SharePro: an accurate and efficient genetic colocalization method accounting for multiple causal signals.

MOTIVATION: Colocalization analysis is commonly used to assess whether two or more traits share the same genetic signals identified in genome-wide association studies (GWAS), and is important for prioritizing targets for functional follow-up of GWAS results. Existing colocalization methods can have suboptimal performance when there are multiple causal variants in one genomic locus. RESULTS: We propose SharePro to extend the COLOC framework for colocalization analysis. SharePro integrates linkage disequilibrium (LD) modeling and colocalization assessment by grouping correlated variants into effect groups. With an efficient variational inference algorithm, posterior colocalization probabilities can be accurately estimated. In simulation studies, SharePro demonstrated increased power with a well-controlled false positive rate at a low computational cost. Compared to existing methods, SharePro provided stronger and more consistent colocalization evidence for known lipid-lowering drug target proteins and their corresponding lipid traits. Through an additional challenging case of the colocalization analysis of the circulating abundance of R-spondin 3 GWAS and estimated bone mineral density GWAS, we demonstrated the utility of SharePro in identifying biologically plausible colocalized signals. AVAILABILITY AND IMPLEMENTATION: SharePro for colocalization analysis is written in Python and openly available at https://github.com/zhwm/SharePro_coloc.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

AIMS/HYPOTHESIS: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion. METHODS: Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging. RESULTS: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation. CONCLUSIONS/INTERPRETATION: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. DATA AVAILABILITY: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Science fiction authors' perspectives on human genetic engineering.

Participants in the human gene editing debate often consider examples from science fiction but have rarely engaged directly with the science fiction community as stakeholders. To understand how science fiction authors develop and spread their views on gene editing, we created an online questionnaire that was answered by 78 authors, including 71 who had previously written about genetic engineering. When asked which ethical issues science fiction should explore, respondents most frequently mentioned affordability, new social divisions, consent and unforeseen safety risks. They rarely advocated exploring psychological effects or religious objections. When asked which works of fiction had influenced their perceptions of gene editing, the most frequent responses were the film Gattaca, the Star Trek franchise and the novels The Island of Doctor Moreau and Brave New World Unlike other stakeholders, they rarely cited Frankenstein as an influence. This article examines several differences between bioethicists, the general public and science fiction authors, and discusses how this community's involvement might benefit proponents and opponents of gene editing. It also provides an overview of works mentioned by our respondents that might serve as useful references in the debate.

Genome-wide landscape establishes novel association signals for metabolic traits in the Arab population.

While the Arabian population has a high prevalence of metabolic disorders, it has not been included in global studies that identify genetic risk loci for metabolic traits. Determining the transferability of such largely Euro-centric established risk loci is essential to transfer the research tools/resources, and drug targets generated by global studies to a broad range of ethnic populations. Further, consideration of populations such as Arabs, that are characterized by consanguinity and a high level of inbreeding, can lead to identification of novel risk loci. We imputed published GWAS data from two Kuwaiti Arab cohorts (n = 1434 and 1298) to the 1000 Genomes Project haplotypes and performed meta-analysis for associations with 13 metabolic traits. We compared the observed association signals with those established for metabolic traits. Our study highlighted 70 variants from 9 different genes, some of which have established links to metabolic disorders. By relaxing the genome-wide significance threshold, we identified 'novel' risk variants from 11 genes for metabolic traits. Many novel risk variant association signals were observed at or borderline to genome-wide significance. Furthermore, 349 previously established variants from 187 genes were validated in our study. Pleiotropic effect of risk variants on multiple metabolic traits were observed. Fine-mapping illuminated rs7838666/CSMD1 rs1864163/CETP and rs112861901/[INTS10,LPL] as candidate causal variants influencing fasting plasma glucose and high-density lipoprotein levels. Computational functional analysis identified a variety of gene regulatory signals around several variants. This study enlarges the population ancestry diversity of available GWAS and elucidates new variants in an ethnic group burdened with metabolic disorders.

Rel-Dependent Immune and Central Nervous System Mechanisms Control Viral Replication and Inflammation during Mouse Herpes Simplex Encephalitis.

