Sequential phosphorylation of insulin receptor substrate-2 by glycogen synthase kinase-3 and c-Jun NH2-terminal kinase plays a role in hepatic insulin signaling

2008 ◽  
Vol 294 (2) ◽  
pp. E307-E315 ◽  
Author(s):  
Hadar Sharfi ◽  
Hagit Eldar-Finkelman
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Insulin Signaling ◽  
Glycogen Synthase ◽  
Serine Phosphorylation ◽  
Glycogen Synthase Kinase ◽  
Regulatory Subunit ◽  
Insulin Receptor Substrate ◽  
Phosphorylation Sites ◽  
Hepatic Insulin Signaling

Serine phosphorylation of insulin receptor substrate (IRS) proteins is a potential inhibitory mechanism in insulin signaling. Here we show that IRS-2 is phosphorylated by glycogen synthase kinase (GSK)-3. Phosphorylation by GSK-3 requires prior phosphorylation of its substrates, prompting us to identify the “priming kinase.” It was found that the stress activator anisomycin enhanced the ability of GSK-3 to phosphorylate IRS-2. Use of a selective c-Jun NH2-terminal kinase (JNK) inhibitor and cells overexpressing JNK implicated JNK as the priming kinase. This allowed us to narrow down the number of potential GSK-3 phosphorylation sites within IRS-2 to four regions that follow the motif SXXXSP. IRS-2 deletion mutants enabled us to localize the GSK-3 and JNK phosphorylation sites to serines 484 and 488, respectively. Mutation at serine 488 reduced JNK phosphorylation of IRS-2, and mutation of each site separately abolished GSK-3 phosphorylation of IRS-2. Treatment of H4IIE liver cells with anisomycin inhibited insulin-induced tyrosine phosphorylation of IRS-2; inhibition was reversed by pretreatment with the JNK and GSK-3 inhibitors. Moreover, overexpression of JNK and GSK-3 in H4IIE cells reduced insulin-induced tyrosine phosphorylation of IRS-2 and its association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. Finally, both GSK-3 and JNK are abnormally upregulated in the diabetic livers of ob/obmice. Together, our data indicate that IRS-2 is sequentially phosphorylated by JNK and GSK-3 at serines 484/488 and provide evidence for their inhibitory role in hepatic insulin signaling.

scholarly journals Insulin Resistance in Human Preeclamptic Placenta Is Mediated by Serine Phosphorylation of Insulin Receptor Substrate-1 and -2

2006 ◽  
Vol 91 (2) ◽  
pp. 709-717 ◽  
Author(s):  
Marco Scioscia ◽  
Khalid Gumaa ◽  
Sirilaksana Kunjara ◽  
Malcolm A. Paine ◽  
Luigi E. Selvaggi ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Insulin Signaling ◽  
Serine Phosphorylation ◽  
Regulatory Subunit ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
Cross Sectional ◽  
Maternal Risk ◽  
Therapeutic Implications

Context: Preeclampsia is a severe complication of human pregnancy often associated with maternal risk factors. Insulin resistance represents a major risk for developing preeclampsia during pregnancy. Objective: A putative second messenger of insulin, inositol phosphoglycan P type (P-IPG), was previously shown to be highly increased during active preeclampsia. Its association with insulin resistance was investigated. Design and Setting: A cross-sectional study was carried out in a referral center. Patients: Nine preeclamptic (PE) and 18 healthy women were recruited and matched for maternal age, body mass index, parity, and ethnicity in a 1:2 ratio. Placental specimens were collected immediately after delivery. Intervention: Placental tissue was incubated with insulin and P-IPG production assessed. Insulin signaling proteins were subsequently studied by immunoblotting. Results: P-IPG extracted from human term placentas upon incubation with insulin was found to be far lower in those with preeclampsia than controls (P < 0.001). Immunoblotting studies revealed serine phosphorylation of insulin receptor substrate-1 and -2 in PE placentas (P < 0.001) with downstream impairment of insulin signaling. The activation of the p85 regulatory subunit of phosphatidylinositol 3- kinase was markedly decreased in PE samples (P < 0.001). Conclusions: These findings highlight the importance of P-IPG in active preeclampsia and demonstrate a substantially different response to the insulin stimulus of human PE placentas. Acquired alterations in activation of proteins involved in insulin signaling may play a role in the complex pathogenesis of preeclampsia, probably as a consequence of the immunological dysfunction that occurs in this syndrome. These results seem to confirm an insulin-resistant state in PE placenta and shed a different light on its role in the pathogenesis of this disease with potential therapeutic implications.

scholarly journals Interleukin-1α Inhibits Insulin Signaling with Phosphorylating Insulin Receptor Substrate-1 on Serine Residues in 3T3-L1 Adipocytes

