[Show abstract][Hide abstract] ABSTRACT: Genotyping microarrays are an important resource for genetic mapping, population genetics and monitoring of the genetic integrity of laboratory stocks. We have developed the third generation of the Mouse Universal Genotyping Array (MUGA) series, GigaMUGA, a 143,259-probe Illumina Infinium II array for the house mouse (Mus musculus). The bulk of the content of GigaMUGA is optimized for genetic mapping in the Collaborative Cross and Diversity Outbred populations and for substrain-level identification of laboratory mice. In addition to 141,090 SNP probes, GigaMUGA contains 2,006 probes for copy number concentrated in structurally polymorphic regions of the mouse genome. The performance of the array is characterized in a set of 500 high-quality reference samples spanning laboratory inbred strains, recombinant inbred lines, outbred stocks, and wild-caught mice. GigaMUGA is highly informative across a wide range of genetically-diverse samples, from laboratory substrains to other Mus species. In addition to describing the content and performance of the array, we provide detailed probe-level annotation and recommendations for quality control.
Full-text · Article · Dec 2015 · G3-Genes Genomes Genetics
[Show abstract][Hide abstract] ABSTRACT: RNA editing refers to post-transcriptional processes that alter the base sequence of RNA. Recently, hundreds of new RNA editing targets have been reported. However, the mechanisms that determine the specificity and degree of editing are not well understood. We examined quantitative variation of site-specific editing in a genetically diverse multiparent population, Diversity Outbred mice, and mapped polymorphic loci that alter editing ratios globally for C-to-U editing and at specific sites for A-to-I editing. An allelic series in the C-to-U editing enzyme Apobec1 influences editing efficiency of Apob and 58 additional C-to-U editing targets. We identified 49 A-to-I editing sites with polymorphisms in the edited transcript that alter editing efficiency. In contrast to the shared genetic control of C-to-U editing, most of the variable A-to-I sites were determined by local nucleotide polymorphisms in proximity to the editing site in the RNA secondary structure. Our results indicate that RNA editing is a quantitative trait subject to genetic variation and that evolutionary constraints have given rise to distinct genetic architectures in the two canonical types of RNA editing.
[Show abstract][Hide abstract] ABSTRACT: Studies of the genetic loci that contribute to variation in gene expression
frequently identify loci with broad effect on gene expression: expression
quantitative trait locus (eQTL) hotspots. We describe a set of exploratory
graphical methods as well as a formal likelihood-based test for assessing
whether a given hotspot is due to one or multiple polymorphisms. We first look
at the pattern of effects of the locus on the expression traits that map to the
locus: the direction of the effects, as well as the degree of dominance. A
second technique is to focus on the individuals that exhibit no recombination
event in the region, apply dimensionality reduction (such as with linear
discriminant analysis) and compare the phenotype distribution in the
non-recombinants to that in the recombinant individuals: If the recombinant
individuals display a different expression pattern than the non-recombinants,
this indicates the presence of multiple causal polymorphisms. In the formal
likelihood-based test, we compare a two-locus model, with each expression trait
affected by one or the other locus, to a single-locus model. We apply our
methods to a large mouse intercross with gene expression microarray data on six
[Show abstract][Hide abstract] ABSTRACT: Bottom-up
proteomics database search algorithms used for peptide
identification cannot comprehensively identify post-translational
modifications (PTMs) in a single-pass because of high false discovery
rates (FDRs). A new approach to database searching enables global
PTM (G-PTM) identification by exclusively looking for curated PTMs,
thereby avoiding the FDR penalty experienced during conventional variable
modification searches. We identified over 2200 unique, high-confidence
modified peptides comprising 26 different PTM types in a single-pass
Preview · Article · Sep 2015 · Journal of Proteome Research
[Show abstract][Hide abstract] ABSTRACT: Glucose stimulation of insulin secretion in pancreatic β cells involves cell depolarization and subsequent opening of voltage-dependent Ca2+ channels to elicit insulin granule exocytosis. This pathway alone does not account for the entire magnitude of the secretory response in β cells. In this issue, Ferdaoussi, Dai, and colleagues reveal that insulin secretion is amplified by cytosolic isocitrate dehydrogenase-dependent transfer of reducing equivalents, which generates NADPH and reduced glutathione, which in turn activates sentrin/SUMO-specific protease-1 (SENP1). β Cell-specific deletion of Senp1 in murine models reduced the amplification of insulin exocytosis, resulting in impaired glucose tolerance. Further, their studies demonstrate that restoring intracellular NADPH or activating SENP1 improves insulin exocytosis in human β cells from donors with type 2 diabetes, suggesting a potential therapeutic target to augment insulin production.
