Attenuation of insulin-stimulated insulin receptor substrate-1 serine 307 phosphorylation in insulin resistance of type 2 diabetes

Insulin resistance is a primary characteristic of type 2 diabetes and likely causally related to the pathogenesis of the disease. It is a result of defects in signal transduction from the cell surface receptor of insulin to target effects. We found that insulin-stimulated phosphorylation of serine 307 (corresponding to serine 302 in the murine sequence) in the immediate downstream mediator protein of the insulin receptor, insulin receptor substrate-1 (IRS1), is required for efficient insulin signaling and that this phosphorylation is attenuated in adipocytes from patients with type 2 diabetes. Inhibition of serine 307 phosphorylation by rapamycin mimicked type 2 diabetes and reduced the sensitivity of IRS1 tyrosine phosphorylation in response to insulin, while stimulation of the phosphorylation by okadaic acid, in cells from patients with type 2 diabetes, rescued cells from insulin resistance. EC(50) for insulin-stimulated phosphorylation of serine 307 was about 0.2 nM with a t(1/2) of about 2 min. The amount of IRS1 was similar in cells from non-diabetic and diabetic subjects. These findings identify a molecular mechanism for insulin resistance in non-selected patients with type 2 diabetes.     

Phosphorylation of IRS1 at Serine 307 in Response to Insulin in Human Adipocytes Is Not Likely to be Catalyzed by p70 Ribosomal S6 Kinase

The insulin receptor substrate-1 (IRS1) is phosphorylated on serine 307 (human sequence, corresponding to murine serine 302) in response to insulin as part of a feedback loop that controls IRS1 phosphorylation on tyrosine residues by the insulin receptor. This in turn directly affects downstream signaling and is in human adipocytes implicated in the pathogenesis of insulin resistance and type 2 diabetes. The phosphorylation is inhibited by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). The mTORC1-downstream p70 ribosomal protein S6 kinase (S6K1), which is activated by insulin, can phosphorylate IRS1 at serine 307 in vitro and is considered the physiological protein kinase. Because the IRS1 serine 307-kinase catalyzes a critical step in the control of insulin signaling and constitutes a potential target for treatment of insulin resistance, it is important to know whether S6K1 is the physiological serine 307-kinase or not. We report that, by several criteria, S6K1 does not phosphorylate IRS1 at serine 307 in response to insulin in intact human primary adipocytes: (i) The time-courses for phosphorylation of S6K1 and its phosphorylation of S6 are not compatible with the phosphorylation of IRS1 at serine 307; (ii) A dominant-negative construct of S6K1 inhibits the phosphorylation of S6, without effect on the phosphorylation of IRS1 at serine 307; (iii) The specific inhibitor of S6K1 PF-4708671 inhibits the phosphorylation of S6, without effect on phosphorylation of IRS1 at serine 307. mTOR-immunoprecipitates from insulin-stimulated adipocytes contains an unidentified protein kinase specific for phosphorylation of IRS1 at serine 307, but it is not mTOR or S6K1.     

Turnover of the active fraction of IRS1 involves raptor-mTOR- and S6K1-dependent serine phosphorylation in cell culture models of tuberous sclerosis

The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.     

Mammalian target of rapamycin regulates IRS-1 serine 307 phosphorylation

Insulin signaling can be negatively regulated by phosphorylation of serine 307 of the insulin receptor substrate (IRS)-1. Rapamycin, an inhibitor of the kinase mTOR, can prevent serine 307 phosphorylation and the development of insulin resistance. We further investigated the role of mTOR in regulating serine 307 phosphorylation, demonstrating that serine 307 phosphorylation in response to insulin, anisomycin, or tumor necrosis factor was quantitatively and temporally associated with activation of mTOR and could be inhibited by rapamycin. Amino acid stimulation activated mTOR and resulted in IRS-1 serine 307 phosphorylation without activating PKB or JNK. Okadaic acid, an inhibitor of the phosphatase PP2A, activated mTOR and stimulated the phosphorylation of serine 307 in a rapamycin-sensitive manner, indicating serine 307 phosphorylation requires mTOR activity but not PP2A, suggesting that mTOR itself may be responsible for phosphorylating serine 307. Finally, we demonstrated that serine 307 phosphorylated IRS-1 is detected primarily in the cytosolic fraction.     

