Barry I. Posner

A genome-wide association study identifies novel risk loci for type 2 diabetes

Type 2 diabetes mellitus results from the interaction of environmental factors with a combination of genetic variants, most of which were hitherto unknown. A systematic search for these variants was recently made possible by the development of high-density arrays that permit the genotyping of hundreds of thousands of polymorphisms. We tested 392,935 single-nucleotide polymorphisms in a French case–control cohort. Markers with the most significant difference in genotype frequencies between cases of type 2 diabetes and controls were fast-tracked for testing in a second cohort. This identified four loci containing variants that confer type 2 diabetes risk, in addition to confirming the known association with the TCF7L2 gene. These loci include a non-synonymous polymorphism in the zinc transporter SLC30A8, which is expressed exclusively in insulin-producing β-cells, and two linkage disequilibrium blocks that contain genes potentially involved in β-cell development or function (IDE–KIF11–HHEX and EXT2–ALX4). These associations explain a substantial portion of disease risk and constitute proof of principle for the genome-wide approach to the elucidation of complex genetic traits.

Genetic variant near IRS1 is associated with type 2 diabetes, insulin resistance and hyperinsulinemia

Genome-wide association studies have identified common variants that only partially explain the genetic risk for type 2 diabetes (T2D). Using genome-wide association data from 1,376 French individuals, we identified 16,360 SNPs nominally associated with T2D and studied these SNPs in an independent sample of 4,977 French individuals. We then selected the 28 best hits for replication in 7,698 Danish subjects and identified 4 SNPs showing strong association with T2D, one of which (rs2943641, P = 9.3 × 10−12, OR = 1.19) was located adjacent to the insulin receptor substrate 1 gene (IRS1). Unlike previously reported T2D risk loci, which predominantly associate with impaired beta cell function, the C allele of rs2943641 was associated with insulin resistance and hyperinsulinemia in 14,358 French, Danish and Finnish participants from population-based cohorts; this allele was also associated with reduced basal levels of IRS1 protein and decreased insulin induction of IRS1-associated phosphatidylinositol-3-OH kinase activity in human skeletal muscle biopsies.

Peroxide(s) of vanadium: a novel and potent insulin-mimetic agent which activates the insulin receptor kinase.

The actions of insulin, vanadate (V) and hydrogen peroxide (H2O2) on IGF-II binding and insulin receptor tyrosine kinase activity were studied in rat adipocytes. Incubating adipocytes with a combination of V plus H2O2 resulted in a potent synergistic effect on both the increase in IGF-II binding and the activation of the insulin receptor kinase. Catalase, which removes H2O2, abolished this synergism if added at the time of mixing of V plus H2O2 but not if added 10 min. later, suggesting that the formation of peroxide(s) of vanadate generated a potent insulin mimicker. The data support a critical role for the insulin receptor kinase in insulin action. The novel insulin-mimetic compound, a presumed peroxide of vanadate, could prove useful for investigating insulin action and may be valuable for treating insulin resistance.

Compartmentalized signal transduction by receptor tyrosine kinases

Signal transduction through receptor tyrosine kinases is believed to occur mainly at the plasma membrane. Ligands bind to their cognate receptors and trigger autophosphorylation events, which are detected by intracellular signalling molecules. However, ligands, such as epidermal growth factor and insulin, induce the rapid internalization of their receptors into endosomes. Although this event is traditionally thought to attenuate the ligand-induced response, in this article the authors discuss an alternative scenario in which selective and regulated signal transduction from receptor tyrosine kinases occurs within the endosome.

MODULATION OF INTERFERON-GAMMA -INDUCED MACROPHAGE ACTIVATION BY PHOSPHOTYROSINE PHOSPHATASES INHIBITION: EFFECT ON MURINE LEISHMANIASIS PROGRESSION

Phagocyte functions are markedly inhibited after infection with the intracellular protozoan parasiteLeishmania. This situation strongly favors the installation and propagation of this pathogen within its mammalian host. Previous findings by us and others have established that alteration of several signaling pathways (protein kinase C-, Ca2+- and protein-tyrosine kinases-dependent signaling events) were directly responsible for Leishmania-induced macrophage (MØ) dysfunctions. Here we report that modulation of phosphotyrosine-dependent events with a protein tyrosine phosphatases (PTP) inhibitor, the peroxovanadium (pV) compound bpV(phen) (potassium bisperoxo(1,10-phenanthroline)oxovanadate(Vi)), can control host-pathogen interactions by different mechanisms. We observed that the inhibition of parasite PTP resulted in an arrest of proliferation and death of the latter in coincidence with cyclin-dependent kinase (CDK1) tyrosine 15 phosphorylation. Moreover the treatment of MØ with bpV(phen) resulted in an increased sensitivity to interferon-γ stimulation, which was reflected by enhanced nitric oxide (NO) production. This enhanced IFN-γ-induced NO generation was accompanied by a marked increase of inducible nitric oxide synthase (iNOS) mRNA gene and protein expression. Finally we have verified the in vivo potency of bpV(phen) over a 6-week period of daily administration of a sub-toxic dose. The results revealed its effectiveness in controlling the progression of visceral and cutaneous leishmaniasis. Therefore PTP inhibition ofLeishmania and MØ by the pV compound bpV(phen) can differentially affect these eukaryotic cells. This strongly suggests that PTP plays an important role in the progression ofLeishmania infection and pathogenesis. The apparent potency of pV compounds along with their relatively simple and versatile structure render them attractive pharmacological agents for the management of parasitic infections.

