Perspectives in Diabetes Diabetes and Suppressors of Signaling Sif G. Rønn,1 Nils Billestrup,1 and Thomas Mandrup-Poulsen1,2

he pathogenesis of type 1 diabetes is not clearly understood, but it is generally accepted that type 1 diabetes is an immune-mediated disease Tcaused by inflammation in the islets of Langer- hans. Infiltrating macrophages release proinflammatory such as interleukin (IL)-1␤ and tumor necrosis factor (TNF)-␣, which are toxic to the ␤-cell. Activated T-cells also produce proinflammatory cytokines such as TNF-␣ and interferon (IFN)-␥ and express the apoptosis- inducing FasL. Moreover, CD8ϩ T-cells induce cell death via the perforin-granzyme pathway. The net effect of these different factors results in specific destruction of the -producing ␤-cells (1). Type 2 diabetes occurs when ␤-cell secretory capacity fails to compensate for insulin resistance. In type 2 diabetes, cytokines are known to be involved in insulin and leptin resistance (2,3), and cyto- kines have also been suggested to contribute to ␤-cell FIG. 1. Schematic structure of the SOCS proteins. The SOCS proteins failure of type 2 diabetes (4). are characterized by a conserved COOH-terminal domain, the SOCS box. Centrally they contain an SH2 domain, while the NH2-terminal In this review we focus on a group of proteins, the (N-TERM) region is of variable length and amino acid composition. The suppressors of cytokine signaling (SOCS), which affect kinase inhibitory region (KIR) is only found in SOCS-1 and SOCS-3. cytokine signaling and appear to play an important role in the pathological processes leading to both type 1 and type 2 diabetes. of 12 amino acids is found immediately NH2-terminal to the SH2 domain in SOCS-1 and SOCS-3 (9,10). In general, the constitutive level of SOCS protein ex- SOCS PROTEINS pression in cells is low, but SOCS protein expression is The SOCS proteins were identified in 1997 and were highly inducible, often in a transient manner, upon stimu- characterized as a family of proteins capable of inhibiting lation with cytokines both in vitro and in vivo (Fig. 2A) (JAK)–signal transducers and activators of (11). IL-1␤, IFN-␥, and TNF-␣ can induce SOCS expression transcription (STAT) (JAK-STAT) signaling in various tis- in the ␤-cell (12,13). Cytokine-induced SOCS expression is sues (5–7). Eight members of the SOCS family have been regulated via activation of STAT proteins. STAT-binding identified, SOCS-1–7 and cytokine-inducible SH2-contain- elements have been identified in the promoters of CIS (14), ing protein (CIS) (8). They all contain a conserved COOH- SOCS-1 (15), and SOCS-3 (16). Mutations of these ele- ϳ terminal region of 40 amino acids termed the SOCS box ments reduce SOCS expression, and expression of domi- (Fig. 1) (5). They have a central SH2 domain, while the nant-negative variants of the STAT proteins blocks NH2-terminal region is of variable length with no recogniz- cytokine-induced SOCS expression (7,16,17). able motif (8). A kinase inhibitory region (KIR) consisting SOCS-mediated downregulation of cytokine-induced JAK-STAT signaling involves different mechanisms (Fig. 2B). Via its SH2 domain, SOCS-1 binds directly to the JAK From the 1Steno Diabetes Center, Gentofte, Denmark; and the 2Department of and inhibits kinase activity (10). SOCS-3 also inhibits JAK Molecular Medicine, Karolinska Institute, Stockholm, Sweden. activity, but in contrast to SOCS-1, this requires binding Address correspondence and reprint requests to Nils Billestrup, Steno between the SH2 domain of SOCS-3 and the phosphory- Diabetes Center, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark. E-mail: [email protected]. lated receptor (9). CIS inhibits cytokine signaling by Received for publication 1 August 2006 and accepted in revised form 16 binding to phosphorylated tyrosine residues on the cyto- November 2006. kine receptor, thereby masking potential docking sites for CIS, cytokine-inducible SH2-containing protein; IL, interleukin; IFN, inter- feron; IRS, insulin