Suppression of insulin production and secretion by a decretin hormone.
2015; 21 (2): 323-333
Decretins, hormones induced by fasting that suppress insulin production and secretion, have been postulated from classical human metabolic studies. From genetic screens, we identified Drosophila Limostatin (Lst), a peptide hormone that suppresses insulin secretion. Lst is induced by nutrient restriction in gut-associated endocrine cells. limostatin deficiency led to hyperinsulinemia, hypoglycemia, and excess adiposity. A conserved 15-residue polypeptide encoded by limostatin suppressed secretion by insulin-producing cells. Targeted knockdown of CG9918, a Drosophila ortholog of Neuromedin U receptors (NMURs), in insulin-producing cells phenocopied limostatin deficiency and attenuated insulin suppression by purified Lst, suggesting CG9918 encodes an Lst receptor. NMUR1 is expressed in islet β cells, and purified NMU suppresses insulin secretion from human islets. A human mutant NMU variant that co-segregates with familial early-onset obesity and hyperinsulinemia fails to suppress insulin secretion. We propose Lst as an index member of an ancient hormone class called decretins, which suppress insulin output.
View details for DOI 10.1016/j.cmet.2015.01.006
View details for PubMedID 25651184
A genetic strategy to measure circulating Drosophila insulin reveals genes regulating insulin production and secretion.
2014; 10 (8)
Insulin is a major regulator of metabolism in metazoans, including the fruit fly Drosophila melanogaster. Genome-wide association studies (GWAS) suggest a genetic basis for reductions of both insulin sensitivity and insulin secretion, phenotypes commonly observed in humans with type 2 diabetes mellitus (T2DM). To identify molecular functions of genes linked to T2DM risk, we developed a genetic tool to measure insulin-like peptide 2 (Ilp2) levels in Drosophila, a model organism with superb experimental genetics. Our system permitted sensitive quantification of circulating Ilp2, including measures of Ilp2 dynamics during fasting and re-feeding, and demonstration of adaptive Ilp2 secretion in response to insulin receptor haploinsufficiency. Tissue specific dissection of this reduced insulin signaling phenotype revealed a critical role for insulin signaling in specific peripheral tissues. Knockdown of the Drosophila orthologues of human T2DM risk genes, including GLIS3 and BCL11A, revealed roles of these Drosophila genes in Ilp2 production or secretion. Discovery of Drosophila mechanisms and regulators controlling in vivo insulin dynamics should accelerate functional dissection of diabetes genetics.
View details for DOI 10.1371/journal.pgen.1004555
View details for PubMedID 25101872
Mouse model of Timothy syndrome recapitulates triad of autistic traits
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (37): 15432-15437
Autism and autism spectrum disorder (ASD) typically arise from a mixture of environmental influences and multiple genetic alterations. In some rare cases, such as Timothy syndrome (TS), a specific mutation in a single gene can be sufficient to generate autism or ASD in most patients, potentially offering insights into the etiology of autism in general. Both variants of TS (the milder TS1 and the more severe TS2) arise from missense mutations in alternatively spliced exons that cause the same G406R replacement in the Ca(V)1.2 L-type calcium channel. We generated a TS2-like mouse but found that heterozygous (and homozygous) animals were not viable. However, heterozygous TS2 mice that were allowed to keep an inverted neomycin cassette (TS2-neo) survived through adulthood. We attribute the survival to lowering of expression of the G406R L-type channel via transcriptional interference, blunting deleterious effects of mutant L-type channel overactivity, and addressed potential effects of altered gene dosage by studying Ca(V)1.2 knockout heterozygotes. Here we present a thorough behavioral phenotyping of the TS2-neo mouse, capitalizing on this unique opportunity to use the TS mutation to model ASD in mice. Along with normal general health, activity, and anxiety level, TS2-neo mice showed markedly restricted, repetitive, and perseverative behavior, altered social behavior, altered ultrasonic vocalization, and enhanced tone-cued and contextual memory following fear conditioning. Our results suggest that when TS mutant channels are expressed at levels low enough to avoid fatality, they are sufficient to cause multiple, distinct behavioral abnormalities, in line with the core aspects of ASD.
