Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis

Melanocortin-4 receptor (MC4R) mutations cause dysregulation of energy balance and hyperinsulinemia. We have used mouse models to study the physiological roles of extrahypothalamic MC4Rs. Re-expression of MC4Rs in cholinergic neurons (ChAT-Cre, loxTB MC4R mice) modestly reduced body weight gain without altering food intake and was sufficient to normalize energy expenditure and attenuate hyperglycemia and hyperinsulinemia. In contrast, restoration of MC4R expression in brainstem neurons including those in the dorsal motor nucleus of the vagus (Phox2b-Cre, loxTB MC4R mice) was sufficient to attenuate hyperinsulinemia, while the hyperglycemia and energy balance were not normalized. Additionally, hepatic insulin action and insulin-mediated suppression of hepatic glucose production were improved in ChAT-Cre, loxTB MC4R mice. These findings suggest that MC4Rs expressed by cholinergic neurons regulate energy expenditure and hepatic glucose production. Our results also provide further evidence of the dissociation in pathways mediating the effects of melanocortins on energy balance and glucose homeostasis.     

Expression,purification, and characterization of proteins from high-quality combinatorial libraries of the mammalian calmodulin central linker

Combinatorial libraries offer an attractive approach towards exploring protein sequence, structure and function. Although several strategies introduce sequence diversity, the likelihood of identifying proteins with novel functions is increased when the library of genes encodes for folded and soluble structures. Here we present the first application of the binary patterning approach of combinatorial protein library design to the unique central linker region of the highly-conserved protein, calmodulin (CaM). We show that this high-quality approach translates very well to the CaM protein scaffold: All library members over-express and are functionally diverse, having a range of conformations in the presence and absence of calcium as determined by circular dichroism spectroscopy. Collectively, these data support that the binary patterning approach, when applied to the highly-conserved protein fold, can yield large collections of folded, soluble and highly-expressible proteins.     

Imatinib potentiates antitumor T cell responses in gastrointestinal stromal tumor through the inhibition of Ido

Imatinib mesylate targets mutated KIT oncoproteins in gastrointestinal stromal tumor (GIST) and produces a clinical response in 80% of patients. The mechanism is believed to depend predominantly on the inhibition of KIT-driven signals for tumor-cell survival and proliferation. Using a mouse model of spontaneous GIST, we found that the immune system contributes substantially to the antitumor effects of imatinib. Imatinib therapy activated CD8(+) T cells and induced regulatory T cell (T(reg) cell) apoptosis within the tumor by reducing tumor-cell expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase (Ido). Concurrent immunotherapy augmented the efficacy of imatinib in mouse GIST. In freshly obtained human GIST specimens, the T cell profile correlated with imatinib sensitivity and IDO expression. Thus, T cells are crucial to the antitumor effects of imatinib in GIST, and concomitant immunotherapy may further improve outcomes in human cancers treated with targeted agents.     

Oceanographic anomalies and sea‐level rise drive mangroves inland in the Pacific coast of Mexico

Question: Although mangrove forests are generally regarded as highly threatened, some studies have shown that mangrove canopies in the Pacific coast of Mexico have been increasing in recent decades. We investigated the possible causes driving this reported mangrove expansion. Location: The mangrove lagoons of Magdalena Bay in Baja California, Mexico. Methods: We used 50‐year‐old aerial photographs and 24‐year‐old satellite images to compare long‐term vegetation change, surveyed a coastal vegetation transect to analyse flooding levels, compiled six decades of tidal and oceanographic information, as well as hurricane data to analyse changes in storm frequency or sea‐level conditions, and used isotopic analysis to date the age of trees along the gradient. Results: A significant increase in mangrove cover has occurred in backwaters of the lagoons during the last 40 years, and especially during the El Niño anomalies of the 1980s and 1990s, while at the same time the mangrove fringe has been receding. Conclusions: The observed change can be attributed to the combined action of the warm surface waters of El Niño events and sea‐level rise. Jointly, these two effects are sufficient to flood large areas of previously non‐flooded salt flats, dispersing mangrove seedlings inland. The inland expansion of mangroves, however, does not ease conservation concerns, as it is the seaward fringes, and not the inland margins, that provide the most valuable environmental services for fisheries and coastal protection.     

Real time analysis of β2-adrenoceptor-mediated signaling kinetics in Human Primary Airway Smooth Muscle Cells reveals both ligand and dose dependent differences

Background

β₂-adrenoceptor agonists elicit bronchodilator responses by binding to β₂-adrenoceptors on airway smooth muscle (ASM). In vivo, the time between drug administration and clinically relevant bronchodilation varies significantly depending on the agonist used. Our aim was to utilise a fluorescent cyclic AMP reporter probe to study the temporal profile of β₂-adrenoceptor-mediated signaling induced by isoproterenol and a range of clinically relevant agonists in human primary ASM (hASM) cells by using a modified Epac protein fused to CFP and a variant of YFP.

Methods

Cells were imaged in real time using a spinning disk confocal system which allowed rapid and direct quantification of emission ratio imaging following direct addition of β₂-adrenoceptor agonists (isoproterenol, salbutamol, salmeterol, indacaterol and formoterol) into the extracellular buffer. For pharmacological comparison a radiolabeling assay for whole cell cyclic AMP formation was used.

Results

Temporal analysis revealed that in hASM cells the β₂-adrenoceptor agonists studied did not vary significantly in the onset of initiation. However, once a response was initiated, significant differences were observed in the rate of this response with indacaterol and isoproterenol inducing a significantly faster response than salmeterol. Contrary to expectation, reducing the concentration of isoproterenol resulted in a significantly faster initiation of response.

Conclusions

We conclude that confocal imaging of the Epac-based probe is a powerful tool to explore β₂-adrenoceptor signaling in primary cells. The ability to analyse the kinetics of clinically used β₂-adrenoceptor agonists in real time and at a single cell level gives an insight into their possible kinetics once they have reached ASM cells in vivo.