The processing of receptor-bound [125I-Tyr11]somatostatin by RINm5F insulinoma cells

The peptide somatostatin (SRIF) is secreted by delta cells of the endocrine pancreas and inhibits the secretion of insulin from pancreatic beta cells. We have previously shown that [125I-Tyr11]SRIF binds to specific, high affinity receptors on RINm5F insulinoma cells and that these receptors mediate the action of SRIF to inhibit insulin release. In the present study we investigated the processing of receptor-bound [125I-Tyr11]SRIF in this clonal cell line. Surface-bound and internalized peptides were distinguished by the ability of an acid/salt solution (0.2 M acetic acid, 0.5 M NaCl, pH 2.5) to dissociate only exposed ligand-receptor complexes. Surprisingly, greater than 80% of saturably bound [125I-Tyr11]SRIF was removed by this acid wash independent of the time or temperature of the binding incubation. In contrast, the processing of receptor-bound [125I]EGF (epidermal growth factor) in RINm5F cells was markedly temperature-dependent. Although over 90% of saturably bound [125I]EGF was dissociated by acid after a 4 degrees C binding incubation, less than 10% was removed by acid treatment after 37 degrees C binding. The radioactivity released upon dissociation of receptor-bound [125I-Tyr11]SRIF was analyzed by high performance liquid chromatography and shown to consist of a mixture of intact peptide (40%) and [125I]tyrosine (60%). However, neither the rate of [125I-Tyr11]SRIF dissociation nor its degradation were affected by NH4Cl, methylamine, or leupeptin at concentrations which inhibited the lysosomal degradation of [125I] EGF. Of 11 other protease inhibitors tested, only the metalloendoprotease inhibitor, phosphoramidon, substantially reduced the degradation of receptor-bound [125I-Tyr11]SRIF. These data indicate that, unlike [125I] EGF, receptor-bound [125I-Tyr11]SRIF is not rapidly internalized by RINm5F cells and is degraded by a nonlysosomal process which may involve a metalloendoprotease.     

Internalization and degradation of receptor bound C-reactive protein by U-937 cells: induction of H2O2 production and tumoricidal activity

The presence of a membrane receptor for C-reactive protein (CRP-R) on the human monocytic cell line U-937 was the basis for determining the metabolic fate of the receptor-bound ligand and the functional response of the cells to CRP. Internalized [125I]CRP was measured by removing cell surface-bound [125I]CRP with pronase. Warming cells to 37 degrees C resulted in the internalization of approx. 50% of the receptor-bound [125I]CRP or receptor-bound [125I]CRP-PC-KLH complexes. U-937 cells degraded about 25% of the internalized [125I]CRP into TCA-soluble radiolabeled products. The lysosomotrophic agents (chloroquine, NH4Cl) greatly decreased the extent of CRP degradation without altering binding or internalization. In addition, a pH less than 4.0 resulted in dissociation of receptor-bound [125I]CRP. Treatment of U-937 cell with monensin, a carboxylic ionophore which prevents receptor recycling, resulted in accumulation of internalized [125I]CRP. Therefore, it appears that the CRP-R complex is internalized into an endosomal compartment where the CRP is uncoupled from its receptor and subsequently degraded. CRP initiated the differentiation of the U-937 cells so that they acquired the ability to produce H2O2 and also display in vitro tumoricidal activity. The results support the concept that internalization and degradation of CRP leads to the activation of monocytes during inflammation.     

Effect of thyroxine-binding globulin (TBG) on thyroxine (T4) uptake by human peripheral mononuclear cells: evidence of TBG-dependent uptake of T4

