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Richard P. Woychik - One of the best experts on this subject based on the ideXlab platform.
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An Agouti mutation lacking the basic domain induces yellow pigmentation but not obesity in transgenic mice
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: R. J. Miltenberger, Randall L. Mynatt, William O. Wilkison, Richard P. Woychik, B. D. Bruce, E. J. MichaudAbstract:Chronic antagonism of melanocortin receptors by the paracrine-acting Agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type Agouti. Functional analysis of Agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for Agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the Agouti basic domain, the entire 29-aa region was deleted from the Agouti cDNA, and the resulting mutation (AgoutiΔbasic) was expressed in transgenic mice under the control of the β-actin promoter (BAPaΔbasic). Three independent lines of BAPaΔbasic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the AgoutiΔbasic protein was functional in vivo. However, none of the BAPaΔbasic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of AgoutiΔbasic expression in relevant tissues exceeded the level of Agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the AgoutiΔbasic protein, rather than insufficient RNA synthesis, accounts for the phenotype of the BAPaΔbasic transgenic mice. These findings implicate a functional role for the Agouti basic domain in vivo, possibly influencing the biogenesis of secreted Agouti protein or modulating protein–protein interactions that contribute to effective antagonism of melanocortin receptors.
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The Role of the Agouti Gene in the Yellow Obese Syndrome
Journal of Nutrition, 1997Co-Authors: R. J. Miltenberger, R. L. Mynatt, John E. Wilkinson, Richard P. WoychikAbstract:The yellow obese syndrome in mice encompasses many pleiotropic effects including yellow fur, maturity-onset obesity, hyperinsulinemia, insulin resistance, hyperglycemia, increased skeletal length and lean body mass, and increased susceptibility to neoplasia. The molecular basis of this syndrome is beginning to be unraveled and may have implications for human obesity and diabetes. Normally, the Agouti gene is expressed during the hair-growth cycle in the neonatal skin where it functions as a paracrine regulator of pigmentation. The secreted Agouti protein antagonizes the binding of the alpha-melanocyte-stimulating hormone to its receptor (melanocortin 1 receptor) on the surface of hair bulb melanocytes, causing alterations in intracellular cAMP levels. Widespread, ectopic expression of the mouse Agouti gene is central to the yellow obese phenotype, as demonstrated by the molecular cloning of several dominant Agouti mutations and the ubiquitous expression of the wild-type Agouti gene in transgenic mice. Recent experiments have revealed that the hypothalamus and adipose tissue are biologically active target sites for Agouti in the yellow obese mutant lines.
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Combined effects of insulin treatment and adipose tissue-specific Agouti expression on the development of obesity
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: R. L. Mynatt, R. J. Miltenberger, Mitchell L. Klebig, Michael B. Zemel, John E. Wilkinson, William O. Wilkison, Richard P. WoychikAbstract:The Agouti gene product is a secreted protein that acts in a paracrine manner to regulate coat color in mammals. Several dominant mutations at the Agouti locus in mice cause the ectopic, ubiquitous expression of Agouti, resulting in a condition similar to adult-onset obesity and non-insulin-dependent diabetes mellitus. The human Agouti protein is 85% homologous to mouse Agouti; however, unlike the mouse Agouti gene, human Agouti is normally expressed in adipose tissue. To address whether expression of Agouti in human adipose tissue is physiologically relevant, transgenic mice were generated that express Agouti in adipose tissue. Similar to most humans, these mice do not become obese or diabetic. However, we found that daily insulin injections significantly increased weight gain in the transgenic lines expressing Agouti in adipose tissue, but not in nontransgenic mice. These results suggest that insulin triggers the onset of obesity and that Agouti expression in adipose tissue potentiates this effect. Accordingly, the investigation of Agouti’s role in obesity and non-insulin-dependent diabetes mellitus in mice holds significant promise for understanding the pathophysiology of human obesity.
