Evaluation of an amplicon-based custom gene panel for the diagnosis of hereditary tumors.

Genetic testing based on next-generation sequencing (NGS) analysis has recently been used to diagnose hereditary diseases. In this study, we explored the usefulness of our custom amplicon panel that targeted 23 genes related to hereditary tumors given in the American College of Medical Genetics and Genomics recommendations. We applied our custom NGS panel to samples from 12 patients previously diagnosed by Sanger sequencing as having the diseases or diagnosed clinically by meeting the diagnostic criteria in this study. Our gene panel not only successfully identified all variants detected by Sanger sequencing but also identified previously unrecognized variants that resulted in confirmation of the disease, or even in the revision of the diagnosis. For instance, a patient identified with an SDHD gene mutation actually had von Hippel-Lindau (VHL) syndrome, as determined by the presence of a pathogenic VHL gene variant. We also identified false-positive results that were generated by amplification of genome regions that are not intended to be investigated. In conclusion, NGS-based amplicon sequencing is a highly effective method to detect germline variants, as long as they are also carefully reviewed by manual inspection.

Measurement of small fibre pain threshold values for the early detection of diabetic polyneuropathy.

AIM: To investigate whether Aδ and C fibre pain threshold values, measured using intra-epidermal electrical stimulation (IES), in people with and without Type 2 diabetes are useful in evaluating diabetic polyneuropathy (DPN) severity. METHODS: Aδ and C fibre pain threshold values were measured in Japanese people with (n = 120) and without (n = 76) Type 2 diabetes by IES. Nerve conduction studies and other tests were performed to evaluate diabetic complications. RESULTS: Aδ and C fibre pain threshold values were high in people with diabetes compared with control subjects (Aδ fibre: 0.050 vs. 0.030 mA, P < 0.01; C fibre: 0.180 vs. 0.070 mA, P < 0.01). Participants with diabetes and neuropathy had significantly higher Aδ and C fibre pain threshold values than participants without neuropathy (Aδ fibres 0.063 vs. 0.039 mA, P < 0.01; C fibres 0.202 vs. 0.098 mA, P < 0.05). C fibre pain threshold values were significantly higher in participants with diabetes and diabetic microvascular complications than in participants without complications. Threshold values increased with complication progression. When DPN was diagnosed according to the Diabetic Neuropathy Study Group in Japan criteria, the cut-off for the C fibre pain threshold values was 0.125 mA (area under the curve 0.758, sensitivity 81.5%, specificity 61.5%). The IES test took less time (P < 0.01) and was less invasive (P < 0.01) than the nerve conduction studies. CONCLUSIONS: Intra-epidermal electrical stimulation is a non-invasive and easy measurement of small fibre pain threshold values. It may be clinically useful for C fibre measurement to diagnose early DPN as defined by the Diabetic Neuropathy Study Group in Japan criteria.

Efficacy and safety of once-weekly dulaglutide in combination with sulphonylurea and/or biguanide compared with once-daily insulin glargine in Japanese patients with type 2 diabetes: a randomized, open-label, phase III, non-inferiority study.

AIMS: To evaluate 0.75 mg of dulaglutide, a once-weekly glucagon-like peptide-1 receptor agonist, compared with once-daily insulin glargine for glycaemic control in Japanese patients with type 2 diabetes (T2D). METHODS: In this phase III, randomized, open-label, parallel-group, 26-week study, 361 patients with inadequately controlled T2D receiving sulphonylureas and/or biguanides, aged ≥20 years, with glycated haemoglobin (HbA1c) levels 7.0-10.0% (53-86 mmol/mol), inclusive, were randomized (1 : 1) to receive dulaglutide or glargine. Participants and investigators were not masked to treatment allocation. The primary measure was change from baseline in HbA1c at 26 weeks, analysed using a mixed-effects model for repeated measures, with a predefined non-inferiority margin of 0.4%. RESULTS: At week 26, least-squares (LS) mean (standard error) reductions in HbA1c were -1.44 (0.05)% [-15.74 (0.55) mmol/mol] in the dulaglutide group and -0.90 (0.05)% [-9.84 (0.55) mmol/mol] in the glargine group. The mean between-group treatment difference in HbA1c was -0.54% (95% CI -0.67, -0.41) [-5.90 mmol/mol (95% CI -7.32, -4.48)]; p < 0.001. Dulaglutide significantly reduced body weight compared with glargine at week 26 (LS mean difference -1.42 kg, 95% CI -1.89, -0.94; p < 0.001). The most frequent adverse events with dulaglutide treatment were nasopharyngitis and gastrointestinal symptoms. The incidence of hypoglycaemia was significantly lower with dulaglutide [47/181 (26%)] compared with glargine [86/180 (48%)], p < 0.001. CONCLUSION: In Japanese patients with T2D uncontrolled on sulphonylureas and/or biguanides, once-weekly dulaglutide was superior to once-daily glargine for reduction in HbA1c at 26 weeks. Although dulaglutide increased gastrointestinal symptoms, it was well tolerated, with an acceptable safety profile.