Herpes simplex encephalitis (HSE), caused by HSV type 1 (HSV-1) infection, is an acute neuroinflammatory condition of the CNS and remains the most common type of sporadic viral encephalitis worldwide. Studies in humans have shown that susceptibility to HSE depends in part on the genetic make-up of the host, with deleterious mutations in the TLR3/type I IFN axis underlying some cases of childhood HSE. Using an in vivo chemical mutagenesis screen for HSV-1 susceptibility in mice, we identified a susceptible pedigree carrying a causal truncating mutation in the Rel gene (RelC307X ), encoding for the NF-κB transcription factor subunit c-Rel. Like Myd88-/- and Irf3-/- mice, RelC307X mice were susceptible to intranasal HSV-1 infection. Reciprocal bone marrow transfers into lethally irradiated hosts suggested that defects in both hematopoietic and CNS-resident cellular compartments contributed together to HSE susceptibility in RelC307X mice. Although the RelC307X mutation maintained cell-intrinsic antiviral control, it drove increased apoptotic cell death in infected fibroblasts. Moreover, reduced numbers of CD4+CD25+Foxp3+ T regulatory cells, and dysregulated NK cell and CD4+ effector T cell responses in infected RelC307X animals, indicated that protective immunity was also compromised in these mice. In the CNS, moribund RelC307X mice failed to control HSV-1 viral replication in the brainstem and cerebellum, triggering cell death and elevated expression of Ccl2, Il6, and Mmp8 characteristic of HSE neuroinflammation and pathology. In summary, our work implicates c-Rel in both CNS-resident cell survival and lymphocyte responses to HSV-1 infection and as a novel cause of HSE disease susceptibility in mice.

Alu repeats as transcriptional regulatory platforms in macrophage responses to M. tuberculosis infection.

To understand the epigenetic regulation of transcriptional response of macrophages during early-stage M. tuberculosis (Mtb) infection, we performed ChIPseq analysis of H3K4 monomethylation (H3K4me1), a marker of poised or active enhancers. De novo H3K4me1 peaks in infected cells were associated with genes implicated in host defenses and apoptosis. Our analysis revealed that 40% of de novo regions contained human/primate-specific Alu transposable elements, enriched in the AluJ and S subtypes. These contained several transcription factor binding sites, including those for members of the MEF2 and ATF families, and LXR and RAR nuclear receptors, all of which have been implicated in macrophage differentiation, survival, and responses to stress and infection. Combining bioinformatics, molecular genetics, and biochemical approaches, we linked genes adjacent to H3K4me1-associated Alu repeats to macrophage metabolic responses against Mtb infection. In particular, we show that LXRα signaling, which reduced Mtb viability 18-fold by altering cholesterol metabolism and enhancing macrophage apoptosis, can be initiated at response elements present in Alu repeats. These studies decipher the mechanism of early macrophage transcriptional responses to Mtb, highlighting the role of Alu element transposition in shaping human transcription programs during innate immunity.

Genome-wide association scan identifies a colorectal cancer susceptibility locus on chromosome 8q24.

Using a multistage genetic association approach comprising 7,480 affected individuals and 7,779 controls, we identified markers in chromosomal region 8q24 associated with colorectal cancer. In stage 1, we genotyped 99,632 SNPs in 1,257 affected individuals and 1,336 controls from Ontario. In stages 2-4, we performed serial replication studies using 4,024 affected individuals and 4,042 controls from Seattle, Newfoundland and Scotland. We identified one locus on chromosome 8q24 and another on 9p24 having combined odds ratios (OR) for stages 1-4 of 1.18 (trend; P = 1.41 x 10(-8)) and 1.14 (trend; P = 1.32 x 10(-5)), respectively. Additional analyses in 2,199 affected individuals and 2,401 controls from France and Europe supported the association at the 8q24 locus (OR = 1.16, trend; 95% confidence interval (c.i.): 1.07-1.26; P = 5.05 x 10(-4)). A summary across all seven studies at the 8q24 locus was highly significant (OR = 1.17, c.i.: 1.12-1.23; P = 3.16 x 10(-11)). This locus has also been implicated in prostate cancer.

Mapping of clinical and expression quantitative trait loci in a sex-dependent effect of host susceptibility to mouse-adapted influenza H3N2/HK/1/68.