2006 ◽  
Vol 20 (1) ◽  
pp. 114-124 ◽  
Author(s):  
Jianying He ◽  
Isao Usui ◽  
Ken Ishizuka ◽  
Yukiko Kanatani ◽  
Kazuyuki Hiratani ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Proinflammatory Cytokines ◽  
Insulin Signaling ◽  
Serine Phosphorylation ◽  
Insulin Receptor Substrate ◽  
Akt Phosphorylation ◽  
Iκb Kinase ◽  
Serine Kinases

Abstract Proinflammatory cytokines are recently reported to inhibit insulin signaling causing insulin resistance. IL-1α is also one of the proinflammatory cytokines; however, it has not been clarified whether IL-1α may also cause insulin resistance. Here, we investigated the effects of IL-1α treatment on insulin signaling in 3T3-L1 adipocytes. IL-1α treatment up to 4 h did not alter insulin-stimulated insulin receptor tyrosine phosphorylation, whereas tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the association with phosphatidylinositol 3-kinase were partially inhibited with the maximal inhibition in around 15 min. IRS-1 was transiently phosphorylated on some serine residues around 15 min after IL-1α stimulation, when several serine kinases, IκB kinase, c-Jun-N-terminal kinase, ERK, and p70S6K were activated. Chemical inhibitors for these kinases inhibited IL-1α-induced serine phosphorylation of IRS-1. Tyrosine phosphorylation of IRS-1 was recovered only by the IKK inhibitor or JNK inhibitor, suggesting specific involvement of these two kinases. Insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake were not inhibited only by IL-1α. Interestingly, Akt phosphorylation was synergistically inhibited by IL-1α in the presence of IL-6. Taken together, short-term IL-1α treatment transiently causes insulin resistance at IRS-1 level with its serine phosphorylation. IL-1α may suppress insulin signaling downstream of IRS-1 in the presence of other cytokines, such as IL-6.

scholarly journals Chronic Tumor Necrosis Factor-α Treatment Causes Insulin Resistance via Insulin Receptor Substrate-1 Serine Phosphorylation and Suppressor of Cytokine Signaling-3 Induction in 3T3-L1 Adipocytes

Endocrinology ◽  
2007 ◽  
Vol 148 (6) ◽  
pp. 2994-3003 ◽  
Author(s):  
Ken Ishizuka ◽  
Isao Usui ◽  
Yukiko Kanatani ◽  
Agussalim Bukhari ◽  
Jianying He ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Insulin Signaling ◽  
Serine Phosphorylation ◽  
Cytokine Signaling ◽  
Insulin Receptor Substrate ◽  
Signal Transducer ◽  
Suppressor Of Cytokine Signaling ◽  
Transcription 3

Serine phosphorylation of insulin receptor substrate (IRS)-1 and the induction of suppressor of cytokine signaling 3 (SOCS3) is recently well documented as the mechanisms for the insulin resistance. However, the relationship between these two mechanisms is not fully understood. In this study, we investigated the involvement of SOCS3 and IRS-1 serine phosphorylation in TNFα-induced insulin resistance in 3T3-L1 adipocytes. TNFα transiently stimulated serine phosphorylation of IRS-1 from 10 min to 1 h, whereas insulin-stimulated IRS-1 tyrosine phosphorylation was inhibited only after TNFα treatment longer than 4 h. These results suggest that serine phosphorylation of IRS-1 alone is not the major mechanism for the inhibited insulin signaling by TNFα. TNFα stimulation longer than 4 h enhanced the expression of SOCS3 and signal transducer and activator of transcription-3 phosphorylation, concomitantly with the production of IL-6. Anti-IL-6 neutralizing antibody ameliorated suppressed insulin signaling by 24 h TNFα treatment, when it partially decreased SOCS3 induction and signal transducer and activator of transcription-3 phosphorylation. These results suggest that SOCS3 induction is involved in inhibited insulin signaling by TNFα. However, low-level expression of SOCS3 by IL-6 or adenovirus vector did not affect insulin-stimulated IRS-1 tyrosine phosphorylation. Interestingly, when IRS-1 serine phosphorylation was enhanced by TNFα or anisomycin in the presence of low-level SOCS3, IRS-1 degradation was remarkably enhanced. Taken together, both IRS-1 serine phosphorylation and SOCS3 induction are necessary, but one of the pair is not sufficient for the inhibited insulin signaling. Chronic TNFα may inhibit insulin signaling effectively because it causes both IRS-1 serine phosphorylation and the following SOCS3 induction in 3T3-L1 adipocytes.

scholarly journals Dynamic Functional Relay between Insulin Receptor Substrate 1 and 2 in Hepatic Insulin Signaling during Fasting and Feeding