Insulin receptor internalization and signalling

The insulin receptor kinase (IRK) is a tyrosine kinase whose activation, subsequent to insulin binding, is essential for insulin-signalling in target tissues. Insulin binding to its cell surface receptor is rapidly followed by internalization of insulin-IRK complexes into the endosomal apparatus (EN) of the cell. Internalization of insulin into target organs, especially liver, is implicated in effecting insulin clearance from the circulation. Internalization mediates IRK downregulation and hence attenuation of insulin sensitivity although most internalized IRKs readily recycle to the plasma membrane at physiological levels of insulin. A role for internalization in insulin signalling is indicated by the accumulation of activated IRKs in ENs. Furthermore, the maximal level of IRK activation has been shown to exceed that attained at the cell surface. Using an in vivo rat liver model in which endosomal IRKs are exclusively activated has revealed that IRKs at this intracellular locus are able by themselves to promote IRS-1 tyrosine phosphorylation and induce hypoglycemia. Furthermore, studies with isolated rat adipocytes reveal the EN to be the principle site of insulin-stimulated IRS-1 tyrosine phosphorylation and associated PI3K activation. Key steps in the termination of the insulin signal are also operative in ENs. Thus, an endosomal acidic insulinase has been identified which limits the extent of IRK activation. Furthermore, IRK dephosphorylation is effected in ENs by an intimately associated phosphotyrosine phosphatase(s) which, in rat liver, appears to regulate IRK activity in both a positive and negative fashion. Thus, insulin-mediated internalization of IRKs into ENs plays a crucial role in effecting and regulating signal transduction in addition to modulating the levels of circulating insulin and the cellular concentration of IRK in target tissues.

Peroxovanadium compounds: Biological actions and mechanism of insulin-mimesis

When used alone, both vanadate and hydrogen peroxide (H2O2) are weakly insulin-mimetic, while in combination they are strongly synergistic due to the formation of aqueous peroxovanadium species pV(aq). Administration of these pV(aq) species leads to activation of the insulin receptor tyrosine kinase (IRK), autophosphorylation at tyrosine residues and inhibition of phosphotyrosine phosphatases (PTPs). We therefore undertook to synthesize a series of peroxovanadium (pV) compounds containing one or two peroxo anions, an oxo anion and an ancillary ligand in the inner co-ordination sphere of vanadium, whose properties and insulin-mimetic potencies could be assessed. These pV compounds were shown to be the most potent inhibitors of PTPs yet described. Their PTP inhibitory potency correlated with their capacity to stimulate IRK activity. Some pV compounds showed much greater potency as inhibitors of insulin receptor (IR) dephosphorylation than epidermal growth factor receptor (EGFR) dephosphorylation, implying relative specificity as PTP inhibitors. Replacement of vanadium with either molybdenum or tungsten resulted in equally potent inhibition of IR dephosphorylation. However IRK activation was reduced by greater than 80% suggesting that these compounds did not access intracellular PTPs. The insulin-like activity of these pV compounds were demonstrablein vivo. Intra venous (i.v.) administration of bpV(pic) and bpV(phen) resulted in the lowaring of plasma glucose concentrations in normal rats in a dose dependent manner. The greater potency of bpV(pic) compared to bpV(phen) was explicable, in part, by the capacity of the former but not the latter to act on skeletal muscle as well as liver. Finally administration of bpV(phen) and insulin led to a synergism, where tyrosine phosphorylation of the IR β-subunit increased by 20-fold and led to the appearance of four insulin-dependentin vivo substrates. The insulin-mimetic properties of they pV compounds raises the possibility for their use as insulin replacements in the management of diabetes mellitus.

