Gpr40 Is Expressed in Enteroendocrine Cells and Mediates Free Fatty Acid Stimulation of Incretin Secretion

OBJECTIVE—The G-protein–coupled receptor Gpr40 is expressed in β-cells where it contributes to free fatty acid (FFA) enhancement of glucose-stimulated insulin secretion (1–4). However, other sites of Gpr40 expression, including the intestine, have been suggested. The transcription factor IPF1/PDX1 was recently shown to bind to an enhancer element within the 5′-flanking region of Gpr40 (5), implying that IPF1/PDX1 might regulate Gpr40 expression. Here, we addressed whether 1) Gpr40 is expressed in the intestine and 2) Ipf1/Pdx1 function is required for Gpr40 expression

Heterologous Stop Codon Readthrough of Metazoan Readthrough Candidates in Yeast

Recent analysis of genomic signatures in mammals, flies, and worms indicates that functional translational stop codon readthrough is considerably more abundant in metazoa than previously recognized, but this analysis provides only limited clues about the function or mechanism of readthrough. If an mRNA known to be read through in one species is also read through in another, perhaps these questions can be studied in a simpler setting. With this end in mind, we have investigated whether some of the readthrough genes in human, fly, and worm also exhibit readthrough when expressed in S. cerevisiae. We found that readthrough was highest in a gene with a post-stop hexamer known to trigger readthrough, while other metazoan readthrough genes exhibit borderline readthrough in S. cerevisiae.National Institutes of Health (U.S.) (5U54HG004555-03

Tramtrack Is Genetically Upstream of Genes Controlling Tracheal Tube Size in Drosophila

The Drosophila transcription factor Tramtrack (Ttk) is involved in a wide range of developmental decisions, ranging from early embryonic patterning to differentiation processes in organogenesis. Given the wide spectrum of functions and pleiotropic effects that hinder a comprehensive characterisation, many of the tissue specific functions of this transcription factor are only poorly understood. We recently discovered multiple roles of Ttk in the development of the tracheal system on the morphogenetic level. Here, we sought to identify some of the underlying genetic components that are responsible for the tracheal phenotypes of Ttk mutants. We therefore profiled gene expression changes after Ttk loss- and gain-of-function in whole embryos and cell populations enriched for tracheal cells. The analysis of the transcriptomes revealed widespread changes in gene expression. Interestingly, one of the most prominent gene classes that showed significant opposing responses to loss- and gain-of-function was annotated with functions in chitin metabolism, along with additional genes that are linked to cellular responses, which are impaired in ttk mutants. The expression changes of these genes were validated by quantitative real-time PCR and further functional analysis of these candidate genes and other genes also expected to control tracheal tube size revealed at least a partial explanation of Ttk's role in tube size regulation. The computational analysis of our tissue-specific gene expression data highlighted the sensitivity of the approach and revealed an interesting set of novel putatively tracheal genes

Composition-based statistics and translated nucleotide searches: Improving the TBLASTN module of BLAST

BACKGROUND: TBLASTN is a mode of operation for BLAST that aligns protein sequences to a nucleotide database translated in all six frames. We present the first description of the modern implementation of TBLASTN, focusing on new techniques that were used to implement composition-based statistics for translated nucleotide searches. Composition-based statistics use the composition of the sequences being aligned to generate more accurate E-values, which allows for a more accurate distinction between true and false matches. Until recently, composition-based statistics were available only for protein-protein searches. They are now available as a command line option for recent versions of TBLASTN and as an option for TBLASTN on the NCBI BLAST web server. RESULTS: We evaluate the statistical and retrieval accuracy of the E-values reported by a baseline version of TBLASTN and by two variants that use different types of composition-based statistics. To test the statistical accuracy of TBLASTN, we ran 1000 searches using scrambled proteins from the mouse genome and a database of human chromosomes. To test retrieval accuracy, we modernize and adapt to translated searches a test set previously used to evaluate the retrieval accuracy of protein-protein searches. We show that composition-based statistics greatly improve the statistical accuracy of TBLASTN, at a small cost to the retrieval accuracy. CONCLUSION: TBLASTN is widely used, as it is common to wish to compare proteins to chromosomes or to libraries of mRNAs. Composition-based statistics improve the statistical accuracy, and therefore the reliability, of TBLASTN results. The algorithms used by TBLASTN are not widely known, and some of the most important are reported here. The data used to test TBLASTN are available for download and may be useful in other studies of translated search algorithms

Selective Small-Molecule Agonists of G Protein–Coupled Receptor 40 Promote Glucose-Dependent Insulin Secretion and Reduce Blood Glucose in Mice

OBJECTIVE— Acute activation of G protein–coupled receptor 40 (GPR40) by free fatty acids (FFAs) or synthetic GPR40 agonists enhances insulin secretion. However, it is still a matter of debate whether activation of GPR40 would be beneficial for the treatment of type 2 diabetes, since chronic exposure to FFAs impairs islet function. We sought to evaluate the specific role of GPR40 in islets and its potential as a therapeutic target using compounds that specifically activate GPR40

The Drosophila FoxA Ortholog Fork Head Regulates Growth and Gene Expression Downstream of Target of Rapamycin

