Cytokine & Growth Factor Reviews Vol. 7, No. 2, pp. 161 173, 1996
Copyright ,(? 1996 Published by Elsevier Science Ltd. All rights reserved PII:
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Inhibition of Insulin Receptor Signaling by TNF: Potential Role in Obesity and Non-Insulin-Dependent Diabetes Mellitus
Edward Y. Skolnik* and Jerom Marcusohn Adipocytes produce a variety of molecules that are capable of functioning in both a paracrine and autocrine fashion. Tumor necrosis factor (TNF) is one of the proteins produced by adipocytes that has been shown to regulate adipocyte function. Interestingly, adipocyte expression of TNF increases with increasing adipocyte mass and expression of TNF is increased in adipocytes isolated from several genetic models of rodent obesity and from obese humans. This finding has led to the idea that TNF produced by adipocytes functions as a local "adipostat" to limit fat accumulation. Increased production of TNF by adipocytes, however, may contribute to insulin resistance in obesity and in non-insulin-dependent diabetes mellitus (NIDDM). TNF has been shown to inhibit insulinsimulated tyrosine phosphorylation of both the insulin receptor (IR) and insulin receptor substrate (IRS)-I and to stimulate downregulation of the insulin-sensitive glucose transporter, GLUT4, in adipocytes. These findings raise the possibility that pharmacological inhibition of TNF may provide a novel therapeutic target to treat patients with NIDDM. Copyright 1996Publishedby ElsevierSci...... Ltd Key words: Inhibition- Insulin receptor. Signaling. TNF" Obesity" Diabetes.
Obesity is among the most common metabolic disorder in Western society, affecting more than 30% of the adult population. The negative impact of obesity on health is evident by the increased morbidity and mortality observed in this patient population. The increased morbidity caused by obesity likely arises from both obesity itself and the metabolic consequences that result from the increase in adipocyte mass [1]. With respect to the metabolic derrangements that contribute to the increase in morbidity and mortality, obesity is associated with atherogenic lipid profiles and, perhaps most importantly, insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM) (also known as type 2 diabetes mellitus) [1]. N I D D M affects about 5% of the population and is characterized by increased blood glucose levels which arise primarily from peripheral resistance to insulin's action in fat and muscle [2-5]. Binding of insulin to its receptor on fat and muscle regulates postprandial blood glucose levels by stimulating the redistribution of a glu-
cose transport, GLUT4, from an intracellular vesicle to the plasma membrane [6, 7]. The importance of obesity as a risk factor for the development of clinical NIDDM (elevated blood glucose levels) is supported by the findings that over 80% of N I D D M patients are obese and that weight reduction in this patient population is associated with decreased insulin resistance and improved blood glucose control. However, while it has long been known that the propensity to develop diabetes is strongly linked not only to genetic factors but also to obesity, the underlying molecular mechanisms responsible for insulin resistance in the majority of patients with NIDDM, as well as the mechanism(s) whereby obesity contributes to insulin resistance in this patient population are still poorly defined. This review focuses on the role of TNF in mediating insulin resistance in NIDDM. THE ADIPOCYTE AS AN ENDOCRINE ORGAN
Although adipocyte hyperplasia and hypertrophy are central to the obese phenotype, there has been little evidence until recently that the adipocyte actively par*To whom correspondence should be addressed at: New York University MedicalCenter, SkirballInstitute, 540 First Avenue, ticipates in the maintenance of the body weight "set New York, NY 10016, U.S.A. Tel.: 212-263-7458; Fax: 212- point" [8]. The prevailing view had been that the adipo263-5711. cyte is a passive organ responsible only for storing triIAI
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glycerides. The recent cloning of the gene responsible for obesity in the ob/ob homozygous mouse has now provided conclusive evidence that the adipocyte participates in a feedback loop to regulate food intake, thermogenesis and energy expenditure [9]. In response to an increase in adipocyte mass, the protein product of the ob gene, leptin, is secreted by adipocytes [9-12]. Leptin then functions as an endocrine signaling molecule to limit body weight by acting on a receptor in the hypothalmus (and elsewhere)to decrease food intake and increase thermogenesis [11, 13-18]. Leptin is unlikely to be the only molecule secreted by adipocytes that directly or indirectly regulates body weight. Several other molecules, such as adipsin, angiotensinogen and TNF have been found to be secreted by adipocytes [8, 19, 20]. However, with the exception of TNF the biological functions ofthese molecules in adipocyte regulation and food intake is still not known. TNF regulates a variety of biological activities in the adipocyte, Treatment of adipocytes with TNF leads to decreased lipoprotein lipase (LPL) activity and stimulation of lipolysis [21-23]. In addition, TNF induces insulin resistance in adipocytes [20, 24, 25]. It has been hypothesized that these and other actions of TNF function to limit lipid accumulation in adipocytes [20]. Thus, whereas ob functions as a systematic "adipostat" to regulate adipocyte size, TNF may function as one of the local "adipostats" to regulate adipocyte cell size. TNF is primarily produced by immune cells and mediates a variety of beneficial effects. These include the stimulation of immune and inflammatory responses and the involution of transplantable tumors [26, 27]. However, soon after its discovery, it was clear that production of TNF was not always beneficial for the host. Overproduction or inappropriate secretion of TNF has been associated with several deleterious outcomes. Cerami and co-workers identified the same molecule, which they termed cachectin, as a potential endogenous mediator causing wasting in infected animals [28]. Moreover, overproduction of TNF has been found to be one of the mediators of circulatory shock and tissue injury in sepsis and the CNS manifestation of malaria infection [27, 29, 30]. TNF secreted by adipocytes also is likely to produce either beneficial or deleterious consequences, depending on the context in which it is produced. On the one hand, insulin resistance induced by TNF may be a risk factor for the development of atherosclerosis and hypertension. However, the finding that insulin resistance, by itself, may function to prevent weight gain suggests that induction of insulin resistance may also be beneficial and serve to limit fat accumulation [31, 32]. In modern society, the negative impact of insulin resistance arising from increased production of TNF by "overfilled" adipocytes appears to outweigh its positive impact, TNF AND ITS RECEPTOR
