Chin K. Sung

OVEREXPRESSION OF MEMBRANE GLYCOPROTEIN PC-1 CAN INFLUENCE INSULIN ACTION AT A POST-RECEPTOR SITE

An elevated content of membrane glycoprotein PC‐1 has been observed in cells and tissues of insulin resistant patients. In addition, in vitro overexpression of PC‐1 in cultured cells induces insulin resistance associated with diminished insulin receptor tyrosine kinase activity. We now find that PC‐1 overexpression also influences insulin receptor signaling at a step downstream of insulin receptor tyrosine kinase, independent of insulin receptor tyrosine kinase. In the present studies, we employed Chinese hamster ovary cells that overexpress the human insulin receptor (CHO IR cells; ∼106 receptors per cell), and transfected them with human PC‐1 c‐DNA (CHO IR PC‐1). In CHO IR PC‐1 cells, insulin receptor tyrosine kinase activity was unchanged, following insulin treatment of cells. However, several biological effects of insulin, including glucose and amino acid uptake, were decreased. In CHO IR PC‐1 cells, insulin stimulation of mitogen‐activated protein (MAP) kinase activity was normal, suggesting that PC‐1 overexpression did not affect insulin receptor activation of Ras, which is upstream of MAP kinase. Also, insulin‐stimulated phosphatidylinositol (PI)‐3‐kinase activity was normal, suggesting that PC‐1 overexpression did not interfere with the activation of this enzyme by insulin receptor substrate‐1. In these cells, however, insulin stimulation of p70 ribosomal S6 kinase activity was diminished. These studies suggest, therefore, that, in addition to blocking insulin receptor tyrosine kinase activation, PC‐1 can also block insulin receptor signaling at a post‐receptor site. J. Cell. Biochem. 68:366–377, 1998.

Regulation of the Akt/Glycogen synthase kinase‐3 axis by insulin‐like growth factor‐II via activation of the human insulin receptor isoform‐A

Insulin‐like growth factor II (IGF‐II) plays a key role in mitogenesis during development and tumorigenesis and is believed to exert its mitogenic functions mainly through the IGF‐I receptor. Recently, we identified the insulin receptor isoform A (IRA) as an additional high affinity receptor for IGF‐II in both fetal and cancer cells. Here we investigated the mitogenic signaling of IGF‐II via the Akt/Glycogen synthase kinase 3 (Gsk3) axis employing R‐IRA cells that are IGF‐I receptor null mouse embryonic fibroblasts expressing the human IRA. IGF‐II induced activation of the proto‐oncogenic serine kinase Akt, reaching maximal at 5–10 min. IGF‐II also caused the rapid and sustained deactivation of glycogen synthase kinase 3‐beta (Gsk3β), reaching maximal at 1–3 min, shortly preceding, therefore, maximal activation of Akt. Under our conditions, IGF‐II and insulin induced 70–80% inhibition of Gsk3βactivity. In these cells IGF‐II also deactivated Gsk3α although less effectively than Gsk3β. In parallel experiments, we found that IGF‐II induced transient activation of extracellular‐signal‐regulated kinases (Erk) reaching maximal at 5–10 min and decreasing thereafter. Time courses and potencies of regulation of both mitogenic pathways (Akt/Gsk3β and Erk) by IGF‐II via IRA were similar to those of insulin. Furthermore, IGF‐II like insulin effectively stimulated cell cycle progression from the G0/G1 to the S and G2/M phases. Interestingly, AP‐1‐mediated gene expression, that was reported to be negatively regulated by Gsk3β was only weakly increased after IGF‐II stimulation. Our present data suggest that the coordinated activation or deactivation of Akt, Gsk3β, and Erk may account for IGF‐II mitogenic effects and support an active role for IRA in IGF‐II action. J. Cell. Biochem. 82: 610–618, 2001.

Insulin-stimulated cell growth in insulin receptor substrate-1-deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase-independent pathway.

In many human breast cancers and cultured cell lines, insulin receptor expression is elevated, and insulin, via its own insulin receptor, can stimulate cell growth. It has recently been demonstrated that the enzyme phosphatidylinositol‐3‐kinase (PI3‐K) mediates various aspects of insulin receptor signaling including cell growth. In order to understand the mechanisms for insulin‐stimulated cell growth in human breast cancer, we measured insulin‐stimulable PI3‐K activity in a non‐transformed breast epithelial cell line, MCF‐10A, and in two malignantly transformed cell lines, ZR‐75‐1 and MDA‐MB157. All three cell lines express comparable amounts of insulin receptors whose tyrosine autophosphorylation is increased by insulin, and in these cell lines insulin stimulates growth. In MDA‐MB157 and MCF‐10A cells, insulin stimulated PI3‐K activity three‐ to fourfold. In ZR‐75‐1 cells, however, insulin did not stimulate PI3‐K activity. In ZR‐75‐1 cells PI3‐K protein was present, and its activity was stimulated by epidermal growth factor, suggesting that there might be a defect in insulin receptor signaling upstream of PI3‐K and downstream of the insulin receptor. Next, we studied insulin receptor substrate‐1 (IRS‐1), a major endogenous substrate for the insulin receptor which, when tyrosine is phosphorylated by the insulin receptor, interacts with and activates PI3‐K. In ZR‐75‐1 cells, there were reduced levels of protein for IRS‐1. In these cells, both Shc tyrosine phosphorylation and mitogen‐activated protein kinase (MAP‐K) activity were increased by the insulin receptor (indicating that the p21ras pathway may account for insulin‐stimulated cell growth in ZR‐75‐1 cells).

