Insulin Receptor Beta-subunit Research Articles

A role for tyrosine phosphorylation in both activation and inhibition of the insulin receptor tyrosine kinase in vivo.

Upon insulin binding, a conformational change in the insulin receptor (IR) leads to IR beta-subunit autophosphorylation, an increase in IR beta-subunit exogenous tyrosine kinase activity, and the rapid endocytosis of the ligand-receptor complex into endosomes. Previous work has shown that upon internalization, rat hepatic endosomal IRs manifest increased autophosphorylating and exogenous tyrosine kinase activity compared to IRs located at the plasma membrane. As this period of enhanced activity is associated with reduced endosomal IR beta-subunit phosphotyrosine content, it has been proposed that partial dephosphorylation of the internalized IR beta-subunit by an endosomally located phosphotyrosine phosphatase(s) [PTPase(s)] mediates this effect. To test whether endosomal PTPase activity was required for internalization-dependent augmentation of IR tyrosine kinase activity, the present study used the peroxovanadium PTPase inhibitor, bisperoxo(1,10-phenanthroline)oxovanadate anion [bpV(phen)], to block IR dephosphorylation within this subcellular compartment. Rats were pretreated with bpV(phen) before receiving insulin (1.5 micrograms/100 g BW). bpV(phen) inhibited the dephosphorylation of 32P-labeled hepatic endosomal IR by approximately 97% at 15 min post-bpV(phen) injection and prevented a decrease in IR beta-subunit phosphotyrosine content after IR internalization. Fifteen-minute bpV(phen) pretreatment produced a significant reduction (75%; P < 0.001) in maximal insulin-stimulated endosomal IR exogenous kinase activity and decreased IR autophosphorylating activity by 4.3-fold in this subcellular fraction. In conclusion, these findings suggest that an hepatic endosomal PTPase(s) regulates internalization-dependent increases in IR exogenous tyrosine kinase activity.

Insulin receptor expression in bone.

Insulin is known to be an important osteotropic hormone. To date, no study has specifically addressed the possibility that insulin receptor expression may be regulated by differentiation in bone. We report a heterogeneous distribution of insulin receptor (IR) within neonatal rat calvaria using a specific monoclonal antibody to the beta-subunit of the rat insulin receptor (CT-1). Specific binding of CT-1 to mature osteoblasts was demonstrated, with little binding over periosteal tissues or osteocytes. Using enzymatically derived subpopulations of calvarial cells, we showed a correlation between alkaline phosphatase activity and insulin-stimulated 2-deoxyglucose (2-DOG) uptake and increased 125I-insulin binding. Since primary calvarial cultures contain many cell types, we compared 125I-insulin binding, insulin-stimulated 2-DOG uptake, and Northern blot analysis of IR mRNA in the clonal preosteoblast-like cell line UMR 201-10B and the mature osteoblast cell line UMR 106-01. It is shown that UMR 106-01 cells possess higher levels of IR mRNA, insulin binding, and insulin-stimulated glucose uptake, and that insulin up-regulated expression of mRNA of the glucose transporter GLUT1 by 3-fold. In contrast, insulin binding was negligible in UMR 201-10B cells, which expressed much lower levels of IR mRNA. UMR 201-10B cells did not possess an insulin-sensitive glucose uptake system, although they express GLUT1 mRNA. These data are consistent with the hypothesis that, as in muscle and fat, insulin receptor expression correlates with the stage of osteoblast differentiation in vivo and in vitro.

