The protein-tyrosine kinase fer associates with signaling complexes containing insulin receptor substrate-1 and phosphatidylinositol 3-kinase.

In a screen for 3T3-F442A adipocyte proteins that bind SH2 domains, we isolated a cDNA encoding Fer, a nonreceptor protein-tyrosine kinase of the Fes/Fps family that contains a functional SH2 domain. A truncated splicing variant, iFer, was also cloned. iFer is devoid of both the tyrosine kinase domain and a functional SH2 domain but displays a unique 42-residue C terminus and retains the ability to form oligomers with Fer. Expression of both Fer and iFer proteins are strikingly increased upon differentiation of 3T3-L1 fibroblasts to adipocytes. Platelet-derived growth factor treatment of the cultured adipocytes caused rapid tyrosine phosphorylation of Fer and its recruitment to complexes containing platelet-derived growth factor receptor and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase. Insulin treatment of 3T3-L1 adipocytes stimulated association of Fer with complexes containing tyrosine phosphorylated IRS-1 and PI 3-kinase but did not stimulate tyrosine phosphorylation of Fer. PI 3-kinase activity in anti-Fer immunoprecipitates was also acutely activated by insulin treatment of cultured adipocytes. These data demonstrate the presence of Fer tyrosine kinase in insulin signaling complexes, suggesting a role of Fer in insulin action.

Effect of glimepiride (HOE 490) on insulin receptors of skeletal muscles from genetically diabetic KK-Ay mouse.

A new sulfonylurea, glimepiride (HOE 490), has been developed for the glycemic control in non-insulin-dependent diabetes mellitus. We examined the effect of glimepiride on glucose and insulin levels in KK-Ay mice, an animal model of non-insulin-dependent diabetes mellitus, which is characterized by hyperglycemia and hyperinsulinemia. Administration of glimepiride (0.5 mg/kg/day) for 8 weeks to KK-Ay mice resulted in decrease in glucose (297 +/- 36 to 250 +/- 51 mg/dl) and insulin (76 +/- 14 to 41 +/- 14 microU/ml) levels. To clarify the mechanism of the agent, we examined the effect of this new drug on insulin receptors in the skeletal muscles. There was no difference in insulin binding to the receptors from both glimepiride-treated and -untreated KK-Ay mice muscles. The insulin-stimulated autophosphorylation of insulin receptors from KK-Ay mice was decreased compared to that from normal mice (5 +/- 1 vs. 39 +/- 13% over basal). Glimepiride did not ameliorate impaired insulin-stimulated insulin receptor autophosphorylation. To determine the effect of glimepiride on post-insulin receptor signaling pathway, we measured 2-[3H]glycerol incorporation into diacylglycerol in the cultured rat fibroblast cell line overexpressing human insulin receptors. Glimepiride (100 microM) as well as insulin (10 nM) significantly stimulated diacylglycerol production. These results suggest that glimepiride has a potent extrapancreatic effect on glucose metabolism and may directly stimulate glucose transport activity through phospholipid signaling pathway, but not through insulin receptor kinase signaling pathway.

Clinical and laboratory characteristics in the families with diabetes and a mitochondrial tRNA(LEU(UUR)) gene mutation.

We identified three families having a mutation in the mitochondrial tRNA(LEU(UUR)) gene at bp 3243 in 300 patients with non-insulin dependent diabetes mellitus (NIDDM), who had first degree relatives of patients with NIDDM. We found six individuals with diabetes, one with impaired glucose tolerance (IGT), and five with normal glucose tolerance (NGT) among three families. Insulin secretory response to oral glucose load was impaired in six diabetics, but was normal in IGT and NGT, and the proportion of mutant DNA in the blood did not always associate with the severity of glucose intolerance. Furthermore, both gender and obesity may influence the clinical expression of diabetes in three pairs with an age-matched brother-sister relationship with similar high mutation rate in blood samples. Thus, although patients with mitochondrial gene mutation had a high frequency of diabetes, the proportion of mutant DNA evaluated by blood samples may not necessarily indicate glucose intolerance in the members with the mutation. Unidentified factors including gender, aging, and obesity may alter the clinical manifestation of diabetes.

