Control of insulin receptor level in 3T3 cells: effect of insulin-induced down-regulation and dexamethasone-induced up-regulation on rate of receptor inactivation.

Abstract

Chronic exposure of 3T3 mouse fibroblasts to insulin or to the glucocorticoid dexamethasone induces down-regulation and up-regulation, respectively, of cell-surface and total cellular insulin binding capacity. Both processes are reversed upon withdrawal of the inducer. Scatchard analysis of insulin binding for receptors in the down-and up-regulated states indicates that the changes in binding capacity result primarily from alterations in insulin receptor level. That these alterations in total receptor level are due to changes in cell-surface receptor level is indicated by the fact that the level of trypsin-insensitive, presumably intracellular, insulin binding sites does not change appreciably upon down-and up-regulation. The effects ofinsulin-induced down-regulation and dexamethasone-induced up-regulation on the rates of insulin receptor synthesis and~decay were assessed by the heavyisotope density-shift technique. Cells were shifted to medium containing heavy (2H, '3C, '5N) amino acids and, at various times after the shift, light and heavy receptors solubilized from total cellular membranes were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that the insulinand dexamethasone-induced alterations in insulin receptor level were due entirely to changes in the rate constant for receptor inactivation. The decrease in the first-order rate constant for receptor decay caused by dexamethasone is unexpected in view of the known action of steroid hormones in the induction of the synthesis of specific proteins.The insulin receptor is a dynamic cellular protein whose equilibrium concentration in the plasma membrane can be altered by physiological perturbants. The effect of chronic treatment ofcells with insulin on cell surface insulin receptor level is well documented. This phenomenon, known as ligand-induced receptor down-regulation, has been demonstrated in vivo with circulating monocytes (1) and fat cells (2) from obese humans and with cultured human IM-9 lymphocytes (3-6), rat hepatocytes (7), and chicken hepatocytes (8). The reciprocal effect-i.e., an increase in cell! surface insulin binding capacity-is observed upon removal ofinsulin from a down-regulated system (3) or by induction of differentiation of 3T3-Ll preadipocytes into adipocytes (9-11).Changes in the level or affinity ofhormone receptors can also be induced by heterologous hormones (6). Glucocorticoids have been shown to affect cellular levels of prolactin receptors (12), epidermal growth factor receptors (13), and insulin, receptors (14-18). It was of interest, therefore, to determine the mechanism by which glucocorticoids induce an increased insulin binding capacity of cells.In this report the effects of two antagonistic perturbants of insulin receptor level in 3T3 cells are described: the effect of insulin, which causes down-regulation, and that of dexamethasone, which causes up-regulation ofinsulin receptor level. The heavy isotope density-shift technique (8,19,20) was employed to determine whether...

Molecular mechanism of bis(maltolato)oxovanadium(IV)-induced insulin signaling in 3T3-L1 and IM9 cells: impact of dexamethasone

“…Consistent with earlier reports (Fantus et al 1982, Knutson et al 1982, McDonald & Goldfine 1988, we have found an increase in the content of IRb in both cell lines in response to dexamethasone treatment. This prompted us to extend our studies in order to check whether the above glucocorticoid may have any impact on insulin signaling in BMOV-treated cells with depleted IRb level.…”

“…In order to judge this probability, dexamethasone was chosen in our experiment to be co-administered with BMOV because the former drug is known to augment the transcription of IR gene and synthesis of IR in IM9 cells (Fantus et al 1982, McDonald & Goldfine 1988, and result in time-and dose-dependent increase in IR protein in mouse 3T3-C2 fibroblasts (Knutson et al 1982). This is also further confirmed by our study where the total cellular IRb content was increased in response to dexamethasone treatment, almost in a dose-dependent manner in 3T3-L1 cells revealing 41% augmentation at 10 mM drug (Fig.…”

“…In order to focus on this issue more deeply, we have examined the effect of monocarboxylic ionophore monensin, an inhibitor of receptor recycling (Whittaker et al 1986), on the soluble IR pool of IM9 cells receiving an exposure to BMOV for 18 h and a brief insulin treatment for 15 min. Consequently, we have also checked whether treatment of dexamethasone which has been shown to increase the cellular content of IR in 3T3 fibroblasts and IM9 cells (Fantus et al 1982, Knutson et al 1982, McDonald & Goldfine 1988) can encounter the above negative impact of BMOV on IR level in both 3T3-L1 and IM9 cells and thereby resulting in an augmentation in the phosphorylation of IR with subsequent modulation in the downstream insulin signaling molecules, including Akt, GSK, and Raf.…”

Up-regulation of insulin receptors with dexamethasone in cultured human urinary bladder carcinoma cells.

“…Similar results were reported by Fantus et al (1981) and Knutson et al (1982). The latter (Knutson et al, 1982) showed that an increase in the number of insulin binding sites with dexamethasone was due to a decrease in the degradation rate of insulin receptors. Furthermore, dexamethasone changed preadipocytes to adipocytes, accompained by an increase in the number of insulin receptors while changing the affinity for insulin (Rubin et al, 1978).…”

“…We know "down-regulation", in which the number of insulin receptors decreases with insulin treatment (Gavin et al, 1974 ;Blackard et al, 1978 ;Livingston et al, 1978 ;Petersen et al, 1978), and "upregulation", in which the number of insulin receptors increases with gulcocorticoid treatment (Fantus et al, 1981 ;Knutson et al, 1982), to regulate insulin receptors. Recently, Stevens et al (1983) showed that insulin increased its binding sites in cultured chondrosarcoma chondrocytes.…”