The role of insulin receptor substrate 2 in hypothalamic and beta cell function

Insulin receptor substrate 2 (Irs2) plays complex roles in energy homeostasis. We generated mice lacking Irs2 in beta cells and a population of hypothalamic neurons (RIPCreIrs2KO), in all neurons (NesCreIrs2KO), and in proopiomelanocortin neurons (POMCCreIrs2KO) to determine the role of Irs2 in the CNS and beta cell. RIPCreIrs2KO mice displayed impaired glucose tolerance and reduced P cell mass. Overt diabetes did not ensue, because beta cells escaping Cre-mediated recombination progressively populated islets. RIPCreIrs2KO and NesCreIrs2KO mice displayed hyperphagia, obesity, and increased body length, which suggests altered melanocortin action. POMCCreIrs2KO mice did not display this phenotype. RIPCreIrs2KO and NesCreIrs2KO mice retained leptin sensitivity, which suggests that CNS Irs2 pathways are not required for leptin action. NesCreIrs2KO and POMCCreIrs2KO mice did not display reduced beta cell mass, but NesCreIrs2KO mice displayed mild abnormalities of glucose homeostasis. RIPCre neurons did not express POMC or neuropeptide Y. Insulin and a melanocortin agonist depolarized RIPCre neurons, whereas leptin was ineffective. Insulin hyperpolarized and leptin depolarized POMC neurons. Our findings demonstrate a critical role for IRS2 in beta cell and hypothalamic function and provide insights into the role of RIPCre neurons, a distinct hypothalamic neuronal population, in growth and energy homeostasis

Missense Mutation in Exon 2 of SLC36A1 Responsible for Champagne Dilution in Horses

Champagne coat color in horses is controlled by a single, autosomal-dominant gene (CH). The phenotype produced by this gene is valued by many horse breeders, but can be difficult to distinguish from the effect produced by the Cream coat color dilution gene (CR). Three sires and their families segregating for CH were tested by genome scanning with microsatellite markers. The CH gene was mapped within a 6 cM region on horse chromosome 14 (LOD = 11.74 for θ = 0.00). Four candidate genes were identified within the region, namely SPARC [Secreted protein, acidic, cysteine-rich (osteonectin)], SLC36A1 (Solute Carrier 36 family A1), SLC36A2 (Solute Carrier 36 family A2), and SLC36A3 (Solute Carrier 36 family A3). SLC36A3 was not expressed in skin tissue and therefore not considered further. The other three genes were sequenced in homozygotes for CH and homozygotes for the absence of the dilution allele (ch). SLC36A1 had a nucleotide substitution in exon 2 for horses with the champagne phenotype, which resulted in a transition from a threonine amino acid to an arginine amino acid (T63R). The association of the single nucleotide polymorphism (SNP) with the champagne dilution phenotype was complete, as determined by the presence of the nucleotide variant among all 85 horses with the champagne dilution phenotype and its absence among all 97 horses without the champagne phenotype. This is the first description of a phenotype associated with the SLC36A1 gene

CA8 Mutations Cause a Novel Syndrome Characterized by Ataxia and Mild Mental Retardation with Predisposition to Quadrupedal Gait

We describe a consanguineous Iraqi family in which affected siblings had mild mental retardation and congenital ataxia characterized by quadrupedal gait. Genome-wide linkage analysis identified a 5.8 Mb interval on chromosome 8q with shared homozygosity among the affected persons. Sequencing of genes contained in the interval revealed a homozygous mutation, S100P, in carbonic anhydrase related protein 8 (CA8), which is highly expressed in cerebellar Purkinje cells and influences inositol triphosphate (ITP) binding to its receptor ITPR1 on the endoplasmatic reticulum and thereby modulates calcium signaling. We demonstrate that the mutation S100P is associated with proteasome-mediated degradation, and thus presumably represents a null mutation comparable to the Ca8 mutation underlying the previously described waddles mouse, which exhibits ataxia and appendicular dystonia. CA8 thus represents the third locus that has been associated with quadrupedal gait in humans, in addition to the VLDLR locus and a locus at chromosome 17p. Our findings underline the importance of ITP-mediated signaling in cerebellar function and provide suggestive evidence that congenital ataxia paired with cerebral dysfunction may, together with unknown contextual factors during development, predispose to quadrupedal gait in humans

Long-term effects of an inpatient weight-loss program in obese children and the role of genetic predisposition-rationale and design of the LOGIC-trial

