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H-35 rat hepatoma cells were labelled with [32P]orthophosphate and their insulin receptors isolated on wheat germ agglutinin (WGA)-agarose and anti-(insulin receptor) serum. The incubation of these cells with 10 mM-H2O2 for 10 min increased the phosphorylation of both the serine and tyrosine residues of the beta subunit of the insulin receptor. Next, insulin receptors were purified on WGA-agarose from control and H2O2-treated H-35 cells and the purified fractions incubated with [gamma-32P]ATP and Mn2+. Phosphorylation of the beta subunit of insulin receptors obtained from H2O2-treated cells was 150% of that of control cells. The kinase activity of the WGA-purified receptor preparation obtained from H2O2-treated cells, as measured by phosphorylation of src-related synthetic peptide, was increased about 4-fold over control cells. These data suggest that in intact cell systems, H2O2 may increase the insulin receptor kinase activity by inducing phosphorylation of the beta subunit of insulin receptor.

The effect of steroid hormones on insulin binding and the amount of insulin-receptor mRNA was examined in IM-9 lymphocytes. Cortisol and cortexolone, but not oestrogen, increased both the binding of insulin and the amount of insulin-receptor mRNA in a time- and dose-dependent manner. Cortisol was most potent, and induced a 2-fold increase in insulin binding and a 4-fold increase in mRNA. The elevation in binding was due to an increased number of insulin receptors at the cell surface. The increase in mRNA involved all four of the insulin-receptor mRNAs and could not be inhibited by cycloheximide. The cortisol-induced increase in mRNA was associated with a 3-4-fold increase in the synthesis of pro-receptor. The relative potency of the three steroids indicated that these effects were mediated by an interaction with the glucocorticoid receptor. The results of this study suggest that cortisol can increase the number of insulin receptors at the cell surface by increasing the amounts of insulin-receptor mRNA and the synthesis de novo of insulin receptors.

Two kinds of experiments have been performed to study a role of apoA-1 in the LCAT reaction of HDL. Antiserum against apoA-1 was prepared in a rabbit, and the globulin fraction was obtained by ammonium sulfate precipitation after ultracentrifugation at D = 1.25 g/ml. Antibody fraction revealed a precipitation line against HDL as well as against apoA-1. Anti-apoA-1 significantly inhibited the esterification of cholesterol in the medium containing lecithin:cholesterol emulsions, apoA-1 and partially purified LCAT On the other hand, when anti-apoA-1 was added to the incubation mixture containing LCAT and apoHDL instead of apoA-1, the inhibitory effect of anti-apoA-1 disappeared; rather the LCAT reaction was slightly stimulated. This slight stimulatory effect of anti-apoA-1 was also demonstrated when heated HDL, or heated plasma was used as a substrate. The normal globulin fraction of rabbit serum when added in the media containing artificial substrate and apoA-1 or apoHDL and in the media with heated HDL, non-specifically slightly inhibited the LCAT reaction. LCAT could bind to HDL-Sepharose. and if the gels were pretreated with anti-apoA-1, the binding of this enzyme was slightly inhibited. The LCAT binding to apo-HDL-Sepharose was extremely low compared with its binding to HDL-Sepharose. These findings are compatible with the concept that, although apoA-1 stimulates the LCAT reaction in the system where only this apolipoprotein is present as a cofactor, it masks other activator(s), when HDL or artificial substrate with apoHDL are incubated with LCAT.

In muscle and fat, glucose transport occurs through the translocation of GLUT4 from an intracellular pool to the cell surface. Phosphatidylinositol (PI) 3-kinase has been shown to be required in this process. Insulin is thought to activate this enzyme by stimulating its association with tyrosine-phosphorylated proteins such as insulin receptor substrate (IRS)-1, IRS-2, Grb2-associated binder-1, and pp60. To study the role of these endogenous substrates in glucose transport, we analyzed adipocytes from IRS-1 null mice that we previously generated (Tamemoto, H., Kadowaki, T., Tobe, K., Yagi, T., Sakura, H., Hayakawa, T., Terauchi, Y., Ueki, K., Kaburagi, Y., Satoh, S., Sekihara, H., Yoshioka, S., Horikoshi, H., Furuta, Y. , Ikawa, Y., Kasuga, M., Yazaki Y., and Aizawa S. (1994) Nature 372, 182-186). In adipocytes from these mice, we showed that: 1) insulin-induced PI 3-kinase activity in the antiphosphotyrosine immunoprecipitates was 54% of wild-type; 2) pp60 was the major tyrosine-phosphorylated protein that associated with PI 3-kinase, whereas tyrosine phosphorylaion of IRS-2 as well as its association with this enzyme was almost undetectable; and 3) glucose transport and GLUT4 translocation at maximal insulin stimulation were decreased to 52 and 68% of those from wild-type. These data suggest that both IRS-1 and pp60 play a major role in insulin-induced glucose transport in adipocytes, and that pp60 is predominantly involved in regulating this process in the absence of IRS-1.

The Trp 64 Arg mutation in the β₃-adrenergic receptor (β₃AR) gene was investigated in 350 Japanese subjects. This mutation was not associated with non-insulin-dependent diabetes mellitus (NIDDM). In 191 subjects without NIDDM, body mass index (BMI) was significantly higher in subjects homozygous for this mutation than in those homozygous for the normal allele (24.7±1.4 vs 22.1±0.2 kg/m2, p=0.009). Moreover, the frequency of the mutant allele in obese subjects (BMI>26.4) was significantly higher than that in non-obese subjects (BMI<22) (0.37 vs 0.15, p=0.009). The presence of this mutation was also accompanied by significantly higher fasting (p=0.000) and 2 hrs (p=0.018) serum insulin levels during an oral glucose tolerance test. The β₃AR may be one of the loci contributing to obesity and hyperinsulinemia/insulin resistance in Japanese subjects.Copyright 1995, 1999 Academic Press, Inc.

