Colorimetric Detection of Glutamine Synthetase-Catalyzed Transferase Activity in Glucocorticoid-Treated Skeletal Muscle Cells

Santoro, Joseph C.; Harris, G.; Sitlani, Ayesha

doi:10.1006/abio.2000.4911

Cited by 39 publications

(27 citation statements)

References 35 publications

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“…6.3.1.2), cultures were washed twice with PBS and stored at Ϫ80°C in 50 mM imidazole (pH 6.8) before colorimetric determination of enzyme activity with hydroxylamine as described previously. 27 Proteolysis was measured by prelabeling of cell protein with L-[2,6 3 H]-Phe (Amersham [Little Chalfont, UK] TRK552) and measurement of release of acid-soluble radioactivity into the medium, 28 expressed as log 10 of the percentage of the total initial cellular 3 H per hour. 28 Cell viability was monitored by measurement of release of acid-precipitable radioactivity into the medium (an indicator of intact protein leakage and cell detachment) 29 and was unaffected by the culture conditions described in this study.…”

Section: Cell Culturementioning

confidence: 99%

Inhibition of SNAT2 by Metabolic Acidosis Enhances Proteolysis in Skeletal Muscle

Evans

Nasim

Brown

et al. 2008

Journal of the American Society of Nephrology

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Insulin resistance is a major cause of muscle wasting in patients with ESRD. Uremic metabolic acidosis impairs insulin signaling, which normally suppresses proteolysis. The low pH may inhibit the SNAT2 L-Glutamine (L-Gln) transporter, which controls protein synthesis via amino acid-dependent insulin signaling through mammalian target of rapamycin (mTOR). Whether SNAT2 also regulates signaling to pathways that control proteolysis is unknown. In this study, inhibition of SNAT2 with the selective competitive substrate methylaminoisobutyrate or metabolic acidosis (pH 7.1) depleted intracellular L-Gln and stimulated proteolysis in cultured L6 myotubes. At pH 7.1, inhibition of the proteasome led to greater depletion of L-Gln, indicating that amino acids liberated by proteolysis sustain L-Gln levels when SNAT2 is inhibited by acidosis. Acidosis shifted the dose-response curve for suppression of proteolysis by insulin to the right, confirming that acid increases proteolysis by inducing insulin resistance. Blocking mTOR or phosphatidylinositol-3-kinase (PI3K) increased proteolysis, indicating that both signaling pathways are involved in its regulation. When both mTOR and PI3K were inhibited, methylaminoisobutyrate or acidosis did not stimulate proteolysis further. Moreover, partial silencing of SNAT2 expression in myotubes and myoblasts with small interfering RNA stimulated proteolysis and impaired insulin signaling through PI3K. In conclusion, SNAT2 not only regulates mTOR but also regulates proteolysis through PI3K and provides a link among acidosis, insulin resistance, and protein wasting in skeletal muscle cells.

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Section: Cell Culturementioning

confidence: 99%

Inhibition of SNAT2 by Metabolic Acidosis Enhances Proteolysis in Skeletal Muscle

Evans

Nasim

Brown

et al. 2008

Journal of the American Society of Nephrology

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“…Cells were pretreated with RU-486 or A-348441 for 30 min followed by incubating overnight with 100 nM prednisolone. Glutamine synthetase assay was performed using the method of Santoro et al (2001). Wells that contained substrate without compound or prednisolone were used as the background, whereas the wells that contained substrate and prednisolone without any compound were considered as maximal signal.…”

Section: Glutamine Synthetase Assaymentioning

confidence: 99%

Hepatic Glucocorticoid Receptor Antagonism Is Sufficient to Reduce Elevated Hepatic Glucose Output and Improve Glucose Control in Animal Models of Type 2 Diabetes

Jacobson

Geldern²,

Öhman³

et al. 2005

J Pharmacol Exp Ther

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Glucocorticoids amplify endogenous glucose production in type 2 diabetes by increasing hepatic glucose output. Systemic glucocorticoid blockade lowers glucose levels in type 2 diabetes, but with several adverse consequences. It has been proposed, but never demonstrated, that a liver-selective glucocorticoid receptor antagonist (LSGRA) would be sufficient to reduce hepatic glucose output (HGO) and restore glucose control to type 2 diabetic patients with minimal systemic side effects. A-348441 [(3b,5b,7a,12a)-7,12-dihydroxy-3-{2-[{4-[(11b,17b)-17-hydroxy-3-oxo-17-prop-1-ynylestra-4,9-dien-11-yl] phenyl}(methyl)amino]ethoxy}cholan-24-oic acid] represents the first LSGRA with significant antidiabetic activity.A-348441 antagonizes glucocorticoid-up-regulated hepatic genes, normalizes postprandial glucose in diabetic mice, and demonstrates synergistic effects on blood glucose in these animals when coadministered with an insulin sensitizer. In insulin-resistant Zucker fa/fa rats and fasted conscious normal dogs, A-348441 reduces HGO with no acute effect on peripheral glucose uptake. A-348441 has no effect on the hypothalamic pituitary adrenal axis or on other measured glucocorticoid-induced extrahepatic responses. Overall, A-348441 demonstrates that an LSGRA is sufficient to reduce elevated HGO and normalize blood glucose and may provide a new therapeutic approach for the treatment of type 2 diabetes.