Forkhead transcription factors of the FoxO subfamily regulate gene expression programs downstream of the insulin signaling network. It is less clear which proteins mediate transcriptional control exerted by Target of rapamycin (TOR) signaling, but recent studies in nematodes suggest a role for FoxA transcription factors downstream of TOR. In this study we present evidence that outlines a similar connection in Drosophila, in which the FoxA protein Fork head (FKH) regulates cellular and organismal size downstream of TOR. We find that ectopic expression and targeted knockdown of FKH in larval tissues elicits different size phenotypes depending on nutrient state and TOR signaling levels. FKH overexpression has a negative effect on growth under fed conditions, and this phenotype is not further exacerbated by inhibition of TOR via rapamycin feeding. Under conditions of starvation or low TOR signaling levels, knockdown of FKH attenuates the size reduction associated with these conditions. Subcellular localization of endogenous FKH protein is shifted from predominantly cytoplasmic on a high-protein diet to a pronounced nuclear accumulation in animals with reduced levels of TOR or fed with rapamycin. Two putative FKH target genes, CG6770 and cabut, are transcriptionally induced by rapamycin or FKH expression, and silenced by FKH knockdown. Induction of both target genes in heterozygous TOR mutant animals is suppressed by mutations in fkh. Furthermore, TOR signaling levels and FKH impact on transcription of the dFOXO target gene d4E-BP, implying a point of crosstalk with the insulin pathway. In summary, our observations show that an alteration of FKH levels has an effect on cellular and organismal size, and that FKH function is required for the growth inhibition and target gene induction caused by low TOR signaling levels

Variants in the FFAR1 Gene Are Associated with Beta Cell Function

The FFAR1 receptor is expressed mainly in pancreatic beta cells and is activated by medium to long chain free fatty acids (FFAs), as well as by thiazolidinediones, resulting in elevated Ca(2+) concentrations and promotion of insulin secretion. These properties suggest that FFAR1 could be a mediator of lipotoxicity and a potential candidate gene for Type 2 diabetes (T2D). We therefore investigated whether variations at the FFAR1 locus are associated with T2D and beta cell function.We re-sequenced the FFAR1 region in 96 subjects (48 healthy and 48 T2D individuals) and found 13 single nucleotide polymorphisms (SNPs) 8 of which were not previously described. Two SNPs located in the upstream region of the FFAR1 gene (rs1978013 and rs1978014) were chosen and genotyped in 1929 patients with T2D and 1405 healthy control subjects. We observed an association of rs1978013 and rs1978014 with insulinogenic index in males (p = 0.024) and females (p = 0.032), respectively. After Bonferroni corrections, no association with T2D was found in the case-control material, however a haplotype consisting of the T-G alleles conferred protection against T2D (p = 0.0010).Variation in the FFAR1 gene may contribute to impaired beta cell function in T2D

Palmitate-Induced β-Cell Dysfunction Is Associated with Excessive NO Production and Is Reversed by Thiazolidinedione-Mediated Inhibition of GPR40 Transduction Mechanisms

BACKGROUND: Type 2 diabetes often displays hyperlipidemia. We examined palmitate effects on pancreatic islet function in relation to FFA receptor GPR40, NO generation, insulin release, and the PPARgamma agonistic thiazolidinedione, rosiglitazone. PRINCIPAL FINDINGS: Rosiglitazone suppressed acute palmitate-stimulated GPR40-transduced PI hydrolysis in HEK293 cells and insulin release from MIN6c cells and mouse islets. Culturing islets 24 h with palmitate at 5 mmol/l glucose induced beta-cell iNOS expression as revealed by confocal microscopy and increased the activities of ncNOS and iNOS associated with suppression of glucose-stimulated insulin response. Rosiglitazone reversed these effects. The expression of iNOS after high-glucose culturing was unaffected by rosiglitazone. Downregulation of GPR40 by antisense treatment abrogated GPR40 expression and suppressed palmitate-induced iNOS activity and insulin release. CONCLUSION: We conclude that, in addition to mediating acute FFA-stimulated insulin release, GPR40 is an important regulator of iNOS expression and dysfunctional insulin release during long-term exposure to FFA. The adverse effects of palmitate were counteracted by rosiglitazone at GPR40, suggesting that thiazolidinediones are beneficial for beta-cell function in hyperlipidemic type 2 diabetes

Systematic Identification of Genes that Regulate Neuronal Wiring in the Drosophila Visual System

Forward genetic screens in model organisms are an attractive means to identify those genes involved in any complex biological process, including neural circuit assembly. Although mutagenesis screens are readily performed to saturation, gene identification rarely is, being limited by the considerable effort generally required for positional cloning. Here, we apply a systematic positional cloning strategy to identify many of the genes required for neuronal wiring in the Drosophila visual system. From a large-scale forward genetic screen selecting for visual system wiring defects with a normal retinal pattern, we recovered 122 mutations in 42 genetic loci. For 6 of these loci, the underlying genetic lesions were previously identified using traditional methods. Using SNP-based mapping approaches, we have now identified 30 additional genes. Neuronal phenotypes have not previously been reported for 20 of these genes, and no mutant phenotype has been previously described for 5 genes. The genes encode a variety of proteins implicated in cellular processes such as gene regulation, cytoskeletal dynamics, axonal transport, and cell signalling. We conducted a comprehensive phenotypic analysis of 35 genes, scoring wiring defects according to 33 criteria. This work demonstrates the feasibility of combining large-scale gene identification with large-scale mutagenesis in Drosophila, and provides a comprehensive overview of the molecular mechanisms that regulate visual system wiring