<p>Abstract</p> <p>Background</p> <p>The prevalence of childhood obesity has increased worldwide, which is a serious concern as obesity is associated with many negative immediate and long-term health consequences. Therefore, the treatment of overweight and obesity in children and adolescents is strongly recommended. Inpatient weight-loss programs have shown to be effective particularly regarding short-term weight-loss, whilst little is known both on the long-term effects of this treatment and the determinants of successful weight-loss and subsequent weight maintenance.</p> <p>The purpose of this study is to evaluate the short, middle and long-term effects of an inpatient weight-loss program for children and adolescents and to investigate the likely determinants of weight changes, whereby the primary focus lies on the potential role of differences in polymorphisms of adiposity-relevant genes.</p> <p>Methods/Design</p> <p>The study involves overweight and obese children and adolescents aged 6 to 19 years, who participate in an inpatient weight-loss program for 4 to 6 weeks. It started in 2006 and it is planned to include 1,500 participants by 2013. The intervention focuses on diet, physical activity and behavior therapy. Measurements are taken at the start and the end of the intervention and comprise blood analyses (DNA, lipid and glucose metabolism, adipokines and inflammatory markers), anthropometry (body weight, height and waist circumference), blood pressure, pubertal stage, and exercise capacity. Physical activity, dietary habits, quality of life, and family background are assessed by questionnaires. Follow-up assessments are performed 6 months, 1, 2, 5 and 10 years after the intervention: Children will complete the same questionnaires at all time points and visit their general practitioner for examination of anthropometric parameters, blood pressure and assessment of pubertal stage. At the 5 and 10 year follow-ups, blood parameters and exercise capacity will be additionally measured.</p> <p>Discussion</p> <p>Apart from illustrating the short, middle and long-term effects of an inpatient weight-loss program, this study will contribute to a better understanding of inter-individual differences in the regulation of body weight, taking into account the role of genetic predisposition and lifestyle factors.</p> <p>Trial Registration</p> <p><a href="http://www.clinicaltrials.gov/ct2/show/NCT01067157">NCT01067157</a>.</p

Mapping of the Disease Locus and Identification of ADAMTS10 As a Candidate Gene in a Canine Model of Primary Open Angle Glaucoma

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide, with elevated intraocular pressure as an important risk factor. Increased resistance to outflow of aqueous humor through the trabecular meshwork causes elevated intraocular pressure, but the specific mechanisms are unknown. In this study, we used genome-wide SNP arrays to map the disease gene in a colony of Beagle dogs with inherited POAG to within a single 4 Mb locus on canine chromosome 20. The Beagle POAG locus is syntenic to a previously mapped human quantitative trait locus for intraocular pressure on human chromosome 19. Sequence capture and next-generation sequencing of the entire canine POAG locus revealed a total of 2,692 SNPs segregating with disease. Of the disease-segregating SNPs, 54 were within exons, 8 of which result in amino acid substitutions. The strongest candidate variant causes a glycine to arginine substitution in a highly conserved region of the metalloproteinase ADAMTS10. Western blotting revealed ADAMTS10 protein is preferentially expressed in the trabecular meshwork, supporting an effect of the variant specific to aqueous humor outflow. The Gly661Arg variant in ADAMTS10 found in the POAG Beagles suggests that altered processing of extracellular matrix and/or defects in microfibril structure or function may be involved in raising intraocular pressure, offering specific biochemical targets for future research and treatment strategies

Probing the Functional Impact of Sequence Variation on p53-DNA Interactions Using a Novel Microsphere Assay for Protein-DNA Binding with Human Cell Extracts

The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation—including polymorphisms—and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks

The CYCLIN-A CYCA1;2/TAM Is Required for the Meiosis I to Meiosis II Transition and Cooperates with OSD1 for the Prophase to First Meiotic Division Transition

Meiosis halves the chromosome number because its two divisions follow a single round of DNA replication. This process involves two cell transitions, the transition from prophase to the first meiotic division (meiosis I) and the unique meiosis I to meiosis II transition. We show here that the A-type cyclin CYCA1;2/TAM plays a major role in both transitions in Arabidopsis. A series of tam mutants failed to enter meiosis II and thus produced diploid spores and functional diploid gametes. These diploid gametes had a recombined genotype produced through the single meiosis I division. In addition, by combining the tam-2 mutation with AtSpo11-1 and Atrec8, we obtained plants producing diploid gametes through a mitotic-like division that were genetically identical to their parents. Thus tam alleles displayed phenotypes very similar to that of the previously described osd1 mutant. Combining tam and osd1 mutations leads to a failure in the prophase to meiosis I transition during male meiosis and to the production of tetraploid spores and gametes. This suggests that TAM and OSD1 are involved in the control of both meiotic transitions

Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum

Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntington's disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtin's normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtin's normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell–cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca2+ or Mg2+ but not pulses of cAMP. Although hd− cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd− cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid organism model for genetic, cell biological, and biochemical studies to delineate the functions of the HD protein