Severe resistance to insulin and insulin-like growth factor-I in cells from a patient with leprechaunism as a result of two mutations in the tyrosine kinase domain of the insulin receptor

We studied the biological properties of insulin receptors (IRs) and insulin-like growth factor-I (IGF-I) receptors in cultured fibroblasts from a patient with leprechaunism (leprechaun Par-1). Patient cells displayed normal insulin binding capacity and affinity. Basal in vivo auto phosphorylation and in vitro exogenous kinase activity of patient IRs were elevated twofold to threefold compared with control receptors, and insulin had no further effect on these processes. Moreover, patient IRs were unable to promote the stimulation of metabolic and mitogenic pathways. IR substrate-1 (IRS-1) and mitogen-activated protein (MAP) kinase tyrosine phosphorylation and glycogen and DNA synthesis were not increased in the basal state in patient fibroblasts and were also insensitive to the stimulatory effect of insulin. As for IGF-I, although binding and receptor kinase activity were normal, the ability to stimulate glycogen and DNA synthesis was altered in patient cells. Two mutant alleles of the IR gene were detected by denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The maternal allele contained a point mutation in exon 18 encoding the tryptophan-for-arginine substitution at position 1092, and the paternal allele had a point mutation in exon 20 substituting lysine for glutamic acid at codon 1179. Thereby, leprechaun Par-1 was a compound heterozygote for two missense mutations located in the IR β-subunit. The present investigation provides the first evidence that leprechaunism can be causally related to structural alterations in the tyrosine kinase domain of the IR. These alterations result in severe impairment of insulin and IGF-I action.     

The dominant negative effect of a kinase-defective insulin receptor on insulin-like growth factor-I-stimulated signaling in Rat-1 fibroblasts

To study the interaction between insulin receptor (IR) and insulin-like growth factor-I (IGF-I) receptor (IGF-IR) tyrosine kinases, we examined IGF-I action in Rat-1 cells expressing a naturally occurring tyrosine kinase-deficient mutant IR (Asp 1048 IR). IGF-I normally stimulated receptor autophosphorylation, IRS-I phosphorylation, and glycogen synthesis in cells expressing Asp 1048 IR. However, the Asp 1048 IR inhibited IGF-I-stimulated thymidine uptake by 45% to 52% and amino acid uptake (aminoisobutyric acid [AIB]) by 58% in Asp 1048 IR cells. Furthermore, IGF-I-stimulated tyrosine kinase activity toward synthetic polymers, Shc phosphorylation, and mitogen-activated protein (MAP) kinase activity was inhibited. The inhibition of mitogenesis and AIB uptake was restored with the amelioration of the impaired tyrosine kinase activity and Shc phosphorylation by the introduction of abundant wild-type IGF-IR in Asp 1048 IR cells. These results suggest that the Asp 1048 IR causes a dominant negative effect on IGF-IR in transmitting signals to Shc and MAP kinase activation, which leads to decreased IGF-I-stimulated DNA synthesis, and that the kinase-defective insulin receptor does not affect IGF-I-stimulated IRS-I phosphorylation, which leads to the normal IGF-I-stimulated glycogen synthesis.     

Inhibition of insulin receptor phosphorylation by peptides derived from major histocompatibility complex class I antigens

Peptides from the alpha 1 region (residues 61-85) of the D and K molecules of the major histocompatibility complex class I antigens inhibit insulin-induced tyrosine kinase activity of the purified human insulin receptors (IRs) as measured both by autophosphorylation and IR-mediated substrate [poly(Glu,Tyr)] phosphorylation. Half-maximal effect of the Dk-(61-85) peptide on IR autophosphorylation is obtained at 1.2 microM, and almost complete inhibition of IR kinase activity is obtained at 10 microM peptide. The corresponding K kappa-(61-85) peptide has a significantly weaker effect on autophosphorylation. No such effects are observed with nine peptides of similar length, but unrelated to major histocompatibility complex class I antigens. Neither of the major histocompatibility complex class I-derived peptides has any effect on the constitutively active kinase of a genetically engineered cytoplasmic IR domain. Further, insulin binding to IR is unaltered in the presence of the major histocompatibility complex class I-derived peptides. The inhibitory activity of the peptides on insulin-induced IR phosphorylation facilitated the observation that IRs require insulin to become substrate for an independent tyrosine kinase. In the presence of an inhibitory peptide, the constitutively active cytoplasmic IR kinase domain only phosphorylates the intact IR in the presence of insulin. We conclude that the tyrosine kinase activity of IRs may be altered by peptide interaction at an allosteric site and, moreover, IRs require insulin to assume a conformation permitting phosphorylation by an independent kinase.     

