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.

DOC2b is a SNARE regulator of glucose-stimulated delayed insulin secretion.

Insulin secretion is precisely regulated by blood glucose with unique biphasic pattern. The regulatory mechanism of the second-phase insulin release is unclear. In this study, we report that DOC2b (double C2 domain protein isoform b), a SNARE related protein, was associated with insulin vesicles and translocated to plasma membrane within several minutes upon high-glucose stimulation followed by an interaction with syntaxin4, but not syntaxin1. This binding specificity and the time course of DOC2b translocation were suitable for the regulation of second-phase insulin release. Increased DOC2b expression enhanced glucose-stimulated insulin secretion. In contrast, silencing DOC2b inhibited delayed release of insulin, without affecting rapid (approximately 7min) phase secretion. Interestingly, DOC2b had no effects on KCl-triggered insulin release. These data suggest that DOC2b may be a regulator for delayed (second-phase) insulin secretion in MIN6 cells.

DOC2B: a novel syntaxin-4 binding protein mediating insulin-regulated GLUT4 vesicle fusion in adipocytes.

OBJECTIVE: Insulin stimulates glucose uptake in skeletal muscle and adipose tissues primarily by stimulating the translocation of vesicles containing a facilitative glucose transporter, GLUT4, from intracellular compartments to the plasma membrane. The formation of stable soluble N-ethyl-maleimide-sensitive fusion protein [NSF] attachment protein receptor (SNARE) complexes between vesicle-associated membrane protein-2 (VAMP-2) and syntaxin-4 initiates GLUT4 vesicle docking and fusion processes. Additional factors such as munc18c and tomosyn were reported to be negative regulators of the SNARE complex assembly involved in GLUT4 vesicle fusion. However, despite numerous investigations, the positive regulators have not been adequately clarified. RESEARCH DESIGN AND METHODS: We determined the intracellular localization of DOC2b by confocal immunoflorescent microscopy in 3T3-L1 adipocytes. Interaction between DOC2b and syntaxin-4 was assessed by the yeast two-hybrid screening system, immunoprecipitation, and in vitro glutathione S-transferase (GST) pull-down experiments. Cell surface externalization of GLUT4 and glucose uptake were measured in the cells expressing DOC2b constructs or silencing DOC2b. RESULTS: Herein, we show that DOC2b, a SNARE-related protein containing double C2 domains but lacking a transmembrane region, is translocated to the plasma membrane upon insulin stimulation and directly associates with syntaxin-4 in an intracellular Ca(2+)-dependent manner. Furthermore, this process is essential for triggering GLUT4 vesicle fusion. Expression of DOC2b in cultured adipocytes enhanced, while expression of the Ca(2+)-interacting domain mutant DCO2b or knockdown of DOC2b inhibited, insulin-stimulated glucose uptake. CONCLUSIONS: These findings indicate that DOC2b is a positive SNARE regulator for GLUT4 vesicle fusion and mediates insulin-stimulated glucose transport in adipocytes.

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.