Seasonal influenza outbreaks and recurrent influenza pandemics present major challenges to public health. By studying immunological responses to influenza in different host species, it may be possible to discover common mechanisms of susceptibility in response to various influenza strains. This could lead to novel therapeutic targets with wide clinical application. Using a mouse-adapted strain of influenza (A/HK/1/68-MA20 [H3N2]), we produced a mouse model of severe influenza that reproduces the hallmark high viral load and overexpression of cytokines associated with susceptibility to severe influenza in humans. We mapped genetic determinants of the host response using a panel of 29 closely related mouse strains (AcB/BcA panel of recombinant congenic strains) created from influenza-susceptible A/J and influenza-resistant C57BL/6J (B6) mice. Combined clinical quantitative trait loci (QTL) and lung expression QTL mapping identified candidate genes for two sex-specific QTL on chromosomes 2 and 17. The former includes the previously described Hc gene, a deficit of which is associated with the susceptibility phenotype in females. The latter includes the phospholipase gene Pla2g7 and Tnfrsf21, a member of the TNFR superfamily. Confirmation of the gene underlying the chromosome 17 QTL may reveal new strategies for influenza treatment.

Silencing ANGPTL8 reduces mouse preadipocyte differentiation and insulin signaling.

ANGPTL8, expressed mainly in the liver and adipose tissue, regulates the activity of lipoprotein lipase (LPL) present in the extracellular space and triglyceride (TG) metabolism through its interaction with ANGPTL3 and ANGPTL4. Whether intracellular ANGPTL8 can also exert effects in tissues where it is expressed is uncertain. ANGPTL8 expression was low in preadipocytes and much increased during differentiation. To better understand the role of intracellular ANGPTL8 in adipocytes and assess whether it may play a role in adipocyte differentiation, we knocked down its expression in normal mouse subcutaneous preadipocytes. ANGPTL8 knockdown reduced adipocyte differentiation, cellular TG accumulation and also isoproterenol-stimulated lipolysis at day 7 of differentiation. RNA-Seq analysis of ANGPTL8 siRNA or control siRNA transfected SC preadipocytes on days 0, 2, 4 and 7 of differentiation showed that ANGPTL8 knockdown impeded the early (day 2) expression of adipogenic and insulin signaling genes, PPARγ, as well as genes related to extracellular matrix and NF-κB signaling. Insulin mediated Akt phosphorylation was reduced at an early stage during adipocyte differentiation. This study based on normal primary cells shows that ANGPTL8 has intracellular actions in addition to effects in the extracellular space, like modulating LPL activity. Preadipocyte ANGPTL8 expression modulates their differentiation possibly via changes in insulin signaling gene expression.

An electrochemically controlled microcantilever biosensor.

An oligonucleotide-based electrochemically controlled gold-coated microcantilever biosensor that can transduce specific biomolecular interactions is reported. The derivatized microcantilever exhibits characteristic surface stress time course patterns in response to an externally applied periodic square wave potential. Experiments demonstrate that control of the surface charge density with an electrode potential is essential to producing a sensor that exhibits large, reproducible surface stress changes. The time course of surface stress changes are proposed to be linked to an electrochemically mediated competition between the adsorption of solution-based ions and the single- or double-stranded oligonucleotides tethered to the gold surface. A similar potential-actuated change in surface stress also results from the interaction between an oligonucleotide aptamer and its cognate ligand, demonstrating the broad applicability of this methodology.

A genome-wide association study identifies novel risk loci for type 2 diabetes.

Type 2 diabetes mellitus results from the interaction of environmental factors with a combination of genetic variants, most of which were hitherto unknown. A systematic search for these variants was recently made possible by the development of high-density arrays that permit the genotyping of hundreds of thousands of polymorphisms. We tested 392,935 single-nucleotide polymorphisms in a French case-control cohort. Markers with the most significant difference in genotype frequencies between cases of type 2 diabetes and controls were fast-tracked for testing in a second cohort. This identified four loci containing variants that confer type 2 diabetes risk, in addition to confirming the known association with the TCF7L2 gene. These loci include a non-synonymous polymorphism in the zinc transporter SLC30A8, which is expressed exclusively in insulin-producing beta-cells, and two linkage disequilibrium blocks that contain genes potentially involved in beta-cell development or function (IDE-KIF11-HHEX and EXT2-ALX4). These associations explain a substantial portion of disease risk and constitute proof of principle for the genome-wide approach to the elucidation of complex genetic traits.

