seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol 10, 1999: pp. 19᎐29 Article No. scdb.1998.0273, available online at http:rrwww.idealibrary.com on

The role of TNF␣ in metabolism U Jaswinder K. Sethi and Gokhan¨ S. Hotamisligil

Tumor necrosis factor-alpha() TNF␣ is a multifunctional almost every aspect of adipose biology. For example, cytokine which exerts a myriad of biological actions in it can directly alter glucose homeostasis and lipid different tissues and species. Many of these actions can metabolism and plays an important role in the devel- perturb the normal regulation of energy metabolism. In opment of resistance. As TNF␣ is also emerg- adipose tissue, in particular, TNF␣ has been demonstrated ing to be a key component in a number of metabolic to regulate or interfere with adipocyte metabolism at diseases, such as obesity, diabetes, dislipidemia and numerous sites including transcriptional regulation, glucose atherosclerosis, it is crucial to understand the molec- and fatty acid metabolism and hormone receptor signaling. ular mechanisms by which its actions are mediated. The implications of these perturbations in disease states and Only then would it be possible to identify specific the current understanding of the molecular mechanisms targets for therapeutic intervention. For these rea- utilised by TNF␣ are discussed herein. sons we have sought to review the current evidence and understanding of the molecular mechanisms Key words: adipose tissue r glucose metabolism r lipid utilised by TNF␣ in adipose tissue. metabolism r insulin resistance r tumor necrosis factor-␣ Ž.TNF␣ ᮊ1999 Academic Press The TNF ligands and receptors TNF␣ is synthesized as a 26-kDa transmembrane pro-hormone, which undergoes proteolytic cleavage to yield a 17-kDa soluble TNF␣ molecule. Despite Introduction the differences in size and location, both forms of TNF␣ are capable of mediating biological SINCE ADIPOSE TISSUE primarily functions to manage responses3 ᎐ 5 and together may be responsible for energy homeostasis, it is not surprising that this spe- both local and systemic actions of this cytokine.6 To cialised organ has received much attention in recent date mostŽ. if not all of the cellular actions of TNF␣ years. It is now apparent that adipose tissue serves not have been attributed to the activities of two distinct only as a storage depot but also as an endocrine receptors: type 1Ž TNFR1, a 55- or 60-kDa peptide in 1,2 organ. It is actively involved in sensing the nutritio- rodents and humans, respectively. ; and a type 2 nal and metabolic status of the organism through ŽTNFR2, a 75- or 80-kDa in rodents and humans, several different signaling pathways and regulates en- respectively. . Both of these receptors are expressed ergy metabolism by secreting molecules in response ubiquitouslyŽ. albeit at different ratios and to these cues. Thus, it is able not only to respond oligomerise upon ligand binding.7,8 The extracellular locally to metabolic stimuli by altering the expression domains of these two receptors exhibit some se- of genes important in the mobilization or storage of quence homology, while the intracellular domains energy, but can also activate signals to other tissues appear to be quite dissimilar. The latter has been thereby regulating energy balance, systemically. One interpreted as an indication that they might signal for signaling molecule produced by adipose tissue is different biological functions.8 Studies, so far, have TNF␣ which has been demonstrated to modulate addressed this possibility only partially and, to date, most known functions of TNF␣ have been ascribed

U to signals transduced by TNFR1 with TNFR2 playing From the Division of Biological Sciences and Department of primarily a modulatory role in ligand passing.9 How- Nutrition, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA ever, both receptors do seem to activate multiple ᮊ1999 Academic Press kinases and phosphoprotein phosphatases and can 1084-9521r99r010019q11 $30.00r0 utilize all major transduction pathways.10