The effect of sialylated TBG and desialylated TBG on thyroxine (T4) uptake by human peripheral mononuclear cells was investigated in vitro. [125I]-T4 uptake was observed when the cells were incubated with free [125I]-T4. The uptake was inhibited in a concentration dependent manner when TBG was added. During the incubation, [125I]-T4 binding to TBG was observed. [125I]-T4 incorporation into cells was also observed when the cells were incubated with [125I]-T4-sialylated TBG or with [125I]-T4-desialylated TBG complex. The uptake was related to the temperature and length of time of the incubation. The amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-sialylated TBG was greater than that into the cells incubated with [125I]-T4-desialylated TBG during the early 0-20 min. incubation, whereas the amount of [125I]-T4 incorporated into the cells incubated with [125I]-T4-desialylated TBG became greater than that into the cells incubated with [125I]-T4-sialylated TBG after 20 min. of incubation. Pretreatment of the cells with methylamine blocked [125I]-T4 uptake in both cases, i.e. incubated with [125I]-T4-sialylated TBG and incubated with [125I]-T4-desialylated TBG. The results suggest that TBG plays a role not only as a carrier protein for T4 in circulation but also as a protein which can transport T4 from the extracellular into the intracellular space, so that the mechanism of T4 transport mediated by desialylated TBG is different from that mediated by sialylated TBG, and that the T4 transport system in both cases, mediated by sialylated TBG and by desialylated TBG, may be related to the internalization of T4-TBG-TBG receptor complex or of T4-T4 receptor complex if TBG receptors are present in the outer surface of the cell membrane.     

The NPEY sequence is not necessary for endocytosis and processing of insulin-receptor complexes.

The tetrameric amino acid sequence AsnProXTyr (NPXY), where X represents any amino acid, is conserved in the intracytoplasmic domains of several membrane proteins and has been postulated to play a role in receptor-mediated endocytosis. The human insulin receptor (hIR) contains a single copy of the sequence AsnProGluTyr (NPEY) in its intracytoplasmic domain. To determine if this putative consensus sequence is necessary for endocytic functions of hIR, we constructed a mutant receptor, hIR delta NPEY, that lacks NPEY sequence, stably expressed this mutant receptor in Chinese hamster ovary cells, and then studied its endocytic functions. When compared to wild type hIR similarly expressed in Chinese hamster ovary cells, the hIR delta NPEY mutant exhibited: 1) normal subunit organization and insulin binding affinity; 2) essentially normal internalization of covalent photoaffinity labeled insulin-receptor complexes; and 3) normal internalization of receptor-bound [125I]insulin as well as normal degradation and release of the internalized insulin. Therefore, we conclude that the NPEY sequence in the juxtamembrane domain of hIR is not necessary for its endocytic function.     

Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical and morphological effects of gentamicin in human proximal tubular cells: effects on lipid and lipoprotein metabolism

The effects of gentamicin, an antibiotic used extensively for antimicrobial therapy on the ultrastructure, binding, internalization, degradation, and cholesterol esterification of low-density lipoproteins, were investigated in cultured human proximal tubular cells. Cells were incubated with 0.3 mM gentamicin for 21 days with the following observations. Cells treated with gentamicin contained numerous "myeloid bodies." The binding, internalization, and degradation of 125I-labeled low-density lipoproteins ([125I]LDL) in cells treated with gentamicin was twofold lower than control cells. Pulse-chase experiments demonstrated that gentamicin did not impair the internalization of receptor-bound LDL and their subsequent transport to the lysosome. The relative amounts of [125I]LDL displaced by increasing concentrations of unlabeled LDL were the same in both gentamicin-treated and control cells. This pattern was reflected in the cell surface binding, internalization, and degradation of [125I]LDL. Gentamicin did not alter the degradation of [125I]LDL in cell homogenates at 4.0. The data suggest that gentamicin decreases the receptor-mediated endocytosis of LDL and subsequent lipid metabolism.     

Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.     

Relationship between the affinity and proteolysis of the insulin receptor. Evidence that higher affinity receptors are preferentially degraded

125I-Insulin binding to rat liver plasma membranes initiated two processes that occurred with similar time courses: an increase of receptor affinity for hormone and degradation of the Mr 135,000 alpha subunit of the insulin receptor to a fragment of Mr 120,000. Inhibitors of serine proteinases prevented alpha subunit degradation without affecting the affinity change. This shows that the change of affinity is not produced by receptor proteolysis and that the intact alpha subunit of the insulin receptor can exist as a higher or lower affinity species. Hormone binding was much more rapid than receptor proteolysis and the initial rate of alpha subunit degradation was independent of the concentration of occupied lower affinity receptors. Only persistent hormone binding and the accumulation of higher affinity insulin-receptor complexes led to significant receptor proteolysis. As the incubation time between 125I-insulin and membranes increased, the rate at which hormone dissociated from Mr 135,000 complexes diminished, whereas hormone dissociated from Mr 120,000 complexes slowly after brief or extended incubations. These observations suggest that 125I-insulin binds to membranes to form low affinity complexes that are not substrates for proteolysis. A slow conformational change produces higher affinity hormone-receptor complexes that are selectively degraded. Thus, the conversion between states of affinity may play a role in the regulation of receptor proteolysis and, consequently, insulin action in cells.     

Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene

The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblast insulin receptors. These cells bind and internalize insulin normally. Biochemical assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 4 degrees C, and internalization of insulin-receptor complexes was initiated by warming the cells to 37 degrees C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The total number of complexes reached a maximum by 5 min and decreased rapidly thereafter with a t 1/2 of approximately 10 min. There was a distinct delay in the appearance, rate of rise, and peak of intracellular free and degraded insulin. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored, based on the ability of dissociated insulin to rebind to receptor upon neutralization of acidic intracellular vesicles with monensin. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation.     

Metabolism of high-density lipoproteins in cultured rat luteal cells

The uptake of cholesterol from high-density lipoproteins (HDL) labeled with 125I and [3H]cholesterol was examined in cultured rat luteal cells. Luteal cells were incubated with labeled HDL, following which the metabolic fate of the apolipoproteins and cholesterol moieties of the receptor-bound HDL were examined. About 50% of the originally bound HDL apolipoproteins were released into the medium in 24 h by a temperature-dependent process while only 5% of the HDL cholesterol was released unmetabolized. Inclusion of unlabeled HDL in the chase incubation resulted in increased release of apolipoprotein-derived radioactive products without significant change in the release of unmetabolized cholesterol. 60% of the apolipoprotein-derived radioactivity could be precipitated with trichloroacetic acid; the remaining trichloroacetic acid-soluble radioactive fraction was identified as [125I]iodotyrosine. Gel filtration chromatography of the chase-released material showed that the trichloroacetic acid-precipitable products, which contained no detectable amounts of cholesterol, eluted over a range of molecular sizes (9-80 kDa). No intact HDL was retroendocytosed. About 80% of trichloroacetic acid-precipitable products could be immunoadsorbed on anti-apolipoprotein A-I antibody immobilized on CNBr-activated Sepharose, suggesting the presence of fragments containing apolipoprotein A-I. This material was also capable of reassociating with native HDL. Lysosomal inhibitors were partially effective in inhibiting the amount of trichloroacetic acid-soluble products formed. The lysosomal degradation appeared to have no role in the uptake of HDL-derived cholesterol. These studies demonstrate preferential and total uptake of HDL cholesterol by luteal cells, with concomitant degradation of the lipoprotein.     

Isolation and characterization of an insulin-degrading enzyme from Drosophila melanogaster

An insulin-degrading enzyme (IDE) from the cytoplasm of Drosophila Kc cells has been purified and characterized. The purified enzyme is a monomer with an s value of 7.2 S, an apparent Km for porcine insulin of 3 microM, and a specific activity of 3.3 nmol of porcine insulin degraded/(min.mg). N-Terminal sequence analysis of the gel-purified enzyme gave a single, serine-rich sequence. The Drosophila IDE shares a number of properties in common with its mammalian counterpart. The enzyme could be specifically affinity-labeled with [125I]insulin, has a molecular weight of 110K, and has a pI of 5.3. Although Drosophila Kc cells grow at room temperature, the optimal enzyme activity assay conditions parallel those of the mammalian IDE: 37 degrees C and a pH range of 7-8. The Drosophila IDE activity, like the mammalian enzymes, is inhibited by bacitracin and sulfhydryl-specific reagents. Similarly, the Drosophila IDE activity is insensitive to glutathione as well as protease inhibitors such as aprotinin and leupeptin. Insulin-like growth factor II, equine insulin, and porcine insulin compete for degradation of [125I]insulin at comparable concentrations (approximately 10(-6) M), whereas insulin-like growth factor I and the individual A and B chains of insulin are less effective. The high degree of evolutionary conservation between the Drosophila and mammalian IDE suggests an important role for this enzyme in the metabolism of insulin and also provides further evidence for the existence of a complete insulin-like system in invertebrate organisms such as Drosophila.     