receptor substrate; JAK, Janus kinase; MAP, mitogen- downstream signaling molecules such as the STAT pro- activated protein; NF-␬B, nuclear factor-␬B; SOCS, suppressors of cytokine teins (18). Finally, SOCS proteins can inhibit signaling by signaling; STAT, signal transducers and activators of transcription; TAK-1 coupling of signaling proteins to degradation via the kinase, transforming growth factor-␤–activated kinase; TNF, tumor necrosis proteasomal machinery (19). factor. DOI: 10.2337/db06-1068 In the context of diabetes, SOCS-1 and -3 are the most © 2007 by the American Diabetes Association. relevant SOCS members, and their effects are discussed The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance below. SOCS-2 influences growth hormone effects, and with 18 U.S.C. Section 1734 solely to indicate this fact. gigantism is seen in mice lacking SOCS-2 (20). Overexpres-

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FIG. 2. The SOCS proteins inhibit cytokine signaling. A: Cytokine-induced activation of the JAK-STAT pathway results in expression of the various SOCS proteins. B: Following, the SOCS proteins downregulate cytokine signaling in different ways. SOCS-1 binds to and inhibits JAK-activity. SOCS-3 binds the phosphorylated (P) and inhibits JAK activity. CIS binds the receptor, thereby masking STAT docking sites. Finally, via their SOCS box, all SOCS proteins may target their bound signaling molecules for ubiquitination (Ub) and degradation via the proteasomal complex. sion and knock-out studies have revealed sparse informa- A central component in the IL-1␤–induced signaling tion on CIS and SOCS-4–7; however, it seems as though pathway is the transforming growth factor-␤–activated both SOCS-6 and -7 are involved in suppression of insulin kinase (TAK-1), as this enzyme is required for activation of signaling (21–23). the MAP kinase and NF-␬B signaling pathways induced by IL-1␤ (Fig. 3). In addition to NF-␬B, SOCS-3 also inhibits SOCS PROTEINS AND INFLAMMATION IL-1␤–induced activation of the MAP kinases JNK and p38 Since the SOCS proteins were discovered based on their (27). SOCS-3 inhibits IL-1␤ signaling at the level of the ability to suppress JAK-STAT signaling, we and others TAK-1 kinase by interfering with the interaction between investigated whether these proteins were able to suppress TAK-1 and TRAF-6, an interaction essential for TAK-1 signaling induced by cytokines in ␤-cells, as this might activation (Fig. 3) (27). prove to be a new target of ␤-cell protection. IFN-␥ Taken together, expression of SOCS-3 in ␤-cells leads to signaling through activation of the JAK-STAT pathway has a prevention of ␤-cell destruction upon cytokine exposure previously been shown to be one of the classical targets of due to reduced NF-␬B and MAP kinase activation and a SOCS-mediated cytokine inhibition, and indeed, we found reduction in NO production (and probably caspase activa- that SOCS-3 could inhibit IFN-␥ signaling in the ␤-cell line tion), thereby preventing cell necrosis and/or apoptosis. INS-1 (24). Similarly, SOCS-1 has been shown to suppress Array analysis suggested that SOCS-3 inhibits IL-1␤–in- IFN-␥ signaling in the ␤-cell (25,26). In addition to IFN-␥ duced activation of involved in the immune/inflam- signaling, SOCS-3 also suppresses IL-1␤ signaling in the matory response such as intracellular adhesion molecule ␤-cell, an unexpected finding, as this cytokine induces and proteasome and complement components and chemo- activation of mainly nuclear factor-␬B (NF-␬B) and mito- kines (28) along with genes involved in apoptosis, for gen-activated protein (MAP) kinases, demonstrating a example the oncogene c-myc, which has been shown to novel mode of action of the SOCS proteins (27). cause apoptosis of ␤-cells in RIP-II/c-myc transgenic mice NF-␬B is generally involved in cell-survival pathways, (28,29). and indeed the exposure of ␤-cells to IL-1␤ activates both Because SOCS proteins can protect ␤-cells in vitro, it is cell