View details for DOI 10.1073/pnas.1112667108
View details for Web of Science ID 000294804900085
View details for PubMedID 21878566
A Novel Inducible Tyrosine Kinase Receptor To Regulate Signal Transduction and Neurite Outgrowth
JOURNAL OF NEUROSCIENCE RESEARCH
2009; 87 (12): 2624-2631
Nervous system growth factor gene delivery can promote axonal growth and prevent cell death in animal models of CNS trauma and neurodegenerative diseases. The ability to regulate growth factor expression or signaling pathways downstream from growth factor receptors remains a desirable goal for in vivo gene transfer. To achieve precise pharmacological modulation of neurotrophin activity, we have generated a chimeric trkA receptor (ItrkA) by fusing the entire intracellular domain of the trkA high-affinity NGF receptor to two intracellular, modified FK506 binding domains for the synthetic small molecule dimerization ligand AP20187. Rat PC12 cells were transduced with lentiviral vectors containing ItrkA and green fluorescent protein (GFP; via an internal ribosome entry site). Treatment of ItrkA-expressing PC12 cells with AP20187 induced neurite outgrowth and differentiation in a time- and dose-dependent fashion, with a half-maximal response at a concentration of 1 nM AP20187. Seventy percent of cells responded to AP20187 by day 3. Western blots demonstrated that AP20187 treatment resulted in phosphorylation of Erk1/2 and Akt in ItrkA-transduced PC12 cells but not in nontransduced, naïve cells. Phosphorylation levels were comparable to levels obtained with 50 ng/ml nerve growth factor (NGF). In addition, ItrkA lentiviral transduction of primary E15 dorsal root ganglion neurons significantly increased neurite growth three- to fourfold in the presence of AP20187 compared with control GFP transduced and naïve neurons. These results demonstrate that small ligand-induced dimerization of the intracellular domain of trkA can efficiently simulate the biological activity of NGF and provide a means to regulate intracellular neurotrophin receptor signaling.
View details for DOI 10.1002/jnr.22101
View details for Web of Science ID 000268699100002
View details for PubMedID 19405107
Murine and HIV-based retroviral vectors for in vitro and in vivo gene transfer.
Methods in molecular medicine
2006; 129: 241-254
The success of experimental gene therapy is dependent on the ability to safely and efficiently introduce transgenes into the target cell or tissue. Retroviral-based vectors, notably those derived from Moloney murine leukemia virus (MLV) and lentiviral vectors derived from HIV, have proven to be valuable gene transfer vehicles as a result of their ease of production and their ability to mediate long-term transgene expression. One of the most widely used methods for viral vector production is based on the transient transfection of viral vector plasmid DNA into a producer cell line. Here, we describe protocols to produce and standardize high quality MLV-based retroviral and HIV-based lentiviral vectors for ex vivo and in vivo gene delivery.
View details for PubMedID 17085815
Regulated lentiviral NGF gene transfer controls rescue of medial septal cholinergic neurons
2005; 11 (6): 916-925
Nerve growth factor (NGF) has been shown to promote survival and function of cholinergic neurons in the basal forebrain in various models of neuronal degeneration in rodents and primates. We examined whether a regulatable in vivo expression system can control the survival of cholinergic neurons after injury, using a tetracycline-regulated promoter ("tet-off" system) to modulate lentiviral NGF gene delivery. Two weeks after lesions to cholinergic neurons, significant cell rescue (65+/-8% neuron survival; P<0.005 compared to controls) was observed when NGF expression was activated. Treatment with the tetracycline analog doxycycline to turn gene expression "off" resulted in a significant loss of cholinergic neurons (only 37+/-5% neurons remained, an amount that did not differ from untreated, lesioned controls). Animals treated with a constitutively active and robust nonregulated NGF expression system showed the same degree of neuronal rescue (73+/-8%) as animals treated with activated tet-regulated vectors. ELISA measurements confirmed that oral treatment of animals with doxycycline reduced NGF protein levels to levels in untreated control subjects. These data demonstrate for the first time that NGF delivery by lentiviral gene transfer using tetracycline-regulated promoters can completely regulate neuronal rescue and protein production in the brain.
View details for DOI 10.1016/j.ymthe.2005.01.007
View details for Web of Science ID 000229732700015
View details for PubMedID 15922962