Regulation of insulin receptor metabolism. Differentiation-induced alteration of receptor synthesis and degradation (original) (raw)
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Degradation of Insulin Receptors in Rat Adipocytes
Diabetes, 1983
Insulin receptors on viable rat adipocytes were affinitylabeled using a biologically active and photosensitive analogue of insulin, 125 l-B2(2-nitro, 4 azidophenylacetyl)-des-Phe B1-insulin (125 l-NAPA-DP-insulin). The radiolabeled proteins were identified by SDS polyacrylamide gel electrophoresis and autoradiography. Binding of
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1986
The bimolecular binding reaction between mono[TyrAI4-t2sI]iodoinsulin and the insulin receptor was investigated at 37°C in intact isolated rat adipocytes in which membrane traffic was inhibited by 1 mM KCN. This treatment decreased the fraction of cell-associated radioactivity resistant to treatment at pH 3 (usually regarded as internalized ligand) from 70% to 17%. The total amount of tracer being ceil-associated at steady state was reduced to about half of the control value partly because of a decreased apparent binding affinity. The tl/2 for the forward reaction was reduced from 414 s in the control cell to 26 s in the KCN treated cell. Likewise, the tt/2 for the dissociation was reduced from 461 s to 67 s. Both rate constants were pH sensitive, the association rate constant being 7-8-fold more than the dissociation rate constant. Since both rate constants for the bimolecular reaction were one order of magnitude greater than those for the uptake and the release of label in the untreated cell, other processes than binding constitute the rate-limiting step(s) in the cellular reaction with insulin.
Receptor binding and biological effect of insulin in human adipocytes
Diabetologia, 1977
The binding of 125I-labelled insulin to human adipocytes was studied at 37 ~ C. The precipitability of the 125I-labelled insulin preparation (0.03 nmol/1) in tfichloroacetic acid and the concentration of biologically active insulin (7.5 nmol/1) remained constant in buffer incubated with human adipocytes (100 ~tl cells/ml suspension) for 30-60 minutes at 37 ~ C, whereas more than half of the insulin was inactivated by rat fat cells under the same conditions. A constant level of binding of 125I-labelled insulin (0.03 nmol/1) to human adipocytes was obtained after 45 minutes. The apparent dissociation constant of receptor binding was about 0.2 nmol/1 as compared to about 2 nmol/l for rat adipocytes. Conversion of [U-14C]glucose to lipids was stimulated halfmaximally by about 0.05 nmol/l of insulin (similar to rat adipocytes). Thus, half-maximal stimulation of human adipocytes was obtained with a receptor occupancy of about 20-30 per cent.
Proceedings of the National Academy of Sciences, 1974
Chronic (5-16 hr) exposure of cultured human lymphocytes to 10 -8 M insulin at 37° in vitro produced a decrease in insulin receptor concentrations unaccounted for by simple occupancy of sites; acute exposure (0-2 hr) was without effect. These results reproduced observations in vivo where chronic hyperinsulinemia (e.g., 10 -8 M insulin in the circulation of obese insulinresistant hyperglycemic mice) is associated with a substantial reduction in the concentration of insulin receptors per cell, while acute hyperinsulinemia in vivo has no effect on receptor concentration. These data suggest a reciprocal relationship between insulin in the extracellular fluid and the concentration of insulin receptors per cell, which is mediated at the target cell itself by intracellular insulin-sensitive regulatory processes and directly affects target-cell sensitivity to hormone.
Internalization and intracellular processing of insulin and insulin receptors in adipocytes
Metabolism, 1982
I'orcinc I~UIII)L'OIII~O~~~~ IIIUIII~ wab gcnerousl~ auppl~cd b> Dr Ronald <'hawe of the Eli I.illy C II. \\;,I "'1 was purchaed frum the Yew t<ngland Nuclwr ('II., bovine xrum ,Ilbumin (i'rnction V 1 from Armour and Cu.. and collagcnasc l'rom Worthlngtun Binchemicalh. and chloroquinc. NaF. and 2.Gdinltrophenol from Sigma Male Sprague-Dawlq rats wcighlng I60 to 715 g \*crL: used lor alI cxpcrimenta. Rats were stunned bq :I blow to the head. decapitated. and epididymal fat pads removed. lbolated f31 cells wcrc prepared by shaking at 37OC l'or 60 min in Krebs-Rinpcr bicarbonate bulrer containing collagenase (3 mg/ml) and albumln (40 mg/ml) according to the method of Rodbell.' Cell\ were then liltcrcd through nylon mesh (350 PM). centrifuged at 400 rpm for 2 min and washed twice in Tris Butler. Adipocytc count5 wcrc performed according tu :i modification ol' method III of Hirsch and Gallian" in which the cells were fixed in 1% osmium tetroxide in 0.05 M collidinc buffer (made Isotonic with saline) l'or 24 hr al 37°C' and then (akcn up in a known volume of 0 I54 M NaC I l'or counting. Counting was performed with a model ZB Coulter Counter with 400 PM aperture. lrlsirlin Pretreutrwnt und l~issoc~iation Pr0wdurr.r Ten ml of adipocytes (4-h x IO' cells/ml) subpended in pH 7.(> bufer containing 35 mM Tris. I20 mM NaCI. I .7 mM MgSO4. 1.0 mM CaCI, 2.5 mM KCI. IO mM dextrose, and 1'~: bovine serum albumin (Tris-BSA bulrer) were incubated with insulin in 2 ml polypropylene flasks. Cells were then gently agitated In a bhaking water bath at the indicated times and temperatures. At the end of the incubation period, cells were transferred to I6 125 mm polystyrene tubes. centrifuged at 200 rpm for 7 min. and the insulin containing buffer removed and replaced with insulin free Tris-BSA butfer. pH 7.0. Adipocytes were again centrifuged, resuspended in
