Free fatty acids (FFA) have been reported to reduce pancreatic ␤-cell mitogenesis and to increase apoptosis. Here we show that the FFA, oleic acid, increased apoptosis 16-fold in the pancreatic ␤-cell line, INS-1, over a 18-h period as assessed by Hoechst 33342/propidium iodide staining and caspase-3 and -9 activation, with negligible necrosis. A parallel analysis of the phosphorylation activation of protein kinase B (PKB) showed this was reduced in the presence of FFA that correlated with the incidence of apoptosis. At stimulatory 15 mM glucose and/or in the added presence of insulin-like growth factor 1, FFA-induced ␤-cell apoptosis was lessened compared with that at a basal 5 mM glucose. However, most strikingly, adenoviral mediated expression of a constitutively active PKB, but not a "kinase-dead" PKB variant, essentially prevented FFA-induced ␤-cell apoptosis under all glucose/insulin-like growth factor 1 conditions. Further analysis of pro-apoptotic downstream targets of PKB, implicated a role for PKB-mediated phosphorylation inhibition of glycogen synthase kinase-3␣/␤ and the forkhead transcription factor, FoxO1, in protection of FFA-induced ␤-cell apoptosis. In addition, down-regulation of the pro-apoptotic tumor suppresser protein, p53, via PKB-mediated phosphorylation of MDM2 might also play a role in partially protecting ␤-cells from FFA-induced apoptosis. Adenoviral mediated expression of wild type p53 potentiated FFA-induced ␤-cell apoptosis, whereas expression of a dominant negative p53 partly inhibited ␤-cell apoptosis by ϳ50%. Hence, these data demonstrate that PKB activation plays an important role in promoting pancreatic ␤-cell survival in part via inhibition of the pro-apoptotic proteins glycogen synthase kinase-3␣/␤, FoxO1, and p53. This, in turn, provides novel insight into the mechanisms involved in FFA-induced ␤-cell apoptosis.

Glucose can activate the mitogen-activated kinases, Erk-1/2, and the ribosomal-S6 kinase, p70 S6K , in ␤-cells, contributing to an increase in mitogenesis. However, the signaling mechanism by which glucose induces Erk-1/2 and p70 S6K phosphorylation activation is undefined.

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.