Yousuke Ebina

IRS1-Independent Defects Define Major Nodes of Insulin Resistance

Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.

Protein Phosphatase 2A Negatively Regulates Insulin's Metabolic Signaling Pathway by Inhibiting Akt (Protein Kinase B) Activity in 3T3-L1 Adipocytes

ABSTRACT Protein phosphatase 2A (PP2A) is a multimeric serine/threonine phosphatase which has multiple functions, including inhibition of the mitogen-activated protein (MAP) kinase pathway. Simian virus 40 small t antigen specifically inhibits PP2A function by binding to the PP2A regulatory subunit, interfering with the ability of PP2A to associate with its cellular substrates. We have reported that the expression of small t antigen inhibits PP2A association with Shc, leading to augmentation of insulin and epidermal growth factor-induced Shc phosphorylation with enhanced activation of the Ras/MAP kinase pathway. However, the potential involvement of PP2A in insulins metabolic signaling pathway is presently unknown. To assess this, we overexpressed small t antigen in 3T3-L1 adipocytes by adenovirus-mediated gene transfer and found that the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3β, were enhanced both in the absence and in the presence of insulin. Furthermore, protein kinase C λ (PKC λ) activity was also augmented in small-t-antigen-expressing 3T3-L1 adipocytes. Consistent with this result, both basal and insulin-stimulated glucose uptake were enhanced in these cells. In support of this result, when inhibitory anti-PP2A antibody was microinjected into 3T3-L1 adipocytes, we found a twofold increase in GLUT4 translocation in the absence of insulin. The small-t-antigen-induced increase in Akt and PKC λ activities was not inhibited by wortmannin, while the ability of small t antigen to enhance glucose transport was inhibited by dominant negative Akt (DN-Akt) expression and Akt small interfering RNA (siRNA) but not by DN-PKC λ expression or PKC λ siRNA. We conclude that PP2A is a negative regulator of insulins metabolic signaling pathway by promoting dephosphorylation and inactivation of Akt and PKC λ and that most of the effects of PP2A to inhibit glucose transport are mediated through Akt.

GLUT4 TRANSLOCATION BY INSULIN IN INTACT MUSCLE CELLS : DETECTION BY A FAST AND QUANTITATIVE ASSAY

We report a rapid and sensitive colorimetric approach to quantitate the amount of glucose transporters exposed at the surface of intact cells, using L6 muscle cells expressing GLUT4 containing an exofacial myc epitope. Unstimulated cells exposed to the surface 5 fmol GLUT4myc per mg protein. This value increased to 10 fmol/mg protein in response to insulin as 2‐deoxyglucose (10 μM) uptake doubled. The results are substantiated by immunofluorescent detection of GLUT4myc in unpermeabilized cells and by subcellular fractionation. We further show that wortmannin and the cytoskeleton disruptors cytochalasin D and latrunculin B completely blocked these insulin effects. The rapid quantitative assay described here could be of high value to study insulin signals and to screen for potential anti‐diabetic drugs.

Impaired glucose tolerance in mice with a targeted impairment of insulin action in muscle and adipose tissue

Type 2 diabetes is a complex metabolic disorder characterized by peripheral insulin resistance and impaired Β cell function1,2. Insulin resistance is inherited as a non-mendelian trait. In genetically predisposed individuals, resistance of skeletal muscle and adipose tissue to insulin action precedes the onset of clinical diabetes, and is thought to contribute to hyperglycaemia by leading to impaired Β cell function and increased hepatic glucose production4,5. It is not clear whether Β cell and liver defects are also genetically determined. To test the hypothesis that insulin resistance in muscle and fat is sufficient to cause type 2 diabetes in the absence of intrinsic Β cell and liver abnormality, we generated transgenic mice that were insulin-resistant in skeletal muscle and adipose tissue. These mice developed all the prodromal features of type 2 diabetes but, despite the compounded effect of peripheral insulin resistance and a mild impairment of Β cell function, failed to become diabetic. These findings indicate the need for a critical re-examination of the primary site(s) of insulin resistance in diabetes.

