No abstract

Insulin resistance results, in part, from impaired insulin signaling in insulin target tissues. Consequently, increased levels of insulin are necessary to control plasma glucose levels. The effects of elevated insulin levels on pancreatic beta (β) cell function, however, are unclear. In this study, we investigated the possibility that insulin may influence survival of pancreatic β cells. Studies were conducted on RINm, RINm5F and Min-6 pancreatic β-cells. Cell death was induced by treatment with H(2)O(2), and was estimated by measurements of LDH levels, viability assay (Cell-Titer Blue), propidium iodide staining and FACS analysis, and mitochondrial membrane potential (JC-1). In addition, levels of cleaved caspase-3 and caspase activity were determined. Treatment with H(2)O(2) increased cell death; this effect was increased by simultaneous treatment of cells with insulin. Insulin treatment alone caused a slight increase in cell death. Inhibition of caspase-3 reduced the effect of insulin to increase H(2)O(2)-induced cell death. Insulin increased ROS production by pancreatic β cells and increased the effect of H(2)O(2). These effects were increased by inhibition of IR signaling, indicative of an effect independent of the IR cascade. We conclude that elevated levels of insulin may act to exacerbate cell death induced by H(2)O(2) and, perhaps, other inducers of apoptosis.

The mechanism underlying the important role of protein kinase C␦ (PKC␦) in the apoptotic effect of etoposide in glioma cells is incompletely understood. Here, we examined the role of PKC␦ in the activation of Erk1/2 by etoposide. We found that etoposide induced persistent activation of Erk1/2 and nuclear translocation of phospho-Erk1/2. MEK1 inhibitors decreased the apoptotic effect of etoposide, whereas inhibitors of p38 and JNK did not. The activation of Erk1/2 by etoposide was downstream of PKC␦ since the phosphorylation of Erk1/2 was inhibited by a PKC␦-KD mutant and PKC␦ small interfering RNA. We recently reported that phosphorylation of PKC␦ on tyrosines 64 and 187 was essential for the apoptotic effect of etoposide. Using PKC␦ tyrosine mutants, we found that the phosphorylation of PKC␦ on these tyrosine residues, but not on tyrosine 155, was also essential for the activation of Erk1/2 by etoposide. In contrast, nuclear translocation of PKC␦ was independent of its tyrosine phosphorylation and not necessary for the phosphorylation of Erk1/2. Etoposide induced down-regulation of kinase phosphatase-1 (MKP-1), which correlated with persistent phosphorylation of Erk1/2 and was dependent on the tyrosine phosphorylation of PKC␦. Moreover, silencing of MKP-1 increased the phosphorylation of Erk1/2 and the apoptotic effect of etoposide. Etoposide induced polyubiquitylation and degradation of MKP-1 that was dependent on PKC␦ and on its tyrosine phosphorylation. These results indicate that distinct phosphorylation of PKC␦ on tyrosines 64 and 187 specifically activates the Erk1/2 pathway by the down-regulation of MKP-1, resulting in the persistent phosphorylation of Erk1/2 and cell apoptosis. PKC␦2 is a novel PKC isoform that plays a major role in apoptosis in a cell-and stimulus-specific manner (1). PKC␦ has been reported to affect both the extrinsic and intrinsic apoptotic pathways and to mediate the apoptotic effect of various stimuli such as etoposide (2, 3), oxidative stress (4), ceramide (5), cisplatin (6), and phorbol 12-myristate 13-acetate (7). Conversely, it has been recently recognized that PKC␦ can act as an anti-apoptotic kinase in some cellular systems including Sindbis virus-infected (8) and TRAIL-treated glioma cells (9), nitric oxide-induced macrophage cell death (10), and cells expressing activated p21 RAS (11). Important factors that regulate the apoptotic function of PKC␦ are phosphorylation on distinct tyrosine residues and its subcellular localization (1). Tyrosine phosphorylation of PKC␦ is now recognized as a critical determinant in the activation, cleavage, localization and substrate affinity of this isoform (12-16). In addition to the tyrosine phosphorylation of PKC␦ by phorbol 12-myristate 13-acetate and various growth factors (12,17,18), PKC␦ undergoes phosphorylation in response to many apoptotic stimuli including etoposide (2), TRAIL (9), oxidative stress (4, 19), ␥-radiation (20), and cisplatin (13). PKC␦ has been shown to activate multiple signaling pathways that are associated with cel...

Abstract. Antiserum to human chorionic gonadotropin (HCG) caused marked inhibition of adventitious rooting of Begonia semperflorens and Chrysanthemum morifolium stem cuttings. Immuno-absorption of crude protein extract from chrvsanthemum foliage through a column of polymerized and unsolubilized HCG antibodies resulted in a significant reduction in adventitious root promoting activity of the extract. These results are discussed in the light of a hypothesis that an endogenous protein growth regulating substance which immunologically resembles HCG exists in plant systems. Further experimentation with HCG suggests that its mode of action is possibly via the regulation of peroxidase enzymatic control of auxin levels.In previous studies Leshem (10) and Leshem and Lunenfeld (11) have shown that HCG, a proteinaceous human sex hormone, has growth stimulating effects on certain morphogenetic processes in plants, especially upon adventitious rooting of Vitis vinifera, Begonia semperflorens and Brassica oleracea. It was shown that HCG possibly acts via regulation of endogenous gibberellin levels, the latter being markedly repressed by the gonadotropin, and a hypothesis was suggested that HCG triggers mevalonic acid conversion to steroids thereby diminishing precursor for gibberellins.In the present study bv use of immunological techniques an attempt has been made to indicate the existence of an endogenous gonadotropin-like proteinaceous growth regulating factor in plants. Fur-ther experimentation was carried out in order to elucidate the mode of HCG action in plant systems, especially in the light of hormone-enzyme interactions which are prevalent both in plant (3,7,13,14,15,21) One ml of the antiserum neutralized 600 International Units (IU) of HCG as determined by the procedure of Loraine and Brown (12). As control active rabbit serum lacking HCG antibodies was tised.The approach used in the immuno-absorption trials was as follows: If a protein extract of plant material were to be passed through a medium conitaining HCG antibodies, the effluent would be depleted of any existent endogenous HCG or any immunologically similar antigenic substances which could "cross react" with the antibodies (1) and resultinglv evoke a weaker growth stimulating response. This system was obtained by insolubilizing HCG antibodies by covalent binding of the molecules to one another with ethyl-chloroformate using the technique described by Avramaes and Ternynck (2).The immunoabsorbent properties of such insolubilized proteins were found by these workers to be specific, stable and effective to isolate specific antigens from mixtures and to remove contaminating antigen proteins from solutions. The protein obtained in this manner was packed in a 50 cm X 3 cm 4 glass column through which protein extract was subsequently passed. In order to determine whether this procedure does actually work in the present trials, various concentrations of HCG solutions were run through test columns and resulting effluents were assayed for presence of HCG by immuno-...