Expression and regulation of transcript for the novel transmembrane protein Tmem182 in the adipocyte and muscle lineage
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White adipose tissue is not only an energy storage organ; it also functions as an endocrine organ. The coordination and integration of numerous gene expression events is required to establish and maintain the adipocyte phenotype.
We previously observed a 45-fold upregulation for a transcript encoding a novel predicted transmembrane protein, Tmem182, upon brown preadipocyte to adipocyte conversion. Here we use real-time PCR analysis to further characterize Tmem182 transcript expression in the adipocyte lineage. Analysis across a panel of 10 murine tissues revealed highest Tmem182 transcript expression in white adipose tissues (WAT), with 10-fold to 20-fold higher levels than in brown adipose tissue (BAT). Tmem182 transcript expression is ~3-fold upregulated in BAT of genetically obese (ob/ob) mice vs. wild type C57BL/6. Analysis of three in vitro models of white adipogenesis indicates markedly enriched expression of Tmem182 transcript in adipocytes vs. preadipocytes. Compared to 3T3-L1 preadipocytes, a 157-fold higher level of Tmem182 transcript is detected at 3 day post-induction of adipogenesis and an ~2500-fold higher level in mature 3T3-L1 adipocytes. TNFα treatment of 3T3-L1 adipocytes resulted in a ~90% decrease in Tmem182 transcript level. As skeletal muscle and heart were also found to express Tmem182 transcript, we assessed expression in C2C12 myogenesis and observed a ~770-fold upregulation upon conversion of myoblasts to myocytes.
WAT is the most prominent site of Tmem182 transcript expression and levels of transcript for Tmem182 are altered in adipose tissues of ob/ob mice and upon exposure of 3T3-L1 adipocytes to the proinflammatory cytokine TNFα. The dramatic upregulation of Tmem182 transcript during in vitro adipogenesis and myogenesis suggests Tmem182 may function in intracellular pathways important in these two cell types.
KeywordsBrown Adipose Tissue White Adipose Tissue Stromal Vascular Fraction Cell Subcutaneous White Adipose Tissue White Adipose Tissue Depot
Polymerase chain reaction
Fetal bovine serum
White adipose tissue
Brown adipose tissue
Peroxisome proliferator-activated receptor
CCAAT/Enhancer binding protein
White adipose tissue (WAT) is the major site for storage of excess energy and these triglyceride stores are mobilized to meet the energy needs of the organism. Adipose tissue is now also recognized as an endocrine organ with synthesis and secretion of a variety of soluble factors such as leptin, resistin, adiponectin, retinol binding protein-4 and TNFα [1, 2, 3, 4]. Adipocytes make up from one-third to two-thirds of the cell population found in adipose tissue, with endothelial cells, nerve cells, macrophages, fibroblast-like interstitial cells and preadipocytes, and perhaps other cell types, comprising the remaining stromal-vascular component . Mature adipocytes form as the result of the differentiation of preadipocyte precursors present in adipose tissue [6, 7, 8, 9, 10]. Established preadipocyte cell lines such as 3T3-L1  have been an extensively used in vitro model to define genes central to the adipocyte phenotype [12, 13]. Adipogenesis is accompanied by increased transcription of genes that encode proteins key to adipocyte function, for example lipogenesis, lipolysis, lipid transport, and hormone responsiveness [7, 14, 15]. In vitro and in vivo studies have uncovered a pivotal role for peroxisome proliferator-activated receptor γ (PPARγ), a member of the ligand-activated steroid hormone receptor family, in the adipogenic program [8, 10, 16, 17, 18, 19]. Studies have also illustrated the important contributions of the CCAAT/enhancer-binding proteins (C/EBPs) and other transcriptional signals to adipogenesis [8, 10, 20].
