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.     

Polylysine increases the number of insulin binding sites in soluble insulin receptor preparations

The effects of cationic polyamino acids on insulin binding to soluble insulin receptor preparations were studied. Incubation of partially or fully purified receptor preparations with polylysine (pLys) increased by several-fold the amount of [125I]insulin that remained associated with the receptor, as determined both by precipitation of receptor-insulin complexes by polyethylene glycol or by separation of the complexes from the free hormone by gel filtration. This elevation in the amount of bound insulin resulted from increased number of insulin binding sites, and could not be attributed to an increased affinity of the receptors to insulin. In fact, pLys reduced 2-3-fold the affinity of insulin binding to its receptor as determined by equilibrium binding studies, and by monitoring the rate of exchange of bound [125I]insulin with unlabeled hormone. pLys induced specific interactions between insulin and its native receptor since other basic compounds such as histone, spermidine, polymixin B, compound 48/80, lysine, and arginine failed to reproduce its effects. pLys did not interact with the free ligand, nor did it promote interactions between insulin and denatured receptor forms. Furthermore, pLys did not induce binding of insulin to other proteins present in the partially purified receptor preparations. The effects of pLys were time and dose-dependent and were proportional to the pLys chain length. The longer the chain, the greater was the effect. Enhanced insulin binding and receptor beta-subunit autophosphorylation (in the presence of insulin) exhibited a similar dependency on the chain length of pLys. pLys effects on insulin binding were associated with formation of large protein aggregates that remained trapped at the top of Sephacryl S-300 columns. These aggregates contained substantial amounts of receptor-insulin complexes. Our results suggest that pLys induces formation of receptor clusters that create de novo insulin binding sites among adjacent receptor tetramers. Alternatively, formation of receptor aggregates might facilitate insulin binding to a soluble receptor subfraction that otherwise fails to bind the hormone.     

Increases of spinal kinin receptor binding sites in two rat models of insulin resistance

An autoradiographic study was conducted to determine whether kinin receptors are altered in the rat spinal cord in two experimental models of chronic hyperglycemia and insulin resistance. Sprague-Dawley rats were given 10% d-glucose in their drinking water alone or with insulin (9 mU/kg/min with osmotic pumps) for 4 weeks. Both groups and control rats were treated either with a normal chow diet or with an alpha-lipoic acid-supplemented diet as antioxidant therapy. After 4 weeks of treatment, glycemia, insulinemia, blood pressure, insulin resistance index, the production of superoxide anion in the aorta and the density of B2 receptor binding sites in the dorsal horn were significantly increased in the two models. These effects were prevented or attenuated by alpha-lipoic acid. In contrast, B2 receptor binding sites of most spinal cord laminae were increased in the glucose group only and were not affected by alpha-lipoic acid. Results show that chronic hyperglycemia associated with insulin resistance increases B1 and B2 receptor binding sites in the rat spinal cord through distinct mechanisms, including the oxidative stress for the B1 receptor.     

Mapping of carbohydrate sites on the human insulin receptor

Prior to investigating the role of individual glycosylation sites in insulin receptor function, we are mapping the sites of glycosylation in the receptor. We report here a generally applicable methodology for the isolation and identification of glycosylation sites in cell surface glycoproteins. Human insulin receptors were labeled with [3H]-sugars using a CHO cell line transfected with the human receptor cDNA. Labelled receptors were mixed with receptors purified from human placental membranes and tryptic peptides prepared. Peptides were fractionated by gel filtration chromatography to limit the number of non-glycopeptides present. Peptides were then separated by reverse phase HPLC and glycopeptides identified by scintillation counting. Using this technique we have shown the insulin receptor to be glycosylated at Asn 397 and Asn 881. This increase the known number of occupied glycosylation sites to five.     

Cell surface receptors for insulin and human growth hormone. Effect of microtubule and microfilament modifiers.

