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.     

Identification and properties of insulin receptors in neuroblastoma C 1300 N 18 cells in various functional states

The presence of insulin receptors in neuroblastoma C 1300 N18 cells was shown by the method of the [125I]insulin binding with cells as well as by the electron-cytochemical methods based on the analysis of the binding of colloid gold-labelled insulin and of agglutinin of wheat embryos with the surface of plasma membrane. It is established that the number and distribution of insulin receptors depend on the functional state of cells. Expression of receptors on the membrane increases after 5'-deoxyuridine-induced differentiation of cells. Due to changes in the lipid composition of cells caused by the incubation with lecithin-cholesterol liposomes (an increase in the content of cholesterol and its esters, as well as of unsaturated fatty acids and appearance of lysolecithin) the quantity of insulin receptors decreases and cell membranes are damaged. The lowering insulin regulation is not observed for insulin receptors of the neuroblastoma C 1300 N18 cells.     

Insulin and insulin-like growth factor receptors in the nervous system

Insulin and the insulin-like growth factors (I and II) are homologous peptides essential to normal metabolism as well as growth. These peptide hormones are present in the brain, and, based on biosynthetic labeling studies as well as evidence for local gene expression, they are synthesized by nervous tissue as well as being taken up by the brain from the peripheral circulation. Furthermore, the presence of insulin and IGF receptors in the brain, on both neuronal and glial cells, also suggests a role for these peptides in the nervous system. Thus, these ligands affect brain electrical activity, either as neurotransmitters or as neuromodulators, altering the release and re-uptake of other neurotransmitters. The insulin and IGF-I and -II receptors found in the brain exhibit a lower molecular weight than corresponding receptors on peripheral tissues, primarily caused by alterations in glycosylation. Despite these alterations, both brain insulin and IGF-I receptors exhibit tyrosine kinase activity in cell-free systems, as do their peripheral counterparts. Brain insulin and IGF-I receptors are developmentally regulated, with the highest levels appearing in fetal or perinatal life. However, the altered glycosylation of brain receptors does not appear until late in fetal development. The receptors are widely distributed in the brain, but especially enriched in the circumventricular organs, choroid plexus, hypothalamus, cerebellum, and olfactory bulb. These studies on the insulin and IGF receptor in brain, add strong support to the suggestion that insulin and IGFs are important neuroactive substances, regulating growth, development, and metabolism in the brain.     

Insulin receptor kinase following internalization in isolated rat adipocytes

We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization.     

The inhibition of a membrane-bound enzyme as a model for anaesthetic action and drug toxicity.

Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.     

Characterization of the insulin and insulin-like growth factor receptors and responsitivity of a fibroblast/adipocyte cell line before and after differentiation

TA1 cells, like 3T3-L1 cells, undergo a differentiation process in vitro from a fibroblast to an adipocyte phenotype. The TA1 pre-adipocytes were found to have low numbers of insulin receptors but high numbers of receptors for insulin-like growth factors (IGF) I and II. Also, the pre-adipocytes were more responsive to IGF than insulin as measured by either stimulation of glucose or amino acid uptake. After differentiation, the adipocytes had higher numbers of insulin receptors and a better responsitivity to insulin than to IGF-I. These results indicate that insulin-like growth factors are the primary regulators of the pre-adipocytes whereas insulin regulates the adipocytes.     

Insulin receptors and bioresponses in a human liver cell line (Hep G-2)

A newly developed human hepatoma cell line, designated Hep G-2, expresses high-affinity insulin receptors meeting all the expected criteria for classic insulin receptors. 125I-insulin binding is time-dependent and temperature-dependent and unlabeled insulin competes for the labeled hormone with a half-maximal displacement of 1-3 ng/ml. This indicates a Kd of about 10(-10) M. Since Scatchard analysis of the binding data results in a curvilinear plot and unlabeled insulin accelerates the dissociation of bound hormone, these receptors exhibit the negative cooperative interactions characteristic of insulin receptors in many other cell and tissue types. Proinsulin and des(Ala, Asp)-insulin compete for 125I-insulin binding with 4% and 2%, respectively, of the potency of insulin. Anti-(insulin receptor) antibody competes fully for insulin binding. The two insulin-like growth factors, multiplication-stimulating activity and IGF-I are 2% as potent as insulin against the Hep G-2 insulin receptor. Furthermore, Hep G-2 cells respond to insulin in several bioassays. Glucose uptake, glycogen synthase, uridine incorporation into RNA and acetate incorporation into lipid are all stimulated to varying degrees by physiological concentrations of insulin. In addition, these cells 'down-regulate' their insulin receptor, internalize 125I-insulin and degrade insulin in a manner similar to freshly isolated rodent hepatocytes. This is the first available human liver cell line in permanent culture in which both insulin receptors and biological responses have been carefully examined.     