19 J. K. Sethi and G. S. Hotamisligel

Both TNF receptors can also be released from the Ž.Figure 1 . Each of these can be altered in response cell surface through proteolytic cleavage and exist in to extracellular stimuli such as insulin, cortisol, cate- soluble form. The circulating levels of both soluble cholamines, growth hormone, testosterone, free fatty TNF receptors are elevated in many pathological acidsŽ. FFA and cytokines.16 There is also an increas- states, including sepsis,11 cancers,12 autoimmune dis- ing body of evidence to support a role for TNF␣ in eases,12 fever,13 chronic lymphocytic leukemia12 and modulating lipid metabolism. First, treatment of tu- obesity14,15, and may serve to modulate TNF␣ bioac- mor-bearingŽ. cachectic rodents with anti-TNF␣ anti- tivity both temporally and spatially. bodies protects against abnormalities in lipid metabolism.17 In obesity, the elevated levels of TNF␣ may also contribute to the elevated basal lipolysis that Role of TNF␣ in lipid metabolism is a characteristic of from obese subjects.16 This is further supported by studies in which the A key function of adipocytes is the regulation of lipid administration of the sTNFR᎐IgG chimera,18,71 but metabolism according to the physiological energy not anti-TNF␣ antibody,19 decreases serum FFA lev- requirements. The three biochemical sites of regula- els of obese rodents. Moreover, TNF␣ deficient mice tion are fatty acid uptake, lipogenesis and lipolysis exhibit lower circulating FFA and triglycerides than

Figure 1. Actions of TNF␣ on lipid metabolism in adipocytes. As indicated by the shaded area, the net action of TNF␣ in adipocytes is to decrease FFA uptake and triglyceride synthesis Ž.lipogenesis whilst increasing lipolysis. LPL, lipoprotein lipase; FFA, free fatty acids; FATP, fatty acid transporter protein; aP2, adipocyte fatty acid binding protein; ACS, acyl-CoA synthetase; ␤ ␤ HSL, hormone sensitive lipase; AR, 3 adrenergic receptor; AC, adenylate cyclase; PKA, protein kinase A; Glut 4, insulin sensitive glucose transporter. An asterisk indicates proteins whose activity andror expression is down-regulated by TNF␣ but upregulated by PPAR␥ and its ligands. This also includes lipogenic enzymes such as acetyl-CoA carboxylase and fatty acid synthetase.