Insulin receptor substrate 1 mediates insulin and insulin-like growth factor I-stimulated maturation of Xenopus oocytes.

Insulin and insulin-like growth factor I (IGF-I) initiate cellular functions by activating their homologous tyrosine kinase receptors. In most mammalian cell types, this results in rapid tyrosine phosphorylation of a high-molecular-weight substrate termed insulin receptor substrate 1 (IRS-1). Previous studies suggest that IRS-1 may act as a "docking" protein that noncovalently associates with certain signal-transducing molecules containing src homology 2 domains; however, direct evidence for the role of IRS-1 in the final biological actions of these hormones is still lacking. We have developed a reconstitution system to study the role of IRS-1 in insulin and IGF-I signaling, taking advantage of the fact that Xenopus oocytes possess endogenous IGF-I receptors but have little or no IRS-1, as determined by immunoblotting with anti-IRS-1 and antiphosphotyrosine antibodies. After microinjection of IRS-1 protein produced in a baculovirus expression system, tyrosyl phosphorylation of injected IRS-1 is stimulated by both insulin and IGF-I in a concentration-dependent manner, with IGF-I more potent than insulin. Furthermore, after IRS-1 injection, both hormones induce a maturation response that correlates well with the amount of injected IRS-1. By contrast, overexpression of human insulin receptors in the Xenopus oocytes does not enhance either IRS-1 phosphorylation or oocyte maturation response upon insulin stimulation. These results demonstrate that IRS-1 serves a critical role in linking IGF-I and insulin to their final cellular responses.     

Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action

The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an “unregulated” mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance.     

Ethanol inhibits insulin receptor tyrosine kinase

BACKGROUND: Ethanol inhibits insulin-like growth factor-I (IGF-I) and insulin signaling in various cell types. The tyrosine autophosphorylation of the IGF-I and insulin receptors appears to be a target for ethanol, as well as other receptor tyrosine kinases. METHODS AND RESULTS: The effect of ethanol on purified recombinant insulin receptor kinase activity was examined. A noncompetitive inhibition was observed at pharmacologically relevant concentrations of ethanol. Both peptide substrate tyrosine phosphorylation and kinase autophosphorylation are inhibited by ethanol. Near equivalent inhibition of kinase activity was noted for 300 mM methanol, 150 mM ethanol, 20 mM 1-propanol, and 10 mM 1-butanol. CONCLUSION: The findings identify a direct protein interaction site of ethanol, and provide insight into the mechanism by which ethanol inhibits receptor tyrosine kinase activity.     

IRS-1 expression and activation are not sufficient to activate downstream pathways and enable IGF-I growth response in estrogen receptor negative breast cancer cells.

IGF-responsive breast cancer cells activate insulin receptor substrate (IRS)-1 after IGF-I treatment. To determine if IRS-1 expression was sufficient to enable IGF-responsiveness, two IGF-I unresponsive breast cancer cell lines (MDA-MB-435A and MDA-MB-468) were transfected with IRS-1. While IGF-I caused tyrosine phosphorylation of IRS-1 in both transfected cell lines, increased MAP kinase activity was not seen. IGF-I treatment of 435A IRS-1 transfected cells resulted in minimal increased PI3 kinase activity associated with IRS-1, while IRS-2/PI3 kinase was greatly reduced. In MDA-MB-468 IRS-1 transfected cells, IGF-I caused increased IRS-1 associated PI3 kinase activity compared to parental cells, but at levels far below those observed in IGF-responsive MCF-7 cells. The transfected cells were also not responsive to IGF-I in monolayer growth. Thus, IRS-1 expression and activation alone are insufficient to mediate a proliferative response to IGF-I in breast cancer cells, and it is likely that maximal activation of downstream signaling pathways must also occur.     