An Insulin Receptor Mutant (Asp707→ Ala), Involved in Leprechaunism, Is Processed and Transported to the Cell Surface but Unable to Bind Insulin

We have identified a homozygous mutation near the carboxyl terminus of the insulin receptor (IR) alpha subunit from a leprechaun patient, changing Asp707 into Ala. Fibroblasts from this patient had no high affinity insulin binding sites. To examine the effect of the mutation on IR properties, the mutant IR was stably expressed in Chinese hamster ovary cells. Western blot analysis and metabolic labeling showed a normal processing of the mutant receptor to alpha and beta subunits. No increase in high affinity insulin binding sites was observed on Chinese hamster ovary cells expressing the mutant receptor, and also, affinity cross-linking of 125I-labeled insulin by disuccinimidyl suberate to these cells failed to label the mutant alpha subunit. Biotinylation of cell surface proteins by biotin succinimidyl ester resulted in efficient biotinylation of the mutant IR alpha and beta subunits, showing its presence on the cell surface. On solubilization of the mutant insulin receptor in Triton X-100-containing buffers, 125I-insulin was efficiently cross-linked to the receptor alpha subunit by disuccinimidyl suberate. These studies demonstrate that Ala707 IR is normally processed and transported to the cell surface and that the mutation distorts the insulin binding site. Detergent restores this site. This is an example of a naturally occurring mutation in the insulin receptor that affects insulin binding without affecting receptor transport and processing. This mutation points to a major contribution of the alpha subunit carboxyl terminus to insulin binding.

Bradykinin enhances GLUT4 translocation through the increase of insulin receptor tyrosine kinase in primary adipocytes: evidence that bradykinin stimulates the insulin signalling pathway

It has been suggested that bradykinin stimulates glucose uptake in experiments in vivo and in cultured cells. However, its mechanism has not yet been fully elucidated. In this study, the effects of bradykinin on the insulin signalling pathway were evaluated in isolated dog adipocytes. The bradykinin receptor binding study revealed that dog adipocytes possessed significant numbers of bradykinin receptors (Kd = 83 pmol/l, binding sites = 1.7 x 10(4) site/ cell). Reverse transcription-polymerase chain reaction amplification showed the mRNA specific for bradykinin B2 receptor in the adipocytes. Bradykinin alone did not increase 2-deoxyglucose uptake in adipocytes; however, in the presence of insulin (10(-7) mol/l) it significantly increased 2-deoxyglucose uptake in a dose-dependent manner. Bradykinin also enhanced insulin stimulated GLUT4 translocation from the intracellular fraction to the cell membrane, and insulin induced phosphorylation of the insulin receptor beta subunit and insulin receptor substrate-1 (IRS-1) without affecting the binding affinities or numbers of cell surface insulin receptors in dog adipocytes. The time-course of insulin stimulated phosphorylation of the insulin receptor beta subunit revealed that phosphorylation reached significantly higher levels at 10 min, and stayed at the higher levels until 120 min in the presence of bradykinin, suggesting that bradykinin delayed the dephosphorylation of the insulin receptor. It is concluded that bradykinin could potentiate insulin induced glucose uptake through GLUT4 translocation. This effect could be explained by the potency of bradykinin to upregulate the insulin receptor tyrosine kinase activity which stimulates phosphorylation of IRS-1, followed by GLUT4 translocation.

Interaction between the Grb10 SH2 Domain and the Insulin Receptor Carboxyl Terminus

Grb10 is a member of a recently identified family of adapter proteins that are thought to play a role in receptor tyrosine kinase-mediated signal transduction. We identified and isolated the Grb10 SH2 domain based on its interaction with the intracellular domain of the insulin receptor beta-subunit using the yeast two-hybrid system. The interaction was specific for the insulin receptor and the insulin-like growth factor-1 receptor, and it required a catalytically active receptor kinase domain and an intact Grb10 SH2 domain. Glutathione S-transferase fusion proteins containing the Grb10 SH2 domain associated in an insulin-dependent manner with insulin receptors from cell lysates and with purified insulin receptors. Co-precipitation experiments revealed the association of cellular Grb10 with hormone-stimulated insulin receptors in cell extracts. The Grb10 SH2 domain did not bind to an insulin receptor lacking 43 amino acids at the carboxyl terminus, and it exhibited highest affinity for a phosphopeptide containing Tyr(P)-1322. Unlike p85 and Syp, which also bind to Tyr(P)-1322, Grb10 was not found to associate with insulin receptor substrate-1. These results suggest that Grb10 is a novel insulin receptor interactive protein and provide direct evidence for an insulin receptor substrate-1-independent function of the insulin receptor carboxyl terminus in protein binding.