20 TNF␣ and adipocyte metabolism their wild-type littermates.20,21 Finally, the exogenous as indomethacin, thiazolidinediones and 15- application of TNF␣ can stimulate lipolysis and in- dPGJ2.23,26,38 ᎐ crease circulating FFA levels in vivo and in vitro.22 26 The rate limiting enzyme, hormone sensitive lipase Since FFA can also mediate insulin resistance,26 the Ž.HSL is a candidate protein whose levels may be actions of TNF␣ on insulin sensitivityŽ see ‘Role of regulated by TNF␣. However, this action remains TNF␣ in mediating insulin resistance’ below. may be controversial since studies have either shown no reg- potentiated by increased lipolysis.27,28 ulation by TNF␣ Žin human cultured adipocytes24 . , In adipocytes, fatty acids are derived predomi- or a significant decreaseŽ in 3T3-L1 adipocytes26 . . It nantly via uptake from the circulation or from intra- remains to be seen if TNF␣ can modulate the activity cellular lipolysis and to a lesser extent by de novo of HSL directly or acts by up-regulating other, as yet synthesis from glucoseŽ. Figure 1 . Fatty acid uptake is unidentified, modulatory proteins. Perilipin is an- facilitated by the extracellular activity of lipoprotein other protein implicated in lipolysis, although its lipaseŽ. LPL which varies with nutritional and en- exact function remains unknown. It is localized at the docrine status.16 TNF␣ has been shown to inhibit surface of lipid droplets39 and downregulated by LPL activity and to down-regulate its protein expres- TNF␣ 26 but not by catecholamines. Intriguingly, sion in vitro, and in vivo.25,29,30 However, this action over-expression of perilipin in 3T3-L1 adipocytes se- in human adipocytes remains controversial.31 More lectively blocks TNF␣- but not catecholamine-stimu- recently, TNF␣ has also been shown to decrease the lated lipolysis.132 expression of FFA transportersŽ such as FATP and Whilst the downregulation of key lipolytic proteins FAT. in adipose tissue.32 Together, these actions of is not consistent with the stimulation of lipolysis TNF␣ could decrease FFA uptake from the circula- by TNF␣, these studies do highlight one problem tionŽ. Figure 1 and contribute to the hyperlipidemia facing mechanistic investigations on differentiated that is observed during infections and also in obesity. adipocytes: since the expression of lipogenic and In addition to inhibiting FFA uptake, TNF␣ also lipolytic proteins is intrinsically linked to adipocyte acts to decrease the expression of key enzymes in- differentiation, and TNF␣ can suppress the expres- volved in lipogenesisŽ. Figure 1 , namely acetyl-CoA sion of adipogenic genes, it is difficult to identify the carboxylase33 and fatty acid synthase.34 However, this direct targets of TNF␣ signaling. Indeed, the obser- may not occur in mature adipocytes.33 Nonetheless, a vations discussed aboveŽ particularly on cultured 3T3- recent report suggests that TNF␣ can also decrease LI adipocytes. may be the causal effects of modifying acyl-CoA synthetaseŽ. ACS mRNA and activity in the differentiation program and this may not be hamster adipose tissue.35 This would result in re- representative of actions in vivo. Nonetheless, the duced re-esterification of FFA and together with the possibility remains that the lipolytic actions of TNF␣ decreased substrate availabilityŽ through inhibition of are mediated not by upregulation of key lipolytic insulin-sensitive glucose uptake. may be the cause of enzymes but by the downregulation of proteins in- suppressed triglyceride accumulation. volved in the trapping of FFA in adipocytesŽ e.g. ACS ␣ ␤ The third target of TNF action is the lipolytic and perilipin. . Interestingly, 2 adrenergic receptor ␤ machinery of adipocytes. While the lipolytic process Ž.2-AR expression has been reported to be up-regu- is predominantly regulated by adrenergic stimulation lated by TNF␣ in adipocytes. However, this is accom- ␤ ␤ and mediated by a cAMP-dependent pathwayŽ Figure panied by a significant decrease in 13and 1. , the mechanism of TNF␣-induced lipolysis appears adrenoceptor expression.40 Whether this differential to be distinct and less well understood. Despite this, regulation contributes to metabolic disorders medi- biochemical and genetic studies have clearly shown ated by TNF␣ in vivo remains to be determined. that the lipolytic actions are mediated by TNFR1.36,37 This is consistent with earlier studies which used human TNF␣ Žselective for TNFR1 in murine sys- Role of TNF␣ in mediating insulin resistance tems. to stimulate lipolysis in murine cultures.22 The actions downstream of TNFR1 may involve transcrip- Elevated levels of TNF␣ have been postulated to tional regulation of key proteins involved in lipolysis, induce insulin resistance in a variety of catabolic since a chronic exposure is required to induce lipoly- disease states, including cancer,41 sepsis42,43 and ᎐ sis in fat cell cultures.23 25 This is supported by the trauma.43,44 This is supported by the observation that demonstration that TNF␣-induced lipolysis is blocked direct exposure of healthy individuals, animals or by activators of the transcription factor, PPAR␥, such isolated cells to TNF␣ induces a state of insulin