protective and deleterious mechanisms. However, in obvious to hypothesize a protective effect of SOCS expres- the ␤-cell the protective response is probably not sufficient sion against diabetes development in vivo, and SOCS-1 has to overcome the destructive mechanisms, and eventually been exploited in this particular context. The T-cell recep- apoptosis is induced (1). Numerous IL-1␤–induced NF-␬B– tor transgenic NOD8.3 mouse is a simple diabetes model in dependent genes, both proapoptotic such as inducible which CD8ϩ T-cell mediated ␤-cell death can be studied nitric oxide synthase (iNOS) and antiapoptotic such as (30). When SOCS-1 is overexpressed in ␤-cells in these manganese superoxide dismutase (MnSOD), are down- mice (RIP-SOCS-1/NOD8.3), diabetes development is com- regulated upon SOCS-3 overexpression in the ␤-cell (28), pletely prevented. Inflammation of the islets is not affected suggesting that SOCS-3 influences NF-␬B activity. SOCS-3 in this model, indicating that SOCS-1 inhibits the effector inhibits IL-1␤–induced NF-␬B DNA binding as well as pathways activated by the 8.3 T-cells in ␤-cells, e.g., IL-1␤–induced activation of NF-␬B–dependent tran- TNF-␣– and IFN-␥–induced Fas expression (31). NOD scription. In addition, SOCS-3 prevents IL-1␤–induced I␬B mice with ␤-cell–specific overexpression of SOCS-1 have a degradation (28). Despite the inhibitory effect on both pro- reduced incidence of diabetes (26,31), correlating with a and antiapoptotic pathways, SOCS expression protects the decreased IFN-␥–induced STAT-1 activation in the SOCS- ␤-cells against the toxic effect of IL-1␤. 1–expressing cells (26). These data support that cytokines

542 DIABETES, VOL. 56, FEBRUARY 2007 S.G. RØNN, N. BILLESTRUP, AND T. MANDRUP-POULSEN

FIG. 3. SOCS-3 inhibits IL-1␤–induced TAK activation. IL-1␤ binding to the IL-1RI results in recruitment of the IL-1 accessory protein (IL-1AcP). Following, a protein complex containing MyD-88, IRAK (IL-1 receptor–associated kinase)-4, Tollip, and IRAK-1 is assembled in the intracellular part. IRAK-4 phosphorylates IRAK-1, which enables binding between IRAK-1 and TRAF (TNF receptor–associated factor)-6. A complex consisting of TRAF-6, TAB (TAK-1 binding protein)1, TAB2, TAK-1, and the ubiquitin ligases Ube13 and Uev1A is formed. TRAF-6 is now activated by ubiquitination, and the kinase activity of TAK-1 initiated. TAK-1 mediates activation of the NF-␬B and MAP kinase (ERK, p38, and JNK) pathways, eventually resulting in gene transcription. SOCS-3 inhibits IL-1␤–induced TAK-1 activity by preventing binding between TAK-1 and TRAF-6, thereby inhibiting the IL-1␤ signaling cascade. are involved in the pathogenic process causing type 1 protective genes, such as the SOCS genes, and the balance diabetes. However, type 1 diabetes was not completely between pro- and antiapoptotic pathways subsequently prevented in the model, illustrating that SOCS-1 overex- determines the fate of the ␤-cell. This balance might in fact pression alone is not sufficient to avert diabetes. This represent one of the explanations why the ␤-cell is more might be explained either because the expression level of vulnerable to inflammatory cytokines than its neighboring SOCS-1 was too low to completely abolish the toxic cells. It could be speculated that the induction of SOCS cytokine effects or because mechanisms not influenced by expression is delayed in the ␤-cell when compared with SOCS expression are also involved in the pathogenesis of other cell types, and the level of cytokine-induced SOCS diabetes. As mentioned, SOCS-1 was shown to inhibit expression in the ␤-cell may be insufficient to overcome IFN-␥ signaling in the transgenic ␤-cells, whereas an the destructive effect of the cytokines. inhibition of other inflammatory cytokines such as IL-1␤ The protective effect of SOCS proteins in ␤-cells could and TNF-␣ was not investigated. Redundant effects caused be exploited, as overexpressing SOCS proteins in pancre- by these cytokines and noncytokine–mediated ␤-cell kill- atic islets or ␤-cells could be used for transplantation of ings, such as the T-cell–mediated perforin pathway, prob- diabetic patients, thereby preventing either recurrence of ably explain why diabetes was not fully prevented. We disease or allograft rejection. In ␤-cells, apart from inhib- have shown that SOCS-3 can inhibit both IL-1␤ and IFN- iting the toxic effects induced by inflammatory cytokines, ␥–mediated ␤-cell death (24). Moreover, array data has SOCS-3 has been shown to suppress growth hormone and shown that SOCS-3 can suppress IL-1␤–induced chemo- insulin signaling (32,33). Growth hormone is known to kine expression in ␤-cells (28), suggesting SOCS-3 as induce ␤-cell proliferation as well as induce insulin gene another ␤-cell protector, since SOCS-3 can inhibit the expression (34,35), and it is a concern that SOCS expres- signaling pathways of several proinflammatory cytokines sion abrogates these effects. However, GLP-1 (glucagon- and perhaps prevent the recruitment of inflammatory like peptide-1) or serum-induced ␤-cell proliferation is not cells, an effect that was not observed in the SOCS-1 affected by SOCS-3 (32). Furthermore, the replication rate transgenic islets. Generation of a NOD-RIP-SOCS-3 mouse of adult ␤-cells is very low (36,37), and the mitogenic effect will help clarify the ability of SOCS-3 as an effective of growth hormone on ␤-cells is more pronounced in protector of the ␤-cell. neonatal islets than in adult islets (38). As transplantation As mentioned, expression of the SOCS proteins is of ␤-cells would normally be performed using adult islets, induced by cytokines, and the physiological role of the the beneficial effects of SOCS-3 against cytotoxic cyto- SOCS proteins is most likely to prevent uncontrolled kines would most likely prevail. In RIP-SOCS-3 tg mice, it cytokine signaling in the cell by negative feedback. In rat was observed that ␤-cell–specific overexpression of islets and NIT-1 insulinoma cells, IFN-␥ induces expres- SOCS-3 had a negative effect on the ␤-cell mass in female sion of CIS, SOCS-1, and SOCS-2 mRNA and protein (12). mice, but apparently the reduced ␤-cell mass did not SOCS-3 mRNA is induced by IL-1␤ and IFN-␥ in primary influence their glucose metabolism, which may be ex- rat islets (unpublished observation), while a combination plained by an increased insulin secreting capacity of the of IL-1␤, IFN-␥, and TNF-␣ induces SOCS-1, -2, and -3 ␤-cells (39). These data further suggest that the potential expression in human islets (13). Thus, in addition to inhibitory effect of SOCS-3 on insulin signaling in ␤-cells induced transcription of proapoptotic genes, the inflam- does not intervene with normal ␤-cell function. SOCS-1– matory cytokines also induce expression of cytokine- expressing islets delay allograft rejection (40), indicating

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FIG. 4. SOCS-1 and -3 inhibit insulin signaling. A: Insulin binding to the insulin receptor initiates autophosphorylation (P) of the receptor and phosphorylation of IRS proteins. Subsequently, intracellular proteins, such as the PI3K, are activated leading to the cellular response. B: Cytokine-induced SOCS-1 and -3 interact with the phosphorylated insulin receptor, thereby preventing binding and activation of the IRS proteins. Moreover, the SOCS proteins target IRS proteins for ubiquitination (Ub) and degradation via the proteasomal complex. Insulin signaling is thereby inhibited and insulin resistance induced. that the SOCS proteins may have a clinical potential in activity of this protein due to conformational changes, transplantation strategies. making it unable to interact with the insulin receptor (45). If endogenous SOCS protein expression in ␤-cells is As mentioned, since the discovery of the SOCS proteins, insufficient to prevent destruction by cytotoxic cytokines it has been clear that their expression is induced by or if overexpression of SOCS proteins in general can cytokines, after