Amplification and analysis of promoter region of insulin receptor gene in a patient with leprechaunism associated with severe insulin resistance.

A patient with leprechaunism associated with severe insulin resistance was studied to identify the molecular and genetic basis for insulin resistance. Insulin binding and surface labeling of transformed lymphocytes prepared from the patient showed a significantly decreased insulin receptor number on the cell surface. Southern blot analysis of the insulin receptor gene showed no evidence of large insertions or deletions. Furthermore, direct sequencing of all 22 exons and exon-intron junctions of the insulin receptor gene failed to show any missense mutations, nonsense mutations, or mutations at exon-intron junctions. However, Northern blot analysis indicated significantly decreased insulin receptor mRNA expression in the patient's cells. Moreover, restriction endonuclease digestion of the amplified cDNA suggested that the expression levels of one allele were less efficient than the other. These findings suggested that the regulatory region of the insulin receptor gene might have abnormalities. Therefore, we examined the 5' flanking region of the insulin receptor gene. Southern blot analysis showed no major deletions or insertions between positions -1,823 and -2 relative to the translation initiation site. A 5' flanking region of the insulin receptor gene spanning positions -881 approximately +7 was amplified by polymerase chain reaction (PCR) and introduced into a reporter plasmid carrying the human growth hormone (hGH) gene. The nucleotide sequence of the amplified fragment showed two polymorphic sites at positions -603 and -500 in the patient, as well as in normal subjects. No other abnormal sequence was found in the patient. Promoter activity measured by hGH expression in transfected mouse L cells was not influenced by the polymorphism at position -603 located in a cluster of GC boxes.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiazolidine derivatives ameliorate high glucose-induced insulin resistance via the normalization of protein-tyrosine phosphatase activities.

The mechanisms for the insulin resistance induced by hyperglycemia were investigated by studying the effect of high glucose concentration (HG) and its modulation by thiazolidine derivatives, on insulin signaling using Rat 1 fibroblasts expressing human insulin receptors (HIRc). Incubating HIRc cells in 27 mM D-glucose for 4 days impaired the insulin-stimulated phosphorylation of pp185 and receptor beta-subunits. Both protein kinase C activities and phorbol dibutyrate binding to intact cells were unchanged; however, cytosolic protein-tyrosine phosphatase (PTPase) activity increased within 1 h prior to the impairment of insulin receptor kinase in HG cells (Maegawa, H., Tachikawa-Ide, R., Ugi, S., Iwanishi, M., Egawa, K., Kikkawa, R., Shigeta, Y., and Kashiwagi, A. (1993) Biochem. Biophys. Res. Commun. 197, 1078-1082). Increased PTPase activity was consistent with a 2-fold increase in the amount of PTP1B, and anti-PTP1B antibody inhibited this increment of cytosolic PTPase activity in HG cells. Co-incubating cells with pioglitazone prevented these abnormalities in cytosolic PTPase, the PTP1B content and the impaired phosphorylation of pp185 and receptor beta subunits in HG cells. Finally, HG cells had impaired insulin-stimulated alpha-amino-isobutyric acid uptake, which was ameliorated by exposure to thiazolidine derivatives. In conclusion, exposing cells to high glucose levels desensitizes insulin receptor function, and thiazolidine derivatives can reverse the process via the normalization of cytosolic PTPase, but not of protein kinase C.

Two naturally occurring mutations in the kinase domain of insulin receptor accelerate degradation of the insulin receptor and impair the kinase activity.

We identified two novel heterozygous missense mutations of the insulin receptor gene: the Asp1179 mutation in one family and the Leu1193 mutation in two unrelated families with extreme insulin resistance. In these patients, the number of insulin receptors on the cell surface was found to be markedly decreased by insulin binding and surface labeling studies in transformed lymphocytes. Insulin binding to the transfected COS 7 cells and Rat-1 cells with both mutant cDNAs was also decreased to 5-31% of normal, and the mutant insulin receptors showed a markedly decreased kinase activity. Although biosynthetic labeling studies revealed that both mutant receptors were synthesized as 190-kDa proreceptors, the degradation of the mutant proreceptors was 2-fold faster than that of the wild type proreceptors. However, the degradation rate of the mutant receptors on the cell surface was comparable to that of wild type insulin receptor. These results suggest that the Asp1179 and Leu1193 mutations in the kinase domain are unique in causing decreased insulin receptor number on the cell surface by accelerated intracellular degradation, and that insulin resistance in these patients is mainly due to the decreased receptor number rather than impaired kinase activity.