Single-Cell RNA Sequencing Reveals a Role for Reactive Oxygen Species and Peroxiredoxins in Fatty Acid-Induced Rat ß-Cell Proliferation.

The functional mass of insulin-secreting pancreatic ß-cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing ß-cells. Type 2 diabetes appears when these compensatory mechanisms fail. Nutrients including glucose and fatty acids are important contributors to the ß-cell compensatory response, but their underlying mechanisms of action remain poorly understood. We investigated the transcriptional mechanisms of ß-cell proliferation in response to fatty acids. Isolated rat islets were exposed to 16.7 mmol/L glucose with or without 0.5 mmol/L oleate (C18:1) or palmitate (C16:0) for 48 h. The islet transcriptome was assessed by single-cell RNA sequencing. ß-Cell proliferation was measured by flow cytometry. Unsupervised clustering of pooled ß-cells identified different subclusters, including proliferating ß-cells. ß-Cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved in energy metabolism and mitochondrial activity. Comparison of proliferating versus nonproliferating ß-cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, N-acetyl cysteine and the peroxiredoxin inhibitor conoidin A both blocked oleate-induced ß-cell proliferation. Our study reveals a key role for ROS signaling through peroxiredoxin activation in oleate-induced ß-cell proliferation.

Molecular phenotyping of single pancreatic islet leader beta cells by "Flash-Seq".

AIMS: Spatially-organized increases in cytosolic Ca2+ within pancreatic beta cells in the pancreatic islet underlie the stimulation of insulin secretion by high glucose. Recent data have revealed the existence of subpopulations of beta cells including "leaders" which initiate Ca2+ waves. Whether leader cells possess unique molecular features, or localisation, is unknown. MAIN METHODS: High speed confocal Ca2+ imaging was used to identify leader cells and connectivity analysis, running under MATLAB and Python, to identify highly connected "hub" cells. To explore transcriptomic differences between beta cell sub-groups, individual leaders or followers were labelled by photo-activation of the cryptic fluorescent protein PA-mCherry and subjected to single cell RNA sequencing ("Flash-Seq"). KEY FINDINGS: Distinct Ca2+ wave types were identified in individual islets, with leader cells present in 73 % (28 of 38 islets imaged). Scale-free, power law-adherent behaviour was also observed in 29 % of islets, though "hub" cells in these islets did not overlap with leaders. Transcripts differentially expressed (295; padj < 0.05) between leader and follower cells included genes involved in cilium biogenesis and transcriptional regulation. Providing some support for these findings, ADCY6 immunoreactivity tended to be higher in leader than follower cells, whereas cilia number and length tended to be lower in the former. Finally, leader cells were located significantly closer to delta, but not alpha, cells in Euclidian space than were follower cells. SIGNIFICANCE: The existence of both a discrete transcriptome and unique localisation implies a role for these features in defining the specialized function of leaders. These data also raise the possibility that localised signalling between delta and leader cells contributes to the initiation and propagation of islet Ca2+ waves.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

Aims/hypothesis: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly-connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility which we explore here by focussing on the imprinted gene neuronatin (Nnat), which is required for normal insulin synthesis and secretion. Methods: Single cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing eGFP under the control of the Nnat enhancer/promoter regions were generated for fluorescence-activated cell (FAC) sorting of beta cells and downstream analysis of CpG methylation by bisulphite and RNA sequencing, respectively. Animals deleted for the de novo methyltransferase, DNMT3A from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 and Cal-590. Insulin secretion was measured using Homogeneous Time Resolved Fluorescence Imaging. Results: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic data sets demonstrated the early establishment of Nnat-positive and negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a sub-population specialised for insulin production, reminiscent of recently-described "ßHI" cells and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialization. Conclusions/interpretation: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may thus contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.

A genome-wide association study reveals variants in ARL15 that influence adiponectin levels.