which the classical target of the SOCS protect ␤-cells, therapeutic approaches manipulating proteins is to inhibit the JAK-STAT signaling pathway SOCS protein expression may be of great value to diabetic (5–7). However, several reports have established that the patients and perhaps to people suffering from other inflam- SOCS proteins can also inhibit other kinds of signaling matory diseases. However, it should be stated that if in the pathways, among these being insulin signaling and insulin- future the SOCS proteins are going to be used in the clinic, like growth factor signaling (46). Based on these findings a major challenge will be to obtain an SOCS effect in and on the fact that SOCS-1 knock-out mice have a low specific target cells or tissues, such as the ␤-cells. This will blood glucose level and increased insulin signaling (47), be absolutely necessary in order to avoid side effects due the SOCS proteins have been suggested to represent the to disturbances of beneficial cytokine effects throughout link between elevated levels of cytokines and insulin the body, the most important being their role in the resistance. inflammatory response against pathogens. NOD mice with Phosphorylation of the main signaling components ac- ␤-cell–specific SOCS-1 expression have a reduced inci- tivated by IRS-1 and-2isinhibited by SOCS expression dence of diabetes but show an enhanced susceptibility to both in vitro and in vivo. SOCS-1 and -3 interact with the virus-induced diabetes because of a decreased IFN-␥ re- insulin receptor, but a direct inhibition of autophosphor- sponse (26,41), illustrating the important balance between ylation of the insulin receptor has not been reported. On beneficial and nonbeneficial cytokine signaling. Moreover, the other hand, SOCS-3 binds phosphorylated Tyr960 on it is important to avoid conditions in which massive SOCS the insulin receptor, which is important for IRS-1 binding, expression may induce other pathologic conditions (ex- proposing one mechanism by which the SOCS proteins amples discussed below). inhibit insulin signaling (Fig. 4) (48,49). Phosphorylated Tyr960 serves as docking site for signaling molecules SOCS AND INSULIN RESISTANCE activated by insulin, among these STAT-5B, and insulin- The insulin receptor belongs to the recep- induced STAT-5B activation is inhibited by SOCS-3 (48). tor family and has intrinsic kinase activity, leading to SOCS-3 inhibits IRS-1 phosphorylation induced by insulin autophosphorylation as well as phosphorylation of intra- in COS-7 cells and also inhibits binding between IRS-1 and cellular substrates such as insulin receptor substrate the down-stream phosphatidylinositol-3 kinase (50). Inhi- (IRS)-1 and -2 (Fig. 4). Subsequently, these phophorylated bition by SOCS-1 is probably mediated through binding to proteins recruit other signaling proteins, leading to activa- the kinase domain of the insulin receptor, preventing tion of different signaling cascades (42). further phosphorylation (49). Finally, targeting of IRS-1 Insulin resistance is associated with an increased level and -2 for ubiquitin-dependent degradation via the protea- of circulation cytokines (e.g., IL-6, growth hormone, somal machinery has been suggested as another mecha- TNF-␣, IFN-␥, IL-1␤, and leptin) (42,43). A reduced level of nism explaining SOCS-1– and SOCS-3–mediated inhibition IRS tyrosine phosphorylation has been observed both in of insulin signaling (51). Thus, when mutations were animal models of type 2 diabetes and in type 2 diabetic introduced in the conserved SOCS box of SOCS-1, its patients (44). Also, TNF-␣ has been found to increase interaction with the elongin BC ubiquitin-ligase complex serine phosphorylation of IRS-1, which decreases the was abrogated and ubiquitination and degradation of IRS-1