The receptors for the polypeptide hormones, insulin and growth hormone, are located on the cell surface. Since the cytoplasmic microtubules and microfilaments are involved in the mobility and distribution of surface receptors for immunoglobulins and lectins, we investigated the role of these structures in the binding of insulin and human growth hormone to their receptors on cultured human lymphocytes (IM-9). Cells preincubated with microfilament modifiers, cytochalasin A, B, and D (10 mug/ml), had decreased binding of insulin (30%) and human growth hormone (60%) under steady state conditions, which was not reversed by removing the cytochalasins from the medium and was due entirely to a reduced number of receptor sites on the cell surfact. The lost receptors were not detected in the medium, suggesting a redistribution within the cell. The cytochalasins failed to alter the affinity of the hormones for their receptors or the negative cooperativity of the insulin receptor. The anti-microtubule agents (vincristine, vinblastine, colchicine) had no effect on the binding of insulin and growth hormone to their receptors. Deuterium oxide, a stabilizer of microtubules and other proteins, decreased the affinity (40%) of insulin for its receptors under steady state conditions and accelerated moderately the spontaneous dissociation of 125I-insulin from its receptors. Since cytochalasin decreases the number of available insulin and human growth hormone receptor sites, cytochalasin-sensitive microfilamentous structures appear to modulate the exposure of cell surface hormone receptors, while microtubules do not seem to be involved.     

Supramolecular recognition: association of palladium molecular clefts with planar platinum complexes

The crystal structures of two new molecular recognition adducts formed between a dicationic, di-terpyridyl-Pd-Cl molecular cleft and two square planar platinum complexes are reported. In both structures, the planar platinum-containing guests are located within the molecular cleft formed by the two parallel disposed terpyridyl-Pd-Cl⁺ units of the receptor. The crystal structure of the adduct formed between the molecular cleft and a neutral platinum complex has interplanar distances between the host and guest of 3.24 Å, a distance shorter than that usually ascribed to π-stacking interaction (∼3.45 Å). The short distance is likely the result of metal-metal interaction between the host and guest. The second adduct, that between the dicationic molecular receptor and an anionic platinum complex, also bears the guest within the molecular cleft. The interplanar distances between the cationic terpyridyl-Pd-Cl units of the host and the anionic guest (3.21 and 3.29 Å) are also shorter than typical π-stacking distances but no metal-metal interaction is present. Coulombic attraction between the host and guest is believed to be responsible for the short interplanar separation. These data are discussed in relation to analogous systems that associate through π-π and metal-metal interaction.     

Effect of experimentally-induced chronic hyperprolactinemia on insulin binding and antilipolytic response in adipocytes from male rats

Male Wistar rats with chronic hyperprolactinemia induced by grafting an anterior pituitary gland under the right kidney capsule were studied as experimental model. In these animals basal plasma glucose and insulin levels were unaltered. Epididymal adipocytes from hyperprolactinemic rats showed a significant increase in insulin binding at low unlabeled insulin concentrations. This increase in insulin binding can be principally attributed to an increase in the high affinity-low capacity binding sites, as demonstrated when Scatchard analysis was interpreted in terms of two types of insulin receptors. The dissociation constants (KD1 and KD2) were not different between the groups. The apparent insulin receptor affinity was also unchanged. Moreover, a decreased sensitivity to the antilipolytic effect of insulin was also obtained in adipocytes from hyperprolactinemic rats. These findings indicate that chronic hyperprolactinemia is able to increase high affinity insulin receptors in epididymal adipocytes, but tends to diminish the antilipolytic response, suggesting a lack of coupling between insulin binding and its biological activity in male adipose tissue. Several possible mechanisms involved in the process are suggested.     

The natural occurrence of insulin receptors in groups on adipocyte plasma membranes as demonstrated with monomeric ferritin-insulin

This study was designed to document whether the reported distribution of insulin receptors in small groups of receptor sites randomly distributed in the glycocalyx of adipocytes and isolated adipocyte plasma membranes was a naturally occurring phenomena or due to artifacts. Possible artifacts include: (1) oligomeric forms of ferritin in the ferritin-insulin preparation, (2) an uneven distribution of the glycocalyx on the plasma membrane, or (3) ligand-induced aggregation of occupied receptor complexes. Biogel A 1.5m chromatography of the ferritin-insulin conjugate revealed the ferritin in the ferritin-insulin complex to consist of 55% monomers, 15% dimers, and 30% oligomers. The monomer peak was purified (> 95%) for use in these studies. Cationic ferritin, a glycocalyx marker, when incubated with paraformaldehyde-fixed plasma membranes, was found to be uniformly distributed on the surface of the plasma membrane indicative of uniformly distributed glycocalyx. The ability to demonstrate and inhibit ligand-induced aggregation on the isolated plasma membrane was established with a multivalent ligand, ferritin-concanavalin A. More than 66% of the ferritin-concanavalin A receptors were found in large clusters of 5 or more and 34% as singletons or clusters of up to 4 when incubated at 24°C with fresh membranes. Only 38% of the ferritin-concanavalin A receptors were in large clusters; 62% were singletons or clusters up to 4 on membranes prefixed with paraformaldehyde before incubation. The distribution of the monomeric ferritin-insulin was similar on both adipocytes and purified adipocyte plasma membranes and was consistent with earlier reports with ferritin-insulin. The quantitative distribution of the monomeric ferritin-insulin as singletons or in groups of 2–6 was comparable between the intact cells and isolated membranes incubated at 24°C. The binding of 500 μUnits monomeric ferritin-insulin per ml to the isolated plasma membranes was studied under incubation conditions similar to those used with ferritin-concanavalin A. Under all three conditions, fresh membranes at 24°C and 0–4°C and prefixed membranes at 24°C, the pattern of distribution of the monomeric ferritin-insulin as singletons or groups of 2–6 was identical, indicating that the ligand was not causing aggregation into clusters as did the concanavalin A. Thus, the occurrence of insulin receptors in small groups appears to be a natural phenomenon in the plasma membrane structure of adipocytes.     