TNF has been used to describe two related cytokines encoded by distinct genes. These molecules have been
termed tumor necrosis factor-alpha (TNF) and tumor necrosis factor-beta [also known as lymphotoxin-7 (LT)]. TNF and LT are highly homologous at the protein level and both molecules engage identical receptors on target cells [27, 33]. It is not surprising, therefore, that these molecules have been found to mediate essentially identical responses. The major difference between these molecules is that, whereas LT is secreted mostly by activated T- and NK-cells, TNF is expressed by a greater variety of cells [20, 27]. This review focuses on TNF since the expression of LT has not been found to be perturbed in obese patients. TNF is synthesized as a 26-kDa prohormone which undergoes cleavage to yield a 17-kDa soluble TNF molecule [27, 34]. Both the 26-kDa and 17-kDa forms of TNF are capable of mediating biological responses. The difference between these two forms is that the 26-kDa form of TNF contains a transmembrane domain and is expressed as an integral membrane protein while the 17kDa product functions as a soluble TNF molecule. Soluble TNF, and presumably its transmembrane form, form non-covalent trimers which function as a unit to engage three receptor molecules leading to receptor oligomerization and activation (see below)[35]. TNF mediates its biological effects by binding to two different receptors on the surface of cells [33, 36]. These receptors have been termed p60 and p80 to coincide with their molecular weights. The p60 and pS0 TNF receptors are part of a growing receptor superfamily that is characterized by multiple cystine-rich domains in the extracellular amino-terminal domain [37]. In contrast to the similarity in the extracellular domains, the cytoplasmic domains of receptors in this group are diverse, implying that different receptors couple to different cytoplasmic signaling molecules. However, although similarities between the cytoplasmic domain of the p60 and p80 TNF receptors have not been found, recent studies have identified domains that are conserved among some family members that function to couple to similar signaling molecules [38, 39]. Both TNF receptors are expressed on most cells [33]. Studies using agonistic antibodies, mutant forms of TNF capable of binding only the p60 or p80 receptor, as well as mice containing targeted disruption of either the p60 or p80 receptors have indicated that activation of the p60 receptor is sufficient to mediate most biological responses by TNF [33, 36, 4(~42]. The role of the p80 receptor in TNF signaling has been more difficult to resolve. Although activation of the p80 receptor has been clearly shown to contribute to signaling by the p60 receptor, activation of the p80 receptor alone has been shown to activate biological responses under only certain conditions [33, 36, 43]. This led to the initial idea that under most circumstances, the pS0 receptor functions as an accessory molecule to facilitate signaling by the p60 receptor [36, 44]. The "ligand passing" model proposes that ligand binding to the p80 receptor does not directly signal the cell, but rather functions solely to increase the local concentration of TNF at the cell surface, thereby
Inhibition of IR Signaling by TNF making more TNF available to bind the p60 receptor which is responsible for transducing the TNF signal, With respect to insulin resistance, activation of the p60 TNF receptor is also sufficient to mediate insulin resistance in insulin-responsive tissues[24, 45, 110]. However, while "ligand passing" may be one function of the p80 receptor, recent experimentalevidence has unequivocally demonstrated that p80 receptor directly signals cells and that activation of the p80 receptor also contributes to the inhibition of insulin signaling by TNF [34, 43, 46, 110]. These findings, coupled with the demonstration that the p80 receptor is upregulated in fat and skeletal muscle in the obese rodent model of insulin resistance [47], suggests that the p80 receptor may play an important role in mediating the insulin resistance induced by TNF.
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ondary changes that contributes to the increase in insulin resistance. Current evidence indicates that the primary as well as secondary defect(s) accounting for insulin resistance in NIDDM occur downstream of insulin binding to its receptor. Thus, before discussing TNF's role in mediating insulin resistance, we shall first review what is known about how the insulin receptor (I R) normally signals cells (Figure 1). The early intracellular signaling events linking the IR to GLUT4 translocation and glucose uptake in fat and muscle are now beginning to be understood. The first step in insulin signaling involves binding of insulin to a cell surface receptor containing tyrosine kinase activity [52]. The IR is a heterotetrameric protein composed of two identical alpha and beta subunits. The alpha subunit is extracellular and binds the ligand, whereas the beta subunit is mostly cytosolic and contains the receptor tyroINSULIN SIGNALING AND THE PATHOGENESIS sine kinase activity. Ligand binding to the alpha subunit OF NIDDM results in autophosphorylation of the IR beta subunit and activation of the intrinsic tyrosine domain. The tyrosine Insulin maintains blood glucose homeostasis by both kinase is critical for the IR to signal cells; any impairment stimulating glucose uptake into insulin-sensitive fat and of the tyrosine kinase leads to a decreased ability of the muscle cells and by suppressing glucose production by receptor to stimulate cell growth and metabolic functions, hepatocytes [2, 5, 48]. Insulin mediates glucose uptake such a glucose uptake and glycogen synthesis [52-54]. into peripheral tissues by stimulating the translocation Once activated, the IR associates with and tyrosine of a specialized glucose transporter, GLUT4, from an phosphorylates a variety ofcytosolic signaling molecules intracellular compartment to the plasma membrane [6, in the cell. IR substrates (IRS) 1 and 2 are two of the 7]. Following insertion of GLUT4 into the plasma mem- targets of the I R and comprise a family of 180-190-kDa brane, GLUT4, then functions to transport glucose into cytoplasmic proteins that are tyrosine phosphorylated by the cell. Only adipose tissue, skeletal muscle and cardiac the IR and the insulinqike growth factor (IGF-I) receptor tissue express GLUT4 and, as a result, these tissues are [52, 55]. IRS-1 and 2 contain over 20 potential tyrosine primarily responsibleforinsulin-stimulatedregulationof phosphorylation sites and over 30 potential serine/ blood glucose levels in the post-absorptive state. The fact threonine phosphorylation sites. Many of the tyrosine that skeletal muscle accounts for the removal of > 80% phosphorylation sites on IRS-1,2 are contained within of insulin-stimulated glucose from blood indicates that consensus SH2 binding motifs. SH2 domains are found insulin resistance in muscle must occur for hyperglycemia in a variety of distinct signaling molecules and mediate to develop. Both longitudinal