The insulin receptor and type I IGF receptor: Comparison of structure and function

The insulin receptor and type I IGF receptor are closely related in structure and function. The receptors are heterotetrameric glycoproteins, of structure αββα, which are widely distributed in mammalian tissues. A third member of this receptor family has been described, the insulin receptor-related receptor, for which a ligand has still to be identified. It has also been demonstrated that the insulin receptor and IGF receptor form αββ′α′ hybrids in cells expressing both receptors.The key elements in the function of any receptor are recognition of ligand and transmission of an intracellular signal. In the insulin and IGF receptors, determinants of binding specificity are contained within amino-terminal and cysteine-rich domains of the extracellular α-subunit. Intracellular signalling is dependent on ligand activated tyrosine kinase activity in the transmembrane β-subunit, which phosphorylates both the receptor itself and the specific substrate insulin receptor substrate-1 (IRS-1). Phosphorylated IRS-1 binds the enzyme phosphatidylinositol 3-kinase and may act as a multivalent docking site for SH2 domains of other proteins involved in signalling. The possibility that some signalling molecules interact directly with the receptors has not been ruled out.The specificity of action of insulin and IGFs in vivo depends on differences between the respective receptors in tissue distribution, ligand binding specificity and intrinsic signalling capacity. However, the detailed aspects of gene and receptor structure which underly these functional differences are still poorly understood. Moreover, the issue of specificity is complicated by the existence of hybrid and atypical receptors, which in principle could bind and respond to both insulin and IGF-I, although the physiological significance of these receptor subtypes is at present unclear.     

The NSILA-s receptor in liver plasma membranes. Characterization and comparison with the insulin receptor.

NSILA-s (nonsuppressible insulin-like activity, soluble in acid ethanol) is a serum peptide that has insulin-like and growth-promoting activities. We have demonstrated previously that liver plasma membranes possess separate receptors for NSILA-s and insulin and have characterized the insulin receptor in detail. In the present study we have characterized the properties and specificity of the NSILA-s receptor and compared them to those of the insulin receptor in the same tissue. Both 125I-NSILA-s and 125I-insulin bind rapidly and reversibly to their receptors in liver membranes; maximal NSILA-s binding occurs at 20 degrees while maximal insulin binding is seen at 1-4 degrees. The pH optimum for NSILA-s binding is broad (6.0 to 8.0), in contrast to the very sharp pH optimum (7.5 to 8.0) for insulin binding. Both receptors exhibit a high degree of specificity. With the insulin receptor, NSILA-s and insulin analogues compete for binding in proportion to their insulin-like potency: insulin greater than proinsulin greater than NSILA-s. With the NSILA-s receptor, NSILA-s is most potent and the order is reversed: NSILA-s greater than proinsulin greater than insulin. Furthermore, six preparations of NSILA-s which varied 70-fold in biological activity competed for 125I-NSILA-s binding in order of their potencies. NSILA-s which had been inactivated biologically by reduction and aminoethylation and growth hormone were less than 1/100,000 as potent as the most purified NSILA-s preparation. Purified preparations of fibroblast growth factor, epidermal growth factor, nerve growth factor, and somatomedins B and C were less than 1% as effective as NSILA-s in competing for the 125I-NSILA-s suggesting that these factors act through other receptors. In contrast, somatomedin A was 10% as active as NSILA-s and multiplication-stimulating activity was fully as active as NSILA-s in competing for the NSILA-s receptor. Analysis of the data suggests that there are approximately 50 times more insulin receptors than NSILA-s receptors per liver cell, while the apparent affinity of NSILA-s receptors is somewhat higher than that of the insulin receptor.     