We studied a simple tandem repeal DNA polymorphism in the glycogen synthase gene and polymorphisms at codon 513 (Ala→Pro) and 972 (Gly→Arg) in the insulin receptor substrate-1 (IRS-1) gene in 197 non-insulin-dependent diabetes mellitus (NIDDM) and 178 control subjects in Japan. Eight alleles (−3G, −2G, −1G, 0G, 1G, 2G, 3G, and 4G) were identified in the tandem repeat polymorphism in the glycogen synthase gene. No difference in the frequencies of these alleles was found between diabetics and controls. The codon 972 polymorphism of IRS-1 gene was observed in 7 diabetics (3.6%) and 8 controls (4.5%), whereas the codon 513 polymorphism was not found in either of the two groups. We conclude that the tandem repeat polymorphism in the glycogen synthase gene and the polymorphisms at codons 513 and 972 of the IRS-1 gene are not associated with a higher risk for the development of NIDDM in Japanese subjects.Copyright 1994, 1999 Academic Press, Inc.

We have prepared site-specific antibodies toward human insulin, insulin-like growth factor-I, and epidermal growth factor receptors with chemically synthesized peptides derived from the cDNA-predicted amino acid sequences of these receptors. Two classes of antibodies were produced toward each receptor: one toward the carboxyl termini and the other against the kinase domains containing sequences homologous to the tyrosyl phosphorylation site of the product of src gene (pp60v-src). Both classes of antibodies specifically immunoprecipitated the appropriate 125I-ligand-receptor complexes and [35S]methionine-labeled receptors with almost equal potencies. Antibodies toward the kinase domains inhibited both autophosphorylation and tyrosine kinase activity of the corresponding receptors in a cell-free system, whereas antibodies toward the carboxyl termini did not. Microinjection of the kinase-inhibitory antibodies into the cytoplasm of human epidermoid carcinoma KB cells blocked the ability of the corresponding ligand to induce membrane ruffling. In contrast, these inhibitory antibodies did not block the ability of noncorresponding ligands to induce the same response. Furthermore, control immunoglobulin and antibodies toward the carboxyl termini did not block this biological response. These results support a role for the tyrosine-specific protein kinase activities of these growth factor receptors in mediating their biological effects and suggest that the regions homologous to the tyrosyl phosphorylation site of pp60v-src are important for these kinase activities both in cell-free and intact cell systems.

It has been documented that streptozotocin-induced diabetes in rats is associated with diminished effects of insulin despite increased insulin binding to its receptor. This paradox led us to examine whether any alterations of insulin receptor-kinase activities occur in this type of insulin resistance. Insulin binding capacity/mg of protein of solubilized, wheat germ agglutinin-purified preparations from livers was increased by 1.8-fold in the streptozotocin (65 mg/kg) diabetic rats. This increase was associated with a parallel increase in receptor protein as measured by an immunoblotting method using anti-insulin receptor antibody. Moreover, no apparent change was observed in the stoichiometry of alpha and beta subunits of the insulin receptor between diabetic and control rats. Insulin-stimulated (10(-7) M) phosphorylation of the beta subunit of the insulin receptor was decreased by 40% in diabetic rats when equal quantities of insulin binding capacity were compared. Phosphorylation of an exogenously added synthetic peptide (similar in sequence to the tyrosine phosphorylation site in pp60src) by the insulin receptor-kinase was also decreased by 25% in diabetic rats. These abnormalities were partially restored by in vivo insulin treatment. These data suggest that diminished insulin receptor autophosphorylation and kinase activity could provide a possible mechanism for the "post-binding insulin resistance" in diabetic rats.

The purified insulin receptor kinase catalyzed the phosphorylation of native tubulin and microtubule-associated proteins (MAPs; MAP2, tau) on tyrosine residues. Insulin (10(-7) M) stimulated the reaction by 4-10-fold by increasing Vmax with little change in Km. alpha-Tubulin was preferred as a substrate for the kinase compared to beta-tubulin. MAP2 was found to be the best substrate among the cytoskeletal proteins tested; in the presence of insulin, the Vmax for MAP2 was 6.3 nmol/min/mg, its Km was 5.1 microM, and 1.7 mol of phosphate were incorporated per mol of MAP2. Under the same conditions used for this phosphorylation of tubulin and MAPs, actin and tropomyosin were very poorly phosphorylated. These data, coupled with previous evidence for potential functional relationships between insulin action and microtubules, raise the possibility that microtubule proteins may be cellular targets for the insulin receptor kinase.

The clinical efficacy of troglitazone, a new oral hypoglycaemic agent was investigated in Type 2 diabetes in combination with sulphonylureas. Two hundred and ninety‐one patients with Type 2 diabetes (age 21–81 years) whose previous glycaemic control by sulphonylureas was judged stable but unsatisfactory (fasting plasma glucose (FPG) > 8.3 mmol l−1) were randomly allocated into the troglitazone treatment group (troglitazone group, n= 145) or the placebo treatment group (placebo group, n= 146). They were treated by test drugs for 12 weeks in combination with the same dose of sulphonylureas before the trial. One hundred and twenty‐two patients who received troglitazone and 126 patients who received placebo were evaluated for efficacy. The baseline characteristics did not differ significantly between the two groups. In the troglitazone group, FPG and HbA1Cdecreased significantly after the treatment (before vs after, FPG: 10.8 ± 2.0 mmol l−1vs 9.2 ± 2.5 mmol l−1, p< 0.001; HbA1C: 9.2 ± 1.4 % vs 8.5 ± 1.5 %, p< 0.001). FPG and HbA1Cdid not change after the treatment in the placebo group (before vs after, FPG: 10.5 ± 1.7 mmol l−1vs 10.7 ± 2.2 mmol l−1; HbA1C: 9.0 ± 1.5 % vs 9.2 ± 1.6 %). Serum total cholesterol and HDL‐cholesterol did not change in either group, however, serum triglyceride significantly decreased in the troglitazone group. No serious adverse events occurred in either group. In conclusion, troglitazone 400 mg day−1had a significant hypoglycaemic effect in combination with sulphonylureas without any serious adverse events. Troglitazone, developed as an insulin action enhancer, can be a useful hypoglycaemic agent in the treatment of patients with Type 2 diabetes who are not well controlled by sulphonylureas alone.