and cross-sectional studies, protein-protein interaction by binding phosphotyrosine as well as studies employing the euglycemicinsulin clamp, moieties in the context of short amino acid sequences have convincingly demonstrated that an increase in per- [56, 57]. Binding of signaling molecules, via their SH2 ipheral resistance to insulin's action in muscle and fat domains, to tyrosine phosphorylated IRS-I,2 couples the initiates the preclinical process of NIDDM [2, 5, 48]. IR to downstream signaling pathways [52]. These findings, coupled with the demonstration that there PI3-kinase is one of the SH2 domain-containing sigis a strong hereditary component that accounts for haling molecules activated by binding IRS-I,2 that is NIDDM [49, 50], have led to the conclusion that the likely to be critical for coupling the IR to glucose uptake primary or inherited lesion in NIDDM is impaired sen- and GLUT4 translocation [58-63]. Once activated, PI3sitivity of peripheral tissues to insulin. While secretion of kinase phosphorylates phosphatidylinositol (PI) PI-4,5insulin by the beta cell of the pancreas initially increases P2 to generate PI-3,4,5-P3 [64]. PI-3,4,5-P~ functions as to maintain euglycemia, over time the beta cell becomes an early second messenger linking the IR to GLUT4 insufficient to maintain euglycemia, and clinical NIDDM translocation and glucose transport, although, the mech(increased blood glucose levels) develops. The transition anism whereby PI3-kinase mediates this function is still from a compensated state of insulin resistance to clinical not known. The importance of IRS-I and PI3-kinase disease is characterized by a variety of additional changes in insulin-stimulated GLUT4 translocation and glucose which may either be inherited or arise as a result of the uptake is supported by the findings that IRS-1 knockout diabetic environment [2, 5, 48, 49, 5l]. These changes mice are insulin-resistant and that inhibition of PI3include; (1) a decrease in beta cell function resulting in a kinase, using pharmacological inhibitors or by expression reduced rate of insulin secretion; (2) increased glucose of dominant/negative PI3-kinase molecules, blocks insuproduction by the liver; and (3) an increase in peripheral lin-stimulated GLUT4 translocation and glucose uptake resistance to insulin in fat and muscle. Increased pro- in adipocytes [65 69]. duction of TNF by adipocytes may be one of the secThe genetic or primary component(s) accounting for
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E.Y. Skolnik and J. Marcusohn INSULIN
Skeletal Muscle Adipocytes
Figure 1. Insulin receptor signaling. Ligand binding to the IR leads to receptor autophosphorylation which functions to activate the tyrosine kinase catalytic domain and to create binding sites for the PTB domains oflRS-1,2 and SHC. Binding of IRS-1,2 and SHC to the IR juxtaposes these moleculesadjacent to tyrosine kinase, thereby enabling them to become tyrosine-phosphorylated. Tyrosine-phosphorylated IRS-1,2 and SHC then bind and activate SH2-containing signaling molecules. Binding of the SH2 domain of GRB2 to SHC results in the activation of the RAS-MAP kinase pathway, whereas binding of the SH2 domains of P85-associated PI3-kinase to IRS1,2 results in the activation of PI3-kinase. PI3-kinase is responsible for coupling the IR to GLUT4 translocation in insulin-responsivetissues such as skeletal muscle and adipocytes. insulin resistance in the "common type" of NIDDM are still not known. Studies have focused on molecular defects in two insulin-stimulated pathways that may be primarily affected in patients with a genetic predisposition to develop NIDDM. These pathways are both involved in glucose disposal. The first pathway includes the activation of GLUT4 translocation and glucose uptake by insulin [3]. The second pathway involves the activation of glycogen synthase by insulin. Abnormalities in the non-oxidative pathways of glucose disposal, i.e. glycogen synthesis, have been found in first degree prediabetic relatives of patients with NIDDM and insulin resistance, leading investigators to propose that defects in intracellular glucose metabolism, rather than GLUT4 translocation, are primarily responsible for decreased glucose uptake in NIDDM [49]. The early intracellular responses activated by the IR are central to the activation of both pathways. However, studies conducted so far have failed to identify mutations in any candidate genes in a high percentage of patients with NIDDM [5, 48, 49 52, 70]. In spite of our lack of knowledge about the primary defect(s) accounting for NIDDM, a variety of abnormalities in insulin signaling have been identified in patients once clinical disease is present. These abnormalities further impair signaling by the IR, thereby leading to a worsening of the insulin resistance and hyperglycemia if increased insulin secretion from the pancreas is unable to compensate for the increased insulin resistance. These abnormalities in IR signaling are thought to be secondary to either the diabetic state or to environmental factors because these changes improve, at least partly, after the institution of better glycemic control, weight reduction, and exercise [2, 5, 48]. Some of the changes that have been identified include: (1) a decreased number of IRs; (2) a decrease in IR tyrosine kinase activity resulting in decreased IR autophosphorylation and decreased tyro-
sine phosphorylation of IRS- 1; (3) a decrease in GLUT4 protein expression in adipocytes; and (4) a decrease in insulin-stimulated GLUT4 translocation and insulinstimulated non-oxidative glucose disposal. The nature of these secondary changes that contribute to insulin resistance has been the focus of several studies. One of the secondary changes that contributes to insulin resistance is hyperglycemia [48]. High glucose levels impair glucose transport by interfering at several steps in the insulin-signaling cascade and have led to the concept of the "glucose toxicity" model [3, 48]. Several other candidate molecules that may contribute to the increase in insulin resistance have also been described [5, 48, 70]. TNF is one of the best characterized of these molecules. TNF AND INSULIN RESISTANCE The first association of TNF, as well as other cytokines with insulin resistance, involved studies of patients with several disease states. It has long been known to physicians that infection and other stresses result in increased insulin resistance; infection triggers clinical diabetes in prediabetic patients and leads to increased insulin requirements in diabetic patients [71]. The finding that several of these pathological states are also associated with increased levels of circulating TNF, together with the finding that chronic administration of TNF to animals induces insulin resistance, supported the idea that TNF contributes to insulin resistance in these diseases [71, 73]. Increased production of TNF by inflammatory cells, such as macrophages, is responsible for high circulation levels of TNF in the diseases described above [21,27, 63]. Even before TNF was identified as a secretory product of adipocytes, experiments, performed in cultured cells and whole animals, demonstrated that TNF modulates adipocyte function and phenotype. TNF (cachegtin) was
