Iodoinsulin Binding Research Articles

Identification and characterization of the ligand-binding domain of insulin receptor by use of an anti-peptide antiserum against amino acid sequence 241-251 of the .alpha. subunit

We previously reported that a 23-kDa receptor proteolytic fragment containing an insulin-binding site was localized within residues 205-316 in the cysteine-rich region of the insulin receptor alpha subunit and postulated that sequence 241-251 plays a major role in insulin binding [Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., & Goldfine, I.D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329]. In the present study, we have used an antiserum raised against a synthetic peptide containing sequence 241-251 to test this postulate and to study the role of sequence 241-251 in insulin binding. The antiserum immunoprecipitated the 23-kDa fragment, confirming our sequence assignment of this fragment. It also immunoprecipitated the intact alpha subunit of the insulin receptor that had been denatured by reduction and alkylation. However, sequence 241-251 in the native receptor was inaccessible to the antiserum since the antiserum did not block [125I]iodoinsulin binding and did not precipitate either photoaffinity-labeled insulin receptors or insulin receptors labeled with 125I. However, using a radioactive photoaffinity probe [( 125I]-AZAP-insulin) that allows cleavage and removal of insulin after photolabeling, we found that sequence 241-251 became accessible to the antiserum after removal of insulin. We conclude therefore that sequence 241-251 forms part of the insulin-binding domain of the insulin receptor and that the binding of insulin to the receptor induces a conformational change that allows exposure of this domain after removal of insulin. Such a conformational change may play a role in activation of the receptor and transmembrane signaling.

Adenosine 3',5'-Cyclic Monophosphate-Dependent Protein Kinase (A Kinase) Regulation of Insulin Receptor Function: Phosphorylation of Insulin Receptor with A Kinase Decreases the Insulin Binding Activity.

The effect of phosphorylation of insulin receptor with adenosine 3',5'-cyclic monophosphate-dependent protein kinase (A kinase) on its insulin binding activity was investigated by using insulin receptors prepared from rat liver in vitro. A 95 KDa protein was phosphorylated by stimulation of insulin receptor kinase. This protein was also phosphorylated by A kinase. Analysis of phosphoamino acid showed that tyrosine residue(s) was phosphorylated by activation of insulin receptor kinase, whereas phosphoserine and phosphothreonine were dominantly generated by activation of A kinase. [125I] Iodoinsulin binding activity was decreased by prior phosphorylation of the receptor with A kinase. Scatchard analysis showed that the affinity for insulin was decreased by the phosphorylation with A kinase. Although the maximal activity of insulin receptor kinase was not affected by phosphorylation with A kinase, the insulin concentration which induced half maximal activity (ED50) of the receptor kinase was increased by the phosphorylation with A kinase. These results suggested that counter regulatory hormones whose actions are mediated by the generation of adenosine 3',5'-cyclic monophosphate regulate the insulin binding to the alpha subunit through phosphorylation of the beta subunit of insulin receptor.

Evidence for separate receptors for insulin and insulin-like growth factor-I in choroid plexus of rat brain by quantitative autoradiography.

Binding of insulin and insulin-like growth factor-I (IGF-I) to the choroid plexus was quantitatively characterized using autoradiography and computer densitometry. Slide-mounted brain slices were incubated in 0.1 nM [125I]-insulin or [125I]-[Thr59]IGF-I. To determine specificity of the binding sites, the labeled peptides were mixed with unlabeled analogues. Autoradiography was done with LKB Ultrofilm and analyzed with a computer image analysis system and program for densitometry. Results showed that binding was time and temperature dependent and reversible. Binding of the iodinated insulin and IGF-I was inhibited by unlabeled peptides in a dose-dependent manner. The rank order of potency of these peptides in competing for the choroid plexus iodoinsulin binding sites was: chicken insulin greater than porcine insulin greater than desoctapeptide insulin greater than IGF-I. IGF-I was more potent than porcine insulin in competing for the choroid plexus iodolGF-I binding sites. Somatostatin was ineffective. Non-linear regression analysis revealed the presence of high- (Kd 1.3 +/- 0.2 nM) and low-affinity (Kd 36 +/- 1.4 nM) binding sites for insulin and a single high-affinity binding site (Kd 3.1 +/- 0.3 nM) for IGF-I in the choroid plexus. There were approximately 50 times more binding sites (Bmax) for IGF-I than for insulin high-affinity sites, whereas the number of low-affinity sites for insulin was about equal to the number of IGF-I high-affinity sites. The results of these binding studies with iodinated insulin and [Thr59]IGF-I support the conclusion that the rat choroid plexus has separate high-affinity receptors for insulin and IGF-I, and that the IGF-I receptors outnumber the insulin receptors.

