Cytochalasin B-induced alterations of insulin binding and microfilament organization in cultured fibroblasts

The effect of cytochalasin B (CB) on insulin binding has been investigated in confluent cultures of chick embryo fibroblasts. Time- and dose-dependent increases in binding of [¹²⁵I]insulin was observed after incubation of fibroblasts with CB. At 10 μg/ml, CB caused a 2-fold increase in binding, due to an increase in the number of binding sites from 9.3 × 10³ to 2.0 × 10⁴ per cell. Removal of CB from the growth medium was accompanied by a decrease in [¹²⁵I]insulin binding to control values in 24 h. Increase in the binding of insulin in CB-treated CEF was also accompanied by enhancement of insulin to stimulation of [³H]thymidine incorporation into acid-insoluble material. CB treatment also caused disorganization and disappearance of microfilament bundles and changes in cell shape from flat, with a few blebs and folds on the cell surface, to round with numerous blebs and folds. The data from this study suggest that changes in the number of surface insulin-binding sites may be related to the state of organization of cytoskeletal structures in chick embryo fibroblasts.     

Studies on the regulation of insulin receptors in cultured BALB/3T3 fibroblasts

The level of [¹²⁵I]insulin binding to BALB/ 3T3 fibroblasts was low in growing cells and high in stationary cells. Since frequent changes of medium (every 2 h) did not modify the hormone binding of the stationary cells, it is unlikely that serum factors directly regulate the number of insulin receptors. Cells were grown to different densities by plating them in different concentrations of serum. Insulin binding was low in dense cultures maintained actively growing by high serum concentration, while binding was high in sparse cultures which were growth-arrested due to serum depletion. Thus, cell density does not directly regulate the insulin receptors. The growth status of the cells is the only factor that explains consistently the variations of insulin binding in these and previous [1, 2] experiments. Synchronization of the cells by two different methods did not show a reproducible cellcycle dependence for the insulin receptors.     

Internalization of insulin and its receptor in the isolated rat adipose cell: Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37°C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [¹²⁵I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [¹²⁵I]insulin to these same membrane fractions. At 37°C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20–30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16°C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.     

Insulin Receptor Sub-Types in a Human Lymphoid-Derived Cell Line (IM-9): Differential Regulation by Insulin,Dexamethasone and Monensin

Abstract

The cells of the human IM-9 lymphocyte-derived line contain a sub-population of insulin binding sites which differ from classical insulin binding sites in their higher binding affinity for insulin-like growth factor II (IGF-II) and insulin-like growth factor I (IGF-I). These atypical insulin binding sites are identified on IM-9 cells by [¹²⁵I]IGF-II binding.

To determine whether the atypical and classical insulin receptors of IM-9 cells were subject to different modes of in vivo regulation, we treated IM-9 cells with agents known to alter the surface expression of insulin receptors - insulin, dexamethasone and monensin. We then measured insulin and IGF-II binding to the surface of the washed cells.

Pretreatment of IM-9 cells with 1 μM insulin for 20 h at 37°C induced a 44–48% decrease in the number of high affinity insulin binding sites, but no change in the number of IGF-II binding sites. In contrast, the surface expression of both insulin and IGF-II binding sites (classical and atypical insulin receptors) increased 1.3 to 1.7-fold after treatment with dexamethasone (200 nM) and decreased 30 to 45% after monensin (1 μM). These results suggest that atypical and classical insulin receptors are differentially susceptible to down-regulation by insulin.     

Vanadate down-regulates cell surface insulin and growth hormone receptors and inhibits insulin receptor degradation in cultured human lymphocytes