Inhibition of IR Signalingby TNF originally isolated, based on its ability to suppress lipoprotein lipase in cultured adipocytes [21]. In addition, TNF was shown to stimulate lipolysis in fat cells, to prevent the differentiation of preadipocytes into adipocytes in cell culture and, when applied at high doses to adipocytes in vitro, to mediate the reversal of the adipocyte phenotype [21]. With this background, Spiegelman and co-workers provided the first insights into a possible role for TNF in mediating insulin resistance in rodent models of obesity and in obese patients with NIDDM. These investigators found that TNF mRNA was elevated five-to-ten-fold in adipose tissue isolated from several rodent models of obesity and insulin resistance compared with lean controls [25]. The increase in TNF mRNA was also associated with about a two-fold increase in TNF protein measured in explanted adipose tissue from obese adipocytes. Moreover, these investigators directly linked TNF to at least some of the insulin resistance observed in these obese rodent models. Neutralization of TNF following 3 days of treatment of zucker fatty rats (fa/fa) with a recombination soluble TNF receptor-Ig (TNFR-Ig) fusion protein led to a reduction in the insulin resistance in this animal model [25, 74]. More recent studies have extended some of the findings in obese rodent models of insulin resistance to obese nondiabetic humans. These studies have confirmed that TNF is expressed in human adipocytes and the expression of adipocyte TNF mRNA and protein, for the most part increases with increasing obesity [32, 75]. Moreover, adipocyte TNF mRNA was found to decrease with weight reduction in the subjects studied. Thus, these preliminary studies indicate that at least with regards to the expression of TNF by obese adipocytes, TNF is regulated similarly in human and rodent adipocytes.
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pression of GLUT4 and C/EBP-c< genes by TNF was rapid with maximal suppression of both genes occurring after only 4 h of TNF treatment [76]. The finding that expression of several adipocyte specific genes is downregulated by TNF in these studies raised the possibility that partial dedifferentiation of adipocytes is responsible for the decrease in GLUT4. In addition whiledownregulation of G L U T 4 b y T N F could certainly account for insulin resistance, decreased GLUT4 unlikely accounts for the insulin resistance observed in obesity and NIDDM. Decreased GLUT4 is found in adipose tissue, but not in skeletal muscle from obese patients with NIDDM [3]. The finding that skeletalmuscle accounts for > 80% of the insulin-stimulated glucose uptake indicates that TNF would also have to induce downregulation of GLUT4 in skeletal muscle to induce insulin resistance in vivo. In addition, more recent evidence has suggested that TNF mediates insulin resistance in adipocytes by mechanisms that are independent of GLUT4 downregulation [25, 45, 74, 77]. First, chronic treatment of the 3T3-F442A and 3T3-L1 adipocyte cell lines with doses of TNF that were much lower than those usedin previous studies inhibited insulin-stimulated glucose uptake, although, at this dose of TNF, GLUT4 protein was not decreased [45]. Second, neutralization of TNF in fa/fa rats using a soluble TNFR-Ig fusion protein led to improved insulin sensitivity, although GLUT4 levels were not normalized by this treatment [25, 74]. Thus, these findings indicate that TNF induces insulin resistance by a mechanism(s) that is independent of GLUT4 downregulation in vivo.
Inhibition of insulin-stimulated phosphorylation
IR tyrosine kinase activity and insulin-stimulated tyrosine phosphorylation of IRS-I are decreased in all three MECHANISM WHEREBY TNF INDUCES INSULIN RESISTANCE insulin-responsive tissues, including adipocytes, skeletal muscle and liver isolated from patients with NIDDM and Downregulation of GLUT4 from animal models of genetic and acquired forms of obesity and insulin resistance [5, 48]. In addition, the The cellular and molecular mechanisms linking TNF decrease in IRS-1 phosphorylation is associated with to insulin resistance are now beginning to be understood, decreased insulin-stimulated activation of PI3-kinase [5], Even before it was recognized that adipocytes secrete The findings that these changes frequently improve folTNF, several studies demonstrated that TNF down- lowing weight reduction and better glycemic controlindiregulates G L U T 4 i n 3T3-LI adipocytes and in L6myo- cate that they are likely secondary to the diabetic blasts in cell cultures [24, 76]. Pekala and co-workers environment [48, 78]. Thus, if TNF is an important showed that treatment of 3T3-L1 adipocytes with TNF mediator of insulin resistance in vivo, treatment of insulinfor 3 days reduced GLUT4 protein by 80% [24, 76]. sensitive tissues with TNF should either inhibit the IR Moreover, downregulation of GLUT4 correlated with tyrosine kinase and/or inhibit insulin-stimulated tyrosine TNF-induced insulin resistance in 3T3-L1 adipocytes, phosphorylation ofIRS-1. Studiesin both hepatoma and TNF inhibited GLUT4 by both destabilizing GLUT4 adipocyte cell lines, as well as in rodent models of insulin mRNA and by decreasing GLUT4 transcription [76]. resistance in vivo, indicate that TNF is capable of mediaTNF inhibition of GLUT4 transcription was mediated, ting both these effects. at least partially, by TNF-induced downregulation of Treatment of the Fao hepatoma cell line with TNF the transcription factor C/EBP-c~; C.EBP-c~ is thought to resulted in about a 60% decrease in insulin-stimulated control the expression of several adipocyte specific genes, phosphorylation of IRS-1, while treatment of 3T3-L1 including GLUT4. Interestingly, transcriptional sup- and 3T3-F442A adipocyte cell lines with TNF led to
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E.Y. Skolnik and J. Marcusohn Enhanced production of other molecules which are responsible for the increase in insulin resistance
decreased insulin-stimulated tyrosine phosphorylation of both the IR and IRS-1 [45, 77, 79, 80]. Interestingly, although hepatoma cells exhibited decreased insulinstimulated phosphorylation after only 15 min of TNF treatment, adipocytes required at least 3 days of treatment with TNF to mediate this effect. The impairment in insulin-stimulated phosphorylation by TNF in adipocytes also correlated with decreased insulin-stimulated glucose uptake. Moreover, low doses of TNF (25 pM vs 5 nM in the previous studies) were found to inhibit insulin signaling in the 3T3-F442A and 3T3-L1 adipocyte cell lines in these studies. At these low doses, TNF inhibited insulin signaling without causing downregulation of GLUT4. Thus, these findings suggest that, at physiologically relevant levels, TNF mediates insulin resistance in adipocytes primarily by blocking insulin-stimulated tyrosine phosphorylation.