Regulation of insulin receptor substrate-2 tyrosine phosphorylation in animal models of insulin resistance

Insulin induces a wide variety of growth and metabolic responses in many cell types. These actions are initiated by insulin binding to its receptor and involve a series of alternative and complementary pathways created by the multiple substrates of the insulin receptor (insulin receptor substrates [IRSs]). We investigated IRS-1 and IRS-2 tyrosine phosphorylation; their association with phosphatidylinositol-3-OH kinase (PI3-K); and the phosphorylation of Akt, a serine-threonine kinase situated downstream of PI3-K, in liver and muscle of two animal models of insulin resistance: epinephrine- or dexamethasone-treated rats. We used in vivo insulin infusion followed by tissue extraction, immunoprecipitation, and immunoblotting. IRS-1 and IRS-2 protein expression did not change in liver and muscle of the epinephrine-treated rats, but in dexamethasone-treated rats IRS-1 presented an increase in liver and a decrease in muscle tissue. PI3-K and Akt protein expression did not change in liver or muscle of the two animal models of insulin resistance. There was a downregulation in insulin-induced IRS-1 and IRS-2 tyrosine phosphorylation and association with PI3-K in both models of insulin resistance. In parallel, insulin-induced Akt phosphorylation was reduced in both tissues of epinephrine-treated rats, and in liver but not in muscle of dexamethasonetreated rats. The reduction in insulin-induced Akt phosphorylation may help to explain the insulin resistance in liver and muscle of epinephrine-treated rats and in the liver of dexamethasone-treated rats.     

Alpha2-HSG, a specific inhibitor of insulin receptor autophosphorylation, interacts with the insulin receptor

Human fetuin, [alpha2-Heremans Schmid Glycoprotein (alpha2-HSG)], is a natural inhibitor of insulin receptor tyrosine kinase activity (IR-TKA). Previously, we have demonstrated that alpha2-HSG inhibits the mitogenic pathway without affecting the metabolic arm of insulin signal transduction. In this study, we demonstrate the time-course and specificity of inhibition, its interaction with IR and probable physiological role. In intact rat1 fibroblasts overexpressing the human insulin receptor (HIRc B), incubation of recombinant human alpha2-HSGbac (1.8 microM) inhibited insulin-induced IR autophosphorylation by over 80%. This inhibitory effect of alpha2-HSGbac on insulin-induced IR autophosphorylation was blunted by half in 60 min. Interestingly, alpha2-HSGbac at similar concentrations (0.9 or 1.8 microM), had no effect on EGF- or IGF-I-induced cognate receptor autophosphorylation. Anti-alpha2-HSG immunoprecipitates of alpha2-HSGbac-treated HIRc B cell lysates demonstrated the presence of IR. Our data suggest that alpha2-HSGbac preferentially interacts with the activated IR. To further characterize the site(s) of interaction, the effect of alpha2-HSGbac on trypsin-treated IR autophosphorylation was studied. Trypsin-treatment of intact HIRc B cells results in proteolysis of the IR alpha-chain and constitutive activation of IR-TKA. We demonstrate that alpha2-HSGbac (0.1 microM) completely inhibited trypsin-activated IR autophosphorylation and TKA in vitro indicating that this effect was not mediated by its interaction with the proximal 576 amino acid residues of the IR alpha-subunit. The physiological relevance of these observations was explored by characterizing the effects of alpha2-HSG injection in rats. Alpha2-HSGbac (2 microM), acutely injected through the portal vein of normal rats, inhibited insulin-stimulated IR autophosphorylation and IRS-1 phosphorylation in liver and hindlimb muscle. Taken together our results suggest that alpha2-HSG, by interacting with IR, specifically inhibits insulin-stimulated IR autophosphorylation and may play a physiological role in the regulation of insulin signaling.     

