The P446L variant in GCKR associated with fasting plasma glucose and triglyceride levels exerts its effect through increased glucokinase activity in liver

Genome-wide association studies have identified a number of signals for both Type 2 Diabetes and related quantitative traits. For the majority of loci, the transition from association signal to mutational mechanism has been difficult to establish. Glucokinase (GCK) regulates glucose storage and disposal in the liver where its activity is regulated by glucokinase regulatory protein (GKRP; gene name GCKR). Fructose-6 and fructose-1 phosphate (F6P and F1P) enhance or reduce GKRP-mediated inhibition, respectively. A common GCKR variant (P446L) is reproducibly associated with triglyceride and fasting plasma glucose levels in the general population. The aim of this study was to determine the mutational mechanism responsible for this genetic association. Recombinant human GCK and both human wild-type (WT) and P446L-GKRP proteins were generated. GCK kinetic activity was observed spectrophotometrically using an NADP+-coupled assay. WT and P446L-GKRP-mediated inhibition of GCK activity and subsequent regulation by phosphate esters were determined. Assays matched for GKRP activity demonstrated no difference in dose-dependent inhibition of GCK activity or F1P-mediated regulation. However, the response to physiologically relevant F6P levels was significantly attenuated with P446L-GKRP (n = 18; P ≤ 0.03). Experiments using equimolar concentrations of both regulatory proteins confirmed these findings (n = 9; P < 0.001). In conclusion, P446L-GKRP has reduced regulation by physiological concentrations of F6P, resulting indirectly in increased GCK activity. Altered GCK regulation in liver is predicted to enhance glycolytic flux, promoting hepatic glucose metabolism and elevating concentrations of malonyl-CoA, a substrate for de novo lipogenesis, providing a mutational mechanism for the reported association of this variant with raised triglycerides and lower glucose levels

Aligning Single-Cell Developmental and Reprogramming Trajectories Identifies Molecular Determinants of Myogenic Reprogramming Outcome

Cellular reprogramming through manipulation of defined factors holds great promise for large-scale production of cell types needed for use in therapy and for revealing principles of gene regulation. However, most reprogramming systems are inefficient, converting only a fraction of cells to the desired state. Here, we analyze MYOD-mediated reprogramming of human fibroblasts to myotubes, a well-characterized model system for direct conversion by defined factors, at pseudotemporal resolution using single-cell RNA-seq. To expose barriers to efficient conversion, we introduce a novel analytic technique, trajectory alignment, which enables quantitative comparison of gene expression kinetics across two biological processes. Reprogrammed cells navigate a trajectory with branch points that correspond to two alternative decision points, with cells that select incorrect branches terminating at aberrant or incomplete reprogramming outcomes. Analysis of these branch points revealed insulin and BMP signaling as crucial molecular determinants of reprogramming. Single-cell trajectory alignment enables rigorous quantitative comparisons between biological trajectories found in diverse processes in development, reprogramming, and other contexts

Race-Ethnic Differences in the Association of Genetic Loci with HbA1c levels and Mortality in U.S. Adults: The Third National Health and Nutrition Examination Survey (NHANES III)

Background: Hemoglobin A1c (HbA1c) levels diagnose diabetes, predict mortality and are associated with ten single nucleotide polymorphisms (SNPs) in white individuals. Genetic associations in other race groups are not known. We tested the hypotheses that there is race-ethnic variation in 1) HbA1c-associated risk allele frequencies (RAFs) for SNPs near SPTA1, HFE, ANK1, HK1, ATP11A, FN3K, TMPRSS6, G6PC2, GCK, MTNR1B; 2) association of SNPs with HbA1c and 3) association of SNPs with mortality. Methods We studied 3,041 non-diabetic individuals in the NHANES (National Health and Nutrition Examination Survey) III. We stratified the analysis by race/ethnicity (NHW: non-Hispanic white; NHB: non-Hispanic black; MA: Mexican American) to calculate RAF, calculated a genotype score by adding risk SNPs, and tested associations with SNPs and the genotype score using an additive genetic model, with type 1 error = 0.05. Results: RAFs varied widely and at six loci race-ethnic differences in RAF were significant (p < 0.0002), with NHB usually the most divergent. For instance, at ATP11A, the SNP RAF was 54% in NHB, 18% in MA and 14% in NHW (p < .0001). The mean genotype score differed by race-ethnicity (NHW: 10.4, NHB: 11.0, MA: 10.7, p < .0001), and was associated with increase in HbA1c in NHW (β = 0.012 HbA1c increase per risk allele, p = 0.04) and MA (β = 0.021, p = 0.005) but not NHB (β = 0.007, p = 0.39). The genotype score was not associated with mortality in any group (NHW: OR (per risk allele increase in mortality) = 1.07, p = 0.09; NHB: OR = 1.04, p = 0.39; MA: OR = 1.03, p = 0.71). Conclusion: At many HbA1c loci in NHANES III there is substantial RAF race-ethnic heterogeneity. The combined impact of common HbA1c-associated variants on HbA1c levels varied by race-ethnicity, but did not influence mortality