2008 ◽  
Vol 8 (1) ◽  
pp. 49-64 ◽  
Author(s):  
Naoto Kubota ◽  
Tetsuya Kubota ◽  
Shinsuke Itoh ◽  
Hiroki Kumagai ◽  
Hideki Kozono ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Insulin Signaling ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
Hepatic Insulin Signaling

scholarly journals Serine Phosphorylation Proximal to Its Phosphotyrosine Binding Domain Inhibits Insulin Receptor Substrate 1 Function and Promotes Insulin Resistance

2004 ◽  
Vol 24 (21) ◽  
pp. 9668-9681 ◽  
Author(s):  
Yan-Fang Liu ◽  
Avia Herschkovitz ◽  
Sigalit Boura-Halfon ◽  
Denise Ronen ◽  
Keren Paz ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Insulin Signaling ◽  
Insulin Treatment ◽  
Binding Domain ◽  
Serine Phosphorylation ◽  
Insulin Receptor Substrate ◽  
Control Mechanisms ◽  
Insulin Receptor Substrate 1 ◽  
Phosphotyrosine Binding Domain

ABSTRACT Ser/Thr phosphorylation of insulin receptor substrate (IRS) proteins negatively modulates insulin signaling. Therefore, the identification of serine sites whose phosphorylation inhibit IRS protein functions is of physiological importance. Here we mutated seven Ser sites located proximal to the phosphotyrosine binding domain of insulin receptor substrate 1 (IRS-1) (S265, S302, S325, S336, S358, S407, and S408) into Ala. When overexpressed in rat hepatoma Fao or CHO cells, the mutated IRS-1 protein in which the seven Ser sites were mutated to Ala (IRS-17A), unlike wild-type IRS-1 (IRS-1WT), maintained its Tyr-phosphorylated active conformation after prolonged insulin treatment or when the cells were challenged with inducers of insulin resistance prior to acute insulin treatment. This was due to the ability of IRS-17A to remain complexed with the insulin receptor (IR), unlike IRS-1WT, which underwent Ser phosphorylation, resulting in its dissociation from IR. Studies of truncated forms of IRS-1 revealed that the region between amino acids 365 to 430 is a main insulin-stimulated Ser phosphorylation domain. Indeed, IRS-1 mutated only at S408, which undergoes phosphorylation in vivo, partially maintained the properties of IRS-17A and conferred protection against selected inducers of insulin resistance. These findings suggest that S408 and additional Ser sites among the seven mutated Ser sites are targets for IRS-1 kinases that play a key negative regulatory role in IRS-1 function and insulin action. These sites presumably serve as points of convergence, where physiological feedback control mechanisms, which are triggered by insulin-stimulated IRS kinases, overlap with IRS kinases triggered by inducers of insulin resistance to terminate insulin signaling.

Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle

2005 ◽  
Vol 288 (6) ◽  
pp. E1188-E1194 ◽  
Author(s):  
Betsy B. Dokken ◽  
Julie A. Sloniger ◽  
Erik J. Henriksen
Keyword(s):  
Skeletal Muscle ◽  
Tyrosine Phosphorylation ◽  
Glucose Transport ◽  
Insulin Signaling ◽  
Insulin Action ◽  
Glycogen Synthase ◽  
Serine Phosphorylation ◽  
Zucker Rats ◽  
Type I ◽  
Insulin Resistant

Glycogen synthase kinase-3 (GSK3) has been implicated in the multifactorial etiology of skeletal muscle insulin resistance in animal models and in human type 2 diabetic subjects. However, the potential molecular mechanisms involved are not yet fully understood. Therefore, we determined if selective GSK3 inhibition in vitro leads to an improvement in insulin action on glucose transport activity in isolated skeletal muscle of insulin-resistant, prediabetic obese Zucker rats and if these effects of GSK3 inhibition are associated with enhanced insulin signaling. Type I soleus and type IIb epitrochlearis muscles from female obese Zucker rats were incubated in the absence or presence of a selective, small organic GSK3 inhibitor (1 μM CT118637, Ki < 10 nM for GSK3α and GSK3β). Maximal insulin stimulation (5 mU/ml) of glucose transport activity, glycogen synthase activity, and selected insulin-signaling factors [tyrosine phosphorylation of insulin receptor (IR) and IRS-1, IRS-1 associated with p85 subunit of phosphatidylinositol 3-kinase, and serine phosphorylation of Akt and GSK3] were assessed. GSK3 inhibition enhanced ( P <0.05) basal glycogen synthase activity and insulin-stimulated glucose transport in obese epitrochlearis (81 and 24%) and soleus (108 and 20%) muscles. GSK3 inhibition did not modify insulin-stimulated tyrosine phosphorylation of IR β-subunit in either muscle type. However, in obese soleus, GSK3 inhibition enhanced (all P < 0.05) insulin-stimulated IRS-1 tyrosine phosphorylation (45%), IRS-1-associated p85 (72%), Akt1/2 serine phosphorylation (30%), and GSK3β serine phosphorylation (39%). Substantially smaller GSK3 inhibitor-mediated enhancements of insulin action on these insulin signaling factors were observed in obese epitrochlearis. These results indicate that selective GSK3 inhibition enhances insulin action in insulin-resistant skeletal muscle of the prediabetic obese Zucker rat, at least in part by relieving the deleterious effects of GSK3 action on post-IR insulin signaling. These effects of GSK3 inhibition on insulin action are greater in type I muscle than in type IIb muscle from these insulin-resistant animals.

