Suppressor of cytokine signaling 3 is a physiological regulator of adipocyte insulin signaling

Many proinflammatory cytokines and hormones have been demonstrated to be involved in insulin resistance. However, the molecular mechanisms whereby these cytokines and hormones inhibit insulin signaling are not completely understood. We observed that several cytokines and hormones that induce insulin resistance also stimulate SOCS3 expression in 3T3-L1 adipocytes and that SOCS3 mRNA is increased in adipose tissue of obese/diabetic mice. We then hypothesized that SOCS3 may mediate cytokine- and hormone-induced insulin resistance. By using SOCS3-deficient adipocytes differentiated from mouse embryonic fibroblasts, we found that SOCS3 deficiency increases insulin-stimulated IRS1 and IRS2 phosphorylation, IRS-associated phosphatidylinositol 3-kinase activity, and insulin-stimulated glucose uptake. Moreover, lack of SOCS3 substantially limits the inhibitory effects of tumor necrosis factor-alpha to suppress IRS1 and IRS2 tyrosine phosphorylation, phosphatidylinositol 3-kinase activity, and glucose uptake in adipocytes. The ameliorated insulin signaling in SOCS3-deficient adipocytes is mainly due to the suppression of tumor necrosis factor-alpha-induced IRS1 and IRS2 protein degradation. Therefore, our data suggest that endogenous SOCS3 expression is a key determinant of basal insulin signaling and is an important molecular mediator of cytokine-induced insulin resistance in adipocytes. We conclude that SOCS3 plays an important role in mediating insulin resistance and may be an excellent target for therapeutic intervention in insulin resistance and type II diabetes.     

Hepatitis B Virus X Protein Impairs Hepatic Insulin Signaling Through Degradation of IRS1 and Induction of SOCS3

Background

Hepatitis B virus (HBV) is a major cause of chronic liver diseases, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. The role of HCV in associations with insulin signaling has been elucidated. However, the pathogenesis of HBV-associated insulin signaling remains to be clearly characterized. Therefore, we have attempted to determine the mechanisms underlying the HBV-associated impairment of insulin signaling.

Methodology

The expressions of insulin signaling components were investigated in HBx-transgenic mice, HBx-constitutive expressing cells, and transiently HBx-transfected cells. Protein and gene expression was examined by Western blot, immunohistochemistry, RT-PCR, and promoter assay. Protein-protein interaction was detected by coimmunoprecipitation.

Principal Findings

HBx induced a reduction in the expression of IRS1, and a potent proteasomal inhibitor blocked the downregulation of IRS1. Additionally, HBx enhanced the expression of SOCS3 and induced IRS1 ubiquitination. Also, C/EBPα and STAT3 were involved in the HBx-induced expression of SOCS3. HBx interfered with insulin signaling activation and recovered the insulin-mediated downregulation of gluconeogenic genes.

Conclusions/Significance

These results provide direct experimental evidences for the contribution of HBx in the impairment of insulin signaling.     

Structural insights into suppressor of cytokine signaling 1 protein‐ identification of new leads for type 2 diabetes mellitus

The study considers the Suppressor of cytokine signaling 1 (SOCS1) protein as a novel Type 2 diabetes mellitus (T2DM) drug target. T2DM in human beings is also triggered by the over expression of SOCS proteins. The SOCS1 acts as a ubiquitin ligase (E3), degrades Insulin Receptor Substrate 1 and 2 (IRS1 and IRS2) proteins, and causes insulin resistance. Therefore, the structure of the SOCS1 protein was evaluated using homology‐modeling and molecular dynamics methods and validated using standard computational protocols. The Protein‐Protein docking study of SOCS1 with its natural substrates, IRS1 and IRS2, and subsequent solvent accessible surface area analysis gave insight into the binding region of the SOCS1 protein. The in silico active site prediction tools highlight the residues Val155 to Ile211 in SOCS1 being implicated in the ubiquitin mediated protein degradation of the proteins IRS1 and IRS2. Virtual screening in the active site region, using large structural databases, results in selective lead structures with 3‐Pyridinol, Xanthine, and Alanine moieties as Pharmacophore. The virtual screening study shows that the residues Glu149, Gly187, Arg188, Leu191, and Ser205 of the SOCS1 are important for binding. The docking study with current anti‐diabetic therapeutics shows that the drugs Glibenclamide and Glyclopyramide have a partial affinity towards SOCS1. The predicted ADMET and IC₅₀ properties for the identified ligands are within the acceptable range with drug‐like properties. The structural data of SOCS1, its active site, and the identified lead structures are expedient in the development of new T2DM therapeutics.     

