Mapping of Early Signaling Events in Tumor Necrosis Factor-α-mediated Lipolysis in Human Fat Cells

Rydén, Mikael; Dicker, Andrea; Harmelen, Vanessa van; Hauner, Hans; Brunnberg, Martin; Perbeck, L.; Lönnqvist, Fredrik

doi:10.1074/jbc.m109498200

Cited by 237 publications

(216 citation statements)

References 39 publications

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“…The former are murine cells that display several distinct metabolic and signal transduction differences compared with human cells. 19 Preadipocytes are obtained from fibroblast-like cells isolated from the stromal portion of human fat tissue which are differentiated in vitro into cells displaying functional traits identical to those of mature fat cells. 11,14 However, they cannot be expanded and display only a limited life span.…”

Section: Discussionmentioning

confidence: 99%

“…For protein measurements cells were lysed in a prechilled protein lysis buffer containing 50 mM Tris pH 7.6, 150 mM NaCl, 1% Triton X-100, 0.1 M PMSF and protease inhibitors as described previously. 19 Protein content was assayed using BCA Protein Assay Reagent Kit (PIERCE, Rockford, IL, USA) on 96-well microtiter plates with BSA as a standard. Measurements were performed on a spectrophotometer in accordance with the guidelines given in the kit.…”

Section: Lipolysismentioning

confidence: 99%

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Differential function of the α2A-adrenoceptor and phosphodiesterase-3B in human adipocytes of different origin

et al. 2005

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OBJECTIVE: Human adipocytes can be obtained in vitro by differentiation of human preadipocytes or mesenchymal stem cells (hMSC). Although functionally similar to freshly isolated cells, no detailed comparison of the different cell types has been performed. The antilipolytic a2A-adrenoceptor (AR) and the cAMP-degrading enzyme Phosphodiesterase-3B (PDE3B) have been implicated in the fine-tuning of lipolysis but little is known regarding their role in human adipocytes nor whether their expression and/or function differs in fat cells from different precursors. METHODS:The effects of a2A-AR and PDE3B inhibition in mature adipocytes was determined and compared to that in differentiated preadipocytes and hMSC-derived fat cells. Gene expression was determined by real-time PCR and protein expression by Western blot. RESULTS: Noradrenaline (NA) stimulated lipolysis in preadipocytes and mature adipocytes but markedly reduced lipolysis in differentiated hMSC derived-adipocytes. This was due to a potent stimulation of a2A-AR since co-incubation with NA and the a2-AR-inhibitor yohimbine restored NA-induced lipolysis. The order of Yohimbine response was hMSC4preadipocytes4mature adipocytes. Although a2-AR mRNA expression was highest in mature adipocytes there was no difference in a2A-AR protein levels between the cell types. In contrast, Ga i 2 mRNA and protein expression was significantly higher in MSC-derived adipocytes, suggesting that differences in the response to a2A-AR inhibition reside at the postreceptor level. Incubation with the cAMPanalog 8-bromo(8b) cAMP increased lipolysis in hMSC-derived fat cells while co-incubation with the PDE3-specific inhibitor OPC3911 did not alter the lipolytic effect. In contrast, OPC3911 increased 8bcAMP-induced lipolysis significantly in preadipocytes and mature adipocytes. The response to PDE3B inhibition was; mature adipocytes4preadipocytes4hMSC a finding that correlated significantly with both PDE3B mRNA expression and enzymatic activity. CONCLUSION: Although differentiated adipocytes of different origins display similar functional characteristics there are important differences in the regulation of lipolysis with a marked a2A-AR and less pronounced PDE3B effect in fat cells from MSCs.

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Section: Discussionmentioning

confidence: 99%

Section: Lipolysismentioning

confidence: 99%

Differential function of the α2A-adrenoceptor and phosphodiesterase-3B in human adipocytes of different origin

et al. 2005

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“…Isolation of protein from in vitro differentiated adipocytes was performed as described earlier. 17 All steps were performed on ice to assure structural integrity of the proteins. Protein content was assayed using Blots were blocked for 1 h at room temperature in Trisbuffered saline containing 0.1% Tween 20 and 2% bovine serum albumin.…”