Long-term treatment with empagliflozin as add-on to oral antidiabetes therapy in Japanese patients with type 2 diabetes mellitus.

AIMS: To evaluate the safety and efficacy of empagliflozin for 52 weeks as add-on to one other oral antidiabetes therapy in Japanese patients with type 2 diabetes mellitus (T2DM). METHODS: Patients on biguanide (n = 133), thiazolidinedione (n = 273), α-glucosidase inhibitor (n = 139), dipeptidyl-peptidase-4 inhibitor (n = 139) or glinide (n = 140) were randomized 1 : 1 to receive empagliflozin 10 or 25 mg double-blind as add-on therapy for 52 weeks. Patients on sulphonylurea (SU; n = 336) were randomized 2 : 2 : 1 to receive empagliflozin 10 or 25 mg double-blind or open-label metformin as add-on therapy for 52 weeks. The primary objective was to evaluate safety. Change from baseline in glycated haemoglobin (HbA1c) at week 52 was a secondary endpoint. RESULTS: Adverse events (AEs) were reported in 67.6-84.6% of patients receiving empagliflozin. Confirmed hypoglycaemic AEs (plasma glucose ≤70 mg/dl and/or requiring assistance) were reported in 4.4 and 6.6%, respectively, of patients receiving empagliflozin 10 and 25 mg as add-on to SU and in 0.0 to 2.9%, respectively, of patients receiving empagliflozin 10 and 25 mg as add-on to other therapies. Baseline mean ± standard deviation HbA1c ranged from 7.51 ± 0.73 to 8.06 ± 0.76% across background therapy groups. At week 52, adjusted mean ± standard error changes from baseline in HbA1c ranged from -0.77 ± 0.06 to -1.00 ± 0.06% in patients receiving empagliflozin. CONCLUSIONS: In Japanese patients with T2DM, empagliflozin 10 and 25 mg as add-on to one other oral antidiabetes therapy for 52 weeks were well tolerated and were associated with clinically meaningful reductions in HbA1c.

Long-term safety of linagliptin monotherapy in Japanese patients with type 2 diabetes.

AIMS: In a phase III study conducted among Japanese patients with type 2 diabetes mellitus (T2DM), linagliptin 5 and 10 mg showed clinically meaningful improvements in glycaemic parameters after 12 and 26 weeks compared with placebo and voglibose, respectively. This extension study assessed long-term tolerability of linagliptin over 52 weeks. METHODS: Japanese patients with T2DM who completed either phase of a 12-week/26-week study comparing linagliptin monotherapy with placebo or voglibose were eligible to enrol. In the extension study, the comparator groups switched to linagliptin 5 or 10 mg, while the linagliptin groups maintained dosage. RESULTS: In all, 540 patients received at least one dose of linagliptin 5 or 10 mg and 494 completed the extension. Long-term treatment with linagliptin was well tolerated; adverse events (AEs) of special interest and serious AEs occurred in small percentages of patients. Drug-related AEs occurred in 10.2 and 10.6% of patients in the linagliptin 5- and 10-mg groups, respectively, and discontinuations due to drug-related AEs occurred in 1.1 and 0.7%, respectively. Only one (0.4%) patient in each dose group experienced investigator-defined hypoglycaemia during the treatment period (both events were non-severe). Body weight was not clinically altered in either group. The glycated haemoglobin A1c profiles over time were similar with linagliptin 5 and 10 mg. CONCLUSIONS: These findings provide evidence for the safety and tolerability of oral linagliptin at either 5 or 10 mg for up to 52 weeks for the treatment of Japanese patients with T2DM, without clinically relevant increase in the risk of hypoglycaemia or weight gain.

