μg total RNA sample, 1 μL of 10 μM oligo-dT(24) was added, and the mixture was denatured at 70 for 2 min, then immediately placed on ice. The reverse transcrip- tion mastermix (2 μL of 5X first strand buffer, 1 μL of 20 mM DTT, 1 μL of 10 mM dNTP, and 1 μL of Powerscript reverse transcriptase {Clontech, BD Biosciences, Oxford, UK}) was then added and incubated for 1 h at 42 . Enzymic activity was stopped by heating to 72 for 10 min. cRNA amplification and labelling Total RNA was extracted and reverse tran- scribed as described above, except that the CDS oligo-dT was replaced with a modified oligo-dT with the T7 promotor sequence incorporated. The first strand cDNA product was subjected to two rounds of PCR amplification, using the SMART