Monoclonal antibodies to receptors for insulin and somatomedin-C

Three monoclonal antibodies, designated alpha IR-1, alpha IR-2, and alpha IR-3, were prepared by fusing FO myeloma cells with spleen cells from a mouse immunized with a partially purified preparation of insulin receptors from human placenta. These antibodies were characterized by their ability to immunoprecipitate solubilized receptors labeled with 125I-insulin or 125I-somatomedin-C in the presence or absence of various concentrations of unlabeled insulin or somatomedin-C. alpha IR-1 preferentially immunoprecipitates insulin receptors and also less effectively immunoprecipitates somatomedin-C receptors, while alpha IR-2 and alph IR-3 preferentially immunoprecipitate somatomedin-C receptors, but may also weakly immunoprecipitate insulin receptors. These three monoclonal antibodies, as well as A410, a rabbit polyclonal antibody, were used to immunoprecipitate insulin and somatomedin-C receptors from solubilized human lymphoid (IM-9) cells and human placenta membranes that had been 125I-labeled with lactoperoxidase. Analysis of the immunoprecipitates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both receptors are composed of alpha and beta subunits. The beta subunit of the insulin receptor (immunoprecipitated by alpha IR-1 and A410) has a slightly more rapid mobility than the corresponding subunit of the somatomedin-C receptor (immunoprecipitated by alpha IR-2 and alpha IR-3). Interestingly, the alpha subunit of the placenta somatomedin-C receptor has a slightly faster mobility than its counterpart from IM-9 cells. Immunoprecipitation of receptor that had been reduced and denatured to generate isolated subunits indicates that alpha IR-2 and alpha IR-3 interact with the alpha subunit of the somatomedin-C receptor while A410 interacts with both subunits of the insulin receptor. alpha IR-1 failed to react with reduced and denatured receptors.     

Differential signaling of insulin and IGF-1 receptors to glycogen synthesis in murine hepatocytes.

We have used SV40-transformed hepatocytes from insulin receptor-deficient mice (-/-) and normal mice (WT) to investigate the different abilities of insulin and IGF-1 receptors to stimulate glycogen synthesis. We report that insulin receptors are more potent than IGF-1 receptors in stimulating glycogen synthesis. Both receptors stimulate glycogen synthesis in a PI 3-kinase-dependent manner, but only the effect of insulin receptors is partially rapamycin-dependent. Insulin and IGF-1 receptors activate Akt to a similar extent, whereas GSK-3 inactivation in response to IGF-1 is considerably lower in both -/- and WT cells, compared to the effect of insulin in WT cells. The findings indicate that (i) the potency of insulin and IGF-1 receptors in stimulating glycogen synthesis correlates with their ability to inactivate GSK-3, (ii) the extent of GSK-3 inactivation does not correlate with the extent of Akt activation mediated by insulin or IGF-1 receptors, indicating that the effect of insulin on GSK-3 requires additional kinases, and (iii) the pathways required for insulin stimulation of glycogen synthesis in mouse hepatocytes are PI 3-kinase-dependent and rapamycin-sensitive.     

Ultrastructural analysis of the organization and distribution of insulin receptors on the surface of 3T3-L1 adipocytes: rapid microaggregation and migration of occupied receptors

Monomeric ferritin-insulin and high-resolution electron microscopic analysis were used to study the organization, distribution, and movement of insulin receptors on differentiated 3T3-L1 adipocytes. Analysis of the binding to prefixed cells showed that insulin initially occupied single and paired receptors preferentially located on microvilli. The majority of receptors (60%) were found as single molecules and 30% were pairs. In 1 min at 37% C, 50% of the receptors on nonfixed cells were found on the intervillous plasma membrane and more than 70% of the total receptors had microaggregated. By 30 min only 7% of the receptors were single or paired molecules on microvilli. The majority were on the intervillous membrane, with 95% of those receptors in groups. The receptor groups on the intervillous plasma membrane could be found in both noncoated invaginations and coated pits. The concentration of occupied receptors in the noncoated invaginations and the coated pits was similar; however, ten times more noncoated invaginations than coated pits contained occupied insulin receptors. The observations in this study contrast with those reported on rat adipocytes using identical techniques (Jarett and Smith, 1977). Insulin receptors on adipocytes were initially grouped and randomly distributed over the entire cell surface and did not microaggregate into larger groups. Insulin receptors on rat adipocytes were found in noncoated invaginations but were excluded from the coated pits. The differences in the organization and behavior of the insulin receptor between rat and 3T3-L1 adipocytes suggest that the mechanisms regulating the initial organization of insulin receptors and the aggregation of occupied receptors may be controlled by tissue-specific processes. Since both of these cell types are equally insulin sensitive, the differences in the initial organization and distribution of the insulin receptors on the cell surface may not be related to the sensitivity or biological responsiveness of these cells to insulin but may affect other processes such as receptor regulation and internalization. On the other hand, the microaggregates of occupied receptors on both cell types may relate to biological responsiveness.     