Recently, a mitochondrial mutation was found to be associated with maternally inherited diabetes mellitus (Kadowaki, T., Kadowaki, H., Mori, Y., Tobe, K., Sakuta, R., Suzuki, Y., Tanabe, Y, Sakura, H., Awata, T., Goto, Y., Hayakawa, T., Matsuoka, K., Kawamori, R., Kamada, T., Horai, S., Nonaka, I., Hagura, R., Akanuma, Y., and Yazaki, Y. (1994) N. Engl. J. Med. 330, 962-968). In order to elucidate its etiology, we have investigated the involvement of mitochondrial function in insulin secretion. Culture of the pancreatic beta-cell line, betaHC9, with low dose ethidium bromide (EB) (0.4 microg/ml) for 2-6 days resulted in a substantial decrease in the transcription level of mitochondrial DNA (to 10-20% of the control cells) without changing its copy number, whereas the transcription of nuclear genes was grossly unaffected. Electron microscopic analysis revealed that treatment by EB caused morphological changes only in mitochondria and not in other organelles such as nuclei, endoplasmic reticula, Golgi bodies, or secretory granules. When the cells were treated with EB for 6 days, glucose (20 mM) could no longer stimulate insulin secretion, while glibenclamide (1 microM) still did. When EB was removed after 3- or 6-day treatment, mitochondrial gene transcription recovered within 2 days, and the profiles of insulin secretion returned to normal within 7 days. Studies with fura-2 indicated that in EB-treated cells, glucose (20 mM) failed to increase intracellular Ca2+, while the effect of glibenclamide (1 microM) was maintained. Our system provides a unique way to investigate the relationship between mitochondrial function and insulin secretion.

Growth hormone (GH) and prolactin (PRL) binding to their receptors, which belong to the cytokine receptor superfamily, activate Janus kinase (JAK) 2 tyrosine kinase, thereby leading to their biological actions. We recently showed that GH mainly stimulated tyrosine phosphorylation of epidermal growth factor receptor and its association with Grb2, and concomitantly stimulated mitogen-activated protein kinase activity in liver, a major target tissue. Using specific antibodies, we now show that GH was also able to induce tyrosine phosphorylation of insulin receptor substrate (IRS)-1/IRS-2 in liver. In addition, the major tyrosine-phosphorylated protein in anti-p85 phosphatidylinositol 3-kinase (PI3-kinase) immunoprecipitate from liver of wild-type mice was IRS-1, and IRS-2 in IRS-1 deficient mice, but not epidermal growth factor receptor. These data suggest that tyrosine phosphorylation of IRS-1 may be a major mechanism for GH-induced PI3-kinase activation in physiological target organ of GH, liver. We also show that PRL was able to induce tyrosine phosphorylation of both IRS-1 and IRS-2 in COS cells transiently transfected with PRLR and in CHO-PRLR cells. Moreover, we show that tyrosine phosphorylation of IRS-3 was induced by both GH and PRL in COS cells transiently transfected with IRS-3 and their cognate receptors. By using the JAK2-deficient cell lines or by expressing a dominant negative JAK2 mutant, we show that JAK2 is required for the GH- and PRL-dependent tyrosine phosphorylation of IRS-1, -2, and -3. Finally, a specific PI3-kinase inhibitor, wortmannin, completely blocked the anti-lipolytic effect of GH in 3T3 L1 adipocytes. Taken together, the role of IRS-1, -2, and -3 in GH and PRL signalings appears to be phosphorylated by JAK2, thereby providing docking sites for p85 PI3-kinase and activating PI3-kinase and its downstream biological effects.

The ATP-sensitive K channel (K-ATP channel) in pancreatic β cells is believed to play a crucial role in glucose stimulated insulin release. We investigated whether defects in the recently cloned gene for a putative subunit of this channel (KATP-2) could be a cause of diabetes in Japanese patients. The coding region of this β-cell type channel gene was investigated in 192 diabetics with a family history of the disorder by single-stranded conformational polymorphism (SSCP) analysis. Two silent polymorphisms were found and confirmed by sequencing, but no missense or nonsense mutations were detected. The allele frequency of the polymorphisms was compared with 96 control subjects without a family history of the disease, and no clear difference was found. These results indicate that genetic defects of the KATP-2 channel may not be a major cause of diabetes in Japan.

Two kinds of experiments have been performed to study a role of apoA-1 in the LCAT reaction of HDL. Antiserum against apoA-1 was prepared in a rabbit, and the globulin fraction was obtained by ammonium sulfate precipitation after ultracentrifugation at D = 1.25 g/ml. Antibody fraction revealed a precipitation line against HDL as well as against apoA-1. Anti-apoA-1 significantly inhibited the esterification of cholesterol in the medium containing lecithin:cholesterol emulsions, apoA-1 and partially purified LCAT On the other hand, when anti-apoA-1 was added to the incubation mixture containing LCAT and apoHDL instead of apoA-1, the inhibitory effect of anti-apoA-1 disappeared; rather the LCAT reaction was slightly stimulated. This slight stimulatory effect of anti-apoA-1 was also demonstrated when heated HDL, or heated plasma was used as a substrate. The normal globulin fraction of rabbit serum when added in the media containing artificial substrate and apoA-1 or apoHDL and in the media with heated HDL, non-specifically slightly inhibited the LCAT reaction. LCAT could bind to HDL-Sepharose. and if the gels were pretreated with anti-apoA-1, the binding of this enzyme was slightly inhibited. The LCAT binding to apo-HDL-Sepharose was extremely low compared with its binding to HDL-Sepharose. These findings are compatible with the concept that, although apoA-1 stimulates the LCAT reaction in the system where only this apolipoprotein is present as a cofactor, it masks other activator(s), when HDL or artificial substrate with apoHDL are incubated with LCAT.