Time course of insulin-receptor binding and insulin-induced lipogenesis in isolated rat fat cells
Journal of Biological Chemistry, 1975
1. Isolated rat fat cells were incubated at 37" with [U-14C]glucose 0.55 mnr and l*SI-labeled insulin. The amount of receptor-bound l*SI-labeled insulin and the rate of insulininduced 14C-lipid synthesis were assessed during association and dissociation of **%labeled insulin. 2. The rate of 14C-lipid synthesis was constant from zero time in the absence of insulin and in the presence of insulin in a high concentration (0.7 PM). With insulin in a low concentration (56 paa) the insulin-induced rate of "C-lipid synthesis was proportional to the receptor occupancy; the receptor binding reached equilibrium and the rate of "C-lipid synthesis reached a constant value after 30 to 45 min. With insulin in a concentration of 0.7 IIM the rate of "C-lipid synthesis reached a steady state before equilibrium of the receptor binding was obtained. 3. After preincubation with 56 PM 12SI-labeled insulin followed by removal of eztracellular insulin the decrease in the rate of insulin induced "C-lipid synthesis followed the decrease in receptor occupancy with a half-time of about 10 min. After preincubation with insulin in concentrations of 0.28, 0.56, and 1.4 nrd a maximum rate of 14C-lipid synthesis was maintained for about 8, 15, and 30 min, respectively. 4. The following model is suggested. Binding of insulin to the previously described receptors with a dissociation constant of about 3 IIM (GAMMELTOFT, S., AND GLIEMANN, J. (1973) Biochim. Siophys Acta 320, 16-32) represents the first step in the action of insulin on lipid synthesis from glucose. The receptor occupancy is rate-determining at low concentrations of insulin, i.e. when the occupancy is small (about 2% or less). At higher insulin concentrations some other step becomes rate-determining and the higher occupancy at equilibrium therefore causes no further increase in the steady state lipogenesis. However, a high receptor occupancy causes a prolonged maintenance of a maximal (or near-maximal) effect after removal of insulin from the medium. One of the major effects of insulin on fat cells is to stimulate the conversion of glucose to neutral lipids. It is generally agreed
Journal of Biological Chemistry
We have used an iodinated, photoreactive analog of insulin, 1251-B2(2-nitro-4-azidophenylacetyl)-des-PheB'-insulin, to covalently label insulin receptors on the cell surface of isolated rat adipocytes. Following internalization of the labeled insulin-receptor complexes at 37 "C, we measured the rate and extent of recycling of these complexes using trypsin to distinguish receptors on the cell surface from those inside the cell. The return of internalized photoaffinity-labeled receptors to the cell surface was very rapid at 37 "C proceeding with an apparent t H of 6 min. About 95% of the labeled receptors present in the cell 20 min after the initiation of endocytosis returned to the cell surface by 40 min. Recycling was slower at 25 and 16 "C compared to 37 "C and essentially negligible at 12 "C or in the presence of energy depleters. Addition of excess unlabeled insulin had no effect on the recycling of photoaffinity-labeled insulin receptor complexes, whereas monensin, chloroquine, and Tris partially inhibited this process. These data indicate that dissociation of insulin from internalized receptors is not necessary for insulin receptor recycling. Furthermore, agents which have been shown to prevent vesicular acidification inhibit the recycling of insulin receptors by a mechanism other than prevention of ligand dissociation. Once insulin binds to its receptors on target tissues, the occupied receptors are internalized via an endocytotic mechanism (1-5). Following internalization, the intracellular receptors can either be sequestered within the cell (6), degraded (7, 8), or recycled back to the plasma membrane (4, 5, 9). These general pathways are applicable to a wide variety of surface-bound ligands (for review, see Ref. lo), although the relative proportions of internalized receptors which are degraded, sequestered, or recycled vary greatly among different cell types and ligand receptor systems. Insulin-induced internalization of insulin receptors has been described (1-5), but relatively little is known about factors which regulate the cellular traffic of insulin receptors once they are internalized. Since insulin is always present in the plasma under normal physiological conditions, there is constant ligand-induced receptor internalization. The constant loss of receptors from
High molecular weight forms of the insulin receptor
Biochemistry, 1986
The insulin receptor of liver, adipose, and placental plasma membranes was photoaffinity labeled with radioiodinated NfB29-(monoazidobenzoyl)insulin. Three specifically labeled bands of 450, 360, and 260 kilodaltons (kDa) were identified in each tissue by polyacrylamide gel electrophoresis of the membranes solubilized in sodium dodecyl sulfate (SDS). The 360-and 260-kDa bands corresponded to partially reduced forms of the 450-kDa band. The distribution of radioactivity between the three insulin receptor bands was dependent on the tissue, the purity of the receptor preparation, and the conditions of solubilization in SDS.