scholarly journals Insulin down-regulates insulin receptor substrate-2 expression through the phosphatidylinositol 3-kinase/Akt pathway

2003 ◽  
Vol 179 (2) ◽  
pp. 253-266 ◽  
Author(s):  
Y Hirashima ◽  
K Tsuruzoe ◽  
S Kodama ◽  
M Igata ◽  
T Toyonaga ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Kinase Inhibitor ◽  
Insulin Response ◽  
Dominant Negative ◽  
Akt Pathway ◽  
Insulin Receptor Substrate ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Gene Assay

Insulin receptor substrate (IRS)-1 and IRS-2 are the major substrates that mediate insulin action. Insulin itself regulates the expression of the IRS protein in the liver, but the underlying mechanisms of IRS-1 and IRS-2 regulation are not fully understood. Here we report that insulin suppressed the expression of both IRS-1 and IRS-2 proteins in Fao hepatoma cells. The decrease in IRS-1 protein occurred via proteasomal degradation without any change in IRS-1 mRNA, whereas the insulin-induced suppression of IRS-2 protein was associated with a parallel decrease in IRS-2 mRNA without changing IRS-2 mRNA half-life. The insulin-induced suppression of IRS-2 mRNA and protein was blocked by the phosphatidylinositol (PI) 3-kinase inhibitor, LY294002, but not by the MAP kinase-ERK kinase (MEK) inhibitor, PD098059. Inhibition of Akt by overexpression of dominant-negative Akt also caused complete attenuation of the insulin-induced decrease in IRS-2 protein and partial attenuation of its mRNA down-regulation. Some nuclear proteins bound to the insulin response element (IRE) sequence on the IRS-2 gene in an insulin-dependent manner in vitro, and the binding was also blocked by the PI 3-kinase inhibitor. Reporter gene assay showed that insulin suppressed the activity of both human and rat IRS-2 gene promoters through the IRE in a PI 3-kinase-dependent manner. Our results indicate that insulin regulates IRS-1 and IRS-2 through different mechanisms and that insulin represses IRS-2 gene expression via a PI 3-kinase/Akt pathway.

scholarly journals Caffeine modulates phosphorylation of insulin receptor substrate-1 and impairs insulin signal transduction in rat skeletal muscle

2011 ◽  
Vol 111 (6) ◽  
pp. 1629-1636 ◽  
Author(s):  
Tatsuro Egawa ◽  
Satoshi Tsuda ◽  
Xiao Ma ◽  
Taku Hamada ◽  
Tatsuya Hayashi
Keyword(s):  
Skeletal Muscle ◽  
Insulin Receptor ◽  
Insulin Signaling ◽  
Kinase Inhibitor ◽  
Glycogen Synthase ◽  
Mammalian Target Of Rapamycin ◽  
Insulin Receptor Substrate ◽  
Dependent Manner ◽  
Insulin Signal Transduction ◽  
Physiological Dose

Caffeine decreases insulin sensitivity and insulin-stimulated glucose transport in skeletal muscle; however, the precise mechanism responsible for this deleterious effect is not understood fully. We investigated the effects of incubation with caffeine on insulin signaling in rat epitrochlearis muscle. Caffeine (≥1 mM, ≥15 min) suppressed insulin-stimulated insulin receptor substrate (IRS)-1 Tyr612 phosphorylation in a dose- and time-dependent manner. These responses were associated with inhibition of the insulin-stimulated phosphorylation of phosphatidylinositol 3-kinase (PI3K) Tyr458, Akt Ser473, and glycogen synthase kinase-3β Ser9 and with inhibition of insulin-stimulated 3- O-methyl-d-glucose (3MG) transport but not with inhibition of the phosphorylation of insulin receptor-β Tyr1158/62/63. Furthermore, caffeine enhanced phosphorylation of IRS-1 Ser307 and an IRS-1 Ser307 kinase, inhibitor-κB kinase (IKK)-α/β Ser176/180. Blockade of IKK/IRS-1 Ser307 by caffeic acid ameliorated the caffeine-induced downregulation of IRS-1 Tyr612 phosphorylation and 3MG transport. Caffeine also increased the phosphorylation of IRS-1 Ser789 and an IRS-1 Ser789 kinase, 5′-AMP-activated protein kinase (AMPK). However, inhibition of IRS-1 Ser789 and AMPK phosphorylation by dantrolene did not rescue the caffeine-induced downregulation of IRS-1 Tyr612 phosphorylation or 3MG transport. In addition, caffeine suppressed the phosphorylation of insulin-stimulated IRS-1 Ser636/639 and upstream kinases, including the mammalian target of rapamycin and p70S6 kinase. Intravenous injection of caffeine at a physiological dose (5 mg/kg) in rats inhibited the phosphorylation of insulin-stimulated IRS-1 Tyr612 and Akt Ser473 in epitrochlearis muscle. Our results indicate that caffeine inhibits insulin signaling partly through the IKK/IRS-1 Ser307 pathway, via a Ca2+- and AMPK-independent mechanism in skeletal muscle.

