Comparison of Glucose Monitoring Methods during Steady-State Exercise in Women

Data from Continuous Glucose Monitoring (CGM) systems may help improve overall daily glycemia; however, the accuracy of CGM during exercise remains questionable. The objective of this single group experimental study was to compare CGM-estimated values to venous plasma glucose (VPG) and capillary plasma glucose (CPG) during steady-state exercise. Twelve recreationally active females without diabetes (aged 21.8 ± 2.4 years), from Central Washington University completed the study. CGM is used by individuals with diabetes, however the purpose of this study was to first validate the use of this device during exercise for anyone. Data were collected between November 2009 and April 2010. Participants performed two identical 45-min steady-state cycling trials (~60% Pmax) on non-consecutive days. Glucose concentrations (CGM-estimated, VPG, and CPG values) were measured every 5 min. Two carbohydrate gel supplements along with 360 mL of water were consumed 15 min into exercise. A product-moment correlation was used to assess the relationship and a Bland-Altman analysis determined error between the three glucose measurement methods. It was found that the CGM system overestimated mean VPG (mean absolute difference 17.4 mg/dL (0.97 mmol/L)) and mean CPG (mean absolute difference 15.5 mg/dL (0.86 mmol/L)). Bland-Altman analysis displayed wide limits of agreement (95% confidence interval) of 44.3 mg/dL (2.46 mmol/L) (VPG compared with CGM) and 41.2 mg/dL (2.29 mmol/L) (CPG compared with CGM). Results from the current study support that data from CGM did not meet accuracy standards from the 15197 International Organization for Standardization (ISO)

Cyclic nucleotide phosphodiesterases in Drosophila melanogaster

Cyclic nucleotide PDEs (phosphodiesterases) are important enzymes that regulate intracellular levels of cAMP and cGMP. In the present study, we identify and characterize novel PDEs in the genetic model, Drosophila melanogaster. The Drosophila genome encodes five novel PDE genes in addition to dunce. Predicted PDE sequences of Drosophila show highly conserved critical domains when compared with human PDEs. Thus PDE-encoding genes of D. melanogaster are CG14940-PDE1C, CG8279-PDE6β, CG5411-PDE8A, CG32648-PDE9 and CG10231-PDE11. Reverse transcriptase–PCRs of adult tissues reveal widespread expression of PDE genes. Drosophila Malpighian (renal) tubules express all the six PDEs: Drosophila PDE1, dunce (PDE4), PDE6, PDE8, PDE9 and PDE11. Antipeptide antibodies were raised against PDE1, PDE6, PDE9 and PDE11. Verification of antibody specificity by Western blotting of cloned and expressed PDE constructs allowed the immunoprecipitation studies of adult Drosophila lysates. Biochemical characterization of immunoprecipitated endogenous PDEs showed that PDE1 is a dual-specificity PDE (Michaelis constant K(m) for cGMP: 15.3±1 μM; K(m) cAMP: 20.5±1.5 μM), PDE6 is a cGMP-specific PDE (K(m) cGMP: 37±13 μM) and PDE11 is a dual-specificity PDE (K(m) cGMP: 6±2 μM; K(m) cAMP: 18.5±5.5 μM). Drosophila PDE1, PDE6 and PDE11 display sensitivity to vertebrate PDE inhibitors, zaprinast (IC(50) was 71±39 μM for PDE1, 0.65±0.015 μM for PDE6 and 1.6±0.5 μM for PDE11) and sildenafil (IC(50) was 1.3±0.9 μM for PDE1, 0.025±0.005 μM for PDE6 and 0.12±0.06 μM for PDE11). We provide the first characterization of a cGMP-specific PDE and two dual-specificity PDEs in Drosophila, and show a high degree of similarity in structure and function between human and Drosophila PDEs

Novel subcellular locations and functions for secretory pathway Ca2+/Mn2+-ATPases

Secretory pathway Ca2+/Mn2+-ATPases (SPCAs) are important for maintenance of cellular Ca2+ and Mn2+ homeostasis, and, to date, all SPCAs have been found to localize to the Golgi apparatus. The single Drosophila SPCA gene (SPoCk) was identified by an in silico screen for novel Ca2+-ATPases. It encoded three SPoCk isoforms with novel, distinct subcellular specificities in the endoplasmic reticulum (ER) and peroxisomes in addition to the Golgi. Furthermore, expression of the peroxisome-associated SPoCk isoform was sexually dimorphic. Overexpression of organelle-specific SPoCk isoforms impacted on cytosolic Ca2+ handling in both cultured Drosophila cells and a transporting epithelium, the Drosophila Malpighian (renal) tubule. Specifically, the ER isoform impacted on inositol (1,4,5)-trisphosphate-mediated Ca2+ signaling and the Golgi isoform impacted on diuresis, whereas the peroxisome isoform colocalized with Ca2+ “spherites” and impacted on calcium storage and transport. Interfering RNA directed against the common exons of the three SPoCk isoforms resulted in aberrant Ca2+ signaling and abolished neuropeptide-stimulated diuresis by the tubule. SPoCk thus contributed to both of the contrasting requirements for Ca2+ in transporting epithelia: to transport or store Ca2+ in bulk without compromising its use as a signal