Interaction between the Insulin Receptor and Its Downstream Effectors: USE OF INDIVIDUALLY EXPRESSED RECEPTOR DOMAINS FOR STRUCTURE/FUNCTION ANALYSIS

A structural analysis has been carried out to determine which part of the intracellular domain of the insulin receptor (IR) beta subunit is involved in direct interaction with the receptor substrates IRS-1 and Shc. Toward this end, the juxtamembrane (JM) domain (amino acids 943-984) and the carboxyl-terminal (CT) region (amino acids 1245-1 331) of IR were expressed in bacteria as (His)6-fusion peptides, and their interaction with IRS-1 and Shc was studied. We could demonstrate that the CT region of IR was sufficient to bind Shc, although significant, but much lower binding of Shc to the JM region could be detected as well. Furthermore, in vitro Tyr phosphorylation of the CT region potentiated its interactions with Shc 2-fold. In contrast, the JM region, but not the CT domain of the IR, was sufficient to mediate interactions between the IR and IRS-1. These interactions did not involve the pleckstrin homology (PH) region of IRS-1, since an IRS-1 mutant, in which four "blocks" of the PH domain (Pro5-Pro65) were deleted, interacted with the JM region of IR with the same efficiency as native IRS-1. These results suggest that the IR interacts with its downstream effectors through distinct receptor regions, and that autophosphorylation of Tyr residues located at the CT domain of the IR can modulate these interactions.

A stable peroxovanadium compound with insulin-like action in human fat cells

Aqueous solutions of peroxovanadium (pV) compounds are potent insulin-mimics in various types of cell. Since chemical instability is a problem with these agents, we studied the insulin-like action in human fat cells of a stable pV complex, bpV(pic). It enhanced 14C-U-glucose uptake in a dose-dependent manner by approximately twofold which was slightly less than the effect of insulin (approximately threefold). The pV complex did not alter cell-surface insulin binding and submaximal concentrations did not influence cellular sensitivity to insulin action on glucose uptake. The bpV(pic) inhibited the lipolytic effect of isoprenaline to the same extent as insulin; however, when the cGMP-inhibitable low-K(m) phosphodiesterase (cGI-PDE) was blocked with the specific inhibitor OPC 3911, the antilipolytic effect of insulin, but not that of bpV(pic), was completely prevented. Moreover, when lipolysis was stimulated by the non-hydrolysable cAMP analogue N6-monobutyryl cAMP, bpV(pic), in contrast to insulin, maintained an antilipolytic effect. These findings indicate that bpV(pic) exerts its antilipolytic effect not only through cGI-PDE activation, similar to the effect of insulin, but also by means of other mechanisms. The tyrosine kinase activity of insulin receptors from human placenta was not altered by the pV compound itself, whereas bpV(pic) clearly enhanced insulin-stimulated activity. In contrast, in situ tyrosine phosphorylation of the insulin receptor beta-subunit as well as that of several other proteins was clearly increased in cells which were treated with bpV(pic), whereas vanadate only amplified insulin-stimulated tyrosine phosphorylation. In conclusion, bpV(pic) exerts powerful insulin-like effects in human fat cells and may be a new and potentially useful agent in the management of insulin-resistant states.

The role of receptor kinase activity and the NPEY960 motif in insulin-accelerated receptor-mediated insulin internalization.