Insulin is able to down-regulate its specific cell surface receptor in cultured human lymphocytes. The effect of vanadate, a known insulinomimetic agent, was examined to determine whether it could mimic insulin to down-regulate the insulin receptor. Exposure of cultured human lymphocytes (IM-9) to vanadate (0-200 microM) resulted in a time- and dose-dependent decrease in cell surface insulin receptors to 60% of control, while insulin (100 nM) down-regulated to 40%. The vanadate effect, in contrast to the rapid effect of insulin, was slow to develop (4-6 h). Surface receptor recovery after 18 h exposure was rapid after vanadate removal (20 min), but it required hours after insulin suggesting the presence of an intracellular (cryptic) pool of receptors after vanadate treatment. Insulin binding to Triton X-100-solubilized whole cells after 18 h treatment revealed that total cell receptors had decreased to 50% of control after insulin but increased to 120 and 189% of control after 100 and 200 microM vanadate, respectively. Furthermore, vanadate inhibited the insulin-mediated loss of total cell receptors from 50 to 28%. Removal of cell surface receptors by trypsin before cell solubilization revealed that 100 microM vanadate increased insulin binding to 321% of control indicating an accumulation of intracellular receptors. Labeling of cell surface proteins with Na125I and lactoperoxidase followed by immunoprecipitation of solubilized receptors with anti-receptor antibody after incubation for various times up to 20 h and quantitation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, while insulin shortened t1/2 from 7.3 to 5.3 h, vanadate prolonged receptor t1/2 to 14 h. No effect of vanadate was detected on insulin receptor tyrosine kinase activity with up to 4 h incubation at the vanadate concentrations used in this study. Furthermore, human growth hormone surface receptors were similarly down-regulated by vanadate. We conclude that 1) vanadate has an apparent insulin-like effect to down-regulate cell surface insulin receptors in cultured human lymphocytes; 2) in contrast to insulin-induced down-regulation which is associated with receptor degradation vanadate causes an accumulation of intracellular (cryptic) receptors and inhibits insulin receptor degradation; and 3) these effects of vanadate may be exerted on other cell surface receptors.     

Chicken Insulin Discriminates Between Receptors for Insulin and Insulin-Like Growth Factor I on Centrally-and Peripherally-Derived Glial Cells

Abstract

Insulin and IGF-I receptors in G26–20 cells, derived from a mouse oligodendroglioma, and in RN-2 cells, derived from a rat Schwannoma, were characterized by specific binding to [¹²⁵I]insulin and [¹²⁵I]IGF-I respectively. In both cell lines, the Kd for insulin was 1.5 nM. Insulin receptor number was 33,000/cell for RN-2 cells and 17,000 receptors/ cell for G26–20 cells. RN-2 cells have 700,000 IGF-I receptors/cell with a Kd of 2 nM while G26–20 cells have 60,000 receptors/cell with an affinity of 4.9 nM. However, the independence of these two receptor populations in each cell type was equivocal since the subunit structure of these receptors appears identical by electrophoresis. In both cell lines, competition with insulin analogs for [¹²⁵I]insulin binding demonstrated chicken insulin>insulin>IGF-I. Competition for [¹²⁵I]IGF-I binding showed that IGF-I was approximately 85-fold more potent than insulin. Chicken insulin was ineffective at all concentrations. Thus, chicken insulin can be used as a specific ligand to unequivocally discriminate between IGF-I and insulin receptors and effects.     

Rapid down regulation of insulin receptors in adipocytes artifact of the incubation buffer

Rat adipocytes were incubated with 15 nM insulin in different buffers at 37°C. The cells were washed and reincubated at 16°C in the presence of 18 pM A14-[¹²⁵I]monoiodoinsulin to determine the insulin receptor concentration. After incubation for 2 h in Tris buffer the binding decreased to about 30 %, whereas no decrease was found after incubation in Hepes, phosphate or bicarbonate buffers. Binding of tracer insulin reached a constant level by 45 min in Hepes buffer at 37°C, whereas it continued to increase in Tris buffer. Washout of tracer insulin after incubation in Tris buffer at 37°C showed a large, slowly dissociable fraction. It is suggested that the rapid down regulation of insulin receptors $\text{in}\text{vitro}$ is an artifact of the Tris buffer and that the phenomenon is due to a slowly reversible occupancy of a receptor pool with unlabelled insulin.     

