scholarly journals Reciprocal feedback regulation of insulin receptor and insulin receptor substrate tyrosine phosphorylation by phosphoinositide 3-kinase in primary adipocytes

2002 ◽  
Vol 368 (3) ◽  
pp. 875-884 ◽  
Author(s):  
Ingeborg HERS ◽  
Christopher J. BELL ◽  
Alastair W. POOLE ◽  
Donyang JIANG ◽  
Richard M. DENTON ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Binding Site ◽  
Tyrosine Phosphorylation ◽  
Binding Sites ◽  
Specific Binding ◽  
Sh2 Domain ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Tyrosine Kinase ◽  
Irs Proteins ◽  
Phosphoinositide 3 Kinase

Signalling by the insulin receptor substrate (IRS) proteins is critically dependent on the tyrosine phosphorylation of specific binding sites that recruit Src homology 2 (SH2)-domain-containing proteins, such as the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase), the tyrosine phosphatase SHP-2 and the adapter protein Grb2. Here we show that stimulation by insulin of freshly isolated primary adipocytes resulted in the expected rapid tyrosine phosphorylation of the insulin receptor, IRS-1 and IRS-3. Inhibition of PI 3-kinase enhanced the insulin-stimulated phosphorylation of IRS-1 on (i) Tyr612 and Tyr941 (p85 binding sites), concomitant with an increased association of the p85 subunit of PI 3-kinase; (ii) Tyr896 (a Grb2 binding site); and (iii) Tyr1229 (an SHP-2 binding site), although little or no binding of SHP-2 to IRS-1 was detectable under any conditions. In contrast, inhibition of PI 3-kinase led to a decrease in insulin-stimulated p85 binding to IRS-3, but had no effect on SHP-2 binding. Furthermore, insulin-induced insulin receptor tyrosine phosphorylation, phosphorylation of Tyr1158 and insulin receptor tyrosine kinase activity were all reduced by inhibition of PI 3-kinase at later time points (20min). The results demonstrate that, in primary adipocytes, PI 3-kinase feedback control of signalling by the insulin receptor and IRS proteins is multifaceted and reciprocal, illustrating the complexity of predicting the net flux of the insulin signal(s) through the IRS proteins.

scholarly journals Insulin Receptor Substrate 3 (IRS-3) and IRS-4 Impair IRS-1- and IRS-2-Mediated Signaling

2001 ◽  
Vol 21 (1) ◽  
pp. 26-38 ◽  
Author(s):  
Kaku Tsuruzoe ◽  
Renee Emkey ◽  
Kristina M. Kriauciunas ◽  
Kohjiro Ueki ◽  
C. Ronald Kahn
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Cell Lines ◽  
Protein Level ◽  
Fibroblast Cell ◽  
Host Cells ◽  
Mitogen Activated Protein Kinase ◽  
Insulin Receptor Substrate ◽  
Akt Phosphorylation ◽  
Irs Proteins

ABSTRACT To investigate the roles of insulin receptor substrate 3 (IRS-3) and IRS-4 in the insulin-like growth factor 1 (IGF-1) signaling cascade, we introduced these proteins into 3T3 embryonic fibroblast cell lines prepared from wild-type (WT) and IRS-1 knockout (KO) mice by using a retroviral system. Following transduction of IRS-3 or IRS-4, the cells showed a significant decrease in IRS-2 mRNA and protein levels without any change in the IRS-1 protein level. In these cell lines, IGF-1 caused the rapid tyrosine phosphorylation of all four IRS proteins. However, IRS-3- or IRS-4-expressing cells also showed a marked decrease in IRS-1 and IRS-2 phosphorylation compared to the host cells. This decrease was accounted for in part by a decrease in the level of IRS-2 protein but occurred with no significant change in the IRS-1 protein level. IRS-3- or IRS-4-overexpressing cells showed an increase in basal phosphatidylinositol 3-kinase activity and basal Akt phosphorylation, while the IGF-1-stimulated levels correlated well with total tyrosine phosphorylation level of all IRS proteins in each cell line. IRS-3 expression in WT cells also caused an increase in IGF-1-induced mitogen-activated protein kinase phosphorylation and egr-1 expression (∼1.8- and ∼2.4-fold with respect to WT). In the IRS-1 KO cells, the impaired mitogenic response to IGF-1 was reconstituted with IRS-1 to supranormal levels and was returned to almost normal by IRS-2 or IRS-3 but was not improved by overexpression of IRS-4. These data suggest that IRS-3 and IRS-4 may act as negative regulators of the IGF-1 signaling pathway by suppressing the function of other IRS proteins at several steps.

scholarly journals The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis.