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Agouti regulation of intracellular calcium role in the insulin resistance of viable yellow mice
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Michael B. Zemel, Richard P. Woychik, I R Patel, Sue H Kadwell, Edward J Michaud, Jung Han Kim, William O. WilkisonAbstract:Abstract Several dominant mutations at the Agouti locus in the mouse cause a syndrome of marked obesity, hyperinsulinemia, and insulin resistance. Although it is known that the Agouti gene is expressed in an ectopic manner in these mutants, the precise mechanism by which the Agouti gene product mediates these effects is unclear. Since intracellular Ca2+ is believed to play a role in mediating insulin action and dysregulation of Ca2+ flux is observed in diabetic animals and humans, we examined the status of intracellular Ca2+ in mice carrying the dominant Agouti allele, viable yellow (Avy). We show here that in mice carrying this mutation, the intracellular free calcium concentration ([Ca2+]i) is elevated in skeletal muscle, and the degree of elevation is closely correlated with the degree to which the mutant traits are expressed in individual animals. Moreover, we demonstrate that the Agouti gene product is capable of inducing increased [Ca2+]i in cultured and freshly isolated skeletal muscle myocytes from wild-type mice. Based on these findings, we present a model in which we propose that the Agouti polypeptide promotes insulin resistance in mutant animals through its ability to increase [Ca2+]i.
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ectopic expression of the Agouti gene in transgenic mice causes obesity features of type ii diabetes and yellow fur
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Mitchell L. Klebig, John E. Wilkinson, J G Geisler, Richard P. WoychikAbstract:Abstract Mice that carry the lethal yellow (Ay) or viable yellow (Avy) mutation, two dominant mutations of the Agouti (a) gene in mouse chromosome 2, exhibit a phenotype that includes yellow fur, marked obesity, a form of type II diabetes associated with insulin resistance, and an increased susceptibility to tumor development. Molecular analyses of these and several other dominant "obese yellow" a-locus mutations suggested that ectopic expression of the normal Agouti protein gives rise to this complex pleiotropic phenotype. We have now tested this hypothesis directly by generating transgenic mice that ectopically express an Agouti cDNA clone encoding the normal Agouti protein in all tissues examined. Transgenic mice of both sexes have yellow fur, become obese, and develop hyperinsulinemia. In addition, male transgenic mice develop hyperglycemia by 12-20 weeks of age. These results demonstrate conclusively that the ectopic Agouti expression is responsible for most, if not all, of the phenotypic traits of the dominant, obese yellow mutants.
Gregory S. Barsh - One of the best experts on this subject based on the ideXlab platform.
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Molecular Pharmacology of Agouti Protein in Vitro and in Vivo
Annals of the New York Academy of Sciences, 2006Co-Authors: Gregory S. Barsh, Michael Martin Ollmann, Brent D Wilson, Kimberly A. Miller, Teresa M. GunnAbstract:Agouti protein and Agouti-related protein (Agrp) are paracrine signaling molecules that act by antagonizing the effects of melanocortins, and several alternatives have been proposed to explain their mechanisms of action. Genetic crosses in a sensitized background uncover a phenotypic difference between overexpression of Agouti and loss of Mc1r function, demonstrate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from alpha-MSH stimulation. In vitro, Agouti protein inhibits melanocortin action by two mechanisms: competitive antagonism that depends on the carboxyterminus of the protein, and downregulation of melanocortin receptor signaling that depends on the aminoterminus. Our findings provide evidence of a novel signaling mechanism whereby alpha-MSH and Agouti protein function as independent ligands that inhibit each other's binding and transduce opposite signals through a single receptor.