Associations of common polymorphisms in GCKR with type 2 diabetes and related traits in a Han Chinese population: a case-control study

<p>Abstract</p> <p>Background</p> <p>Several studies have shown that variants in the glucokinase regulatory protein gene (<it>GCKR</it>) were associated with type 2 diabetes and dyslipidemia. The purpose of this study was to examine whether tag single nucleotide polymorphisms (SNPs) in the <it>GCKR </it>region were associated with type 2 diabetes and related traits in a Han Chinese population and to identify the potential mechanisms underlying these associations.</p> <p>Methods</p> <p>We investigated the association of polymorphisms in the <it>GCKR </it>gene with type 2 diabetes by employing a case-control study design (1118 cases and 1161 controls). Four tag SNPs (rs8179206, rs2293572, rs3817588 and rs780094) with pairwise r²> 0.8 and minor allele frequency > 0.05 across the <it>GCKR </it>gene and its flanking regions were studied and haplotypes were constructed. Genotyping was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy using a MassARRAY platform.</p> <p>Results</p> <p>The G alleles of <it>GCKR </it>rs3817588 and rs780094 were associated with an increased risk of type 2 diabetes after adjustment for year of birth, sex and BMI (OR = 1.24, 95% CI 1.08-1.43, p = 0.002 and OR = 1.22, 95% CI 1.07-1.38, p = 0.002, respectively). In the non-diabetic controls, the GG carriers of rs3817588 and rs780094 were nominally associated with a lower plasma triglyceride level compared to the AA carriers after adjustment for year of birth, sex and BMI (p for trend = 0.00004 and 0.03, respectively). Furthermore, the association of rs3817588 with plasma triglyceride level was still significant after correcting for multiple testing.</p> <p>Conclusions</p> <p>The rs3817588 A/G polymorphism of the <it>GCKR </it>gene was associated with type 2 diabetes and plasma triglyceride level in the Han Chinese population.</p

Whole-genome and multisector exome sequencing of primary and post-treatment glioblastoma reveals patterns of tumor evolution

Glioblastoma (GBM) is a prototypical heterogeneous brain tumor refractory to conventional therapy. A small residual population of cells escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence ~7 mo after diagnosis. Understanding the molecular architecture of this residual population is critical for the development of successful therapies. We used whole-genome sequencing and whole-exome sequencing of multiple sectors from primary and paired recurrent GBM tumors to reconstruct the genomic profile of residual, therapy resistant tumor initiating cells. We found that genetic alteration of the p53 pathway is a primary molecular event predictive of a high number of subclonal mutations in glioblastoma. The genomic road leading to recurrence is highly idiosyncratic but can be broadly classified into linear recurrences that share extensive genetic similarity with the primary tumor and can be directly traced to one of its specific sectors, and divergent recurrences that share few genetic alterations with the primary tumor and originate from cells that branched off early during tumorigenesis. Our study provides mechanistic insights into how genetic alterations in primary tumors impact the ensuing evolution of tumor cells and the emergence of subclonal heterogeneity

Cellular characterisation of the GCKR P446L variant associated with type 2 diabetes risk

Aims/hypothesis Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP; gene symbol GCKR) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intracellular localisation. Methods Fluorescently tagged rat and human wild-type (WT)- or P446L-GCKR and GCK were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low- or high-glucose conditions (5.5 or 25 mmol/l glucose, respectively). Interaction between GCK and GKRP was measured by sensitised emission-based fluorescence resonance energy transfer (FRET) efficiency

