Insulin Receptors In Rat Adipocytes Research Articles

Long lasting cadmium intake is associated with reduction of insulin receptors in rat adipocytes.

The effects of chronic cadmium exposure on adipose tissue have not been extensively reported. In adult Wistar male rats we investigated in vivo effect of 6 weeks lasting cadmium intake in drinking tap water (CdCl2 9,7 mg/l). Insulin receptors in isolated adipocytes from epididymal fat and glucose transporter protein GLUT4 content in fat tissue plasma membranes were determined. Control and Cd treated rats had similar water intake with subsequent heavy augmentation of Cd content in liver of experimental animals. In comparison with controls, Cd intake did not influence body mass increment and fat cell size, but significantly increased serum glycemia and moderately elevated insulinemia. Cadmium intake significantly reduced (approximately 50%) both, total insulin receptors number and density of the receptors in fat cells. No differences in the content of GLUT4 in crude plasma membranes of adipose tissue were observed. Diminished insulin receptors in adipocytes could account for diabetogenic effect of long lasting cadmium intake.

The upregulating effect of insulin and vanadate on cell surface insulin receptors in rat adipocytes is modulated by glucose and energy availability.

The aim of this study was to further characterize the rapid effects of insulin and the tyrosine phosphatase inhibitor vanadate to amplify cell surface insulin binding capacity in isolated rat adipocytes. The effect of 20 min insulin treatment (1000 microU/ml) was 2- to 3-fold (p < 0.01) when cells were treated in medium containing 5.6 mM D-glucose, but it was totally absent in glucose-free medium. Other carbon energy sources, such as fructose and pyruvate, could only partly substitute for D-glucose, with an approximately 1.5-fold insulin effect. Moreover, inhibiting transmembrane glucose transport with cytochalasin B completely blocked the effect of insulin to enhance cell surface binding. The effect of vanadate was only partly glucose-dependent, since a submaximal effect (1.5- to 2-fold, p<0.05) was seen also in the absence of glucose. The tyrosine kinase inhibitor genistein markedly blunted the effect of vanadate (from 3- to 4-fold to approximately 2-fold, p < 0.05) also indicating the importance of tyrosine phosphorylation-related mechanisms in the upregulation of cell surface insulin binding. Glycosylation of insulin receptors as a mechanism for this effect appears unlikely since neither the effect of insulin nor that of vanadate was altered by the glycosylation inhibitor tunicamycin. The time course for the insulin effect displayed a long duration (at least 6 h), suggesting a maintenance role of insulin keeping its receptors accessible for ligand binding at the cell surface. In conclusion, the effect of insulin and vanadate to upregulate cell-surface insulin receptors is energy-dependent and to some extent specifically glucose-dependent.

Synergistic activation of a family of phosphoinositide 3-kinase via G-protein coupled and tyrosine kinase-related receptors

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in a variety of receptor-stimulated cell responses. Stimulation of receptors possessing (or coupling to) protein-tyrosine kinase activates heterodimeric PI 3-kinases, which consist of an 85-kDa regulatory subunit (p85) containing Src-homology 2 (SH2) domains and a 110-kDa catalytic subunit (p110α or p110β). Thus, this form of PI 3-kinases could be activated in vitro by a phosphotyrosyl peptide containing a YMXM motif that binds to the SH2 domains of p85. Receptors coupling to αβγ-trimeric G proteins also stimulate the lipid kinase activity of a novel p110γ isoform, which is not associated with p85, and thereby is not activated by tyrosine kinase receptors. The activation of p110γ PI 3-kinase appears to be mediated through the βγ subunits of the G protein (Gβγ). In addition, rat liver heterodimeric PI 3-kinases containing the p110β catalytic subunit are synergistically activated by the phosphotyrosyl peptide plus Gβγ. Such enzymatic properties were also observed with a recombinant p110β/p85α expressed in COS-7 cells. In contrast, another heterodimeric PI 3-kinase consisting of p110α and p85 in the same rat liver, together with a recombinant p110α/p85α, was not activated by Gβγ, though their activities were stimulated by the phosphotyrosyl peptide. Synergistic activation of PI 3-kinase by the stimulation of the two major receptor types was indeed observed in intact cells, such as chemotactic peptide ( N-formyl–Met–Leu–Phe) plus insulin (or Fcγ II) receptors in differentiated THP-1 and CHO cells and adenosine (A1) plus insulin receptors in rat adipocytes. Thus, PI 3-kinase isoforms consisting of p110β catalytic and SH2-containing (p85 or its related) regulatory subunits appeared to function as a ‘cross-talk’ enzyme between the two signal transduction pathways mediated through tyrosine kinase and G protein-coupled receptors.

