NF-kB is important for TNF-a-induced lipolysis in human adipocytes

Tumor necrosis factor-a (TNF-a) promotes lipolysis in mammal adipocytes via the mitogen-activated protein kinase (MAPK) family, resulting in reduced expression/ function of perilipin (PLIN). The role of another pivotal intracellular messenger activated by TNF-a, nuclear factorkB (NF-kB), has not been recognized. We explored the role of NF-kB in TNF-a-induced lipolysis of human fat cells. Primary cultures of human adipocytes were incubated in the presence of a cell-permeable peptide that inhibits NF-kB signaling (WP). Incubation with WP, but not with a biologically inactive peptide (MP), abolished the nuclear translocation of NF-kB and effectively abrogated TNF-a-induced lipolysis in a concentration-dependent manner. Western blot analysis demonstrated that although TNF-a per se reduced mainly PLIN protein expression, TNF-a in the presence of WP resulted in a pronounced combined reduction of both hormone-sensitive lipase (HSL) and PLIN protein. The expression of a set of other lipolytic or adipocyte-specific proteins was not affected. The regulation was presumably at the transcriptional level, because mRNA expression for HSL and PLIN was markedly reduced with TNF-a in the presence of NF-kB inhibition. This was confirmed in gene reporter assays using human PLIN and HSL promoter constructs. We conclude that in the presence of NF-kB inhibition, TNF-a-mediated lipolysis is reduced, which suggests that NF-kB is essential for retained human fat cell lipolysis.— Laurencikiene, J., V. van Harmelen, E. Arvidsson Nordström, A. Dicker, L. Blomqvist, E. Näslund, D. Langin, P. Arner, and M. Rydén. NF-kB is important for TNF-a-induced lipolysis in human adipocytes. J. Lipid Res. 2007. 48: 1069–1077. Supplementary key words tumor necrosis factor-a & nuclear factor-kB & signal & differentiation The release of energy-rich FFAs through lipolysis is a key event in adipocyte function, and increased circulating FFA levels promote insulin resistance (1). In obesity and cachexia as well as other clinical conditions, the lipolytic activity of fat cells is increased, resulting in reduced insulin sensitivity. Although a number of hormones and other external factors enhance lipolysis in fat cells, abnormal autocrine regulation could be a common pathological factor for obesity and cachexia, as reviewed (2, 3). Adipocytes as well as stromal cells in adipose tissue produce the cytokine tumor necrosis factor-a (TNF-a). In vitro studies have demonstrated that TNF-a stimulates adipocyte lipolysis in murine and human adipocytes (4). TNF-a expression is increased in obesity in both rodent and human adipose tissue (5, 6) and is also enhanced in animal models of cachexia (7). TNF-a promotes lipolysis via several mechanisms. For instance, TNF-a downregulates phosphodiesterase-3B (PDE3B) expression in 3T3-L1 cells (8) and human adipocytes (9). PDE3B is the major hydrolytic enzyme of cAMP activated by insulin, which thereby mediates the antilipolytic effect of this hormone. Another proposed mechanism of TNF-a is downregulation of the GTP binding protein Gai, which mediates antilipolytic signals. However, the expression of Gai is downregulated in rodent (10) but not human adipocytes (11). In both rodent and human cells, TNF-a activation promotes phosphorylation (9) and the downregulation of perilipin (PLIN) expression (11, 12). PLIN is located on the surface of the intracellular triglyceride lipid droplet and regulates the access of hormone-sensitive lipase (HSL), the main hydrolytic Manuscript received 25 October 2006 and in revised form 19 January 2007. Published, JLR Papers in Press, February 1, 2007. DOI 10.1194/jlr.M600471-JLR200 1 J. Laurencikiene and V. van Harmelen contributed equally to this work. 2 To whom correspondence should be addressed. e-mail: [email protected] Copyright D 2007 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 48, 2007 1069 by gest, on N ovem er 7, 2017 w w w .j.org D ow nladed fom enzyme of stored triglycerides in human fat cells. The alterations at the levels of PLIN, PDE3B, and Gai lead to increased basal (spontaneous) lipolysis. To date, the PLIN effect is the best studied