Autophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic beta cells and glucose homeostasis has not been described. We produced mice with beta cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. beta cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of beta cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient beta cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in beta cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic beta cells, and its impairment causes insulin deficiency and hyperglycemia because of abnormal turnover and function of cellular organelles.

The cardiac Na+/Ca2+ exchanger (NCX1; ref. 2) is a bi-directional Ca2+ transporter that contributes to the electrical activity of the heart. When, and if, Ca2+ is exported or imported depends on the Na+/Ca2+ exchange ratio. Whereas a ratio of 3:1 (Na+:Ca2+) has been indicated by Ca2+ flux equilibrium studies, a ratio closer to 4:1 has been indicated by exchange current reversal potentials. Here we show, using an ion-selective electrode technique to quantify ion fluxes in giant patches, that ion flux ratios are approximately 3.2 for maximal transport in either direction. With Na+ and Ca2+ on both sides of the membrane, net current and Ca2+ flux can reverse at different membrane potentials, and inward current can be generated in the absence of cytoplasmic Ca2+, but not Na+. We propose that NCX1 can transport not only 1 Ca2+ or 3 Na+ ions, but also 1 Ca2+ with 1 Na+ ion at a low rate. Therefore, in addition to the major 3:1 transport mode, import of 1 Na+ with 1 Ca2+ defines a Na+-conducting mode that exports 1 Ca2+, and an electroneutral Ca2+ influx mode that exports 3 Na+. The two minor transport modes can potentially determine resting free Ca2+ and background inward current in heart.

No abstract

Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1’s role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.

Through in vivo analyses of mTOR deficiency and in vitro studies of human and mouse pancreatic islets, Chau et al. show that mTOR plays a critical role in β cell survival in diabetes. mTOR associates with and inhibits the transcriptional ChREBP–Mlx complex, suppressing TXNIP expression and β cell death.

Multifunctional carbon nanotube (CNT) composite fibers are currently of considerable interest in applications where actuation and energy-storage functions are highly desirable, such as electronic textiles. CNT fibers have been shown to function as excellent electrochemical supercapacitors giving specific capacitances of 100 F g -1 [1] . CNT assemblies can also produce useful actuation strains when electrochemically charged [2] and can potentially operate to high stresses because of the excellent mechanical properties of individual CNTs. The development of CNT fibers that simultaneously produce a high capacitance and useful actuation performance remains a challenge however, because the high surface area needed for high capacitance significantly reduces the strength and compromises actuation performance. To date, it has not been possible to develop an ion-conducting binder that mechanically stabilizes the CNT assembly, maintains electrical connectivity between nanotubes, and allows free transport of ions between the nanotubes and an external electrolyte. Pioneering work by Poulin [3] and Baughman [4] have established processing methods for preparing continuous fibers of CNTs and CNT composites that are ideal for electronic textiles. Various studies on these fibers and other CNT assemblies have highlighted the difficulties involved in producing mechanically robust, high-conductivity and high-surface-area electrodes. While single wall carbon nanotube (SWNT) fibers containing ∼40 % poly(vinyl alcohol) (PVA) binder give exceptional mechanical properties, their conductivity is very low at 0.2 S cm -1 .[1] The binder can be removed by pyrolysis to improve conductivity so that the fibers can be operated as electromechancial actuators. While quite high actuation stresses were obtained in these thermally annealed CNT fibers, their low flexibility and high creep during charge and discharge were noted as significant problems.[5] Similarly, fibers spun without the aid of a polymer binder [1] produce high conductivities (140 S cm -1 after thermal annealing) and capacitances (100 F g -1 ) but are mechanically fragile. To resolve these problems, crosslinked DNA has been chosen as a binder for CNT fibers. DNA is a good candidate for improved electrical conductivity for electrochemical devices with CNTs, as DNA has electrical characteristics similar to those of semiconducting diodes in that current flows in one direction only. [6][7][8] In addition, DNA more effectively coats, separates, and solubilizes CNTs than other surfactants because of the large surface area of its phosphate backbone, which interacts with water, and there are many bases in DNA that can bind to CNTs.[9] Therefore, DNA wrapping can debundle CNTs in high concentration CNT dispersions. Consequently, DNA wrapping may improve electrochemical actuation and capacitance of nanotubes in composite fibers by its improved electrical conductivity, high CNT surface area and enhanced mechanical stability due to the p-p interaction between the DNA and the CNT sidewall. We rep...

No abstract

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2–3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10–15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion–coupled transporters in giant patches.