21 J. K. Sethi and G. S. Hotamisligel

᎐ resistance.18,25,45 49 Recent studies have also firmly insulin action including glucose transport, pro- established TNF␣ as an important mediator of obe- duction, insulin receptor signaling and improved lipid ᎐ sity-related insulin resistance.18,20,48,50 53 There now metabolism. However, in the absence of TNF␣ func- exists a substantial body of evidence to support this tion, the extent of protection from obesity-related latter hypothesis. First, in obese individuals and ro- insulin resistance is not complete and may depend dent models of obesity, TNF␣ is over-expressed in on other, as yet unidentified, factors or on the sever- ᎐ adipose and muscle tissue.50,52 58 This has been de- ity of the obesity model. For example, a recent report monstrated at both mRNA and cellular protein levels could not demonstrate the effects of the lack of both and is consistent with recent reports which suggest TNFRs in a dietary model of rodent obesity where that circulating levels of TNF␣ are increased in indi- the leptin system is intact.74 Further investigations are ᎐ viduals with NIDDM.59 64 Furthermore, the levels of required to definitively resolve this issue. TNF␣ expression strongly correlate with hyperinsu- In normal insulin sensitive tissues, insulin binding linemia and decreased insulin sensitivity. to its receptor triggers signaling cascades which result Dietary and chemical treatment of obesity im- in a number of cellular responses. One of these end proves insulin sensitivity and correlates with a de- responses is the increased translocation of Glut 4Ž the creased production of TNF␣.53,57,58,61,65 The inhibi- insulin sensitive glucose transporter. to the plasma tory actions of TNF␣ can also be reversed by insul- membrane which facilitates insulin-stimulated glu- in sensitizing drugs such as thiazolidined- cose uptakeŽ. Figure 2 . A number of in vitro studies, ᎐ iones.27,28,52,66 69 In vivo, these actions coincide with in adipocytes, suggest that TNF␣ may act directly to a decrease TNF␣ expression.28,52,69 Interestingly, in down-regulate Glut 4 gene expressionŽ. Figure 2 , obese mice deficient in the adipocyte-specific fatty thereby decreasing insulin-stimulated glucose acid binding protein, aP2, there is substantial protec- transport.25,75,76 Intriguingly, the Glut 4 content is tion from obesity-related insulin resistance and in reduced in adipocytes from obese NIDDM subjects.77 this model, adipose TNF␣ level does not increase Whether this is the main mode of TNF␣ action in with weight gain.70 human adipose tissue in obesity remains unclear. The neutralisation of endogenous TNF␣ with the However, in obese rodents lacking TNF␣, adipose sTNFR᎐IgG chimera also appears to improve insulin levels of Glut 4 remain similar to wild-type animals.20 sensitivity in obese and insulin resistant rats.18,50,71 In This suggests that in adipose tissue Ž.in vivo TNF␣ contrast, one report suggests that the administration acts via a mechanism independent of Glut 4 expres- of a neutralizing anti-TNF␣ antibody fails to improve sion. insulin sensitivity in obese NIDDM subjects.72 Al- TNF␣ has also been shown to act on the proximal though it is possible that the actions of TNF␣ may steps of insulin signaling. In cultured adipocytes TNF␣ differ among species, these divergent results also increases the phosphorylation of insulin receptor highlight the disadvantage of studies using neutraliz- substrate-1Ž. IRS-1 at serine residues Ž Figure 2 . . This ing antibodies or binding proteins, since it is difficult converts this multi-functional docking protein into to ascertain whether the injected substance has com- an inhibitor of the insulin receptorŽ. IR tyrosine pletely antagonised the antigen at all sites within the kinase.48,78,79 Such a modification of IRS-1 has been body before being degraded. However, a recent study observed in obesity and burn-induced insulin resis- has successfully illustrated that these problems may tance44,49 and is sufficient to block the downstream be overcome by using an effective delivery system to events of IR signaling, including the association of neutralize TNF␣ action in obesity.71 IRS-1 with phosphotidylinositolŽ. PI 3-kinase.47 ᎐ 49 Perhaps the most definitive evidence of a role for That these actions of TNF␣ play a role in insulin TNF␣ in obesity-linked insulin resistance has come resistance in vivo is supported by investigations in from recent studies in ligand or receptor deficient which both pharmacological and genetic blockade of mice.20,21,73 These independent reports demonstrate TNF␣ function results in increased signaling capacity that the absence of either TNF␣ or its functional of insulin receptors.18,20,71 Conversely, in 3T3-L1 receptors affords considerable protection from the adipocytes, acute treatment with TNF␣ Ž.30 min can insulin resistance that occurs in at least three models enhance IRS-1 tyrosine phosphorylation and its asso- of rodent obesityŽ namely, genetic, dietary and ciation to the p85 PI3-kinase subunit.80 Currently, chemically-induced obesity. . These studies also de- very little is known about the mediators of the signal monstrate that TNF␣ influences insulin sensitivity transduction pathway that forms this crosstalk through its actions on multiple targets involved in between TNFR and insulin receptors. Since neither

