Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

GLUT4, the major insulin-responsive glucose transporter isoform in rat adipocytes, rapidly recycles between the cell surface and an intracellular pool with two first order rate constants, one for internalization (kin) and the other for externalization (kex). Insulin decreases kin by 2.8-fold and increases kex by 3.3-fold, thus increasing the steady-state cell surface GLUT4 level by approximately 8-fold (Jhun, B. H., Rampal, A. L., Liu, H., Lachaal, M., and Jung, C. (1992) J. Biol. Chem. 267, 17710-17715). To gain an insight into the biochemical mechanisms that modulate these rate constants, we studied the effects upon them of okadaic acid (OKA), a phosphatase inhibitor that exerts a insulin-like effect on glucose transport in adipocytes. OKA stimulated 3-O-methylglucose transport maximally 3.1-fold and increased the cell surface GLUT4 level 3.4-fold. When adipocytes were pulse-labeled with an impermeant, covalently reactive glucose analog, [3H]1,3-bis-(3-deoxy-D-glucopyranose-3-yloxy)-2-propyl 4-benzoylbenzoate, and the time course of labeled GLUT4 recycling was followed, the kex was found to increase 2.8-fold upon maximal stimulation by OKA, whereas the kin remained unchanged within experimental error. These findings demonstrate that OKA mimics the insulin effect on only GLUT4 externalization and suggest that insulin stimulates GLUT4 externalization by increasing the phosphorylation state of a serine/threonine phosphoprotein, probably by inhibiting protein phosphatase 1 or 2A.
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PMID:Okadaic acid stimulates glucose transport in rat adipocytes by increasing the externalization rate constant of GLUT4 recycling. 787 40

The roles of the glucose transporter isoforms, GLUT1 and GLUT4, in mediating insulin-stimulated glucose transport were investigated by stably overexpressing the transporters in L6 myoblasts. Levels of GLUT1 and GLUT4 in myoblasts from the cell lines having the highest content of these transporters were approximately 16- and 30-fold higher, respectively, than levels in nontransfected cells. The basal rate of 2-deoxy[3H]glucose uptake was severalfold higher in cells overexpressing GLUT1 than in the parent L6 myoblasts or in control cell lines that were generated by transfecting cells with expression vectors lacking transporter insert. The basal rate was not elevated in any of the lines expressing GLUT4. The net increase in 2-deoxy[3H]glucose uptake produced by insulin was larger in both the GLUT1 and GLUT4 cells than in the control cells. Insulin increased uptake in GLUT4 cells by as much as 6-fold; whereas, the fold increase over basal uptake produced by insulin in GLUT1 cells was comparable to that (2-fold) observed in the control myocytes. Thus, both GLUT1 and GLUT4 can mediate insulin-stimulated glucose transport in L6 myoblasts, although GLUT4 is needed to observe large percentage increases comparable to those observed in skeletal muscle fibers in vivo. In contrast to insulin, the protein phosphatase inhibitors, okadaic acid and calyculin A, inhibited glucose transport in cells expressing either GLUT1 or GLUT4. Calyculin A, which produced a half-maximum effect at 10 nM, was approximately 100 times more potent than okadaic acid in decreasing both basal and insulin-stimulated 2-deoxyglucose uptake. Inhibition of uptake by calyculin A was associated with a decrease in the cell surface concentration of both GLUT1 and GLUT4. These results indicate that increased protein phosphorylation can lead to inhibition of transport mediated by both GLUT1 and GLUT4.
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PMID:Glucose transport in L6 myoblasts overexpressing GLUT1 and GLUT4. 840 71

In adipose and muscle, insulin stimulates glucose uptake and glycogen synthase activity. Phosphatidylinositol 3-kinase (PI3K) activation is necessary but not sufficient for these metabolic actions of insulin. The insulin-stimulated translocation of phospho-c-Cbl to lipid rafts, via its association with CAP, comprises a second pathway regulating GLUT4 translocation. In 3T3-L1 adipocytes, overexpression of a dominant negative CAP mutant (CAP Delta SH3) completely blocked the insulin-stimulated glucose transport and glycogen synthesis but only partially inhibited glycogen synthase activation. In contrast, CAP Delta SH3 expression did not affect glycogen synthase activation by insulin in the absence of extracellular glucose. Moreover, CAP Delta SH3 has no effect on the PI3K-dependent activation of protein phosphatase-1 or phosphorylation of glycogen synthase kinase-3. These results indicate blockade of the c-Cbl/CAP pathway directly inhibits insulin-stimulated glucose uptake, which results in secondary inhibition of glycogen synthase activation and glycogen synthesis.
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PMID:Activation of glycogen synthase by insulin in 3T3-L1 adipocytes involves c-Cbl-associating protein (CAP)-dependent and CAP-independent signaling pathways. 1122 22