Selective degradation of insulin within rat liver endosomes

To characterize the role of the endosome in the degradation of insulin in liver, we employed a cell-free system in which the degradation of internalized 125I-insulin within isolated intact endosomes was evaluated. Incubation of endosomes containing internalized 125I-insulin in the cell-free system resulted in a rapid generation of TCA soluble radiolabeled products (t1/2, 6 min). Sephadex G-50 chromatography of radioactivity extracted from endosomes during the incubation showed a time dependent increase in material eluting as radioiodotyrosine. The apparent Vmax of the insulin degrading activity was 4 ng insulin degraded.min-1.mg cell fraction protein-1 and the apparent Km was 60 ng insulin.mg cell fraction protein-1. The endosomal protease(s) was insulin-specific since neither internalized 125I-epidermal growth factor (EGF) nor 125I-prolactin was degraded within isolated endosomes as assessed by TCA precipitation and Sephadex G-50 chromatography. Significant inhibition of degradation was observed after inclusion of p-chloromercuribenzoic acid (PCMB), 1,10-phenanthroline, bacitracin, or 0.1% Triton X-100 into the system. Maximal insulin degradation required the addition of ATP to the cell-free system that resulted in acidification as measured by acridine orange accumulation. Endosomal insulin degradation was inhibited markedly in the presence of pH dissipating agents such as nigericin, monensin, and chloroquine or the proton translocase inhibitors N-ethylmaleimide (NEM) and dicyclohexylcarbodiimide (DCCD). Polyethylene glycol (PEG) precipitation of insulin-receptor complexes revealed that endosomal degradation augmented the dissociation of insulin from its receptor and that dissociated insulin was serving as substrate to the endosomal protease(s). The results suggest that as insulin is internalized it rapidly but incompletely dissociates from its receptor. Dissociated insulin is then degraded by an insulin specific protease(s) leading to further dissociation and degradation.     

An insulin epidermal growth factor-binding protein from Drosophila has insulin-degrading activity

We have recently described the purification and characterization of an insulin-degrading enzyme (IDE) from Drosophila melanogaster that can cleave porcine insulin, is highly conserved through evolution and is developmentally regulated. We now report that the IDE is, in fact, an insulin EGF-binding protein (dp100) that we had isolated previously from Drosophila using an antihuman EGF receptor antiserum. This conclusion is based upon the following evidence. (a) dp100, identified by its ability to cross-link to labeled insulin, EGF, and transforming growth factor-alpha (TGF-alpha), and to be immunoprecipitated by anti-EGF receptor antisera, copurifies with the IDE activity. Thus, the purified IDE can be affinity labeled with either 125I-insulin, 125I-EGF, or 125I-TGF-alpha, and this labeling is specifically inhibited with unlabeled insulin, EGF, and the insulin B chain. (b) The antiserum to the human EGF receptor, which recognizes dp100, is able to specifically immunoprecipitate the insulin-degrading activity. (c) The purified IDE preparation contains a single protein of 110 kD which is recognized by both the anti-EGF receptor antiserum and anti-Drosophila IDE antiserum. (d) Polyclonal antiserum to the purified IDE, which specifically recognized only the 110-kD band in Drosophila Kc cells, immunoprecipitates dp100 cross-linked to 125I-TGF-alpha and dp100 cross-linked to 125I-insulin from the purified IDE preparation. (e) EGF, which competes with insulin for binding to dp100, also inhibits the degradation of insulin by the purified IDE. These results raise the possibility that a functional interaction between the insulin and EGF growth factor families can occur which is mediated by the insulin-degrading enzyme.     

Affinity purification of insulin-degrading enzyme and its endogenous inhibitor from rat liver.

A metallothiol protease called insulin-degrading enzyme (IDE) seems to be implicated in insulin metabolism to terminate the response of cells to hormone, as well as in other biological functions, including muscle differentiation, regulation of growth factor levels, and antigen processing. In order to obtain highly pure and biologically active IDE, we have developed an immunoaffinity method using a monoclonal antibody to this enzyme (9B12). When the cytosolic fraction of rat liver was first applied to a 9B12-coupled Affi-Gel 10 column, more than 97% of the insulin-degrading activity was absorbed. Among various kinds of buffers successfully eluting the enzyme, only the buffer with a high pH (pH 11) could retain the full biological activity of this enzyme. IDE was further purified via two steps of chromatography using Mono Q anion exchange and Superose 12 molecular sieve columns. The final preparation showed a single band at 110 kDa on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the eluate from the immunoaffinity column, the inhibitory activity associated with the enzyme was also observed. To better recover this endogenous inhibitor, heat-treated cytosolic fraction was fractionated by ammonium sulfate precipitation and applied to the immunoaffinity column on which IDE had been adsorbed. Then, IDE and its inhibitor could be co-eluted with pH 11 as a complex form. After heat treatment of this fraction, the inhibitor was further purified using the same series of chromatography as IDE to more than 20,000-fold; it showed a 14 kDa band on SDS-PAGE. It inhibited both the insulin degradation by IDE in a competitive manner and the cross-linking of 125I-insulin to IDE. Highly purified IDE and the endogenous inhibitor will be useful tools for better understanding the various biological functions of this enzyme.     