22 TNF␣ and adipocyte metabolism

Figure 2. Potential mechanisms of TNF␣-induced insulin resistance in adipocytes. TNF␣ has been demonstrated to disrupt insulin signaling at a number of peripheral sites which are dependent on IRS phosphorylation. As indicated, some of these actions can be mimicked by protein kinase CŽ. PKC isoforms, cell permeable ceramides, neutral sphingomylinase Ž nSMase . and the protein phosphatase inhibitor, okadaic acidŽ. OKA . In addition, chronic exposure to TNF␣ can suppress the expression of many adipocyte-specific genes including the insulin-sensi- tive glucose transporterŽ. Glut 4 . Pre-treatment with the insulin sensitizing thiazolidinediones Ž.TZD has been shown to be reverse at least two actions of TNF␣. TNF␣ can also stimulate the production of additional mediators such as free fatty acidsŽ. FFA and leptin which may act to potentiate its actions on insulin resistance in adipocytes. IR, insulin receptor; IRS, insulin receptor substrate; PI3K, phosphotidylinositol 3-kinase.

TNFR1 nor TNFR2 exhibits intrinsic kinase activity, ate the endogenous downregulationŽ negative feed- the search is on for receptor associated proteins that back. of adipose IR activity in pathological states such couple TNFR activation to IRS-1 phosphorylation. To as obesity and NIDDM.86 this end, a few candidate kinases have been impli- Sphingolipid metabolism may also play a role in cated. Of particular interest is PKC␧ which can facili- TNF␣-induced insulin resistanceŽ. Figure 2 . TNF␣ tate TNF␣-induced inhibition of IR signaling in hu- increases intracellular ceramide levels and the treat- man embryonic kidneyŽ. HEK293 cells and whose ment of cells with neutral sphingomyelinase and syn- translocation is stimulated by TNF␣.81 Other PKC thetic analogs of ceramides can mimic the actions of ␣ ␦ ␤ ␪ ␣ 87,88 isoenzymes, namely , , 2 and , have recently TNF on IR and IRS-1 phosphorylation. Whether been shown to inhibit IR kinase activity in an IRS-1 ceramides then act directly on IR signaling or down- dependent manner.82 Alternatively, TNF␣ may be regulate Glut 4 gene expression remains controver- inhibiting IR signaling by inhibiting serinerthreonine sial.89 However, Ceramide generation is known to phosphatases83 andror recruiting the activity of pro- regulate the activities of numerous other seriner tein tyrosine phosphatasesŽ. PTPases .84,85 However, it threonine protein kinases, such as PKC ␨ 90 and Raf1 remains to be seen whether any of these enzymes are kinase,91 as well as phosphatases, such as protein involved in TNF␣-induced inhibition or simply medi- phosphatase 2A.92,93 It is likely that one or more of