scholarly journals Glycogen synthase kinase 3β mediates high glucose-induced ubiquitination and proteasome degradation of insulin receptor substrate 1

2010 ◽  
Vol 206 (2) ◽  
pp. 171-181 ◽  
Author(s):  
Sanhua Leng ◽  
Wenshuo Zhang ◽  
Yanbin Zheng ◽  
Ziva Liberman ◽  
Christopher J Rhodes ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Molecular Mechanism ◽  
High Glucose ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Insulin Receptor Substrate ◽  
Glycogen Synthase Kinase 3Β ◽  
Proteasome Degradation ◽  
Protein Levels

High glucose (HG) has been shown to induce insulin resistance in both type 1 and type 2 diabetes. However, the molecular mechanism behind this phenomenon is unknown. Insulin receptor substrate (IRS) proteins are the key signaling molecules that mediate insulin's intracellular actions. Genetic and biological studies have shown that reductions in IRS1 and/or IRS2 protein levels are associated with insulin resistance. In this study we have shown that proteasome degradation of IRS1, but not of IRS2, is involved in HG-induced insulin resistance in Chinese hamster ovary (CHO) cells as well as in primary hepatocytes. To further investigate the molecular mechanism by which HG induces insulin resistance, we examined various molecular candidates with respect to their involvement in the reduction in IRS1 protein levels. In contrast to the insulin-induced degradation of IRS1, HG-induced degradation of IRS1 did not require IR signaling or phosphatidylinositol 3-kinase/Akt activity. We have identified glycogen synthase kinase 3β (GSK3β or GSK3B as listed in the MGI Database) as a kinase required for HG-induced serine332 phosphorylation, ubiquitination, and degradation of IRS1. Overexpression of IRS1 with mutation of serine332 to alanine partially prevents HG-induced IRS1 degradation. Furthermore, overexpression of constitutively active GSK3β was sufficient to induce IRS1 degradation. Our data reveal the molecular mechanism of HG-induced insulin resistance, and support the notion that activation of GSK3β contributes to the induction of insulin resistance via phosphorylation of IRS1, triggering the ubiquitination and degradation of IRS1.

Molecular and functional resistance to insulin in hypothalamus of rats exposed to cold

2003 ◽  
Vol 285 (1) ◽  
pp. E216-E223 ◽  
Author(s):  
Márcio A. Torsoni ◽  
José B. Carvalheira ◽  
Márcio Pereira-Da-Silva ◽  
Marco A. de Carvalho-Filho ◽  
Mário J. A. Saad ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Insulin Signaling ◽  
Room Temperature ◽  
Arcuate Nucleus ◽  
Insulin Receptor Substrate ◽  
Food Ingestion ◽  
Phosphatidylinositol 3 Kinase ◽  
Resistance To Insulin ◽  
Low Levels

Insulin and leptin act in the hypothalamus, providing robust anorexigenic signals. The exposure of homeothermic animals to a cold environment leads to increased feeding, accompanied by sustained low levels of insulin and leptin. In the present study, the initial and intermediate steps of the insulin-signaling cascade were evaluated in the hypothalamus of cold-exposed Wistar rats. By immunohistochemistry, most insulin receptor (IR) and insulin receptor substrate-2 (IRS-2) immunoreactivity localized to the arcuate nucleus. Basal levels of tyrosine phosphorylation of IR and IRS-2 were increased in cold-exposed rats compared with rats maintained at room temperature. However, after an acute, peripheral infusion of exogenous insulin, significantly lower increases of IR and IRS-2 tyrosine phosphorylation were detected in the hypothalamus of cold-exposed rats. Insulin-induced association of p85/phosphatidylinositol 3-kinase with IRS-2, Ser473 phosphorylation of Akt, and tyrosine phosphorylation of ERK was significantly reduced in the hypothalamus of cold-exposed rats. To test the hypothesis of functional impairment of insulin signaling in the hypothalamus, intracerebroventricularly cannulated rats were acutely treated with insulin, and food ingestion was measured over a period of 12 h. Cold-exposed animals presented a significantly lower insulin-induced reduction in food consumption compared with animals maintained at room temperature. Hence, the present studies reveal that animals exposed to cold are resistant, both at the molecular and the functional level, to the actions of insulin in the hypothalamus.

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