Insulin metabolism in human adipocytes from subcutaneous and visceral depots.

Subjects with the metabolic syndrome (insulin resistance, glucose intolerance, dyslipidemia, hypertension, etc.) have a relative increase in abdominal fat tissue compared to normal individuals and obesity has also been shown to be associated with a decrease in insulin clearance. The majority of the clearance of insulin is due to the action of insulin-degrading enzyme (IDE) and IDE is present throughout all tissues. Since abdominal fat is increased in obesity we hypothesized that IDE may be altered in the different fat depots. Adipocytes were isolated from fat samples obtained from subjects during elective abdominal surgery. Fat samples were taken from subcutaneous (SQ) and visceral (VIS) sites. Insulin metabolism was compared in adipocytes isolated from SQ and VIS fat tissue. Adipocytes from the VIS site degraded more insulin that those from SQ fat tissue. Inhibitors of cathepsins B and D has no effect on the degradation of insulin, while bacitracin, an inhibitor of IDE, inhibited degradation by approx. 33% in both SQ and VIS adipocytes. These data show that insulin metabolism is relatively greater in VIS than in SQ fat tissue and potentially due to IDE.

Stereospecific induction of nuclear factor-kappaB activation by isochamaejasmin.

The root of Stellera chamaejasme L. is a traditional Chinese herb termed Rui Xiang Lang Du and has been used to treat solid tumors, tuberculosis and psoriasis. Exactly how S. chamaejasme L. regulates cellular responses remains unclear. We examined four biflavonoids isolated from S. chamaejasme L., including isochamaejasmin, two of its stereo-isomers and a methyl derivative, in functional assays originally designed to screen ligands for the G protein-coupled formyl peptide receptor-like 1 (FPRL1). Isochamaejasmin was found to induce the expression of a nuclear factor (NF)-kappaB-directed reporter gene in transfected HeLa cells with an EC50 of 3.23 microM, independently of FPRL1. The isochamaejasmin-stimulated NF-kappaB reporter activity was accompanied by nuclear translocation of NF-kappaB proteins and was blocked by a dominant-negative construct of IkappaBalpha. Isochamaejasmin also induced time-dependent phosphorylation of the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 and p38, and a novel protein kinase C (PKCdelta). Likewise, inhibition of these kinases with the respective pharmacological inhibitors significantly reduced the isochamaejasmin-stimulated NF-kappaB activation. It is noteworthy that the two stereoisomers and the methyl derivative did not induce detectable activation of NF-kappaB and were more cytotoxic than isochamaejasmin, which could partially rescue cycloheximide-induced apoptosis. Inhibition of NF-kappaB activation reversed the anti-apoptotic effect of isochamaejasmin. These results provide the first evidence for a potential mechanism of action by S. chamaejasme L., and indicate that structurally similar compounds derived from S. chamaejasme L. may have different pharmacological properties.

Reconstitution of chemotactic peptide-induced nicotinamide adenine dinucleotide phosphate (reduced) oxidase activation in transgenic COS-phox cells.

A whole-cell-based reconstitution system was developed to study the signaling mechanisms underlying chemoattractant-induced activation of NADPH oxidase. This system takes advantage of the lack of formyl peptide receptor-mediated response in COS-phox cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox), which respond to phorbol ester and arachidonic acid with O()(2) production. By exogenous expression of signaling molecules enriched in neutrophils, we have identified several critical components for fMLP-induced NADPH oxidase activation. Expression of PKCdelta, but not PKCalpha, -betaII, and -zeta, is necessary for the COS-phox cells to respond to fMLP. A role of PKCdelta in neutrophil NADPH oxidase was confirmed based on the ability of fMLP to induce PKCdelta translocation and the sensitivity of fMLP-induced O()(2) production to rottlerin, a PKCdelta-selective inhibitor. Optimal reconstitution also requires phospholipase C-beta2 and PI3K-gamma. We found that formyl peptide receptor could use the endogenous Rac1 as well as exogenous Rac1 and Rac2 for NADPH oxidase activation. Exogenous expression of p40(phox) potentiated fMLP-induced O()(2) production and raised the level of O()(2) in unstimulated cells. Collectively, these results provide first direct evidence for reconstituting fMLP-induced O()(2) production in a nonhemopoietic cell line, and demonstrate the requirement of multiple signaling components for optimal activation of NADPH oxidase by a chemoattractant.

A novel nonpeptide ligand for formyl peptide receptor-like 1.

Formyl peptide receptor-like 1 (FPRL1) is a G protein-coupled receptor that binds natural and synthetic peptides as well as lipoxin A(4) and mediates important biological functions. To facilitate its pharmacological characterization, we screened a compound library and identified a substituted quinazolinone (Quin-C1, 4-butoxy-N-[2-(4-methoxy-phenyl)-4-oxo-1,4-dihydro-2H-quinazolin-3-yl]-benzamide) as a ligand for FPRL1. Quin-C1 induces chemotaxis and secretion of beta-glucuronidase in peripheral blood neutrophils with a potency of approximately 1/1000 of that of the peptide agonist WKYMVm. In studies using transfected rat basophilic leukemia (RBL) cell lines expressing either formyl peptide receptor or FPRL1, Quin-C1 induced enzyme release from RBL-FPRL1 but not RBL-FPR cells. Likewise, Quin-C1 selectively stimulates calcium mobilization in RBL-FPRL1 cells, a response that was markedly inhibited by pertussis toxin. Quin-C1 also stimulates phosphorylation of extracellular signal-regulated protein kinases 1 and 2 and induces internalization of an FPRL1 fused to green fluorescent protein. In degranulation assays, both the FPRL1-selective peptide agonist MMK1 and Quin-C1 exhibited lower efficacy and potency than WKYMVm, with EC(50) values of 7.17 x 10(-8) M and 1.88 x 10(-6) M, respectively, compared with the EC(50) value for WKYMVm (2.29 x 10(-8) M). However, Quin-C1 did not induce neutrophil superoxide generation at up to 100 microM. Based on these results, we conclude that Quin-C1 is a novel nonpeptide ligand that binds to FPRL1 and selectively stimulates FPRL1-mediated functions. Quin-C1 is a prototype of substituted quinazolinones based on which further structural modifications may be made to improve its efficacy and potency for FPRL1.