Decreased insulin receptor (IR) autophosphorylation in fibroblasts from patients with PCOS: effects of serine kinase inhibitors and IR activators

Insulin resistance is characteristic of many patients with polycystic ovary syndrome (PCOS). Several studies have suggested that a decrease in insulin receptor (IR) autophosphorylation is a significant component of this resistance. In this study, we have used a highly sensitive ELISA to measure IR tyrosine phosphorylation in fibroblasts from patients with PCOS and healthy control women. After the stimulation of intact fibroblasts with insulin, IR tyrosine phosphorylation in cells from the PCOS patients was decreased by approximately 40% when compared with controls. However, when IR were first immunocaptured from fibroblasts and then stimulated with insulin, neither basal nor insulin-stimulated IR autophosphorylation was different between the two groups, suggesting that a factor independent of the IR was involved. To examine the role of increased serine kinase activity in decreased IR autophosphorylation in PCOS, fibroblasts from PCOS patients were pretreated with inhibitors of serine kinases before insulin stimulation. Pretreatment with H7, a nonspecific protein kinase inhibitor, completely reversed the decrease in insulin-stimulated IR autophosphorylation. Pretreatment with H89, an inhibitor of protein kinase A, partially reversed this function, whereas pretreatment with G?6983, an inhibitor of protein kinase C, was without effect. We next studied the effects of two small molecule activators of the IR tyrosine kinase: TLK16998 and Merck L7. Both TLK16998 and Merck L7 were able to reverse the impaired insulin-stimulated IR autophosphorylation. In summary, a factor(s) extrinsic to the IR cause impaired IR signaling in fibroblasts from patients with PCOS. Reversal of the impaired IR signaling by inhibitors of serine kinase activity suggests that serine kinase-mediated pathways may be involved in the insulin resistance. Moreover, the observation that TLK16998 and Merck L7 improved IR tyrosine phosphorylation in fibroblasts from patients with PCOS suggests that specific pharmacological therapies might be developed to treat the insulin resistance in PCOS.     

Bradykinin enhances insulin receptor tyrosine kinase in 32D cells reconstituted with bradykinin and insulin signaling pathways

We have previously shown that bradykinin potentiated insulin-induced glucose uptake through GLUT4 translocation in canine adipocytes and skeletal muscles. The aim of this study was to determine the molecular mechanism of bradykinin enhancement of the insulin signal. For this purpose, 32D cells, which express a limited number of insulin receptors and lack endogenous bradykinin B2 receptor (BK2R) or insulin receptor substrate (IRS)-1 were transfected with BK2R cDNA and/or insulin receptor cDNA and/or IRS-1 cDNA, and analyzed. In 32D cells that expressed BK2R and insulin receptor (32D-BKR/IR), bradykinin alone had no effect on the phosphorylation of the insulin receptor, but it enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor. In 32D cells that expressed BK2R, insulin receptor and IRS-1 (32D-BKR/IR/IRS1), bradykinin also enhanced insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1. An increase in insulin-stimulated phosphorylation of IRS-1 by treatment with bradykinin in 32D-BKR/IR/IRS1 cell was associated with increased binding of 85 kD subunit of phosphatidylinositol 3 (PI 3)-kinase and increased IRS-1 associated PI 3-kinase activity. These effects of bradykinin were not observed in 32D cells which lack the expression of BK2R (32D-IR/IRS1) or insulin receptor (32D-BKR/IRS1). Furthermore, tyrosine phosphatase activity against insulin receptor beta-subunit in plasma membrane fraction of 32D-BKR/IR cells was significantly reduced by bradykinin, suggesting that the effect of bradykinin was in part mediated by inhibition of protein tyrosine phosphatase(s). Our results clearly demonstrated that bradykinin enhanced insulin-stimulated tyrosine kinase activity of the insulin receptor and downstream insulin signal cascade through the BK2R mediated signal pathway.     