Insulin-Induced Circular Membrane Ruffling on Rat 1 Cells Expressing a High Number of Human Insulin Receptors: Circular Ruffles Caused by Rapid Actin Reorganization Exhibit High Density of Insulin Receptors and Phosphotyrosines

The early effects of insulin on morphological changes were examined using Rat 1 cells expressing one million human insulin receptors (Rat1HIR cells). Insulin transiently induced circular membrane ruffling on Rat1HIR cells. The circular ruffles were detected by phase microscopy as well as by fluorescence microscopy when actin was stained with rhodamine-conjugated phalloidin. The circular ruffles were also visualized by fluorescence microscopy when stained with either antiinsulin receptor monoclonal antibody, αIR-1, or antiphosphotyrosine antibody, followed by fluorescein isothiocyanate-labeled second antibodies. Control Rat1 cells or Rat1HIR A/K1018 cells expressing kinase-defective insulin receptors did not show any circular ruffles when treated with insulin. These results suggest that the circular ruffles are formed by actin reorganization induced by insulin and that this process requires the protein-tyrosine kinase activity of the receptor. Detection of insulin receptors and phosphotyrosines in the circular ruffles suggests the possibility that the insulin receptors in the ruffles may be highly active and phosphorylating either the receptors themselves or proteins associated with the ruffles. These results are consistent with our previous in vitro observations that a large aggregate form of the purified insulin receptor is a highly active protein-tyrosine kinase.     

Internalization and recycling of insulin receptors in hepatoma cells. Absence of regulation by receptor occupancy.

Insulin receptors of Fao hepatoma cells were labelled with a 125I-labelled photoreactive insulin analogue or by surface iodination catalysed by lactoperoxidase. Cells were then incubated at 37 degrees C, and the cellular localization of the labelled receptors was assessed by limited exposure of intact cells to trypsin. The results show that: (1) photolabelled insulin-receptor complexes are internalized and recycled in Fao hepatoma cells; (2) the dynamics of photolabelled insulin receptors (internalization and recycling) is similar before and after down-regulation; (3) the unoccupied receptors labelled by surface iodination are internalized and recycled similarly to covalent insulin-receptor complexes; (4) insulin does not induce internalization of surface-iodinated insulin receptors. We conclude that internalization and recycling of insulin receptors are independent of receptor occupancy by insulin in Fao hepatoma cells.     

The Role of IGF-system in Vascular Insulin Resistance

Insulin and IGF-I are closely related peptides, which interact by several mechanisms. In high supraphysiological concentrations (>/=10 (-8) M), they cross-react with each other's receptors with 100- to 1000-fold lower affinity than with their cognate receptors. This can cause confusion, since in many in vitro studies, insulin has been used in high unphysiological concentrations, which activate IGF-I receptors. Due to the differences in affinity, insulin and IGF-I probably do not activate each other's receptors in vivo. IGF-I receptors are several-fold more abundant than insulin receptors in human micro- and macrovascular endothelial cells and in human vascular smooth muscle cells. Both insulin and IGF-I receptor protein can be demonstrated and they are activated by their cognate ligand at physiological concentrations of 10 (-9)-10 (-10) M. In vascular smooth muscle cells, IGF-I but not insulin stimulates metabolism and growth. IGF-I stimulates DNA-synthesis and growth in microvascular endothelial cells, but neither insulin nor IGF-I have any effect on macrovascular endothelial cells. Both insulin and IGF-I have been shown to stimulate nitric oxide production in endothelial cells, but only the effect of IGF-I was obtained at a physiological concentration. In both endothelial and vascular smooth muscle cells, insulin and IGF-I receptors occur as insulin/IGF-I hybrid receptors with high affinity to IGF-I and low for insulin. Due to the low number of insulin receptors and the presence of hybrid receptors the insulin receptor signal is probably too attenuated to elicit biological effects, explaining the insulin resistance of vascular cells in vitro. In vivo both insulin and IGF-I have been reported to increase muscle blood flow in physiological concentrations. Whether this is due to direct effects on endothelial cells or indirectly induced is not clear. The effect of insulin is attenuated by insulin resistance. In conclusion, the in vitro data suggest that endothelial cells and vascular smooth muscle cells are sensitive to IGF-I, but insensitive to insulin, and this is due to a preponderance of IGF-I receptors and the presence of insulin/IGF-I hybrid receptors.     