TNF INHIBITS INSULIN-STIMULATED PHOSPHORYLATIONBY ENHANCING SERINE PHOSPHORYLATION OF IRS-1
To demonstrate that TNF production by adipocytes contributes to insulin resistance in vivo, these same investigators determined whether neutralization of TNF by a soluble TNFR-Ig fusion protein improved insulin sensitivity and insulin-stimulated phosphorylation in fa/fa rats [25, 74]. fa and its murine homologue db have now been shown to encode the ob (leptin) receptor [16, 17, 81]. Mutations in the ob receptor in fatty rats and db mice lead to defective ob receptor signaling, fa/fa rats are a good genetic model of obesity and insulin resistance, and marked increases in TNF mRNA can be detected in adipocytes from these animals by 7-8 weeks of life [25]. Treatment of fa/fa rats with soluble TNFR-IgG fusion protein for 3 days led to decreased insulin resistance as demonstrated by the improvement in plasma glucose, insulin and free fatty acid levels [25, 74]. The decrease in insulin resistance after treatment was associated with improved insulin-stimulated phosphorylation of the IR and IRS-1 in skeletal muscle and fat to levels that were similar to lean control animal. In contrast to the improvement in insulin-stimulated phosphorylation, treatment with TNFR-Ig did not affect the GLUT4 protein levels in either skeletal muscle or fat. Thus, two conclusions can be drawn from these experiments. First, TNF contributes to the insulin resistance in vivo in at least one generic rodent model of obesity and insulin resistance, Second, these findings suggest that inhibition of insulinstimulated tyrosine phosphorylation in both skeletal muscle and fat, rather than downregulation of GLUT4, is the primary mechanism whereby TNF mediates insulin resistance in vivo. Surprisingly, treatment of fa/fa rats with soluble TNFR-Ig did not improve insulin-stimulated phosphorylation in the liver [74]. These results could be interpreted to indicate that TNF does not mediate insulin resistance in the liver of these animals. However, in contrast to other studies [82], these investigators did not observe a significant decrease in insulin-stimulated phosphorylation in livers isolated from untreated fa/fa rats compared to control lean animals after normalizing for the number of IRs.
Treatment of Fao hepatoma cells and adipocytes with TNF results in serine phosphorylation of IRS-1 [77, 80]. From a variety of different studies, it is now clear that serine/threonine phosphorylation of IRS-1, and possibly the IR itself, negatively regulates insulin signaling, although the mechanism whereby these phosphorylations inhibit insulin signaling is still poorly defined (Figure 2). Previous studies have shown inhibition of IR signaling after treatment of cells with the serine/threonine phosphatase inhibitors okadaic acid and calyculin or by overexpressing some isoforms of the serine/threonine protein kinase, protein kinase C [84, 85]. Interestingly, okadaic acid inhibited insulin-signaling by stimulating the serine/ threonine phosphorylation of IRS-1 [85]. While treatment of cells with okadaic acid did not impair the function of the IR tyrosine kinase, okadaic acid markedly impaired the ability of the IR to tyrosine phosphorylate IRS-1 in vivo and in reconstitution experiments in vitro. These findings suggested that serine/threonine phosphorylation induced a conformational change in IRS- 1 that may have prevented IRS-1 from interacting with the IR. This suggestion is consistent with the finding that binding of IRS-1 to the IR is required for IRS-1 phosphorylation by the IR both in vivo and in vitro [52]. IRS1 and IRS-2 contain a domain that is distinct from SH2 domains that bind phosphotyrosine, termed phosphotyrosine binding (PTB), which is responsible for the association of IRS-1 and 2 as well as another signaling molecule SHC with the autophosphorylated IR [55, 8690] (Figure 1). Thus, serine/threonine phosphorylation of IRS-1,2 may inhibit tyrosine phosphorylation of IRS-1,2 by interfering with the binding of IRS-1,2's PTB domain to the activated IR. A recent study, designed to understand the mechanism whereby TNF inhibits insulin signaling, has also demonstrated that serine/threonine phosphorylation of IRS1 is central to the inhibitory effect of TNF on insulin signaling [77]. However, the findings in this report suggest that serine phosphorylated IRS-1, may not only uncouple an active IR from IRS-1, but may also directly inhibit the IR tyrosine kinase. In contrast to the finding in oka-
TNF may also mediate insulin resistance via indirect mechanisms. For example, TNF-stimulated lipolysis in the adipocyte may also contribute to insulin resistance in vivo by TNF. Several studies have demonstrated that TNF leads to the stimulation of lipase activity in adipocytes [23]. Increase in adipocyte lipolysis could then lead to increased plasma levels of fatty acids, which, by providing an alternative source of energy for skeletal muscle, would lead to decreased insulin-stimulated glucose uptake [83].