We report a homozygous missense mutation at position 1092 (substitution of glutamine for arginine) in the tyrosine kinase domain of the insulin receptor in a patient with leprechaunism associated with severe insulin resistance and intrauterine growth retardation. Site-directed mutagenesis as well as analyses of the patient's lymphocytes revealed that this mutation causes a marked decrease in tyrosine kinase activity of the insulin receptor without any defect in insulin binding, which causes severe defects in insulin-stimulated glucose transport, glycogen synthesis and DNA synthesis. Thus, this is the first homozygous mutation resulting in a selective-kinase defect of the insulin receptor. Interestingly, the parents who are cousins and are heterozygous for the mutation have type A insulin resistance syndrome. This correlation between genotype and phenotype in a single pedigree suggests that the severity of the mutation will determine the phenotype. Based upon this assumption, we have been successful in prenatal diagnosis of the fifth child. Furthermore, we have demonstrated the effectiveness of clinical administration of insulin-like growth factor-I (IGF-I) in this patient and in vitro analysis of the patient's skin fibroblasts, suggesting that IGF-I can compensate for insulin action via the IGF-I receptor in a patient almost lacking functional insulin receptors. [Diabetologia (1997) 40: 412–420]We report a homozygous missense mutation at position 1092 (substitution of glutamine for arginine) in the tyrosine kinase domain of the insulin receptor in a patient with leprechaunism associated with severe insulin resistance and intrauterine growth retardation. Site-directed mutagenesis as well as analyses of the patient's lymphocytes revealed that this mutation causes a marked decrease in tyrosine kinase activity of the insulin receptor without any defect in insulin binding, which causes severe defects in insulin-stimulated glucose transport, glycogen synthesis and DNA synthesis. Thus, this is the first homozygous mutation resulting in a selective-kinase defect of the insulin receptor. Interestingly, the parents who are cousins and are heterozygous for the mutation have type A insulin resistance syndrome. This correlation between genotype and phenotype in a single pedigree suggests that the severity of the mutation will determine the phenotype. Based upon this assumption, we have been successful in prenatal diagnosis of the fifth child. Furthermore, we have demonstrated the effectiveness of clinical administration of insulin-like growth factor-I (IGF-I) in this patient and in vitro analysis of the patient's skin fibroblasts, suggesting that IGF-I can compensate for insulin action via the IGF-I receptor in a patient almost lacking functional insulin receptors. [Diabetologia (1997) 40: 412–420]

Insulin treatment of rat H-35 hepatoma cells causes rapid tyrosine phosphorylation of a high molecular weight protein termed pp185 besides autophosphorylation of the β-subunit of the insulin receptor (IR) in an intact cell system. To elucidate the molecular basis for tyrosine phosphorylation of pp185, cell-free phosphorylation of pp185 was performed using phosphotyrosine-containing proteins (PYPs) purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibody. After insulin treatment of cells, marked increases of tyrosine phosphorylation of pp185 and IR were observed compared to noninsulin-treated cells. Site-specific antibodies that specifically inactivate IR kinase inhibited tyrosine phosphorylation of pp185 as well as the β-subunit of IR. PYPs purified from detergent-free cell extracts contained pp185 but little IR; tyrosine phosphorylation of pp185 did not occur. Addition of IR kinase purified from human placenta to these PYPs restored insulin-dependent tyrosine phosphorylation of pp185. These results suggest that tyrosine phosphorylation of pp185 is catalyzed directly by IR kinase in this cell-free system.

Apolipoprotein E (apoE) is known to play an important role in lipoprotein metabolism. We have studied the effect of apoE on the metabolism of plasma cholesterol by injecting apoE intravenously into rabbits deficient in low density lipoprotein receptors [Watanabe heritable hyperlipidemic (WHHL) rabbits]. Approximately 30 mg of apoE was injected per rabbit; a total of five WHHL rabbits were used. One hour later, plasma cholesterol levels fell 8.3% (from 488 +/- 192 to 446 +/- 174 mg/dl). After 3 hr, cholesterol levels had fallen by 19% (to 392 +/- 152 mg/dl). The reduced levels were maintained for at least 8 hr after injection of apoE. Cholesterol in very low density lipoproteins (VLDLs) and intermediate density lipoproteins fell rapidly during the first 2 hr after injection, followed by a reduction in the low density lipoprotein cholesterol level. Changes in apolipoprotein B levels in each lipoprotein fraction were very similar to those of cholesterol. Plasma apoE levels 3 min after injection were elevated 3-fold to 22.8 +/- 6.3 mg/dl and returned to initial levels 8 hr after injection. The rate of removal of intravenously injected 125I-labeled VLDL that had been incubated with apoE was 3-fold higher than that of unmodified VLDL. From these results, we conclude that the injected apoE is incorporated into VLDLs and that VLDL particles carrying more apoE are removed from the blood more rapidly, resulting in reduced formation of low density lipoprotein and lowered cholesterol levels.

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.