Common elements in interleukin 4 and insulin signaling pathways in factor-dependent hematopoietic cells.

Interleukin 4 (IL-4), insulin, and insulin-like growth factor I (IGF-I) efficiently induced DNA synthesis in the IL-3-dependent murine myeloid cell lines FDC-P1 and FDC-P2. Although these factors could not individually sustain long-term growth of these lines, a combination of IL-4 with either insulin or IGF-I did support continuous growth. The principal tyrosine-phosphorylated substrate observed in FDC cells stimulated with IL-4, previously designated 4PS, was of the same size (170 kDa) as the major substrate phosphorylated in response to insulin or IGF-I. These substrates had phosphopeptides of the same size when analyzed by digestion with Staphylococcus aureus V8 protease, and each tightly associated with the 85-kDa component of phosphatidylinositol 3-kinase after factor stimulation. IRS-1, the principal substrate phosphorylated in response to insulin or IGF-I stimulation in nonhematopoietic cells, is similar in size to 4PS. However, anti-IRS-1 antibodies failed to efficiently precipitate 4PS, and some phosphopeptides generated by V8 protease digestion of IRS-1 were distinct in size from the phosphopeptides of 4PS. Nevertheless, IL-4, insulin, and IGF-I were capable of stimulating tyrosine phosphorylation of IRS-1 in FDC cells that expressed this substrate as a result of transfection. These findings indicate that (i) IL-4, insulin, and IGF-I use signal transduction pathways in FDC lines that have at least one major feature in common, the rapid tyrosine phosphorylation of 4PS, and (ii) insulin and IGF-I stimulation of hematopoietic cell lines leads to the phosphorylation of a substrate that may be related to but is not identical to IRS-1.     

The G972R variant of the insulin receptor substrate-1 gene impairs insulin signaling and cell differentiation in 3T3L1 adipocytes; treatment with a PPARgamma agonist restores normal cell signaling and differentiation

The insulin receptor substrate-1 (IRS-1) plays a central role in insulin sensitivity, and association studies have shown that the IRS-1 G972R variant is a risk factor for insulin resistance. However, how this mutation may lead to impaired insulin sensitivity is still to be determined. Our study aimed to evaluate, after transfection of the IRS-1 G972R variant in 3T3L1 adipocytes, the effect of this mutation on insulin signaling and on cell differentiation. The 3T3L1 cells were transfected with pcDNA3 expression vector containing either the human wild-type IRS-1 or the G972R variant. After induction of differentiation, the 3T3L1 transfected with wild-type IRS-1 differentiated in 6-8 days, while the cells transfected with G972R variant did not differentiate. To determine whether the defect in IRS-1 was responsible for this, we analyzed the expression of several genes involved in the insulin signaling pathway. Results showed that PPARgamma expression was significantly reduced in cells transfected with the mutated IRS-1, together with a significant decrease in binding of phosphatidylinositol-3 kinase (PI 3-kinase) to IRS-1 G972R and in PI 3-kinase activity. In addition, we observed that the interaction between the insulin receptor (IR) and the IRS-1 G972R protein was increased and that the autophosphorylation of the IR was significantly inhibited in 3T3L1-G972R cells compared with 3T3L1-WT. Treatment of the 3T3L1-G972R cells with pioglitazone (PIO), a PPARgamma agonist, restored differentiation with higher level of PPARgamma expression and restoration of PI 3-kinase binding to IRS-1 G972R and PI 3-kinase activity. IR autophosphorylation was also increased. Withdrawal of PIO in fully differentiated 3T3L1-G972R cells determined the reappearance of the insulin signaling defect. Finally, we observed higher levels of IRS-2 expression, suggesting that IRS-2 may play a more important role in adipocyte insulin signaling. In conclusion, IRS-1 G972R variant impairs insulin signaling, and treatment with PPARgamma agonist restores the normal phenotype of 3T3L1 cells.     