544 DIABETES, VOL. 56, FEBRUARY 2007 S.G. RØNN, N. BILLESTRUP, AND T. MANDRUP-POULSEN and -2 prevented (51). Hepatic levels of IRS-1 and -2 are conflicting results. In one study, a SOCS-3 promoter poly- reduced in mice with hepatic overexpression of SOCS-1; morphism was associated with increased whole-body in- moreover, these animals have an impaired glucose toler- sulin sensitivity (55), thereby supporting the theory of ance. In contrast, when an SOCS-1 protein with a deletion SOCS-3 as a mediator of insulin resistance, whereas a in the SOCS box region was expressed, IRS levels were correlation between variants in the SOCS-3 gene and unaffected and the animals had a normal glucose response insulin resistance could not be detected in a U.K. popula- (51), further illustrating the importance of SOCS-1 in tion of female twins (56). insulin resistance. Elevated levels of SOCS-3 expression were found in the adipose tissue of ob/ob and db/db fat mice when compared SOCS AND LEPTIN RESISTANCE with lean control animals, supporting the hypothesis of Leptin is an adipocyte-derived cytokine controlling food SOCS-3 as a mediator of insulin resistance (50). Moreover, intake, energy balance, and neuroendocrine function via SOCS-1 and -3 liver mRNA levels are increased in different actions in the hypothalamus. Leptin has significant effects insulin-resistant mouse models such as db/db mice, ob/ob on insulin sensitivity. For example, in conditions of lipo- mice, and mice on high-fat diet (HFD) (52). TNF-␣ causes dystrophia, characterized by the absence of adipose tissue an increased level of SOCS-3 in the adipose tissue of OF1 and consequently leptin, severe insulin resistance is seen. mice (50), and ob/ob TNF-␣ receptor–deficient mice are However, the insulin sensitivity is restored upon leptin more insulin sensitive than wild-type ob/ob mice (53). infusion (57). Like most cytokines, leptin signals through Since the level of SOCS-3 was reduced so much in adipose the classical JAK-STAT pathway (Fig. 2). It binds the long tissue of these mice, TNF-␣–induced SOCS-3 expression isoform of the leptin receptor, which recruits JAK-2 upon could be a plausible explanation of insulin resistance in activation. Subsequently, JAK-2 is activated and facilitates obese animals (50). tyrosine phosphorylation of JAK-2 itself and of Tyr985 and The involvement of SOCS-3 and -1 in insulin resistance Tyr1,138 of the leptin receptor (58). Phosphorylated Tyr985 has been further established by studying their influence on binds the SH-2 domain containing tyrosine phosphatase, insulin signaling in mouse liver. Adenoviral-mediated he- and Tyr1,138 leads to STAT-3 activation (3), thereby medi- patic overexpression of the SOCS proteins induced a state ating effects of leptin in the cell. of insulin resistance in C57BL/6 mice (52), demonstrated Ob/ob mice lack leptin expression, db/db mice lack a by reduced phosphatidylinositol-3 kinase activation, re- functional leptin receptor, and both of these models are duced expression, and phosphorylation of IRS-1 and -2 in characterized by an obese phenotype and insulin resis- the liver upon insulin treatment (49). Further, when insu- tance (58). In obese humans and rodent models of obesity, lin-resistant db/db mice were treated with antisense oligo- leptin resistance is often observed, characterized by a high nucleotides directed against SOCS-1 and -3, the otherwise circulating level of leptin in the blood that nevertheless repressed phosphorylation of IRS-1 and -2 was partially fails to mediate its normal effects, such as reduction in restored, and insulin sensitivity was greatly improved, food intake and increased energy expenditure, thereby particularly after reducing SOCS-3 expression in the liver resulting in weight gain (3). The mechanism behind leptin (52). resistance is not clearly understood, but defects in intra- In humans, a link between elevated levels of inflamma- cellular leptin signaling may represent one explanation. As tory cytokines, SOCS expression, and insulin resistance leptin signals through the JAK-STAT pathway, it has been has also been suggested. Elevated IL-6 correlates with investigated whether the SOCS proteins are able