Gsk-3-Mediated Proteasomal Degradation of ATF4 Is a Proapoptotic Mechanism in Mouse Pancreatic ß-Cells.

Endoplasmic reticulum (ER) stress is a key pathogenic factor in type 1 and 2 diabetes. Glycogen synthase kinase 3 (Gsk-3) contributes to ß-cell loss in mice. However, the mechanism by which Gsk-3 leads ß-cell death remains unclear. ER stress was pharmacologically induced in mouse primary islets and insulinoma cells. We used insulinoma cells derived from Akita mice as a model of genetic ER stress. Gsk-3 activity was blocked by treating with Gsk-3 inhibitors or by introducing catalytically inactive Gsk-3ß. Gsk-3 inhibition prevented proteasomal degradation of activating transcriptional factor 4 (ATF4) and alleviated apoptosis. We found that ATF4-S214 was phosphorylated by Gsk-3, and that this was required for a binding of ATF4 with ßTrCP, which mediates polyubiquitination. The anti-apoptotic effect of Gsk-3 inhibition was attenuated by introducing DN-ATF4 or by knockdown of ATF4. Mechanistically, Gsk-3 inhibition modulated transcription targets of ATF4 and in turn facilitated dephosphorylation of eIF2α, altering the protein translational dynamism under ER stress. These observations were reproduced in the Akita mouse-derived cells. Thus, these results reveal the role of Gsk-3 in the regulation of the integrated stress response, and provide a rationale for inhibiting this enzyme to prevent ß-cell death under ER stress conditions.

Estrogen receptor alpha regulates insulin sensitivity through IRS-1 tyrosine phosphorylation in mature 3T3-L1 adipocytes.

There are many clinical and experimental reports demonstrating that estrogens and insulin interact when affecting their target organs. Estrogen receptors consist of two isoforms, estrogen receptors-alpha (ER-alpha) and -beta (ER-beta), but their roles in insulin-induced glucose uptake in mature adipose tissue have yet to be clarified. To evaluate the roles of ER-alpha, expressed predominantly in adipocytes, we have investigated the effects of estradiol (E2), an ER-alpha selective agonist (PPT), and its selective antagonist (MPP) on glucose uptake and insulin action in 3T3-L1 adipocytes. 3T3-L1 adipocytes were exposed to E2 or PPT and/or MPP at different concentrations. The cells were then subjected to 2-deoxy-D-glucose transport assay, western blot analysis, or RT-PCR analysis. Treatment of these cells with E2 or PPT resulted in biphasic effects on glucose transport, that is high (10(-5) M or 3 x 10(-6) M each) and low (10(-8) M) doses produced inhibition and stimulation, respectively. The favorable effect observed at 10(-8) M of E2 was diminished by treatment with MPP. Western bolt analysis revealed that these effects of E2, PPT and MPP paralleled the level of IRS-1 tyrosine phosphorylation. However, IRS-1 serine phosphorylation, suppressor of cytokine signaling (SOCS)-1,-2,-3 and protein tyrosine phosphatase 1B (PTP1B) expression did not change compared to control subjects. Our data clearly show that ER-alpha contributes to insulin stimulated glucose uptake through regulation of the tyrosine phosphorylation of IRS-1 protein.

Myosin motor Myo1c and its receptor NEMO/IKK-gamma promote TNF-alpha-induced serine307 phosphorylation of IRS-1.

Tumor necrosis factor-alpha (TNF-alpha) signaling through the IkappaB kinase (IKK) complex attenuates insulin action via the phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser307. However, the precise molecular mechanism by which the IKK complex phosphorylates IRS-1 is unknown. In this study, we report nuclear factor kappaB essential modulator (NEMO)/IKK-gamma subunit accumulation in membrane ruffles followed by an interaction with IRS-1. This intracellular trafficking of NEMO requires insulin, an intact actin cytoskeletal network, and the motor protein Myo1c. Increased Myo1c expression enhanced the NEMO-IRS-1 interaction, which is essential for TNF-alpha- induced phosphorylation of Ser307-IRS-1. In contrast, dominant inhibitory Myo1c cargo domain expression diminished this interaction and inhibited IRS-1 phosphorylation. NEMO expression also enhanced TNF-alpha-induced Ser307-IRS-1 phosphorylation and inhibited glucose uptake. In contrast, a deletion mutant of NEMO lacking the IKK-beta-binding domain or silencing NEMO blocked the TNF-alpha signal. Thus, motor protein Myo1c and its receptor protein NEMO act cooperatively to form the IKK-IRS-1 complex and function in TNF-alpha-induced insulin resistance.