A Role for Nuclear Factor κB in the Antiapoptotic Function of Insulin

Abstract

We previously reported that insulin activates nuclear factor B (NF-B) in Chinese hamster ovary (CHO)-R cells overexpressing wild-type insulin receptors (IRs) through a pathway requiring IR tyrosine kinase and Raf-1 kinase activities. We now investigated whether the activation of NF-B by insulin could serve an antiapoptotic function. Insulin (10 ؊9 -10 ؊7 M) inhibited apoptosis induced by serum withdrawal in CHO-R cells in a concentration-dependent manner. Insulin antiapoptotic signaling: (i) was dependent on IR number and IR tyrosine kinase activity since it was reduced in parental CHO cells and was abolished in CHO-Y2 cells overexpressing IRs mutated at Tyr 1162/1163 ; (ii) was, like insulin activation of NF-B, dependent on Raf-1 but not on mitogen-activated protein kinase activity since both processes were decreased by the dominant-negative Raf-1 mutant Raf-C4 whereas they persisted in mitogen-activated protein kinase-depleted cells; and (iii) required NF-B activation since it was decreased by proteasome inhibitors and the dominant-negative IB-␣ (A32/36) mutant and was mimicked by overexpression of the NF-B c-Rel subunit. We also show that insulin antiapoptotic signaling but not insulin activation of NF-B involved phosphatidylinositol 3-kinase (PI 3-kinase), as supported by the inhibition of the former but not of the latter process by the PI 3-kinase inhibitor LY294002. Inhibition of both NF-B and PI 3-kinase totally abolished insulin antiapoptotic signaling. Thus insulin exerts a specific antiapoptotic function which is dependent on IR tyrosine kinase activity and is mediated by both a Raf-1-dependent pathway that leads to NF-B activation and a PI 3-kinase-dependent pathway.

Effects of insulin on in vitro vascular cell adhesion molecule-1 expression and in vivo soluble VCAM-1 release

“…Our in vitro data do not completely agree with recent findings showing a 3.5±fold NF-kB induction in Chinese hamster ovary cells after insulin stimulation [5]. Ovary cell transfectants stably overexpressing wild-type insulin receptors were, however, used in that study [5].…”

“…Thus, discrepancies between our and previous data [5] simply reflects differences in cellular systems, i. e. in insulin sensitivity between HUVECs and transfected ovary cells. Accordingly, NFkB was not stimulated by insulin in parental, i. e. untransfected and therefore not overexpressing insulin receptors, Chinese hamster ovary cells [5]. Our data seems also to be in contrast with recent data showing that NF-kB induced mdr1 b, a membrane-bound Pglycoprotein, expression in rat hepatoma cells stimulated by insulin [7].…”

“…Ovary cell transfectants stably overexpressing wild-type insulin receptors were, however, used in that study [5]. Thus, discrepancies between our and previous data [5] simply reflects differences in cellular systems, i. e. in insulin sensitivity between HUVECs and transfected ovary cells.…”

“…Ovary cell transfectants stably overexpressing wild-type insulin receptors were, however, used in that study [5]. Thus, discrepancies between our and previous data [5] simply reflects differences in cellular systems, i. e. in insulin sensitivity between HUVECs and transfected ovary cells. Accordingly, NFkB was not stimulated by insulin in parental, i. e. untransfected and therefore not overexpressing insulin receptors, Chinese hamster ovary cells [5].…”

“…In this regard, cytokine-induced VCAM-1 expression is tightly transcriptionally regulated and requires the activation and nuclear translocation of nuclear factor-kB (NF-kB), i. e. a cytoplasmic multisubunit transcription factor which is essential to induce VCAM-1 promoter gene activation as well as interferon-b regulatory factor-1 [4]. Activation of NF-kB by insulin has been reported in Chinese hamster ovary cells overexpressing wild-type insulin receptors [5]. Thus, in our experiments we used two different promoter-reporter gene constructs (an NF-kB and a VCAM-1 reporter) to study the effects of insulin on VCAM-1 transcriptional activation.…”