1996 ◽  
Vol 16 (5) ◽  
pp. 2509-2517 ◽  
Author(s):  
L Yenush ◽  
R Fernandez ◽  
M G Myers ◽  
T C Grammer ◽  
X J Sun ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Human Insulin ◽  
Mammalian Cells ◽  
Insulin Response ◽  
Mitogen Activated Protein Kinase ◽  
Insulin Receptor Substrate ◽  
Human Insulin Receptor ◽  
Irs Proteins ◽  
Drosophila Insulin Receptor

The Drosophila insulin receptor (DIR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins which binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric DIR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulated tyrosine autophosphorylation of the human insulin receptor and hDIR, and both receptors mediated tyrosine phosphorylation of Shc and activated mitogen-activated protein kinase. IRS-1 was required by the human insulin receptor to activate PI 3-kinase and p70s6k, whereas hDIR associated with PI 3-kinase and activated p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the DIR possesses additional signaling capabilities compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells.

scholarly journals Identification of SH2B2β as an Inhibitor for SH2B1- and SH2B2α-Promoted Janus Kinase-2 Activation and Insulin Signaling

Endocrinology ◽  
2007 ◽  
Vol 148 (4) ◽  
pp. 1615-1621 ◽  
Author(s):  
Minghua Li ◽  
Zhiqin Li ◽  
David L. Morris ◽  
Liangyou Rui
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Insulin Signaling ◽  
Sh2 Domain ◽  
Janus Kinase ◽  
Pleckstrin Homology Domain ◽  
Insulin Receptor Substrate ◽  
Janus Kinase 2 ◽  
Pleckstrin Homology ◽  
Insulin Receptor Substrate 1

The SH2B family has three members (SH2B1, SH2B2, and SH2B3) that contain conserved dimerization (DD), pleckstrin homology, and SH2 domains. The DD domain mediates the formation of homo- and heterodimers between members of the SH2B family. The SH2 domain of SH2B1 (previously named SH2-B) or SH2B2 (previously named APS) binds to phosphorylated tyrosines in a variety of tyrosine kinases, including Janus kinase-2 (JAK2) and the insulin receptor, thereby promoting the activation of JAK2 or the insulin receptor, respectively. JAK2 binds to various members of the cytokine receptor family, including receptors for GH and leptin, to mediate cytokine responses. In mice, SH2B1 regulates energy and glucose homeostasis by enhancing leptin and insulin sensitivity. In this work, we identify SH2B2β as a new isoform of SH2B2 (designated as SH2B2α) derived from the SH2B2 gene by alternative mRNA splicing. SH2B2β has a DD and pleckstrin homology domain but lacks a SH2 domain. SH2B2β bound to both SH2B1 and SH2B2α, as demonstrated by both the interaction of glutathione S-transferase-SH2B2β fusion protein with SH2B1 or SH2B2α in vitro and coimmunoprecipitation of SH2B2β with SH2B1 or SH2B2α in intact cells. SH2B2β markedly attenuated the ability of SH2B1 to promote JAK2 activation and subsequent tyrosine phosphorylation of insulin receptor substrate-1 by JAK2. SH2B2β also significantly inhibited SH2B1- or SH2B2α-promoted insulin signaling, including insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1. These data suggest that SH2B2β is an endogenous inhibitor of SH2B1 and/or SH2B2α, negatively regulating insulin signaling and/or JAK2-mediated cellular responses.

scholarly journals Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1.

1993 ◽  
Vol 13 (12) ◽  
pp. 7418-7428 ◽  
Author(s):  
X J Sun ◽  
D L Crimmins ◽  
M G Myers ◽  
M Miralpeix ◽  
M F White
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Regulatory Elements ◽  
Sh2 Domain ◽  
Insulin Receptor Substrate ◽  
Insulin Stimulation ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
Amino Terminal

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.

scholarly journals Mechanism of feedback regulation of insulin receptor substrate-1 phosphorylation in primary adipocytes

2005 ◽  
Vol 388 (2) ◽  
pp. 713-720 ◽  
Author(s):  
Ingeborg HERS ◽  
Jeremy M. TAVARÉ
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Mammalian Target Of Rapamycin ◽  
Feedback Regulation ◽  
Serine Phosphorylation ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
P70s6 Kinase ◽  
Mitogen Activated Protein ◽  
Phosphoinositide 3 Kinase

