Regulation of Insulin Receptor Metabolism - Semantic Scholar

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Differentiation of 3T3-Ll preadipocytes into mature adipocytes is accompanied by a 10- to 20-fold rise in the level of insulin receptor extractable from total cellular.
THRJOURNALOF BIOLOGICAL CHEMISTRY Vol. 256, No. 8, Issue of Aprll 25,pp 3917-3925, 1981 Prmted in U . S A

Regulation of Insulin Receptor Metabolism DIFFERENTIATION-INDUCED ALTERATION OF RECEPTOR SYNTHESIS AND DEGRADATION* (Received for publication, October 28, 1980, and in revised form, January 14, 1981)

Brent C. Reed$, Gabriele V. Ronnettg, Peter R. Clements, and M. Daniel Lane From the Department of. Physiological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 2j205

Conversely, elevated levels of cell surface insulin receptors have been observed, e.g. during reversal of the down regulated state caused by decreased insulin levels (1,4), uponexposure to certaindrugs, (8,9), and in certain cases, whencell function is altered as in the differentiation of.thymocytes and 3T3-Ll preadipocytes. Thymocytes acquire insulin receptorsupon activation by mitogens (10) while 3T3-Ll preadipocytes, during differentiation into adipocytes, exhibit a 10- to 20-fold elevation in the number of surface insulin receptors (11-14) and acquire an increased sensitivity of glucose transport rate to insulin (13, 14). The mechanism(s) by which the number of cell surface insulin receptors is controlled is not understood. Severalpossibilities exist includingthe activation or inactivation of receptors, shifts in the distribution of receptors between the cell surface and intracellular compartment, and alteration of the rates of receptor synthesis and/or degradation. The low level of insulin receptor in most cell types and the lack of a suitable procedure for distinguishing newly synthesized from old (previously synthesized) receptorsdiscouraged studies aimed a t measuring rates of receptor synthesis and degradation. We have recently adapteda heavy isotope “density shift” method to measure the synthesis and degradation of the insulin receptor. This procedure was originally described by Devreotes and Fambroughfor studying acetylcholine receptor turnover (15-17). The density shift approach entails exposing cells to medium containing amino acids enriched in 13C, “N,and ‘H. Newly synthesizeddense and previously existing light insulin receptors are then extracted from cell membranes with detergent and are separated by isopycnic banding on a CsCl gradient. The dense and light receptor peaks are located and quantitated by determining The level of surface insulin receptors is an important factor the insulin binding activity of each fraction of the gradient. incontrolling the cells’ response to insulin. The lowered Measurement of theamount of denseandlightreceptor sensitivity of cells to insulin in certain pathological states can present a t increasing times of exposure of the cells to dense be attributed to a reduced numberof surface insulin receptors. amino acids provides results for computing the rate of new In addition, the hyperinsulinemia accompanying some insulin- dense receptor synthesis andold light receptor degradation. resistant states has beenidentified as anassociated factor for This approach providesasensitive means to determine lowered receptor levels (1-3). This phenomenon known as whether changes in receptor synthetic or degradative rates “insulin-induced receptor down regulation” has been demon- regulate cell surface receptor levels. Our initial report (18) strated in vivo and in cell culturewithseveral cell types described the applicationof this method to the measurement including IM-9 lymphocytes (4), liver cells (5, 6), and adipo- of rates of insulin receptor turnover in differentiated 3T3-Ll cytes (7). adipocytes. T o determine whether anincreased rate of receptor synthesis or degradation is responsible for the differentia* This investigation was supported by grants from the National Institutes of Health (AM-14574), the American Heart Association tion-linked increase in insulin receptor level, we have now (78822), and the Kroc Foundation. The costs of publication of this measured the turnover of insulin receptor in 3T3-CZ fibroarticle were defrayed in part by the payment of page charges. This blasts and in both undifferentiated and differentiated 3T3-Ll article must therefore be hereby marked “advertisement” in accord- cells and have compared their relative rates of receptor synance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 Supported by a Postdoctoral Fellowship (AM-05506) from the thesis and degradation. The results demonstrate that an increased rate of receptor synthesis upondifferentiation acNational Institutes of Health. 5 Supported by Medical ScientistTraining Program Grant counts entirely for the rise in receptor number. High levels of GM07309. insulin receptor are also shown to have relatively little effect

Differentiation of 3T3-Ll preadipocytes into mature adipocytes is accompanied by a 10- to 20-fold rise in the level of insulin receptor extractable from total cellular membranes with Triton X-100.A heavy isotope density shift technique is utilized to measure relative synthetic and degradation rates of insulin receptor in undifferentiated and differentiated 3T3-Ll cells and in nondifferentiating 3T3-C2 cells. Comparison of the rates of new dense receptor synthesis and light receptor decay in the differentiated and undifferentiated states revealed that the 10- to 20-fold rise in steady state receptor level accompanying differentiation is due solely to a 20- to IO-fold increase in the rate of insulin receptor synthesis. A 2-fold increase in the first order rate constant f o r degradation, kd,also accompanied differentiation. For the three cell types studied, the half-lives of the receptor were r e m a r k a b l y similar ranging from 713 hr. In differentiated 3T3-Ll adipocytes insulin had little or no effect on receptor level or the rate of insulin receptor degradation. W e were unable, therefore, to demonstrate insulin-induced down regulation of total cellular insulin receptors in the adipocyte form of3T3L 1 cells. The molecular weight of the receptor, banded isopycnically on CsCl gradients, was approximately 700,000 by gel filtration in thepresence of CsC1. Mixing experiments between totally dense and totally light receptor extracts indicated that no receptor subunit interchange occurs under the conditions of the turnover study.