The E3 ubiquitin ligase HOIL-1 induces the polyubiquitination and degradation of SOCS6 associated proteins

The suppressor of cytokine signaling (SOCS) proteins are thought to exert their function through the recruitment of interacting-proteins to the ubiquitin/proteasome degradation pathway. All SOCS proteins bind an Elongin BC E3 ubiquitin ligase complex through the common Socs-box. Here, we show that haem-oxidized IRP2 ubiquitin ligase-1 (HOIL-1), another E3 ubiquitin ligase, interacts with SOCS6. The Ubl domain of HOIL-1 and the SH2 and Socs-box domains of SOCS6 are required for the interaction. HOIL-1 expression stabilizes SOCS6 and induces the ubiquitination and degradation of proteins associated with SOCS6. These data suggest that SOCS proteins may interact with different E3 ubiquitin ligases in addition to a common Elongin BC E3 complex.     

Suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 cause insulin resistance through inhibition of tyrosine phosphorylation of insulin receptor substrate proteins by discrete mechanisms

Insulin resistance is a pathophysiological component of type 2 diabetes and obesity and also occurs in states of stress, infection, and inflammation associated with an upregulation of cytokines. Here we show that in both obesity and lipopolysaccharide (LPS)-induced endotoxemia there is an increase in suppressor of cytokine signaling (SOCS) proteins, SOCS-1 and SOCS-3, in liver, muscle, and, to a lesser extent, fat. In concordance with these increases by LPS, tyrosine phosphorylation of the insulin receptor (IR) is partially impaired and phosphorylation of the insulin receptor substrate (IRS) proteins is almost completely suppressed. Direct overexpression of SOCS-3 in liver by adenoviral-mediated gene transfer markedly decreases tyrosine phosphorylation of both IRS-1 and IRS-2, while SOCS-1 overexpression preferentially inhibits IRS-2 phosphorylation. Neither affects IR phosphorylation, although both SOCS-1 and SOCS-3 bind to the insulin receptor in vivo in an insulin-dependent fashion. Experiments with cultured cells expressing mutant insulin receptors reveal that SOCS-3 binds to Tyr960 of IR, a key residue for the recognition of IRS-1 and IRS-2, whereas SOCS-1 binds to the domain in the catalytic loop essential for IRS-2 recognition in vitro. Moreover, overexpression of either SOCS-1 or SOCS-3 attenuates insulin-induced glycogen synthesis in L6 myotubes and activation of glucose uptake in 3T3L1 adipocytes. By contrast, a reduction of SOCS-1 or SOCS-3 by antisense treatment partially restores tumor necrosis factor alpha-induced downregulation of tyrosine phosphorylation of IRS proteins in 3T3L1 adipocytes. These data indicate that SOCS-1 and SOCS-3 act as negative regulators in insulin signaling and serve as one of the missing links between insulin resistance and cytokine signaling.     

SOCS-1 deficiency does not prevent diet-induced insulin resistance

Obesity is associated with inflammation and increased expression of suppressor of cytokine signaling (SOCS) proteins, which inhibit cytokine and insulin signaling. Thus, reducing SOCS expression could prevent the development of obesity-induced insulin resistance. Using SOCS-1 knockout mice, we investigated the contribution of SOCS-1 in the development of insulin resistance induced by a high-fat diet (HFD). SOCS-1 knockout mice on HFD gained 70% more weight, displayed a 2.3-fold increase in epididymal fat pads mass and increased hepatic lipid content. This was accompanied by increased mRNA expression of leptin and the macrophage marker CD68 in white adipose tissue and of SREBP1c and FAS in liver. HFD also induced hyperglycemia in SOCS-1 deficient mice with impairment of glucose and insulin tolerance tests. Thus, despite the role of SOCS proteins in obesity-related insulin resistance, SOCS-1 deficiency alone is not able to prevent insulin resistance induced by a diet rich in fat.     