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Association of an Agouti allele with fawn or sable coat color in domestic dogs
Mammalian Genome, 2005Co-Authors: Tom G. Berryere, Gregory S. Barsh, Julie A. Kerns, Sheila M. SchmutzAbstract:The type of pigment synthesized in mammalian hair, yellow–red pheomelanin or black–brown eumelanin, depends on the interaction between Agouti protein and the Melanocortin 1 receptor. Although the genetics of pigmentation is broadly conserved across most mammalian species, pigment type-switching in domestic dogs is unusual because a yellow–tan coat with variable amounts of dark hair is thought to be caused by an allele of the Agouti locus referred to as fawn or sable ( a ^ y ). In a large survey covering thirty seven breeds, we identified an Agouti allele with two missense alterations, A82S and R83H, which was present (heterozygous or homozygous) in 41 dogs (22 breeds) with a fawn or sable coat, but was absent from 16 dogs (8 breeds) with a black-and-tan or tricolor phenotype. In an additional 33 dogs (14 breeds) with a eumelanic coat, 8 (German Shepherd Dogs, Groenendaels, Schipperkes, or Shetland Sheepdogs) were homozygous for a previously reported mutation, non-Agouti R96C; the remainder are likely to have carried dominant black, which is independent of and epistatic to Agouti . This work resolves some of the complexity in dog coat color genetics and provides diagnostic opportunities and practical guidelines for breeders.
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Induction of brain-region-specific forms of obesity by Agouti.
The Journal of Neuroscience, 2004Co-Authors: Martien J H Kas, Gregory S. Barsh, Birgitte Tiesjema, Gertjan Van Dijk, Keith M. Garner, Olivier Ter Brake, Joost Verhaagen, Roger A.h. AdanAbstract:Disruption of melanocortin (MC) signaling, such as by ectopic Agouti overexpression, leads to an obesity syndrome with hyperphagia, obesity, and accelerated body weight gain during high-fat diet. To investigate where in the brain disruption of MC signaling results in obesity, long-term Agouti expression was induced after local injections of recombinant adeno-associated viral particles in selected brain nuclei of adult rats. Agouti expression in the paraventricular nucleus, a hypothalamic region with a high density of MC receptors, induced acute onset hyperphagia and rapid weight gain that persisted for at least 6 weeks. In contrast, obesity and hyperphagia developed with a 3 week delay when Agouti was expressed in the dorsal medial hypothalamus. Agouti expression in the lateral hypothalamus (LH) did not affect food intake and body weight during regular diet, despite the presence of MC receptors in this region. However, during exposure to a high-fat diet, animals with Agouti expression in the LH exhibited a marked increase in body weight. Here we show that the LH is important for the protection against diet-induced obesity by controlling caloric intake during consumption of a high-fat diet. Together, this study provides evidence that different aspects of the Agouti-induced obesity syndrome, such as hyperphagia and diet responsiveness, are mediated by distinct brain regions and opens challenging opportunities for further understanding of pathophysiological processes in the development of the obesity syndrome.
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Genetic and biochemical studies of the Agouti-attractin system.
Journal of Receptors and Signal Transduction, 2002Co-Authors: Gregory S. Barsh, Teresa M. GunnAbstract:ABSTRACTPleiotropic effects of melanocortin signaling were first described nearly 100 years ago when mice carrying the lethal yellow (Ay) allele of the Agouti coat color gene were recognized to develop increased growth and adiposity. Work from our laboratory and others over the last several years has demonstrated that the non-pigmentary effects of A y are caused by ectopic expression of Agouti protein, a paracrine signaling molecule whose normal function is to inhibit signaling through the melanocortin 1 receptor (Mc1r), but which can mimic the effects of Agouti-related protein (Agrp), a homologous neuropeptide produced in the medial portion of the arcuate nucleus that acts as a potent antagonist of the Mc3r and Mc4r. Recently we have used the genetics of pigmentation as an in vivo screening system to analyze other mutations in the Agouti–melanocortin pathway, leading to the identification of Attractin (Atrn), a widely expressed type I transmembrane protein that serves as an accessory receptor for Agouti ...
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The melanocortin 1 receptor is the principal mediator of the effects of Agouti signaling protein on mammalian melanocytes.