Insulin receptor serine kinase activation by casein kinase 2 and a membrane tyrosine kinase

The insulin receptor (IR) tyrosine kinase can apparently directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor serine (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphorylation of the membrane glycoproteins by casein kinase-1, casein kinase-2, or casein kinase-3 (Biochem Biophys Res Commun 171:75–83, 1990). In this study, IRS kinase was further characterized. The presence of vanadate or phosphotyrosine in reaction mixtures was required for activation to be observed. Phosphoserine and phosphothreonine are only about 25% as effective as phosphotyrosine, whereas sodium fluoride and molybdate were ineffective in supporting activation. Vanadate and phosphotyrosine support IRS kinase activation by apparently inhibiting phosphotyrosine protein phosphatases present among the membrane glycoproteins. IR -subunit, myelin basic protein, and microtubule-associated protein-2 are good substrates for IRS kinase. The kinase prefers Mn²⁺ (K_a=1.3 mM) as a metal cofactor. Mg²⁺ (K_a=3.3 mM) is only 30% as effective as Mn²⁺. The kinase activity is stimulated by basic polypeptides, with greater than 30-fold activation achieved with polylysine and protamine. Our results suggest that both serine/threonine and tyrosine phosphorylation are required for activation of IRS kinase. Serine phosphorylation is catalyzed by one of the casein kinases, whereas tyrosine phosphorylation is catalyzed by a membrane tyrosine kinase, possibly IR tyrosine kinase. (Mol Cell Biochem121: 167–174, 1993)     

Regulation of insulin receptor substrate 1 pleckstrin homology domain by protein kinase C: role of serine 24 phosphorylation

Phosphorylation of insulin receptor substrate (IRS) proteins on serine residues is an important posttranslational modification that is linked to insulin resistance. Several phosphoserine sites on IRS1 have been identified; the majority are located proximal to the phosphotryosine-binding domain or near key receptor tyrosine kinase substrate- and/or Src-homology 2 domain-binding sites. Here we report on the characterization of a serine phosphorylation site in the N-terminal pleckstrin homology (PH) domain of IRS1. Bioinformatic tools identify serine 24 (Ser24) as a putative substrate site for the protein kinase C (PKC) family of serine kinases. We demonstrate that this site is indeed a bona fide substrate for conventional PKC. In vivo, IRS-1 is also phosphorylated on Ser24 after phorbol 12-myristate 13-acetate treatment of cells, and isoform-selective inhibitor studies suggest the involvement of PKCalpha. By comparing the pharmacological characteristics of phorbol 12-myristate 13-acetate-stimulated Ser24 phosphorylation with phosphorylation at two other sites previously linked to PKC activity (Ser307 and Ser612), we show that PKCalpha is likely to be directly involved in Ser24 phosphorylation, but indirectly involved in Ser307 and Ser612 phosphorylation. Using Ser24Asp IRS-1 mutants to mimic the phosphorylated residue, we demonstrate that the phosphorylation status of Ser24 does play an important role in regulating phosphoinositide binding to, and the intracellular localization of, the IRS1-PH domain, which can ultimately impinge on insulin-stimulated glucose uptake. Hence we provide evidence that IRS1-PH domain function is important for normal insulin signaling and is regulated by serine phosphorylation in a manner that could contribute to insulin resistance.     

Effect of hyperglycemia on insulin receptor signaling in the cultured retinal Müller glial cells

Hyperglycemia and impaired insulin signaling are considered as major factors in the retinal pathology in diabetic retinopathy (DR). Numerous reports support that these two factors damage retinal glial as well as neuronal cells early in diabetes. However, it is not known whether diabetic induced hyperglycemia causes a depression to the insulin signaling. In this study we utilized a well characterized cultured Muller cells (TR-MUL) where we found a high expression of insulin receptor molecules. TR-MUL Cells were treated with high glucose, glutamate and hydrogen peroxide, and activated with insulin. Following treatments, cell lysates were analyzed by immunoblotting experiments for insulin receptor (IRβ) and insulin receptor substrate (IRS1). In addition, cell lysates were immunoprecipitated using antibodies against insulin receptor proteins to analyze tyrosine phosphorylation and serine phosphorylation of insulin receptor proteins. Results indicate that hyperglycemia did not affect the expression of insulin receptor proteins in cultured TR-MUL cells. Although, hyperglycemia seems to inhibit the interaction between IRS1 and IRβ. Hydrogen peroxide increased the tyrosine phosphorylation of insulin receptor proteins but excess glutamate could not affect the insulin receptor proteins indicating that glutamate may not cause oxidative stress in TR-MUL cells. Hyperglycemia lowered serine phosphorylation of IRS^ser632 and IRS^ser1101 however, IRS^ser307 was not affected. Thus, hyperglycemia may not affect insulin signaling through tyrosine phosphorylation of insulin receptor proteins but may inhibit the interactions between insulin receptor proteins. Hyperglycemia induced phosphorylation of various serine residues of IRS1 and their influence on insulin signaling needs further investigation in TR-MUL cells.     