We and others recently generated mice with a targeted disruption of the insulin receptor substrate 1 (IRS-1) gene and demonstrated that they exhibited growth retardation and had resistance to the glucose-lowering effect of insulin. Insulin initiates its biological effects by activating at least two major signalling pathways, one involving phosphatidylinositol 3-kinase (PI3-kinase) and the other involving a ras/mitogen-activated protein kinase (MAP kinase) cascade. In this study, we investigated the roles of IRS-1 and IRS-2 in the biological action in the physiological target organs of insulin by comparing the effects of insulin in wild-type and IRS-1-deficient mice. In muscles from IRS-1-deficient mice, the responses to insulin-induced PI3-kinase activation, glucose transport, p70 S6 kinase and MAP kinase activation, mRNA translation, and protein synthesis were significantly impaired compared with those in wild-type mice. Insulin-induced protein synthesis was both wortmannin sensitive and insensitive in wild-type and IRS-1 deficient mice. However, in another target organ, the liver, the responses to insulin-induced PI3-kinase and MAP kinase activation were not significantly reduced. The amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) was roughly equal to that of IRS-1 (in wild-type mice) in the liver, whereas it only 20 to 30% of that of IRS-1 in the muscles. In conclusion, (i) IRS-1 plays central roles in two major biological actions of insulin in muscles, glucose transport and protein synthesis; (ii) the insulin resistance of IRS-1-deficient mice is mainly due to resistance in the muscles; and (iii) the degree of compensation for IRS-1 deficiency appears to be correlated with the amount of tyrosine-phosphorylated IRS-2 (in IRS-1-deficient mice) relative to that of IRS-1 (in wild-type mice).

Transfection of Chinese hamster ovary cells with the expression vector containing rabbit brain HepG2-type glucose transporter cDNA resulted in a dramatic over-expression (approximately 10-fold) of glucose transporter as assessed by either immunoblotting with antipeptide antibody against rabbit brain glucose transporter or photoaffinity labeling with [3H]cytochalasin B. 2-Deoxyglucose uptake was also increased 4-fold in the transfected cells, while no increase in transport activity or transporter amount was observed in cells that were transfected with the expression vector alone without glucose transporter cDNA. Significantly, insulin (10−7M) increased 2-deoxyglucose uptake in both control and transfected cells, but the increased amount of the transported 2-deoxyglucose by insulin in the transfected cells was 4.2-fold greater than that in control cells, indicating that the expressed rabbit brain HepG2-type glucose transporter responded to insulin. In addition, we have recently demonstrated that the HepG2-type glucose transporter exists in rat adipocytes and responds to insulin in a fashion similar to a majority of other types of glucose transporters (Oka, Y., Asano, T., Shibasaki, Y., Kasuga, M., Kanazawa, Y., and Takaku, F. (1988) J. Biol. Chem. 263, 13432–13439). In contrast, insulin did not stimulate glucose transport activity in HepG2 cells or IM-9 lymphocytes that have a significant amount of the HepG2-type glucose transporter. Thus, the results in this study further support the notion that insulin regulation of glucose transport activity depends on a tissue-specific signaling mechanism.

Concanavalin A (ConA) stimulated the phosphorylation of the beta-subunit of the insulin receptor and an Mr-185,000 protein on serine and tyrosine residues in intact H-35 rat hepatoma cells. This Mr-185,000 protein whose phosphorylation was stimulated by ConA was identical to pp185, a protein reported previously to be a putative endogenous substrate for the insulin receptor tyrosine kinase in rat hepatoma cells. In Chinese hamster ovary (CHO) cells transfected with cDNA of the human insulin receptor, tyrosine-phosphorylation of pp185 was strongly enhanced by ConA compared with the controls, suggesting that the induction of tyrosine-phosphorylation of pp185 was due to stimulation of the insulin receptor kinase by ConA. Moreover, monovalent ConA only slightly induced the tyrosine-phosphorylation of pp185, which was enhanced by the addition of anti-ConA IgG, suggesting that ConA stimulated the insulin receptor kinase mainly by the receptor cross-linking or aggregation in intact cells. These data suggest that the insulin-mimetic action of ConA is related to the autophosphorylation and activation of the insulin receptor tyrosine kinase, as well as the subsequent phosphorylation of pp185 in intact cells.

Various biological responses stimulated by insulin have been thought to be regulated by phosphatidylinositol 3-kinase, including glucose transport, glycogen synthesis, and protein synthesis. However, the molecular link between phosphatidylinositol 3-kinase and these biological responses has been poorly understood. Recently, it has been shown that protein kinase B (PKB/c-Akt/Rac) lies immediately downstream from phosphatidylinositol 3-kinase. Here, we show that expression of a constitutively active form of PKB induced glucose uptake, glycogen synthesis, and protein synthesis in L6 myotubes downstream of phosphatidylinositol 3-kinase and independent of Ras and mitogen-activated protein kinase activation. Introduction of constitutively active PKB induced glucose uptake and protein synthesis but not glycogen synthesis in 3T3L-1 adipocytes, which lack expression of glycogen synthase kinase 3 different from L6 myotubes. Furthermore, we show that deactivation of glycogen synthase kinase 3 and activation of rapamycin-sensitive serine/threonine kinase by PKB in L6 myotubes might be involved in the enhancement of glycogen synthesis and protein synthesis, respectively. These results suggest that PKB acts as a key enzyme linking phosphatidylinositol 3-kinase activation to multiple biological functions of insulin through regulation of downstream kinases in skeletal muscle, a major target tissue of insulin.

Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on multiple tyrosine residues in response to insulin and binds several Src homology 2 domain-containing proteins, thereby initiating downstream signaling. To assess the tyrosine phosphorylation sites that mediate relevant downstream signaling and biological effects, we created site-directed mutants of IRS-1 and overexpressed them in the Xenopus laevis oocyte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced with phenylalanine), insulin activated phosphatidylinositol (PI) 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and induced oocyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr-460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanine), insulin did not activate PI 3-kinase, p70 S6 kinase, and mitogen-activated protein kinase and failed to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound protein 2) with IRS-1 plays a major role in insulin-induced oocyte maturation. Activation of PI 3-kinase may lie upstream of mitogen-activated protein kinase activation and p70 S6 kinase activation in response to insulin.