in human lipolysis (11). However, it is still not entirely clear how PLIN regulates lipolysis. Two different models have been proposed. In the barrier/translocation model introduced by Londos et al. (13), PLIN constitutes a physical barrier to HSL. Upon PLIN phosphorylation and downregulation, lipid surface accessibility for HSL is enhanced. This fits with data from PLIN knockout mice, which display an increased basal adipocyte lipolysis. In contrast, a series of recent studies have proposed a second model in which PLIN functions as a scaffold protein. In this model, PLIN phosphorylation leads to an altered structure and/or interaction with regulatory proteins. PLIN thereby recruits HSL and probably increases lipase access to the lipid surface (14 –17). It is important to stress that neither of these models has been demonstrated in human adipocytes; rather, both are based on studies in rodent fat cells and immortalized cell lines. The intracellular signaling pathways elicited by TNF-a form a complicated and intricate web that has mainly been characterized in nonfat cells (18). Some of the signals that mediate the TNF-a effect on lipolysis have been dissected in adipocytes (9, 19). These involve p44/42 and c-jun NH2terminal kinase ( JNK) of the mitogen-activated protein kinase (MAPK) family, which phosphorylate and activate downstream transcription factors and thereby regulate the expression of genes involved in lipolysis. Most importantly, p44/42 and JNK relay the effects on PLIN (11, 20) and PDE3B (9). Additional pathways besides MAPKs could mediate TNF-a effects on adipocyte lipolysis. One candidate is nuclear factor-kB (NF-kB) (21), which is a homodimer or heterodimer composed from five related transcription factors. These contain a conserved sequence that is responsible for dimerization and DNA binding; the best characterized members are p65 and p50 (22). In the cytoplasm, NF-kB is bound to inhibitors of NF-kB (IkBs), which prevent NF-kB from entering the cell nucleus. TNF-a activates a set of proteins termed IkB kinases (IKKs). IKKs are trimeric complexes formed by a, b, and g subunits in which the a and b subunits phosphorylate IkB. This leads to an altered conformation of IkB, which releases NF-kB to enter the nucleus while IkB is targeted to the proteasome degradation pathway. The function of NF-kB in human adipocytes is unclear, although a few reports in murine cells indicate that NF-kB inhibits adipogenesis (23) and that TNF-a downregulates adipocyte-specific genes via NF-kB (24). Studies of NF-kB have to some extent been hampered by the lack of selective and specific inhibitory compounds. However, a 28 amino acid long peptide derived from the IKKg sequence was recently developed. This peptide inhibits trimerization of the IKK complex and the subsequent activation of NF-kB (25). The peptide includes a short sequence derived from the Drosophila antennapedia homeodomain protein, which enables cell membrane translocation. It is an effective NF-kB inhibitor and inflammatory antagonist in different in vitro (26) and in vivo (27) models. To study the role of NF-kB in adipocyte lipolysis, we treated primary cultures of human adipocytes with this inhibitor (herein referred to as WP) and TNF-a. Parallel experiments were performed with a similar but biologically inactive (mutated) peptide lacking the membrane permeabilization sequence (MP). MATERIALS AND METHODS Subjects and adipose tissue Subcutaneous adipose tissue was obtained from otherwise healthy subjects who underwent gastric banding surgery for obesity or abdominal liposuction for cosmetic reasons. No subject was on any regular medication. No selection was made for age, gender, or body mass index. The study was approved by the Ethics Committee at Karolinska University Hospital. All subjects gave their informed consent to participate in the study. Specimens from subcutaneous adipose tissue were obtained within 30– 45 min after the onset of surgery. All subjects fasted overnight before surgery, and only saline was administered intravenously until the tissue samples were taken. In general, 5–40 g of adipose tissue was obtained, which yielded two to three 24-well plates of preadipocytes.