The pectoralis minor length test: a study of the intra-rater reliability and diagnostic accuracy in subjects with and without shoulder symptoms

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly citedBackground. Postural abnormality and muscle imbalance are thought to contribute to pain and a loss of normal function in the upper body. A shortened pectoralis minor muscle is commonly identified as part of this imbalance. Clinical tests have been recommended to test for shortening of this muscle. The aim of this study was to evaluate the intra-rater reliability and diagnostic accuracy of the pectoralis minor length test. Methods. Measurements were made in 45 subjects with and 45 subjects without shoulder symptoms. Measurements were made with the subjects lying in supine. In this position the linear distance from the treatment table to the posterior aspect of the acromion was measured on two occasions (separated by a minimum of 30 minutes and additional data collection on other subjects to reduce bias) by one rater. The reliability of the measurements was analyzed using intraclass correlation coefficients (ICC), 95% confidence intervals (CI) and standard error of measurement (SEM). The diagnostic accuracy of the test was investigated by determining the sensitivity, specificity, positive and negative likelihood ratios of the test against a 'gold standard' reference. The assessor remained 'blinded' to data input and the measurements were staggered to reduce examiner bias. Results. The pectoralis minor length test was found to have excellent intra-rater reliability for dominant and non-dominant side of the subjects without symptoms, and for the painfree and painful side of the subjects with symptoms. The values calculated for the sensitivity, specificity, positive and negative likelihood ratios suggest this test performed in the manner investigated in this study and recommended in the literature, lacks diagnostic accuracy. Conclusion. The findings of this study suggest that although the pectoralis minor length test demonstrates acceptable clinical reliability, its lack of specificity suggests that clinicians using this test to inform the clinical reasoning process with regard treatment planning must do so with caution. Trial registration. National Research Register: N0060148286.Peer reviewe

Central Role of SREBP-2 in the Pathogenesis of Osteoarthritis

Background: Recent studies have implied that osteoarthritis (OA) is a metabolic disease linked to deregulation of genes involved in lipid metabolism and cholesterol efflux. Sterol Regulatory Element Binding Proteins (SREBPs) are transcription factors regulating lipid metabolism with so far no association with OA. Our aim was to test the hypothesis that SREBP-2, a gene that plays a key role in cholesterol homeostasis, is crucially involved in OA pathogenesis and to identify possible mechanisms of action. Methodology/Principal Findings: We performed a genetic association analysis using a cohort of 1,410 Greek OA patients and healthy controls and found significant association between single nucleotide polymorphism (SNP) 1784G>C in SREBP-2 gene and OA development. Moreover, the above SNP was functionally active, as normal chondrocytes’ transfection with SREBP-2-G/C plasmid resulted in interleukin-1β and metalloproteinase-13 (MMP-13) upregulation. We also evaluated SREBP-2, its target gene 3-hydroxy-3-methylglutaryl-coenzymeA reductase (HMGCR), phospho-phosphoinositide3-kinase (PI3K), phospho-Akt, integrin-alphaV (ITGAV) and transforming growth factor-$\beta$ (TGF-$\beta$) mRNA and protein expression levels in osteoarthritic and normal chondrocytes and found that they were all significantly elevated in OA chondrocytes. To test whether TGF-$\beta$ alone can induce SREBP-2, we treated normal chondrocytes with TGF-$\beta$ and found significant upregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13. We also showed that TGF-$\beta$ activated aggrecan (ACAN) in chondrocytes only through Smad3, which interacts with SREBP-2. Finally, we examined the effect of an integrin inhibitor, cyclo-RGDFV peptide, on osteoarthritic chondrocytes, and found that it resulted in significant upregulation of ACAN and downregulation of SREBP-2, HMGCR, phospho-PI3K and MMP-13 expression levels. Conclusions/Significance: We demonstrated, for the first time, the association of SREBP-2 with OA pathogenesis and provided evidence on the molecular mechanism involved. We suggest that TGF-$\beta$ induces SREBP-2 pathway activation through ITGAV and PI3K playing a key role in OA and that integrin blockage may be a potential molecular target for OA treatment