The Trp64Arg mutation of the β3-adrenergic receptor (β3AR) is prevalent in several ethnic groups and is associated with weight gain, and some features of syndrome X such as insulin resistance and dyslipidaemia. Nevertheless, it is not known at present whether this mutation is associated with visceral obesity, which is an important risk factor for the development of hypertension, dyslipidaemia, insulin resistance, non-insulin-dependent diabetes mellitus, and atherosclerosis. To investigate whether this mutation may contribute to visceral obesity, we studied the relationships between β3AR genotypes and clinical phenotypes. The Trp64Arg allele of β3AR was examined in 278 Japanese men with respect to variables relating to visceral obesity assessed by computerised tomography. To detect the Trp64Arg mutation, polymerase chain reaction-restriction fragment length polymorphism analysis using Bst NI digestion was performed. This mutation was more frequently observed in subjects with higher body mass index (BMI) (p= 0.02). Moreover, in 120 subjects with a moderate degree of obesity (22 M BMI < 26.4 kg/m2), the mutation (homozygotes and heterozygotes) was associated with visceral obesity (higher ratio of visceral to subcutaneous fat area; V/S) (p= 0.03). Furthermore, the Trp64Arg allele was more frequent in subjects with lower serum triglyceride levels (p= 0.02) and the Trp64Arg homozygotes, but not heterozygotes, exhibited lower triglyceride levels. Thus, this mutation appears to be associated with visceral obesity but with lower serum triglyceride. It is suggested that those with the mutation may describe a subset of subjects characterized by decreased lipolysis in visceral adipose tissue. [Diabetologia (1997) 40: 469–472]The Trp64Arg mutation of the β3-adrenergic receptor (β3AR) is prevalent in several ethnic groups and is associated with weight gain, and some features of syndrome X such as insulin resistance and dyslipidaemia. Nevertheless, it is not known at present whether this mutation is associated with visceral obesity, which is an important risk factor for the development of hypertension, dyslipidaemia, insulin resistance, non-insulin-dependent diabetes mellitus, and atherosclerosis. To investigate whether this mutation may contribute to visceral obesity, we studied the relationships between β3AR genotypes and clinical phenotypes. The Trp64Arg allele of β3AR was examined in 278 Japanese men with respect to variables relating to visceral obesity assessed by computerised tomography. To detect the Trp64Arg mutation, polymerase chain reaction-restriction fragment length polymorphism analysis using Bst NI digestion was performed. This mutation was more frequently observed in subjects with higher body mass index (BMI) (p= 0.02). Moreover, in 120 subjects with a moderate degree of obesity (22 M BMI < 26.4 kg/m2), the mutation (homozygotes and heterozygotes) was associated with visceral obesity (higher ratio of visceral to subcutaneous fat area; V/S) (p= 0.03). Furthermore, the Trp64Arg allele was more frequent in subjects with lower serum triglyceride levels (p= 0.02) and the Trp64Arg homozygotes, but not heterozygotes, exhibited lower triglyceride levels. Thus, this mutation appears to be associated with visceral obesity but with lower serum triglyceride. It is suggested that those with the mutation may describe a subset of subjects characterized by decreased lipolysis in visceral adipose tissue. [Diabetologia (1997) 40: 469–472]

Insulin has pleiotropic effects on the regulation of cell physiology through binding to its receptor. The wide variety of tyrosine phosphorylation motifs of insulin receptor substrate 1 (IRS-1), a substrate for the activated insulin receptor tyrosine kinase, may account for the multiple functions of insulin. Recent studies have shown that activation of the insulin receptor leads to the regulation of focal adhesion proteins, such as a dephosphorylation of focal adhesion kinase (pp125FAK). We show here that C-terminal Src kinase (Csk), which phosphorylates C-terminal tyrosine residues of Src family protein tyrosine kinases and suppresses their kinase activities, is involved in this insulin-stimulated dephosphorylation of focal adhesion proteins. We demonstrated that the overexpression of Csk enhanced and prolonged the insulin-induced dephosphorylation of pp125FAK. Another focal adhesion protein, paxillin, was also dephosphorylated upon insulin stimulation, and a kinase-negative mutant of Csk was able to inhibit the insulin-induced dephosphorylation of pp125FAK and paxillin. Although we have shown that the Csk Src homology 2 domain can bind to several tyrosine-phosphorylated proteins, including pp125FAK and paxillin, a majority of protein which bound to Csk was IRS-1 when cells were stimulated by insulin. Our data also indicated that tyrosine phosphorylation levels of IRS-1 appear to be paralleled by the dephosphorylation of the focal adhesion proteins. We therefore propose that the kinase activity of Csk, through the insulin-induced complex formation of Csk with IRS-1, is involved in insulin's regulation of the phosphorylation levels of the focal adhesion proteins, possibly through inactivation of the kinase activity of c-Src family kinases.

GLUT1 glucose transporter cDNA was modified to introduce a single amino acid substitution of leucine for tryptophan 412, a putative cytochalasin B photo-affinity labeling site. Although the mutated transporter was expressed into plasma membranes of Chinese hamster ovary cells, glucose transport activity of the mutated transporter was observed to be only 15-30% of that of the wild-type GLUT1 when glucose transport activity was assessed by 2-deoxyglucose uptake at 0.1-10 mM concentrations. Analysis of glucose uptake kinetics depict that a mutation induced a 3-fold decrease in turnover number and a 2.5-fold increase in Km compared with the wild-type GLUT1. Importantly, cytochalasin B labeling was not abolished but decreased by 40%, and cytochalasin B binding was also decreased. In addition, the results obtained with side-specific glucose analogs suggested that the outer glucose binding site of the mutant appeared intact but the inner binding site was modulated. These results indicate 1) tryptophan 412 is not a cytochalasin B labeling site(s), although this residue is located in or close to the inner glucose binding site of the GLUT1 glucose transporter, 2) substitution of leucine for tryptophan 412 decreases the intrinsic activity of GLUT1 glucose transporter, which is definable as the turnover number/Km, to approximately 15% of that of the wild-type.