c-Jun N-terminal kinase (JNK) mediates feedback inhibition of the insulin signaling cascade

Activation of the c-Jun N-terminal kinase (JNK) by proinflammatory cytokines inhibits insulin signaling, at least in part, by stimulating phosphorylation of rat/mouse insulin receptor substrate 1 (Irs1) at Ser(307) (Ser(312) in human IRS1). Here we show that JNK mediated feedback inhibition of the insulin signal in mouse embryo fibroblasts, 3T3-L1 adipocytes, and 32D(IR) cells. Insulin stimulation of JNK activity required phosphatidylinositol 3-kinase and Grb2 signaling. Moreover, activation of JNK by insulin was inhibited by a cell-permeable peptide that disrupted the interaction of JNK with cellular proteins. However, the direct binding of JNK to Irs1 was not required for its activation by insulin, whereas direct binding was required for Ser(307) phosphorylation of Irs1. Insulin-stimulated Ser(307) phosphorylation was reduced 80% in cells lacking JNK1 and JNK2 or in cells expressing a mutant Irs1 protein lacking the JNK binding site. Reduced Ser(307) phosphorylation was directly related to increased insulin-stimulated tyrosine phosphorylation, Akt phosphorylation, and glucose uptake. These results support the hypothesis that JNK is a negative feedback regulator of insulin action by phosphorylating Ser(307) in Irs1.     

RhoA/Rho kinase blocks muscle differentiation via serine phosphorylation of insulin receptor substrate-1 and -2

Although the RhoA/Rho kinase (RhoA/ROK) pathway has been extensively investigated, its roles and downstream signaling pathways are still not well understood in myogenic processes. Therefore, we examined the effects of RhoA/ROK on myogenic processes and their signaling molecules using H9c2 and C2C12 cells. Increases in RhoA/ROK activities and serine phosphorylation levels of insulin receptor substrate (IRS)-1 (Ser307 and Ser636/639) and IRS-2 were found in proliferating myoblasts, whereas IRS-1/2 tyrosine phosphorylation and phosphatidylinositol (PI) 3-kinase activity increased during the differentiation process. ROK strongly bound to IRS-1/2 in proliferation medium but dissociated from them in differentiation medium (DM). ROK inactivation by a ROK inhibitor, Y27632, or a dominant-negative ROK, decreased IRS-1/2 serine phosphorylation with increases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity, which led to muscle differentiation even in proliferation medium. Inhibition of ROK also enhanced differentiation in DM. ROK activation by a constitutive active ROK blocked muscle differentiation with the increased IRS-1/2 serine phosphorylation, followed by decreases in IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity in DM. Interestingly, fibroblast growth factor-2 added to DM also blocked muscle differentiation through RhoA/ROK activation. Fibroblast growth factor-2 blockage of muscle differentiation was reversed by Y27632. Collectively, these results suggest that the RhoA/ROK pathway blocks muscle differentiation by phosphorylating IRS proteins at serine residues, resulting in the decreased IRS-1/2 tyrosine phosphorylation and PI 3-kinase activity. The absence of the inhibitory effects of RhoA/ROK in DM due to low concentrations of myogenic inhibitory growth factors seems to allow IRS-1/2 tyrosine phosphorylation, which stimulates muscle differentiation via transducing normal myogenic signaling.     

