27 research outputs found

    Optimization and validation of HTRF-based detection of the GCK-GKRP interaction.

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    <p>(A) Titration of GCK and GKRP. Results are presented as the ratio of the acceptor emission (665 nm) and donor emission (615 nm) for eight different GCK and GKRP concentrations measured at t = 60 minutes. Each data point is mean ± SEM for n = 6. (B) Effect of S6P on HTRF detection of the GCK-GKRP interaction. The experiment was run in the presence of 5 nM GCK and 5 nM GKRP. Values shown are mean ± SEM (n = 8 for each [S6P]). (C–F) Effect of known inhibitors glucose (C), F1P (D), and two GKAs (E–F) on HTRF detection of the GCK-GKRP interaction in the presence of 5 nM GCK, 5 nM GKRP, and 2 ”M S6P. Values shown are mean ± SEM (n = 8 for each concentration).</p

    Optimization and validation of diaphorase-coupled detection of the GCK enzymatic reaction.

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    <p>(A) Reaction progress of the dual-coupled diaphorase reaction at various GCK and GKRP concentrations. Each data point is mean ± SEM for n = 8. Dashed lines are NADP<sup>+</sup>/NADPH controls, with the %NADPH increasing from the bottom (0%) to the top (100%) dashed line. (B) Effect of GKRP modulators on the dual-coupled diaphorase reaction. The reaction included 10 nM GCK and 10 nM GKRP. Results are shown for t = 10 minutes. Each data point is mean ± SEM for n = 2–4.</p

    Localization of GCK in cryopreserved hepatocytes.

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    <p>Overlay of GCK (FITC channel; green) localization with Hoechst nuclear stain (blue) in cryopreserved hepatocytes from images collected at 10X magnification. The contrast was held fixed for all images, and the full dynamic range of all the 12 bit images has been maintained. (A–C) GCK localization in male Sprague-Dawley rat hepatocytes at (A) 2.5 mM glucose, (B) 16.7 mM glucose, and (C) 2.5 mM glucose and 31.6 ”M GKA-EMD. (D–H) GCK localization in cryopreserved human hepatocytes. (D) Translocation of GCK in GKRP-positive cells in two separate lots (TRZ: closed symbols; FOS: open symbols) of human hepatocytes at various concentrations of glucose. (E–F) Translocation of GCK in the presence of GKA-EMD at various concentrations of glucose for donor TRZ and FOS, respectively. (G–H) Representative images from donor TRZ at (G) 2.5 mM glucose and (H) 2.5 mM glucose with 31.6 ”M GKA. Data points are mean ± SEM for n = 4–8.</p

    Parallel assay development strategy to interrogate recombinant GCK and GKRP.

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    <p>Human GCK and GKRP were affinity-purified using GST and FLAG tags, respectively. (A) FRET-based (HTRF) detection. Antibodies recognizing the affinity tags are conjugated to FRET donor and acceptor molecules. Excitation of the donor results in energy transfer (FRET; red dashed oval) to the acceptor only if the acceptor and donor are in close proximity. (B) Reaction scheme for G6PDH/diaphorase dual-coupled assay. The generation of the fluorescent product resorufin (red dashed oval) is measured as the reaction progresses in real time by excitation at 525 nm with emission at 590 nm. (C) Reaction scheme for coupling of ADP generation by GCK to luminescence-based detection. The GCK reaction is allowed to run for a set period of time, and the reaction is then terminated and a two-step reaction utilizes Ultra-Gloℱ firefly luciferase to generate bioluminescence (red dashed oval). Reagent 1: ADP-Gloℱ Reagent; Reagent 2: Kinase Detection Reagent.</p

    Quality metrics for the HTRF assay with the LOPAC<sup>1280</sup> library in 1536-well plates.

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    <p>(A) Z’ factor, (B) signal/background, (C) % variance, and (D) calculated IC<sub>50</sub> of the control compound GKA-EMD as a function of assay plate.</p

    <i>GCK</i> mutations identified in Slovakian probands with a phenotype of GCK-MODY.

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    <p>All sequence information is based on GenBank reference sequence NM_000162.3. Nucleotide numbering reflects cDNA position, with +1 corresponding to the A of the major start codon of exon 1a (present in the pancreatic isoform). Y = yes, N = no.</p

    Inhibition of wild type, I436N and L315H glucokinase proteins by human GKRP.

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    <p>Data are shown as mean ±SEM, and were obtained from 4 independent measurements. Independent t-tests were used to ascertain differences between GKRP-mediated inhibition of both mutants versus that obtained with the wild-type GCK enzyme.</p

    Clinical and biochemical parameters of Slovakian <i>GCK</i> mutation carriers.

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    <p>Data are presented as median values (range).</p>*<p>Data only available for 35 probands.</p>#<p>Data only available for 33 probands.</p>$<p>For the remaining 32 subjects, diabetes/impaired glucose tolerance was not detected prior to genetic testing.</p><p>BMI = body mass index, HbA1c = glycated hemoglobin A1c, HDL = High Density Lipoprotein, OHA = oral hypoglyceamic agents.</p

    Evaluation of Common Type 2 Diabetes Risk Variants in a South Asian Population of Sri Lankan Descent

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    <div><p>Introduction</p><p>Most studies seeking common variant associations with type 2 diabetes (T2D) have focused on individuals of European ancestry. These discoveries need to be evaluated in other major ancestral groups, to understand ethnic differences in predisposition, and establish whether these contribute to variation in T2D prevalence and presentation. This study aims to establish whether common variants conferring T2D-risk in Europeans contribute to T2D-susceptibility in the South Asian population of Sri Lanka.</p><p>Methodology</p><p>Lead single nucleotide polymorphism (SNPs) at 37 T2D-risk loci attaining genome-wide significance in Europeans were genotyped in 878 T2D cases and 1523 normoglycaemic controls from Sri Lanka. Association testing was performed by logistic regression adjusting for age and sex and by the Cochran-Mantel-Haenszel test after stratifying according to self-identified ethnolinguistic subgroup. A weighted genetic risk score was generated to examine the combined effect of these SNPs on T2D-risk in the Sri Lankan population.</p><p>Results</p><p>Of the 36 SNPs passing quality control, sixteen showed nominal (p<0.05) association in Sri Lankan samples, fifteen of those directionally-consistent with the original signal. Overall, these association findings were robust to analyses that accounted for membership of ethnolinguistic subgroups. Overall, the odds ratios for 31 of the 36 SNPs were directionally-consistent with those observed in Europeans (p = 3.2×10<sup>−6</sup>). Allelic odds ratios and risk allele frequencies in Sri Lankan subjects were not systematically different to those reported in Europeans. Genetic risk score and risk of T2D were strongly related in Sri Lankans (per allele OR 1.10 [95%CI 1.08–1.13], p = 1.2×10<sup>−17</sup>).</p><p>Conclusion</p><p>Our data indicate that most T2D-risk variants identified in Europeans have similar effects in South Asians from Sri Lanka, and that systematic difference in common variant associations are unlikely to explain inter-ethnic differences in prevalence or presentation of T2D.</p></div

    Comparison of allelic ORs between South Asians from Sri Lanka and Europeans.

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    <p>Loci labelled in bold are variants that showed nominal significance (p<0.05) in Sri Lankan subjects. European ORs used were derived from previously reported studies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098608#pone.0098608.s001" target="_blank">Table S1</a>).</p
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