We analyzed single-stranded conformational poly morphisms to screen for mutations and polymorphisms in the insulin receptor gene in subjects with or without insulin resistance. Using this new technique, we demonstrated the existence of mutations in the insulin receptor gene which we had identified previously. In addition, a new mutation was found in exon 20 of the insulin receptor gene in a patient with moderate insulin resistance associated with morbid obesity, acanthosis nigricans, and polycystic ovary syndrome. The patient was heterozygous for a mutation substituting Leu (CTG) for Pro (CCG) at codon 1178. Pro1178is a part of a characteristic sequence motif (D1150F1151G1152-A1177P1178E1179) common to many protein kinases. Analysis of single-stranded conformational polymorphisms was also used to estimate the frequency of a polymorphism at codon 1058. The two codons CAC (1058 His) and CAT (1058 His) both had a prevalence of 50% in 30 Japanese subjects. These data demonstrate that analysis of single-stranded conformational polymorphisms is a simple and sensitive screening method for mutations and polymorphisms in the insulin receptor gene in subjects with or without insulin resistance. Identification of a mutation in the insulin receptor gene in a patient with a moderate degree of insulin resistance associated with morbid obesity suggests that insulin receptor mutations may exist in patients with Type 2 (non-insulin-dependent) diabetes mellitus associated with a moderate degree of insulin resistance.

The urinary excretion of 1,5-anhydro-D-glucitol, a pyranoid polyol, in humans was studied. The plasma of nondiabetic human subjects contained high concentrations of this polyol (>110 μmol/l), and there was a tendency for the 24-h excretion of it to become more variable in direct proportion to its plasma concentration. In contrast, diabetic patients showed lower plasma concentrations of this polyol, and the variation in the 24-h excretion of 1,5-anhydro-D-glucitol was especially notable among the patients with an extremely low plasma concentration of the polyol. This diabetic group showed a statistically significant correlation (p<0.01), between the urinary 1,5-anhydro-D-glucitol and urinary glucose. This correlation was more markedly demonstrated during a 100-g oral glucose tolerance test: parallel changes were observed in the concentrations of 1,5-anhydro-D-glucitol and glucose in the urine collected every hour after the glucose load. These observations led to the proposal that low plasma concentration of this polyol, which is observed in diabetes mellitus, may be the result of a frequent and/or prolonged high blood glucose concentration beyond the renal threshold for glucose excretion.

This study was undertaken to define intracellular signaling pathways upstream to glycogen synthase activation. First, we examined the role of the two pathways of insulin signaling, Ras-dependent and wortmannin/LY294002-sensitive, in glycogen synthase activation. Although negative dominant Ras (Ras17N) induction in PC12 cells markedly decreased activities of mitogen-activated protein kinase (MAP) and pp90 S6 kinase in response to insulin or insulin-like growth factor I (IGF-I), activation of glycogen synthase by these agents was unaffected by negative dominant Ras induction. In contrast, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), inhibitors of phosphatidylinositol 3-kinase, antagonized glycogen synthase activation in response to insulin or IGF-I. Next, we examined the contribution of pp70 S6 kinase, one of the wortmannin/LY294002-sensitive signaling molecules on glycogen synthase activation. Immunosuppressant rapamycin completely blocked activation of pp70 S6 kinase by insulin or IGF-I, but rapamycin alone or in combination with induction of negative dominant Ras failed to antagonize glycogen synthase activation by these hormones. These data suggest that 1) activation of Ras-MAP kinase is not necessary for stimulation of glycogen synthase and 2) activation of wortmannin/LY294002-sensitive pathway, independent of pp70 S6 kinase, plays a key role in glycogen synthase regulation in PC12 cells.

The notion of an insulin-dependent translocation of the glucose transporter in rat adipocytes was confirmed by immunoblotting and reconstitution of glucose transport activity of subcellular fractions. Quantitatively, however, significantly different results were obtained with these two techniques; when compared with reconstitution, immunoblotting detected translocation of a larger amount of the transporter from a low density microsome fraction to a plasma membrane fraction. The acidtropic agents chloroquine and dibucaine, which have been reported to inhibit the recycling of various receptors, were utilized to study the detailed translocation mechanism of the glucose transporter and the insulin receptor. These acidtropic agents caused accumulation of 125I-insulin in a subcellular fraction probably corresponding to lysosomes. They did not, however, significantly affect either the insulin-induced activation of glucose transport or the recycling of the transporter and the insulin receptor as detected by immunoblotting. About 50% of radioactivity released from adipocytes which were allowed to internalize insulin was due to intact insulin, and chloroquine did not change the release rate of intact insulin, raising the possibility of receptor-mediated exocytosis of insulin. The release of degraded insulin decreased with chloroquine treatment. The results are consistent with the idea that these acidtropic agents mainly act to inhibit degradation of insulin in lysosomes, and their effect on the recycling of the glucose transporter and the insulin receptor is minimal, indicating that the recycling of these membrane proteins proceeds irrespective of organelle acidification. Electron micrographs showed vesicles underneath the plasma membranes, with sizes similar to those of the low density microsome fraction where the internalized glucose transporter and the insulin receptor were located.

The type I insulin-like growth factor (IGF) receptor, like the insulin receptor, contains a ligand-stimulated protein-tyrosine kinase activity in its beta-subunit. However, in vivo, no substrates have been identified. We used anti-phosphotyrosine antibodies to identify phosphotyrosine-containing proteins which occur during IGF-I stimulation of normal rat kidney and Madin-Darby canine kidney cells labeled with ortho[32P]phosphate. Both cells provide a good system to study the function of the type I IGF receptors because they contain high concentrations of these receptors but no insulin receptors. In addition, physiological levels of IGF-I, but not insulin, stimulated DNA synthesis in growth-arrested cells. IGF-I stimulated within 1 min of tyrosine phosphorylation of two proteins. One of them, with a molecular mass between 97 and 102 kDa, was supposed to be the beta-subunit of the type I IGF receptor previously identified. The other protein had an approximate molecular mass of 185 kDa, which resembled, by several criteria, pp 185, originally identified during the initial response of Fao cells to insulin binding (White, M. F., Maron, R., and Kahn, C. R. (1985) Nature 318, 183-186). These data suggest that tyrosine phosphorylation of pp 185 may occur during activation of both the type I IGF receptor and the insulin receptor, and it could be a common substrate that transmits important metabolic signals during ligand binding.