Serine and threonine phosphorylation of IRS-1 (insulin receptor substrate-1) has been reported to decrease its ability to be tyrosine-phosphorylated by the insulin receptor. Insulin itself may negatively regulate tyrosine phosphorylation of IRS-1 through a PI3K (phosphoinositide 3-kinase)-dependent feedback pathway. In the present study, we examined the regulation and role of IRS-1 serine phosphorylation in the modulation of IRS-1 tyrosine phosphorylation in physiologically relevant cells, namely freshly isolated primary adipocytes. We show that insulin-stimulated phosphorylation of Ser312 and Ser616 in IRS-1 was relatively slow, with maximal phosphorylation achieved after 20 and 5 min respectively. The effect of insulin on phosphorylation of both these sites required the activation of PI3K and the MAPKs (mitogen-activated protein kinases) ERK1/2 (extracellular-signal-regulated kinase 1 and 2), but not the activation of mTOR (mammalian target of rapamycin)/p70S6 kinase, JNK (c-Jun N-terminal kinase) or p38MAPK. Although inhibition of PI3K and ERK1/2 both substantially decreased insulin-stimulated phosphorylation of Ser312 and Ser616, only wortmannin enhanced insulin-stimulated tyrosine phosphorylation of IRS-1. Furthermore, inhibition of mTOR/p70S6 kinase, JNK or p38MAPK had no effect on insulin-stimulated IRS-1 tyrosine phosphorylation. The differential effect of inhibition of ERK1/2 on insulin-stimulated IRS-1 phosphorylation of Ser312/Ser616 and tyrosine indicates that these events are independent of each other and that phosphorylation of Ser312/Ser616 is not responsible for the negative regulation of IRS-1 tyrosine phosphorylation mediated by PI3K in primary adipocytes.

scholarly journals Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1

1993 ◽  
Vol 13 (12) ◽  
pp. 7418-7428
Author(s):  
X J Sun ◽  
D L Crimmins ◽  
M G Myers ◽  
M Miralpeix ◽  
M F White
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Regulatory Elements ◽  
Sh2 Domain ◽  
Insulin Receptor Substrate ◽  
Insulin Stimulation ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Sh2 Domains ◽  
Amino Terminal

IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85 alpha) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively. These results confirm the p85 alpha recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.

scholarly journals Mechanisms of inhibition of lipolysis by insulin, vanadate and peroxovanadate in rat adipocytes

1999 ◽  
Vol 339 (2) ◽  
pp. 281-289 ◽  
Author(s):  
Isabelle CASTAN ◽  
Jonny WIJKANDER ◽  
Vincent MANGANIELLO ◽  
Eva DEGERMAN
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Regulatory Subunit ◽  
Insulin Receptor Substrate ◽  
Insulin Receptor Substrate 1 ◽  
Rat Adipocytes ◽  
Lipolytic Action ◽  
Dependent Protein ◽  
Phosphodiesterase Type ◽  
Phosphoinositide 3 Kinase

Vanadate and peroxovanadate (pV), potent inhibitors of tyrosine phosphatases, mimic several of the metabolic actions of insulin. Here we compare the mechanisms for the anti-lipolytic action of insulin, vanadate and pV in rat adipocytes. Vanadate (5 mM) and pV (0.01 mM) inhibited lipolysis induced by 0.01–1 µM isoprenaline, vanadate being more and pV less efficient than insulin (1 nM). A loss of anti-lipolytic effect of pV was observed by increasing the concentration of isoprenaline and/or pV. pV induced tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 to a greater extent than insulin, whereas vanadate affected these components little if at all. In addition, only a higher concentration (0.1 mM) of pV induced the tyrosine phosphorylation of p85, the 85 kDa regulatory subunit of phosphoinositide 3-kinase (PI-3K). Vanadate activated PI-3K-independent (in the presence of 10 nM isoprenaline) and PI-3K-dependent (in the presence of 100 nM isoprenaline) anti-lipolytic pathways, both of which were found to be independent of phosphodiesterase type 3B (PDE3B). pV (0.01 mM), like insulin, activated PI-3K- and PDE3B-dependent pathways. However, the anti-lipolytic pathway of 0.1 mM pV did not seem to require insulin receptor substrate-1-associated PI-3K and was found to be partly independent of PDE3B. Vanadate and pV (only at 0.01 mM), like insulin, decreased the isoprenaline-induced activation of cAMP-dependent protein kinase. Overall, these results underline the complexity and the diversity in the mechanisms that regulate lipolysis.