Inhibition of IR Signaling by TNF
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TNFR
ISer,ne,..reon,ne
IPhosphorylation I
Adipocytes
j
i
of IRS-1 i
I Inhibit IR kinase I
I? Inhibit tyrosine phosphorylation 1
Iof IRS1,2 by inhibiting binding of /
IIRS-1,2 PTB domain to activated IRJ
Figure 2. Inhibition of IR signaling by TNF. TNF inhibits insulin signaling by stimulating the serine phosphorylation of IRS-l. Serine phosphorylated IRS-1 inhibits signaling by the IR by inhibiting the tyrosine phosphorylation of IRS-I by the IR. In addition, TNF may also decrease the expression of GLUT4 in adipocytes. daic acid treated adipocytes, IR tyrosine kinase activity assayed in vitro is decreased in adipocytes treated with TNF [74, 77, 85]. Interestingly, the decrease in IR tyrosine kinase activity is mediated by IRS-1 that is serine phosphorylated as result of TNF treatment. This conclusion was based on the observation that TNF did not inhibit the IR tyrosine kinase in 32D cells which lack endogenous IRS-1,2 [77]. However, in contrast to the findings in the parental 32D cells, TNF inhibited the IR in these same cells following transfection with a plasmid encoding the IRS-1 cDNA. Moreover, IRS-1 that was serine phosphorylated inhibited the IR tyrosine kinase activity in vitro. The IR tyrosine kinase activity was inhibited in vitro by IRS-1 that was isolated either from adipocytes stimulated in cell culture with TNF or from IRS-1 isolated from skeletal muscle or adipocytes of fa/fa rats. The inhibition of the IR kinase depended upon serine phosphorylation of IRS-I because dephosphorylation of IRS-1 with calf intestinal alkaline phosphatase reversed the inhibitory effects of IRS- 1. Although it is still not clear whether serine phosphorylated IRS-I directly or indirectly inhibits the IR tyrosine kinase via the association with another molecule, these findings suggests a second possible mechanism whereby serine phosphorylated IRS-1 inhibits insulin signaling, While it is clear that TNF inhibits insulin-stimulated phosphorylation in a variety of different cells, not all studies have demonstrated an inhibitory effect of TNF on IR tyrosine kinase activity. Treatment of the Fao hepatoma cell line with TNF led to decreased tyrosine phosphorylation of IRS-1 without inhibiting the IR tyrosine kinase [80]. In addition, whereas serine phosphorylated IRS-1 isolated from TNF-treated adipocytes inhibited the IR tyrosine kinase, serine/threonine phosphorylated IRS-I isolated from okadaic acid treated adipocytes or from cells overexpressing protein kinase C
did not inhibit the IR tyrosine kinase & vitro [85, 91]. It is possible that the discrepancies observed are due to the fact that serine/threonine phosphorylation of IRS- 1 leads to different outcomes depending upon the sites phosphorylated on IRS-I. For example, TNF may activate distinct signaling pathways in liver and fat which result in the serine phosphorylation of different sites on IRS-I in each cell line. The finding that liver and fat must be treated with TNF for different lengths of time before TNF inhibits insulin signaling in each cell line is consistent with the idea that TNF activates distinct signaling pathways in these two ceils. In contrast to the findings by Hotamisligil et al. [74, 77], a recent study has suggested that downregulation of GLUT4 by TNF in L1-3T3 adipocytes, rather than impaired insulin-stimulated phosphorylation, is the primary mechanism whereby TNF inhibits glucose transport in this cell line [92]. These investigators found that treatment of LI-3T3 adipocytes with low doses of TNF led to a marked reduction in GLUT4 protein levels after 3 days of TNF treatment. However, the signaling pathway connecting the IR to GLUT4 translocation appeared intact in these cells since insulin was still capable of stimulating GLUT4 translocation in these cells. The decrease in glucose uptake was attributed to the decrease in total pool of GLUT4 available for translocation rather than changes in insulin-stimulated tyrosine phosphorylation. At present, the reason for the discrepancy in these results is not clear. SIGNALING PATHWAYS ACTIVATED BY
TNF
THAT MEDIATE SERINE PHOSPHORYLATION IRS1 AND INSULIN RESISTANCE The signaling pathway(s) activated by the TNF receptor that mediates insulin resistance are not yet known.
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However, as discussed above, the mechanism whereby TNF stimulates serinephosphorylation of lRS-1 is likely to be central in confering insulin resistance. TNF could increase serine phosphorylation of IRS-I by either activating a kinase and/or inhibiting a phosphatase. The finding that ceils treated with the phosphatase inhibitor okadaic acid exhibit both an increase in serine/threonine phosphorylated IRS-1 and insulin resistance indicates that an active kinase that is able to phosphorylate IRS-1 is present in unstimulated cells and that this kinase must be kept in check by a phosphatase [85]. However, as of now, it is still not known whether TNF stimulates IRS-1 phosphorylation by regulating a kinase or a phosphatase. Moreover, the different length of time in which adipocytes and hepatocytes must be treated with TNF before they manifest insulin resistance, suggests that TNF mediates insulin resistance by distinct mechanisms in different cell lines [45, 80]. The finding that insulin resistance in Fao hepatoma cells occurs after only 15 min of TNF treatment indicates that TNF mediates insulin resistance in this cell via post-translational modification of an existing protein. However, the extended length of time that adipocytes must be treated before they manifest insulin resistance suggests that TNF mediates insulin resistance in adipocytes by regulating the transcription of a gene that either directly or indirectly stimulates IRS1 phosphorylation, Unlike the IR, which contains intrinsic tyrosine kinase activity, neither the p60 not the p80 TNF receptors conrains intrinsic catalytic activity, and thus it has been difficult to determine the mechanism whereby these receptors signal cells. As of now, the TNF receptors have been shown to utilize two distinct mechanisms to couple to proximal cytoplasmic signaling molecules (Figure 3). First, the p60 TNF receptor has been shown to activate at least two lipid second messengers. TNF activates sphingomylinase, leading to the generation of the lipid second messenger ceramide and also a phosphatidylcholine-specific phospholipase C (PC-PLC)leading to the generation of diacylglycerol (DAG) [93-96]. Second, both TNF receptors have now been shown to associate with a variety of signaling molecules in cells, [97-99]. Oligomerization of the receptors is thought to activate these molecules, although the mechanism whereby these molecules are activated is still poorly defined [100]. Ceramide is a.component of all sphingolipids and over the past several years it has become evident that the p60 TNF receptor activates both an acidic and neutral sphingomylinase, which hydrolyses sphingolipid to generate the lipid second messenger ceramide [93, 94 96]. Using cell permeable analogs of ceramide, ceramide has been linked to the regulation of a variety of biological responses. Some of these responses include the stimulation of apoptosis, monocyte differentiation and activation of NF-xB [93, 94]. Ceramide may also mediate some of the insulin resistance induced by the p60 TNF receptor; treatment of hepatoma cells and 3T3-L1 adipocytes with sphingomylinase or cell-permeable analogs of