This study used biochemical and quantitative ultrastructural approaches to examine the roles that insulin receptor beta subunit kinase activity, the NPEY motif in the juxtamembrane region, and tyrosine phosphorylation within that domain plays in insulin-accelerated receptor-mediated insulin internalization in CHO cells. Internalization of insulin in cells that expressed kinase-deficient receptors (CHOA1018) or receptors lacking the NPEY Ala954-Asp965 domain (CHO delta 960) was reduced by 80% compared to cells expressing wild-type human insulin receptors (CHOHIRc). Ultrastructural analysis revealed that the decreased internalization in CHOA1018 cells was due to the reduced ability of the kinase deficient receptor to migrate from the microvilli of cultured cells and aggregate on the cell surface. Deletion of the NPEY motif in the juxtamembrane region of the beta subunit severely reduced receptor migration, interfered with the normal aggregation of receptors on the cell surface, and virtually eliminated accumulation of the occupied receptors in the coated invaginations. Replacement of Tyr960 in the NPEY domain/with phenylalanine (CHOF960) had no significant effect on insulin internalization, receptor mobility, aggregation or accumulation in coated invaginations. In contrast, replacement of Tyr960 with alanine (CHOA960) decreased insulin internalization, slowed migration, receptor aggregation and accumulation in coated invaginations. These studies document that kinase activity is required, but not sufficient, for receptor movement from the microvilli and aggregation of occupied receptors on the non-villous surface. An intact NPEY motif or surrounding amino acids, but not the phosphorylation of Tyr960 plays a role in receptor mobility and aggregation and is essential for the accumulation of insulin receptors in coated invaginations.

Insulin Resistance Is Mediated by a Proteolytic Fragment of the Insulin Receptor

Insulin resistance is a common clinical feature of obesity and non-insulin-dependent diabetes mellitus, and is characterized by elevated serum levels of glucose, insulin, and lipids. The mechanism by which insulin resistance is acquired is unknown. We have previously demonstrated that upon chronic treatment of fibroblasts with insulin, conditions that mimic the hyperinsulinemia associated with insulin resistance, the membrane-associated insulin receptor beta subunit is proteolytically cleaved, resulting in the generation of a cytosolic fragment of the beta subunit, beta', and that the generation of beta' is inhibited by the thiol protease inhibitor E64 (Knutson, V. P. (1991) J. Biol. Chem. 266, 15656-15662). In this report, we demonstrate that in 3T3-L1 adipocytes: 1) cytosolic beta' is generated by chronic insulin administration to the cells, and that E64 inhibits the production of beta'; 2) chronic administration of insulin to the adipocytes leads to an insulin-resistant state, as measured by lipogenesis and glycogen synthesis, and E64 totally prevents the generation of this insulin-induced cellular insulin resistance; 3) E64 has no effect on the insulin-induced down-regulation of insulin receptor substrate-1, and therefore insulin resistance is not mediated by the down-regulation of insulin receptor substrate-1; 4) under in vitro conditions, partially purified beta' stoichiometrically inhibits the insulin-induced autophosphorylation of the insulin receptor beta subunit; and 5) administration of E64 to obese Zucker fatty rats improves the insulin resistance of the rats compared to saline-treated animals. These data indicate that beta' is a mediator of insulin resistance, and the mechanism of action of beta' is the inhibition of the insulin-induced autophosphorylation of the beta subunit of the insulin receptor.

Ethanol inhibits insulin receptor substrate-1 tyrosine phosphorylation and insulin-stimulated neuronal thread protein gene expression.

Neuronal thread proteins (NTPs) are molecules that accumulate in the brains of patients with Alzheimer's disease, and may play a key role in both normal and neurodegenerative neuritic sprouting. In this investigation we determined whether NTP expression is up-regulated by insulin, an important neurotrophic factor that stimulates differentiation-associated neurite outgrowth, and studied the effects of ethanol, a known inhibitor of growth factor receptor tyrosine phosphorylation, on NTP expression and insulin-mediated signal transduction cascade in neuronal [primitive neuroectodermal tumour cell line 2; (PNET2)] cells. PNET2 cells were treated with 50 m-units/ml insulin in the presence or absence of 100 mM ethanol for 0.2-96 h, and cell proliferation and expression of NTP molecules were investigated by metabolic labelling, immunoprecipitation and immunohistochemical staining. Insulin stimulation resulted in an immediate increase in the levels of three (38, 18 and 15 kDa) of five NTP species (the others were of 26 and 21 kDa), followed by a decline in expression within 120 min; however, studies performed up to 96 h of culture demonstrated up-regulation by insulin of all five NTP species. Ethanol either abolished or severely muted the short- and long-term insulin-mediated upregulation of NTP expression, and substantially reduced insulin-mediated neuronal differentiation. The effects of ethanol on NTP gene expression were associated with impaired insulin-mediated tyrosine phosphorylation of both the insulin receptor beta subunit and the insulin receptor substrate-1 (IRS-1), resulting in decreased association of phosphatidylinositol 3-kinase with IRS-1. The findings suggest that ethanol may inhibit NTP expression associated with central nervous system neuronal differentiation by uncoupling the IRS-1-mediated insulin signal transduction pathway.