A novel role for a Drosophila homologue of cGMP-specific phosphodiesterase in the active transport of cGMP

cGMP was first discovered in urine, demonstrating that kidney cells extrude this cyclic nucleotide. Drosophila Malpighian tubules provide a model renal system in which a homologue of mammalian PDE (phosphodiesterase) 6 is expressed. In humans, this cG-PDE (cGMP-specific PDE) is specifically expressed in the retinal system, where it controls visual signal transduction. In order to gain insight into the functional role of DmPDE6 (Drosophila PDE6-like enzyme) in epithelial function, we generated transgenic animals with targeted expression of DmPDE6 to tubule Type I (principal) cells. This revealed localization of DmPDE6 primarily at the apical membranes. As expected, overexpression of DmPDE6 resulted in elevated cG-PDE activity and decreased tubule cGMP content. However, such targeted overexpression of DmPDE6 creates a novel phenotype that manifests itself in inhibition of the active transport and efflux of cGMP by tubules. This effect is specific to DmPDE6 action, as no effect on cGMP transport is observed in tubules from a bovine PDE5 transgenic line which display reduced rates of fluid secretion, an effect not seen in DmPDE6 transgenic animals. Specific ablation of DmPDE6 in tubule principal cells, via expression of a targeted DmPDE6 RNAi (RNA interference) transgene, conferred increased active transport of cGMP, confirming a direct role for DmPDE6 in regulating cGMP transport in tubule principal cells. Pharmacological inhibition of DmPDE6 in wild-type tubules using the cG-PDE inhibitor, zaprinast, similarly results in stimulated cGMP transport. We provide the first demonstration of a novel role for a cG-PDE in modulating cGMP transport and efflux

Capa, Neuromedin and Hugin receptor <i>(CG8795)</i>-associated calcium signatures.

<p>(<b>A</b>) Typical cytoplasmic Ca²⁺ response in S2 cells expressing capaR and apoaequorin challenged with Drm-capa-1, Drm-PK-1, hugg, Drm-PK-2 and NMU-25 at a concentration of 10⁻⁷ M. (<b>B</b>) Typical cytoplasmic Ca²⁺ response in S2 cells expressing NMUR 2 and apoaequorin challenged with Drm-capa-1, Drm-PK-1, hugg, Drm-PK-2 and NMU-25 at a concentration of 10⁻⁷ M. (<b>C</b>) Typical cytoplasmic Ca²⁺ response in S2 cells expressing <i>CG8795</i>and apoaequorin challenged with Drm-capa-1, Drm-PK-1, hugg, Drm-PK-2 and NMU-25 at a concentration of 10⁻⁷ M. (<b>D</b>) Human NMU-25 dose-response curve in S2 cells and intact tubule. NMU-25 peptide stimulation of NMUR 2- or capaR- and apoaequorin-co-transfected S2 cells; and of tubule principal cells expressing apoaequorin transgene. Cells or tubules were challenged with increasing concentrations of agonist, and [Ca²⁺]_i was measured. Values were expressed as maximal (nM) - background (nM) (mean ± S.E.M., <i>N</i> = 3).</p

capaR is tubule-specific and localized to principal cells; manipulation of capaR expression levels modulates [Ca²⁺]_i and fluid transport rates.

<p>(<b>A</b>) Mean mRNA expression data ± SEM were collated from Affymetrix tissue-specific array datasets described in flyatlas.org <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029897#pone.0029897-Chintapalli1" target="_blank">[14]</a> for adult and larval tissues as indicated. Blue shading (dark-adult; light-larvae) indicates epithelial tissues; whereas green shading (dark-adult; light-larvae) indicates fat body or tissues containing fat body <i>eg.</i>, adult head and carcass. ‘mRNA signal’ indicates how abundant <i>capaR</i> mRNA is; and for each tissue, <i>capaR</i> mRNA was detectably expressed in 4 out of 4 arrays (flyatlas.org). In order to assess the expression pattern of capaR <i>in vivo</i>, the capaR promoter-driven GAL4 line, capaR-GAL4, was generated and crossed with UAS-GFP, and fluorescence examined by GFP histochemistry in tissues from progeny of the cross (top left panel). For orientation, tubule regions are indicated by M (main segment); I (initial segment); L (lower tubule). Expression of capaR-driven GFP occurs in the principal cells in the tubule main segment, exclusion of a stellate cell (arrowed, top right panel). (<b>B–D</b>) <i>Drosophila</i> capa receptor is expressed in principal cells of the Malpighian tubule. (<b>B</b>) Tubules were processed with pre-immune serum and only low-level non-specific staining of intracellular vesicles was observed, confirming the specificity of the antibody. (<b>C</b>) Immunocytochemistry using anti-capaR rabbit polyclonal antibody and anti-Rabbit IgG-Texas Red conjugate reveal basolateral membrane localization of capaR in tubule principal cells. (<b>D</b>) Merge of z-stacks from (<b>B</b>) picture reveals exclusion of a stellate cell (arrowed). In panels A, B–D, nuclei are labelled blue with DAPI, scale bar represents 30 µM. (<b>E</b>) Manipulation of capaR affects cytosolic [Ca²⁺]_i levels in intact tubules. Tubules were dissected from c42>UAS-apoaequorin flies (c42aeq), c42aeq>UAS-capaR RNAi flies and c42aeq>UAS-capaR. Resting cytoslic [Ca²⁺]_i levels were measured, after which tubules were stimulated with 10⁻⁷ M capa-1 to obtain stimulated cytosolic [Ca²⁺]_i readings. Primary and secondary pooled data for cytosolic [Ca²⁺]_i levels are shown as nM [Ca²⁺]_i ± SEM, <i>N</i> = 6, where * P<0.05, Student's <i>t</i>-test. (<b>F</b>) Fluid transport by <i>Drosophila</i> c42-GAL4>capaR RNAi renal tubules is significantly decreased (as determined using a Student's <i>t</i>-test (*P<0.05)) compared to the parental GAL4 line when the tubule is stimulated by application of capa-1 (10⁻⁷ M). Secretion rates are expressed as nl/min ± SEM (<i>N</i> = 6).</p