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  • Phosphorylation of insulin-like growth factor (IGF) binding protein-3 by breast cancer cell membranes enhances IGF-I binding. 12933678

    Cross-linking of nonglycosylated biotinylated IGF binding protein (IGFBP)-3 to T-47D cell membranes identifies complexes with Mr of 32, 50, 70, and 100 kDa. Nonbiotinylated glycosylated IGFBP-3 competed for binding to each of these sites. The 32-kDa band approximated the size of intact nonglycosylated IGFBP-3, but its abundance was enhanced by cross-linking, and it had a more acidic isoelectric point on isoelectric focusing, suggesting that it had undergone phosphorylation. Immobilized IGFBP-3 was phosphorylated in the presence of (32)P-gamma ATP by both T-47D cell membranes and by intact cells treated with phenylarsine oxide to inhibit internalization. MCF-7 and COS-1 cells were also able to bind and phosphorylated IGFBP-3. IGF-I inhibited both IGFBP-3 binding to membranes and phosphorylation. However, incubation of T-47D cells with IGFBP-3 enhanced binding of (125)I-IGF-I to the cell monolayer indicating that membrane bound IGFBP-3 was able to bind IGF-I. Immobilized IGFBP-3 when phosphorylated by T-47D membranes bound significantly more (125)I-IGF-I than nonphosphorylated IGFBP-3. Treatment with alkaline phosphatase significantly reduced (125)I-IGF-I binding to phosphorylated immobilized IGFBP-3 and also reduced (125)I-IGF-I to T-47D cell monolayers preincubated with IGFBP-3. Phosphorylation of IGFBP-3 by T-47D membranes was partially blocked by inhibitors of both protein kinase A and C. These data demonstrate that binding of IGFBP-3 to breast cancer membranes is accompanied by phosphorylation at the plasma membrane and that both processes are inhibited by IGF-I. However, once phosphorylated the ability of IGFBP-3 to bind IGF-I is enhanced, resulting in increased association of the IGF-I with the cell membrane.
    Document Type:
    Reference
    Product Catalog Number:
    06-108

  • Transforming growth factor-alpha: a major human serum factor that promotes human keratinocyte migration. 16691197

    In unwounded skin, human keratinocytes (HKs) are in contact with a plasma filtrate. In an acute wound, HKs come in contact with serum for the first time. Because human serum (HS), but not plasma, promotes HK migration, we speculated that a major HK pro-motility factor in vivo comes from serum. In this study, we compared all of the published growth factors (GFs), reported to promote HK migration, with HS. No single GF could duplicate the HK pro-motility activity in HS. Among these GFs, transforming growth factor-alpha [corrected] showed the highest HK pro-motility activity, reaching approximately 80% of the activity in HS. The order of potency was: TGFalpha greater than insulin greater than EGF greater than heparin binding (HB)-EGF greater than IGF-1 greater than basic fibroblast growth factor greater than IL-8 greater than HGF greater than IL-1 greater than KGFgreater than TGFbeta. Interestingly, the combination of TGFalpha and insulin could duplicate the HK pro-motility activity in HS, although only the TGFalpha, but not insulin, levels increase in serum over plasma. Addition of neutralizing antibodies against TGFalpha to serum or depletion of TGFalpha from serum by immunoprecipitation significantly abolished its HK pro-motility activity. Plasma with added TGFalpha stimulated HK migration that reached more than 80% of the serum stimulation. Since insulin levels are identical between plasma and serum, we propose that TGFalpha is the physiologic HK pro-motility factor in HS.
    Document Type:
    Reference
    Product Catalog Number:
    AB3849
    Product Catalog Name:

  • Activation of p38 mitogen-activated protein kinase abolishes insulin-mediated myocardial protection against ischemia-reperfusion injury. 18003719

    Myocardial ischemia-reperfusion injury contributes significantly to morbidity and mortality in patients with diabetes. Insulin decreases myocardial infarct size in animals and the rate of apoptosis in cultured cells. Ischemia-reperfusion activates p38 mitogen-activated protein kinase (MAPK), which regulates cellular apoptosis. To examine whether p38 MAPK affects insulin's cardioprotection against ischemia-reperfusion injury, we studied overnight-fasted adult male rats by use of an in vivo rat model of myocardial ischemia-reperfusion. A euglycemic clamp (3 mU.min(-1).kg(-1)) was begun either 10 min before ischemia (InsulinBI), 5 min before reperfusion (InsulinBR), or 30 min after the onset of reperfusion (InsulinAR), and continued until the end of the study. Compared with saline control, insulin decreased the infarct size in both InsulinBI (P less than 0.001) and InsulinBR (P less than 0.02) rats but not in InsulinAR rats. The ischemic area showed markedly increased phosphorylation of p38 MAPK compared with the nonischemic area in saline animals. Acute activation of p38 MAPK with anisomycin (2 mg/kg iv 10 min before ischemia) had no effect on infarct size in saline rats. However, it completely abolished insulin's protective effect in InsulinBI and InsulinBR rats. Activation of p38 MAPK by anisomycin was associated with marked and persistent elevation in IRS-1 serine phosphorylation. Treatment of animals with SB-239063, a potent and specific inhibitor of p38 MAPK, 10 min before reperfusion enabled insulin-mediated myocardial protection in InsulinAR rats. We conclude that insulin protects myocardium against ischemia-reperfusion injury when given prior to ischemia or reperfusion, and activation of p38 MAPK abolishes insulin's cardioprotective effect.
    Document Type:
    Reference
    Product Catalog Number:
    06-248
    Product Catalog Name:
    Anti-IRS1 Antibody