Section: Protein Expression Analysismentioning

confidence: 99%

Endothelin-1 stimulates human adipocyte lipolysis through the ETA receptor

et al. 2008

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Objective: Levels of the vascular peptide endothelin-1 (ET-1) are significantly elevated in obesity. Adipose tissue-derived ET-1 attenuates insulin-mediated antilipolysis in human visceral adipocytes through the activation of the ET receptor B (ET B R), thereby linking ET-1 to insulin resistance. Whether ET-1 has direct effects on lipolysis in human adipocytes is not known. Research design and subjects: Endothelin-1 receptor (ETR) mRNA expression was determined by quatitative PCR in 130 nonobese and obese subjects. ET-1 mRNA in different adipose tissue regions was also assessed. ETR protein expression was analyzed by western blotting in 37 subjects. The effect of ET-1 on lipolysis was assessed in freshly isolated adipocytes and in vitro differentiated adipocytes from human donors. Results: Freshly isolated human adipocytes incubated with different concentrations of ET-1 showed no acute effect on lipolysis. In contrast, a 24 h incubation in primary cultures of human adipocytes resulted in a significant 50% increase in lipolysis. This effect was concentration dependent and could be mimicked by an agonist of the ET A receptor but not with a selective ET B R agonist. Adipocyte differentiation was not affected by any of the agonists. In subcutaneous (s.c.) adipose tissue from 19 nonobese and 18 obese subjects, the protein expression of ET A R was significantly higher in obese subjects whereas there was no difference in ET B R expression. Interestingly, the differences in protein expression were not observed at the mRNA level as ET A R expression was similar between lean and obese subjects. Conclusion: Long-term but not acute incubation of human adipocytes with ET-1 results in a significant increase in lipolysis. This appears to be mediated through the activation of ET A R, demonstrating a yet another receptor-specific effect of ET-1. In addition, the protein expression of ET A R is increased in s.c. adipose tissue in obesity, possibly through post-transcriptional mechanisms. An increased effect of ET-1 could be a mechanism that contributes to increased basal lipolysis in human obesity.

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“…We and others have previously shown that TNF-a promotes a slight increase in human adipocyte apoptosis in culture. 13,14 This effect is somewhat more pronounced in cells subjected to concomitant transfections which preclude firm conclusions when differences among promoter constructs are studied. We therefore used the wellestablished murine cell line 3T3-L1 to investigate the TNF-amediated regulation of the human CIDEA promoter in more detail.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the human CIDEA promoter in fat cells

et al. 2008

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Background: Cell death-inducing DFFA (DNA fragmentation factor-a)-like effector A (CIDEA) is a protein that regulates lipolysis in human adipocytes through cross-talk involving tumor necrosis factor-a (TNF-a). TNF-a downregulates CIDEA mRNA although it is unclear whether this is mediated through transcriptional or post-transcriptional mechanisms. CIDEA has important metabolic effects in human fat cells and genetic variations in the human CIDEA gene have been correlated to the development of obesity. However, little is known about the factors regulating CIDEA expression in human adipocytes. We set out to describe the transcriptional control of human CIDEA. Methods: A 1.1-kb genomic fragment upstream of the transcriptional start site (TSS) of human CIDEA was cloned and deletion fragments were generated. Transcriptional activity of the promoter was analyzed by luciferase reporter assays in in vitrodifferentiated human adipocytes. The effect of TNF-a was assessed in human adipocytes and murine 3T3-L1 cells transfected with deletion fragments of the CIDEA promoter. Protein-DNA interactions were analyzed by electrophoretic mobility shift assays (EMSA). Results: Basal transcriptional activity was found in a 97-bp region upstream of the TSS. We studied the effect of three common haplotypes in the promoter region but found no significant difference in transcriptional activity among them. Incubation of in vitro-differentiated human adipocytes as well as 3T3-L1 cells with TNF-a reduced the transcriptional activity of the human CIDEA promoter, demonstrating a direct effect on CIDEA transcription. EMSAs and mutational analysis indicated that this was mediated by a nuclear factor-kB (NF-kB) site at position À163/À151. Conclusion: We demonstrate that basal transcription of the human CIDEA gene is confined to the 97 first bases upstream of TSS and that TNF-a negatively regulates transcription of this gene, which at least in part involves NF-kB activation.

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Mapping of Early Signaling Events in Tumor Necrosis Factor-α-mediated Lipolysis in Human Fat Cells

Cited by 237 publications

References 39 publications

Differential function of the α2A-adrenoceptor and phosphodiesterase-3B in human adipocytes of different origin

Differential function of the α2A-adrenoceptor and phosphodiesterase-3B in human adipocytes of different origin

Endothelin-1 stimulates human adipocyte lipolysis through the ETA receptor

Characterization of the human CIDEA promoter in fat cells

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