Positive and negative regulation of insulin signaling through IRS-1 phosphorylation

This review will provide insight on the current understanding of the regulation of insulin signaling in both physiological and pathological conditions through modulations that occur with regards to the functions of the insulin receptor substrate 1 (IRS1). While the phosphorylation of IRS1 on tyrosine residue is required for insulin-stimulated responses, the phosphorylation of IRS1 on serine residues has a dual role, either to enhance or to terminate the insulin effects. The activation of PKB in response to insulin propagates insulin signaling and promotes the phosphorylation of IRS1 on serine residue in turn generating a positive-feedback loop for insulin action. Insulin also activates several kinases and these kinases act to induce the phosphorylation of IRS1 on specific sites and inhibit its functions. This is part of the negative-feedback control mechanism induced by insulin that leads to termination of its action. Agents such as free fatty acids, cytokines, angiotensin II, endothelin-1, amino acids, cellular stress and hyperinsulinemia, which induce insulin resistance, lead to both activation of several serine/threonine kinases and phosphorylation of IRS1. These agents negatively regulate the IRS1 functions by phosphorylation but also via others molecular mechanisms (SOCS expression, IRS degradation, O-linked glycosylation) as summarized in this review. Understanding how these agents inhibit IRS1 functions as well as identification of kinases involved in these inhibitory effects may provide novel targets for development of strategies to prevent insulin resistance.     

IRS proteins and the common path to diabetes

Although a full understanding of insulin/insulin-like growth factor (IGF) action is evolving, the discovery of insulin receptor substrate (IRS) proteins and their role to link cell surface receptors to the intracellular signaling cascades provided an important step forward. Moreover, Insulin/IGF receptors use common signaling pathways to accomplish many tasks, the IRS proteins add a unique layer of specificity and control. Importantly, the IRS-2 branch of the insulin/IGF-signaling pathway is a common element in peripheral insulin response and pancreatic beta-cell growth and function. Failure of IRS-2 signaling might explain the eventual loss of compensatory hyperinsulinemia during prolonged periods of peripheral insulin resistance. Moreover, short-term inhibition of IRS protein functions by serine phosphorylation, or sustained inhibition by ubiquitin-targeted proteosome-mediated degradation suggests a common molecular mechanism for insulin resistance during acute injury or infection, or the sensitivity of beta-cells to autoimmune destruction. The broad role of IRS-1 and IRS-2 in cell growth and survival reveals a common regulatory pathway linking development, somatic growth, fertility, neuronal proliferation, and aging to the core mechanisms used by vertebrates for nutrient sensing.     

SOCS2 can enhance interleukin-2 (IL-2) and IL-3 signaling by accelerating SOCS3 degradation

Cytokine responses can be regulated by a family of proteins termed suppressors of cytokine signaling (SOCS) which can inhibit the JAK/STAT pathway in a classical negative-feedback manner. While the SOCS are thought to target signaling intermediates for degradation, relatively little is known about how their turnover is regulated. Unlike other SOCS family members, we find that SOCS2 can enhance interleukin-2 (IL-2)- and IL-3-induced STAT phosphorylation following and potentiate proliferation in response to cytokine stimulation. As a clear mechanism for these effects, we demonstrate that expression of SOCS2 results in marked proteasome-dependent reduction of SOCS3 and SOCS1 protein expression. Furthermore, we provide evidence that this degradation is dependent on the presence of an intact SOCS box and that the loss of SOCS3 is enhanced by coexpression of elongin B/C. This suggests that SOCS2 can bind to SOCS3 and elongin B/C to form an E3 ligase complex resulting in the degradation of SOCS3. Therefore, SOCS2 can enhance cytokine responses by accelerating proteasome-dependent turnover of SOCS3, suggesting a mechanism for the gigantism observed in SOCS2 transgenic mice.     