Transmembrane signaling by an insulin receptor lacking a cytoplasmic beta-subunit domain.

To assess the function of the cytoplasmic domain of the insulin receptor (IR) beta subunit, we have studied a mutant IR truncated by 365 aa (HIR delta 978), thereby deleting > 90% of the cytoplasmic domain. HIR delta 978 receptors were processed normally to homodimers that were expressed at the cell surface where they bind insulin with normal affinity. Although these truncated IRs were inactive with respect to ligand-induced internalization and autophosphorylation, insulin stimulated endogenous substrate (pp185) phosphorylation significantly more in HIR delta 978 cells than in untransfected Rat1 cells. Importantly, despite absence of the beta-subunit cytoplasmic domain, fibroblasts expressing HIR delta 978 receptors displayed enhanced sensitivity to insulin for stimulation of glucose incorporation into glycogen, alpha-aminoisobutyric acid uptake, thymidine incorporation, and S6 kinase activity compared with parental fibroblasts. Insulin also induced the expression of the protooncogene c-fos and the early growth response gene Egr-1 in HIR delta 978 cells far greater than in parental Rat1 fibroblasts. Furthermore, an agonistic monoclonal antibody specific for the human IR stimulated insulin action in fibroblasts expressing wild-type human IR but had no effect on HIR delta 978 cells. In conclusion, the HIR delta 978 truncated IRs appear to confer enhanced insulin sensitivity by augmenting the signaling properties of the endogenous rodent IRs.     

Insulin receptor substrate-4 is expressed in muscle tissue without acting as a substrate for the insulin receptor

Insulin receptor substrate (IRS) proteins represent key elements of the insulin-signaling cascade. IRS-4 is the most recently characterized member of the IRS family with an undefined in vivo function. In contrast to IRS-1 and IRS-2, IRS-4 exhibits a limited tissue expression, and IRS-4 protein has not been detected in any mouse or primary human tissue so far. The purpose of the present study was to analyze the expression of IRS-4 in rat muscle and human skeletal muscle cells and assess involvement of IRS-4 in initial insulin signaling. Using immunoblotting and immunoprecipitation, the specific expression of IRS-4 protein could be demonstrated in rat soleus and cardiac muscle and human skeletal muscle cells, but it was not significantly detectable in quadriceps and gastrocnemius. A prominent down-regulation of IRS-4 was observed in heart and soleus muscle of WOKW rats, an animal model of the metabolic syndrome. In human skeletal muscle cells, both IRS-1 and IRS-2 are rapidly phosphorylated on tyrosine in response to insulin, whereas essentially no tyrosine phosphorylation of IRS-4 was observed in response to both insulin and IGF-I. Instead, a 2-fold increase in IRS-4 tyrosine phosphorylation was observed in myocytes subjected to osmotic stress. In conclusion, IRS-4 protein is expressed in heart and skeletal muscle in a fiber type specific fashion. Our data suggest that IRS-4 does not function as a substrate of the insulin and the IGF-I receptor in primary muscle cells but may be involved in nonreceptor tyrosine kinase signaling.     

Role of insulin receptor substrate-1 serine 307 phosphorylation and adiponectin in adipose tissue insulin resistance in late pregnancy