Insulin as a regulator of androgen biosynthesis by cultured rat ovarian cells: cellular mechanism(s) underlying physiological and pharmacological hormonal actions.

It is the objective of the in vitro studies reported herein to further evaluate the role of insulin in the regulation of ovarian androgen biosynthesis, to assess its dose requirements, and to elucidate the cellular mechanism(s) underlying its high dose action. To this end, use was made of recently developed primary culture systems of ovarian androgen-producing cells, the differentiation of which is subject to regulation by gonadotropic and insulinotropic signaling. Treatment of collagenase-processed whole ovarian dispersates or highly enriched (greater than 90%) thecal-interstitial cells from immature rats with insulin (1 microgram/ml) or hCG (1 ng/ml), resulted in 1.5- and 2.6-fold increments in the accumulation of androsterone (3 alpha-hydroxy-5 alpha-androstan-17-one), the main androgenic steroid identified in culture medium by HPLC. However, combined treatment with both agents unmasked a synergistic interaction resulting in 5.7-fold amplification of hCG action, the increase in androsterone accumulation representing enhanced biosynthesis rather than diminished degradation. Unaccounted for by cellular growth and independent of the cellular density of plating (1 X 10(4)-1 X 10(6) viable cells/culture) or the hCG dose (0.1-10 ng/ml) employed, the insulin effect proved time and dose dependent with a minimal time requirement of 72 h. [125I-TyrA14]Iodoinsulin binding to untreated highly enriched thecal-interstitial cells proved highly specific, saturable, and reversible, displaying a single class (Hill coefficient = 0.93 +/- 0.07) of high affinity (Kd = 1.7 X 10(-10) M), low capacity (4746 +/- 283 sites/cell) binding sites. Treatment with physiological concentrations (10 ng/ml) of insulin produced limited, albeit measureable, down-regulation of the insulin receptor. In contrast, provision of relatively high concentrations (1 microgram/ml) of insulin resulted (despite marked adsorption/degradation) in substantial (greater than 60%) down-regulation of the insulin receptor, but not the type I insulin-like growth factor receptor, the ligand of which has also been shown to amplify hCG-supported androgen biosynthesis. These findings suggest that the thecal-interstitial cell is a site of insulin reception and action, that physiological concentrations of insulin are capable of participating in the regulation of ovarian androgen biosynthesis, and that this effect is probably mediated via high affinity insulin receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Metabolic basis for the diabetogenic action of growth hormone in the obese (ob/ob) mouse.

The ob/ob mouse responds predictably to chronic treatment with large doses of pituitary GH with marked hyperglycemia and decreased glucose tolerance. The purpose of the present study was to characterize the metabolic alterations produced by GH that lead to this diabetogenic response in the ob/ob mouse in order to determine whether this animal might serve as a useful model for the study of the cellular mechanisms involved in the diabetogenic action of GH. Female ob/ob mice were treated sc for 3 days with either saline or 200 micrograms/day S-carboxymethylated human GH (RCM-hGH), a diabetogenic GH derivative lacking significant growth-promoting or insulin-like activities. Six hours before the start of the experiment, the animals were given a sc injection of 2 micrograms dexamethasone and deprived of food. RCM-hGH treatment produced marked increases in fasting blood glucose and plasma insulin concentrations, but had no effect on plasma glucagon or serum insulin-like growth factor I levels. It had no effect on liver glycogen level or in vitro hepatic glucose production in the absence or presence of pyruvate and lactate added to the incubation medium. By contrast, the in vitro stimulatory effects of insulin on [14C] glucose oxidation by isolated soleus muscle or segments of parametrial fat were greatly attenuated by RCM-hGH treatment, without changes in rates of basal glucose oxidation. This change in peripheral tissue responsiveness to insulin does not appear to involve glucose transport, since the in vitro stimulation by insulin of 3-O-[14C]methylglucose transport into isolated diaphragm muscle was not altered by RCM-hGH treatment. Moreover, the RCM-hGH-induced reduction in adipose tissue responsiveness to insulin does not appear to be mediated by a reduction in insulin binding, since [125I]iodoinsulin binding to adipocytes isolated from RCM-hGH-treated mice was similar to that to cells from saline-treated animals. Interestingly, the reduction in responsiveness to insulin seen with segments of adipose tissue from RCM-hGH-treated animals was not found with isolated adipocytes prepared from such tissue by collagenase digestion. These results suggest that the hyperglycemia and glucose intolerance produced in ob/ob mice by chronic GH treatment result primarily from increased peripheral tissue insulin resistance. Therefore, the ob/ob mouse provides a useful model to elucidate the cellular mechanism(s) of this aspect of the diabetogenic action of GH.

