ABSTRACT
Oxazolidinediones are a class of oral antidiabetic agents that are closely related structurally and pharmacologically to thiazolidinediones. The thiazolidinediones have been shown to partially reverse the loss in insulin-responsive glucose uptake caused by chronic treatment with dexamethasone. This study was conducted to determine certain aspects of the mechanism of thiazolidinedione and oxazolidinedione action. We selected the oxazolidinedione CP-92,768-2 (5-[2-[(5-methyl2-phenyl-4-oxazolyl)methyl]5-benzofuranyl methyl]2,4- oxazolidinedione) to determine whether these agents could reverse the dexamethasone-induced down-regulation of IRS-1, the insulin receptor substrate-1. In 3T3-L1 adipocytes, dexamethasone treatment resulted in down-regulation of IRS-1 to 60% of control values. Simultaneous treatment with CP-92,768-2 significantly increased IRS-1 to 78% of the control value (EC50, < 10 nM), although it did not completely reverse the dexamethasone effect at any concentration tested. CP-92,768-2 alone did not have any effect on IRS-1. CP-92,768-2 did not affect the stability of IRS-1 protein in the presence or absence of dexamethasone, as measured by [35S]methionine pulse-chase labeling. Dexamethasone decreased messenger RNA (mRNA) for IRS-1 after 24 h of treatment to 40% of the control value. CP-92,768-2 partially reversed this decrease in IRS-1 mRNA to 65% of the control value after 24 h of treatment, but had no effect on IRS-1 mRNA in the absence of dexamethasone. Dexamethasone down-regulated the insulin stimulation of [3H]thymidine incorporation to 68% of the control value. Dexamethasone in the presence of CP-92,768-2 down-regulated insulin stimulation of thymidine incorporation by only 9%. Dexamethasone also down-regulated the expression of phosphoenolpyruvate carboxykinase (PEPCK) protein by 50%. CP-92,768-2 partially protected PEPCK from the dexamethasone down-regulation. Conversely, the up-regulation of expression of PEPCK and IRS-1 produced by dexamethasone in KRC-7 hepatoma cells was not affected by CP-92,768-2. One contribution of oxazolidinediones to an increase in insulin responsiveness in the presence of glucocorticoids may be the up-regulation of IRS-1 in adipose cells.
Subject(s)
Adipocytes/metabolism , Benzofurans/pharmacology , Dexamethasone/pharmacology , Oxazoles/pharmacology , Oxazolidinones , Phosphoproteins/metabolism , 3T3 Cells , Animals , Down-Regulation , Gene Expression Regulation/drug effects , Insulin/pharmacology , Insulin Receptor Substrate Proteins , Mice , Mifepristone/pharmacology , Phosphoenolpyruvate Carboxykinase (GTP)/metabolism , Phosphoproteins/genetics , RNA, Messenger/metabolism , Receptors, Glucocorticoid/metabolismABSTRACT
Insulin resistance resulting from prolonged exposure of intact animals or cultured cells to glucocorticoids is often attributed to postreceptor signaling defects. To better understand the specific effects of glucocorticoids on insulin signaling, we have characterized the effect of dexamethasone on the expression of an insulin signaling intermediate, the insulin receptor substrate-1 (IRS-1) in 3T3-L1 adipocytes. Addition of dexamethasone resulted in a 40-70% decline in steady-state IRS-1 protein over 24-48 h of treatment. Dexamethasone did not significantly change the degradation rate of IRS-1 protein but decreased the net rate of amino acid incorporation into IRS-1 by 87%. Between 1 and 2.5 h of treatment with dexamethasone, actinomycin D, or both drugs given simultaneously, the concentration of IRS-1 mRNA declined with a half-life of 0.7-1.0 h. However, after 4 h of dexamethasone treatment, IRS-1 mRNA concentrations stabilized at approximately 35% of the control level. The dexamethasone-induced decline in IRS-1 protein could be prevented by simultaneous administration of the glucocorticoid antagonist mifepristone, RU38486. These results suggest that in 3T3-L1 adipocytes the loss of IRS-1 protein after dexamethasone treatment can be accounted for chiefly by inhibition of the synthesis of IRS-1 mRNA.
Subject(s)
Adipocytes/metabolism , Dexamethasone/pharmacology , Phosphoproteins/metabolism , RNA, Messenger/metabolism , 3T3 Cells , Adipocytes/drug effects , Amino Acid Sequence , Animals , Antibodies , Autoradiography , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Insulin Receptor Substrate Proteins , Kinetics , Mice , Molecular Sequence Data , Peptides/chemistry , Peptides/immunology , Phosphoproteins/biosynthesis , Phosphoproteins/isolation & purification , RNA, Messenger/biosynthesis , Rabbits/immunology , Sulfur Radioisotopes , Time Factors , Transcription, Genetic/drug effectsABSTRACT
A nonradioactive modification of the traditional ribonuclease protection assay is described and applied to the detection of two messenger RNAs. The biotinylated probes are stable and easy to handle, which permits the processing of large numbers of samples. Chemiluminescence is generated with streptavidin-conjugated alkaline phosphatase and provides sensitivity equal to or greater than that achieved with the radioactive detection method.
Subject(s)
RNA Probes , RNA, Messenger/analysis , Ribonucleases/pharmacology , Actins/genetics , Animals , Biotin , Insulin Receptor Substrate Proteins , Mice , Phosphoproteins/geneticsABSTRACT
One of the first steps that follows insulin receptor activation is the tyrosine phosphorylation of the 160-185 kDa insulin receptor substrate IRS-1. In 3T3-L1 adipocytes, expression of IRS-1 is down-regulated by chronic exposure to insulin. Expression of IRS-1 mRNA is essentially unchanged. However, [35S]Met pulse-chase labeling demonstrates that the rate of degradation of IRS-1 protein is about 10 times faster in insulin-treated cells than in basal cells. The down-regulation occurs in the presence of cycloheximide or actinomycin D and therefore is not dependent upon protein synthesis. Chloroquine does not inhibit the insulin-induced degradation, suggesting that the site of proteolysis is an extra-lysosomal compartment. The insulin-regulated proteolysis of IRS-1 may contribute to the insulin resistance seen in these cells following chronic exposure to insulin.
