β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1

FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins G_q/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses (“biased agonism”). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of G_q/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of G_q activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to G_q/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.     

Thirty-eight blood,tissue and consumptive measures from rats exposed perinatally and as adults to 0.5 Hz magnetic fields

In a factorial design, 40 male rats 195 ± 15 days of age that had been exposed: (1) perinatally for 5 days to either 10^–6T to 10^–3T variation, 0.5 Hz rotating magnetic fields (RMFs) or to sham field conditions, and (2) to one of two typical postweaning caging conditions were exposed for 5 days to either 10^–6T, 10^–7T, 10^–8T, 0.5 Hz RMFs or sham fields (<10^–9T variation in an experimental room) or to colony room control conditions. Consumptive behaviors, selected tissue weights, differential white blood cell counts and 20 constituents from blood sera (analyzed by sequential multiple analyses computer) were measured. Except for marginally significant differences in Na, Cl, and Ca, there were no significant differences between adult RMF intensities for any of the measures; a significant caging by adult intensity interaction was noted for GOT. Rats that had been exposed to the experimental room (continuous noise and light) displayed significant decreases in serum albumin, globulin, glucose and phosphorus levels as well as food and water consumption. Rats that had been exposed perinatally to the RMFs displayed significant 20% reductions in UREA, GOT, and LDH activity, 4% increases in testicle weights and 17% decreases in thymus weights relative to perinatal sham field controls. The absence of significant perinatal condition by adult condition interactions did not support the hypothesis that perinatal RMF exposure might enhance responsiveness to more natural, less intense field variations. Caging differences were associated with highly significant alterations in body weight, several tissue weights, Na, Ca, protein, cholesterol, triglycerides, glucose, CO₂ and LDH.     

Effect of Azoles on the Secretion of a Candida albicans Metallopeptidase

Azole antifungals act by inhibiting the activity of a lanosterol demethylase involved in the generation of the ergosterol of the cellular membrane of fungi. These drugs could also have action on other yeast components, like secreted aspartyl proteases. We demonstrate in this study that the in vitro secretion of a metallopeptidase could be modified during the growth of Candida albicans with subinhibitory concentrations of some azoles. Eight isolates of this yeast have been cultivated in presence of MIC, MIC/2 and MIC/4 of voriconazole, fluconazole and itraconazole. The presence of voriconazole and fluconazole decreased the secretion of the metallopeptidase in the culture medium, whereas itraconazole increased this secretion for three isolates. This study points to the fact that some antifungals, given in prophylaxis, could act in an unfavourable way on some potential factors of pathogenicity.     

The NALCN ion channel is activated by M3 muscarinic receptors in a pancreatic β-cell line

A previously uncharacterized putative ion channel, NALCN (sodium leak channel, non-selective), has been recently shown to be responsible for the tetrodotoxin (TTX)-resistant sodium leak current implicated in the regulation of neuronal excitability. Here, we show that NALCN encodes a current that is activated by M3 muscarinic receptors (M3R) in a pancreatic β-cell line. This current is primarily permeant to sodium ions, independent of intracellular calcium stores and G proteins but dependent on Src activation, and resistant to TTX. The current is recapitulated by co-expression of NALCN and M3R in human embryonic kidney-293 cells and in Xenopus oocytes. We also show that NALCN and M3R belong to the same protein complex, involving the intracellular I–II loop of NALCN and the intracellular i3 loop of M3R. Taken together, our data show the molecular basis of a muscarinic-activated inward sodium current that is independent of G-protein activation, and provide new insights into the properties of NALCN channels.     

GLP-1 Mediates Antiapoptotic Effect by Phosphorylating Bad through a ��-Arrestin 1-mediated ERK1/2 Activation in Pancreatic ��-Cells

