Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P31749 (AKT)
 22,954 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glucosamine, which enters the hexosamine pathway downstream of the rate-limiting step, has been routinely used to mimic the insulin resistance caused by high glucose and insulin. We investigated the effect of glucosamine on insulin-stimulated glucose transport in 3T3-L1 adipocytes. The Delta-insulin (insulin-stimulated minus basal) value for 2-deoxyglucose uptake was dramatically inhibited with increasing concentrations of glucosamine with an ED50 of 1.95 mM. Subcellular fractionation experiments demonstrated that reduction in insulin-stimulated 2-deoxyglucose uptake by glucosamine was due to an inhibition of translocation of both Glut 1 and Glut 4 from the low density microsomes (LDM) to the plasma membrane. Analysis of the insulin signaling cascade revealed that glucosamine impaired insulin receptor autophosphorylation, insulin receptor substrate (IRS-1) phosphorylation, IRS-1-associated PI 3-kinase activity in the LDM, and AKT-1 activation by insulin. Measurement of intracellular ATP demonstrated that the effects of glucosamine were highly correlated with its ability to reduce ATP levels. Reduction of intracellular ATP using azide inhibited Glut 1 and Glut 4 translocation from the LDM to the plasma membrane, insulin receptor autophosphorylation, and IRS-1 tyrosine phosphorylation. Additionally, both the reduction in intracellular ATP and the effects on insulin action caused by glucosamine could be prevented by the addition of inosine, which served as an alternative energy source in the medium. We conclude that direct administration of glucosamine can rapidly lower cellular ATP levels and affect insulin action in fat cells by mechanisms independent of increased intracellular UDP-N-acetylhexosamines and that increased metabolism of glucose via the hexosamine pathway may not represent the mechanism of glucose toxicity in fat cells.
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PMID:Glucosamine-induced insulin resistance in 3T3-L1 adipocytes is caused by depletion of intracellular ATP. 968 25

Gene 33 (g33) is a non-tissue-specific gene regulated in rat liver and hepatoma cells by insulin and other agents. It is thought to participate in the transition from quiescence to proliferation in mitogen-treated cells. The mechanism(s) by which insulin exerts its action on g33 are not totally understood; it is unclear whether a functional insulin receptor is required for this action. In this study, we evaluate the mechanism for insulin induction of g33 mRNA in Chinese hamster ovary (CHO) cells transfected with the neomycin-resistant plasmid (CHONeoB), human insulin receptor (CHONewIRa), and a kinase-defective insulin receptor mutated at the ATP-binding site (CHOK1018A). Transfected cells had higher levels of insulin binding than that of CHONeoB cells; insulin-induced phosphorylation of the insulin receptor and its intracellular substrates were impaired in CHOK1018A cells. Maximal insulin induction of mRNA(g33) occurred 3 h after hormonal exposure in all cell lines. The degree of insulin stimulation of g33 mRNA levels was four- to sixfold higher in CHONewIRa than in CHONeoB or CHOK1018A cells, which had minimal levels of insulin-stimulated g33 mRNA levels. Half-maximal stimulation of g33 mRNA levels was observed at 0.06 +/- 0.01 nM in CHONewIRa cells, consistent with insulin interaction with its own receptor. Wortmannin, an inhibitor of phosphatidyl inositol 3-kinase (PI3K), had some effects on insulin stimulation of g33 mRNA in CHO NewIRa cells. PD98059, an inhibitor of mitogen-activated kinase kinase (MAPKK), and rapamycin, a p70 S6 kinase inhibitor, had minimal effect on insulin stimulation of g33 mRNA in all cells tested. By contrast, hydroxy-2-naphthalenylmethyl)phosphonic acid triacetoxymethyl ester (HNMPA(AM)(3), a selective inhibitor of the insulin receptor tyrosine kinase, caused complete inhibition of insulin stimulation of g33 mRNA levels. These data indicate that the insulin receptor with intact kinase activity is required for insulin stimulation of g33 mRNA levels. They also suggest that AKT, a PI 3-kinase downstream effector molecule, could mediate insulin stimulation of g33 mRNA. The mechanism(s) of insulin regulation of g33 expression downstream of receptor do not seem to rely entirely on the classic insulin receptor transduction pathway, as a minor effect was observed upon inhibition of MAPKK, suggesting that multiple pathways may be involved.
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PMID:Insulin-induced gene 33 mRNA expression in Chinese hamster ovary cells is insulin receptor dependent. 1076 Sep 51