The c-Jun NH(2)-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser(307)

Tumor necrosis factor alpha (TNFalpha) inhibits insulin action, in part, through serine phosphorylation of IRS proteins; however, the phosphorylation sites that mediate the inhibition are unknown. TNFalpha promotes multipotential signal transduction cascades, including the activation of the Jun NH(2)-terminal kinase (JNK). Endogenous JNK associates with IRS-1 in Chinese hamster ovary cells. Anisomycin, a strong activator of JNK in these cells, stimulates the activity of JNK bound to IRS-1 and inhibits the insulin-stimulated tyrosine phosphorylation of IRS-1. Serine 307 is a major site of JNK phosphorylation in IRS-1. Mutation of serine 307 to alanine eliminates phosphorylation of IRS-1 by JNK and abrogates the inhibitory effect of TNFalpha on insulin-stimulated tyrosine phosphorylation of IRS-1. These results suggest that phosphorylation of serine 307 might mediate, at least partially, the inhibitory effect of proinflammatory cytokines like TNFalpha on IRS-1 function.     

The effects of intracellular calcium depletion on insulin signaling in 3T3-L1 adipocytes

We have examined the requirement for intracellular calcium (Ca(2+)) in insulin signal transduction in 3T3-L1 adipocytes. Using the Ca(2+) chelator 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, sodium (BAPTA-AM), we find both augmentation and inhibition of insulin signaling phenomena. Pretreatment of cells with 50 microM BAPTA-AM did not affect tyrosine phosphorylation of insulin receptor substrate (IRS)1/2 or insulin receptor (IR)beta. The decreased mobility of IRS1 normally observed after chronic stimulation with insulin, due to serine phosphorylation, was completely eliminated by Ca(2+) chelation. Correlating with decreased insulin-induced serine phosphorylation of IRS1, phosphotyrosine-mediated protein-protein interactions involving p85, IRS1, IRbeta, and phosphotyrosine-specific antibody were greatly enhanced by pretreatment of cells with BAPTA-AM. As a result, insulin-mediated, phosphotyrosine-associated PI3K activity was also enhanced. BAPTA-AM pretreatment inhibited other insulin-induced phosphorylation events including phosphorylation of Akt, MAPK (ERK1 and 2) and p70 S6K. Phosphorylation of Akt on threonine-308 was more sensitive to Ca(2+) depletion than phosphorylation of Akt on serine-473 at the same insulin dose (10 nM). In vitro 3'-phosphatidylinositol-dependent kinase 1 activity was unaffected by BAPTA-AM. Insulin-stimulated insulin-responsive glucose transporter isoform translocation and glucose uptake were both inhibited by calcium depletion. In summary, these data demonstrate a positive role for intracellular Ca(2+) in distal insulin signaling events, including initiation/maintenance of Akt phosphorylation, insulin-responsive glucose transporter isoform translocation, and glucose transport. A negative role for Ca(2+) is also indicated in proximal insulin signaling steps, in that, depletion of intracellular Ca(2+) blocks IRS1 serine/threonine phosphorylation and enhances insulin-stimulated protein-protein interaction and PI3K activity.     

IκB kinase β (IKKβ) does not mediate feedback inhibition of the insulin signalling cascade

In the present study, we have examined whether IKKβ [IκB (inhibitor of nuclear factor κB) kinase β] plays a role in feedback inhibition of the insulin signalling cascade. Insulin induces the phosphorylation of IKKβ, in vitro and in vivo, and this effect is dependent on intact signalling via PI3K (phosphoinositide 3-kinase), but not PKB (protein kinase B). To test the hypothesis that insulin activates IKKβ as a means of negative feedback, we employed a variety of experimental approaches. First, pharmacological inhibition of IKKβ via BMS-345541 did not potentiate insulin-induced IRS1 (insulin receptor substrate 1) tyrosine phosphorylation, PKB phosphorylation or 2-deoxyglucose uptake in differentiated 3T3-L1 adipocytes. BMS-345541 did not prevent insulin-induced IRS1 serine phosphorylation on known IKKβ target sites. Secondly, adenovirus-mediated overexpression of wild-type IKKβ in differentiated 3T3-L1 adipocytes did not suppress insulin-stimulated 2-deoxyglucose uptake, IRS1 tyrosine phosphorylation, IRS1 association with the p85 regulatory subunit of PI3K or PKB phosphorylation. Thirdly, insulin signalling was not potentiated in mouse embryonic fibroblasts lacking IKKβ. Finally, insulin treatment of 3T3-L1 adipocytes did not promote the recruitment of IKKβ to IRS1, supporting our findings that IKKβ, although activated by insulin, does not promote direct serine phosphorylation of IRS1 and does not contribute to the feedback inhibition of the insulin signalling cascade.     