23 J. K. Sethi and G. S. Hotamisligel these enzymes could be involved in modulating IRS-1 body fat, can modulate insulin sensitivity, can de- in response to TNF␣. crease food intake and regulate other aspects of In addition to adipocytes, TNF␣-induced insulin energy metabolismŽ. as cited in ref 104 . It is therefore resistance has also been demonstrated in a number tempting to speculate that TNF␣ may recruit leptin of other cell types including; hepatoma cellsŽ FAO to potentiate its insulin resistance effects through an and KRC-7. ,47,78,85,88 fibroblasts, 94 myeloid cells autocrine or paracrine action. However, since obese Ž.32D49,87 and rat muscle cellsŽ. L6 .95 The factors TNF␣-deficient mice still exhibit higher leptin levels recruited to mediate its actions appear to vary de- than their lean counterparts,104 TNF␣ may not be pending not only on the duration and concentration the master regulator of adipose derived leptin. of TNF␣ but may also be specific to the tissue under TNF␣-mediated release of leptin from adipose tis- investigation. For example, in fetal brown adipocytes, sue may be part of the adipostat mechanism that TNF␣ inhibition of IR phosphorylation is mediated relates circulating leptin concentrations to triglyc- via IRS-2 96 whilst in cultured white adipocytes and eride stores. It was postulated that cytokine-induced 32D cells, IRS-1 is the major substrate that is modu- anorexia which occurs during infection and inflam- lated.45,49 It has also been suggested that chronic mation, may be mediated by enhanced leptin secre- treatment with TNF␣ can quantitatively regulate IR tion.102,103 However, recent studies using mice defi- and IRS-1 in cultured adipocytes.97 Furthermore, cient in either leptin Ž.obrob or its functional recep- TNF␣ may employ additional extracellular mediators tor Ž.dbrdb , show that leptin is unlikely to be the sole by altering free fatty acid and leptin secretion from mediator of LPS-induced appetite suppression.107 adipose tissue. Whether these act directly with TNF␣ We have used TNF receptor deficient mice to signaling pathways or indirectly as a means of poten- identify the receptor responsible for TNF␣-induced tiating its effects on adiposeŽ. or other tissues remains leptin production in vivo. This function of TNF␣ to be determined. appears to be predominantly attributed to the activity of TNFR1.108 However, unlike most other actions of TNF␣, transcriptional regulation does not seem to be Role of TNF␣ in regulating leptin production the main mode of action. Indeed, the increased levels of leptin secreted in response to TNF␣ are not re- Like TNF␣, leptin also has significant effects on flected by increased leptin mRNA expression levels in energy metabolism and appetite. Adipose tissue is adipose tissue.104 This discordance has been demon- considered to be the major source of circulating strated in vitro in 3T3-LI adipocytes, and in vivo in leptin.98 An increasing number of reportsŽ from ro- obese rodents104 and humans.106 Further pharmaco- dents and humans. suggest that leptin expression and logical characterization with 3T3-L1 adipocytes has secretion can be positively regulated by insulin, glu- lent some clues as to how TNF␣ may influence leptin ᎐ cocorticoids and glucosamine.99 101 Cytokines includ- production.104 These have demonstrated that the ing TNF␣ 60,102,103 also regulate leptin production. TNF␣-stimulated leptin production is insensitive to Recently, we and others have demonstrated that TNF␣ the protein synthesis inhibitor, cyclohexamide, but ᎐ treatment increases leptin production in vivo.102 104 can be blocked by the secretion inhibitor, brefeldin In contrast, treatment of cultured adipocytes pro- A. This indicates that TNF␣ is acting post-translation- duces a transient response, with a chronic treatment ally perhaps by mobilizing preformed pools of leptin. inhibiting leptin gene expression and decreasing se- creted protein.104,105 Nevertheless, obese mice lack- ing TNF␣ or TNF receptors exhibit significantly lower Role of TNF␣ in regulating PAI-1 circulating leptin levels.21,74,104 This strongly suggests biosynthesis that TNF␣ does have a physiological role in positively regulating leptin production in rodent models of Adipose tissue expression and circulating levels of obesity. That TNF␣ plays a similar role in human plasminogen activator inhibitorŽ. PAI-1 is elevated in obesity has also recently been suggested.60,106 Indeed obese individuals109 and may represent the main a significant correlation exists between circulating source of the elevated circulating plasma PAI-1 leptin, TNF␣ and body mass indexŽ. BMI in observed in obesity.110 This hypothesis is supported humans.60,106 Interestingly, both TNF␣ and circulat- by previous studies which demonstrate that PAI-1 ing leptin exhibit numerous parallels in their biology; levels can be reduced significantly in parallel to weight they both show a strong correlation with percent loss.110 The expression of TNF␣ also shows a strong