Ala1048-->Asp mutation in the kinase domain of insulin receptor causes defective kinase activity and insulin resistance.

We identified a heterozygous missense mutation that substituted aspartic acid (GAC) for alanine (GCC) at codon 1048 of the insulin receptor gene in a patient who displayed typical symptoms of Type A syndrome of insulin resistance. The proband's mother and younger brother were also found to be heterozygous for the mutation. We constructed the identified mutant insulin receptor cDNA by site-directed mutagenesis, transfected the mutant cDNA into COS 7 cells, and found that kinase activity of the mutant insulin receptors was markedly impaired. Ala1048 is located in the kinase domain of the insulin receptor beta-subunit and is conserved in most of protein-tyrosine kinases. Besides, neighboring Glu1047 is invariant in all protein kinases and is thought to be involved in interaction with ATP. Photoaffinity labeling of the mutant insulin receptor with ATP analogue, 8-azido (alpha-32P)ATP was not influenced by the mutation, suggesting that the mutation did not inhibit ATP binding but possibly interfered with subsequent phosphoryl transfer. Insulin-stimulated phosphorylation of exogenous substrate by partially purified insulin receptors prepared from COS 7 cells that were cotransfected with wild-type and mutant insulin receptor cDNAs was markedly impaired, whereas autophosphorylation was decreased by approximately 50% of wild-type receptors. These results indicated that the identified heterozygous substitution of Asp for Ala1048 in insulin receptor was responsible for insulin resistance of this patient.

Importance of Asp1191 for tyrosine kinase activity of the insulin receptor: functional difference of universally conserved Asp between tyrosine kinase and c-AMP dependent serine/threonine protein kinase.

Asp1191 of the tyrosine kinase domain in the insulin receptor is located in the almost perfectly conserved Ser-Asp-X-Trp motif of all tyrosine kinases, and the function of the motif has not been clarified. In this motif, Asp1191 is universally conserved in all protein kinases and corresponds to Asp220 in catalytic subunit of c-AMP dependent protein kinase. However, while other universally conserved amino acid residues are involved in catalysis, only Asp220 is not. To determine whether this motif was not also involved in catalysis, Asp1191 was replaced either by alanine, asparagine, or glutamic acid and Met1192 was replaced by valine. Asp1191-->Ala, Asn or Glu receptor lacked tyrosine kinase activity, while Met1192-->Val receptor showed normal kinase activity. These mutant receptors had no effect on insulin binding. These results suggested that the universally conserved Asp1191 in all protein kinases, which was not required for catalytic action of c-AMP dependent protein kinase, was essential for the tyrosine kinase activity of the insulin receptor.

Pioglitazone ameliorates high glucose induced desensitization of insulin receptor kinase in Rat 1 fibroblasts in culture.

A new oral agent, pioglitazone, increases insulin sensitivity by activating receptor kinase in insulin-resistant rats. To clarify the mechanism, we studied in vitro effects of glucose and pioglitazone on the insulin receptor function using Rat 1 fibroblasts which expressed human insulin receptors. Insulin receptor kinase activity was impaired by incubating cells for 4 days in the presence of 27mM D-glucose. The glucose effect was time- and dose-dependent and also specific for D-glucose, since D-raffinose incubation had no effect. Pioglitazone treatment did not have any effect on intact receptor kinase. However, exposure of both 27mM D-glucose and 0.1 microM pioglitazone to the cells completely prevented the glucose-induced impairment of insulin receptor kinase activity, suggesting that pioglitazone might reverse the processes which are critical for the glucose-induced desensitization of insulin receptor kinase.