Ontogeny of insulin-receptor interaction: Correlation with circulatory insulin levels

Interaction of [¹²⁵I]-insulin with intact hepatocytes and its correlation with circulatory insulin level was examined. The hepatocytes from new-born rats bound lowest amount of [¹²⁵I]-insulin (1.39±0.41 pM/mg cell protein) when circulatory insulin level was high (8±1.5 ΜU/ml). Hepatocytes from 7 day and 21 day old animals demonstrated a more or less similar relationship, Cells from 31 day old animals exhibited maximum insulin binding, activity (5.13±0.18.pM/mg cell protein) against a low serum insulin level (4.25±0.25 ΜU/ ml). Scatchard analysis of insulin binding shows that the affinity is higher in the hepatocytes from new-born animals than in the hepatocytes of 31 day old animals. Higher binding observed in the latter case may be due to a greater number of binding sites. Hepatocytes from one year old rats bound very little insulin (2.50±0.36 pM/mg cell protein) against a high circulatory insulin level (9.25±0.85 ΜU/ml). In view of these results, it appears that the down-regulation hypothesis holds true during ontogeny too.     

Insulin effect on GABA uptake in astroglial primary cultures

Astroglial cultures from newborn mouse cerebral cortex contain [¹²⁵I]insulin binding sites. Binding was specific reversible, time dependent and reached equilibrium after 45 min. Insulin analogues compete for this [¹²⁵I]Insulin binding. Incubation of cerebral cortex astroglial cultures with insulin induced a time-and dose-dependent inhibition of the [³H]GABA high affinity uptake. A decrease in theV _max rather than, an effect on theK _m was observed. This effect was dose-dependent and effective at 10^–10 M. Autoradiographic observations on the cell monolayer showed the presence of two groups of cells: one which strongly takes up [³H]GABA and consist in smaller GFAP positive process-bearing cells and another group of much flatter and larger GFAP positive cells which uptake was lower. The smaller stellate cells were apparently the most sensitive to insulin effect. These results: 1) confirm the presence of insulin binding sites on astroglial primary cultures, 2) show an effect of insulin of [³H]GABA high affinity uptake of these cells; this effect being optimal on a stellate-like population of astrocytes, and 3) indicate, that insulin may interfere in neuromodulation through astroglial signals.     

Effects of mild heat shock on glycogenesis and its regulation by insulin in cultured fetal hepatocytes

The effects of a mild heat shock were investigated using cultured 15-day-old fetal rat hepatocytes in which an acute glucocorticoid-dependent glycogenic response to insulin was present. After exposure from 15 min to 2 h at 42.5°C, cell surface [¹²⁵I]insulin binding progressively decreased down to 60% of the value shown in cells kept at 37°C, due to a decrease in the apparent number of insulin binding sites with little change in insulin receptor affinity. In parallel cultures, protein labeling with [³⁵S]methionine exhibited stimulated synthesis of specific proteins, in particular, 73-kDa Hsc (heat shock cognate) and 72-kDa Hsp (heat shock protein). When cells were returned to 37°C after 2 h at 42.5°C, cell surface insulin binding showed a two-third restoration within 3 h (insulin receptor half-life = 13 h), with similar concomitant return of Hsps72,73 synthesis to preinduction levels. The rate of [¹⁴C]glucose incorporation into glycogen measured at 37°C after 1- to 2-h heat treatment revealed a striking yet transient increase in basal glycogenesis (up to 5-fold). At the same time, the glycogenesis stimulation by insulin was reduced (from 3.2 to 1.4—fold), whereas that induced by a glucose load was maintained. Induction of thermotolerance after a first heating was obtained for the heat shock-dependent events except for the enhanced basal glycogenesis. In insulin-unresponsive cells grown in the absence of glucocorticoids, heat shock decreased the glycogenic capacity without modifying the glucose load stimulation, supporting the hypothesis that insulin and thermal stimulation of glycogenesis share at least part of the same pathway. Inverse variations were observed between Hsps72,73 synthesis and both cell surface insulin receptor level and insulin glycogenic response in fetal hepatocytes experiencing heat stress. © 1995 Wiley-Liss, Inc.     