Linagliptin monotherapy provides superior glycaemic control versus placebo or voglibose with comparable safety in Japanese patients with type 2 diabetes: a randomized, placebo and active comparator-controlled, double-blind study.

AIMS: To evaluate the efficacy and safety of linagliptin 5 and 10 mg vs. placebo and voglibose in Japanese patients with type 2 diabetes mellitus (T2DM). METHODS: This study enrolled patients with inadequately controlled T2DM who were previously treated with one or two oral antidiabetics or were drug naÏve. After a 2 to 4-week washout and placebo run-in, 561 patients were randomized (2 : 2 : 2 : 1) to double-blind treatment with linagliptin 5 or 10 mg qd, voglibose 0.2 mg tid or placebo. The primary endpoint was the change from baseline in haemoglobin A1c (HbA1c) with linagliptin vs. placebo after 12 weeks and vs. voglibose after 26 weeks. RESULTS: Baseline characteristics were well balanced across treatment groups (overall mean HbA1c was 8.01%). The adjusted mean (95% confidence interval) treatment differences at week 12 were -0.87% (-1.04, -0.70; p < 0.0001) and -0.88% (-1.05, -0.71; p < 0.0001) for linagliptin 5 and 10 mg vs. placebo and at week 26 were -0.32% (-0.49, -0.15; p = 0.0003) and -0.39% (-0.56, -0.21; p < 0.0001) for linagliptin 5 and 10 mg vs. voglibose. At week 12, mean HbA1c was 7.58, 7.48 and 8.34% in patients receiving linagliptin 5 mg, linagliptin 10 mg and placebo, respectively. At week 26, mean HbA1c was 7.63% with linagliptin 5 mg, 7.50% with linagliptin 10 mg and 7.91% with voglibose. Drug-related adverse event rates were comparable across treatment groups over 12 weeks (9.4% linagliptin 5 mg, 8.8% linagliptin 10 mg and 10.0% placebo) and 26 weeks (11.3% linagliptin 5 mg, 10.6% linagliptin 10 mg and 18.5% voglibose). There were no documented cases of hypoglycaemia. CONCLUSIONS: Linagliptin showed superior glucose-lowering efficacy and comparable safety and tolerability to both placebo and voglibose in Japanese patients with T2DM.

CHOP deletion does not impact the development of diabetes but suppresses the early production of insulin autoantibody in the NOD mouse.

C/EBP homologous protein (CHOP) has been proposed as a key transcription factor for endoplasmic reticulum (ER) stress-mediated ß-cell death induced by inflammatory cytokines in vitro. However, the contribution of CHOP induction to the pathogenesis of type 1 diabetes is not yet clear. To evaluate the relevance of CHOP in the pathogenesis of type 1 diabetes in vivo, we generated CHOP-deficient non-obese diabetic (NOD.Chop (-/-)) mice. CHOP deficiency did not affect the development of insulitis and diabetes and apoptosis in ß-cells. Interestingly, NOD.Chop (-/-) mice exhibited a delayed appearance of insulin autoantibodies compared to wild-type (wt) mice. Adoptive transfer with the diabetogenic, whole or CD8(+)-depleted splenocytes induced ß-cell apoptosis and the rapid onset of diabetes in the irradiated NOD.Chop (-/-) recipients with similar kinetics as in wt mice. Expression of ER stress-associated genes was not significantly up-regulated in the islets from NOD.Chop (-/-) compared to those from wt mice or NOD-scid mice. These findings suggest that CHOP expression is independent of the development of insulitis and diabetes but might affect the early production of insulin autoantibodies in the NOD mouse.

The effect of group-based lifestyle interventions on risk factors and insulin resistance in subjects at risk for metabolic syndrome: the Tabaruzaka Study 1.