Journal of Cell Science, 2001Co-Authors: Zalfa A Abdel-malek, M. Lynn Lamoreux, Michael Martin Ollmann, Gregory S. Barsh, M C Scott, Minao Furumura, Vincent J. HearingAbstract:The Agouti gene codes for Agouti signaling protein (ASP), which is temporally expressed in wild-type mouse follicular melanocytes where it induces pheomelanin synthesis. Studies using purified full-length Agouti signaling protein has shown that it competes with (α)-melanocyte stimulating hormone for binding to the melanocortin 1 receptor. We have investigated whether ASP binds exclusively to the melanocortin 1 receptor expressed on mouse melanocytes in primary culture, or additionally activates a receptor that has not been identified yet. We have compared the responses of congenic mouse melanocytes derived from C57 BL/6J-E(+)/E(+), e/e, or E(so)/E(so) mice to (alpha)-MSH and/or ASP. E(+)/E(+) melanocytes express the wild-type melanocortin 1 receptor, e/e melanocytes express a loss-of-function mutation in the melanocortin 1 receptor that results in a yellow coat color, and E(so)/E(so) is a mutation that causes constitutive activation of the melanocortin 1 receptor and renders melanocytes unresponsive to (alpha)-melanocyte stimulating hormone. Mouse E(+)/E(+) melanocytes, but not e/e or E(so)/E(so) melanocytes, respond to Agouti signaling protein with decreased basal tyrosinase activity, and reduction in levels of tyrosinase and tyrosinase-related proteins 1 and 2. Only in E(+)/E(+) melanocytes does Agouti signaling protein abrogate the stimulatory effects of (alpha)-melanocyte stimulating hormone on cAMP formation and tyrosinase activity. These results indicate that a functional melanocortin 1 receptor is obligatory for the response of mammalian melanocytes to Agouti signaling protein.
William O. Wilkison - One of the best experts on this subject based on the ideXlab platform.
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Interactions of alpha-melanotropin and Agouti on B16 melanoma cells: evidence for inverse agonism of Agouti.
Journal of Receptors and Signal Transduction, 2009Co-Authors: Walter Siegrist, William O. Wilkison, Roma Drozdz, Renato Cotti, Derril H. Willard, Alex N. EberleAbstract:Abstractα-Melanocyte-stimulating hormone (α-MSH, α-melanotropin) and Agouti control the switch between eumelanin and pheomelanin synthesis in mammalian melanocytes. Here we investigated interactions between α-MSH, Agouti protein, cAMP elevating agents and phorbol ester on mouse B16 melanoma cells. Agouti (Kd 3.7nmol/l) and α-MSH (Kd 2.3 nmol/l) had similar affinities to the MC1 melanocortin receptor. Both α-MSH and Agouti induced MC1 receptor down-regulation. Agouti antagonized melanogenesis induced by α-MSH, forskolin, cholera toxin (CT), and pertussis toxin (PT). It also reduced the constitutive melanin formation of long-term cultures. Cell proliferation was inhibited by Agouti (43% at 100 nM). This effect was reversed by α-MSH, forskolin, or CT. B16-G4F cells, a cell variant that lacks the MC1 receptor, did not respond to Agouti. From these results we conclude that Agouti shows the characteristics of an inverse agonist acting through the MC1 receptor.
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Regulation of the Melanocortin Receptors by Agouti
The Melanocortin Receptors, 2000Co-Authors: William O. WilkisonAbstract:The melanocortin family of receptors has been implicated in the regulation of a number of physiologic systems. Despite the cloning and characterization of these receptors, little is known about their regulation. I will summarize in this chapter what is known about a novel regulator of melanocortin receptor activity, the Agouti gene product. Not only does the action of Agouti on these receptors explain or clarify the physiologic role of some of these receptors, Agouti function and regulation also imparts new possibilities for these receptors having a role in processes such as energy homeostasis.