Glucagon alters insulin binding to isolated rat epididymal adipocytes: possible role of adenosine 3',5'-monophosphate in modification of insulin action.

Effects of glucagon on insulin action in rat epididymal adipocytes were studied in vitro. [125I]iodoinsulin binding to isolated adipocytes was inhibited by preincubation of the cells with isoproterenol, epinephrine, or glucagon. Fifty percent inhibition was observed with 2 X 10(-8) M glucagon, 10(-6) M isoproterenol, and 10(-7) M epinephrine. Maximal (90%) inhibition induced by glucagon was observed at 10(-6) M. In Scatchard analysis, [125I]iodoinsulin competition data generated curvilinear plots in buffer- and glucagon-treated cells. Pretreatment of the cells with (Bu)2cAMP reduced insulin binding activity. However, the simultaneous addition of (Bu)2cAMP with [125I]iodoinsulin did not produce the inhibition of the binding when the cells were not pretreated with the agent. cAMP level in the cells was increased by incubation with glucagon. 3-O-[Methyl-3H]methylglucose uptake by isolated adipocytes was inhibited by pretreatment of the cells with glucagon. These results suggest that glucagon regulates insulin action through decrease in insulin receptor activity, and that it is possible that the inhibition is mediated by cAMP production in adipocytes.

Quantitative autoradiographic evidence for insulin receptors in the choroid plexus of the rat brain.

Techniques of in vitro receptor autoradiography were used to visualize binding of 125I-insulin on slices of frozen rat brain. Slide-mounted sections of frozen rat brain were incubated in 0.05 nM porcine 125I-monoiodoinsulin, alone or mixed with 1 microM unlabeled porcine insulin, ribonuclease, or glucagon, for 2 h at 22 degrees C. The labeled brain slices were apposed to LKB Ultrofilm to generate autoradiograms. The method permitted equal access of labeled insulin to both sides of the blood-brain barrier and localization of insulin binding sites in small anatomic regions. Quantitative estimates of specific iodoinsulin binding were made by computer digital image densitometry of the autoradiographic film images. High concentrations of specific binding sites for iodoinsulin were present in the choroid plexus of the lateral (26.9 +/- 2.0 X 10(-3) fmol/mm2), fourth (18.3 +/- 3.0 X 10(-3) fmol/mm2), and third (13.2 +/- 1.5 X 10(-3) fmol/mm2) ventricles (insulin binding is expressed per unit area of autoradiographic image). Binding to the third ventricular choroid plexus was similar to the concentrations observed for liver slices and the external plexiform layer of the olfactory bulb. Specific binding of iodoinsulin in the cingulate cortex and other surrounding regions was less than in choroid plexus. Ribonuclease or glucagon had no measurable effect on binding when mixed with labeled insulin. The results support the hypothesis that the choroid plexus has a high density of receptors for insulin, and suggests that the choroid plexus may be a target of CSF insulin action and/or a site of insulin transport into the CSF.

The development of insulin receptors and responsiveness is an early marker of differentiation in the muscle cell line L6.