  • The malate-aspartate NADH shuttle member Aralar1 determines glucose metabolic fate, mitochondrial activity, and insulin secretion in beta cells. 15494407

    The NADH shuttle system, which transports reducing equivalents from the cytosol to the mitochondria, is essential for the coupling of glucose metabolism to insulin secretion in pancreatic beta cells. Aralar1 and citrin are two isoforms of the mitochondrial aspartate/glutamate carrier, one key constituent of the malate-aspartate NADH shuttle. Here, the effects of Aralar1 overexpression in INS-1E beta cells and isolated rat islets were investigated for the first time. We prepared a recombinant adenovirus encoding for human Aralar1 (AdCA-Aralar1), tagged with the small FLAG epitope. Transduction of INS-1E cells and isolated rat islets with AdCA-Aralar1 increased aralar1 protein levels and immunostaining revealed mitochondrial localization. Compared with control INS-1E cells, overexpression of Aralar1 potentiated metabolism secretion coupling stimulated by 15 mm glucose. In particular, there was an increase of NAD(P)H generation, of mitochondrial membrane hyperpolarization, ATP levels, glucose oxidation, and insulin secretion (+45%, p < 0.01). Remarkably, this was accompanied by reduced lactate production. Rat islets overexpressing Aralar1 secreted more insulin at 16.7 mm glucose (+65%, p < 0.05) compared with controls. These results show that aspartate-glutamate carrier capacity limits glucose-stimulated insulin secretion and that Aralar1 overexpression enhances mitochondrial metabolism.
    Document Type:
    Reference
    Product Catalog Number:
    12-343
    Product Catalog Name:
    Acetyl-Histone H4 (Lys5) Peptide

  • Dynamic regulation of estrogen receptor-alpha isoform expression in the mouse fallopian tube: mechanistic insight into estrogen-dependent production and secretion of insu ... 17848632

    Estrogen receptors (ERs) are members of the nuclear receptor superfamily and are involved in regulation of fallopian tube functions (i.e., enhancement of protein secretion, formation of tubal fluid, and regulation of gamete transport). However, the ER subtype-mediated mechanisms underlying these processes have not been completely clarified. Recently, we identified ERbeta expression and localization in rat fallopian tubes, suggesting a potential biological function of ERbeta related to calcium-dependent ciliated beating. Here we provide for the first time insight into the less studied ERalpha isoforms, which mediate estrogen-dependent production and secretion of IGFs in vivo. First, Western blot studies revealed that three ERalpha isoforms were expressed in mouse fallopian tubes. Subsequent immunohistochemical analysis showed that ERalpha was detected in all cell types, whereas ERbeta was mainly localized in ciliated epithelial cells. Second, ERalpha isoform levels were dramatically downregulated in mouse fallopian tubes by treatment with E(2) or PPT, an ERalpha agonist, in a time-dependent manner. Third, the presence of ICI 182,780, an ER antagonist, blocked the E(2)- or PPT-induced downregulation of tubal ERalpha isoform expression in mice. However, alteration of ERalpha immunoreactivity following ICI 182,780 treatment was only detected in epithelial cells of the ampullary region. Fourth, changes in ERalpha isoform expression were found to be coupled to multiple E(2) effects on tubal growth, protein synthesis, and secretion in mouse fallopian tube tissues and fluid. In particular, E(2) exhibited positive regulation of IGF-I and IGF-II protein levels. Finally, using growth hormone receptor (GHR) gene-disrupted mice, we showed that regulation by E(2) of IGF production was independent of GH-induced GHR signaling in mouse fallopian tubes in vivo. These data, together with previous studies from our laboratory, suggest that the long-term effects of estrogen agonist promote IGF synthesis and secretion in mouse tubal epithelial cells and fallopian tube fluid via stimulation of ERalpha.
    Document Type:
    Reference
    Product Catalog Number:
    06-629

  • DHEA treatment reduces fat accumulation and protects against insulin resistance in male rats. 9467418

    The purpose of this study was to determine whether administration of dehydroepiandrosterone (DHEA) protects male rats against the accumulation of body fat the development of insulin resistance with advancing age. We found that supplementation of the diet with 0.3% DHEA between the ages of 5 months and approximately 25 months resulted in a significantly lower final body weight (DHEA, 593 +/- 18 g vs control, 668 +/- 12 g, p 0.02), despite no decrease in food intake. Lean body mass was unaffected by the DHEA, and the lower body weight was due to a approximately 25% reduction in body fat. The rate of glucose disposal during a euglycemic, hyperinsulinemic clamp was 30% higher in the DHEA group than in the sedentary controls due to a greater insulin responsiveness. The DHEA administration was as effective in reducing body fat content and maintaining insulin responsiveness as exercise in the form of voluntary wheel running. The DHEA had no significant effect on muscle GLUT4 content. A preliminary experiment provided evidence suggesting that muscle insulin signaling, as reflected in binding of phosphatidylinositol 3-kinase to the insulin receptor substrate-1, was enhanced in the DHEA-treated and wheel running groups as compared to controls. These results provide evidence that DHEA, like exercise, protects against excess fat accumulation and development of insulin resistance in rats.
    Document Type:
    Reference
    Product Catalog Number:
    06-248
    Product Catalog Name:
    Anti-IRS1 Antibody