Sedimentation characteristics of subcellular vesicles associated with internalized insulin and those bound with intracellular glucose transport activity

Comparative studies were made on the sedimentation characteristics of microsomal vesicles associated with internalized [¹²⁵I]iodoinsulin and those bound with intracellular glucose transport activity. Upon linear sucrose density gradient centrifugation, the internalized hormone formed a peak slightly, but significantly, on the higher density side of the peak of intracellular glucose transport activity. After a long centrifugation, the peak of ¹²⁵I activity became lower and broader than that of glucose transport activity. Internalized ¹²⁵I activity was also found in the medium-density microsomal fraction, which had little glucose transport activity. Accumulation of ¹²⁵I activity in the medium-density fraction and that in the low-density fraction were both completed in approximately 10 min. Under basal conditions, little, if any, insulin binding activity was detectable in either the medium- or low-density microsomal fractions; in contrast, some glucose transport activity was always present in the low-density fraction. These results indicate that the subcellular distribution of internalized insulin and of intracellular glucose transport activity are different, suggesting that the pathways of intracellular processing of the insulin receptor and the glucose transport mechanism are different.     

Unmasking of insulin receptors in rat submaxillary gland microsomes: effect of high ionic strength, phospholipase C and S-adenosyl-L-methionine

The specific binding of [125I]insulin to submaxillary gland microsomes was significantly enhanced by increasing the ionic strength of the incubation medium. This effect was neither related to changes in receptor or hormone degradation nor in the polymerization of the tracer. When equilibrium binding data from competition curves of unlabelled insulin versus [125I]insulin were analyzed, a marked increase in total binding capacity in high ionic strength was observed (from 890 to 2440 fmol/mg protein), with no change in binding affinity. Phospholipase C digestion was also able to increase specific [125I]insulin binding to microsomes. These results suggest the presence of masked receptors in submaxillary gland microsomes. Methylation of rat submaxillary gland microsomes by using S-adenosyl-L-methionine as the methyl donor significantly increased [125I]insulin binding. Scatchard analysis of the equilibrium binding data showed that addition of S-adenosyl-L-methionine (0.46 mM) to microsomes resulted in an enhancement of the total binding capacity (from 990 to 1520 fmol/mg protein) with no change in the affinity constants, which suggests the exposure of masked insulin receptors under such conditions. Both the methyl group incorporation into membrane phospholipids and the effect on insulin binding were dependent on the S-adenosyl-L-methionine concentration used and were partially suppressed in the presence of S-adenosyl-L-homocysteine, a specific competitive inhibitor of the methyltransferases activity. When microsomes were treated with S-adenosyl-L-[methyl-3H]methionine, the 3H-labelled methyl groups incorporated were found mainly in the lipid fraction associated to phosphatidylcholine, suggesting in this case that the unmasking of insulin receptors could be a consequence of alterations produced in membrane composition. The effects of phospholipase C, S-adenosyl-L-methionine and high ionic strength on insulin binding were not additive, suggesting that these procedures unmask receptors from the same pool.     

Comparison of Insulin Binding Proteins In Plasma and Nuclear Membranes of Obese and Lean Mouse Liver

Abstract

Plasma membranes obtained from obese (ob/ob) and lean (+/+ or +/ob) mouse livers were chemically crosslinked to [¹²⁵I] -insulin and examined by electrophoresis and autoradiography. The pattern of crosslinked hormone was qualitatively similar in obese and lean plasma membranes. A major insulin binding protein of approximately M 120,000 was observed. Two additional bands were apparent, one which remained near the top of the gel and one about M 90,000. A minor band at approximately M 50,000 was also detected. For each of the insulin binding proteins a reduction in the amount of [¹²⁵I]-insulin bound was observed with obese plasma membranes as compared with lean. For all proteins the insulin binding was specific as determined by competition with unlabeled hormone. In addition to plasma membrane receptors, insulin has also been reported to bind to nuclear membranes. The autoradiographic patterns of gels of [¹²⁵]-insulin bound and crosslinked to nuclear membranes from obese and lean mouse livers indicated the presence of proteins of the same M as those described for plasma membranes. Nuclear membrane proteins bound less insulin than plasma membranes and, again, the obese was decreased relative to the lean. Contamination of the nuclear membrane fraction by plasma membranes was ruled out. Scatchard analyses of [¹²⁵]-insul in bound to plasma and nuclear membranes indicated that the decrease in hormone binding in the obese mouse is a result of a reduction in the absolute number of receptors. The findings presented in this study provide additional support for this conclusion by demonstrating that membranes from obese mice are comprised of the same set of apparently unaltered insulin binding proteins. Further, the presence of similar insulin binding proteins in both nuclear and plasma membranes suggests a physiological relationship between these structures with respect to hormone binding and/or in the mechanism of action of insulin.     