After reaching confluence, mononucleated L6 myoblasts fuse into multinucleated contracting myotubes. This process is accompanied by the synthesis of characteristic skeletal muscle proteins, such as myosin heavy chain and the MM isoenzyme of creatine kinase. We have studied the development of insulin receptors and insulin responsiveness during differentiation in the L6 cells. Insulin was bound to high affinity receptors in both myoblasts and differentiated myotubes. The binding showed characteristics typical for insulin binding in other cell types, including high affinity, appropriate specificity, an upwardly concave Scatchard plot, and down-regulation. In the logarithmic growth phase, the myoblasts exhibited a low level of insulin binding, but on initiation of cell fusion, the resulting myotubes progressively developed a 2-fold increase in specific [125I]iodoinsulin binding as a result of a 2-fold increase in receptor number. The increase in insulin binding was an early differentiation event, preceding the accumulation of creatine kinase by 24 h. The development of insulin binding during differentiation correlated closely with an increased ability of the hormone to stimulate maximal 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake at physiological concentrations. The L6 cells are a useful model for studying the binding and effects of physiological insulin concentrations in skeletal muscle before and after differentiation.

Insulin binding, glucose oxidation, and methylglucose transport in isolated adipocytes from pregnant rats near term.

To elucidate the mechanism of insulin resistance during late pregnancy, we studied [125I]iodoinsulin binding, [1-14C]glucose oxidation, and 3-O-[methyl-14C]glucose transport in adipocytes isolated from pregnant rats on day 19 or 20 of gestation. Neither the affinity or number of insulin receptors on pregnant rat adipocytes differed from those on age-matched nonpregnant female rats. Insulin-stimulated glucose oxidation was reduced in the pregnant rat adipocytes. The maximum velocity of insulin-stimulated methylglucose transport was also significantly reduced in the pregnant rat adipocytes. These results suggest that insulin resistance in isolated adipocytes from pregnant rats near term is caused by some postreceptor changes, one of which is a reduction in the number and/or mobility of insulin-stimulated hexose transporters.

Receptor binding and biological activity of specifically labeled [125I]- and [127I]monoiodoinsulin isomers in isolated rat adipocytes.

The receptor binding characteristics and biological activity of single site monoiodinated insulin was investigated using isolated rat adipocytes. Pork insulin was iodinated by the lactoperoxidase method, and each of the four monoiodo derivatives (iodotyr-A14, iodotyr-A19, iodotyr-B16, and iodotyr-B26) was isolated by high performance liquid chromatography. Both radioactive 125I-labeled and nonradioactive 127I-labeled iodoinsulins were prepared. In competitive binding experiments in which each 125I-labeled iodoisomer competed for receptor binding at 15 C with its homologous 127I-labeled iodoisomer, the concentrations of homolog required for 50% inhibition of tracer binding were 1.67 +/- 0.06, 2.14 +/- 0.23, and 2.35 +/- 0.27 X 10(-9) M for the B26, A14, and B16 iodoisomers, respectively (mean +/- SEM; n = 3). Scatchard analysis of the homologous competition curves using a two-site model indicated that there was no difference in the total number of specific sites to which each isomer could bind or in their affinity for binding to the low affinity site. However, a significantly (P less than 0.05) higher affinity for (B26)iodoinsulin binding to the high affinity site was observed compared to either the (A14)- or (B16)iodoisomers (Kd values of 1.09 +/- 0.18, 2.09 +/- 0.40, and 2.24 +/- 0.15 X 10(-9) M for B26, A14, and B16, respectively). Degradation of the isomers by adipocytes incubated at 37 C occurred at a rate proportional to their receptor binding affinity. The biological activity of iodoinsulins was also evaluated, based on the ability either to stimulate glucose oxidation or to exert an antilipolytic effect. The 127I-labeled (B26)iodoisomer exhibited the greatest potency in both assays compared to either (A14)- or (B16)iodoinsulins, consistent with its higher apparent affinity for receptor binding. The receptor-binding activity and biological potency of unmodified native insulin and 127I-labeled (A14)-iodoinsulin were directly compared. Identical results were obtained for each in both types of assay, suggesting that (A14)iodoinsulin is a valid tracer for insulin with isolated adipocytes. We conclude that in isolated rat adipocytes, (B26)iodoinsulin has greater activity than either unlabeled insulin or the other iodinated derivatives. (A14)Iodoinsulin is indistinguishable from native insulin; (B16)iodoinsulin has slightly reduced activity, while that of (A19)iodoinsulin is considerably reduced.