Human diabetes associated with a deletion of the tyrosine kinase domain of the insulin receptor

The insulin receptor has an intrinsic tyrosine kinase activity that is essential for signal transduction. A mutant insulin receptor gene lacking almost the entire kinase domain has been identified in an individual with type A insulin resistance and acanthosis nigricans. Insulin binding to the erythrocytes or cultured fibroblasts from this individual was normal. However receptor autophosphorylation and tyrosine kinase activity toward an exogenous substrate were reduced in partially purified insulin receptors from the probands lymphocytes that had been transformed by Epstein-Barr virus. The insulin resistance associated with this mutated gene was inherited by the proband from her mother as an apparently autosomal dominant trait. Thus a deletion in one allele of the insulin receptor gene may be at least partly responsible for some instances of insulin-resistant diabetes.

GLUT-4myc ectopic expression in L6 myoblasts generates a GLUT-4-specific pool conferring insulin sensitivity

Insulin stimulates glucose uptake into muscle and fat cells via recruitment of the glucose transporter 4 (GLUT-4) from intracellular store(s) to the cell surface. Robust stimulation of glucose uptake by insulin coincides with the expression of GLUT-4 during differentiation of muscle and fat cells, but it is not known if GLUT-4 expression suffices to confer insulin sensitivity to glucose uptake. We have therefore examined the effect of expression of a myc epitope-tagged GLUT-4 (GLUT-4myc) into L6 myoblasts, which do not express endogenous GLUT-4 until differentiated into myotubes. Ectopic expression of GLUT-4myc markedly improved insulin sensitivity of glucose uptake in L6 myoblasts. The GLUT-4myc protein distributed equally to the cell surface and intracellular compartments in myoblasts, and the intracellular fraction of GLUT-4myc further increased in myotubes. In myoblasts, the intracellular GLUT-4myc compartment contained the majority of the insulin-regulatable amino peptidase (IRAP) but less than half of the GLUT-1, suggesting segregation of GLUT-4myc and IRAP to a specific cellular locus. Insulin stimulation of phosphatidylinositol 3-kinase and protein kinase B-α activities was similar for L6-GLUT-4myc myoblasts and myotubes. At both stages, GLUT-4myc responded to insulin by translocating to the cell surface. These results suggest that GLUT-4myc segregates into a specific compartment in L6 myoblasts and confers insulin sensitivity to these cells. L6-GLUT-4myc myoblasts, which are easily transfectable with various constructs, are a useful resource to study insulin action.

Insulin-resistant diabetes associated with partial deletion of insulin-receptor gene

The insulin-receptor genes from a 16-year-old girl with type A insulin resistance, who presented with fasting hyperinsulinaemia, acanthosis nigricans, and reduced insulin binding, and from her family were examined. One allele of her insulin-receptor gene inherited from her mother contained a 1.2 kb deletion arising from a recombination between two Alu elements. The deletion removed the 14th exon in the beta subunit and altered the reading frame, to produce a stop codon after aminoacid 867. Pedigree analysis indicated that this mutation alone will not cause diabetes, and the proband is possibly a compound heterozygote. 4 other members of her family were heterozygous for the same mutation; all 4 had a decrease in insulin binding and slight impairment of glucose tolerance. Perhaps the same mutation is an underlying feature of some cases of non-insulin-dependent diabetes mellitus.

Site-directed mutagenesis studies on the iron-binding domain and the determinant for the substrate oxygenation site of porcine leukocyte arachidonate 12-lipoxygenase.

cDNA for arachidonate 12-lipoxygenase of porcine leukocytes was expressed in Escherichia coli. The recombinant 12-lipoxygenase was purified by immunoaffinity chromatography to near homogeneity with a specific activity of about 1.5 mumol/min per mg protein. Each of eight histidine residues, which were well-conserved among various mammalian lipoxygenases and presumed as ligands for non-heme iron, was substituted with leucine by site-directed mutagenesis. Each mutant enzyme was immunoaffinity-purified to near homogeneity. Mutations of His-361, -366 and -541 caused a total loss of enzyme activity, and the iron content was much lower (0.10, 0.06 and 0.06 g atom/mol protein) than that of the wild-type enzyme (0.53). Mutations of His-128 and -356 gave 159% and 162% specific activity of the wild-type enzyme, and the iron contents were 0.55 and 0.52 g atom/mol protein. Substitution of His-426 decreased the activity to 5%, but the iron content was 0.4 g atom/mol protein. The expression level of mutants at His-384 and -393 was too low to precisely determine the iron content. Taken together, His-361, -366 and -541 may play important roles for iron-binding in catalytically active 12-lipoxygenase. Since a high homology of amino acid sequence was known between porcine leukocyte 12-lipoxygenase and mammalian 15-lipoxygenases, we attempted to convert the 12-lipoxygenase to a 15-lipoxygenase. A double mutation of Val-418 and -419 to Ile and Met increased the ratio of 15- and 12-lipoxygenase activities from 0.1 to 5.7.