  • A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular sm ... 10400906

    PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.
    Document Type:
    Reference
    Product Catalog Number:
    06-498

  • Association of insulin receptor substrate 3 with SH2 domain-containing proteins in rat adipocytes. 9642156

    We have recently purified and cloned a new member of the insulin receptor substrate family, designated insulin receptor substrate 3 (IRS-3), from rat adipocytes. The amino acid sequence of IRS-3 shows multiple potential sites for tyrosine phosphorylation in motifs which engage specific SH2 domain-containing proteins. In order to determine which SH2 domain proteins complex with IRS-3, we have searched for coimmunoprecipitation from lysates of untreated and insulin-stimulated adipocytes. Phosphatidylinositol 3-kinase and the tyrosine phosphatase SHP-2 complexed with the tyrosine phosphorylated form of IRS-3, whereas the phospholipase Cgamma did not, and the adaptor Grb2 did so to a much lesser extent. These findings complete the survey of SH2 domain proteins associated with each of the four known members of the IRS family and provide the framework for further analysis of the role of IRS-3 in insulin signaling.
    Document Type:
    Reference
    Product Catalog Number:
    16-101
    Product Catalog Name:
    Anti-Phosphotyrosine Antibody, clone 4G10®, agarose conjugate

  • Puerarin attenuates endothelial insulin resistance through inhibition of inflammatory response in an IKKβ/IRS-1-dependent manner. 22314193

    Puerarin is an isoflavonoid isolated from the root of the plant Pueraria lobata and has been used as a prescribed drug in China for the treatment of cardiovascular diseases in the clinical practice. Puerarin possesses potential therapeutic activities for metabolic and cardiovascular disorders. However, little is yet known about its bioprotection against endothelial dysfunction insulin resistance involved. In this study, we established insulin resistance by palmitate stimulation in the endothelium and investigated the action of puerarin on the modulation of insulin sensitivity under the insulin resistant condition. Palmitate stimulation impaired insulin-mediated vasodilation in the rat aorta and puerarin treatment effectively restored the impaired vasodilation in a concentration-dependent manner (1, 10 and 50 μM). Palmitate stimulation evoked inflammatory response in endothelial cells. Puerarin inhibited IKKβ/NF-κB activation and decreased TNF-α and IL-6 production with the downregulation of relative gene overexpression. Palmitate stimulation impaired the insulin PI3K signaling pathway and reduced insulin-mediated NO production in endothelial cells. Puerarin attenuated PA-induced phosphorylation of insulin receptor substrate-1 (IRS-1) at S307 and effectively ameliorated insulin-mediated tyrosine phosphorylation of IRS-1. The beneficial modification of serine/tyrosine phosphorylation of IRS-1 restored downstream Akt/eNOS activation, and thereby increased insulin-mediated NO production. These results suggest that puerarin inhibits inflammation and attenuates endothelial insulin resistance in an IKKβ/IRS-1-dependent manner.
    Document Type:
    Reference
    Product Catalog Number:
    CBL2117
    Product Catalog Name:
    Anti-Interleukin-6 Antibody, clone B-E8

  • Combined magnetic fields increase insulin-like growth factor-II in TE-85 human osteosarcoma bone cell cultures. 7789337

    In vitro exposure to low-energy, combined magnetic fields (CMF) increased the release of insulin-like growth factor (IGF)-II from human TE-85 osteosarcoma cells. Short-term CMF exposure of only 10 min increased IGF-II levels in conditioned medium 1 h post CMF exposure. IGF-II levels were measured with a radioreceptor assay using H-35 cells that contain abundant IGF-II but not IGF-I receptors. This assay also uses a recently validated BioGel P-10 acid gel filtration method to remove IGF binding protein before quantitation of either IGF-I or IGF-II. In addition to an increase in IGF-II levels, DNA synthesis, as an index of cell proliferation, was increased during the 24-h period post CMF exposure. A monoclonal antibody against IGF-II blocked the increase in cell proliferation following CMF exposure, whereas a control monoclonal antibody against osteocalcin did not attenuate the mitogenic action of CMF exposure. The effect of CMF exposure to increase both cell proliferation and IGF-II was cell-density dependent with greater stimulation by CMF observed at lower densities. Together, these data are consistent with the hypothesis that CMF exposure stimulates release/production of IGF-II from bone cells and that increased IGF-II then promotes an increase in cell proliferation.
    Document Type:
    Reference
    Product Catalog Number:
    05-166
    Product Catalog Name:
    Anti-IGF-II Antibody, clone S1F2