Changes in tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) and association of p85 subunit of phosphatidylinositol 3-kinase with IRS-1 after feeding in rat liver in vivo

1997 ◽  
Vol 154 (2) ◽  
pp. 267-273 ◽  
Author(s):  
Y Ito ◽  
M Ariga ◽  
S-I Takahashi ◽  
A Takenaka ◽  
T Hidaka ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Rat Liver ◽  
Insulin Receptor Substrate ◽  
Β Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Substrate 1 ◽  
Insulin Receptor Tyrosine Kinase ◽  
Phosphatidylinositol 3

Abstract The binding of insulin to its receptor rapidly induces intrinsic insulin receptor tyrosine kinase activity, resulting in tyrosine phosphorylation of various cytosolic substrates, such as insulin receptor substrate-1 (IRS-1) which, in turn, associates with a p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) followed by activation of this enzyme. In the present study, we have examined these early steps of insulin signalling in rat liver in vivo after food ingestion. After fasting for 22 h, a 12% casein diet was available ad libitum throughout the 8-h experimental period. Plasma insulin concentrations increased within 45 min after feeding, reached a maximum at 1·5 h and gradually decreased until 8 h. Autophosphorylation of the insulin receptor β-subunit in liver was detected even during fasting and increased about 1·5-fold at 1·5 h after feeding. Basal tyrosine phosphorylation of IRS-1 was detectable during starvation, increased about twofold at 3 h after feeding and levels were maintained until 8 h. The content of the p85 subunit of PI 3-kinase associated with IRS-1 also increased after feeding in parallel with the changes in tyrosine phosphorylation of IRS-1. Because tyrosine phosphorylation of the insulin receptor β-subunit and IRS-1 and the association of the p85 subunit of PI 3-kinase with IRS-1 in liver were closely correlated with the changes in the plasma concentration of insulin, we concluded that endogenous insulin secreted in response to eating caused these insulin-dependent intracellular changes in the liver. Journal of Endocrinology (1997) 154, 267–273

scholarly journals Insulin stimulates the tyrosine dephosphorylation of docking protein p130cas (Crk-associated substrate), promoting the switch of the adaptor protein Crk from p130cas to newly phosphorylated insulin receptor substrate-1

1998 ◽  
Vol 334 (3) ◽  
pp. 595-600 ◽  
Author(s):  
Andrey SOROKIN ◽  
Eleanor REED
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Sh2 Domain ◽  
Stable Complex ◽  
Insulin Receptor Substrate ◽  
Dependent Manner ◽  
Insulin Receptor Substrate 1 ◽  
Docking Protein

The docking protein p130cas (Crk-associated substrate) forms a stable complex with the adaptor protein CrkII in a tyrosine-phosphorylation-dependent manner. Insulin-induced tyrosine phosphorylation of insulin receptor substrates results in the redistribution of CrkII between p130cas and insulin receptor substrate-1. A decrease in the association between CrkII and p130cas in response to insulin stimulation was detected in CHO cells stably expressing insulin receptor or insulin receptor substrate-1, and in L6 rat myoblasts. Along with the decrease in the association of CrkII with p130cas, the amount of tyrosine-phosphorylated insulin receptor substrate-1 co-precipitated with CrkII increased in all cell types studied. The insulin-induced decrease in the CrkII–p130cas association was further confirmed by Far Western Blot analysis with the Src homology 2 (SH2) domain of CrkII. Insulin regulates the association of CrkII with p130cas by tyrosine dephosphorylation of p130cas and co-ordinated tyrosine phosphorylation of insulin receptor substrate-1. Tyrosine-phosphorylated insulin receptor substrate-1 serves as a docking protein for multiple adaptor proteins and competes with p130cas for CrkII.

scholarly journals 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

2004 ◽  
Vol 24 (12) ◽  
pp. 5434-5446 ◽  
Author(s):  
Kohjiro Ueki ◽  
Tatsuya Kondo ◽  
C. Ronald Kahn
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Cultured Cells ◽  
Glycogen Synthesis ◽  
Cytokine Signaling ◽  
Insulin Receptor Substrate ◽  
Suppressor Of Cytokine Signaling ◽  
Necrosis Factor Alpha ◽  
Irs Proteins

ABSTRACT 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.

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