scholarly journals JAK2 Activates TFII-I and Regulates Its Interaction with Extracellular Signal-Regulated Kinase

2001 ◽  
Vol 21 (10) ◽  
pp. 3387-3397 ◽  
Author(s):  
Dae-Won Kim ◽  
Brent H. Cochran
Keyword(s):  
Transcription Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Serum Stimulation

ABSTRACT TFII-I is a transcription factor that shuttles between the cytoplasm and nucleus and is regulated by serine and tyrosine phosphorylation. Tyrosine phosphorylation of TFII-I can be regulated in a signal-dependent manner in various cell types. In B lymphocytes, Bruton's tyrosine kinase has been identified as a TFII-I tyrosine kinase. Here we report that JAK2 can phosphorylate and regulate TFII-I in nonlymphoid cells. The activity of TFII-I on the c-fospromoter in response to serum can be abolished by dominant negative JAK2 or the specific JAK2 kinase inhibitor AG490. Consistent with this, we have also found that JAK2 is activated by serum stimulation of fibroblasts. Tyrosine 248 of TFII-I is phosphorylated in vivo upon serum stimulation or JAK2 overexpression, and mutation of tyrosine 248 to phenylalanine inhibits the ability of JAK2 to phosphorylate TFII-I in vitro. Tyrosine 248 of TFII-I is required for its interaction with and phosphorylation by ERK and its in vivo activity on the c-fos promoter. These results indicate that the interaction between TFII-I and ERK, which is essential for its activity, can be regulated by JAK2 through phosphorylation of TFII-I at tyrosine 248. Thus, like the STAT factors, TFII-I is a direct substrate of JAK2 and a signal-dependent transcription factor that integrates signals from both tyrosine kinase and mitogen-activated protein kinase pathways to regulate transcription.

scholarly journals Extracellular Signal-Regulated Kinase Binds to TFII-I and Regulates Its Activation of the c-fosPromoter

2000 ◽  
Vol 20 (4) ◽  
pp. 1140-1148 ◽  
Author(s):  
Dae-Won Kim ◽  
Brent H. Cochran
Keyword(s):  
Map Kinase ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Binding Motif ◽  
Dependent Manner ◽  
Consensus Map ◽  
Interaction Domain ◽  
Extracellular Signal

ABSTRACT We have previously shown that TFII-I enhances transcriptional activation of the c-fos promoter through interactions with upstream elements in a signal-dependent manner. Here we demonstrate that activated Ras and RhoA synergize with TFII-I for c-fospromoter activation, whereas dominant-negative Ras and RhoA inhibit these effects of TFII-I. The Mek1 inhibitor, PD98059 abrogates the enhancement of the c-fos promoter by TFII-I, indicating that TFII-I function is dependent on an active mitogen-activated protein (MAP) kinase pathway. Analysis of the TFII-I protein sequence revealed that TFII-I contains a consensus MAP kinase interaction domain (D box). Consistent with this, we have found that TFII-I forms an in vivo complex with extracellular signal-related kinase (ERK). Point mutations within the consensus MAP kinase binding motif of TFII-I inhibit its ability to bind ERK and its ability to enhance the c-fos promoter. Therefore, the D box of TFII-I is required for its activity on the c-fos promoter. Moreover, the interaction between TFII-I and ERK can be regulated. Serum stimulation enhances complex formation between TFII-I and ERK, and dominant-negative Ras abrogates this interaction. In addition, TFII-I can be phosphorylated in vitro by ERK and mutation of consensus MAP kinase substrate sites at serines 627 and 633 impairs the phosphorylation of TFII-I by ERK and its activity on the c-fos promoter. These results suggest that ERK regulates the activity of TFII-I by direct phosphorylation.