Distribution of receptors for insulin and insulin-like growth factor I (somatomedin C) in the adrenal gland

Rat adrenal glands contain cell surface high-affinity receptors for several peptide hormones. Receptors for IGF-I were abundant in this tissue, but receptors for insulin were relatively scarce. The behavior of adrenal membrane IGF-I receptors in radioligand binding assays was similar to the behavior of IGF-I receptors from other tissues, with a KD congruent to 6.2 x 10(-9) M. Covalent cross-linking studies with [125I]IGF-I revealed an IGF-I receptor alpha-subunit with Mr congruent to 135,000 on dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions, as well as a smaller radiolabeled peptide, Mr = 116,000. In contrast, little binding of [125I]insulin to adrenal membranes was observed and no labeling occurred in cross-linking studies using [125I]insulin. These results contrast with the findings of whole-body autoradiographic studies that indicated substantial binding of [125I]insulin to adrenal glands and suggest that IGF-I, rather than insulin, may play a critical role in the growth and development of the adrenal gland.     

Construction of a toxic insulin molecule: Selection and partial characterization of cells resistant to its killing effects

We have constructed an insulin-diphtheria hormono-toxin which migrates as a single 29 kd band on 10% SDS polyacrylamide gel electrophoresis. This corresponds to a one to one molar ratio of the diphtheria A-chain (23 kDa) and insulin (6 kDa) molecules. The diphtheria A-chain: insulin (DTaI) hormono-toxin demonstrates cytotoxicity in V-79 Chinese hamster cells exhibiting an LD₅₀ of 1.1×10^–8M, which is 22 x more potent than whole diphtheria toxin. Also, DTaI can competitively displace [¹²⁵I]-insulin with an ED₅₀ of 1.1×10^–8 M, which is identical to the ED₅₀ of insulin (1.1×10^–8M) and showed limited cross-reactivity with the IGF-1 receptor (12% displacement of [¹²⁵I]-IGF-1 with a DTaI concentration of 1.1×10^–8 M). We have used DTaI to select conjugate-resistant clones from the V-79 Chinese hamster fibroblast parental cell line. Conjugate-resistant variants expressed insulin binding levels ranging from 8.0±2.0 fmoles/mg protein down to 3.6±0.5 fmoles/mg protein while insulin binding in the V-79 parental cell line was 11.2±0.2 fmoles/mg protein. Additionally, a number of conjugate resistant clones expressed variable ability to grow in medium containing 5% serum. The altered ability of these clones to grow in a serum-containing medium did not correlate directly with the changes observed for insulin binding. One mutant, IV-A1-j, did not grow in a serum-free defined medium containing insulin as the predominant mitogen. This IV-A1-j mutant had a lower number of insulin receptors, no change in insulin binding affinity, no change in the rate of internalization of [¹²⁵I]-insulin and no apparent difference in [¹²⁵I]-IGF-1 binding. Further, insulin-stimulated sugar transport was similar to that observed in the parental cell line. Based on these observations we suggest that 1) DTaI elicits its cytotoxicological effects through the insulin receptor trafficking pathway, 2) DTaI can be used to isolate cells altered at the level of insulin binding and/or action, and 3) signal transduction mechanisms responsible for mediating insulin-dependent cell growth can be pursued using mutants such as IV-A1-j.     