The plasma concentration of 1,5-anhydro-D-glucitol (AG)(1-deoxyglucose) is known to decrease in diabetic patients. In order to evaluate the usefulness of this polyol as a diabetic marker, we examined the specificity of the plasma AG reduction in various diseases: the plasma AG level was determined in 108 newly diagnosed diabetic patients, 229 normal subjects and 200 patients with various other disorders. The mean plasma AG concentration in diabetes mellitus was 1.9±1.8 μg/ml (mean±SD), which was definitely lower than that in healthy subjects and patients with other diseases including some metabolic and hormonal diseases (mean value range: 13.4–28.3 μg/ml). Only the “malignancies” group showed statistically different mean values from that in normal subjects; however, these values were much higher than those of diabetic patients. The AG concentration seemed to be relatively low in some severe by uraemic patients, but is likely to be little influenced by the glomerular filtration rate. Upon adjustment for sex and age, AG concentration was not found to be correlated with the degree of obesity in both healthy subjects and diabetic patients. The plasma AG concentration showed a tendency to be higher in healthy males than in healthy females in all age-matched groups; however, statistically significant differences were not seen. Also, no significant influence of age was observed.

In a 5–12 year follow-up study of 288 subjects with impaired glucose tolerance after a 100-g glucose load, 48 worsened to overt Type 2 (non-insulin-dependent) diabetes with the elevation of fasting blood glucose. The initial level of blood glucose was a major predictor of subsequent worsening to diabetes. In addition, subjects with a lower insulin response to glucose showed a higher incidence of worsening to the disease, irrespective of blood glucose levels. Multivariate analysis indicated that a diminished insulin response and a high maximal body weight index, as well as a high level of fasting and 2-h glucose values at the initial 100-g oral glucose tolerance test were significant independent risk factors for the development of diabetes in Japanese subjects.

To elucidate the mechanisms whereby changes in dietary composition affect the action of insulin on glucose metabolism, insulin binding and glucose uptake and oxidation have been studied in epididymal fat pad adipocytes from rats fed high glucose diets for 5 and 10 days. After 5 days, insulin binding was increased, due mainly to an increased number of receptors (3.4×105vs. 2.4×105sites per cell) in spite of increased plasma insulin levels (3.0±0.2 vs. 2.1±0.1 μg/l; p<0.05). The maximal response of glucose oxidation to insulin was increased (925±55 vs. 510±58 n moles/2×105cells/2h; p<0.01) and the dose-response curve of glucose uptake was shifted to the left. After 10 days, receptor number decreased to the control level and the effect of insulin on glucose uptake and oxidation (% basal) were similar to controls. Thus, in the early stage of high glucose feeding, insulin receptor number, insulin sensitivity of glucose uptake, and insulin responsiveness of glucose oxidation were increased.

The changes in insulin response to a 100 g glucose tolerance test after treatment by diet, sulphonylurea and insulin were compared in non-ketotic diabetic patients who had fasting blood glucose concentrations higher than 160 mg/100 ml. Patients were selected so that their pre-treatment and post-treatment blood glucose levels were comparable between different treatment groups. Their insulin responses were poor initially but increased significantly when the diabetic state was improved by each treatment. The degree of improvement of insulin response was similar between different treatment groups, when their fasting blood glucose decreased below 140 mg/100 ml and the glucose tolerance curves were improved to a similar extent. Preand post-treatment ∑ IRI values (sum of insulin values during glucose tolerance test, mean±SD) were 102±50 and 200±37 μU/ml in diet-treated group (n = 28), 90±40 and 195±53 μU/ml in sulphonylurea-treated-group (n=48), and 83±28 and 193±38 μU/ml in insulin-treated group (n = 13), respectively. The data suggest that the poor insulin response in overt diabetes results not only from an inherent insensitivity of B-cells to glucose but also from the metabolic derangement of diabetes. Poor insulin response and overtly diabetic metabolism seems to form a vicious cycle.

Insulin binding and degrading activities were solubilized by a nonionic detergent. Triton X100, from guinea pig kidney particulate fractions (100,000 × g pellet). The solubilized insulin binding activity appeared as a single peak on Sepharose 6B gel filtration with a Stokes radius of 73 A. The pI of the solubilized insulin binding activity determined by flat-bed isoelectric focusing was 5.6. On the other hand, the Stokes radius of the solubilized molecule with insulin degrading activity was 54 Å by the same column with a pI of 5.2. More than 98% of the insulin binding activity could be adsorbed to a column of concanavalin A-agarose, while about 94% of the insulin degrading activity could not be adsorbed to this column. These results strongly suggest that the macromolecule for the insulin binding activity is not identical to that for the insulin degrading activity.

Glucose transporter (GT) has been suggested to be involved in the insulin biosynthesis. However, the functional relationship between GT and insulin biosynthesis is not well understood. In this report, we have generated rat pancreatic B cell lines (RINr) that stably overexpress a cDNA encoding the brain type GT. These cell lines showed 3- to 4-fold increase in insulin mRNA and protein. These results suggest that GT might have some relationship to the insulin biosynthesis in the pancreatic B cells.

Glucose transporter (GT) has been suggested to be involved in the insulin biosynthesis. However, the functional relationship between GT and insulin biosynthesis is not well understood. In this report, we have generated rat pancreatic B cell lines (RINr) that stably overexpress a cDNA encoding the brain type GT. These cell lines showed 3‐ to 4‐fold increase in insulin mRNA and protein. These results suggest that GT might have some relationship to the insulin biosynthesis in the pancreatic B cells.