Effect of pioglitazone on insulin receptors of skeletal muscles from high-fat-fed rats.

A new oral agent, 5-[4-[2-(5-ethyl-12-pyridyl)ethoxy]-benzyl]-2,4-thiazolidinedione, or pioglitazone, has been developed for the treatment of non-insulin-dependent diabetes mellitus (NIDDM). We examined its effectiveness in high-fat-fed rats resistant to insulin. Administration of the agent (10 mg.kg-1 x d-1) for 2 weeks resulted in decreases in hyperlipidemia and hyperinsulinemia, indicating that insulin sensitivity had increased in vivo in high-fat-fed rats. To clarify the mechanism of the drug, we examined insulin binding and kinase activity of insulin receptors from muscles of both untreated and treated high-fat-fed rats. Pioglitazone treatment did not change insulin binding in high-fat-fed rats, but increased insulin-stimulated autophosphorylation of insulin receptors to the level of control animals. Kinase activity toward an exogenous substrate, poly Glu4-Tyr1, in pioglitazone-treated high-fat-fed rats was also increased to the level of control animals. These results suggest that pioglitazone increases insulin sensitivity by activating tyrosine kinase activity of receptors in high-fat-fed rats, and this drug appears to be a useful one with a new mode of action for the treatment of NIDDM with insulin resistance.

A mutation (Trp1193-->Leu1193) in the tyrosine kinase domain of the insulin receptor associated with type A syndrome of insulin resistance.

We evaluated a 35-year-old diabetic male patient with type A insulin resistance, showing acanthosis nigricans. Insulin binding to the patient's Epstein-Barr-virus transformed lymphocytes was mildly reduced. The maximal insulin-stimulated autophosphorylation of the insulin receptor from the patient's transformed lymphocytes was decreased to 45% of that from the control subjects. On examination, the biological activities of insulin and insulin-like growth factor I in the patient's cultured fibroblasts, insulin sensitivity of amino isobutyric acid uptake and thymidine incorporation was decreased, but insulin-like growth factor I action was normal. The sequence analysis of amplified genomic DNA revealed that the patient was heterozygous for a mutation substituting Leu for Trp at codon 1193 in exon 20 of the insulin receptor gene. The patient's mother and sister were also heterozygous for a mutation in the insulin receptor gene that substituted Leu for Trp1193 in the beta subunit of the receptor. Therefore, the mutation causes insulin resistance in a dominant fashion. They were less hyperglycaemic and more hyperinsulinaemic than the proband after glucose loading. The mother had diabetes mellitus but did not show acanthosis nigricans, while the sister did not have diabetes and showed acanthosis nigricans. These results suggest that this mutation causes defective tyrosine kinase activity of the insulin receptor, which results in insulin resistance. Insulin action and phenotypic appearance may be mediated by different factors.

[A case of Cushing syndrome caused by ACTH producing atypical lung carcinoid tumor].

A forty-one-year-old male who was found to have lung carcinoid tumor showed clinical features of Cushing syndrome. At the age of 38 years during a regular check-up, a chest roentgenogram showed multiple nodular shadows in the bilateral lung, and he was admitted to hospital. By open lung biopsy, he was diagnosed as having malignant carcinoid or small cell carcinoma of the lung. The abnormal lung shadows did not respond to chemotherapy (CDDP + VDS). The clinical course after discharge was uneventful until the age of 41 years, when he was readmitted to our hospital because of brain metastasis. Physical examination revealed moon face and central obesity. Plasma ACTH level was high and dexamethasone suppression test showed no cortisol suppression on 8 mg dexamethasone administration. Therefore, it was thought that the patient had ACTH producing ectopic tumor which led to Cushing syndrome. We measured the molecular weight of ACTH by column chromatography and found he had a big ACTH (molecular weight about 22,000). Reexamining him clinically and histologically, we concluded that the patient had atypical carcinoid tumor in the lung which might produce ACTH causing Cushing syndrome with metastasis to the brain.

Pioglitazone increases insulin sensitivity by activating insulin receptor kinase.