Receptor-mediated degradation by rat adipocytes: comparison of A-14 with A-19 125I-labelled insulin

We compared A-14 and A-19 125I-labelled insulin in receptor-binding and degradation. Percent receptor-binding of A-14 and A-19 125I-labelled insulin to 2.4 X 10(9)/ml erythrocytes after 210 min incubation at 15 degrees C was 7.8 and 4.9%, respectively. Percent insulin-receptor binding of A-14 insulin was 1.6 times greater than that of A-19 insulin. A similar result was obtained in an adipocytes insulin binding study. Percent receptor-binding of A-14 and A-19 insulin to 2 X 10(5)/ml fat cells after 30 min incubation in the above buffer was 3.9 and 2.4%, respectively. Degradation of A-14 and A-19 insulin in rat adipocytes was also studied by molecular sieve column chromatography. Isolated rat adipocytes were allowed to associate with A-14 and A-19 125I-insulin for 60 min at 37 degrees C, pH 8.0 in a HEPES-phosphate buffer, and then cells were separated from the buffer by centrifugation. After solubilization with triton X-100, both the solubilized cells and the incubation medium were applied to the Bio-Gel P-30 column to assess the insulin degradation. Degradation of A-14 125I-insulin by the isolated rat adipocytes was 1.6 times greater than that of A-19 125I-insulin. Furthermore, the peak which was thought to be intermediate degradation products of insulin was obtained between the peak of intact insulin and that of 125I-tyrosine. Such a peak of intermediates was much smaller in the incubation media than in the cell-associated materials.(ABSTRACT TRUNCATED AT 250 WORDS)     

蛋白激酶C抑制剂对U937细胞清道夫受体功能的影响

为了解细胞内蛋白质磷酸化水平对清道夫受体功能的影响,用蛋白激酶Ｃ抑掉剂形孢菌素（ｓｔａｕｒｏｓｐｏｒｉｎｅ,ＳＴＡ）处理人Ｕ９３７细胞,分别测定对照组和处理组细胞对碘标记的氧化低密度脂蛋白（＾１２５Ｉ）ｏｘ－ＬＤＬ的降解,结合,细胞表面受体复合物的内移以及细胞内脂质蓄积的程度,并利用放射自显影方法观察药物对细胞表面受体表达的影响,结果发现ＳＴＡ可以促进细胞结合（＾１２５Ｉ）ｏｘ－ＬＤＬ增加细胞表面     

Evidence for an early degradative event to the insulin molecule following binding to hepatocyte receptors

We have used photoreactive insulin analogues to investigate as related processes, early structural modification of the receptor-bound insulin molecule and internalisation of the insulin-receptor complex. In isolated rat hepatocytes an initial modification of bound insulin leads to the generation of a molecular species unchanged in molecular weight but with reduced receptor and antibody binding affinities and altered electrophoretic mobility. Using photoreactive insulin analogues and density gradient cell fractionation the insulin receptor complex has been shown to undergo internalisation from the plasma membrane to a low density vesicular fraction, the endosome. No labelled material was found in lysosomal fractions after up to 10 min incubation at 37 degrees C. The degree of labelling of the endosome fraction depended on the position of the photoreactive group within the insulin molecule. The data suggest that before or during endocytosis, a small peptide is proteolytically cleaved from the C terminus of the insulin B chain.     

Leprechaunism: an inherited defect in a high-affinity insulin receptor.

