It has been shown that IGF-1-induced pancreatic -cell proliferation is glucose-dependent; however, the mechanisms responsible for this glucose dependence are not known. Adenoviral mediated expression of constitutively active phosphatidylinositol 3-kinase (PI3K) in the pancreatic -cells, INS-1, suggested that PI3K was not necessary for glucose-induced -cell proliferation but was required for IGF-1-induced mitogenesis. Examination of the signaling components downstream of PI3K, 3-phosphoinositide-dependent kinase 1, protein kinase B (PKB), glycogen synthase kinase-3, and p70-kDa-S6-kinase (p70 S6K ), suggested that a major part of glucose-dependent -cell proliferation requires activation of mammalian target of rapamycin/p70 S6K , independent of phosphoinositide-dependent kinase 1/PKB activation. Adenoviral expression of the kinase-dead form of PKB in INS-1 cells decreased IGF-1-induced -cell proliferation. However, a surprisingly similar decrease was also observed in adenoviral wild type and constitutively active PKB-infected cells. Upon analysis of extracellular signal-regulated protein kinase 1 and 2 (ERK1/ERK2), an increase in ERK1/ERK2 phosphorylation activation by glucose and IGF-1 was observed in kinase-dead PKB-infected cells, but this phosphorylation activation was inhibited in the constitutively active PKB-infected cells. Hence, there is a requirement for the activation of both ERK1/ERK2 and mammalian target of rapamycin/p70 S6K signal transduction pathways for a full commitment to glucose-induced pancreatic -cell mitogenesis. However, for IGF-1-induced activation, these pathways must be carefully balanced, because chronic activation of one (PI3K/PKB) can lead to dampening of the other (ERK1/2), reducing the mitogenic response.
glucose and IGF-1 was more prolonged (>24 h) and, though not as robust as TGF-␣/EGF, did increase -cell proliferation. Phosphorylation of p70 S6K was also increased by IGF-1/glucose, but not by TGF-␣/EGF, despite upstream PKB activation. It was found that IGF-1 induced phosphatidylinositol 3-kinase (PI3K) association with insulin receptor substrate (IRS)-1 and -2 in a glucosedependent manner, whereas TGF-␣/EGF did not. The importance of specific IRS-2-mediated signaling events was emphasized in that adenoviral-mediated overexpression of IRS-2 further increased glucose/IGF-1-induced -cell proliferation (more than twofold; P < 0.05) compared with control or adenoviral-mediated IRS-1 overexpressing INS-1 cells. Neither IRS-1 nor IRS-2 overexpression induced a -cell proliferative response to TGF-␣/EGF. Thus, a prolonged activation of Erk1/2 and PI3K signaling pathways is important in committing a -cell to a mitogenic event, and it is likely that this sustained activation is instigated by signal transduction occurring specifically through IRS-2. Diabetes 51: 966 -976, 2002
Regulation of insulin receptor substrate (IRS)-2 expression is critical to -cell survival, but the mechanisms that control this are complex and undefined. Here in pancreatic -cells (INS-1), chronic exposure (>8 h) to 15 mM glucose and/or 5 nM IGF-1, increased Ser/Thr phosphorylation of IRS-2, which correlated with decreased IRS-2 levels. This glucose/IGF-1-induced decrease in IRS-2 levels was prevented by the proteasomal inhibitor, lactacystin. In addition, the glucose/IGF-1-induced increase in Ser/Thr phosphorylation of IRS-2 and the subsequent decrease in INS-1 cell IRS-2 protein levels was thwarted by the mammalian target of rapamycin(mTOR) inhibitor, rapamycin. Moreover, adenoviral-mediated expression of constitutively active mTOR (mTOR⌬) further increased glucose/IGF-1-induced Ser/Thr phosphorylation of IRS-2 and decreased IRS-2 protein levels, whereas adenoviralmediated expression of "kinase-dead" mTOR (mTOR-KD) conversely reduced Ser/Thr phosphorylation of IRS-2 and maintained IRS-2 protein levels. In adenoviral-infected -cells expressing mTOR⌬, the decrease in IRS-2 protein levels was also prevented by rapamycin or lactacystin, further indicating a proteasomal mediated degradation of IRS-2 mediated via mTOR-induced Ser/Thr phosphorylation of IRS-2. Finally, we found that chronic activation of mTOR leading to decreased levels of IRS-2 in INS-1 cells led to a significant decrease in PKB activation and consequently increased -cell apoptosis. Thus, chronic activation of mTOR by glucose (and/or IGF-1) in -cells leads to increased Ser/Thr phosphorylation of IRS-2 that targets it for proteasomal degradation, resulting in decreased IRS-2 expression and increased -cell apoptosis. This may be a contributing mechanism as to how -cell mass is decreased by chronic hyperglycemia in the pathogenesis of type-2 diabetes.
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