SOCS3 inhibits insulin signaling in porcine primary adipocytes

Insulin resistance is a major player in the pathogenesis of type II diabetes, the metabolic syndrome, and obesity. SOCS3 plays an important role in the development of insulin resistance. To investigate the role of SOCS3 in porcine adipocyte insulin signaling, we first detected the effect of insulin on SOCS3 mRNA and protein expression in porcine primary adipocytes by real-time RT-PCR and Western blotting. Then, we constructed a recombinant adenovirus encoding SOCS3 gene (Ad-SOCS3) which was used to infect differentiated porcine primary adipocytes for 3 days. The expression and phosphorylation of main insulin signaling components were detected by Western blotting. The results showed that 100 nM insulin could induce SOCS3 mRNA expression but not protein expression, and overexpression of SOCS3 decreased IRS1 protein level, insulin-stimulated IRS1 tyrosine phosphorylation, PI3K activation, and Akt phosphorylation, but increased IRS1 serine phosphorylation in porcine primary adipocytes. These results indicate that SOCS3 is an important negative regulator of insulin signaling in porcine adipocytes. Thus, SOCS3 may be a novel therapeutic target for the prevention or treatment of insulin resistance and type II diabetes.     

SOCS-6 binds to insulin receptor substrate 4, and mice lacking the SOCS-6 gene exhibit mild growth retardation

SOCS-6 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCS-1 to SOCS-7 and CIS) which each contain a central SH2 domain and a carboxyl-terminal SOCS box. SOCS-1, SOCS-2, SOCS-3, and CIS act to negatively regulate cytokine-induced signaling pathways; however, the actions of SOCS-4, SOCS-5, SOCS-6, and SOCS-7 remain less clear. Here we have used both biochemical and genetic approaches to examine the action of SOCS-6. We found that SOCS-6 and SOCS-7 are expressed ubiquitously in murine tissues. Like other SOCS family members, SOCS-6 binds to elongins B and C through its SOCS box, suggesting that it might act as an E3 ubiquitin ligase that targets proteins bound to its SH2 domain for ubiquitination and proteasomal degradation. We investigated the binding specificity of the SOCS-6 and SOCS-7 SH2 domains and found that they preferentially bound to phosphopeptides containing a valine in the phosphotyrosine (pY) +1 position and a hydrophobic residue in the pY +2 and pY +3 positions. In addition, these SH2 domains interacted with a protein complex consisting of insulin receptor substrate 4 (IRS-4), IRS-2, and the p85 regulatory subunit of phosphatidylinositol 3-kinase. To investigate the physiological role of SOCS-6, we generated mice lacking the SOCS-6 gene. SOCS-6(-/-) mice were born in a normal Mendelian ratio, were fertile, developed normally, and did not exhibit defects in hematopoiesis or glucose homeostasis. However, both male and female SOCS-6(-/-) mice weighed approximately 10% less than wild-type littermates.     

Tyrosine phosphorylation disrupts elongin interaction and accelerates SOCS3 degradation

The suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor-induced signal transduction. The C-terminal SOCS box region is thought to regulate SOCS protein stability most likely via an elongin C interaction. In the present study, we have found that phosphorylation of SOCS3 at two tyrosine residues in the conserved SOCS box, Tyr204 and Tyr221, can inhibit the SOCS3-elongin C interaction and activate proteasome-mediated SOCS3 degradation. Jak-mediated phosphorylation of SOCS3 decreased SOCS3 protein half-life, and phosphorylation of both Tyr204 and Tyr221 was required to fully destabilize SOCS3. In contrast, a phosphorylation-deficient mutant of SOCS3, Y204F,Y221F, remained stable in the presence of activated Jak2 and receptor tyrosine kinases. SOCS3 stability correlated with the relative amount that bound elongin C, because in vitro phosphorylation of a SOCS3-glutathione S-transferase fusion protein abolished its ability to interact with elongin C. In addition, a SOCS3/SOCS1 chimera that co-precipitates with markedly increased elongin C, was significantly more stable than wild-type SOCS3. The data suggest that interaction with elongin C stabilizes SOCS3 protein expression and that phosphorylation of SOCS box tyrosine residues disrupts the complex and enhances proteasome-mediated degradation of SOCS3.     