Tumor necrosis factor (TNF)-alpha causes insulin resistance on glucose uptake in fetal brown adipocytes. We explored the hypothesis that some effects of TNF-alpha could be mediated by the generation of ceramide, given that TNF-alpha treatment induced the production of ceramide in these primary cells. A short-chain ceramide analog, C2-ceramide, completely precluded insulin-stimulated glucose uptake and insulin-induced GLUT4 translocation to plasma membrane, as determined by Western blot or immunofluorescent localization of GLUT4. These effects were not produced in the presence of a biologically inactive ceramide analog, C2-dihydroceramide. Analysis of the phosphatidylinositol (PI) 3-kinase signaling pathway indicated that C2-ceramide precluded insulin stimulation of Akt kinase activity, but not of PI-3 kinase or protein kinase C-zeta activity. C2-ceramide completely abolished insulin-stimulated Akt/protein kinase B phosphorylation on regulatory residues Thr 308 and Ser 473, as did TNF-alpha, and inhibited insulin-induced mobility shift in Akt1 and Akt2 separated in PAGE. Moreover, C2-ceramide seemed to activate a protein phosphatase (PP) involved in dephosphorylating Akt because 1) PP2A activity was increased in C2-ceramide- and TNF-alpha-treated cells, 2) treatment with okadaic acid concomitantly with C2-ceramide completely restored Akt phosphorylation by insulin, and 3) transient transfection of a constitutively active form of Akt did not restore Akt activity. Our results indicate that ceramide produced by TNF-alpha induces insulin resistance in brown adipocytes by maintaining Akt in an inactive dephosphorylated state.
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PMID:Ceramide mediates insulin resistance by tumor necrosis factor-alpha in brown adipocytes by maintaining Akt in an inactive dephosphorylated state. 1167 35

During differentiation, expression of protein phosphatase-2Calpha (PP2Calpha) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Calpha in insulin signaling, we overexpressed wild-type (WT) PP2Calpha by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Calpha-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Calpha-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Calpha(R174G) did not produce such effects. Furthermore, overexpression of PP2Calpha-WT (but not PP2Calpha(R174G)) decreased the (32)P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Calpha activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Calpha by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Calpha antibody or PP2Calpha small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Calpha is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.
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PMID:Protein phosphatase-2C alpha as a positive regulator of insulin sensitivity through direct activation of phosphatidylinositol 3-kinase in 3T3-L1 adipocytes. 1501 18

Glucose transport into muscle is important for the maintenance of normoglycemia. Thus, understanding mechanisms that regulate expression of GLUT4, the main glucose transporter in skeletal muscle, is important to identify targets for the treatment of diabetes. Exercise increases the expression of GLUT4 mRNA and protein, and we have been investigating the mechanisms involved. Transcription of the GLUT4 gene is transiently activated after an acute bout of exercise and GLUT4 protein can be increased as much as two- to threefold after a few days of repeated exercise bouts. Studies of the GLUT4 promoter have identified two sets of DNA sequences that are important for metabolic regulation and also for increased transcription of the gene in response to exercise. These DNA elements have been shown to bind the transcription factors myocyte enhancer factor 2 (MEF2) and GLUT4 enhancer factor (GEF). The mechanisms that activate these proteins remain one of the important areas of research in this field. Signals that link muscle contraction to the activation of transcription factors (MEF2, GEF) involved in increased expression of GLUT4 during exercise is another area needing further research. Two signals that show promise are changes in the energy charge (acting through AMP activated kinase [AMPK]) and changes in intracellular calcium (acting through calcineurin [a calcium-calmodulin activated phosphatase] and calcium-calmodulin activated kinase [CAMK]). There is good evidence that both increased AMPK activity and increased CAMK activity cause increased transcription of the GLUT4 gene. It remains to be demonstrated that exercise is acting through one or both of these mechanisms.
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PMID:Regulation of GLUT4 gene expression during exercise. 1523 26

Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulin's metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3beta, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C lambda (PKC lambda) activity was also augmented in small-t-antigen-expressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC lambda activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC lambda expression or PKC lambda siRNA. We conclude that PP2A is a negative regulator of insulin's metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC lambda and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.
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PMID:Protein phosphatase 2A negatively regulates insulin's metabolic signaling pathway by inhibiting Akt (protein kinase B) activity in 3T3-L1 adipocytes. 1536 94