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“…Electrophoretic Mobility Shift Assay (EMSA)-For EMSA experiments, 0.1 nmol of oligonucleotides GRE1-TOP, GRE2-TOP, GRE3-TOP, and GRE4-TOP were end-labeled with 2 l of [␥- 33 P]ATP using T4-polynucleotide kinase (Invitrogen) in a 20-l reaction. The labeled oligonucleotides were purified to remove unincorporated labels using a Qiaquick nucleotide removal kit (Qiagen, Valencia, CA).…”

Section: Materials and Plasmid Preparation-allmentioning

confidence: 99%

“…33) was incubated with 5 nM labeled GRE duplex in a buffer containing 40 mM Tris-Cl (pH 7.5), 0.2 mM EDTA, 8 mM dithiothreitol, 80 mM NaCl, 0.1% bovine serum albumin, 20% glycerol, 0.25% Nonidet P-40, and 12-25 g of poly(dI-dC). For the comparison of DNA binding by GR, GR-Dex, and GR-RU486 extracts, the amount of extract and protein used in each lane was normalized by measuring the total protein concentration in each extract by a Coomassie Plus protein assay reagent kit (Pierce, Rockford, IL).…”

Section: Materials and Plasmid Preparation-allmentioning

confidence: 99%

Allosteric Effects of Dexamethasone and RU486 on Glucocorticoid Receptor-DNA Interactions

Pandit

Geissler

Harris

et al. 2002

Journal of Biological Chemistry

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The glucocorticoid receptor (GR) is a DNA-binding protein that can regulate the transcription of a large number of genes in a ligand-dependent fashion. Although much progress has been made on the mechanism of transcriptional regulation by GR, a potential allosteric effect of GR-binding ligands on specific GR-DNA interactions is controversial. In this study, gel-shift methods are used to measure the effects of a classical agonist dexamethasone and a prototypical antagonist RU486 on the in vitro interactions of GR with DNA substrates, which contain glucocorticoid response elements (GREs) from promoters of GR-regulated genes. These studies show that cell extracts containing human GR bind specifically and with high affinity to GREs in the absence of ligand. An agonist dexamethasone and antagonist RU486 do not affect the affinity of GR for DNA but subtly alter the electrophoretic mobility of the GR-DNA complex. Importantly, the dissociation rate of GR from DNA increases as a function of the concentration of GRE-containing DNA. At a fixed DNA concentration, dexamethasone-bound GR dissociates from DNA significantly faster than does ligand-free GR or RU486-bound GR. These results are consistent with a model for transcriptional activation in which a dynamic complex is formed between agonist-bound GR and DNA. The glucocorticoid receptor (GR)1 is a member of the steroid hormone receptor family of transcription factors that regulates a large number of genes in a hormone-or ligand-dependent manner. GR is predominantly cytosolic and requires association with heat shock proteins for ligand binding, a feature that has complicated in vitro structural studies of GR (1-6). The molecular mechanism by which hormones and small molecule ligands regulate GR-mediated gene expression is not clearly understood. A favored model is that hormone binding induces dissociation of heat shock proteins from the ligand-bound GR complex, which then translocates to the nucleus for sequencespecific DNA binding to GREs (7-10). However, the underlying mechanism for the expression of agonist versus antagonist activity for the steroid-bound GR-DNA complex is not clear.Attempts to study the allosteric effects of hormones on GR-DNA interactions in vitro have led to contradictory observations. In general, studies indicate that hormones are not required for stable GR binding to DNA in vitro (11-16). Specifically, complexes of agonist triamcinolone acetonide and antagonist RU486 bound to GR have a comparable affinity for glucocorticoid response elements from different promoters (14 -16). In contrast to these studies done on asymmetric GR binding sites, gel-shift experiments reported with GR and an artificial palindromic GRE suggest that ligands directly affect GR-DNA binding (17). The agonist dexamethasone enhances GR-DNA binding severalfold, whereas antagonists including RU486 have no effect on GR-DNA binding. However, this observation is contested by a report that also implements gelshift methods with a palindromic GRE substrate but uses in vitro transcribed...

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Colorimetric Detection of Glutamine Synthetase-Catalyzed Transferase Activity in Glucocorticoid-Treated Skeletal Muscle Cells

Cited by 39 publications

References 35 publications

Inhibition of SNAT2 by Metabolic Acidosis Enhances Proteolysis in Skeletal Muscle

Inhibition of SNAT2 by Metabolic Acidosis Enhances Proteolysis in Skeletal Muscle

Hepatic Glucocorticoid Receptor Antagonism Is Sufficient to Reduce Elevated Hepatic Glucose Output and Improve Glucose Control in Animal Models of Type 2 Diabetes

Allosteric Effects of Dexamethasone and RU486 on Glucocorticoid Receptor-DNA Interactions

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