Expression of insulin receptors and insulin action in cell hybrids and cybrids.

The expression of insulin receptors and insulin action was studied in cell hybrids and cybrids produced by fusion of the BWIJ mouse hepatoma cell line with nucleated and enucleated mouse L-cells (LEA-2A) respectively. The BWIJ parent and the cybrids expressed high numbers of insulin receptors, whereas the hybrids resembled the L-cell parent with low numbers of receptors. Likewise, the hybrids resembled the LEA-2A cells with high levels of glycogen synthase, whereas the BWIJ cells and cybrids had much lower levels. Both parents, the cybrids, and the hybrids, expressed insulin stimulation of alpha-aminoisobutyric acid influx, but the dose-response curves indicated an increased insulin sensitivity in the cells with the higher receptor concentration. Insulin also stimulated 86Rb+ uptake in the hepatoma parent, hybrids and cybrids, but not in the L-cell parent. These data suggest that insulin receptors, like other hepatoma-specific properties, behave as a 'luxury function' of the hepatoma cell line and are extinguished when the hepatoma cell is fused with a less differentiated cell type. The biological activities associated with insulin action, on the other hand, are much more complex in their expression and probably the result of the interaction of multiple factors that vary in their expression in cell hybrids and cybrids.     

Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 and partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor alpha subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-[gamma-32P]triphosphate, a protein of 95 kilodaltons representing the insulin receptor beta subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins.     

Fasting and refeeding alter the insulin receptor tyrosine kinase in chicken liver but fail to affect brain insulin receptors

Insulin receptors from chicken liver and brain were studied following alterations in the nutritional state. Chickens were either fasted for 48 h, fasted for 48 h and then refed for 24 h, or fed a regular diet ad libitum. 125I-Porcine insulin binding was significantly elevated in liver membranes from the fasted animals and lowered in refed chickens when compared to preparations from ad libitum fed chickens. These changes in 125I-insulin binding were inversely related to the levels of plasma insulin and since receptor affinities for insulin were similar in each group, they probably represent alterations in receptor number. Apparent Mr of alpha subunits of the insulin receptors was unaffected by alterations in the nutritional states. The presence of ATPase-like activities that co-eluted with liver insulin receptors from wheat germ agglutinin lectin columns but not from pea lectin columns necessitated the use of both pea and wheat germ agglutinin for liver insulin receptor purification. The insulin receptors purified from both lectin columns were recognized by anti-insulin receptor antiserum and had similar affinities for insulin which were unaltered by the nutritional state. Insulin-stimulatable autophosphorylation of the beta subunit of the insulin receptor was lower in livers from fasted chickens and intermediate in refed chickens. Furthermore, basal and insulin-induced phosphorylation of the artificial substrate poly(Glu,Tyr) 4:1 was significantly less in the fasting state and intermediate in the refed state compared to the ad libitum fed state. Insulin sensitivity (measured as the dose of insulin required for 50% maximal stimulation of kinase activity) was similar in all three states suggesting that the differences in insulin-induced phosphorylation are due to a change in maximal stimulation and not a change in insulin sensitivity. In contrast to the alterations seen with liver receptors, brain insulin receptors were unaffected by these alterations in nutritional state. These findings suggest that: liver insulin receptors are affected by altering the nutritional state; insulin binding to liver membranes is inversely related to plasma insulin levels; and tyrosine kinase is decreased both in fasted and refed animals suggesting an uncoupling of the normal interaction between alpha subunit and beta subunit in liver insulin receptors.     