ceramide induces insulin resistance in these cells [109, 110]. Ceramide has been shown to activate a serine/ threonine protein kinase termed ceramide activated protein kinase (CAP) [94]. Although CAP kinase is not yet cloned, in vitro reconstitution experiments have suggested that this kinase couples TNF to the RAS-RAFMAP kinase signaling pathway in HL60 cells [101]. However, while MAP kinase (also known as ERK) can phosphorylate IRS-1 in vitro activation of MAP kinase is unlikely to be responsible for the insulin resistance mediated by TNF [52]; other receptors that activate MAP kinase, such as PDGF, do not induce insulin resistance. Nevertheless, the finding that CAP kinase activates a well known kinase cascade indicates that CAP kinase is a candidate kinase that may directly or indirectly stimulate serine/threonine phosphorylation of IRS-1. Two additional pieces of evidence suggest that ceramide may mediate insulin resistance caused by TNF. First, several other cytokines have been shown to mediate insulin resistance in adipocytes in culture. Interestingly, these cytokines, which include interleukin (IL)-I and interferon 7, have been shown to stimulate the production ofceramide in cells [45, 93]. Second, ceramide regulates the transcription of a variety of proteins in cells [94, 96]. These findings raise the possibility that ceramide activates a signaling pathway, which regulates the transcription of a still yet to be defined gene that is responsible for the increase in IRS-1 phosphorylation and insulin resistance in insulin-responsive tissues, such as adipocytes. Treatment of insulin-responsive tissues with cell permeable analogs of ceramide should shed light on the possible role of ceramide in mediating insulin resistance by TNF. TNF also activates PC-PLC which generates DAG by stimulating hydrolysisofphosphatidylcholine[95]. DAG is likely an important second messenger responsible for mediating cellular responses by TNF. DAG is a wellestablished activator of the serine/threonine kinase protein kinase C (PKC). In addition, DAG activates an acidic sphingomyelinase, which has been implicated in mediating the activation of NF-xB by TNF [95, 96]. PKC has received a great deal of interest as a potential negative regulator of insulin-stimulated phosphorylation. Activation of endogenous PKC, using phorbol esters or overexpression of some PKC isoforms inhibits insulinstimulated phosphorylation in cells [84, 91, 102]. Some studies, although not all, have shown that activation of protein kinase C leads to serine/threonine phosphorylation of the IR resulting in decreased IR kinase activity [52, 91, 102]. In addition, overexpression of PKC~ has been shown to stimulate serine/threonine phosphorylation of IRS-1 in CHO cells [91]. However, IRS-1 isolated from CHO cells overexpressing PKC~ is phosphorylated normally by the IR in vitro [91]. This is in contrast to the decreased in vitro phosphorylation of serine/threonine phosphorylated IRS-1 isolated from okadaic acid-treated or TNF-treated adipocytes [77, 85]. Moreover, staurosporin, a non-specific inhibitor of protein kinase C, did not abrogate the ability of TNF to inhibit insulin-stimulated phosphorylation in Fao hepa-
Inhibition of IR Signaling by TNF
p 8 0 TNFRI I [ I
~
~
TNFR I I I I neutral [ ceramld-eI ~SMase (~).
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169
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NF-•B activation ? Other effects otherTNFR associatedproteins
Figure3. TNF receptor signaling. See text for details. Ligand binding of the TNF receptor leads to receptor oligomerization and activation of several intracellular signaling pathways. The p60 TNF receptor activates sphingomylinase, leading to the generation of the lipid second messenger ceramide and also a phosphatidylcholine-specific phospholipase C (PC-PLC) leading to the generation of diacylglycerol (DAG). In addition, both TNF receptors have now been shown to associate with a variety of signaling molecules in cells. The signaling pathway, activated by TNF that mediates insulin resistance, is still not known.
tocytes [80]. Thus, these findings suggest that PKC is unlikely to mediate insulin resistance caused by TNF. A variety of other serine/threonine kinases are activated by the p60 T N F receptor. These kinases include the stress activated protein kinase (SAP, also known as N-terminal Jun kinase), p38, protein kinase A and /% casein kinase [103-105]. SAP kinase and p38 are members of the MAP kinase family and are strongly activated in cells treated with TNF and ceramide [111]. However, SAP kinase is also unlikely to mediate the inhibition of insulin-stimulated phosphorylation in TNF-treatedcells; overexpression of activated p21 Rac in 3T3-L1 adipocytes results in constitutive activation of SAP kinase, yet insulin-stimulated phosphorylation and glucose uptake are normal [106]. In addition to generating the lipid second messenger described above, both the p60 and p80 TNF receptors have been shown to associate with signaling molecules that can mediate several TNF-stimulated biological responses. The cytoplasmic domain of the p60 T N F receptor contains a domain of about 100 amino acids that has been termed the death domain [38]. This domain is responsible for both receptor oligomerization and the association of the p60 T N F receptor with a variety of signaling molecules which are capable of stimulating apoptosis when overexpressed in cells [100]. Similarly, several proteins that associate with cytoplasmic domain of the p80 receptor have been identified [46]. One of these
proteins, TRAF2, activates NFkB when overexpressed in cells [98]. These findings raise the intriguing possibility that one of the proximal signaling molecules responsible for stimulating insulin resistance by T N F will be found to directly associate with the TNF receptor. Structure/function studies, in which mutant TNF receptors are expressed in insulin-responsive tissues, should allow the identification of the cytoplasmic portion of the TNF receptor responsible for mediating insulin resistance. These studies should be a valuable first step to help uncover signaling molecules that are responsible for stimulating insulin resistance by TNF. HOW IS THE TNF mRNA REGULATED IN ADIPOCYTES? It is still not known what signals the "overfilled" adipocyte to increase production of TNF mRNA. The finding that TNF m R N A is increased in adipocytes isolated from a variety of different genetic models of obesity suggests that the signal to increase T N F m R N A may be intrinsic to the adipocyte [25]. Specifically, the adipocyte may directly regulate its own production of TNF m R N A based upon the amount of stored triglycerides. Alternatively, obesity itself may lead to the secretion of a common factor(s), which in turn stimulates the adipocyte to produce TNF mRNA. Leptin, however, cannot be the secreted factor responsible for increasing adipocyte TNF
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mRNA. This is based on the fact that adipocyte T N F m R N A is increased in homozygous ob mice which lack leptin and that exogenous administration of leptin to these animals corrects the insulin resistance [14].