The adipocyte-derived protein adiponectin is highly heritable and inversely associated with risk of type 2 diabetes mellitus (T2D) and coronary heart disease (CHD). We meta-analyzed 3 genome-wide association studies for circulating adiponectin levels (n = 8,531) and sought validation of the lead single nucleotide polymorphisms (SNPs) in 5 additional cohorts (n = 6,202). Five SNPs were genome-wide significant in their relationship with adiponectin (P< or =5x10(-8)). We then tested whether these 5 SNPs were associated with risk of T2D and CHD using a Bonferroni-corrected threshold of P< or =0.011 to declare statistical significance for these disease associations. SNPs at the adiponectin-encoding ADIPOQ locus demonstrated the strongest associations with adiponectin levels (P-combined = 9.2x10(-19) for lead SNP, rs266717, n = 14,733). A novel variant in the ARL15 (ADP-ribosylation factor-like 15) gene was associated with lower circulating levels of adiponectin (rs4311394-G, P-combined = 2.9x10(-8), n = 14,733). This same risk allele at ARL15 was also associated with a higher risk of CHD (odds ratio [OR] = 1.12, P = 8.5x10(-6), n = 22,421) more nominally, an increased risk of T2D (OR = 1.11, P = 3.2x10(-3), n = 10,128), and several metabolic traits. Expression studies in humans indicated that ARL15 is well-expressed in skeletal muscle. These findings identify a novel protein, ARL15, which influences circulating adiponectin levels and may impact upon CHD risk.

Discriminating protein tags on a dsDNA construct using a Dual Nanopore Device.

We report Brownian dynamics simulation results with the specific goal to identify key parameters controlling the experimentally measurable characteristics of protein tags on a dsDNA construct translocating through a double nanopore setup. First, we validate the simulation scheme in silico by reproducing and explaining the physical origin of the asymmetric experimental dwell time distributions of the oligonucleotide flap markers on a 48 kbp long dsDNA at the left and the right pore. We study the effect of the electric field inside and beyond the pores, critical to discriminate the protein tags based on their effective charges and masses revealed through a generic power-law dependence of the average dwell time at each pore. The simulation protocols monitor piecewise dynamics at a sub-nanometer length scale and explain the disparate velocity using the concepts of nonequilibrium tension propagation theory. We further justify the model and the chosen simulation parameters by calculating the Péclet number which is in close agreement with the experiment. We demonstrate that our carefully chosen simulation strategies can serve as a powerful tool to discriminate different types of neutral and charged tags of different origins on a dsDNA construct in terms of their physical characteristics and can provide insights to increase both the efficiency and accuracy of an experimental dual-nanopore setup.

Glycerol-3-phosphate phosphatase operates a glycerol shunt in pancreatic ß-cells that controls insulin secretion and metabolic stress.

OBJECTIVE: The recently identified glycerol-3-phosphate (Gro3P) phosphatase (G3PP) in mammalian cells, encoded by the PGP gene, was shown to regulate glucose, lipid and energy metabolism by hydrolyzing Gro3P and to control glucose-stimulated insulin secretion (GSIS) in ß-cells, in vitro. However, whether G3PP regulates ß-cell function and insulin secretion in vivo is not known. METHODS: We now examined the role of G3PP in the control of insulin secretion in vivo, ß-cell function and glucotoxicity in inducible ß-cell specific G3PP-KO (BKO) mice. Inducible BKO mice were generated by crossing floxed-G3PP mice with Mip-Cre-ERT (MCre) mice. All the in vivo studies were done using BKO and control mice fed normal diet and the ex vivo studies were done using pancreatic islets from these mice. RESULTS: BKO mice, compared to MCre controls, showed increased body weight, adiposity, fed insulinemia, enhanced in vivo GSIS, reduced plasma triglycerides and mild glucose intolerance. Isolated BKO mouse islets incubated at high (16.7 mM), but not at low or intermediate glucose (3 and 8 mM), showed elevated GSIS, Gro3P content as well as increased levels of metabolites and signaling coupling factors known to reflect ß-cell activation for insulin secretion. BKO islets also showed reduced glycerol release and increased O2 consumption and ATP production at high glucose only. BKO islets chronically exposed to elevated glucose levels showed increased apoptosis, reduced insulin content and decreased mRNA expression of ß-cell differentiation markers, Pdx-1, MafA and Ins-2. CONCLUSIONS: The results demonstrate that ß-cells are endowed with a "glycerol shunt", operated by G3PP that regulates ß-cell metabolism, signaling and insulin secretion in vivo, primarily at elevated glucose concentrations. We propose that the glycerol shunt plays a role in preventing insulin hypersecretion and excess body weight gain and contributes to ß-cell mass preservation in the face of hyperglycemia.