Culture of cell lines and adipogenic conversion for 3T3-L1, ScAP-23 and wt-BAT, for the fractionation of adipose tissues, and for culture and differentiation of murine preadipocytes from subcutaneous (SC) WAT was as described [21, 22]. C2C12 cells were maintained and passaged as subconfluent cultures in DMEM with10% FBS. For differentiation, cultures at 70% confluence were switched to DMEM with 2% horse serum and 10 μg/ml insulin, and were cultured under these conditions for 7 days. For treatment of 3T3-L1 adipocytes with TNFα and various pharmacological inhibitors the method was as described [23, 24, 25]. After serum-starvation for 6 h, 3T3-L1 adipocytes were pretreated with either 50 μM LY294002, 50 μM PD98059, 20 μM SB203580, 100 nM wortmannin, 1 μM rapamycin (Sigma-Aldrich, St. Louis, MO), or DMSO vehicle for 1 h and then cultured in 10 ng/ml of TNFα for 16 h in the presence of inhibitors. RNA was purified using TriZol Reagent (Invitrogen Corp.) according to manufacturer's instruction. For studies of Tmem182 transcript expression in murine tissues, 8 wk old C57BL/6 or ob/ob male mice were utilized, with all animal treatments conducted with approval of the University of Toledo Health Science Campus Institutional Animal Care and Use Committee.
Real-time PCR analysis was as previously described [23, 24, 26]. For this, total RNA was subject to purification with an RNeasy RNA purification kit with DNase I treatment (Qiagen Corp., Valencia, CA) and 5 μg used for first strand cDNA synthesis with SuperScript II RNase H-reverse transcriptase (Invitrogen Corp.) and an oligo(dT)-22 primer. Real time PCR was conducted with an ABI 7500 Real Time PCR System. Target cDNA levels were analyzed by SYBR green-based real-time PCR in 25 μl reactions containing 1× SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA), 100 nM each forward and reverse primers, and 10 ng of cDNA. Analyses were performed in triplicate and expression of each gene was normalized against Gapdh transcript level. The cycle threshold value was generated using ABI PRISM 7500 SDS software version 1.2 and exported to an Excel spreadsheet to calculate fold differences. Sequence of PCR primers used were: Tmem182 (5'-ACACCAATCAGCCACCATCC-3' and 5'-GCCACGGTAAATAATTGCGGAG-3'); Gapdh (5'-GGCAAATTCAACGGCACAG-3' and 5'-CGGAGATGATGACCCTTTTG-3'); and Myogenin (5'-GCCATCCAGTACATTGAGC-3'and 5'-GTAAGGGAGTGCAGATTGTG-3'). Primers were designed to span introns.
Properties of Tmem182 gene and sequence
Tissue distribution of Tmem182 transcript expression
To begin to address the modulation of Tmem182 transcript levels in regard to the pathophysiology of adipocytes, we compared transcript expression in BAT and WAT from wild type C57BL/6 mice and ob/ob mice, the latter a well-studied murine model of genetic obesity. For Tmem182 transcript, we find a slight increase (~1.7-fold, p < 0.001) in ob/ob WAT for the SC depot (Figure 2C). Furthermore, compared to wt BAT, we find a 6.3-fold upregulation (p < 0.001) of Tmem182 transcript level in ob/ob vs. wt BAT, suggestive of a role for dysregulation of Tmem182 in the obese state (Figure 2C). It would thus appear that in the ob/ob genetic model, BAT shifts to a level of Tmem182 transcript expression that is more similar to that found in WAT.
Differentiation-dependent expression of Tmem182 transcript in adipogenesis
Regulation of Tmem182 transcript by TNFα
Differentiation-dependent expression of Tmem182 transcript in myogenesis
The primary amino acid sequence of Tmem182 predicts an evolutionarily conserved novel transmembrane protein. Tmem182 protein sequence lacks homologies with previously defined protein families and Tmem182 function is currently unknown. Enrichment of Tmem182 transcript in WAT, alteration in obesity, differentiation-dependent upregulation in adipogenesis and regulation by TNFα suggests that expression of Tmem182 may be integral to the adipocyte phenotype. Interestingly, Tmem182 transcript is also enriched in muscle tissue and it is markedly upregulated during in vitro myogenesis of C2C12 myoblasts to myocytes. This suggests Tmem182 may function in cellular pathways shared by adipocytes and myocytes but not by their respective precursor cell types. Future studies will further examine the in vitro and in vivo regulation and the function of Tmem182 in adipocytes and muscle cells.
We thank Dr. J.Y. Kim for kindly providing adipocyte RNA samples.
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