Regulation of ERK5 by insulin and angiotensin-II in vascular smooth muscle cells

ERK5 is involved in proliferation of vascular smooth muscle cells (VSMC). The proliferative actions of insulin and angiotensin-II (A-II) in VSMC are mediated in part by ERK1/2. We hypothesized that insulin and A-II also regulate ERK5 activity in VSMC. Acute treatment (<60min) with insulin or A-II increased phosphorylation of ERK1/2 at 15min and ERK5 at 5min. Chronic treatment (< or = 8h) with insulin increased ERK1/2 phosphorylation by 4h and ERK5 by 8h. A-II-stimulated phosphorylation of ERK1/2 by 8h and ERK5 by 4h. The EC(50) for insulin treatment effecting ERK1/2 and ERK5 phosphorylation was 1.5 and 0.1nM, whereas the EC(50) for A-II was 2nM, each. Insulin plus A-II induced an additive effect only on ERK5 phosphorylation. Inhibition of insulin- and A-II-stimulated phosphorylation of ERK5 and ERK1/2 by PD98059 and Wortmannin exhibited differential and time-dependent effects. Taken together, these data indicate that insulin and A-II regulate the activity of ERK5, but different from that seen for ERK1/2.     

Aspirin inhibits serine phosphorylation of IRS-1 in muscle and adipose tissue of septic rats

Whole body insulin resistance has been demonstrated in septic patients and in infected animals. In this study, we demonstrate that sepsis induces insulin resistance and that pretreatment with aspirin inhibits sepsis-induced insulin resistance. Sepsis was observed to lead to serine phosphorylation of IRS-1, a phenomenon which was reversed by aspirin in muscle and WAT, in parallel with a reduction in JNK activity. In addition, our data show an impairment of insulin activation of IR and IRS-1 tyrosine phosphorylation in septic rats and, consistent with the reduction of IRS-1 serine phosphorylation observed in septic animals pretreated with aspirin, there was an increase in IRS-1 protein levels and tyrosine phosphorylation in muscle and WAT. Overall, these results provide important new insights into the mechanism of sepsis-induced insulin resistance.     

Intracellular zinc fluctuations modulate protein tyrosine phosphatase activity in insulin/insulin-like growth factor-1 signaling

Zinc is an effector of insulin/IGF-1 signaling and has insulinomimetic effects, the molecular basis of which is not understood. The present study establishes the capacity of zinc to inhibit protein tyrosine phosphatases (PTPs) as a cause for these effects and, moreover, demonstrates modulation of the insulin response by changes in intracellular zinc. The inhibition of PTPs by zinc occurs at significantly lower concentrations than previously reported. In vitro, zinc inhibits PTPs 1B and SHP-1 with IC(50) values of 17 and 93 nM, respectively. A fluorescent probe with a similar binding constant [FluoZin-3, K(D)(Zn) = 15 nM] detects corresponding concentrations of zinc within cells. Increase of cellular zinc after incubation with both zinc and the ionophore pyrithione augments protein tyrosine phosphorylation, and in particular the phosphorylation of three activating tyrosine residues of the insulin/IGF-1 receptor. Vice versa, specific chelation of cellular zinc with the membrane-permeable N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine suppresses insulin- and IGF-1-stimulated phosphorylation. In the context of the emerging concept that intracellular zinc is tightly regulated and fluctuates dynamically, these results suggest that a pool of cellular zinc modulates phosphorylation signaling.     