Hepatic glycerol shunt and glycerol-3-phosphate phosphatase control liver metabolism and glucodetoxification under hyperglycemia.

OBJECTIVE: Glycerol-3-phosphate (Gro3P) phosphatase (G3PP) hydrolyzes Gro3P to glycerol that exits the cell, thereby operating a "glycerol shunt", a metabolic pathway that we identified recently in mammalian cells. We have investigated the role of G3PP and the glycerol shunt in the regulation of glucose metabolism and lipogenesis in mouse liver. METHODS: We generated hepatocyte-specific G3PP-KO mice (LKO), by injecting AAV8-TBG-iCre to male G3PPfl/fl mice. Controls received AAV8-TBG-eGFP. Both groups were fed chow diet for 10 weeks. Hyperglycemia (16-20 mM) was induced by glucose infusion for 55 h. Hepatocytes were isolated from normoglycemic mice for ex vivo studies and targeted metabolomics were measured in mice liver after glucose infusion. RESULTS: LKO mice showed no change in body weight, food intake, fed and fasted glycemia but had increased fed plasma triglycerides. Hepatic glucose production from glycerol was increased in fasted LKO mice. LKO mouse hepatocytes displayed reduced glycerol production, elevated triglyceride and lactate production at high glucose concentration. Hyperglycemia in LKO mice led to increased liver weight and accumulation of triglycerides, glycogen and cholesterol together with elevated levels of Gro3P, dihydroxyacetone phosphate, acetyl-CoA and some Krebs cycle intermediates in liver. Hyperglycemic LKO mouse liver showed elevated expression of proinflammatory cytokines and M1-macrophage markers accompanied by increased plasma triglycerides, LDL/VLDL, urea and uric acid and myocardial triglycerides. CONCLUSIONS: The glycerol shunt orchestrated by G3PP acts as a glucose excess detoxification pathway in hepatocytes by preventing metabolic disturbances that contribute to enhanced liver fat, glycogen storage, inflammation and lipid build-up in the heart. We propose G3PP as a novel therapeutic target for hepatic disorders linked to nutrient excess.

Differential allelic expression in the human genome: a robust approach to identify genetic and epigenetic cis-acting mechanisms regulating gene expression.

The recent development of whole genome association studies has lead to the robust identification of several loci involved in different common human diseases. Interestingly, some of the strongest signals of association observed in these studies arise from non-coding regions located in very large introns or far away from any annotated genes, raising the possibility that these regions are involved in the etiology of the disease through some unidentified regulatory mechanisms. These findings highlight the importance of better understanding the mechanisms leading to inter-individual differences in gene expression in humans. Most of the existing approaches developed to identify common regulatory polymorphisms are based on linkage/association mapping of gene expression to genotypes. However, these methods have some limitations, notably their cost and the requirement of extensive genotyping information from all the individuals studied which limits their applications to a specific cohort or tissue. Here we describe a robust and high-throughput method to directly measure differences in allelic expression for a large number of genes using the Illumina Allele-Specific Expression BeadArray platform and quantitative sequencing of RT-PCR products. We show that this approach allows reliable identification of differences in the relative expression of the two alleles larger than 1.5-fold (i.e., deviations of the allelic ratio larger than 60:40) and offers several advantages over the mapping of total gene expression, particularly for studying humans or outbred populations. Our analysis of more than 80 individuals for 2,968 SNPs located in 1,380 genes confirms that differential allelic expression is a widespread phenomenon affecting the expression of 20% of human genes and shows that our method successfully captures expression differences resulting from both genetic and epigenetic cis-acting mechanisms.