No preview · Article · Sep 2015 · The Journal of clinical investigation
[Show abstract][Hide abstract] ABSTRACT: We surveyed gene expression in six tissues in an F2 intercross between mouse strains C57BL/6J (abbreviated B6) and BTBR T(+) tf /J (abbreviated BTBR) made genetically obese with the Leptin(ob) mutation. We identified a number of expression quantitative trait loci (eQTL) affecting the expression of numerous genes distal to the locus, called trans-eQTL hotspots. Some of these trans-eQTL hotspots showed effects in multiple tissues, whereas some were specific to a single tissue. An unusually large number of transcripts (~8% of genes) mapped in trans to a hotspot on chromosome 6, specifically in pancreatic islets. By considering the first two principal components of the expression of genes mapping to this region, we were able to convert the multivariate phenotype into a simple Mendelian trait. Fine-mapping the locus by traditional methods reduced the QTL interval to a 298 kb region containing only three genes, including Slco1a6, one member of a large family of organic anion transporters. Direct genomic sequencing of all Slco1a6 exons identified a non-synonymous coding SNP that converts a highly conserved proline residue at amino acid position 564 to serine. Molecular modeling suggests that Pro564 faces an aqueous pore within this 12-transmembrane domain-spanning protein. When transiently overexpressed in HEK293 cells, BTBR OATP1A6-mediated cellular uptake of the bile acid taurocholic acid (TCA) was enhanced compared to B6 OATP1A6. Our results suggest that genetic variation in Slco1a6 leads to altered transport of TCA (and potentially other bile acids) by pancreatic islets, resulting in broad gene regulation.
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVE- Better understanding how glucagon-like peptide 1 (GLP-1) promotes pancreatic β-cell function and/or mass may uncover new treatment for type 2 diabetes. In this study we investigated the potential involvement of microRNAs (miRNAs) in the effect of GLP-1 on glucose-stimulated insulin secretion (GSIS). Research Design and methods- MiRNA levels in INS-1 cells and isolated rodent and human islets treated with GLP-1 in vitro and in vivo (with osmotic pumps) were measured by real-time quantitative PCR. The role of miRNAs on insulin secretion was studied by transfecting INS-1 cells with either precursors or antisense inhibitors of miRNAs. RESULTS- Among the 250 miRNAs surveyed, miR-132 and miR-212 were significantly up-regulated by GLP-1 by greater than 2 fold in INS-1 832/3 cells, which were subsequently reproduced in freshly-isolated rat, mouse, and human islets, as well as the islets from GLP-1 infusion in vivo in mice. The inductions of miR-132 and miR-212 by GLP-1 were correlated with cAMP production and were blocked by the PKA inhibitor H-89, but not affected by the Epac activator 8-pCPT-2`-O-Me-cAMP. GLP-1 failed to increase miR-132 or miR-212 expression levels in the 832/13 line of INS-1 cells, which lacks robust cAMP and insulin responses to GLP-1 treatment. Overexpression of miR-132 or miR-212 significantly enhanced GSIS in both 832/3 and 832/13 cells, and restored insulin responses to GLP-1 in INS-1 832/13 cells. CONCLUSION- GLP-1 increases the expression of miRNAs 132 and 212 via a cAMP/PKA-dependent pathway in pancreatic β-cells. Overexpression of miR-132 or -212 enhances glucose and GLP-1 stimulated insulin secretion.