Strategies based on activating GLP-1 receptor (GLP-1R) are intensively developed for the treatment of type 2 diabetes. The exhaustive knowledge of the signaling pathways linked to activated GLP-1R within the β-cells is of major importance. In β-cells, GLP-1 activates the ERK1/2 cascade by diverse pathways dependent on either Gα_s/cAMP/cAMP-dependent protein kinase (PKA) or β-arrestin 1, a scaffold protein. Using pharmacological inhibitors, β-arrestin 1 small interfering RNA, and islets isolated from β-arrestin 1 knock-out mice, we demonstrate that GLP-1 stimulates ERK1/2 by two temporally distinct pathways. The PKA-dependent pathway mediates rapid and transient ERK1/2 phosphorylation that leads to nuclear translocation of the activated kinases. In contrast, the β-arrestin 1-dependent pathway produces a late ERK1/2 activity that is restricted to the β-cell cytoplasm. We further observe that GLP-1 phosphorylates the cytoplasmic proapoptotic protein Bad at Ser-112 but not at Ser-155. We find that the β-arrestin 1-dependent ERK1/2 activation engaged by GLP-1 mediates the Ser-112 phosphorylation of Bad, through p90RSK activation, allowing the association of Bad with the scaffold protein 14-3-3, leading to its inactivation. β-Arrestin 1 is further found to mediate the antiapoptotic effect of GLP-1 in β-cells through the ERK1/2-p90RSK-phosphorylation of Bad. This new regulatory mechanism engaged by activated GLP-1R involving a β-arrestin 1-dependent spatiotemporal regulation of the ERK1/2-p90RSK activity is now suspected to participate in the protection of β-cells against apoptosis. Such signaling mechanism may serve as a prototype to generate new therapeutic GLP-1R ligands.     

The elevation of glutamate content and the amplification of insulin secretion in glucose-stimulated pancreatic islets are not causally related

Glucose increases insulin secretion by raising cytoplasmic Ca(2+) ([Ca(2+)](i)) in beta-cells (triggering pathway) and augmenting the efficacy of Ca(2+) on exocytosis (amplifying pathway). It has been suggested that glutamate formed from alpha-ketoglutarate is a messenger of the amplifying pathway (Maechler, P., and Wollheim, C. B. (1999) Nature 402, 685-689). This hypothesis was tested with mouse islets depolarized with 30 mm KCl (+ diazoxide) or with a saturating concentration of sulfonylurea. Because [Ca(2+)](i) was elevated under these conditions, insulin secretion was stimulated already in 0 mm glucose. The amplification of secretion produced by glucose was accompanied by an increase in islet glutamate. However, glutamine (0.5-2 mm) markedly augmented islet glutamate without affecting insulin secretion, whereas glucose augmented secretion without influencing glutamate levels when these were elevated by glutamine. Allosteric activation of glutamate dehydrogenase by BCH (2-amino 2-norbornane carboxylic acid) lowered islet glutamate but increased insulin secretion. Similar insulin secretion thus occurred at very different cellular glutamate levels. Glutamine did not affect islet [Ca(2+)](i) and pH(i), whereas glucose and BCH slightly raised pH(i) and either slightly decreased (30 mm KCl) or increased (tolbutamide) [Ca(2+)](i). The general dissociation between changes in islet glutamate and insulin secretion refutes a role of beta-cell glutamate in the amplification of insulin secretion by glucose.     

A mannose binding protein is involved in the adherence of Acanthamoeba species to inert surfaces

Some carbohydrates are known to decrease the attachment of Acanthamoeba sp. to biological surfaces. By a method based on the reduction of a tetrazolium salt (XTT) by the mitochondrial dehydrogenases of the parasites, d-mannose and alpha-d-mannopyranoside have been shown to reduce Acanthamoeba attachment to inert surfaces, indicating that the mannose binding protein of Acanthamoeba trophozoites is involved in adherence to inert surfaces. The reduction in attachment is dose dependant and is not linked with a potential toxicity of the carbohydrates. All the species of Acanthamoeba tested were concerned by this mannose binding protein, but the adhesion of A. culbertsoni was also reduced by the presence of glucose.     

A Candida albicans metallopeptidase degrades constitutive proteins of extracellular matrix

A soluble c-type cytochrome was first purified from Geobacter metallireducens to an electrophoretically homogeneous state. The purified cytochrome c showed absorption peaks at 530 and 409 nm in the oxidized form and 552, 522, and 418 nm in the reduced form. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate allowed us to calculate the molecular mass at 9.5 kDa. It contained 3 mol of heme c per molecule of the protein on the basis of heme c and protein concentration. The mid-point redox potential at pH 7.0 was determined to be -190 mV. Although the N-terminal amino acid sequence of the first 17 residues was similar to that of Desulfuromonas acetoxidans cytochrome c7, G. metallireducens cytochrome c did not show Fe(III)-reducing activity.