to sup- SOCS-3 expression in skeletal muscle of type 2 diabetic press leptin signaling, thereby representing an explanation patients when compared with control subjects. Moreover, of leptin resistance. SOCS-3 inhibits leptin signaling in IL-6 induced SOCS-3 expression and consequently inhib- mammalian cell lines (59–61), and leptin signaling is ited insulin signaling in human differentiated myotubes enhanced when SOCS-3 is knocked down in RNA interfer- grown in vitro (54). SOCS-3 expression was not elevated in ence experiments (61). Leptin-induced STAT-3 activation muscle from nondiabetic obese individuals, despite an mediates activation of the SOCS-3 promoter and thereby enhanced IL-6 expression and insulin resistance in these production of SOCS-3 protein in leptin-sensitive tissues subjects. SOCS-3 expression in skeletal muscle of type 1 such as the hypothalamus, indicating that SOCS-3 is a diabetic patients is not elevated, indicating that the high classical feedback mechanism regulating leptin signaling SOCS expression in type 2 diabetic patients cannot be (59,61–64). The molecular mechanism behind SOCS-3– explained by hyperglycemia. However, high glucose con- mediated inhibition of leptin signaling involves binding of centrations enhanced IL-6–induced SOCS-3 mRNA expres- SOCS-3 to phosphorylated tyrosine residues on the acti- sion in cultured human muscle cells, suggesting that the vated JAK-2 and leptin receptor, thereby preventing acti- increased SOCS-3 expression in type 2 diabetics may be vation of downstream signaling components (60,61). explained by the combination of high glucose and IL-6 Several observations in vivo now suggest SOCS-3 as a levels in the blood of these individuals (54). Though, it mediator of leptin resistance in obesity and type 2 diabe- cannot be excluded that the high level of SOCS-3 expres- tes. In the lethal yellow (Ay/a) mouse, which is an obese sion in muscle of type 2 diabetic patients may also be mouse model of leptin resistance, obesity correlates with induced by other cytokines or hormones. an increased SOCS-3 expression in the leptin-sensitive To summarize, both in vitro and in vivo data support the sites in the hypothalamus, indicating that the increased hypothesis that high levels of inflammatory cytokines lead leptin level seen in obesity leads to enhanced SOCS-3 to increased expression of SOCS-1 and -3 in insulin- expression, which subsequently inhibits leptin signaling sensitive tissues, which induce insulin resistance via inhi- and thereby confers leptin resistance (59). In another bition of the insulin signaling pathway (Fig. 4). Based on study, leptin resistance, measured by the level of STAT-3 these findings, SOCS-1 and -3 are obvious candidate genes phosphorylation in diet-induced obese mice, was likewise coding for the development of type 2 diabetes. However, found to correlate with elevated SOCS-3 expression in the mutation analysis of the human SOCS-3 gene revealed hypothalamus (65). SOCS-3 knock-out mice are embryon-

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TABLE 1 SOCS proteins as potential targets for the treatment of diabetes Disease Pathogenesis Target Advantage Disadvantage Type 1 diabetes Cytokines induce ␤-cell Increase SOCS Protection against ␤-Cell becomes resistant death expression in deleterious to beneficial the ␤-cell immune attack hormones such as growth hormone and insulin Beneficial cytokine signaling such as IL-1␤–induced antiapoptotic pathways or IFN-␥–induced protection against viral attacks may be inhibited Type 2 diabetes/ Cytokines induce SOCS Decrease SOCS Increased insulin Hyper-responsiveness to obesity expression in insulin- expression in sensitivity other actions of sensitive tissues3 insulin- cytokines in insulin resistance sensitive insulin-sensitive tissues tissues Type 2 diabetes/ Leptin induces SOCS Decrease SOCS Increased leptin Hyper-responsiveness to obesity expression in expression in sensitivity other actions of leptin-sensitive leptin- cytokines