Biological activity of immunoreactive insulin-like activity extracted from rat submandibular gland

Earlier studies indicate the presence of an insulin-like immunoreactivity (ILI) in rat submandibular salivary glands (SSG). Previous observations also showed that streptozotocin (STZ)-induced diabetes was accompanied by an increase in SSG ILI concentrations. In the present work we studied the effect of SSG ILI from normal and STZ diabetic rats (ILI-N and ILI-D, respectively) on insulin receptor binding and function in LMH cell line. ILI-N and ILI-D inhibited 125I-insulin binding to intact cells and wheat germ agglutinin (WGA)-purified insulin receptors with a high affinity. Furthermore, ILI-N and ILI-D activated, although weakly, the beta-subunit autophosphorylation of solubilized and WGA-purified insulin receptors. An ATP hydrolytic activity was present in ILI-N and, to a greater extent, in ILI-D extracts, which can at least in part explain their low potency for activating autophosphorylation and kinase activity of insulin receptors in vitro. However, after ILI treatment of intact cells and immunoprecipitation of insulin receptors, ILI induced a dose-dependent tyrosine phosphorylation of the insulin receptor beta-subunit. Finally, ILI-N and ILI-D stimulated amino acid uptake and lipogenesis in LMH cells. These findings suggest that SSG ILI is biologically active and can participate in metabolic regulations.

Insulin-induced differentiation and modulation of neuronal thread protein expression in primitive neuroectodermal tumor cells is linked to phosphorylation of insulin receptor substrate-1.

Neuronal thread proteins (NTPs) are a family of developmentally regulated molecules expressed in central nervous system (CNS) neurons and primitive neuroectodermal tumor (PNET) cell lines. NTP gene expression is modulated with DNA synthesis, neuritic sprouting, and neuronal differentiation. The present study explores the mechanism of insulin modulation of NTP gene expression during neuronal differentiation using PNET cell lines of CNS origin. PNET2 cells underwent neuronal differentiation with neurite outgrowth coupled with transient up-regulation of several species of NTP. In contrast, PNET1 cells failed to differentiate in response to insulin stimulation, although insulin receptors were more abundant than in PNET2 cells. Analysis of the insulin-mediated signal transduction pathway demonstrated that the lack of insulin responsiveness in PNET1 cells was primarily caused by impaired insulin-mediated tyrosyl phosphorylation of the insulin receptor substrate-1 (IRS-1). Correspondingly, the association between phosphatidyl-inositol 3 (PI3) kinase and phosphorylated IRS-1 was reduced in PNET1 compared with PNET2 cells. In contrast, the levels of IRS-1 protein were similar in PNET1 and PNET2 cells, and expression of the insulin receptor beta subunit (Ir beta) and insulin-mediated tyrosyl phosphorylation of the Ir beta were greater in PNET1 than PNET2 cells. The findings suggest that insulin effected neuronal differentiation and modulation of NTP gene expression in PNET cells utilizes a signal transduction cascade that requires tyrosyl phosphorylation of IRS-1.

Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.