The effect of high-protein and high-carbohydrate diets on [125I]iodoinsulin binding in skeletal muscle plasma membranes and isolated hepatocytes of rainbow trout (Salmo gairdneri).

[125I]iodoinsulin-binding studies in the presence of a concentration range of bovine insulin were conducted to establish specific insulin-binding levels in skeletal muscle plasma membranes and isolated hepatocytes of rainbow trout (Salmo gairdneri) reared on control, high-protein or high-carbohydrate diets. Negative co-operativity was observed and receptor concentrations and apparent dissociation constants established for each preparation. No differences of specific binding attributed to diet were detected in skeletal muscle plasma membrane preparations; however, the receptor concentration of isolated hepatocytes from high-carbohydrate-reared trout was increased. This contrasted to comparable mammalian studies.

Preparation of isolated bovine adipocytes: validation of use for studies characterizing insulin sensitivity and binding.

The present study was undertaken to develop techniques to isolate bovine adipocytes, to compare their rates of glucose metabolism and insulin sensitivity with adipocytes in adipose tissue, and to determine if isolated bovine adipocytes specifically bind insulin. Cell size and diameter distributions were the same for adipocytes fixed with OsO4 after isolation with collagenase and adipocytes liberated from OsO4-fixed adipose tissue slices. On a per cell basis, lipogenic rates were greater for isolated adipocytes compared with intact adipose tissue. Similar differences were found for glucose oxidation. In short term incubations, glucose oxidation and lipid synthesis were not stimulated by insulin (0-100 ng/ml) in either isolated adipocytes or tissue. Specific binding of [125I]iodoinsulin at 30 C was low (0.8%) in the first group of six beef cattle sampled, but increased with increasing cell concentration. Insulin degradation after 90 min was less than 5%. The specificity of [125I]iodoinsulin binding was studied in a second group of six animals. There was no specific binding of insulin in this group. In summary, bovine adipocytes can be isolated which are metabolically active and provide a valid system for studying hormone binding and action. In the present study, glucose metabolism in bovine adipocytes was not stimulated by insulin in vitro. This insensitivity to insulin was associated with a negligible capacity for insulin binding. These findings suggest that the lack of insulin sensitivity in bovine adipose tissue may be due to an inability to specifically bind insulin. This may be related to the unique metabolism of ruminant adipose tissue, which is less dependent upon glucose for fatty acid synthesis than is adipose tissue from nonruminant species.

Insulin receptors of human term placental cells and choriocarcinoma (JEG-3) cells: characteristics and regulation.

Insulin has been implicated as a regulator of placental growth and function. In order to probe hormoneplacental interactions, an in vitro human placental cell culture model was employed in conjunction with the human choriocarcinoma cell line, JEG-3. Short term monolayer cultures of human term placentae were established from trypsinized chorionic villi and were presumed to be predominantly of syncytial origin on the basis of their electron microscopic morphology, their capacity to produce placental hormones, and their loss of replicative potential. Specific binding of [125I]iodoinsulin to both cell types was examined using whole cell radioreceptor assays, and was found to be a saturable process, time and temperature dependent, and proportional to cell protein and ligand concentration. At 4 C, a steady state was reached by 18 h and was maintained for 6 h. Specificity of the insulin receptor was evidenced by the inability of structurally unrelated hormones to inhibit [125I]iodoinsulin binding. Scatchard plot...

Modulation of hepatic insulin receptors by a human growth hormone fragment (hGH 6–13)

A synthetic amino-terminal fragment of human growth hormone (hGH) containing the sequence H 2N-Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-COOH (hGH 6–13) was shown to increase [ 125I]iodoinsulin binding to rat hepatic receptors in vivo. Analysis of the binding data indicated an increase in the capacity of the insulin receptors or the number of available receptors. In vitro experiments with isolated hepatocytes and hepatic plasma membranes revealed no direct interaction between the hGH 6–13 and the hepatic insulin receptors. When the isolated hepatocytes were preincubated with the synthetic hGH fragment (10 μg/ml) at 37°C for 30 min prior to tests for insulin binding, the binding of [ 125I]iodoinsulin to the hepatic receptors was significantly enhanced. The variance between the in vivo and in vitro findings was considered in terms of the mechanism of hormonal actions mediated through a secondary cellular mediator.