24 TNF␣ and adipocyte metabolism correlation with PAI-1 expression, particularly in the ligands, such as troglitazone and indomethacin, can context of obesity.110 Indeed, treatment with TNF␣ prevent the actions of TNF␣ in cultured adipocytes stimulates PAI-1 biosynthesis in numerous cell types, as well as in whole animals.28,66 Interestingly, TNF␣ including 3T3-L1 adipocytes,111 adventitial cells and has recently been shown to directly modulate 110 r vascular smooth muscle cells in adipose tissue. Fur- prostaglandin synthesis at the level of PGD2 and or ␣ 119 thermore, inhibiting TNF synthesis with pentoxi- PGE2 synthesis in murine macrophages. Since, fylline treatment also reduces PAI-1 expression. These PGD2 is metabolized to PGJ2 and its derivatives, the observations are in line with the hypothesis that lo- putative endogenous ligands for PPAR␥, it is tempt- cally elevated TNF␣ in adipose tissue acts in an ing to speculate that this may be the primary target autocrine fashion to stimulate PAI-1 production. for TNF␣ action in adipocytes. However, since Thus, in obesity, the increased levels of TNF␣ may be TNF␣-responsive kinasesŽ. MAPK and PKC have been contributing to the elevated plasma PAI-1 levels and implicated in blocking adipogenesis120 and their hence increased risk of coronary heart disease. Addi- phosphorylation cascades can regulate transcription tional factors may also potentiate the actions of TNF␣ factor expression, the possibility remains that TNF␣ on PAI-1 synthesis. These include TGF␤, insulin, may also be acting through these alternative path- triglycerides and FFA.110,112 At least the latter three ways. The TNF␣ receptor primarily involved in medi- are known to be elevated in obesity and modulated ating the effects of TNF␣ on adipocyte differentia- by TNF␣. Studies are currently underway using lig- tion is clearly TNFR1. Recent studies with cell lines and and receptor knockout obese mice to further deficient only in this TNF receptor have definitively address the role of TNF␣ in PAI-1 production in demonstrated that this is the case with both soluble obesity. and transmembrane TNF␣.5,121 These experimental systems should be instrumental in dissecting the sig- naling pathways involved in this action. Role of TNF␣ in adipocyte differentiation

TNF␣ has been shown not only to be a potent TNF␣-induced apoptosis of adipose tissue inhibitor of adipocyte differentiation but capable of suppressing the expression of some adipocyte-specific While the cytotoxic actions of TNF␣ toward certain ᎐ genes in fully differentiated adipocytes.113 115 Inter- cells and tumors are well documented,122,123 the po- estingly, the anti-adipogenic action of TNF␣ also tential apoptotic action on adipocytes has received appears to be reversed by subsequent treatment with little attention. However, reports are beginning to adipogenic agents such as dexamethasone and in- emerge which suggest that, under culture conditions, domethacin.116 This raises the intriguing possibility TNF␣ can also be cytotoxic to both adipocytes and that adipocyte differentiation may be a reversible pre-adipocytes.124,125 Most recently, TNF␣-induced process in vivo and under the control of both positive apoptosis has also been implicated in rodent brown and negative regulators.113,116 This long-standing de- adipocytes126,127 and increased brown fat apoptosis is bate has yet to be resolved. correlated to obesity.126 This, together with the ac- As is the case for the action of other growth factors tions of TNF␣ on adipocyte differentiation, could on development and differentiation, the exact mech- represent a possible mechanism by which TNF␣ may anismŽ. s by which TNF␣ alters the adipocyte differ- be acting to minimize adipose tissue mass. entiation program is not yet clear. However, much Since both TNF receptors are capable of initiating attention has been focused on its effects on transcrip- apoptotic signals,128 ᎐ 130 they are both equally likely tional regulation. TNF␣ has been shown to down- to be involved in adipocyte apoptosis. With the avail- regulate the expression of transcription factors, such ability of receptor deficient mice, studies are cur- as PPAR␥ Žperoxisome proliferator activated-recep- rently underway to identify which receptor is involved tor ␥ .Žand CrEBP␣ CCAATrenhancer-binding pro- in adipocyte apoptosis in vivo and whether this action tein ␣ ., which are involved in the early stages of is involved in obesity. Although very little is known adipocyte conversion.115,117,118 This may explain the about TNF␣-induced cytotoxic signaling in parallel downregulation of those adipose-specific adipocytes, a substantial amount of research has been genesŽ. such as Glut 4 and aP2 that contain binding conducted on other cells and tissues. Here the apop- sites for PPAR␥ or CrEBP␣ in their promoters76,117 totic signaling cascades emanating from each TNF and is consistent with the observation that PPAR␥ receptor have been extensively studied. The reader is