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Regulation of Insulin Receptor Metabolism

on the rate of receptor degradation in differentiated 3T3-Ll cells.

Banded Isopycnically onCsCl Gradients by Gel Filtration on Sepharose 4B-CL-Soluble receptor extracted from 35-50 X lo6 differentiated 3T3-Ll adipocytes was banded isopycnically on CsCl gradients, and the peak fractions were combined as described above. One-half EXPERIMENTALPROCEDURES ml of receptor solution was applied to a column (1.0 cm X 44.5 cm) of Culture Conditions-3T3-LI and 3T3-C2 cells were cultured in 6- Sepharose 4B-CL equilibrated in buffer (2.59 M CsCl in 50 mM Triscm dishes(FalconProducts)as described (19) butwithfetal calf HCI, pH 7.4) a t 4 "C. Four-tenths ml fractions were collected and serum replacing calf serum. Differentiation of 3T3-Ll adipocytes was assayed for '"I-insulin binding activity (200 pl/assay).Molecular induced by a modification of the method of Rubin et al. (13). Two to weight standards were subjectedto gel fitration in an identical three days postconfluence, medium (3.5 m l ) supplemented with 0.5 manner. Ferritin andthyroglobulin were detected by their absorbance mM isobutylrnethylxanthine, 1 p~dexamethasone, and1Opg of insulin a t 280 nm while aldolase and catalasewere assayed enzymatically (23, (Elanco)per ml was addedto inducedifferentiation.On day 3, 24). isobutylmethylxanthine and dexamethasone were removed, and the cells were fed every 2 days with medium containing 10 pg of insulin RESULTS per ml. By day 7, 8040%of the cells in the monolayer expressed the Isopycnic Banding and Insulin BindingProperties of Inadipocyte phenotype. Undifferentiated 3T3-Ll and3T3-C2 cells were maintained in an identical manner but in the absence of drugs or sulin Receptors from Differentiated andUndifferentiated insulin. Unlike 3T3-Ll cells which express increased insulin receptor 3T3-Ll Cells andNondifferentiating 3T3-C2 Cells-The levels due to treatmentwith isobutylmethylxanthine, dexamethasone, properties of the insulin receptors from three cell types were and insulin, 3T3-C2 cells exhibit no significant change in "'I-insulin compared in the studies to be described. The three cell types binding activity when treated with these agents. Density Shift Experiments-Monolayers were incubated at 37 "C included undifferentiated 3T3-Ll preadipocytes, differentiated 3T3-Ll adipocytes, and nondifferentiating 3T3-C2 fiin 10% C02:90% air with 3.5 ml of "heavy"medium, each 100 ml containing 10 ml of 10%dialyzed (against phosphate-buffered saline) broblasts. The latter cell line was derived from the same fetal calf serum, 0.8 mg of tryptophan, 3 mg of cystine,6mg of uncloned stock as 3T3-Llcells and exhibits an extremely low glutamine, 100 mg of a dense amino acid mixture (95% enriched in frequency of adipocyte conversion. Insulinreceptors were 1 mg of insulin, '"C, "N, and 'H (Merck Sharp and Dohme, Canada)), and 90 ml of Dulbecco's modified Eagle's minimal essential medium extracted with Triton X-100 from the total cellular mem(without amino acids). Dense amino acids were dissolved in phos- branes of the three cell types, and the solubilized receptors phate-buffered saline, passed through a UM-2 filter (Amicon, Lexing- were subjected to isopycnic centrifugation on CsCl density ton, MA), andfilter sterilized prior to use. gradients (see under"ExperimentalProcedures").Typical isopycnic Banding of Solubilized Insulin Receptor in CsCl Gra- isopycnic banding profiles for the insulin receptors are shown dients-To remove bound unlabeled insulin, cell monolayers were in Fig. 1. washedtwo times with Krebs-Ringerphosphate buffer (pH 7.4) containing one-half the usual level of calcium, 1% bovine serum albumin, and25 mM glucose. The monolayers were rinsed three times with buffer. A 20-min incubation a t 37 "C followed each of the last 80 three washes. Thisprocedure completely removes cell-associated insulin since exposure of 3T3-Ll cells to insulin-containing medium at 37 "C for several h affects "'I-insulin binding to solubilized receptor 60 t by