Absence of down-regulation of the insulin receptor by insulin. A possible mechanism of insulin resistance in the rat.

Insulin resistance occurs in rat adipocytes during pregnancy and lactation despite increased or normal insulin binding respectively; this suggests that a post-receptor defect exists. The possibility has been examined that, although insulin binding occurs normally, internalization of insulin or its receptor may be impaired in these states. Insulin produced a dose-dependent reduction in the number of insulin receptors on adipocytes from virgin rats maintained in culture medium, probably due to internalization of the hormone-receptor complex. In contrast, adipocytes from pregnant and lactating rats did not exhibit this 'down-regulation' phenomenon. Down regulation was, however, apparent in all groups when the experiments were performed in Tris buffer (where receptor recycling is inhibited), suggesting that in pregnant and lactating rats insulin receptors are rapidly recycled back to the plasma membrane, whereas in virgin rats this recycling process is less effective. Internalization of insulin was also determined by using 125I-labelled insulin. Adipocytes from pregnant and lactating rats appeared to internalize similar amounts of insulin to virgin rats. In the presence of the lysosomal inhibitor chloroquine, adipocytes from pregnant rats internalized more insulin than virgin or lactating rats. These results suggest that adipocytes from pregnant and lactating rats internalize insulin and its receptor normally, whereas intracellular processing of the insulin receptor may differ from that in virgin rats. In addition the rate of lysosomal degradation of insulin may be altered in adipocytes from pregnant rats.     

Differences in signaling properties of the cytoplasmic domains of the insulin receptor and insulin-like growth factor receptor in 3T3-L1 adipocytes.

Insulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase.     

Insulin-mimetic effect of trypsin on the insulin receptor tyrosine kinase in intact adipocytes

It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.     

Intracellular location of the Ah receptor

The subcellular distribution of the Ah receptor from the mouse hepatoma line, Hepa-1, was investigated following cytochalasin B treatment and cell enucleation. Probing the resultant cytoplast and nucleoplast fractions with radiolabelled tetrachlorodibenzo-p-dioxin (TCDD) revealed the presence of a specifically bound peak of receptor only in the cytoplast fraction. However, the quantity of receptor recovered in these experiments was only 10–12% of the expected value. We therefore undertook an investigation to determine the fate of the Ah receptor in the presence of cytochalasin B. Incubation of Hepa-1 cells with this compound resulted in a rapid loss or inactivation of cytosolic binding activity with a concomitant decrease in the amount of receptor partitioned into the nucleus at all time periods examined. Control experiments indicated that cytochalasin B did not compete with TCDD for binding to the Ah receptor and furthermore, that its mechanism of action could not be attributed to a non-specific effect on all cytosolic proteins. The results obtained are discussed in relation to the proposed models for induction by the estrogen and glucocorticoid binding receptors.     

Insulin induces chloroquine-sensitive recycling of insulin-like growth factor II receptors but not of glucose transporters in rat adipocytes