Characterization of the Hepatic Receptor for Insulin in the Perinatal Rat*

The fetus is in a constant anabolic state and responsive to insulin. To examine the possible role of the insulin cell surface receptor in mediating these effects, we have studied insulin-receptor interactions in the developing rat liver. Compared to adult hepatic membranes, specific [125I]iodoinsulin binding was approximately 2 times greater in liver tissues from 21-day-old fetal and 1-day-old neonatal rats. By several criteria (pH, temperature, and time dependencies; specificity; receptor degradation properties; and insulin dissociation characteristics), insulin receptors from perinatal and adult hepatic membranes were similar. At 20 C, however, adult hepatic membranes degraded approximately 3 times as much insulin as liver tissue from 1-day-old neonatal rats. When insulin degradation by adult hepatic membranes was minimized by incubation with N-ethylmaleimide (NEM) or at 4 C, [125I]iodoinsulin binding in perinatal liver membranes remained significantly greater. Scatchard analysis revealed curvilinear pl...

Insulin radioreceptor assay on murine splenic leukocytes and peripheral erythrocytes.

Insulin radioreceptor assays were developed using splenic leukocytes and peripheral erythrocytes from individual mice. Splenic leukocytes were prepared using an NH4Cl buffer which did not alter insulin binding, but gave much higher yields than density gradient methods. Mouse erythrocytes were isolated from heparinized blood by three passages over a Boyum gradient, and a similar buffer was used to separate cells from free [125I]iodoinsulin at the end of the binding incubation. Insulin binding to both splenic leukocytes and peripheral erythrocytes had typical pH, temperature, and time dependencies, and increased linearly with an increased number of cells. Optimal conditions for the splenic leukocytes (6 X 10(7)/ml) consisted of incubation with [125I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.0. In cells from 20 individual mice, the specific [125I]iodoinsulin binding was 2.6 +/- 0.1% (SEM), and nonspecific binding was 0.3 +/- 0.04% (10.6% of total binding). Erythrocytes (2.8 X 10(9)/ml) were incubated with [125I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.2. In cells from 25 individual mice, the specific [125I]iodoinsulin binding was 4.5 +/- 0.2%, and nonspecific binding was 0.7 +/- 0.03% (13.6% of total binding). In both splenic leukocytes and peripheral erythrocytes, analysis of equilibrium binding data produced curvilinear Scatchard plots with approximately 3500 binding sites/leukocyte and 20 binding sites/erythrocyte. These data demonstrate that adequate numbers of splenic leukocytes and peripheral erythrocytes can be obtained from individual mice to study insulin binding in a precise and reproducible manner. This should facilitate direct comparisons of these cells with classical target cells in a variety of mouse model diseases.

Specific binding and internalization of blood-borne [125I]-iodoinsulin by neurons of the rat area postrema.

The specificity and cellular location of binding sites for blood-borne [125I]iodoinsulin in the area postrema and adjacent paravagal region of the rat brain was determined by means of quantitative light and electron microscope radioautography. In both regions coinjected unlabeled insulin inhibited the binding of [125I]iodoinsulin in a dose-dependent manner, and insulin analogs blocked [125I]iodoinsulin binding in rank order of their known in vitro bioactivity. Electron microscope radioautography of this region showed that insulin specifically bound to neuronal dendrites and cell bodies and that some of the specifically bound radioactivity was internalized and concentrated in a variety of subcellular vacuoles. These observations demonstrate that certain neurons of the area postrema are endowed with the receptive capacity common to all known insulin-sensitive cells. Anatomic considerations suggest that insulin target neurons in the area postrema could mediate insulin feedback on parasympathetic function by relaying information regarding circulating insulin status to central autonomic circuits governing vagal activity. This study represents the first demonstration of a receptor-mediated uptake and concentration of a blood-borne polypeptide hormone by central neurons.

A direct in vitro demonstration of insulin binding to isolated brain microvessels.

