Eiichi Araki

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.

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 5ʹ-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 Mr 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. Compared with rat protein, human IRS-1 contains more potential Ser/Thr phosphorylation sites and only one nucleotide binding site. The entire protein coding sequence is contained within a single exon.

The insulin receptor and its substrate: molecular determinants of early events in insulin action.

Publisher Summary This chapter describes insulin receptor and its substrate. It discusses the two earliest molecular events, binding and activation of the insulin receptor kinase and phosphorylation and subsequent signal transduction by the insulin receptor substrate IRS-1. Defining these events has helped add a whole new dimension to the understanding of insulin action. At the same time, studies defining the molecular events at the end of the insulin action cascade, such as activation of glucose transport and regulation of gene expression, have also begun to clarify the specific components required for these signaling events. Although a black box remains between the early and late events in insulin action, it is becoming smaller.

A mitochondrial DNA variant at position 16189 is associated with type 2 diabetes mellitus in Asians

Aims/hypothesisThis multinational study was conducted to investigate the association between a mitochondrial DNA (mtDNA) T16189C polymorphism and type 2 diabetes in Asians. The mtDNA 16189C variant has been reported to be associated with insulin resistance and type 2 diabetes. However, a recent meta-analysis concluded that it is negatively associated with type 2 diabetes in Europids. Since the phenotype of an mtDNA mutant may be influenced by environmental factors and ethnic differences in the nuclear and mitochondrial genomes, we investigated the association between the 16189C variant and type 2 diabetes in Asians.MethodsThe presence of the mtDNA 16189C variant was determined in 2,469 patients with type 2 diabetes and 1,205 non-diabetic individuals from Korea, Japan, Taiwan, Hong Kong and China. An additional meta-analysis including previously published Asian studies was performed. Since mtDNA nucleotide position 16189 is very close to the mtDNA origin of replication, we performed DNA-linked affinity chromatography and reverse-phase liquid chromatography/tandem mass spectrometry and chromatin immunoprecipitation to identify protein bound to the 16189 region.ResultsAnalysis of participants from five Asian countries confirmed the association between the 16189C variant and type 2 diabetes [odds ratio (OR) 1.256, 95% CI 1.08–1.46, pu2009=u20090.003]. Inclusion of data from three previously published Asian studies (type 2 diabetes nu2009=u20093,283, controls nu2009=u20092,176) in a meta-analysis showed similar results (OR 1.335, 95% CI 1.18–1.51, pu2009=u20090.000003). Mitochondrial single-stranded DNA-binding protein (mtSSB) was identified as a candidate protein bound to the 16189 region. Chromatin immunoprecipitation in cybrid cells showed that mtSSB has a lower binding affinity for the 16189C variant than the wild-type sequence.Conclusions/interpretationThe mtDNA 16189C variant is associated with an increased risk of type 2 diabetes in Asians.

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.