Incubation of insulin-treated rat adipocytes with chloroquine, in a time- and concentration-dependent manner, was observed to inhibit the insulin-stimulated increase in insulin-like growth factor II (IGF-II) binding activity, whereas no significant change in IGF-II binding was observed in the absence of insulin. The incremental increase of insulin-stimulated IGF-II binding was inhibited 50% by 0.2 mM chloroquine within 15 min and was nearly completely abolished by 60 min. Interestingly, IGF-II binding was never observed to decrease below the binding value in cells without insulin treatment even when incubation was extended to 180 min. Scatchard analysis of IGF-II binding as well as the specific binding of an anti-IGF-II receptor antibody demonstrated that the loss of IGF-II binding in the insulin-stimulated chloroquine-treated adipocytes was due to a decrease in the number of cell-surface IGF-II receptors, whereas the total number of cellular IGF-II receptors was unaltered. The effect of chloroquine was observed to be reversible, temperature-dependent, and sensitive to the metabolic poison KCN. Furthermore, NH4Cl was also observed to inhibit insulin-stimulated increase in IGF-II binding. In contrast, chloroquine or NH4Cl did not inhibit the basal or insulin-stimulated glucose transport activity. Photoaffinity labeling of the glucose transporter with [3H]cytochalasin B also demonstrated that the basal and insulin-stimulated subcellular distribution of the glucose transporters was unaltered by chloroquine treatment. These results suggest that 1) insulin induces a constitutive, acidotropic agent-sensitive recycling of IGF-II receptor and 2) the glucose transporter and IGF-II receptor do not share the same insulin-regulated intracellular trafficking pathways.     

Effects of vesicle-adipocyte interaction on regulation of insulin receptor recycling

The effect of interacting isolated rat adipocytes with small, unilammelar vesicles on insulin receptor internalization and processing was studied. Treatment of freshly isolated cells with vesicles containing phosphatidylcholine and phosphatidylserine followed by incubation in 35 mM Tris-containing buffer considerably reduced the chloroquine-induced increase in cell-associated 125I-insulin and significantly inhibited the time and insulin dependent loss of surface insulin receptors. The internal receptor pool, as measured by insulin binding to detergent solubilized adipocytes, was relatively smaller in vesicle-treated cells. Concomitant with a slower rate of receptor internalization, insulin-sensitive hexose uptake also demonstrated significantly slower kinetics of decreased response with time. These results support the conclusion that pretreatment of fat cells with phospholipid vesicles inhibits normal insulin receptor cycling.     

An analysis of the relationship between the cellular distribution and the rate of turnover for the separate classes of unoccupied, noncovalently occupied, and covalently occupied insulin receptor

To further investigate insulin's role in regulating the turnover of insulin receptor during down-regulation in 3T3-L1 adipocytes, the relationship between the cellular distribution and turnover of unoccupied, noncovalently occupied, and covalently occupied receptor was examined. At steady-state 12% of the unoccupied receptors and 46% of covalently occupied receptors are intracellular. The apparent first-order rate constant (Kapp) for turnover of the total pool of covalently occupied receptors (0.16 h-1) is 3.8-fold higher than that for unoccupied receptors (0.042 h-1). When unlabeled insulin is added, identical values for both Kapp (0.10 h-1) and distribution (26% internal) are measured for noncovalently and covalently occupied receptors. The rate constant (Kdeg), describing the relative sensitivity of internalized receptor to degradation, is identical (0.36-0.41 h-1) for unoccupied, noncovalently occupied, and permanently occupied pools of internal receptor. Mechanisms for down-regulation postulating: (a) an occupancy-dependent alteration in the conformation of internal receptor increasing receptor sensitivity to internal proteases, (b) a preferential sorting of internal occupied receptor to degradative pathways, or (c) induction of intracellular proteases by insulin, would all reflect a substantial change in Kdeg for occupied receptor and thus are unlikely mechanisms by which insulin increases the rate of receptor turnover. The turnover of insulin receptor in 3T3-L1 adipocytes is regulated primarily by its intracellular concentration and not by the state of occupancy of internalized receptor.     

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.     

Insulin and angiotensin II induce the translocation of scavenger receptor class B, type I from intracellular sites to the plasma membrane of adipocytes

Scavenger receptor class B, type I (SR-BI) mediates the selective uptake of lipids from high density lipoproteins and is expressed in several types of tissues. However, to date little is known about its role in adipocytes. In this study, we investigated the cellular distribution of SR-BI in 3T3-L1 adipocytes and its regulation by hormones known to increase lipid storage such as angiotensin II (Ang II) and insulin. SR-BI was mainly distributed in the cytoplasm as determined by laser-scanning confocal analysis of the immunofluorescence labeling of SR-BI or the study of an enhanced green fluorescent protein-tagged SR-BI fusion protein. Exposure of cells to either insulin or Ang II (1-2 h) induced the mobilization of SR-BI from intracellular pools to the plasma membrane. This was further confirmed by Western blotting on purified plasma membrane and by fluorescence-activated cell sorter analysis of the SR-BI receptor. Similar results were also observed in primary adipocytes. We also demonstrated that, in the presence of either insulin or Ang II, SR-BI translocation to the cell membrane is functional, because insulin and Ang II induced a significant increase in the high density lipoprotein-delivered 22-(N-7-nitrobenz-2-oxa-1,3-diazo-4-yl)-amino-23,24-bisnor-5-cholen-3-ol uptake and in total cholesterol content. These data demonstrate that SR-BI can be acutely mobilized from intracellular stores to the cell surface by insulin or Ang II, two hormones that exert lipogenic effects in adipocytes. This suggests that SR-BI might participate in the storage of lipids in the adipose tissue.     