AIM: The aim of this study was to evaluate the efficacy of two group-based lifestyle interventions in ameliorating the risk factors of metabolic syndrome (MS) and insulin resistance. METHODS: Ninety-eight subjects who had at least one component of MS were randomized into standard intervention (SI) (4-month intervention; n = 50) and extended intervention (EI) (10-month intervention; n = 48) groups, and 39 subjects were followed up for a control group. The effects of intervention were evaluated after 10, 22 and 34 months. RESULTS: At month 10, the standard and EI groups showed improved body mass index (BMI) (SI, -0.28; EI, -0.47; control, -0.09), high-density lipoprotein (HDL) cholesterol, fasting plasma glucose and A1c and a decreased mean number of components of MS (SI, -0.37; EI, -0.51; control, 0.08). At month 34, the effects on BMI (SI, -0.66; EI, -0.60; control, -0.05) and HDL-cholesterol were sustained for both the intervention groups. In controls, the increases in fasting plasma glucose and the mean number of components of MS from the baseline to month 34 were greater than those in the standard and EI groups. Whole body insulin sensitivity index and hepatic insulin resistance index were also improved at month 10. CONCLUSIONS: Group-based lifestyle intervention could be an efficient way to prevent MS. Its effects were sustainable, at least in part, for 2 years. These effects may be mediated by an improvement in insulin sensitivity.

Extensive skin necrosis of the arm in a patient with complex regional pain syndrome.

We report a 36-year-old woman with complex regional pain syndrome (CRPS) type 1 presenting with extensive skin necrosis of the left arm. The patient cooled her arm with ice packs to ease severe pain due to CRPS, in spite of repeated cautions against frostbite injury. The regions of skin necrosis corresponded with the sites where she had applied ice packs. We considered that the severe skin necrosis in our case was due to a self-induced frostbite injury.

[Aortic root replacement and pulmonary vein isolation; report of a case].

A 60-year-old female was admitted to our hospital who suffered from palpitation and dyspnea. Echocardiography revealed severe aortic regurgitation and enlargement of ascending aorta. Electrocardiogram showed tachycardia due to atrial fibrillation. We performed the aortic root replacement with Carboseal composite graft and pulmonary vein isolation using Cardioblate BiPolar (BP) system. Histopathologic diagnosis was giant isolated aortitis. The post operative course was uneventful. And the patient was discharged in normal sinus rhythm.

[Traumatic mitral regurgitation with acute pulmonary edema].

A case of a successful surgical treatment for traumatic mitral valve regurgitation is reported. A 44-year-old, small-statured female with cretinism had a traffic accident. Eleven days after the accident, she was admitted to our hospital with severe respiratory distress syndrome by acute pulmonary edema. Echocardiography showed severe mitral regurgitation due to tendon rupture of posterior leaflet. Mitral valve plasty was performed successfully.

Regulation of insulin receptor substrate-1 in liver and muscle of animal models of insulin resistance.

Insulin rapidly stimulates tyrosine phosphorylation of a protein of approximately 185 kD in most cell types. This protein, termed insulin receptor substrate-1 (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state of insulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction (approximately 50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) reduced IRS-1 phosphorylation in liver and muscle may play a role in insulin-resistant states, especially of the ob/ob mice.

A carboxy-terminal truncation of human alpha-galactosidase A in a heterozygous female with Fabry disease and modification of the enzymatic activity by the carboxy-terminal domain. Increased, reduced, or absent enzyme activity depending on number of amino acid residues deleted.

Fabry disease is an X-linked disorder of glycosphingolipid metabolism caused by a deficiency of alpha-galactosidase A (alpha-Gal A). We identified a novel mutation of alpha-Gal A gene in a family with Fabry disease, which converted a tyrosine at codon 365 to a stop and resulted in a truncation of the carboxy (C) terminus by 65 amino acid (AA) residues. In a heterozygote of this family, although the mutant and normal alleles were equally transcribed in cultured fibroblasts, lymphocyte alpha-Gal A activity was approximately 30% of the normal control and severe clinical symptoms were apparent. COS-1 cells transfected with this mutant cDNA showed a complete loss of its enzymatic activity. Furthermore, those cotransfected with mutant and wildtype cDNAs showed a lower alpha-Gal A activity than those with wild type alone (approximately 30% of wild type alone), which suggested the dominant negative effect of this mutation and implied the importance of the C terminus for its activity. Thus, we generated mutant cDNAs with various deletion of the C terminus, and analyzed. Unexpectedly, alpha-Gal A activity was enhanced by up to sixfold compared with wild-type when from 2 to 10 AA residues were deleted. In contrast, deletion of 12 or more AA acid residues resulted in a complete loss of enzyme activity. Our data suggest that the C-terminal region of alpha-Gal A plays an important role in the regulation of its enzyme activity.

Elevated activity of beta-hexosaminidase and sulfhydryl modification in the B-variant of human lung cancer.