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An Agouti mutation lacking the basic domain induces yellow pigmentation but not obesity in transgenic mice
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: R. J. Miltenberger, Randall L. Mynatt, William O. Wilkison, Richard P. Woychik, B. D. Bruce, E. J. MichaudAbstract:Chronic antagonism of melanocortin receptors by the paracrine-acting Agouti gene product induces both yellow fur and a maturity-onset obesity syndrome in mice that ubiquitously express wild-type Agouti. Functional analysis of Agouti mutations in transgenic mice indicate that the cysteine-rich C terminus, signal peptide, and glycosylation site are required for Agouti activity in vivo. In contrast, no biological activity has been ascribed to the conserved basic domain. To examine the functional significance of the Agouti basic domain, the entire 29-aa region was deleted from the Agouti cDNA, and the resulting mutation (AgoutiΔbasic) was expressed in transgenic mice under the control of the β-actin promoter (BAPaΔbasic). Three independent lines of BAPaΔbasic transgenic mice all developed some degree of yellow pigment in the fur, indicating that the AgoutiΔbasic protein was functional in vivo. However, none of the BAPaΔbasic transgenic mice developed completely yellow fur, obesity, hyperinsulinemia, or hyperglycemia. High levels of AgoutiΔbasic expression in relevant tissues exceeded the level of Agouti expression in obese viable yellow mice, suggesting that suboptimal activity or synthesis of the AgoutiΔbasic protein, rather than insufficient RNA synthesis, accounts for the phenotype of the BAPaΔbasic transgenic mice. These findings implicate a functional role for the Agouti basic domain in vivo, possibly influencing the biogenesis of secreted Agouti protein or modulating protein–protein interactions that contribute to effective antagonism of melanocortin receptors.
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THE Agouti GENE PRODUCT INHIBITS LIPOLYSIS IN HUMAN ADIPOCYTES VIA A CA2+-DEPENDENT MECHANISM
The FASEB Journal, 1998Co-Authors: Bingzhong Xue, William O. Wilkison, Naima Moustaid-moussa, Michael B. ZemelAbstract:Overexpression of the murine Agouti gene results in obesity. The human homologue of Agouti is expressed primarily in human adipocytes, and we have shown recombinant Agouti protein to increase adipo...
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Melanocortin Receptor Binding Determinants in the Agouti Protein
Biochemistry, 1998Co-Authors: Laura L. Kiefer, James M. Veal, Kathleen G. Mountjoy, William O. WilkisonAbstract:The Agouti protein plays an important role in the development of diabetes and obesity in rodents and has been shown to be a potent antagonist of melanocortin receptors. For this reason alanine-scanning mutagenesis was performed on the Agouti protein carboxyl terminus to locate residues important for melanocortin receptor binding inhibition. When Agouti residues Arg116 and Phe118 are changed to alanine, very large decreases in Agouti affinity for melanocortin receptor 1, 3, and 4 result. Mutation of Phe117 to alanine causes a similar increase in Agouti KI app at melanocortin receptor 4. Substitution of Agouti residue Asp108 with alanine results in large increases in KI app for all three melanocortin receptors examined. All of these residues are conserved in the Agouti-related transcript, ART, whose expression is up-regulated in animal models of obesity. The three-dimensional structure of the Agouti carboxyl terminus was modeled, and residues which decrease receptor binding by a factor of ≥15 when mutated t...
Edward J Michaud - One of the best experts on this subject based on the ideXlab platform.
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Agouti regulation of intracellular calcium role in the insulin resistance of viable yellow mice
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Michael B. Zemel, Richard P. Woychik, I R Patel, Sue H Kadwell, Edward J Michaud, Jung Han Kim, William O. WilkisonAbstract:Abstract Several dominant mutations at the Agouti locus in the mouse cause a syndrome of marked obesity, hyperinsulinemia, and insulin resistance. Although it is known that the Agouti gene is expressed in an ectopic manner in these mutants, the precise mechanism by which the Agouti gene product mediates these effects is unclear. Since intracellular Ca2+ is believed to play a role in mediating insulin action and dysregulation of Ca2+ flux is observed in diabetic animals and humans, we examined the status of intracellular Ca2+ in mice carrying the dominant Agouti allele, viable yellow (Avy). We show here that in mice carrying this mutation, the intracellular free calcium concentration ([Ca2+]i) is elevated in skeletal muscle, and the degree of elevation is closely correlated with the degree to which the mutant traits are expressed in individual animals. Moreover, we demonstrate that the Agouti gene product is capable of inducing increased [Ca2+]i in cultured and freshly isolated skeletal muscle myocytes from wild-type mice. Based on these findings, we present a model in which we propose that the Agouti polypeptide promotes insulin resistance in mutant animals through its ability to increase [Ca2+]i.