No preview · Article · Jul 2015 · Molecular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: Many of our insights into obesity and diabetes come from studies in mice carrying natural or induced mutations. In parallel, genome-wide association studies (GWAS) in humans have identified numerous genes that are causally associated with obesity and diabetes, but discovering the underlying mechanisms required in-depth studies in mice. We discuss the advantages of studying natural variation in mice and summarize several examples where the combination of human and mouse genetics opened windows into fundamental physiological pathways. A noteworthy example is the melanocortin-4 receptor (MC4R) and its role in energy balance. The pathway was delineated by discovering the gene responsible for the Agouti mutation in mice. With more targeted phenotyping, we predict that additional pathways relevant to human pathophysiology will be discovered.
No preview · Article · Oct 2014 · Trends in Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: Background:
An unbiased systems approach was used to define energy metabolic events that occur during the pathological cardiac remodeling en route to heart failure (HF).
Methods and results:
Combined myocardial transcriptomic and metabolomic profiling were conducted in a well-defined mouse model of HF that allows comparative assessment of compensated and decompensated (HF) forms of cardiac hypertrophy because of pressure overload. The pressure overload data sets were also compared with the myocardial transcriptome and metabolome for an adaptive (physiological) form of cardiac hypertrophy because of endurance exercise training. Comparative analysis of the data sets led to the following conclusions: (1) expression of most genes involved in mitochondrial energy transduction were not significantly changed in the hypertrophied or failing heart, with the notable exception of a progressive downregulation of transcripts encoding proteins and enzymes involved in myocyte fatty acid transport and oxidation during the development of HF; (2) tissue metabolite profiles were more broadly regulated than corresponding metabolic gene regulatory changes, suggesting significant regulation at the post-transcriptional level; (3) metabolomic signatures distinguished pathological and physiological forms of cardiac hypertrophy and served as robust markers for the onset of HF; and (4) the pattern of metabolite derangements in the failing heart suggests bottlenecks of carbon substrate flux into the Krebs cycle.
Mitochondrial energy metabolic derangements that occur during the early development of pressure overload-induced HF involve both transcriptional and post-transcriptional events. A subset of the myocardial metabolomic profile robustly distinguished pathological and physiological cardiac remodeling.
[Show abstract][Hide abstract] ABSTRACT: Glucagon-like peptide-1 (GLP-1) is
a natural agonist for GLP-1R,
a G protein-coupled receptor (GPCR) on the surface of pancreatic β
cells. GLP-1R agoinsts are attractive for treatment of type 2 diabetes,
but GLP-1 itself is rapidly degraded by peptidases in vivo. We describe a design strategy for retaining GLP-1-like activity
while engendering prolonged activity in vivo, based
on strategic replacement of native α residues with conformationally
constrained β-amino acid residues. This backbone-modification
approach may be useful for developing stabilized analogues of other
Preview · Article · Sep 2014 · Journal of the American Chemical Society
[Show abstract][Hide abstract] ABSTRACT: Massively parallel RNA sequencing (RNA-seq) has yielded a wealth of new insights into transcriptional regulation. A first step in the analysis of RNA-seq data is the alignment of short sequence reads to a common reference genome or transcriptome. Genetic variants that distinguish individual genomes from the reference sequence can cause reads to be misaligned, resulting in biased estimates of transcript abundance. Fine-tuning of read alignment algorithms does not correct this problem. We have developed Seqnature software to construct individualized diploid genomes and transcriptomes for multiparent populations and have implemented a complete analysis pipeline that incorporates other existing software tools. We demonstrate in simulated and real data sets that alignment to individualized transcriptomes increases read mapping accuracy, improves estimation of transcript abundance, and enables the direct estimation of allele-specific expression. Moreover, when applied to expression QTL mapping we find that our individualized alignment strategy corrects false-positive linkage signals and unmasks hidden associations. We recommend the use of individualized diploid genomes over reference sequence alignment for all applications of high-throughput sequencing technology in genetically diverse populations.