Deficiency of Cbl-b gene enhances infiltration and activation of macrophages in adipose tissue and causes peripheral insulin resistance in mice.

OBJECTIVE—c-Cbl plays an important role in whole-body fuel homeostasis by regulating insulin action. In the present study, we examined the role of Cbl-b, another member of the Cbl family, in insulin action. RESEARCH DESIGN AND METHODS—C57BL/6 (Cbl-b+/+) or Cbl-b-deficient (Cbl-b−/−) mice were subjected to insulin and glucose tolerance tests and a hyperinsulinemic-euglycemic clamp test. Infiltration of macrophages into white adipose tissue (WAT) was assessed by immunohistochemistry and flow cytometry. We examined macrophage activation using co-cultures of 3T3-L1 adipocytes and peritoneal macrophages. RESULTS—Elderly Cbl-b−/− mice developed glucose intolerance and peripheral insulin resistance; serum insulin concentrations after a glucose challenge were always higher in elderly Cbl-b−/− mice than age-matched Cbl-b+/+ mice. Deficiency of the Cbl-b gene significantly decreased the uptake of 2-deoxyglucose into WAT and glucose infusion rate, whereas fatty liver was apparent in elderly Cbl-b−/− mice. Cbl-b deficiency was associated with infiltration of macrophages into the WAT and expression of cytokines, such as tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein (MCP)-1. Co-culture of Cbl-b−/− macrophages with 3T3-L1 adipocytes induced leptin expression and dephosphorylation of insulin receptor substrate 1, leading to impaired glucose uptake in adipocytes. Furthermore, Vav1, a key factor in macrophage activation, was highly phosphorylated in peritoneal Cbl-b−/− macrophages compared with Cbl-b+/+ macrophages. Treatment with a neutralizing anti–MCP-1 antibody improved peripheral insulin resistance and macrophage infiltration into WAT in elderly Cbl-b−/− mice. CONCLUSIONS—Cbl-b is a negative regulator of macrophage infiltration and activation, and macrophage activation by Cbl-b deficiency contributes to the peripheral insulin resistance and glucose intolerance via cytokines secreted from macrophages.

Transcriptional Activation of the Glut1 Gene in Response to Oxidative Stress in L6 Myotubes

Exposure of L6 myotubes to prolonged low grade oxidative stress results in increased Glut1 expression at both the protein and mRNA levels, leading to elevated glucose transport activity. To further understand the cellular mechanisms responsible for this adaptive response, the Glut1 transcription rate and mRNA stability were assessed. Nuclear run-on assays revealed 2.0- and 2.4-fold increases in Glut1 transcription rates in glucose oxidase- and xanthine/xanthine oxidase-pretreated cells, respectively. Glut1 mRNA stability was increased with both treatments compared with the control (t½ = 7.8 ± 1.3, 6.0 ± 2.0, and 2.4 ± 0.5 h, respectively). The serum-responsive element and AP-1 (but not the cAMP-responsive element) showed increased binding capacity following oxidative stress. Both activation of AP-1 binding and elevation ofGlut1 mRNA were prevented by cycloheximide. The involvement of enhancer 1 of the Glut1 gene was demonstrated using transfected 293 cells. Induction ofGlut1 mRNA in response to oxidative stress differed from its activation by chronic insulin exposure as demonstrated by the ability of rapamycin to inhibit the latter without an effect on the former. In conclusion, oxidative stress increases the Glut1transcription rate by mechanisms that may involve activation of AP-1 binding to enhancer 1 of the Glut1 gene.