Mechanisms of Enhanced Transmembrane Signaling by an Insulin Receptor Lacking a Cytoplasmic β-Subunit Domain

We have recently characterized a mutant insulin receptor (HIR delta 978) in which the insulin receptor beta-subunit was truncated at amino acid residue 978. Compared with parental Rat1 cells, the cells expressing the truncated receptor exhibited enhanced sensitivity to insulin's biologic actions. All of these effects are now extended to transcriptional events, since we now show enhanced sensitivity to insulin stimulation of c-fos mRNA expression. These effects were insulin-specific, since insulin-like growth factor-1 stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, and thymidine incorporation into DNA were normal. In addition, the truncated receptor exhibited enhanced sensitivity only in vivo, but not in vitro, since the kinase activity of wheat germ agglutinin-purified receptor preparations was comparable between HIR delta 978 and parental Rat1 insulin receptors. Parental rat endogenous insulin-like growth factor-1 receptors and transfected human insulin receptors form hybrid receptors as well as homologous tetrameric receptors. The normal heterotetrameric receptors possess kinase activity in vivo leading to tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and its association with the p85 regulatory subunit of phosphatidyl inositol 3-kinase. Interestingly, preincubation with human-specific anti-insulin receptor antibody abolished the increased insulin sensitivity in glucose incorporation into glycogen in HIR delta 978 cells. Furthermore, microinjection of anti-IRS-1 antibody into HIR delta 978 cells inhibited insulin stimulation of DNA synthesis. In summary: 1) truncated receptors on the cell surface confer enhanced insulin sensitivity in vivo; 2) the normal heterotetrameric receptors are functionally active and couple to IRS-1 efficiently; and 3) IRS-1 is an important molecule transmitting insulin's biological signals in HIR delta 978 cells.

Two mutations in the insulin receptor gene of a patient with leprechaunism: application to prenatal diagnosis.

Leprechaunism is an autosomal recessive disorder caused by mutations in the insulin receptor gene and characterized by intrauterine and postnatal growth restriction, abnormal glucose homeostasis, and severe insulin-resistance. Here we report the biochemical and molecular characterization of a male patient, NZ, who died at 2 yr of age with this syndrome. 125I-Insulin binding to fibroblasts from the proband, his mother, father, and unaffected sister was reduced to 8, 53, 38, and 35% of controls, respectively. Analysis of the insulin receptor gene by polymerase chain reaction amplification using primers flanking each of the 22 exons and direct DNA sequencing identified 2 different mutations in the proband. The paternal mutation was an in-frame deletion of base pairs 1159-1161 in exon 3, which resulted in the loss of the codon for Asn-281. The maternal mutation was a G-->A transition in the first nucleotide of the splice-donor junction in intron 13. The maternal mutation activated a cryptic splice site 27 base pairs upstream in exon 13 and caused an in-frame deletion of amino acids 859-867 of the extracellular domain of the insulin receptor beta subunit. Identification of both mutations enabled prenatal diagnosis in 2 subsequent pregnancies. In the first pregnancy, DNA from cells cultured from chorionic villus (CV) biopsies carried both mutations in the insulin receptor gene. In the second pregnancy, DNA from the CV biopsy cells was negative for both mutations, indicating that the fetus was unaffected by leprechaunism. Insulin binding could not be used in prenatal diagnosis because cells cultured from some control CV biopsies failed to bind insulin. These data indicate that patient NZ with leprechaunism was a compound heterozygote for 2 novel mutations in the insulin receptor gene and that direct DNA sequencing enables prenatal diagnosis for this lethal disorder.

Early alteration of insulin stimulation of PI 3-kinase in muscle and adipocyte from gold thioglucose obese mice.