scholarly journals Decreased IGF Type 1 Receptor Signaling in Mammary Epithelium during Pregnancy Leads to Reduced Proliferation, Alveolar Differentiation, and Expression of Insulin Receptor Substrate (IRS)-1 and IRS-2

Endocrinology ◽  
2011 ◽  
Vol 152 (8) ◽  
pp. 3233-3245 ◽  
Author(s):  
Zhaoyu Sun ◽  
Sain Shushanov ◽  
Derek LeRoith ◽  
Teresa L. Wood
Keyword(s):  
Insulin Receptor ◽  
Mammary Epithelium ◽  
Whey Acidic Protein ◽  
Dominant Negative ◽  
Acidic Protein ◽  
Normal Mammary Gland ◽  
Insulin Receptor Substrate ◽  
Alveolar Development ◽  
Pregnancy And Lactation

The IGFs and the IGF type 1 receptor (IGF-1R) are essential mediators of normal mammary gland development in mice. IGF-I and the IGF-1R have demonstrated functions in formation and proliferation of terminal end buds and in ductal outgrowth and branching during puberty. To study the functions of IGF-1R during pregnancy and lactation, we established transgenic mouse lines expressing a human dominant-negative kinase dead IGF-1R (dnhIGF-1R) under the control of the whey acidic protein promoter. We provide evidence that the IGF-1R pathway is necessary for normal epithelial proliferation and alveolar formation during pregnancy. Furthermore, we demonstrate that the whey acidic protein-dnhIGF-1R transgene causes a delay in alveolar differentiation including lipid droplet formation, lumen expansion, and β-casein protein expression. Analysis of IGF-1R signaling pathways showed a decrease in P-IGF-1R and P-Akt resulting from expression of the dnhIGF-1R. We further demonstrate that disruption of the IGF-1R decreases mammary epithelial cell expression of the signaling intermediates insulin receptor substrate (IRS)-1 and IRS-2. No alterations were observed in downstream signaling targets of prolactin and progesterone, suggesting that activation of the IGF-1R may directly regulate expression of IRS-1/2 during alveolar development and differentiation. These data show that IGF-1R signaling is necessary for normal alveolar proliferation and differentiation, in part, through induction of signaling intermediates that mediate alveolar development.

scholarly journals 4-Hydroxyisoleucine: experimental evidence of its insulinotropic and antidiabetic properties

1999 ◽  
Vol 277 (4) ◽  
pp. E617-E623 ◽  
Author(s):  
Christophe Broca ◽  
René Gross ◽  
Pierre Petit ◽  
Yves Sauvaire ◽  
Michèle Manteghetti ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Insulin Response ◽  
Oral Glucose ◽  
Dependent Manner ◽  
Insulin Dependent ◽  
Glucose Tolerance Tests ◽  
Insulin Dependent Diabetic ◽  
Single Intravenous Administration

We have recently shown in vitro that 4-hydroxyisoleucine (4-OH-Ile), an amino acid extracted from fenugreek seeds, potentiates insulin secretion in a glucose-dependent manner. The present study was designed to investigate whether 4-OH-Ile could exert in vivo insulinotropic and antidiabetic properties. For this purpose, intravenous or oral glucose tolerance tests (IVGTTs and OGTTs, respectively) were performed not only in normal animals but also in a type II diabetes rat model. During IVGTT in normal rats or OGTT in normal dogs, 4-OH-Ile (18 mg/kg) improved glucose tolerance. The lactonic form of 4-OH-Ile was ineffective in normal rats. In non-insulin-dependent diabetic (NIDD) rats, a single intravenous administration of 4-OH-Ile (50 mg/kg) partially restored glucose-induced insulin response without affecting glucose tolerance; a 6-day subchronic administration of 4-OH-Ile (50 mg/kg, daily) reduced basal hyperglycemia, decreased basal insulinemia, and slightly, but significantly, improved glucose tolerance. In vitro, 4-OH-Ile (200 μM) potentiated glucose (16.7 mM)-induced insulin release from NIDD rat-isolated islets. So, the antidiabetic effects of 4-OH-Ile on NIDD rats result, at least in part, from a direct pancreatic B cell stimulation.