FUTURE QUESTIONS The above findings raise the intriguing possibility that T N F comprises one of the central molecules responsible for mediating insulin resistance in obesity and N I D D M . However, many questions remain to be answered before the role of T N F as an endogenous mediator of insulin resistance in these diseases becomes clear. One critical question that remains unanswered is whether the findings in genetic models of rodent obesity and insulin resistance can be extrapolated to N I D D M in humans. At least with respect to T N F m R N A by adipocytes, both humans and rodents are similar; adipocytes isolated from obese patients express increased T N F m R N A and protein [32, 75]. However, other findings in humans are not entirely consistent with experimental results obtained either from adipocytes treated in cell culture with T N F or from rodent models of obesity and insulin resistance. Defects in IR tyrosine kinase activity, for the most part, have not been observed in skeletal muscle or fat isolated from obese insulin resistant patients prior to the onset ofclinical diabetes, despite the fact that T N F m R N A is increased in these patients [48, 107]. Moreover, a recent report has demonstrated a decrease in protein levels of the IR, IRS-1 and P85 associated with PI3-kinase in skeletal muscle isolated from obese insulin-resistant subjects [108]. These findings led these investigators to conclude that decreased levels of these proteins, rather than impairment in insulin-stimulated phosphorylation of IRS-1, is primarily responsible for insulin resistance in these patients. As of yet, T N F has not been shown to mediate a decrease in these protein levels in skeletal muscle. These findings, together with the demonstration that insulin resistance must be accompanied with pancreatic beta cell failure for clinical diabetes to develop, suggests that TNF-independent events are likely critical for N I D D M to occur [51]. In addition, very little is known about the mechanism whereby T N F m R N A expression is regulated in adipocytes or the mechanism whereby T N F mediates insulin resistance either in cell culture or in obese rodent models. For example, neither the signaling pathways activated by T N F that are responsible for serine phosphorylating IRS1 nor the mechanism whereby serine-phosphorylated IRS-1 inhibits the IR tyrosine kinase are known. The mechanism whereby T N F produced by adipocytes blocks glucose transport in skeletal muscle is also not known. The fact that increased circulating plasma levels of T N F are observed in only a small percentage of obese fatty rats and not at all in obese humans suggests that T N F must be produced locally within skeletal muscles to mediate its effects. Production of T N F by skeletal muscle has not been easy to demonstrate. Therefore, it has been
proposed that adipocytes found in skeletal muscle of obese subjects are responsible for the increased production of T N F in muscle. However, as of yet, increased expression of T N F by adipocytes infiltrating skeletal muscle has not been shown. It remains a possibility that T N F produced by adipocytes indirectly stimulates insulin resistance in skeletal muscle by stimulating the production of a circulating factor, which is then responsible for inhibiting insulin-stimulated phosphorylation in muscle. Future studies will no doubt more directly test TNF's role in both mediating insulin resistance and in functioning as a local "adipostat" to limit fat accumulation. These studies will likely include the crossing of insulin resistance rodent models of obesity with mice containing targeted disruption of the p60 and p80 T N F receptors or of TNF. The role of T N F in mediating insulin resistance in humans with N I D D M will likely be more difficult to resolve. Nevertheless, these findings raise the intriguing possibility that treatment of N I D D M may, one day, include therapies designed to block T N F production by adipocytes and/or TNF's effects on insulinsensitive target cells. REFERENCES 1. Flier JS. Obesity. In Kahn CRandWeirGC, eds. Diabetes Mellitus. Lea and Febiger, Malverne, PA, 1994, 351-362. 2. DeFronzo RA, Bonadonna RC, Ferrannini E. Pathogenesis of NIDDM. A balanced overview. Diabetes Care 1992, 15, 318-368. 3. Garvey WT. Glucose transport and NIDDM. Diabetes Care 1992, 15, 396-417. 4. Haring HU, Mehnert H. Pathogenesis of Type 2 diabetes mellitus: candidates for a signal transmitter defect causing insulin resistance of the skeletal muscle. Diabetologia 1993, 36, 176-182. 5. Kahn CR. Insulin action, diabetogenes, and the cause of type II diabetes. Diabetes 1994, 43, 1066-1084. 6. Birnbaum MJ. The insulin-sensitive glucose transporter. Int Rev Cytol 1992, 13, 239-97. 7. James DE, Piper RC. Targeting of mammalian glucose transporters. J Cell Sci 1993, 104, 607-612. 8. Flier JS. The adipocyte: Storage depot or node on the energy information superhighway? Cell 1995, 80, 15-18. 9. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994, 372, 425-431. 10. Maffei M. Increased expression in adipocytes of ob RNA in mice with lesions of the hypothalmus and with mutations in the db locus. Proc Natl Acad Sci USA 1995, 92, 6957-6960. 11. Hallaas JL, Gajiwala KS, Maffei Met al. Weight reducing effects of the plasma protein encoded by the obese gene. Science 1995, 269, 543-546. 12. Frederich RC, Lollmann B, Hamann Aet al. Expression of ob mRNA and its encoded protein in rodents. J Clin Invest 1995, 96, 1658 1663. 13. McGarry JD. Does leptin lighten the problem of obesity? Curr Biol 1995, 5, 1342 1344. 14. Pellymounter MA, Cullen MJ, Baker MB et al. Effects of the obese gene product on body regulation in ob/ob mice. Science 1995, 269, 540-543. 15. Campfield LA, Smith F J, Guisez Y, Devos R, Burn P. Recombinant mouse ob protein: evidence for peripheral signal linking adiposity and central neural networks. Science 1995, 269, 546~59.
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