Activities of beta-hexosaminidase A and beta-hexosaminidase B (Hex B) were measured both in human lung carcinoma and the adjacent normal tissues of 47 patients. The specific activity of total beta-hexosaminidase in the tumors was considerably higher than in the adjacent normal tissues, irrespective of histological types. In isoelectric focusing experiments, Hex B purified from normal lung exhibited a single peak with an isoelectric point (pI) of 7.9, while Hex B purified from adenocarcinoma contained two forms with pI 7.6 and 7.9. With respect to heat stability, Hex B from the normal lung was very stable at 52 degrees, while the tumor Hex B (mixture of pI 7.6 and 7.9 forms) was unstable. After treatment of the tumor enzyme with dithiothreitol, heat stability was restored. When the tumor pI 7.6 form was treated with dithiothreitol and subjected to polyacrylamide gel electrophoresis, the enzyme converted to a pI 7.9 form similar to that of the normal lung. Determination of the sulfhydryl group of the tumor pI 7.6 form under nondenaturing conditions showed that the enzyme had some easily reducible disulfide bonds on its surface. These findings indicate that the formation of mixed disulfide bonds in the tumor Hex B increases the net negative charge and results in the appearance of a heat-labile form.

Elevated activities and properties of arylsulfatases A and B and B-variant in human lung tumors.

The activities of arylsulfatases A and B were determined in human primary and secondary tumor tissues (total, 53 cases) of various histological types. Significantly higher activities of these sulfatases were found in almost all the primary lung carcinomas as compared to their corresponding uninvolved tissues. No significant correlation was demonstrated between the enzyme activities and histological figures (stroma amounts, etc.). Lung adenocarcinoma and squamous cell carcinoma showed the presence of an additional arylsulfatase component (B1) which was not detected in normal human lung. The tumor arylsulfatase B1 had an isoelectric point (pI) of 6.7 and was clearly distinguished from arylsulfatase A (pI 4.9) and arylsulfatase B (pI 9.1 to 9.2) in normal lung and lung tumor. The tumor B1 enzyme was demonstrated to be most probably an isoenzyme of arylsulfatase B, since this unusual enzyme was indistinguishable from arylsulfatase B in terms of Ag+ inhibition; its kinetic parameters of Km for p-nitrocatechol sulfate, which was 2.9 mM with B1; optimum pH of 6.3 for B1; heat stability; and substrate specificity for three synthetic and two physiological substrates.

Elevation of glycoprotein galactosyltransferase activity in human lung cancer related to histological types.

Uridine diphosphogalactose:glycoprotein galactosyltransferases were examined in human lung adenocarcinoma and squamous cell carcinoma. The galactosyltransferase activities in tissue homogenates from both carcinomas were higher than in adjacent normal control with asialoagalactofetuin as a substrate. This activity in adenocarcinoma (27 cases) was two times higher than that in squamous cell carcinoma (19 cases) with statistical significance (p less than 0.001). Using Triton-solubilized enzymes from a particulate fraction, similar differences in the activity were observed with ovalbumin, asialoagalactofetuin, and its beta-eliminated derivative as acceptors but not with bovine submaxillary mucin. These observations mean that the higher activity of galactosyltransferase(s) in lung carcinomas (especially in adenocarcinoma) is mainly responsible for galactosylation of carbohydrate chains in N-glycoside-type but not O-glycoside-type glycoproteins.

Cyclooxygenase-2 contributes to functional hyperemia in whisker-barrel cortex.

The prostanoid-synthesizing enzyme cyclooxygenase-2 (COX-2) is expressed in selected cerebral cortical neurons and is involved in synaptic signaling. We sought to determine whether COX-2 participates in the increase in cerebral blood flow produced by synaptic activity in the somatosensory cortex. In anesthetized mice, the vibrissae were stimulated mechanically, and cerebral blood flow was recorded in the contralateral somatosensory cortex by a laser-Doppler probe. We found that the COX-2 inhibitor NS-398 attenuates the increase in somatosensory cortex blood flow produced by vibrissal stimulation. Furthermore, the flow response was impaired in mice lacking the COX-2 gene, whereas the associated increase in whisker-barrel cortex glucose use was not affected. The increases in cerebral blood flow produced by hypercapnia, acetylcholine, or bradykinin were not attenuated by NS-398, nor did they differ between wild-type and COX-2 null mice. The findings provide evidence for a previously unrecognized role of COX-2 in the mechanisms coupling synaptic activity to neocortical blood flow and provide an insight into one of the functions of constitutive COX-2 in the CNS.