Exercise induces a rapid increase in expression of the GLUT4 isoform of the glucose transporter in skeletal muscle. One of the signals responsible for this adaptation appears to be an increase in cytosolic Ca(2+). Myocyte enhancer factor 2A (MEF2A) is a transcription factor that is involved in the regulation of GLUT4 expression. It has been reported that the Ca(2+)-regulated phosphatase calcineurin mediates the activation of MEF2 by exercise. It has also been shown that the expression of activated calcineurin in mouse skeletal muscle results in an increase in GLUT4. These findings suggest that increases in cytosolic Ca(2+) induce increased GLUT4 expression by activating calcineurin. However, we have obtained evidence that this response is mediated by a Ca(2+)-calmodulin-dependent protein kinase. The purpose of this study was to test the hypothesis that calcineurin is involved in mediating exercise-induced increases in GLUT4. Rats were exercised on 5 successive days using a swimming protocol. One group of swimmers was given 20 mg/kg body weight of cyclosporin, a calcineurin inhibitor, 2 h before exercise. A second group was given vehicle. GLUT4 protein was increased approximately 80%, GLUT4 mRNA was increased approximately 2.5-fold, MEF2A protein was increased twofold, and hexokinase II protein was increased approximately 2.5-fold 18 h after the last exercise bout. The cyclosporin treatment completely inhibited calcineurin activity but did not affect the adaptive increases in GLUT4, MEF2A, or hexokinase expression. We conclude that calcineurin activation does not mediate the adaptive increase in GLUT4 expression induced in skeletal muscle by exercise.
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PMID:Calcineurin does not mediate exercise-induced increase in muscle GLUT4. 1573 36

Overexpression of the protein phosphatase 1 (PP1) subunit protein targeting to glycogen (PTG) markedly enhances cellular glycogen levels. In order to disrupt the endogenous PTG-PP1 complex, small interfering RNA (siRNA) constructs against PTG were identified. Infection of 3T3-L1 adipocytes with PTG siRNA adenovirus decreased PTG mRNA and protein levels by >90%. In parallel, PTG reduction resulted in a >85% decrease in glycogen levels 4 days after infection, supporting a critical role for PTG in glycogen metabolism. Total PP1, glycogen synthase, and GLUT4 levels, as well as insulin-stimulated signaling cascades, were unaffected. However, PTG knockdown reduced glycogen-targeted PP1 protein levels, corresponding to decreased cellular glycogen synthase- and phosphorylase-directed PP1 activity. Interestingly, GLUT1 levels and acute insulin-stimulated glycogen synthesis rates were increased two- to threefold, and glycogen synthase activation in the presence of extracellular glucose was maintained. In contrast, glycogenolysis rates were markedly increased, suggesting that PTG primarily acts to suppress glycogen breakdown. Cumulatively, these data indicate that disruption of PTG expression resulted in the uncoupling of PP1 activity from glycogen metabolizing enzymes, the enhancement of glycogenolysis, and a dramatic decrease in cellular glycogen levels. Further, they suggest that reduction of glycogen stores induced cellular compensation by several mechanisms, but ultimately these changes could not overcome the loss of PTG expression.
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PMID:Central role for protein targeting to glycogen in the maintenance of cellular glycogen stores in 3T3-L1 adipocytes. 1635 3

Emerging evidence illustrates the importance of the positive transcription elongation factor (P-TEF)b in control of global RNA synthesis, which constitutes a major feature of the compensatory response to diverse hypertrophic stimuli in cardiomyocytes. P-TEFb complex, composed of cyclin T and cdk9, is critical for elongation of nascent RNA chains via phosphorylation of the carboxyl-terminal domain of RNA polymerase (Pol) II. We and others have shown that the activity of P-TEFb is inhibited by its association with cardiac lineage protein (CLP)-1, the mouse homolog of human HEXIM1, in various physiological and pathological conditions. To investigate the mechanism of control of P-TEFb activity by CLP-1 in cardiac hypertrophy, we used a transgenic mouse model of hypertrophy caused by overexpression of calcineurin in the heart. We observed that the level of CLP-1 associated with P-TEFb was reduced markedly in hypertrophic hearts. We also generated bigenic mice (MHC-cyclin T1/CLP-1(+/-)) by crossing MHC-cyclin T1 transgenic mice with CLP-1 heterozygote. The bigenic mice exhibit enhanced susceptibility to hypertrophy that is accompanied with an increase in cdk9 activity via an increase in serine 2 phosphorylation of carboxyl-terminal domain and an increase in GLUT1/GLUT4 ratio. These mice have compensated systolic function without evidence of fibrosis and reduced lifespan. These data suggest that the reduced level of CLP-1 introduced in the background of elevated levels of cyclin T1 elevates derepression of P-TEFb activity and emphasizes the importance of the role of CLP-1 in the mechanism governing compensatory hypertrophy in cardiomyocytes.
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PMID:Positive transcription elongation factor b activity in compensatory myocardial hypertrophy is regulated by cardiac lineage protein-1. 1954 17


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