A new oral agent, 5-[4-(2-(5-ethyl 12-pyridyl)ethoxy]- benzoyl]-2,4-thiazolidinedione (pioglitazone), has been developed for treatment of non-insulin-dependent diabetes mellitus (NIDDM). This agent increases insulin sensitivity in vivo in genetically obese Wistar fatty rats. Administration of the agent (3 mg/kg/day) for 10 days to the rats ameliorated hyperglycemia and hyperinsulinemia, indicating that it decreased insulin resistance. To clarify the mechanism of the drug to increase insulin sensitivity, we examined insulin binding and kinase activity of insulin receptors from muscles of both untreated and treated rats. Pioglitazone treatment did not change insulin binding in Wistar fatty rats but increased insulin-stimulated autophosphorylation of insulin receptors to 78% over the level in the control but not the basal state. Kinase activity toward exogenous substrate, poly Glu4Tyr1, was also increased to 87% over the level of untreated control obese rats. In contrast, in lean rats, pioglitazone treatment did not increase autophosphorylation and kinase activity toward exogenous substrates. To further elucidate the mechanism, we incubated insulin receptors with the agent and measured kinase activity. Incubation of solubilized receptors with the agent did not increase kinase activity. However, the receptors from IM-9 cells, which were incubated with 10(-8) M pioglitazone for 7 days, showed a 46% increase over the control in insulin-stimulated autophosphorylation and kinase activity. These results suggested that pioglitazone increased insulin sensitivity in part by activating kinase of the receptors through indirect effect on insulin receptors and that the drug may have useful benefits in insulin resistance of NIDDM.

[A rare case of allergic granulomatous angitis (Churg Strauss syndrome) with positive anti-glomerular basement membrane (GBM) antibody in serum].

A 33-year-old man with a 6-month history of rhinitis and bronchial asthma was referred to our hospital with polyarthralgia, severe anemia, hypoxemia, mononeuropathy multiplex, and renal insufficiency with hematuria. Marked eosinophilia was observed in his sputum, peripheral blood, and bronchoalveolar lavage fluid (BALF). In addition, his sputum contained many hemosiderin-laden macrophages, indicative of pulmonary hemorrhage. His chest roentgenogram on admission showed diffuse ground grass appearance. High resolution computed tomography (HRCT) demonstrated diffuse high density areas throughout the lung fields and characteristic irregularity and enlargement of the peripheral pulmonary arteries. His general condition rapidly deteriorated, but dramatically improved with oral steroid administration, and his major symptoms disappeared within a few days. Examination of the biopsied lung tissue revealed unequivocal evidence of pulmonary angitis with marked eosinophilic infiltration and perivascular granulomas. Bone marrow biopsy showed hyperplasia of eosinophilic leukocytes in contrast to the low cellularity. Suppression of erythroid hemopoiesis was thought to be the primary cause for his rapidly progressive anemia. Serum anti-GBM antibody titer returned to within the normal range soon after the initiation of steroid therapy.

Receptor binding and biologic activity of biosynthetic human insulin and mini-proinsulin produced by recombinant gene technology.

Human insulin and its precursor, mini-proinsulin, made with a new biosynthetic method, were tested for their receptor binding, biologic action, and antibody binding ability. The structure of mini-proinsulin is similar to that of proinsulin with a shortened C-peptide, B(1-29)-Ala-Ala-Lys-A(1-21) insulin. The ability of biosynthetic human insulin to bind to receptors, to stimulate 2-deoxyglucose uptake in isolated adipocytes, and to bind to insulin antibody was comparable to that of semisynthetic human insulin. The ability of mini-proinsulin to bind to insulin receptors and to stimulate 2-deoxyglucose uptake in adipocytes was 0.5 and 0.2% that of human insulin, whereas the corresponding abilities of proinsulin were 5 and 3%, respectively. Despite having less receptor binding and biologic activity, mini-proinsulin demonstrated higher affinity for the insulin antibody than did proinsulin. These results suggest that biosynthetic human insulin behaves similarly to semisynthetic human insulin in its receptor binding and biologic activity, and that the shortened C-peptide region reduces receptor binding by fixing or covering the N-terminal region of the A chain, which is important for receptor binding.