The activation of phosphatidylinositol 3-kinase (PIK) was studied in vivo and in vitro in soleus muscle and adipocytes from young (8 wk) and old (30 wk) gold thioglucose obese mice. Insulin resistance assessed from muscle glucose transport and glycogen synthesis was present both in young and old obese mice. Adipocyte lipid synthesis and muscle glycolysis or glucose oxidation are not defective in young obese mice but become resistant later on. After incubation with 50 nM insulin, muscle antiphosphotyrosine-immunoprecipitable PIK activity was stimulated 5- to 10-fold in both young and old animals. This response was impaired by 56 and 75% in muscles from young and old obese mice, respectively. Insulin stimulation of receptor tyrosine kinase activity was only slightly decreased in muscle of young obese mice, whereas insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation was blunted. The altered PIK stimulation in muscle, which is present both in vivo and in vitro, is thus characterized by a reduced association of PIK activity with IRS-1 and appears to result from a diminished IRS-1 tyrosine phosphorylation. In adipocytes isolated from lean mice, antiphosphotyrosine-immunoprecipitable PIK increased 25-fold within 10 min of incubation with insulin. This stimulation was markedly altered both in young and old obese mice, whereas lipogenesis was insulin resistant only in old obese animals. In adipocytes from young obese mice, insulin's stimulatory effect on the phosphorylation of insulin receptor beta-subunit, pp60, and an exogenous substrate was normal, whereas IRS-1 tyrosine phosphorylation was markedly depressed.(ABSTRACT TRUNCATED AT 250 WORDS)

C-terminus or juxtamembrane deletions in the insulin receptor do not affect the glucose-dependent inhibition of the tyrosine kinase activity.

We have previously shown, in rat-1 fibroblasts which stably overexpress high levels of human insulin receptor (HIR), that high glucose levels induce an inhibition of insulin receptor tyrosine kinase (IRK) activity [Berti, L., Mosthaf, L., Kellerer, M., Tippmer, S., Mushack, J., Seffer, E., Seedorf, K., Häring, H. (1994) J. Biol. Chem. 269, 3381-3386]. This effect appears to be mediated through activation of protein kinase C and phosphorylation of the receptor beta-subunit on threonine or serine residues. The aim of the present study was to determine whether the juxtamembrane region or the C-terminus tail of the receptor are involved in the IRK modulation by glucose. In these domains increased serine and threonine phosphorylation was observed after phorbol ester or insulin stimulation of cells, and a regulatory function for IRK activity seems conceivable. We used an antibody directed against one potential regulatory site in the C-terminus tail, i.e. PSer1315, to study the effect of glucose. An increased signal was detected in HIR from rat-1 fibroblasts treated with phorbol 12-myristate 13-acetate or glucose (25 mM). To investigate whether this site in the C-terminus is essential for glucose-dependent IRK inhibition, rat-1 fibroblasts stably overexpressing a C-terminus-truncated human insulin receptor lacking 43 amino acids (HIR delta CT) were studied in parallel with cells expressing the wild-type receptor. As described earlier, HIR delta CT has lost the ability to stimulate glucose uptake. Glucose (25 mM) inhibited the insulin effect on the autophosphorylation of both receptors to a similar extent. Thus, glucose (25 mM) stimulates phosphorylation of Ser1315, however, this appears not to mediate the inhibitory effect on IRK. To test whether serine residues 955/956 and 962/964 in the juxtamembrane region of the insulin receptor are involved in the inhibitory effect of glucose, 293 cells transiently transfected either with wild-type HIR or HIR with a juxtamembrane deletion spanning amino acids 954-965 [des-(954-965)-HIR] were studied in parallel. As described earlier, the des-(954-965)-HIR has lost the ability to stimulate PI-3 kinase. However, 25 mM glucose equally inhibited the insulin effect on tyrosine phosphorylation of the receptor. Together, the data suggest that the regulatory serine or threonine phosphorylation site(s) involved in the inhibitory effect of hyperglycemia are neither located in the C-terminus nor in the juxtamembrane region of the insulin receptor beta subunit.

