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

Nutrients and certain growth factors stimulate pancreatic ␤-cell mitogenesis, however, the appropriate mitogenic signal transduction pathways have not been defined. In the glucose-sensitive pancreatic ␤-cell line, INS-1, it was found that glucose (6 -18 mM) independently increased INS-1 cell proliferation (>20-fold at 15 mM glucose). Insulin-like growth factor I (IGF-I)-induced INS-1 cell proliferation was glucose-dependent only in the physiologically relevant concentration range (6 -18 mM glucose). The combination of IGF-I and glucose was synergistic, increasing INS-1 cell proliferation >50-fold at 15 mM glucose ؉ 10 nM IGF-I. Glucose metabolism and phosphatidylinositol 3-kinase (PI 3-kinase) activation were necessary for both glucose and IGF-I-stimulated INS-1 cell proliferation. IGF-I and 15 mM glucose increased tyrosine phosphorylation mediated recruitment of Grb2/mSOS and PI 3-kinase to IRS-2 and pp60. Glucose and IGF-I also induced Shc association with Grb2/ mSOS. Glucose (3-18 mM) and IGF-I, independently of glucose, activated mitogen-activated protein kinase but this did not correlate with IGF-I-induced ␤-cell proliferation. In contrast, p70 S6K was activated with increasing glucose concentration (between 6 and 18 mM), and potentiated by IGF-I in the same glucose concentration range which correlated with INS-1 cell proliferation rate. Thus, glucose and IGF-I-induced ␤-cell proliferation were mediated via a signaling mechanism that was facilitated by mitogen-activated protein kinase but dependent on IRS-mediated induction of PI 3-kinase activity and downstream activation of p70 S6K . The glucose dependence of IGF-I mediated INS-1 cell proliferation emphasizes ␤-cell signaling mechanisms are rather unique in being tightly linked to glycolytic metabolic flux.