Aspirin inhibits serine phosphorylation of insulin receptor substrate 1 in tumor necrosis factor-treated cells through targeting multiple serine kinases

The hypoglycemic effects of high dose salicylates in the treatment of diabetes were documented before the advent of insulin. However, the molecular mechanisms by which salicylates exert these anti-diabetic effects are not well understood. In this study, we analyzed the effects of aspirin (acetylsalicylic acid) on serine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells treated with tumor necrosis factor (TNF)-alpha. Phosphorylation of IRS-1 at Ser307, Ser267, and Ser612 was monitored by immunoblotting with phospho-specific IRS-1 antibodies. In 3T3-L1 and Hep G2 cells, phosphorylation of IRS-1 at Ser307 in response to TNF-alpha treatment correlated with phosphorylation of JNK, c-Jun, and degradation of IkappaBalpha. Moreover, phosphorylation of IRS-1 at Ser307 in embryo fibroblasts derived from either JNK or IKK knockout mice was reduced when compared with that in the wild-type controls. Taken together, these data suggest that serine phosphorylation of IRS-1 in response to TNF-alpha is mediated, in part, by JNK and IKK. Interestingly, aspirin treatment inhibited the phosphorylation of IRS-1 at Ser307 as well as the phosphorylation of JNK, c-Jun, and degradation of IkappaBalpha. Furthermore, other serine kinases including Akt, extracellular regulated kinase, mammalian target of rapamycin, and PKCzeta were also activated by TNF-alpha (as assessed by phospho-specific antibodies). Phosphorylation of IRS-1 at Ser267 and Ser612 correlated with the activation of these kinases. Phosphorylation of Akt and the mammalian target of rapamycin (but not extracellular regulated kinase or PKCzeta) in response to TNF-alpha was inhibited by aspirin treatment. Finally, aspirin rescued insulin-induced glucose uptake in 3T3-L1 adipocytes pretreated with TNF-alpha. We conclude that aspirin may enhance insulin sensitivity by protecting IRS proteins from serine phosphorylation catalyzed by multiple kinases.     

Insulin and Metabolic Stress Stimulate Multisite Serine/Threonine Phosphorylation of Insulin Receptor Substrate 1 and Inhibit Tyrosine Phosphorylation

IRS1 and IRS2 are key substrates of the insulin receptor tyrosine kinase. Mass spectrometry reveals more than 50 phosphorylated IRS1 serine and threonine residues (Ser(P)/Thr(P) residues) in IRS1 from insulin-stimulated cells or human tissues. We investigated a subset of IRS1 Ser(P)/Thr(P) residues using a newly developed panel of 25 phospho-specific monoclonal antibodies (αpS/TmAb^Irs1). CHO cells overexpressing the human insulin receptor and rat IRS1 were stimulated with insulin in the absence or presence of inhibitors of the PI3K → Akt → mechanistic target of rapamycin (mTOR) → S6 kinase or MEK pathways. Nearly all IRS1 Ser(P)/Thr(P) residues were stimulated by insulin and significantly suppressed by PI3K inhibition; fewer were suppressed by Akt or mTOR inhibition, and none were suppressed by MEK inhibition. Insulin-stimulated Irs1 tyrosine phosphorylation (Tyr(P)^Irs1) was enhanced by inhibition of the PI3K → Akt → mTOR pathway and correlated with decreased Ser(P)-302^Irs1, Ser(P)-307^Irs1, Ser(P)-318^Irs1, Ser(P)-325^Irs1, and Ser(P)-346^Irs1. Metabolic stress modeled by anisomycin, thapsigargin, or tunicamycin increased many of the same Ser(P)/Thr(P) residues as insulin, some of which (Ser(P)-302^Irs1, Ser(P)-307^Irs1, and four others) correlated significantly with impaired insulin-stimulated Tyr(P)^Irs1. Thus, IRS1 Ser(P)/Thr(P) is an integrated response to insulin stimulation and metabolic stress, which associates with reduced Tyr(P)^Irs1 in CHO^IR/IRS1 cells.     

Indinavir and nelfinavir inhibit proximal insulin receptor signaling and salicylate abrogates inhibition: Potential role of the NFkappa B pathway