Identification, partial purification, and characterization of two guanosine triphosphate-binding proteins associated with insulin receptors

We have previously suggested that at least two different G-proteins are involved in mediating insulin receptor functions. Here we identify and partially purify two G-proteins with apparent molecular masses of 41 and 67 kilodaltons (kDa) that interact with insulin receptors in rat adipocytes and human placenta. Treatment of isolated rat adipocytes with insulin inhibited pertussis toxin-catalyzed ADP-ribosylation of a 41-kDa G-protein in subsequently isolated plasma membranes by 30.2 +/- 3.0% and in partially purified insulin receptor preparations by 35.6 +/- 5.7%. There was no associated decrease in the concentration of the 41-kDa G-protein in the plasma membranes, as determined by immunoblot with a common G alpha antibody. The common G alpha antibody also recognized a 67-kDa protein in the plasma membranes, the concentration of which was not affected by insulin. However, the 67-kDa protein was enriched in partially purified solubilized insulin receptor preparations. Two similar, 41- and 67-kDa G-proteins were identified in the wheat germ-purified insulin receptor preparations obtained from human placenta. Removal of these two G-proteins from insulin receptor preparations results in loss of the ability of insulin to stimulate receptor kinase activity. Addition of a fraction enriched with 41- and 67-kDa G-proteins to the G-protein-depleted insulin receptor restores the insulin sensitivity of the insulin receptor kinase activity. Furthermore, addition of G-protein-depleted insulin receptors to the fraction containing partially purified 41- and 67-kDa G-proteins enhances pertussis toxin-catalyzed ADP-ribosylation of the 41-kDa G-protein. These results indicate that either the 41- or 67-kDa G-protein, or both, interact with the insulin receptor mediating insulin receptor kinase activity. Such mutual interaction and regulation between the insulin receptor and G-proteins could be an important component of the signal transduction mechanism for insulin.

Identification, partial purification, and characterization of two guanosine triphosphate-binding proteins associated with insulin receptors.

We have previously suggested that at least two different G-proteins are involved in mediating insulin receptor functions. Here we identify and partially purify two G-proteins with apparent molecular masses of 41 and 67 kilodaltons (kDa) that interact with insulin receptors in rat adipocytes and human placenta. Treatment of isolated rat adipocytes with insulin inhibited pertussis toxin-catalyzed ADP-ribosylation of a 41-kDa G-protein in subsequently isolated plasma membranes by 30.2 +/- 3.0% and in partially purified insulin receptor preparations by 35.6 +/- 5.7%. There was no associated decrease in the concentration of the 41-kDa G-protein in the plasma membranes, as determined by immunoblot with a common G alpha antibody. The common G alpha antibody also recognized a 67-kDa protein in the plasma membranes, the concentration of which was not affected by insulin. However, the 67-kDa protein was enriched in partially purified solubilized insulin receptor preparations. Two similar, 41- and 67-kDa G-proteins were identified in the wheat germ-purified insulin receptor preparations obtained from human placenta. Removal of these two G-proteins from insulin receptor preparations results in loss of the ability of insulin to stimulate receptor kinase activity. Addition of a fraction enriched with 41- and 67-kDa G-proteins to the G-protein-depleted insulin receptor restores the insulin sensitivity of the insulin receptor kinase activity. Furthermore, addition of G-protein-depleted insulin receptors to the fraction containing partially purified 41- and 67-kDa G-proteins enhances pertussis toxin-catalyzed ADP-ribosylation of the 41-kDa G-protein. These results indicate that either the 41- or 67-kDa G-protein, or both, interact with the insulin receptor mediating insulin receptor kinase activity. Such mutual interaction and regulation between the insulin receptor and G-proteins could be an important component of the signal transduction mechanism for insulin.

Biochemical Characteristics of an Initial Degradation Product of Insulin by Insulin-Degrading Enzyme