Kinetics of insulin receptor internalization and recycling in adipocytes. Shunting of receptors to a degradative pathway by inhibitors of recycling

The kinetics of receptor internalization and recycling was directly determined in adipocytes by measuring 125I-insulin binding to total, intracellular, and cell-surface insulin receptors. In the absence of insulin 90% of all receptors were on the cell-surface and 10% were intracellular. Insulin (100 ng/ml) rapidly altered this distribution by translocating surface receptors to the cell-interior through a temperature and energy dependent process. Surface-derived receptors were seen within cells as early as 30 s and accumulated intracellularly at the rate of approximately 20,000/min (t 1/2 = 2.7 min). After 6 min the size of the intracellular receptor pool plateaued (for up to 2 h), with 30% of surface receptors residing within the cell. This plateau was due to the attainment of an equilibrium between receptor uptake and recycling, since removal of insulin (to stop receptor uptake) was followed by both a rapid depletion of intracellular receptors and a a concomitant and stoichiometric reappearance of receptors on the cell-surface. Receptors were efficiently recycled, with little or no net loss observed even after 4 h of insulin treatment; however, recycling could be partially inhibited (approximately 10%) by several agents (e.g. chloroquine and Tris). Tris treatment of adipocytes in the presence of insulin led to 50% loss of surface and total receptors at 2 and 4 h, respectively. Since chloroquine prevented the decrease in total receptors, but not the loss of surface receptors, it appears that Tris impairs recycling by diverting a portion of incoming receptors to a chloroquine-inhibitable degradative site. From these results we conclude that: 1) insulin triggers endocytotic uptake of insulin-receptor complexes; 2) internalized receptors are then rapidly reinserted into the plasma membrane, and the receptors can traverse this recycling pathway within 6 min; 3) prolonged recycling does not normally result in measurable receptor loss, but when receptors are prevented from recycling, they become trapped intracellularly and are shunted to a chloroquine-sensitive degradative pathway; and 4) chloroquine and Tris are only partially effective inhibitors of receptor recycling.     

Identification and characterization of glucose transport proteins in plasma membrane- and Golgi vesicle-enriched fractions prepared from lactating rat mammary gland.

Previous studies have indicated that turkey erythrocyte and rat liver membranes contain endogenous alpha beta heterodimeric insulin receptors in addition to the disulphide-linked alpha 2 beta 2 heterotetrameric complexes characteristic of most cell types. We utilized 125I-insulin affinity cross-linking to examine the structural properties of insulin receptors from rat liver and turkey erythrocyte membranes prepared in the absence and presence of sulphydryl alkylating agents. Rat liver membranes prepared in the absence of sulphydryl alkylating agents displayed specific labelling of Mr 400,000 and 200,000 bands, corresponding to the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes respectively. In contrast, affinity cross-linking of membranes prepared with iodoacetamide (IAN) or N-ethylmaleimide identified predominantly the alpha 2 beta 2 heterotetrameric insulin receptor complex. Similarly, affinity cross-linking and solubilization of intact turkey erythrocytes in the presence of IAN resulted in exclusive labelling of the alpha 2 beta 2 heterotetrameric insulin receptor complex, whereas in the absence of IAN both alpha 2 beta 2 and alpha beta species were observed. Turkey erythrocyte alpha 2 beta 2 heterotetrameric insulin receptors from IAN-protected membranes displayed a 3-4-fold stimulation of beta subunit autophosphorylation and substrate phosphorylation by insulin, equivalent to that observed in intact human placenta insulin receptors. Turkey erythrocyte alpha beta heterodimeric insulin receptors, prepared by defined pH/dithiothreitol treatment of IAN-protected membranes, were also fully competent in insulin-stimulated protein kinase activity compared with alpha beta heterodimeric human placenta receptors. In contrast, endogenous turkey erythrocyte alpha beta heterodimeric insulin receptors displayed basal protein kinase activity which was insulin-insensitive. These data indicate that native turkey erythrocyte and rat liver insulin receptors are structurally and functionally similar to alpha 2 beta 2 heterotetrameric human placenta insulin receptors. The alpha beta heterodimeric insulin receptors previously identified in these tissues most likely resulted from disulphide bond reduction and denaturation of the alpha 2 beta 2 holoreceptor complexes during membrane preparation.