The SOCS box of SOCS-1 accelerates ubiquitin-dependent proteolysis of TEL-JAK2

Fusion of the TEL gene on 12p13 to the JAK2 tyrosine kinase gene on 9p24 has been found in human leukemia. TEL-mediated oligomerization of JAK2 results in constitutive activation of the tyrosine kinase (JH1) domain and confers cytokine-independent proliferation on interleukin-3-dependent Ba/F3 cells. Forced expression of the JAK inhibitor gene SOCS1/JAB/SSI-1 induced apoptosis of TEL-JAK2-transformed Ba/F3 cells. This suppression of TEL-JAK2 activity was dependent on SOCS box-mediated proteasomal degradation of TEL-JAK2 rather than on kinase inhibition. Degradation of JAK2 depended on its phosphorylation and its high affinity binding with SOCS1 through the kinase inhibitory region and the SH2 domain. It has been demonstrated that von Hippel-Lindau disease (VHL) tumor-suppressor gene product possesses the SOCS box that forms a complex with Elongin B and C and Cullin-2, and it functions as a ubiquitin ligase. The SOCS box of SOCS1/JAB has also been shown to interact with Elongins; however, ubiquitin ligase activity has not been demonstrated. We found that the SOCS box interacted with Cullin-2 and promoted ubiquitination of TEL-JAK2. Furthermore, overexpression of dominant negative Cullin-2 suppressed SOCS1-dependent TEL-JAK2 degradation. Our study demonstrates the substrate-specific E3 ubiquitin-ligase-like activity of SOCS1 for activated JAK2 and may provide a novel strategy for the suppression of oncogenic tyrosine kinases.     

Two new substrates in insulin signaling,IRS5/DOK4 and IRS6/DOK5

We have identified two new human genes that encode proteins with tandem pleckstrin homology-phosphotyrosine binding (PH-PTB) domains at their amino termini. Because the other known PH-PTB proteins (insulin receptor substrates: IRS-1, IRS-2, IRS-3, and IRS-4, and the downstream of kinases: DOK-1, DOK-2, and DOK-3) are substrates of insulin and insulin-like growth factor (IGF)-1 receptors, we asked whether these new proteins, termed IRS5/DOK4 and IRS6/DOK5, might also have roles in insulin and IGF-1 signaling. Northern analyses indicate that IRS5/DOK4 is ubiquitously expressed but most abundant in kidney and liver. IRS6/DOK5 expression is highest in skeletal muscle. Both proteins are tyrosine-phosphorylated in response to insulin and IGF-1 in transfected cells, although the kinetics differ. Insulin receptor-phosphorylated IRS5/DOK4 associates with RasGAP, Crk, Src, and Fyn, but not phosphatidylinositol 3-kinase p85, Grb2, SHP-2, Nck, or phospholipase Cgamma Src homology 2 domains, and activates MAPK in cells. IRS6/DOK5 neither associates with these Src homology 2 domains nor activates MAPK. IRS5/DOK4 and IRS6/DOK5 represent two new signaling proteins with potential roles in insulin and IGF-1 action.     