in tissues3 leptin sensitive leptin-sensitive tissues resistance tissues ically lethal, but the haplo-insufficient SOCS-3 mouse has a SOCS-3 terminates leptin signaling, thereby giving rise to marked reduction of SOCS-3 expression. Interestingly, leptin resistance. As elevated levels of SOCS-3 expression these mice have increased leptin sensitivity when com- have been found in various mouse models of obesity and pared with their wild-type littermates, demonstrated by leptin resistance, suppression of SOCS-3 effects might be enhanced weight loss and enhanced tyrosine phosphory- of therapeutic interest in order to treat or prevent leptin lation of STAT3 in the hypothalamus upon exogenous and insulin resistance as well as obesity (Table 1). leptin administration (66). When mice are fed HFD, they develop obesity and leptin resistance. However, food consumption and weight gain were comparable in SOCS-3 haplo-insufficient mice, whether they were fed normal diet CONCLUSION AND PERSPECTIVES or HFD, and they ingested less food and gained less weight Since the discovery of the SOCS proteins, it has become than their wild-type littermates (66). Wild-type mice on clear that they are important negative regulators of cyto- HFD developed insulin resistance, whereas this was pre- kine signaling in various tissues. Numerous studies have vented in the SOCS-3 haplo-insufficient mice. The exact contributed to a better understanding of the physiological mechanism behind this is not known, but since leptin is role of the SOCS proteins, and SOCS proteins possess known to increase insulin sensitivity, the leptin resistance interesting properties in relation to both type 1 and type 2 seen in the wild-type mice fed HFD may constitute one diabetes. The SOCS proteins are able to suppress cyto- explanation of their insulin resistance. Interestingly, the toxic cytokine signaling, leading to ␤-cell death and mak- SOCS-3 haplo-insufficient mice have normal insulin and ing these proteins interesting targets in attempts to blood glucose levels upon being fed HFD, implying that suppress the inflammatory response leading to type 1 the increased leptin sensitivity in these mice is accompa- diabetes. It has recently been shown that recombinant nied by normal insulin sensitivity (66). These results were cell-penetrating SOCS-3 can protect mice from pathogen- reproduced in mice with conditional SOCS-3 knock-out in induced acute inflammation by suppressing cytokine sig- the brain. These mice also have increased leptin-induced naling (68); thus, this concept is testable in clinical islet STAT-3 phosphorylation in the hypothalamus, associated with increased weight loss, reduced food intake, and transplantation. On the other hand, overindulgence of resistance to HFD-induced obesity (67). Likewise, HFD- SOCS protein expression seems to be one of the mecha- induced insulin resistance was prevented in these mice. nisms behind insulin and leptin resistance in type 2 The data illustrate that mice with a low SOCS-3 expression diabetes, suggesting that inhibitors of SOCS action in level possess an obesity-resistant phenotype, including insulin- and/or leptin-sensitive tissues might be valuable normal insulin sensitivity, and suggest SOCS-3 as an agents in the treatment of this disease (Table 1). Further important inhibitor of leptin effects in vivo. studies on the role of SOCS proteins in diabetes might help To summarize, SOCS-3 is a negative feedback inhibitor elucidate important mechanisms involved in the pathogen- of leptin signaling. Leptin production is proportionate to esis of diabetes and thereby possibly support the SOCS body fat mass, and in conditions of obesity the enhanced proteins as interesting targets for the development of level of leptin in the blood results in an elevated produc- novel therapeutics for the prevention of diabetes and tion of SOCS-3 in leptin-sensitive tissues. Subsequently, perhaps other inflammatory diseases.

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