We previously reported on the detection and HPLC separation of the initial degradation product (peak VI) of native insulin from the reaction of monocomponent porcine insulin with affinity-purified pig skeletal muscle insulin-degrading enzyme (IDE). In the present study, we investigated the biological characteristics of the initial degradation product. Structural analysis of peak VI by amino acid composition and glucose oxidation revealed that peak VI was composed of intact B-chain and a fragment of A-chain. In vivo, peak VI exhibited a hypoglycemic effect on rats. In vitro, this peptide had the binding capacity to insulin receptor of rat adipocytes and the ability to stimulate the glucose oxidation on rat adipocytes and the activity of insulin receptor kinase. However, the biological potencies of peak VI were 1/40 to 1/160 of those of insulin proper. Its reduced biological potencies were probably due to a decrease of affinity for insulin receptor, because both biological potencies of peak VI to bind to insulin receptor and to stimulate the glucose oxidation were 2.5% of insulin. Moreover peak VI showed the same full agonistic effect as insulin on the glucose oxidation at higher concentration. On the other hand, a cross-linking study suggested that peak VI preserves almost the same affinity to IDE as insulin. These findings may indicate the possibility that pig skeletal muscle IDE cleaves peptide bonds within A-chain at an early step of degradation of native porcine insulin and generates peak VI, which is the next substrate to insulin for IDE and keeps reduced insulin-like biological potencies, and then peak VI is converted into several relatively low molecular weight products.

Recycling of photoaffinity-labeled insulin receptors in rat adipocytes. Dissociation of insulin-receptor complexes is not required for receptor recycling.

We have used an iodinated, photoreactive analog of insulin, 125I-B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin, to covalently label insulin receptors on the cell surface of isolated rat adipocytes. Following internalization of the labeled insulin-receptor complexes at 37 degrees C, we measured the rate and extent of recycling of these complexes using trypsin to distinguish receptors on the cell surface from those inside the cell. The return of internalized photoaffinity-labeled receptors to the cell surface was very rapid at 37 degrees C proceeding with an apparent t 1/2 of 6 min. About 95% of the labeled receptors present in the cell 20 min after the initiation of endocytosis returned to the cell surface by 40 min. Recycling was slower at 25 and 16 degrees C compared to 37 degrees C and essentially negligible at 12 degrees C or in the presence of energy depleters. Addition of excess unlabeled insulin had no effect on the recycling of photoaffinity-labeled insulin receptor complexes, whereas monensin, chloroquine, and Tris partially inhibited this process. These data indicate that dissociation of insulin from internalized receptors is not necessary for insulin receptor recycling. Furthermore, agents which have been shown to prevent vesicular acidification inhibit the recycling of insulin receptors by a mechanism other than prevention of ligand dissociation.

Degradation of insulin receptors in rat adipocytes

Degradation of insulin receptors in rat adipocytes

Degradation of insulin receptors in rat adipocytes.

Insulin receptors on viable rat adipocytes were affinity-labeled using a biologically active and photosensitive analogue of insulin, 125I-B2(2-nitro, 4 azidophenylacetyl)-des-PheB1-insulin (125I-NAPA-DP-insulin). The radiolabeled proteins were identified by SDS polyacrylamide gel electrophoresis and autoradiography. Binding of 125I-NAPA-DP-insulin (40 ng/ml) to rat adipocytes at 16 degrees C, followed by photolysis, resulted in the specific labeling of essentially one protein with an apparent molecular weight of 430-450,000 daltons. When this radiolabeled protein was treated with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a 125,000-dalton protein and to a lesser extent in a 90,000-dalton protein. In addition, there was a minimal amount of specific labeling of a 115,000-dalton protein. Under certain experimental conditions, the nonreduced form of the photoaffinity-labeled receptor appeared as a heterogeneous population of proteins having apparent molecular weights of 430,000, 350,000, and 270,000 daltons. Subsequent to photoaffinity labeling of insulin receptors at 16 degrees C, adipocytes were incubated at 37 degrees C for various periods of time to allow for internalization. This resulted in an initial rapid loss of radioactivity in the 430,000- and 125,000-dalton bands. At 60 min the amount of radioactivity in each of these bands was approximately 50% of that present before incubation at 37 degrees C and stayed constant for 120 min. A first-order plot of the decline in receptor-associated radioactivity was biphasic with the initial phase having a half-life of 1.4 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of the insulin receptor of rat adipocytes. The three interconvertible redox forms.