Fatty livers are sensitive to lipopolysaccharide (LPS) damage. This study tests the hypothesis that this vulnerability occurs because protective, antiapoptotic mechanisms are not upregulated appropriately. Genetically obese, leptin-deficient ob/ob mice, a model for nonalcoholic fatty liver disease, and their lean litter mates were treated with a small dose of LPS. General measures of liver injury, early (i.e., cytochrome c release) and late (i.e., activation of caspase 3) events that occur during hepatocyte apoptosis, and various aspects of the signal transduction pathways that induce nuclear factor-kappaB (NF-kappaB) and several of its antiapoptotic transcriptional targets (e.g., inducible nitric oxide synthase, bfl-1, and bcl-xL) were compared. Within 0.5-6 h after LPS exposure, cytochrome c begins to accumulate in the cytosol of normal livers, and procaspase 3 cleavage increases. Coincident with these events, kinases (e.g., AKT and Erk-1 and -2) that result in the degradation of inhibitor kappa-B are activated; NF-kappaB activity is induced, and NF-kappaB-regulated gene products accumulate. Throughout this period, there is negligible histological evidence of liver damage, and serum alanine aminotransferase values barely increase over baseline values. Although ob/ob livers have significant histological liver injury and 11-fold greater serum alanine aminotransferase values than those of lean mice by 6 h post-LPS, they exhibit greater activation of AKT and Erk, more profound reductions in inhibitor kappa-B, enhanced activation of NF-kappaB, and greater induction of NF-kappaB-regulated genes. Consistent with this heightened antiapoptotic response, increases in cytochrome c and procaspase 3 cleavage products are inhibited. Together with evidence that ob/ob hepatocytes have a reduced ATP content and undergo increased lysis after in vitro exposure to tumor necrosis factor-alpha, these findings suggest that fatty livers are sensitive to LPS damage because of vulnerability to necrosis, rather than because of apoptosis.
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PMID:Fatty liver vulnerability to endotoxin-induced damage despite NF-kappaB induction and inhibited caspase 3 activation. 1144 19

Alveolar macrophages (AM) are the first line of defense against infection in the lungs. We previously showed that the production of superoxide and hydrogen peroxide, i.e., the respiratory burst, is stimulated by adenine nucleotides (ADP >> ATP) in rat AM through signaling pathways involving calcium and protein kinase C. Here, we further show that ADP induces a rapid increase in the tyrosine phosphorylation of several proteins that was reduced by the tyrosine kinase inhibitor genistein, which also inhibited the respiratory burst. Interestingly, ADP did not trigger the activation of the mitogen-activated protein kinases ERK1 and ERK2, or that of protein kinase B/AKT, a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway. This is in contrast to another stimulus of the respiratory burst, zymosan-activated serum (ZAS), which activates both the ERK and PI3K pathways. Thus, this study demonstrates that the receptor for ADP in rat AM is not coupled to the ERK and AKT pathways and, that neither the ERK pathway nor AKT is essential to induce the activation of the NAPDH oxidase by ADP in rat AM while tyrosine kinases appeared to be required. The rate and amount of hydrogen peroxide released by the ADP-stimulated respiratory burst was similar to that produced by ZAS stimulation. The absence of ERK activation after ADP stimulation therefore suggests that hydrogen peroxide is not sufficient to activate the ERK pathway in rat AM. Nonetheless, as hydrogen peroxide was necessary for ERK activation by ZAS, this indicates that, in contrast to ADP, ZAS stimulates a pathway that is targeted by hydrogen peroxide and leads to ERK activation.
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PMID:ADP stimulates the respiratory burst without activation of ERK and AKT in rat alveolar macrophages. 1152 53