Recent in vivo autoradiographic studies have suggested that circulating insulin may bind to the capillary wall, i.e., the blood-brain barrier. In the present study the blood-brain barrier insulin receptor was examined directly by measuring [125I]-iodoinsulin binding to capillaries isolated from fresh bovine cerebral cortex. Microvessels were prepared by gentle hand homogenization and trapping on nylon mesh. The binding was rapid, specific, and reversible with one-half maximal binding attained in 7 min and maximal binding achieved in 45 min at room temperature. The high affinity site has an affinity constant of 2.3 +/- 0.3 nM-1, and 50% displacement of [125I]-iodoinsulin occurred at approximately 9 ng/ml. [125I]-Iodoinsulin was not displaced by excess thyrotropin, prolactin, or growth hormone, and proinsulin was much less potent than porcine insulin. These studies confirm the presence of a specific insulin receptor on brain microvessels. Although insulin does not cross the blood-brain barrier, the presence of an insulin receptor provides a possible mechanism by which blood-borne insulin can influence brain cell metabolism.

Regenerating Rat Liver: Insulin and Glucagon Serum Levels and Receptor Binding*

Serum insulin and glucagon levels and liver plasma membrane receptor binding were studied in rats after partial hepatectomy. To clarify whether the surgical stress and decreased food intake that accompanies partial hepatectomy influenced these parameters, sham-operated rats were also studied. When sham-operated rats were compared to nonoperated controls, there was a 30% fall in insulin levels and a significant rise in the number of insulin receptors. In contrast, glucagon levels and glucagon receptor binding were unchanged. When partially hepatectomized rats were compared to sham-operated rats, there was no significant change in either insulin levels or the number of plasma membrane insulin receptors. Insulin degradative activity, however, was decreased in liver plasma membranes from partially hepatectomized animals, causing an apparent increase in [125I]iodoinsulin binding to this organelle. Bacitracin, an inhibitor of insulin degradation, abolished this difference in insulin binding. Glucagon levels rose by 65% after partial hepatectomy, whereas the number of glucagon receptors decreased significantly. The studies demonstrate, therefore, that after partial hepatectomy, serum insulin levels and insulin receptor binding in liver are altered, and these alterations are due to surgical stress and decreased food intake. Glucagon levels and glucagon receptor binding are also altered after partial hepatectomy, but these alterations are due to liver regeneration per se.

The insulin receptor of embryonic chicken cartilage.

Highly purified plasma membranes have been obtained from embryonic chicken cartilage by physical means rather than enzymatic digestion. Rapid and reversible binding of [125I]iodoinsulin to these membranes is demonstrated. Similar to the insulin-binding properties of rat liver and adipocytes and human mononuclear cells, optimal specific binding of insulin to chondrocyte plasma membranes has a sharp pH optimum at 8.0, and maximal binding occurs at 2--4 C. Analysis of equilibrium binding reveals a curvilinear Scatchard plot, whose high affinity segment generates a maximum affinity of 1.0 X 10(9) M-1, and a receptor concentration of 0.4 pmol/mg membrane protein. This affinity constant is similar to those generated for insulin binding to membranes prepared from embryonic chicken liver (2.5 X 10(9) M-1), rat liver (1.4 X 10(9) M-1), and mouse liver (0.6 X 10(9) M-1), whereas the receptor concentration is less than that of embryonic chicken liver membranes (1.1 pmol/mg), which in turn was less than those of rat liver membranes (2.8 pmol/mg) and mouse liver membranes (3.5 pmol/mg). Kinetic studies show augmentation of insulin-receptor dissociation by excess insulin when initial receptor occupancy, is low, suggesting that negative cooperativity is present. There is little or no interaction of other hormones with the chondrocyte insulin receptor, with the exception of proinsulin and the insulin-like growth factors. Porcine proinsulin, bovine proinsulin, somatomedin C, and nonsuppressible insulin-like protein prevent [125I]iodoinsulin binding to chondrocyte plasma membranes with dose-response curves which are parallel to that of unlabeled porcine insulin itself, but with molar potencies relative to porcine insulin of 15%, 9%, 2.5%, and 1.4%, respectively. Porcine insulin and proinsulin both prevent binding of [125I]iodosomatomedin C to chondrocyte plasma membranes but with molar potencies less than 1% that of unlabeled somatomedin C. These observations are consistent with the presence of a specific independent insulin receptor in embryonic chicken cartilage which is similar in its characteristics to the insulin receptor in previously described tissues. Insulin has a weak interaction with the chondrocyte receptor for somatomedin C. Interaction with the somatomedin receptor may be the mechanism by which insulin exerts anabolic effects on cartilage when used in pharmacological amounts.