Insulin receptor substrate 1 and 2 (IRS1 and IRS2): what a tangled web we weave

The insulin receptor is a transmembrane tyrosine kinase that is essential for mediating multiple intracellular signalling cascades that lead ultimately to the biological actions of insulin Tyrosine phosphorylation o f the cytosolic proteins insulin receptor substrate 1 and 2 (IRS1 and IRS2) produces protein 'scaffolding' for the assembly of effector proteins containing Src homology 2 (SH2) domains, thereby generating multisubunit signalling complexes. Although IRS1 was originally isolated as a specific insulin receptor substrate, both IRS1 and IRS2 appear to play a broader role, functioning also as proximal substrates in growth hormone and cytokine receptor signalling. Current data establish IRS1 and IRS2 as critical effectors integrating various cell-type-specific signals into distinct, but overlapping, biological responses.     

Cloning and characterization of the genes encoding the ankyrin repeat and SOCS box-containing proteins Asb-1, Asb-2, Asb-3 and Asb-4

Members of the suppressor of cytokine signalling (SOCS) family of proteins have been shown to inhibit cytokine signalling via direct interactions with JAK kinases or activated cytokine receptors. In addition to their novel amino-terminal regions and SH2 domains that mediate these interactions, the SOCS proteins also contain carboxy-terminal regions of homology called the SOCS box. The SOCS box serves to couple SOCS proteins and their binding partners with the elongin B and C complex, possibly targeting them for degradation. Several other families of proteins also contain SOCS boxes but differ from the SOCS proteins in the type of domain or motif they contain upstream of the SOCS box. We report here the cloning, characterization, mapping and expression analysis of four members of the ankyrin repeat and SOCS box-containing (Asb) protein family.     

SOCS-1 and SOCS-3 inhibit IFN-alpha-induced expression of the antiviral proteins 2,5-OAS and MxA

Although the use of IFN-alpha in combination with ribavirin has improved the treatment efficacy of chronic hepatitis C virus (HCV) infection, 20-50% of patients still fail to eradicate the virus depending on the HCV genotype. Recently, overexpression of HCV core protein has been shown to inhibit IFN signaling and induce SOCS-3 expression. Aim of this study was to examine the putative role of SOCS proteins in IFN resistance. By Western blot analysis, a 4-fold induction of STAT-1/3 phosphorylation by IFN-alpha was observed in mock-transfected HepG2 clones. In contrast, IFN-induced STAT-1/3 phosphorylation was considerably downregulated by SOCS-1/3 overexpression. In mock-transfected cells, IFN-alpha induced 2',5'-OAS and myxovirus resistance A (MxA) promoter activity 40- to 80-fold and 10- to 35-fold, respectively, and this effect was abrogated in SOCS-1/3 overexpressing cells. As detected by Northern blot technique, IFN-alpha potently induced 2',5'-OAS and MxA mRNA expression in the control clones. Overexpression of SOCS-1 completely abolished both 2',5'-OAS and MxA mRNA expression, whereas SOCS-3 mainly inhibited 2',5'-OAS mRNA expression. Our results demonstrate that SOCS-1 and SOCS-3 proteins inhibit IFN-alpha-induced activation of the Jak-STAT pathway and expression of the antiviral proteins 2',5'-OAS and MxA. These data suggest a potential role of SOCS proteins in IFN resistance during antiviral treatment.     

Phosphorylation of IRS proteins, insulin action, and insulin resistance

Insulin signaling at target tissues is essential for growth and development and for normal homeostasis of glucose, fat, and protein metabolism. Control over this process is therefore tightly regulated. It can be achieved by a negative feedback control mechanism whereby downstream components inhibit upstream elements along the insulin-signaling pathway (autoregulation) or by signals from apparently unrelated pathways that inhibit insulin signaling thus leading to insulin resistance. Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues has emerged as a key step in these control processes under both physiological and pathological conditions. The list of IRS kinases implicated in the development of insulin resistance is growing rapidly, concomitant with the list of potential Ser/Thr phosphorylation sites in IRS proteins. Here, we review a range of conditions that activate IRS kinases to phosphorylate IRS proteins on "hot spot" domains. The flexibility vs. specificity features of this reaction is discussed and its characteristic as an "array" phosphorylation is suggested. Finally, its implications on insulin signaling, insulin resistance and type 2 diabetes, an emerging epidemic of the 21st century are outlined.