Normal cell surface expression and selective loss of functions resulting from Phe110 to Ser and Cys126 to Trp substitutions in the formyl peptide receptor.

The N-formyl peptide receptor (FPR) is a G protein-coupled chemoattractant receptor that mediates diverse leukocyte functions when stimulated by bacteria-derived N-formyl peptides such as fMet-Leu-Phe (fMLF). Impaired neutrophil responsiveness to fMLF parallels increased susceptibility to periodontal microorganisms among patients with localized juvenile periodontitis (LJP). To determine whether the recently identified FPR mutations in LJP patients are responsible for selective loss of receptor-mediated functions, we prepared and analyzed RBL-2H3 cells expressing FPR bearing Phe110 to Ser (FPR-F110S) or Cys-126 to Trp (FPR-C126W) replacement as well as a FPR double mutant (FPR-FSCW). All mutant receptors were expressed normally on the cell surface, but were unable to mediate release of beta-hexosaminidase upon fMLF stimulation. FPR-C126W effectively mediated fMLF uptake, an indication of receptor-mediated endocytosis, whereas FPR-F110S and FSCW exhibited markedly reduced ability to uptake fMLF. Both FPR-F110S and FPR-C126W were defective in chemotaxis and displayed reduced Ca2+ mobilization, but mutation at both positions partially restored the ability to respond to fMLF in chemotaxis assay and was nearly normal in Ca2+ mobilization assay. All mutants exhibited diminished accumulation of inositol phosphates. FPR-F110S displayed a delayed and significantly reduced ERK phosphorylation whereas FPR-FSCW nearly lost the ability to phosphorylate ERK. Taken together, these results indicate compromised signaling capabilities due to the FPR mutations, but the loss of function is selective and could be partially rescued by mutations at both positions.

Emodin enhances arsenic trioxide-induced apoptosis via generation of reactive oxygen species and inhibition of survival signaling.

Although arsenic trioxide (As(2)O(3)) induces apoptosis in a relatively wide spectrum of tumors, the sensitivity of different cell types to this treatment varies to a great extent. Because reactive oxygen species (ROS) are critically involved in As(2)O(3)-induced apoptosis, we attempted to explore the possibility that elevating the cellular ROS level might be an approach to facilitate As(2)O(3)-induced apoptosis. Emodin, a natural anthraquinone derivative, was selected because its semiquinone structure is likely to increase the generation of intracellular ROS. Its independent and synergistic effects with As(2)O(3) in cytotoxicity were studied, and the plausible signaling mechanism was investigated in HeLa cells. Cell Proliferation Assay and flow cytometry were used to assess cell viability and apoptosis. Electrophoretic mobility shift assay, luciferase reporter assay, and Western blotting were performed to analyze signaling alteration. The results demonstrated that coadministration of emodin, at low doses of 0.5-10 micro M, with As(2)O(3) enhanced As(2)O(3)-rendered cytotoxicity on tumor cells, whereas these treatments caused no detectable proproliferative or proapoptotic effects on nontumor cells. ROS generation was increased, and activation of nuclear factor kappaB and activator protein 1 was suppressed by coadministration. All enhancements by emodin could be abolished by the antioxidant N-acetyl-L-cysteine. Therefore, we concluded that emodin sensitized HeLa cells to As(2)O(3) via generation of ROS and ROS-mediated inhibition on two major prosurvival transcription factors, nuclear factor kappaB and activator protein 1. This result allows us to propose a novel strategy in chemotherapy that uses mild ROS generators to facilitate apoptosis-inducing drugs whose efficacy depends on ROS.

Serum amyloid A induces IL-8 secretion through a G protein-coupled receptor, FPRL1/LXA4R.

Host response to injury and infection is accompanied by a rapid rise in the blood of acute-phase proteins such as serum amyloid A (SAA). Although SAA has been used as a marker for inflammatory diseases, its role in the modulation of inflammation and immunity has not been defined. Human neutrophils respond to SAA with secretion of the proinflammatory cytokines interleukin 8 (IL-8) and, to a lesser extent, tumor necrosis factor alpha (TNF-alpha). The induction of IL-8 secretion by SAA involves both transcription and translation and correlates with activation of nuclear factor kappaB (NF-kappaB). The proximal signaling events induced by SAA include mobilization of intracellular Ca(2+) and activation of the mitogen-activated protein kinases ERK1/2 and p38, both required for the induced IL-8 secretion. Pertussis toxin effectively blocks SAA-induced IL-8 secretion indicating involvement of a Gi-coupled receptor. Overexpression of FPRL1/LXA4R in HeLa cells results in a significant increase of the expression of NF-kappaB and IL-8 luciferase reporters by SAA, and an antibody against the N-terminal domain of FPRL1/LXA4R inhibits IL-8 secretion. Lipoxin A4, which binds to FPRL1/LXA4R specifically, decreases SAA-induced IL-8 secretion significantly. Collectively, these results indicate that the cytokine-like property of SAA is manifested through activation of the Gi-coupled FPRL1/LXA4R, which has been known to mediate the anti-inflammatory effects of lipoxin A4. The ability of FPRL1/LXA4R to mediate 2 drastically different and opposite functions suggests that it plays a role in the modulation of inflammatory and immune responses.