[Show abstract][Hide abstract] ABSTRACT: We previously positionally cloned Sorcs1 as a diabetes quantitative trait locus. Sorcs1 belongs to the Vacuolar protein sorting-10 (Vps10) gene family. In yeast, Vps10 transports enzymes from the trans-Golgi network (TGN) to the vacuole. Whole-body Sorcs1 KO mice, when made obese with the leptinob mutation (ob/ob), developed diabetes. β-Cells from these mice had a severe deficiency of secretory granules (SGs) and insulin. Interestingly, a single secretagogue challenge failed to consistently elicit an insulin secretory dysfunction. However, multiple challenges of the Sorcs1 KO ob/ob islets consistently revealed an insulin secretion defect. The luminal domain of SORCS1 (Lum-Sorcs1), when expressed in a β-cell line, acted as a dominant-negative, leading to SG and insulin deficiency. Using syncollin-dsRed5TIMER adenovirus, we found that the loss of Sorcs1 function greatly impairs the rapid replenishment of SGs following secretagogue challenge. Chronic exposure of islets from lean Sorcs1 KO mice to high glucose and palmitate depleted insulin content and evoked an insulin secretion defect. Thus, in metabolically stressed mice, Sorcs1 is important for SG replenishment, and under chronic challenge by insulin secretagogues, loss of Sorcs1 leads to diabetes. Overexpression of full-length SORCS1 led to a 2-fold increase in SG content, suggesting that SORCS1 is sufficient to promote SG biogenesis.
Full-text · Article · Aug 2014 · Journal of Clinical Investigation
[Show abstract][Hide abstract] ABSTRACT: The abundance and functional activity of proteins involved in the formation of the SNARE complex are tightly regulated for efficient exocytosis. Tomosyn proteins are negative regulators of exocytosis. Tomosyn causes an attenuation of insulin secretion by limiting the formation of the SNARE complex. We hypothesized that glucose-dependent stimulation of insulin secretion from β-cells must involve reversing the inhibitory action of tomosyn. Here we show that glucose increases tomosyn protein turnover. Within 1 h of exposure to 15 mM glucose, ~50% of tomosyn was degraded. The degradation of tomosyn in response to high glucose was blocked by inhibitors of the proteasomal pathway. Using 32P-labeling and mass spectrometry, we showed that tomosyn-2 is phosphorylated in response to high glucose, phorbol esters, and analogs of cAMP, all key insulin secretagouges. We identified eleven phosphorylation sites in tomosyn-2. Site-directed mutagenesis was used to generate phosphomimetic (S→D) and loss-of-function (S→A) mutants. The S→D mutant had enhanced protein turnover compared to the S→A mutant and wild type tomosyn-2. Additionally, the S→D tomosyn-2 mutant was ineffective at inhibiting insulin secretion. Using a proteomic screen for tomosyn-2 binding proteins we identified Hrd-1, an E3-ubiquitin ligase. We showed that tomosyn-2 ubiquitination is increased by Hrd-1 and knockdown of Hrd-1 by short-hairpin RNA resulted in increased abundance in tomosyn-2 protein levels. Taken together, our results reveal a mechanism by which enhanced phosphorylation of a negative regulator of secretion, tomosyn-2, in response to insulin secretagogues targets it to degradation by the Hrd-1 E3-ubiquitin ligase.
No preview · Article · Jul 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: We previously demonstrated that micro-RNAs 132 and 212 are differentially upregulated in response to obesity in two mouse strains that differ in their susceptibility to obesity-induced diabetes. Here we show the overexpression of micro-RNAs 132 and 212 enhances insulin secretion (IS) in response to glucose and other secretagogues including non-fuel stimuli. We determined that carnitine acyl-carnitine translocase (CACT, Slc25a20) is a direct target of these miRNAs. CACT is responsible for transporting long-chain acyl-carnitines into the mitochondria for β-oxidation. SiRNA mediated knockdown of CACT in β-cells led to the accumulation of fatty acyl-carnitines, and enhanced IS. The addition of long-chain fatty acyl-carnitines promoted IS from INS-1 β-cells as well as primary mouse islets. The effect in INS-1 cells was augmented in response to suppression of CACT. A non-hydrolyzable ether analog of palmitoyl-carnitine stimulated IS, showing that β-oxidation of palmitoyl-carnitine is not required for its stimulation of IS. These studies establish a link between miRNA-dependent regulation of CACT and fatty acyl-carnitine mediated regulation of IS.