25 J. K. Sethi and G. S. Hotamisligel referred to the excellent recent reviews by Hale et TNF for signaling by the 55-kDa TNF receptor. J Biol Chem 131 129 268:18542᎐18548 al and Wallach. 10. Vilcek J, Lee THŽ. 1991 Tumor necrosis factor. New insights into the molecular mechanisms of its multiple actions. J Biol Chem 266:7313᎐7316 11. van Zee KJ, Kohno T, Fischer E, Rock CS, Moldawer LL, Conclusions Lowry SFŽ. 1992 Tumour necrosis factor soluble receptors circulate during experimental and clinical inflammation and TNF␣ plays a key role in regulating energy can protect against excessive tumor necrosis factor alpha in vitro and in vivo. Proc Natl Acad Sci USA 89:4845᎐4849 metabolism under both physiological and pathologi- 12. Aderka D, Engelmann H, Wallach D.Ž. 1993 Soluble tumor cal states. Recent developments of several transgenic necrosis factor receptors in health and disease, in: Tumor models, with functionally-modified TNF␣ systems, Necrosis Factor: Molecular and Cellular Biology and Clinical RelevanceŽ. Fiers W, Buurman WA, eds. pp. 191᎐198. S. have illustrated the definitive involvement of this Karger AG, Basel cytokine in lipid and glucose metabolism, in vivo. 13. Liabakk NB, Sundan A, Waage A, Brockhaus M, Loetcher H, These together with newly developed in vitro systems Lesslauer W, Espevik TŽ. 1991 Development of immunoas- ␣ says for the detection of soluble tumour necrosis factor should aid future mechanistic studies of TNF bi- receptors. J Immunol Methods 141:237᎐243 ology. The molecular dissection of the signaling 14. Hotamisligil GS, Arner P, Atkinson RL, Spiegelman BM events mediating these actions promises to offer novel Ž.1997 Differential regulation of the p80 tumor necrosis factor receptor in human obesity and insulin resistance. targets for treatment of metabolic disorders in which Diabetes 46:451᎐455 TNF␣ plays a key role. 15. Tsigos C, Kyrou I, Economidou C, Tsapogas P, Kantzos N, Mantas P, Katsilambros NŽ. 1998 Elevated circulating tumor necrosis factor alpha receptor 1 and 2Ž. TNFR1, TNFR2 concentrations in human obesity. Int J Obes 21:S9 Acknowledgements 16. Ramsay TGŽ. 1996 Fat cells. Endocrinol Metab Clin N Am 25:847᎐870 17. Carbo N, Costelli P, Tessitore L, Bagby GJ, Lopez-Soriano We thank the members of the Hotamisligil laboratory for FJ, Baccino FM, Argiles JMŽ. 1994 Anti-tumour necrosis their contribution to the work described herein and for factor-alpha treatment interferes with changes in lipid input to the manuscript. Due to space limitations we were metabolism in a cachectic tumor model. Clin Sci 87:349᎐355 unable to cite many valuable reports related to the topics 18. Hotamisligil GS, Budavari A, Murray D, Spiegelman BM covered in this review and apologize for their inadvertent Ž.1994 Reduced tyrosine kinase activity of the insulin recep- tor in obesity᎐diabetes. Central role of tumor necrosis fac- omission. Financial support is provided by the NIH, ADA tor-alpha. J Clin Invest 94:1543᎐1549 and PewŽ. to GSH and The Wellcome Trust Ž. to JKS . 19. Lopezsoriano FJ, Bagby GJ, Williamson DH, Argiles JMŽ. 1997 Anti-TNF treatment does not reverse the abnormalities in Lipid metabolism of the obese Zucker rat. Am J Physiol 35:E656᎐E660 References 20. Uysal KT, Wiesbrock SM, Marino MW, Hotamisligil GS Ž.1997 Protection from obesity-induced insulin resistance in ᎐ 1. Flier JSŽ. 1995 The adipocyte: storage depot or node on the mice lacking TNF-alpha function. 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