Ca2+-independent contraction of longitudinal ileal smooth muscle is potentiated by a zipper-interacting protein kinase pseudosubstrate peptide

2009 ◽  
Vol 297 (2) ◽  
pp. G361-G370 ◽  
Author(s):  
Eikichi Ihara ◽  
Lori Moffat ◽  
Meredith A. Borman ◽  
Jennifer E. Amon ◽  
Michael P. Walsh ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Conformational Changes ◽  
Kinase Inhibitor ◽  
Smooth Muscles ◽  
Dominant Negative ◽  
Myosin Phosphatase ◽  
Interacting Protein ◽  
Zipper Interacting Protein Kinase

As a regulator of smooth muscle contraction, zipper-interacting protein kinase (ZIPK) can directly phosphorylate the myosin regulatory light chains (LC20) and produce contractile force. Synthetic peptides (SM-1 and AV25) derived from the autoinhibitory region of smooth muscle myosin light chain kinase can inhibit ZIPK activity in vitro. Paradoxically, treatment of Triton-skinned ileal smooth muscle strips with AV25, but not SM-1, potentiated Ca2+-independent, microcystin- and ZIPK-induced contractions. The AV25-induced potentiation was limited to ileal and colonic smooth muscles and was not observed in rat caudal artery. Thus the potentiation of Ca2+-independent contractions by AV25 appeared to be mediated by a mechanism unique to intestinal smooth muscle. AV25 treatment elicited increased phosphorylation of LC20 (both Ser-19 and Thr-18) and myosin phosphatase-targeting subunit (MYPT1, inhibitory Thr-697 site), suggesting involvement of a Ca2+-independent LC20 kinase with coincident inhibition of myosin phosphatase. The phosphorylation of the inhibitor of myosin phosphatase, CPI-17, was not affected. The AV25-induced potentiation was abolished by pretreatment with staurosporine, a broad-specificity kinase inhibitor, but specific inhibitors of Rho-associated kinase, PKC, and MAPK pathways had no effect. When a dominant-negative ZIPK [kinase-dead ZIPK(1–320)-D161A] was added to skinned ileal smooth muscle, the potentiation of microcystin-induced contraction by AV25 was blocked. Furthermore, pretreatment of skinned ileal muscle with SM-1 abolished AV25-induced potentiation. We conclude, therefore, that, even though AV25 is an in vitro inhibitor of ZIPK, activation of the ZIPK pathway occurs following application of AV25 to permeabilized ileal smooth muscle. Finally, we propose a mechanism whereby conformational changes in the pseudosubstrate region of ZIPK permit augmentation of ZIPK activity toward LC20 and MYPT1 in situ. AV25 or molecules based on its structure could be used in therapeutic situations to induce contractility in diseases of the gastrointestinal tract associated with hypomotility.

14-3-3 Facilitates Insulin-Stimulated Intracellular Trafficking of Insulin Receptor Substrate 1

2002 ◽  
Vol 16 (3) ◽  
pp. 552-562 ◽  
Author(s):  
Xiaoqin Xiang ◽  
Mingsheng Yuan ◽  
Ying Song ◽  
Neil Ruderman ◽  
Rong Wen ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Intracellular Trafficking ◽  
High Speed ◽  
Insulin Treatment ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
Integral Role ◽  
Endogenous Proteins

Abstract The appearance of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and PI3K in a high-speed pellet fraction (HSP) is thought to be a key event in insulin action. Conversely, the disappearance of the IRS-1/PI3K complex from this fraction has been linked to insulin desensitization. The present study examines the role of 14-3-3, a specific phospho-serine binding protein, in mediating the disappearance of IRS-1 from the HSP after insulin treatment. An in vitro pull-down assay using recombinant 14-3-3 revealed that insulin enhances the association of 14-3-3 with IRS-1 in cultured adipocytes and that this is completely inhibited by wortmannin. An association of IRS-1 and 14-3-3 was also observed and was maximal after stimulation by insulin, when endogenous proteins were immunoprecipitated. Epidermal growth factor (EGF), 12-O-tetradecanoylphorbol-13-acetate, and okadaic acid, other agents that cause serine/threonine phosphorylation of IRS-1, also stimulated IRS binding to 14-3-3. The enhancement of IRS-1 binding to 14-3-3 by insulin was accompanied by movement of IRS-1 and the p85 subunit of PI3K from the HSP to the cytosol. In keeping with a key role of 14-3-3 in mediating this redistribution of IRS-1, the complexes of IRS-1 and 14-3-3 were found in the cytosol but not in the HSP of insulin-treated cells. In addition, colocalization of IRS-1 and 14-3-3 was observed in the cytoplasm after insulin treatment by confocal microscopy. Finally, the addition of a phosphorylated 14-3-3 binding peptide to an adipocyte homogenate (to remove 14-3-3 from IRS-1) increased the abundance of IRS-1/PI3K complexes in the HSP and decreased their abundance in the cytosol. These findings strongly suggest that 14-3-3 participates in the intracellular trafficking of IRS-1 by promoting the displacement of serine-phosphorylated IRS-1 from particular structures. They also suggest that 14-3-3 proteins could play an integral role in the process of insulin desensitization.

