Abstract
Overexpression of the Src homology 2 domain protein Shb in beta-cells of transgenic mice has been shown to promote an increased beta-cell mass. To investigate the mechanisms by which Shb controls the beta-cell mass, we have presently studied the effects of Shb overexpression on the IRS-1-induced signaling pathway in mouse islet beta-cells and in insulin-producing RINm5F cells and correlated these effects to growth and death patterns. Shb overexpression was achieved in RINm5F cells by selection of stable clones or by FACS purification of transiently transfected cells. For Shb overexpression in primary mouse islet cells, a Shb-transgene mouse was used. Cell proliferation and death rates were determined using flow cytometry. Serum-, insulin-, and IGF-1-stimulated signaling events were studied by immunoblot, immunoprecipitation, and in vitro kinase procedures. Transient Shb overexpression in RINm5F cells resulted in increased proliferation. Both Shb-overexpressing RINm5F cells and islet cells from transgenic mice (islet Shb) exhibited increased basal tyrosine phosphorylation of IRS-1. Shb overexpression resulted also in the assembly and activation of a multiunit complex consisting of at least Shb, IRS-1, IRS-2, FAK, and PI3K. Consequently, the phosphorylation of Akt was enhanced under basal conditions in Shb overexpressing cells. Finally, Shb overexpression did not affect insulin-induced phosphorylation of the PI3K-antagonist PTEN. It is concluded that the Shb-induced alterations in the IRS-1/PI3K/Akt pathway may be relevant to the understanding of growth and death patterns of insulin-producing cells.
Highlights
The development of diabetes mellitus depends on the balance between -cell proliferation and death [1]
Because the supply of islet tissue is insufficient for careful studies of IRS-1–mediated signaling events, we utilized the RINm5F cell line with stable or transient Shb overexpression as a model
We presently describe major perturbations in IRS-1 and IRS-2 signaling in insulin producing -cells as a consequence of overexpression of the Src homology 2 (SH2) domain adapter protein Shb
Summary
The development of diabetes mellitus depends on the balance between -cell proliferation and death [1]. Recent investigations have implicated insulin or insulin-like growth factor-1–dependent (IGF) signaling via the insulin receptor substrates (IRS1, IRS-2) of paramount importance for -cell proliferation, survival, and insulin gene expression [2,3,4,5]. The reduced -cell mass in the IRS-2 knockout mice implies a significant role for this signaling protein in -cell replication or survival. Increased -cell apoptosis was noted in IGF-1 receptor/IRS-2 knockout mice [4]. To investigate the mechanisms by which Shb controls the -cell mass, we have presently studied the effects of Shb overexpression on the IRS-1–induced signaling pathway in mouse islet -cells and in insulin-producing RINm5F cells and correlated these effects to growth and death patterns. Serum-, insulin-, and IGF-1stimulated signaling events were studied by immunoblot, immunoprecipitation, and in vitro kinase procedures
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