A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3alpha Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [(33)P]ATP and a biotinylated substrate, also developed in our laboratory. The IC(50) values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening.
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PMID:Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody. 1172 83

AMP-activated protein kinase (AMPK) functions as an energy sensor to provide metabolic adaptations under the ATP-deprived conditions such as hypoxia. In the present study, we considered a role of AMPK in the adaptive response to hypoxia by examining whether AMPK is involved in the regulation of hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor that is critical for hypoxic induction of physiologically important genes. We demonstrate that hypoxia or CoCl2 rapidly activated AMPK in DU145 human prostate cancer cells, and its activation preceded the induction of HIF-1 alpha expression. Under these conditions, blockade of AMPK activity by a pharmacological or molecular approach significantly attenuated hypoxia-induced responses such as HIF-1 target gene expression, secretion of vascular endothelial growth factor, glucose uptake, and HIF-1-dependent reporter gene expression, indicating that AMPK is critical for the HIF-1 transcriptional activity and its target gene expression. Its functional requirement for HIF-1 activity was also demonstrated in several different cancer cell lines, but AMPK activation alone was not sufficient to stimulate the HIF-1 transcriptional activity. We further present data showing that AMPK transmits a positive signal for HIF-1 activity via a signaling pathway that is independent of phosphatidylinositol 3-kinase/AKT and several mitogen-activated protein kinases. Taken together, our results suggest that AMPK is a novel and critical component of HIF-1 regulation, implying its new roles in oxygen-regulated cellular phenomena.
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PMID:AMP-activated protein kinase activity is critical for hypoxia-inducible factor-1 transcriptional activity and its target gene expression under hypoxic conditions in DU145 cells. 3044 3

Histone deacetylase inhibitors induce hyperacetylation of the amino-terminal lysine residues of the core nucleosomal histones, which results in chromatin remodeling and altered gene expression. Present studies demonstrate that exposure to a novel hydroxamic acid analogue histone deacetylase inhibitor, LAQ824, induced p21WAF1 and p27KIP1 and caused growth arrest and apoptosis of human breast cancer SKBR-3 and BT-474 cells that possess amplification and overexpression of Her-2/neu. Treatment with LAQ824 depleted the mRNA and protein levels of Her-2/neu-encoded Her-2, which was associated with attenuation of pAKT, c-Raf-1, and phosphorylated mitogen-activated protein kinase levels. LAQ824 also induced the acetylation of heat shock protein (hsp) 90, resulting in inhibition of its binding to ATP, which has been shown to impair the chaperone association of hsp 90 with its client proteins, Her-2, AKT, and c-Raf-1. Consistent with this, treatment with LAQ824 shifted the binding of Her-2 from hsp 90 to hsp 70, promoting proteasomal degradation of Her-2. Thus, LAQ824 depletes Her-2 through two mechanisms: attenuation of its mRNA levels and promotion of its degradation by the proteasome. Following LAQ824 treatment, the cell membrane association, autotyrosine phosphorylation, and colocalization of Her-2 with HER-3 also declined. Cotreatment with LAQ824 significantly increased trastuzumab-induced apoptosis of BT-474 and SKBR-3 cells. This was associated with greater attenuation of Her-2, c-Raf-1, and pAKT levels. LAQ824 also enhanced taxotere-induced, epothilone B-induced, and gemcitabine-induced apoptosis of BT-474 and SKBR-3 cells. These findings suggest that LAQ824 is active against human breast cancer cells and has the potential to improve the efficacy of trastuzumab, taxotere, gemcitabine, and epothilone B against breast cancer with Her-2/neuamplification.
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PMID:Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizes human breast cancer cells to trastuzumab, taxotere, gemcitabine, and epothilone B. 1457 62