We examined in vivo oral glucose tolerance tests and in vitro insulin binding, cellular response, and insulin-receptor structure of fibroblasts cultured from the skin of a patient with leprechaun syndrome and her parents. In response to oral glucose, the proband exhibited marked hyperinsulinism (maximum plasma insulin = 4,120 microU/ml), the father had mild hyperinsulinism (maximum plasma insulin = 240 microU/ml), and the mother was normal. [125I]insulin binding to monolayers of intact fibroblasts demonstrated complex kinetics that were interpreted using a two-receptor model. Normal high-affinity binding had an apparent KA of 1.6 X 10(10)/molar with 1,100 sites/cell. The proposed low-affinity state receptor had an apparent KA of 6.8 X 10(7)/molar with approximately 30,000 sites/cell. Insulin binding to the proband's cells had no high-affinity binding but had normal low-affinity binding. Cells from the mother had 60%, and cells from the father, 2%, of control insulin binding to the high-affinity receptor, but normal, low-affinity site binding. Two different, insulin-stimulable responses were evaluated under experimental conditions identical with those used for insulin binding. Insulin stimulation of 2-methylaminoisobutyric acid uptake occurred with half-maximal responses between 25 and 50 ng/ml insulin. This response was similar in cells from controls and the patient. By contrast, the uptake and phosphorylation of 2-deoxy-D-glucose was stimulated at half-maximal insulin concentrations between 1 and 10 ng/ml in control cells but was not significantly increased in the proband's cells until 1,000 ng/ml concentrations of insulin were attained. In affinity crosslinking experiments, [125I]insulin was covalently bound to insulin receptors of fibroblast membranes using disuccinimidylsuberate. [125I]insulin specifically bound to 125,000 dalton monomeric subunits and 250,000 dalton dimers. In control cells, the ratio of monomer to dimer was approximately one, but significantly fewer dimers were crosslinked in insulin receptors from the patient's cells. We conclude that in this family two different recessive mutations impair high-affinity insulin-receptor binding and that the proband with leprechaunism is a compound heterozygote for these mutations. The two mutations produced structural changes in the receptor that altered subunit interactions and loss of high-affinity binding and cellular responsivity.     

In vitro effects of glucocorticoid on glucose transport in rat adipocytes: evidence of a post-receptor coupling defect in insulin action

The in vitro effect of glucocorticoid on insulin binding and glucose transport was studied with rat adipocytes. Isolated rat adipocytes were incubated with or without 0.70 microgram/ml (1.9 mumol) of hydrocortisone in TCM 199 medium at 37 degrees C, 5% CO2/95% air (v/v), pH 7.4, for 2, 4, and 8 h, and then fat cell insulin binding and insulin-stimulated 3-O-methylglucose transport were measured. Hydrocortisone did not affect insulin binding in terms of affinity or receptor number. Glucose transport in the absence of insulin was significantly decreased at the incubation time of 2 h and continued to decrease up to 8 h of incubation with hydrocortisone. Decreased insulin sensitivity of glucose transport (i.e., a right-ward shift of the dose response curve) was also demonstrated after 2 h incubation with hydrocortisone, and the ED50 of insulin was maximally increased at 4 h of incubation (0.53 ng/ml for treated vs. 0.22 ng/ml for control cells). Maximal insulin responsiveness was also significantly decreased in treated cells after 8 h incubation with hydrocortisone. When percent maximum glucose transport was expressed relative to receptor-bound insulin, the ED50 values of treated and control cells were 10.5 and 7.2 pg of bound insulin, per 2 X 10(5) cells, respectively. Thus, it was evident that glucocorticoid induced a post-receptor coupling defect in the signal transmission of insulin-receptor complex.     

An assay procedure for solubilized thyroid hormone receptor: use of Lipidex

A method using Lipidex-1000 is reported for the assay of thyroid hormone receptor activity. The receptor was extracted from rat liver nuclei, incubated with [125I]T3, and applied to 1-ml disposable pipet tip columns containing a small volume of Lipidex. The resin absorbed the free hormone, and the receptor-bound hormone was recovered in the eluate. The method allowed accurate assay of the receptor activity, satisfying a linear relationship between the activity and the receptor protein concentration. The usefulness of this method was demonstrated by determining the pH optimum and the Kd and Bmax of T3 binding by the receptor. The Lipidex column can be used for the preparation of an unoccupied receptor after dissociation of the endogenously bound hormone.