Cloning of the mouse insulin receptor substrate-1 (IRS-1) gene and complete sequence of mouse IRS-1.

The mouse IRS-1 gene has been cloned and its structure determined. Mouse IRS-1 differs from rat by the absence of the potential C-terminal nucleotide binding site. Otherwise, the predicted IRS-1 protein is highly conserved between mouse, rat and humans, especially in the possible phosphorylation sites. The highly conserved nature of IRS-1 suggests the importance of these domains in the function of IRS-1 or its association with other proteins.

Human skeletal muscle insulin receptor substrate-1. Characterization of the cDNA, gene, and chromosomal localization.

Insulin receptor substrate-1 is a major substrate of insulin receptor Tyr kinase. We have now cloned the IRS-1 cDNA from human skeletal muscle, one of the most important target tissues of insulin action, localized and cloned the human IRS-1 gene, and studied the expression of the protein in Chinese hamster ovary cells. Human IRS-1 cDNA encodes a 1242 amino acid sequence that is 88% identical with rat liver IRS-1. The 14 potential Tyr phosphorylation sites include 6 Tyr-Met-X-Met motifs and 3 Tyr-X-X-Met motifs that are completely conserved in human IRS-1. Human IRS-1 has > 50 possible Ser/Thr phosphorylation sites and one potential ATP-binding site close to the NH2-terminal. The human IRS-1 gene contains the entire 5'-untranslated region and protein coding region in a single exon and was localized on chromosome 2 q36-37 by in situ hybridization. By Northern blot analysis, IRS-1 mRNA is rare and consists of two species of 6.9 and 6 kilobase. By using quantitative polymerase chain reaction after reverse transcription of total RNA from human fetal tissues, IRS-1 mRNA could be identified in all tissues. When human IRS-1 cDNA was expressed in Chinese hamster ovary cells, the protein migrated between 170,000-180,000 M(r) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was rapidly Tyr phosphorylated upon insulin stimulation. Thus, IRS-1 is widely expressed and highly conserved across species and tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Impact of natural IRS-1 mutations on insulin signals: mutations of IRS-1 in the PTB domain and near SH2 protein binding sites result in impaired function at different steps of IRS-1 signaling.

Insulin receptor substrate-1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates various insulin signals downstream. In this study, we have examined the impact of three natural IRS-1 mutations identified in NIDDM patients (G971R, P170R, and M209T) on insulin signaling. G971R is located near src homology 2 protein binding sites, and P170R and M209T are located in the phosphotyrosine binding domain of IRS-1. 32D-IR cells, stably overexpressing human insulin receptor, were transfected with wild-type human IRS-1 cDNA (WT) or three mutant IRS-1 cDNAs and analyzed. All the cell lines expressing mutant IRS-1 showed a significant reduction in [3H]thymidine incorporation compared with WT. Upon insulin stimulation, cells expressing G971R showed a 39% decrease (P < 0.005) in phosphatidylinositol 3-kinase (PI 3-kinase) activity, a 43% decrease (P < 0.01) in binding of the 85-kDa regulatory subunit of PI 3-kinase, and a 22% decrease (P < 0.05) in mitogen-activated protein kinase activity compared with those expressing WT. Cells expressing P170R and M209T showed slight but significant decreases in PI 3-kinase activity (17 and 14%, respectively; both P < 0.05) and in binding of p85 (22 and 16%, respectively; both P < 0.05) and a greater decrease in mitogen-activated protein kinase activity (41 and 43%, respectively; both P < 0.005) compared with WT. After insulin stimulation, cells expressing P170R and M209T showed significant decreases in IRS-1 phosphorylation (37 and 42%, respectively; both P < 0.05) and in IRS-1 binding to the insulin receptor (48 and 53%, respectively; P < 0.01) compared with WT. G971R showed no changes in IRS-1 phosphorylation and in IRS-1 binding to the insulin receptor compared with WT. These data suggest that the impaired mitogenic response of P170R and M209T was mainly due to reduced binding to the insulin receptor, whereas the impaired response of G971R was mainly due to reduced association with PI 3-kinase p85.