[Show abstract][Hide abstract] ABSTRACT: Increasing evidence has shown that proper control of mitochondrial dynamics (fusion and fission) is required for high capacity ATP production in heart. The transcriptional coactivators, peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) α and β have been shown to regulate mitochondrial biogenesis in heart at the time of birth. The function of the PGC-1 coactivators in heart after birth is incompletely understood.
To assess the role of the PGC-1 coactivators during postnatal cardiac development and in the adult heart in mice.
Conditional gene targeting was used in mice to explore the role of the PGC-1 coactivators during postnatal cardiac development and in adult heart. Marked mitochondrial structural derangements were observed in hearts of PGC-1α/β-deficient mice during postnatal growth, including fragmentation and elongation, associated with the development of a lethal cardiomyopathy. The expression of genes involved in mitochondrial fusion [mitofusin 1 (Mfn1), optic atrophy 1 (Opa1)] and fission [dynamin-related protein 1 (Drp1), fission protein 1 (Fis1)] was altered in hearts of PGC-1α/β-deficient mice. PGC-lα was shown to directly regulate Mfn1 gene transcription by coactivating the estrogen-related receptor α (ERRα upon a conserved DNA element. Surprisingly, PGC-1α/β deficiency in the adult heart did not result in evidence of abnormal mitochondrial dynamics or heart failure. However, transcriptional profiling demonstrated that the PGC-1 coactivators are required for high level expression of nuclear- and mitochondrial-encoded genes involved in mitochondrial dynamics and energy transduction in adult heart.
These results reveal distinct developmental stage-specific programs involved in cardiac mitochondrial dynamics.
Preview · Article · Dec 2013 · Circulation Research
[Show abstract][Hide abstract] ABSTRACT: We describe a chemical tag for duplex proteome quantification using neutron encoding (NeuCode). The method utilizes the straightforward, efficient, and inexpensive carbamylation reaction. We demonstrate the utility of NeuCode carbamylation by accurately measuring quantitative ratios from tagged yeast lysates mixed in known ratios and by applying this method to quantify differential protein expression in mice fed a either control or high-fat diet.
No preview · Article · Oct 2013 · Journal of the American Society for Mass Spectrometry
[Show abstract][Hide abstract] ABSTRACT: Insulin and Zn2+ enjoy a multivalent relationship. Zn2+ binds insulin in pancreatic β cells to form crystalline aggregates in dense core vesicles (DCVs), which are released in response to physiological signals such as increased blood glucose. This transition metal is an essential cofactor in insulin-degrading enzyme and several key Zn2+ finger transcription factors that are required for β cell development and insulin gene expression. Studies are increasingly revealing that fluctuations in Zn2+ concentration can mediate signaling events, including dynamic roles that extend beyond that of a static structural or catalytic cofactor. In this issue of the JCI, Tamaki et al. propose an additional function for Zn2+ in relation to insulin: regulation of insulin clearance from the bloodstream.
Full-text · Article · Sep 2013 · The Journal of clinical investigation
[Show abstract][Hide abstract] ABSTRACT: Lysine acetylation is rapidly becoming established as a key post-translational modification for regulating mitochondrial metabolism.
Nonetheless, distinguishing regulatory sites from among the thousands identified by mass spectrometry and elucidating how
these modifications alter enzyme function remain primary challenges. Here, we performed multiplexed quantitative mass spectrometry
to measure changes in the mouse liver mitochondrial acetylproteome in response to acute and chronic alterations in nutritional
status, and integrated these data sets with our compendium of predicted Sirt3 targets. These analyses highlight a subset of
mitochondrial proteins with dynamic acetylation sites, including acetyl-CoA acetyltransferase 1 (Acat1), an enzyme central
to multiple metabolic pathways. We performed in vitro biochemistry and molecular modeling to demonstrate that acetylation of Acat1 decreases its activity by disrupting the binding
of coenzyme A. Collectively, our data reveal an important new target of regulatory acetylation and provide a foundation for
investigating the role of select mitochondrial protein acetylation sites in mediating acute and chronic metabolic transitions.