Interaction of the Grb10 Adapter Protein with the Raf1 and MEK1 Kinases

Grb10 and its close homologues Grb7 and Grb14, belong to a family of adapter proteins characterized by a proline-rich region, a central PH domain, and a carboxyl-terminal Src homology 2 (SH2) domain. Their interaction with a variety of activated tyrosine kinase receptors is well documented, but their actual function remains a mystery. The Grb10 SH2 domain was isolated from a two-hybrid screen using the MEK1 kinase as a bait. We show that this unusual SH2 domain interacts, in a phosphotyrosine-independent manner, with both the Raf1 and MEK1 kinases. Mutation of the MEK1 Thr-386 residue, which is phosphorylated by mitogen-activated protein kinase in vitro, reduces binding to Grb10 in a two-hybrid assay. Interaction of Grb10 with Raf1 is constitutive, while interaction between Grb10 and MEK1 needs insulin treatment of the cells and follows mitogen-activated protein kinase activation. Random mutagenesis of the SH2 domain demonstrated that the Arg-βB5 and Asp-EF2 residues are necessary for binding to the epidermal growth factor and insulin receptors as well as to the two kinases. In addition, we show that a mutation in Ser-βB7 affects binding only to the receptors, while a mutation in Thr-βC5 abrogates binding only to MEK1. Finally, transfection of Grb10 genes with specific mutations in their SH2 domains induces apoptosis in HTC-IR and COS-7 cells. These effects can be competed by co-expression of the wild type protein, suggesting that these mutants act by sequestering necessary signaling components.

Activation of HIV-1 Long Terminal Repeat Transcription and Virus Replication via NF-kB-dependent and -independent Pathways by Potent Phosphotyrosine Phosphatase Inhibitors, the Peroxovanadium Compounds*

Replication of human immunodeficiency virus type 1 (HIV-1) is increased by different cytokines and T cell activators, also known to modulate tyrosine phosphorylation levels. A novel class of protein tyrosine phosphatase (PTP) inhibitors, peroxovanadium (pV) compounds, were tested for a putative effect on HIV-1 long terminal repeat (LTR) activity. We found that these PTP inhibitors markedly enhanced HIV-1 LTR activity in 1G5 cells, a stably transfected cell line that harbors an HIV-1 LTR-driven luciferase construct. A direct correlation between the extent of tyrosine phosphorylation and the level of HIV-1 LTR inducibility was seen after treatment with three different pV compounds. Transient transfection experiments were carried out in several T cell lines, and after addition of pV, a marked increase in HIV-1 LTR activity was measured. Monocytoid cells were tested using U937-derived cell lines and were also found to be sensitive to the pV-mediated potentiating effect on HIV-1 LTR activity. A significant reduction of the pV-mediated increase in HIV-1 LTR activity was seen in cells transiently transfected with an HIV-1 LTR-driven luciferase construct bearing a mutation in both NF-κB binding sites although detectable levels of induction remained. Electrophoretic mobility shift assays allowed the identification of the nuclear translocation of the NF-κB p50·p65 heterodimer complex induced by pV compounds. A dominant negative version of the repressor IκBα mutated on serines 32 and 36 impeded pV-induced NF-κB-dependent luciferase activity. Western blot analysis showed a clear diminution in the protein level of IκBα starting 30 min after pV treatment of Jurkat E6.1 cells which is indicative of its degradation. On the other hand, no increase in tyrosine phosphorylation was observed on IκBα itself. Finally, we tested the PTP inhibitors on four cell lines latently infected with HIV-1 and showed a consistent pV-mediated increase in virion production. Thus, our studies suggest that pV-mediated activation of HIV-1 LTR activity is controlled by the nuclear translocation of the NF-κB transcription factor, which is mediated by IκBα serine phosphorylation and degradation, but also by a still undefined NF-κB-independent pathway.

Insulin receptor-associated protein tyrosine phosphatase(s): Role in insulin action

Protein tyrosine phosphatases (PTPs) play a critical role in regulating insulin action in part through dephosphorylation of the active (autophosphorylated) form of the insulin receptor (IRK) and attenuation of its tyrosine kinase activity. Following insulin binding the activated IRK is rapidly internalized into the endosomal apparatus, a major site at which the IRK is dephosphorylated in vivo. Studies in rat liver suggest a complex regulatory process whereby PTPs may act, via selective IRK tyrosine dephosphorylation, to modulate IRK activity in both a positive and negative manner. Use of peroxovanadium (pV) compounds, shown to be powerful PTP inhibitors, has been critical in delineating a close relationship between the IRK and its associated PTP(s) in vivo. Indeed the in vivo administration of pV compounds effected activation of IRK in parallel with an inhibition of IRK-associated PTP activity. This process was accompanied by a lowering of blood glucose levels in both normal and diabetic rats thus implicating the IRK-associated PTP(s) as a suitable target for defining a novel class of insulin mimetic agents. Identification of the physiologically relevant IRK-associated PTP(s) should facilitate the development of drugs suitable for managing diabetes mellitus.