Effect of acute zinc deficiency on insulin receptor binding in rat adipocytes

Considerable data have been reported on the relationship between insulin resistance and zinc deficiency. In this study, insulin receptor binding was measured in isolated rat adipocytes. Two assays were carried out at 37°C (binding and internalization) and 16°C (binding) using¹²⁵I insulin 0.05–20 nM. A decreased insulin receptor binding was observed in zinc-deficient rat adipocytes, but we could not make any distinction between the specific zinc depletion effects and the effects of the caloric restriction induced by zinc deficiency.     

Adenosine receptor down-regulation and insulin resistance following prolonged incubation of adipocytes with an A1 adenosine receptor agonist

Adenosine, via interaction with A1 adenosine receptors, increases insulin sensitivity and inhibits lipolysis in adipocytes. To investigate regulation of this system, adipocytes were incubated for up to 72 h with the nonmetabolizable adenosine receptor agonist, N6-phenylisopropyl adenosine (PIA). Adenosine receptors were measured by the binding of 125I-hydroxyphenylisopropyl adenosine to membranes. PIA down-regulated adenosine receptors, decreasing the number of binding sites with no change in affinity. Adipocytes were incubated for 48 h without or with 100 nM PIA to down-regulate the A1 receptors by approximately 60%. The cells were washed, and lipolysis and glucose transport were assessed. The ability of PIA to inhibit lipolysis was markedly attenuated in the down-regulated cells. Furthermore, the EC50 of insulin was increased approximately 3-fold in the PIA-treated cells. 125I-Insulin binding to the PIA-treated cells was unchanged, demonstrating that the decreased insulin sensitivity is not due to decreased insulin receptor binding. Pertussis toxin catalyzed ADP-ribosylation of a 41-kDa protein thought to be the alpha-subunit of Gi. This 41-kDa protein was decreased in membranes from cells treated with PIA, with a maximal 50% loss. This suggests that Gi is down-regulated and that loss of both the A1 adenosine receptor and Gi are involved in the metabolic changes observed after PIA treatment.     

Mutagenesis of lysine 460 in the human insulin receptor. Effects upon receptor recycling and cooperative interactions among binding sites

Mutations in the insulin receptor gene can cause insulin resistance. Previously, we have identified a mutation substituting glutamic acid for lysine at position 460 in the alpha-subunit of the insulin receptor in a patient with a genetic form of insulin resistance. In the present work, we have investigated the effect upon receptor function of amino acid substitutions at position 460. Decreasing the pH from 8.0 to 5.5 caused a progressive acceleration of the dissociation of 125I-insulin from the wild-type insulin receptor. Substitution of acidic amino acids (Glu or Asp) for Lys460 decreased the ability of acid pH to accelerate dissociation of 125I-insulin. In contrast, substitution of Arg or neutral amino acids (Val, Met, Thr, or Gln) had no effect upon the sensitivity to acid pH. Correlated with decreased sensitivity to acid pH, substitution of Glu or Asp at position 460 retarded the dissociation of 125I-insulin from intracellular receptors subsequent to receptor-mediated endocytosis. Furthermore, retardation of dissociation of 125I-insulin from the internalized receptor was associated with a decreased half-life of the receptor. In summary, the Glu460 mutation appears to cause insulin resistance by accelerating receptor degradation and, thereby, decreasing the number of insulin receptors on the cell surface. Additional studies suggested that Lys460 may provide the amino groups whereby disuccinimidyl suberate cross-links the two alpha-subunits to each other. Consistent with the hypothesis that Lys460 is located at the interface between adjacent alpha-subunits, substitutions at position 460 impair cooperative interactions among insulin binding sites. The Glu460 mutation decreases positively cooperative binding interactions; the Arg460 mutation impairs negative cooperativity. Mutations at position 460 in the alpha-subunit did not decrease the ability of insulin to stimulate receptor tyrosine kinase.