Isolated intact rat adipocytes were photoaffinity labeled with radioactive and photoreactive N alpha B1-monoazidobenzoyl insulin (B1-MABI) or N epsilon B29-monoazidobenzoyl insulin (B29-MABI). Polyacrylamide gel electrophoresis of the labeled plasma membranes solubilized in sodium dodecyl sulfate revealed the specific labeling of three receptor species of 380 kDa, 300 kDa, and 230 kDa. Reduction of each species individually produced the subunits of 130 kDa, 90 kDa, and 40 kDa. Exposure of the adipocytes or plasma membranes after photolabeling to sulfhydryl alkylating agents such as N-ethylmaleimide or p-chloromercuriphenylsulfonate resulted in the appearance of the receptor quantitatively in the 380-kDa form. The effect of the sulfhydryl reagent was concentration dependent and in the case of p-chloromercuriphenylsulfonate the three receptor species reappeared when high concentrations of the reagent were used. Incubation of the adipocytes with low concentrations of dithiothreitol before photolabeling reduced these receptors to discrete lower-molecular-weight forms. In addition, an 85-kDa subunit was now photolabeled by B1-MABI. This subunit was demonstrated to be different from the 90-kDa subunit normally labeled by B29-MABI. We conclude that on the cell surface of the adipocyte, there is one molecular-weight form of insulin receptor of 380 kDa composed of one 130-kDa, one 90-kDa, one 85-kDa, and two 40-kDa subunits. The 300 kDa and 230 kDa are partially reduced forms of the 380-kDa species. We further postulate that a membrane factor or factors sensitive to sulfhydryl alkylating reagents may be involved in the partial reduction and oxidation of these three redox receptor species. The distribution of these redox receptor species may be related to the cellular or tissue sensitivity to insulin.

Structure of the insulin receptor of rat adipocytes. The three interconvertible redox forms

Structure of the insulin receptor of rat adipocytes. The three interconvertible redox forms

Corrections - Subunit Structure of Insulin REceptor of Rat Adipocytes As Demonstrated by Photoaffinity Labeling

Corrections - Subunit Structure of Insulin REceptor of Rat Adipocytes As Demonstrated by Photoaffinity Labeling

Subunit structure of insulin receptor of rat adipocytes as demonstrated by photoaffinity labeling.

Isolated rat adipocytes were incubated in the dark with either one or two radioiodinated photoreactive insulin derivatives, N epsilon B29-(azidobenzoyl) insulin (B29-MABI) and N alpha B1-(azidobenzoyl) insulin (B1-MABI), and were then exposed to light. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and radioautography of the crude plasma membrane fraction after reduction showed that B29-MABI labeled specifically three proteins of Mr 130 000, 90 000, and 40 000 whereas B1-MABI labeled specifically two proteins of Mr 130 000 and 40 000. B1-MABI also variably labeled some bands of intermediate Mr between 130 000 and 90 000. In contrast, the labeling of the 40-kilodalton protein was not observed in our previous studies in which photolabeling was carried our on isolated plasma membrane preparations [Yip, C. C., Yeung, C. W. T. & Moule, M. L. (1980) Biochemistry 19, 70-76; Yeung, C. W. T., Moule, M. L., & Yip, C. C. (1980) Biochemistry 19, 2196-2203]. Without reduction, an Mr 300 000 band and a larger band which barely entered a 5-15% gradient gel were specifically labeled by both photoreactive insulins. Reduction of these two high molecular weight bands gave rise to the 130-, 90-, and 40-kilodalton bands. The labeling of these proteins was affected neither by the time or temperature of incubation nor by the addition of methylamine, chloroquine, bacitracin, phenylmethanesulfonyl fluoride, p-(chloromercuri) benzenesulfonic acid, Trasylol, N-ethylmaleimide, or benzamidine. The labeling of these proteins by the photoreactive insulin derivatives was inhibited by first incubating the adipocytes with a human autoimmune serum to insulin receptor. We therefore conclude that these proteins are subunits of the insulin receptor in intact adipocytes.

Evidence for insulin-induced internalization and degradation of insulin receptors in rat adipocytes.

We have investigated the theory that the insulin-induced loss of insulin binding from adipocytes is due to internalization of insulin receptors. Cell-surface receptors were assessed by the binding capacity of intact cells at 16 degrees C. Total (i.e., cell-surface plus intracellular) receptors were assessed by solubilizing the cells in 1% Triton X-100 and then measuring binding by the solubilized extract. Intracellular receptors were measured by treating the cells with trypsin before solubilizing them. The trypsin treatment removed greater than 90% of the cell-surface binding, so that any significant binding by soluble extracts of these cells must represent intracellular receptors. Adipocytes were incubated with insulin (100 ng/ml) with or without chloroquine (0.2 mM) for 4 hr. Insulin alone resulted in a 62% loss of cell-surface receptors, but only a 46% loss of total receptors, and a 170% increase in intracellular receptors, suggesting that the lost cell-surface receptors were internalized, where some were degraded. Insulin in the presence of chloroquine resulted in a 34% loss of cell-surface receptors, but no loss of total receptors, and a 300% increase in intracellular receptors. Thus, in the presence of chloroquine receptors were internalized but not degraded. The loss of cell-surface receptors and appearance of intracellular receptors were time and dose dependent and were linearly related. These results demonstrate that insulin causes translocation of insulin receptors from the cell surface to the cell interior, where they can be degraded (or inactivated) by a chloroquine-sensitive process.