Kinases are an important therapeutic target for drug discovery, and many cancer chemotherapeutic agents have been derived from natural product sources. Natural product samples, however, have the likelihood of assay interference, particularly at elevated test concentrations. The authors developed a competitive fluorescence polarization (FP) assay using red-shifted fluorophores for the AKT kinase and demonstrated utility for testing concentrated natural product extracts. A set of 7 actinomycetes cultures containing indolocarbazoles, known nonselective kinase inhibitors, and a control set of 22 nonproducing indolocarbazole cultures were evaluated. Using red-shifted dyes (Cy3B or Cy5), the authors identified active samples with minimal interference up to the extract concentrations that are 3 times nonextracted culture levels. In contrast, a significant number of interferences were observed using either a fluorescein competitive FP assay or a [33P]ATP Flashplate assay. This work demonstrates that one can screen natural product extracts at high concentrations successfully using FP technology with red-shifted dyes.
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PMID:Use of red-shifted dyes in a fluorescence polarization AKT kinase assay for detection of biological activity in natural product extracts. 1500 49

A bead-based FP assay methodology, termed IMAP trade mark, has been developed for the serine/threonine kinase, AKT, that allows for direct measurement of product formation. The assay design utilizes a fluoresceinated peptide substrate that, when phosphorylated by the kinase, binds to nanoparticles derivatized with trivalent metal cations through a metal-phospholigand interaction. The result of this bound fluoresceinated phosphorylated product is an increase in polarization signal caused by a decrease in the molecular mobility of the bound product. The AKT IMAP FP assay has been formatted in a 384-well microtiter plate with a Z' of 0.75, suitable for HTS. The assay was validated with six known kinase inhibitors. The IC(50) values generated were comparable to previously reported values using a competitive antibody-based FP assay and a radioactive [(33)P]ATP Flashplate transfer assay. The IMAP assay was further evaluated by screening the biologically active sample set, LOPAC trade mark. It was found that no fluorescent samples interfered with the assay's performance and that one could identify appropriate inhibitors. The advantages of IMAP technology are that it does not require the use of antibodies, the polarization signal generated is large in comparison with most FP assays based on antibodies, and IMAP captures and measures the product formed directly. The higher concentrations of fluorophore used in the assay versus competition assays increase the precision of the data obtained and reduce sample interference from compounds. This work demonstrates that IMAP is a valuable technology that may be used in developing numerous kinase assays.
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PMID:Development of a fluorescence polarization AKT serine/threonine kinase assay using an immobilized metal ion affinity-based technology. 1509 Feb 51

Insulin-like growth factor receptor 1 (IGF-1R) plays a critical role in oncogenic transformation (1). IGF-1R is overexpressed in some tumors including breast, lung, cervical, and Wilms' tumors (2-6). Upon binding of IGF-I or IGF-II, IGF-1R, a tyrosine kinase, phosphorylates tyrosine residues on two major substrates, IRS-1 and Shc, which subsequently signal through the Ras/Raf and PI 3-kinase/AKT pathways (7). Extensive literature has shown that when the IGF-1R signaling pathway is blocked by antisense, dominant negative truncation or neutralizing antibodies, cellular transformation and tumor formation in mice is inhibited (8-18). Small molecule kinase inhibitors represent a valid approach to inhibit activity and downstream signalling of IGF-1R. To date, few potent and selective small molecule inhibitors of IGF-1R kinase activity have been reported. We expressed the tyrosine kinase domain of IGF-1R (IGF-1R/TK) in insect cells and subsequently purified the partially activated IGF-1R/TK. A compound library has been screened using a homogeneous time-resolved fluorescence (HTRF) assay. The hits generated by HTRF were then evaluated in a 33P ATP streptavidin-Flashplate assay (Flashplate). There was approximately 78% hit congruence between the two assay formats. One compound, C100, inhibited the IGF-1R kinase activity with an IC50 of 1 microM. C100 also inhibited IGF-1R autophosphorylation, AKT and MAPK activations in cells. This inhibitor provides a useful tool for studying IGF-1R in cells.
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PMID:Screening for small molecule inhibitors of insulin-like growth factor receptor (IGF-1R) kinase: comparison of homogeneous time-resolved fluorescence and 33P-ATP plate assay formats. 1550 6


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