Insulin-like and non-insulin-like selenium actions in 3T3-L1 adipocytes

In insulin‐sensitive 3T3‐L1 adipocytes, selenium stimulates glucose transport and antilipolysis and these actions of selenium, like insulin actions, are sensitive to wortmanin, an inhibitor of phosphatidylinositol‐3‐kinase (PI3K). Selenium stimulates PI3K activity that is sustained up to 24 h. Selenium after 5–10 min increases tyrosine phosphorylation of selective cellular proteins, but after 24 h overall tyrosine phosphorylation is increased. Tyrosine phosphorylation of insulin receptor substrate 1 is detected when enriched by immunoprecipitation with anti‐PI3K antibody. Selenium, however, does not stimulate insulin receptor tyrosine kinase activity. Selenium also increases phosphorylation of other insulin signaling proteins, including Akt and extracellular signal regulated kinases. Selenium‐stimulated glucose transport is accompanied by increases in glucose transporter‐1 content in the plasma membrane. These data are consistent with similar selenium action in glucose transport in 3T3‐L1 fibroblasts expressing mainly GLUT1. In chronic insulin‐induced insulin resistant cells, selenium unlike insulin fully stimulates glucose transport. In summary, selenium stimulates glucose transport and antilipolysis in a PI3K‐dependent manner, but independent of insulin receptor activation. Selenium exerts both insulin‐like and non‐insulin‐like actions in cells.

Insulin-Stimulated Glycogen Synthesis in Cultured Hepatoma Cells: Differential Effects of Inhibitors of Insulin Signaling Molecules

In rat HTC hepatoma cells overexpressing human insulin receptors, insulin stimulated glycogen synthesis by 55-70%. To study postreceptor signaling events leading to insulin-stimulated glycogen synthesis in these cells, we have employed pathway-specific chemical inhibitors such as LY294002, rapamycin and PD98059 to inhibit phosphatidylinositol-3-kinase (PI3K), p70 ribosomal S6 kinase and mitogen-activated protein kinase (MAPK) kinase/MAPK, respectively. LY294002 (50 microM) completely abolished insulin-stimulated glycogen synthesis whereas rapamycin (2-20 nM) partially inhibited it. Neither LY294002 nor rapamycin significantly affected the basal glycogen synthesis. However, PD98059 (100 microM) significantly inhibited the basal glycogen synthesis without affecting insulin-stimulated glycogen synthesis. In these cells, insulin at 100 nM decreased glycogen synthase kinase 3 alpha (GSK3 alpha) activity by 30-35%. LY294002, but neither rapamycin nor PD98059, abolished insulin-induced inactivation of GSK3 alpha. These data suggest that insulin-stimulated glycogen synthesis in rat HTC hepatoma cells is mediated mainly by PI3K-dependent mechanism. In these cells, inactivation of GSK3 alpha, downstream of PI3K, may play a role in insulin-stimulated glycogen synthesis.

Inhibition of Phosphatidylinositol-3-kinase Enhances Insulin Stimulation of Insulin Receptor Substrate 1 Tyrosine Phosphorylation and Extracellular Signal-Regulated Kinases in Mouse R− Fibroblasts

Insulin stimulates phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated kinases (ERK) in various mammalian cells. To study the role of PI3K in insulin stimulation of ERK, we employed PI3K inhibitor LY294002 and mouse embryonic R− fibroblasts lacking IGF-1 receptors. In these R− cells, PI3K inhibition by LY294002 enhanced insulin stimulation of ERK phosphorylation whereas LY294002 inhibited insulin stimulation of Akt phosphorylation. The enhanced insulin stimulation of ERK phosphorylation was accompanied by increased IRS-1 tyrosine phosphorylation. Insulin stimulation of insulin receptor tyrosine phosphorylation was not altered. PI3K inhibition increased IRS-1–Grb2 complex formation and ras activity following insulin treatment of cells. Increased insulin stimulation of ERK by PI3K inhibition was mediated by the MEK/ERK pathway, but did not involve inhibitory Ser259 phosphorylation of raf that was reported to be mediated by Akt. In summary, PI3K inhibition in R− cells enhanced insulin stimulation of ERK phosphorylation by mechanisms involving enhancement of IRS-1 tyrosine phosphorylation, IRS-1–Grb2 complex formation and the ras/MEK/ERK pathway.