The molecular basis of insulin resistance induced by HIV protease inhibitors (HPIs) remains unclear. In this study, Chinese hamster ovary cells transfected with high levels of human insulin receptor (CHO‐IR) and 3T3‐L1 adipocytes were used to elucidate the mechanism of this side effect. Indinavir and nelfinavir induced a significant decrease in tyrosine phosphorylation of the insulin receptor β‐subunit. Indinavir caused a significant increase in the phosphorylation of insulin receptor substrate‐1 (IRS‐1) on serine 307 (S307) in both CHO‐IR cells and 3T3‐L1 adipocytes. Nelfinavir also inhibited phosphorylation of Map/ERK kinase without affecting insulin‐stimulated Akt phosphorylation. Concomitantly, levels of protein tyrosine phosphatase 1B (PTP1B), suppressor of cytokines signaling‐1 and ‐3 (SOCS‐1 and ‐3), Src homology 2B (SH2B) and adapter protein with a pleckstrin homology domain and an SH2 domain (APS) were not altered significantly. When CHO‐IR cells were pre‐treated with sodium salicylate (NaSal), the effects of indinavir on tyrosine phosphorylation of the IR β‐subunit and phosphorylation of IRS‐1 at S307 were abrogated. These data suggest a potential role for the NFκB pathway in insulin resistance induced by HPIs. J. Cell. Biochem. 114: 1729–1737, 2013. © 2013 Wiley Periodicals, Inc.     

Insulin receptor substrates 1 and 2 but not Shc can activate the insulin receptor independent of insulin and induce proliferation in CHO-IR cells

Ligand-activated insulin receptor (IR) attracts and phosphorylates various substrates such as insulin receptor substrates 1-4 (IRS) and Shc. To investigate how binding affinity for substrate affects signalling we generated chimeric receptors with the beta-chain of the insulin receptor containing NPXY motives with different affinities for receptor substrates. We found that the extent of receptor tyrosine phosphorylation positively correlates with binding affinity towards IRS1/2 but not towards Shc. Moreover, overexpression of IRS1 or IRS2 but not of Shc increased IR tyrosine phosphorylation in a dose-dependent manner, also independent of insulin. Molecular truncations of IRS1 revealed that neither the isolated PH and PTB domains nor the C-terminus with the tyrosine phosphorylation sites alone are sufficient for substrate-dependent receptor activation. Overexpression of IRS1 and IRS2 impaired insulin-induced internalization of the IR in a dose-dependent manner suggesting that IRS proteins prevent endosome-associated receptor dephosphorylation/inactivation. IRS1 and IRS2 could therefore target the activated IR to different cellular compartments. Overexpression of IRS1 and IRS2 inhibited insulin-stimulated activation of the MAP kinases Erk1/2 while it increased/induced activation of Akt/PKB. Finally, overexpression of IRS1 and IRS2 but not of Shc induced DNA synthesis in starved CHO-IR cells independent of exogenous growth factors. Our results demonstrate that variations in cellular IRS1 and IRS2 concentration affect insulin signalling both upstream and downstream and that IRS proteins could play instructive rather than just permissive roles in signal transmission.     

Transactivation of ErbB2 and ErbB3 by tumor necrosis factor-alpha and anisomycin leads to impaired insulin signaling through serine/threonine phosphorylation of IRS proteins.

The cellular pathways involved in the impairment of insulin signaling by cellular stress, triggered by the inflammatory cytokine tumor necrosis factor-alpha (TNF) or by translational inhibitors like cycloheximide and anisomycin were studied. Similar to TNF, cycloheximide and anisomycin stimulated serine phosphorylation of IRS-1 and IRS-2, reduced their ability to interact with the insulin receptor, inhibited the insulin-induced tyrosine phosphorylation of IRS proteins, and diminished their association with phosphatidylinositol 3-kinase (PI3K). These defects were partially reversed by wortmannin and LY294002, indicating that a PI3K-regulated step is critical for the impairment of insulin signaling by cellular stress. Induction of cellular stress resulted in complex formation between PI3K and ErbB2/ErbB3 and enhanced PI3K activity, implicating ErbB proteins as downstream effectors of stress-induced insulin resistance. Indeed, stimulation of ErbB2/ErbB3 by NDFbeta1, the ErbB3 ligand, inhibited IRS protein tyrosine phosphorylation and recruitment of downstream effectors. Specific inhibitors of the ErbB2 tyrosine kinase abrogated the activation of ErbB2/ErbB3 and in parallel prevented the reduction in IRS protein functions. Taken together, our results suggest a novel mechanism by which cellular stress induces cross-talk between two different signaling pathways. Stress-dependent transactivation of ErbB2/ErbB3 receptors triggers a PI3K cascade that induces serine phosphorylation of IRS proteins culminating in insulin resistance.