Neutrophil Elastase Increases Vascular Permeability and Leukocyte Transmigration in Cultured Endothelial Cells and Obese Mice.

Neutrophil elastase (NE) plays a pivotal role in inflammation. However, the mechanism underlying NE-mediated inflammation in obesity remains unclear. Here, we report that NE activates protease-activated receptor-2 (PAR2), stimulates actin filament (F-actin) formation, decreases intercellular junction molecule VE-cadherin expression, and increases the permeability of human arterial endothelial cells (hECs).

Neutrophil Elastase Regulates Emergency Myelopoiesis Preceding Systemic Inflammation in Diet-induced Obesity.

Inflammation plays a significant role in the development of obesity-related complications, but the molecular events that initiate and propagate such inflammation remain unclear. Here, we report that mice fed a high-fat diet (HFD) for as little as 1-3 days show increased differentiation of myeloid progenitors into neutrophils and monocytes but reduced B lymphocyte production in the bone marrow. Levels of neutrophil elastase (NE) and the nuclear factors CCAAT/enhancer-binding protein α (C/EBPα) and growth factor-independent 1 (GFI-1) are elevated in hematopoietic stem and progenitor cells from HFD-fed mice, but mice lacking either NE or C/EBPα are resistant to HFD-induced myelopoiesis.

Imbalance between neutrophil elastase and its inhibitor α1-antitrypsin in obesity alters insulin sensitivity, inflammation, and energy expenditure.

The molecular mechanisms involved in the development of obesity and related complications remain unclear. Here, we report that obese mice and human subjects have increased activity of neutrophil elastase (NE) and decreased serum levels of the NE inhibitor α1-antitrypsin (A1AT, SerpinA1). NE null (Ela2(-/-)) mice and A1AT transgenic mice were resistant to high-fat diet (HFD)-induced body weight gain, insulin resistance, inflammation, and fatty liver.

C2 domain-containing phosphoprotein CDP138 regulates GLUT4 insertion into the plasma membrane.

The protein kinase B(β) (Akt2) pathway is known to mediate insulin-stimulated glucose transport through increasing glucose transporter GLUT4 translocation from intracellular stores to the plasma membrane (PM). Combining quantitative phosphoproteomics with RNAi-based functional analyses, we show that a previously uncharacterized 138 kDa C2 domain-containing phosphoprotein (CDP138) is a substrate for Akt2, and is required for optimal insulin-stimulated glucose transport, GLUT4 translocation, and fusion of GLUT4 vesicles with the PM in live adipocytes. The purified C2 domain is capable of binding Ca(2+) and lipid membranes.

A novel pleckstrin homology domain-containing protein enhances insulin-stimulated Akt phosphorylation and GLUT4 translocation in adipocytes.

Protein kinase B/Akt protein kinases control an array of diverse functions, including cell growth, survival, proliferation, and metabolism. We report here the identification of pleckstrin homology-like domain family B member 1 (PHLDB1) as an insulin-responsive protein that enhances Akt activation. PHLDB1 contains a pleckstrin homology domain, which we show binds phosphatidylinositol PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), as well as a Forkhead-associated domain and coiled coil regions.

Isoform-specific regulation of Akt signaling by the endosomal protein WDFY2.

Recent work has led to the identification of novel endocytic compartments with functional roles in both protein trafficking and growth factor signal transduction. The phosphatidylinositol 3-phosphate binding, FYVE domain-containing protein WDFY2 is localized to a distinct subset of early endosomes, which are localized close to the plasma membrane. Here, we find that the serine/threonine kinase Akt interacts with these endosomes in an isoform-specific manner.

Akt substrate TBC1D1 regulates GLUT1 expression through the mTOR pathway in 3T3-L1 adipocytes.

Multiple studies have suggested that the protein kinase Akt/PKB (protein kinase B) is required for insulin-stimulated glucose transport in skeletal muscle and adipose cells. In an attempt to understand links between Akt activation and glucose transport regulation, we applied mass spectrometry-based proteomics and bioinformatics approaches to identify potential Akt substrates containing the phospho-Akt substrate motif RXRXXpS/T. The present study describes the identification of the Rab GAP (GTPase-activating protein)-domain containing protein TBC1D1 [TBC (Tre-2/Bub2/Cdc16) domain family, member 1], which is closely related to TBC1D4 [TBC domain family, member 4, also denoted AS160 (Akt substrate of 160 kDa)], as an Akt substrate that is phosphorylated at Thr(590).

Photoactivated 3-azioctanol irreversibly desensitizes muscle nicotinic ACh receptors via interactions at alphaE262.

3-Azioctanol is a photoactivatable analogue of octanol that noncompetitively inhibits nicotinic acetylcholine receptors (nAChRs). Photolabeling studies using [3H]-3-azioctanol in Torpedo nAChR identified alphaE262 as a site of desensitization-dependent incorporation. However, it is unknown whether photolabeling of alphaE262 causes functional effects in nAChRs and what other roles this residue plays in gating, desensitization, and channel block.

Identification of WNK1 as a substrate of Akt/protein kinase B and a negative regulator of insulin-stimulated mitogenesis in 3T3-L1 cells.

Insulin signaling through protein kinase Akt/protein kinase B (PKB), a downstream element of the phosphatidylinositol 3-kinase (PI3K) pathway, regulates diverse cellular functions including metabolic pathways, apoptosis, mitogenesis, and membrane trafficking. To identify Akt/PKB substrates that mediate these effects, we used antibodies that recognize phosphopeptide sites containing the Akt/PKB substrate motif (RXRXX(p)S/T) to immunoprecipitate proteins from insulin-stimulated adipocytes. Tryptic peptides from a 250-kDa immunoprecipitated protein were identified as the protein kinase WNK1 (with no lysine) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, consistent with a recent report that WNK1 is phosphorylated on Thr60 in response to insulin-like growth factor I.

Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing.

Glucose homeostasis is controlled by insulin in part through the translocation of intracellular glucose transporter 4 to the plasma membrane in muscle and fat cells. Akt/protein kinase B downstream of phosphatidylinositol 3-kinase has been implicated in this insulin-signaling pathway, but results with a variety of reagents including Akt1-/- and Akt2-/- mice have been equivocal. Here we report the application of small interfering RNA-directed gene silencing to deplete both Akt1 and Akt2 in cultured 3T3-L1 adipocytes.

Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c.

Insulin stimulates glucose uptake in muscle and adipocytes by signalling the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. The translocation of GLUT4 may involve signalling pathways that are both independent of and dependent on phosphatidylinositol-3-OH kinase (PI(3)K). This translocation also requires the actin cytoskeleton, and the rapid movement of GLUT4 along linear tracks may be mediated by molecular motors.