Identification of liver cell membrane galactoglycoproteins involved in the process of insulin binding

The glycoproteinic nature of the insulin receptor was indicated using two different approaches: 1. [¹²⁵I]insulin binding to soluble receptors from mouse liver was inhibited by digestion with β-galactosidase or pretreatment with Ricinus communis I or concanavalin A. An other enzyme (neuraminidase) and lectins (wheat germ agglutinin, Dolichos biflorus) did not affect the binding reaction. These data confirmed that insulin directly interacts with the galactoglycoproteins of liver membranes. 2. The galactose oxidase-sodium boro[³H]hydride technique, previously used for labeling accessible membrane galactoglycoproteins, was again utilized to discern the components that interact with insulin. When liver membranes were equilibrated with 10^?7 M insulin prior to labeling, the SDS gel radioactive profiles were specifically modified within two galactoglycoproteins of apparent molecular sizes 195 000 and 145 000, compatible with their participation in the insulin binding interaction. Membrane pretreatment with β-galactosidase or Sophora japonica lectin reduced the labeling in most peaks, thus supporting the argument for labeling sensitivity. Preincubation of membranes with 10^?7 M proinsulin slightly hindered labeling while pretreatment with 10^?7 M glucagon was ineffective, suggesting a specificity of the insulin effect. These data indicate the glycoprotein nature of the insulin receptor for two reasons: alteration of insulin binding after modification of the galactoglycoproteins, and alteration of galactoglycoprotein labeling after insulin binding. Two galactoglycoproteins, with apparent molecular weights 145 000 and 195 000, respectively, were identified and they are suggested to have insulin binding properties.     

Binding of insulin and insulin-like growth factor I in bovine chromaffin cells in primary culture

• 1.1. Insulin and insulin-like growth factor I (IGF-I) receptors were studied in bovine chromaffin cells isolated from the medulla by collagenase digestion and kept in primary culture.
• 2.2. Specific ¹²⁵I-labelled insulin binding increased with time in culture with no significant change in the dissociation constant, K_d~0.3nM. Insulin was nearly 100-fold more potent than IGF-I in displacing ¹²⁵I-labelled insulin.
• 3.3. Affinity crosslinking and SDS gel electrophoresis revealed increased binding of ¹²⁵I-labelled insulin and ¹²⁵I-IGF-I with time in culture, the densities of the labelling indicating relatively a much higher expression of IGF-I than insulin receptors in the cells. The apparent molecular weight of both the hormone binding subunits were 135,000, suggesting that the insulin and IGF-I receptors in the adrenal medulla are of the peripheral types.
• 4.4. Both receptors thus appeared to be affected by the collagenase treatment but with a subsequent recovery when cells were kept in culture.

     

Characterization of the binding of a rat somatomedin to receptors in human placental cell membranes.

The somatomedins presumably initiate their growth promoting effects by first binding to specific cell surface receptors in responsive tissues. The specific and high affinity binding of [¹²⁵I]-rat somatomedin to human placental membranes was saturable and reversible with a dissociation constant of 4.5 × 10^?9 M calculated from Scatchard analysis of competitive binding experiments. Competition for [¹²⁵I]-rat somatomedin binding to placental receptors by other somatomedins and growth factors suggest a close structural relationship between rat somatomedin and the human somatomedin, insulin-like growth factor I.     

Receptor-mediated degradation of insulin in isolated rat adipocytes: Formation of a degradation product slightly smaller than insulin

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [Tyr^A14-¹²⁵I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [¹²⁵I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the ‘insulin’ peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.     

Internalization of insulin receptors and HLA antigens in human hepatoma cells.

Human HepG2 hepatoma cells express a high number of insulin receptors. Growing cells exhibit 70% of their insulin receptors on the plasma membrane. Moreover, cell-surface insulin receptors form molecular complexes with class I major histocompatibility antigens, as determined by co-immunoprecipitation of the receptors by anti-class I monoclonal antibodies. On exposure to saturating concentrations of insulin, the hormone is rapidly internalized into a Pronase-resistant compartment. Internalization of insulin is accompanied by a rapid (t1/2 = 2-3 min) redistribution of insulin receptors from the cell surface to an intracellular compartment. On removal of insulin from the medium, functional receptors recycle back to the plasma membrane, where they can bind insulin again. With chronic exposure of HepG2 cells to insulin, the initial redistribution of receptors is followed by a slow (t1/2 = 9 h) down-regulation of the receptors. Finally, notwithstanding their interaction at the cell surface, insulin receptors and class I major histocompatibility antigens are internalized at different rates and with independent regulation.