Insulin resistance is a hallmark of late pregnancy both in human and rat. Adipose tissue is one of the tissues that most actively contributes to this reduced insulin sensitivity. The aim of the present study was to characterize the molecular mechanisms of insulin resistance in adipose tissue at late pregnancy. To this end, we analyzed the insulin signaling cascade in lumbar adipose tissue of nonpregnant and pregnant (d 20) rats both under basal and insulin-stimulated conditions. We found that the levels of relevant signaling proteins, such as insulin receptor (IR), IR substrate-1 (IRS-1), phosphatidylinositol 3-kinase, 3-phosphoinositide-dependent kinase-1, ERK1/2, and phosphatase and tensin homolog (PTEN) did not change at late pregnancy. However, insulin-stimulated tyrosine phosphorylation of both IR and IRS-1 were significantly decreased, coincident with decreased IRS-1/p85 association and impaired phosphorylation of AKR mouse thymoma viral protooncogene (Akt) and ERK1/2. This impaired activation of IRS-1 occurred together with an increase of IRS-1 phosphorylation at serine 307 and a decrease in adiponectin levels. To corroborate the role of IRS-1 in adipose tissue insulin resistance during pregnancy, we treated pregnant rats with the antidiabetic drug englitazone. Englitazone improved glucose tolerance, and this pharmacological reversal of insulin resistance was paralleled by an increase of adiponectin levels in adipose tissue as well as by a reduction of IRS-1 serine phosphorylation. Furthermore, the impaired insulin-stimulated tyrosine phosphorylation of IRS-1 in adipose tissue of pregnant animals could be restored ex vivo by treating isolated adipocytes with adiponectin. Together, our findings support a role for adiponectin and serine phosphorylation of IRS-1 in the modulation of insulin resistance in adipose tissue at late pregnancy.     

Decrease in the insulin receptor protein level by anti-insulin receptor antibodies: roles of tyrosine kinase activity and receptor internalization

To investigate the mechanism of severe impairment of insulin action in type B insulin resistance, we extracted IgG from the serum of a patient with type B insulin resistance (B-IgG) and analyzed the inhibiting effect of B-IgG not only on insulin signaling but also on IGF-I signaling in Chinese hamster ovary (CHO) cells expressing human insulin receptor or human IGF-I receptor. Preincubation with 1 mg/ml B-IgG prevented insulin-induced phosphorylation of insulin receptor and insulin receptor substrate-1 (IRS-1) but did not alter the IGF-I-induced phosphorylation of the IGF-I receptor and IRS-1. ¹²⁵I-insulin binding was inhibited by 93% after preincubation with BigG at 37° C and was recovered up to 50% of the control value by acid washing. However, when cells were preincubated with B-IgG at 4° C, the insulin binding completely recovered the control value by acid washing. ¹²⁵I-IGF-I binding was not altered by B-IgG preincubation. Immunoblot study revealed that the protein level of the insulin receptor was strongly decreased by preincubation with 1 mg/ml B-IgG at 37° C, but never at 4° C. The IRS-1 protein level did not change by B-IgG preincubation. In order to know the role of the insulin receptor internalization in the inhibiting effect of B-IgG, we employed CHO cells expressing mutant insulin receptors which do not undergo internalization (CHO-K1018R). B-IgG incubation of CHOK1018R at 37° C failed to decrease the protein level of the insulin receptor. The present data indicate that IgG from the diabetic patient with type B insulin resistance decreased insulin receptor protein level, probably due to the enhanced degradation rate of the insulin receptor, in which insulin receptor tyrosine kinase activity and internalization are required for this process. This effect of B-IgG was specific for the insulin receptor with no effect on either IGF-I receptor or IRS-1, as reflected by the IGF-I effectiveness on glycemic control in this patient. Received: 26 September 2001 / Accepted in revised form: 12 July 2002 Correspondence to K. Eguchi     

Elevated plasma cell membrane glycoprotein levels and diminished insulin receptor autophosphorylation in obese, insulin-resistant rhesus monkeys