An extracellular domain of the β subunit is essential for processing, transport and kinase activity of insulin receptor

The extracellular portion of the insulin receptor (IR) beta-subunit has four cysteine and four asparagine residues which are potentially involved in disulphide bond formation between the alpha- and beta-subunits and N-linked glycosylation respectively. However, the function of this portion is not fully understood. In order to investigate the role of the extracellular domain of beta-subunit, we created a deletion mutant of IR cDNA which lacked 47 amino acid residues encoded by 141 bp corresponding to exon 13 of the IR gene. Insulin binding and surface labelling of COS 7 cells transiently expressing the mutant insulin receptors (IR delta Ex13) showed that the mutated receptors were not expressed on the cell surface. However, immunoblot analysis showed that uncleaved form (190 kDa) of the mutant receptors were intracellularly expressed. Deglycosylation with endoglycosidase H showed that the mutant receptors had mainly high-mannose oligosaccharide chains. The mutant IRs bound with high affinity to lentil lectin but with low affinity to wheat germ agglutinin. Therefore, it is suggested that misfolding of the mutant receptors inhibits transport to the Golgi apparatus where processing of oligosaccharide chains, as well as proteolytic cleavage into subunits, takes place. The binding affinity of the mutant receptors for insulin was 50% of normal. Furthermore, insulin-stimulated autophosphorylation of IR delta Ex13 was markedly impaired. These data provide the evidence for a critical role of the extracellular domain of IR beta-subunit for processing and transport as well as the intramolecular signal transduction to activate IR tyrosine kinase.

In vivo effects of vanadate on hepatic glycogen metabolizing and lipogenic enzymes in insulin-dependent and insulin-resistant diabetic animals.

The insulin-mimetic action of vanadate is well established but the exact mechanism by which it exerts this effect is still not clearly understood. The role of insulin in the regulation of hepatic glycogen metabolizing and lipogenic enzymes is well known. In our study, we have, therefore, examined the effects of vanadate on these hepatic enzymes using four different models of diabetic and insulin-resistant animals. Vanadate normalized the blood glucose levels in all animal models. In streptozotocin-induced diabetic rats, the amount of liver glycogen and the activities of the active-form of glycogen synthase, both active and inactive-forms of phosphorylase, and lipogenic enzymes like glucose 6-phosphate dehydrogenase and malic enzyme were decreased and vanadate treatment normalized all of these to near normal levels. The other three animal models (db/db mouse, sucrose-fed rats and fa/fa obese Zucker rats) were characterized by hyperinsulinemia, hypertriglyceridemia, increases in activities of lipogenic enzymes, and marginal changes in glycogen metabolizing enzymes. Vanadate treatment brought all of these values towards normal levels. It should be noted that vanadate shows differential effects in the modulation of lipogenic enzymes activities in type I and type II diabetic animals. It increases the activities of lipogenic enzymes in streptozotocin-induced diabetic animals and prevents the evaluation of activities of these enzymes in hyperinsulinemic animals. The insulin-stimulated phosphorylation of insulin receptor beta subunit and its tyrosine kinase activity was increased in streptozotocin-induced diabetic rats after treatment with vanadate. Our results support the view that insulin receptor is one of the sites involved in the insulin-mimetic actions of vanadate.

Unique and selective mitogenic effects of vanadate on SV40-transformed cells.

Vanadate and insulin both function as unique complete mitogens for SV40-transformed 3T3T cells, designated CSV3-1, but not for nontransformed 3T3T cells. The mitogenic effects induced by vanadate and insulin in CSV3-1 cells are mediated by different signaling mechanisms. For example, vanadate does not stimulate the tyrosine phosphorylation of the insulin receptor beta-subunit nor the 170 kDa insulin receptor substrate-1. Instead, vanadate induces a marked increase in tyrosine phosphorylation of 55 and 64 kDa proteins that is not observed in insulin-stimulated CSV3-1 cells. Perhaps most interestingly, vandate-induced mitogenesis is associated with the selective induction of c-jun and junB expression without significantly inducing c-fos or c-myc. Furthermore, treatment of CSV3-1 cells with genistein abolishes the effects of vanadate on protein tyrosine phosphorylation and c-jun induction. These and related data suggest that modulation of protein tyrosine phosphorylation and c-jun and junB expression may serve the critical roles in mediating vandate-induced mitogenesis in SV40-transformed cells.