Selective in vitro reversal of the insulin resistance of glucose transport in denervated rat skeletal muscle

1989 ◽  
Vol 257 (3) ◽  
pp. E418-E425 ◽  
Author(s):  
M. O. Sowell ◽  
S. L. Dutton ◽  
M. G. Buse
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Insulin Receptor ◽  
Glucose Transport ◽  
Glycogen Synthesis ◽  
Insulin Response ◽  
Medium Composition ◽  
Insulin Resistant ◽  
Denervated Muscles

Denervation (24 h) of skeletal muscle causes severe postreceptor insulin resistance of glucose transport and glycogen synthesis that is demonstrable in isolated muscles after short (30 min) preincubations. After longer preincubations (2-4 h), the insulin response of glucose transport increased to normal, whereas glycogen synthesis remained insulin resistant. Basal and insulin-stimulated amino acid transport were significantly lower in denervated muscles than in controls after short or long incubations, although the percentage stimulation of transport by insulin was not significantly different. The development of glucose transport insulin resistance after denervation was not attributable to increased sensitivity to glucocorticoids or adenosine. The selective in vitro reversal of glucose transport insulin resistance was not dependent on medium composition, did not require protein or prostaglandin synthesis, and could not be attributed to release of a positive regulator into the medium. The data suggest 1) the insulin receptor in muscle stimulates glucose transport by a signaling pathway that is not shared by other insulin-sensitive effector systems, and 2) denervation may affect insulin receptor signal transduction at more than one site.

Epithelial cell spreading induced by hepatocyte growth factor influences paxillin protein synthesis and posttranslational modification

2004 ◽  
Vol 287 (4) ◽  
pp. G886-G898 ◽  
Author(s):  
Ann M. Hopkins ◽  
Matthias Bruewer ◽  
G. Thomas Brown ◽  
A’Drian A. Pineda ◽  
Julie J. Ha ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cell ◽  
Hepatocyte Growth Factor ◽  
Fluorescent Protein ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Epithelial Migration ◽  
Cell Matrix ◽  
Hepatocyte Growth

Superficial wounds in the gastrointestinal tract rapidly reseal by coordinated epithelial cell migration facilitated by cytokines such as hepatocyte growth factor (HGF)/scatter factor released in the wound vicinity. However, the mechanisms by which HGF promotes physiological and pathophysiologic epithelial migration are incompletely understood. Using in vitro models of polarized T84 and Caco-2 intestinal epithelia, we report that HGF promoted epithelial spreading and RhoA GTPase activation in a time-dependent manner. Inducible expression of enhanced green fluorescent protein-tagged dominant-negative RhoA significantly attenuated HGF-induced spreading. HGF expanded a zone of partially flattened cells behind the wound edge containing basal F-actin fibers aligned in the direction of spreading. Concomitantly, plaques positive for the focal adhesion protein paxillin were enhanced. HGF induced an increase in the translation of paxillin and, to a lesser extent, β1-integrin. This was independent of cell-matrix adhesion through β1-integrin. Subcellular fractionation revealed increased cosedimentation of paxillin with plasma membrane-containing fractions following HGF stimulation, without corresponding enhancements in paxillin coassociation with β1 integrin or actin. Tyrosine phosphorylation of paxillin was reduced by HGF and was sensitive to the Src kinase inhibitor PP2. With these taken together, we propose that HGF upregulates a free cytosolic pool of paxillin that is unaffiliated with either the cytoskeleton or focal cell-matrix contacts. Thus early spreading responses to HGF may partly relate to increased paxillin availability for incorporation into, and turnover within, dynamic cytoskeletal/membrane complexes whose rapid and transient adhesion to the matrix drives migration.

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