In obese humans, insulin resistance is accompanied by elevated levels of plasma cell membrane glycoprotein (PC-1) and decreased insulin receptor (IR) tyrosine kinase activity in skeletal muscle. PC-1 overexpression inhibits IR tyrosine kinase and possibly other downstream signaling events. The rhesus monkey in captivity is susceptible to obesity with concomitant insulin resistance. In the present study we analyzed obese (n = 10, 29.4% +/- 1.2% body fat) and non-obese (n = 12, 19.4% +/- 1.9% body fat) rhesus monkeys. Glucose clearance during an euglycemic hyperinsulinemic (400 mU/m(2) body surface area/min) clamp was lower for the obese group (non-obese, 9.7 +/- 0.9; obese, 3.2 +/- 0.7 mg/kg fat-free mass [FFM]/min; P <.01). We performed vastus lateralis muscle biopsies prior to and during the clamp. We measured PC-1 levels in these muscle samples to determine whether PC-1 content is elevated in this primate model of insulin resistance. PC-1 levels were determined by assay of phosphodiesterase activity and specific PC-1 enzyme-linked immunosorbent assay (ELISA). In the obese group, both PC-1 content and activity were 2-fold higher than in the non-obese group (P <.05). In order to investigate the ability of insulin to stimulate IR signaling in vivo in these 2 groups of monkeys, we then measured tyrosine autophosphorylation of the IR by specific ELISA. The increase in IR autophosphorylation in the non-obese group was twice that of the obese group (fold increase over basal: non-obese, 3.7 +/- 0.3; obese, 1.9 +/- 0.6; P <.05). We conclude that insulin resistance secondary to obesity in rhesus monkeys is associated with increased levels of PC-1 and decreased IR signaling capacity in skeletal muscle.     

Human biliverdin reductase: a member of the insulin receptor substrate family with serine/threonine/tyrosine kinase activity

We describe here the tyrosine kinase activity of human biliverdin reductase (BVR) and its potential role in the insulin-signaling pathway. BVR is both a substrate for insulin receptor (IR) tyrosine kinase (IRK) activity and a kinase for serine phosphorylation of IR substrate 1 (IRS-1). Our previous studies have revealed serine/threonine kinase activity of BVR. Y198, in the YMKM motif found in the C-terminal domain of BVR, is shown to be a substrate for insulin-activated IRK. This motif in IRS proteins provides a docking site for proteins that contain a Src homology 2 domain. Additionally, Y228 in the YLSF sequence and Y291 are IRK substrates; the former sequence provides optimum recognition motif in the tyrosine phosphatase, SHP-1, and for SHC (Src homology 2 domain containing transfroming protein 1). BVR autophosphorylates N-terminal tyrosines Y72 and Y83. Serine residues in IRS-1 are targets for BVR phosphorylation, and point mutation of serine residues in the kinase domain of the reductase inhibits phosphotransferase activity. Because tyrosine phosphorylation of IRS-1 activates the insulin signaling pathway and serine phosphorylation of IRS-1 blocks insulin action, our findings that insulin increases BVR tyrosine phosphorylation and that there is an increase in glucose uptake in response to insulin when expression of BVR is "knocked down" by small interfering RNA suggest a potential role for BVR in the insulin signaling pathway.     

Insulin signaling through insulin receptor substrate 1 and 2 in normal liver development

BACKGROUND & AIMS: The insulin growth factor signal transduction pathway is an important regulator of adult hepatocyte proliferation. The purpose of this study was to determine the roles of the insulin receptor substrate (IRS-1 and IRS-2)-mediated growth cascades in rapidly growing fetal rat liver. METHODS: We determined the expression and tyrosyl phosphorylation of the insulin receptor beta subunit (IRbeta), IRS-1 and IRS-2, the binding of phosphatidylinositol 3-kinase (PI3K), and activation of the mitogen-activated protein kinase (MAPK) pathway in the presence or absence of insulin stimulation in vivo during development and in the adult liver. In addition, activation of other downstream components including PI3K, Akt, GSK3beta, Bad, and p70S6 kinase was studied. RESULTS: We observed reduced expression and tyrosyl phosphorylation of IRS-1 in the fetal liver compared with the adult liver. These developmental changes resulted in a lack of sensitivity to insulin stimulation and subsequent downstream activation of the PI3K and MAPK cascades until the postneonatal period. In contrast, there was a high level of IRS-2 expression and insulin-stimulated tyrosyl phosphorylation as early as embryonic day 15 with robust PI3K binding and activation, which may enhance hepatocyte survival during the rapid growth phase of the liver. CONCLUSIONS: The IRS-1 signal transduction pathway does not play a major role in fetal liver growth because IRS-2 functions as the major insulin responsive molecule in early development. However, insulin-mediated IRS-1/MAPK cascade activation contributes to growth in the adult.