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differential expression of a new dominant Agouti allele aiapy is correlated with methylation state and is influenced by parental lineage
Genes & Development, 1994Co-Authors: Edward J Michaud, Scott J. Bultman, M J Van Vugt, H O Sweet, M T Davisson, Richard P. WoychikAbstract:The Agouti gene normally confers the wild-type coat color of mice. Dominant mutations at the Agouti locus result in a pleiotropic syndrome that is characterized by excessive amounts of yellow pigment in the coat, obesity, a non-insulin-dependent diabetic-like condition, and the propensity to form a variety of tumors. Here, we describe a new dominant mutation at the Agouti locus in which an intracisternal A-particle (IAP) has integrated in an antisense orientation immediately 5' of the first coding exon of the gene. This mutation, which we have named Aiapy, results in the ectopic expression of the Agouti gene through the utilization of a cryptic promoter within the IAP 5' long terminal repeat (LTR). The coat color of Aiapy/-mice ranges from solid yellow to a pigment pattern that is similar to wild type (pseudoAgouti), and the expressivity of this mutant phenotype varies with parental inheritance. Those offspring with a yellow coat ectopically express Agouti mRNA at high levels and exhibit marked obesity, whereas pseudoAgouti mice express Agouti mRNA at a very low level and their weights do not differ from wild-type littermates. Data are presented to show that the differential expressivity of the Aiapy allele is correlated with the methylation status of the inserted IAP 5' LTR. These data further support the hypothesis that in dominant yellow mutations at the Agouti locus, it is the ubiquitous expression of the wild-type Agouti coding sequence that is responsible for the yellow coat color, obesity, diabetes, and tumorigenesis.
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a molecular model for the genetic and phenotypic characteristics of the mouse lethal yellow ay mutation
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Edward J Michaud, Liane B Russell, Mitchell L. Klebig, Scott J. Bultman, M J Van Vugt, Lisa Stubbs, Richard P. WoychikAbstract:Abstract Lethal yellow (Ay) is a mutation at the mouse Agouti locus in chromosome 2 that causes a number of dominant pleiotropic effects, including a completely yellow coat color, obesity, an insulin-resistant type II diabetic condition, and an increased propensity to develop a variety of spontaneous and induced tumors. Additionally, homozygosity for Ay results in preimplantation lethality, which terminates development by the blastocyst stage. The Ay mutation is the result of a 170-kb deletion that removes all but the promoter and noncoding first exon of another gene called Raly, which lies in the same transcriptional orientation as Agouti and maps 280 kb proximal to the 3' end of the Agouti gene. We present a model for the structure of the Ay allele that can explain the dominant pleiotropic effects associated with this mutation, as well as the recessive lethality, which is unrelated to the Agouti gene.
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Molecular analysis of reverse mutations from nonAgouti (a) to black-and-tan (a(t)) and white-bellied Agouti (Aw) reveals alternative forms of Agouti transcripts
Genes & Development, 1994Co-Authors: Scott J. Bultman, Mitchell L. Klebig, Edward J Michaud, Hope O. Sweet, Muriel T. Davisson, Richard P. WoychikAbstract:The Agouti gene regulates the differential production of eumelanin (black or brown) and phaeomelanin (yellow) pigment granules by melanocytes in the hair follicles of mice. The original nonAgouti [a] allele, which confers a predominantly black coat color, has been shown to revert to two other more dominant Agouti alleles, black-and-tan {a*) and white-bellied Agouti (A**^, with an exceptionally high frequency. The a' and A^ alleles confer phenotypes in which the pigmentation is not uniformly distributed over the dorsal and ventral surfaces of the animal; in both cases the ventral surface of the animal is markedly lighter than the dorsal surface due to an increase in phaeomelanin production. To understand the unusually high reversion rate of a to a' or A^, and to decipher the molecular events associated with the different pigmentation patterns associated with these three Agouti alleles, we have characterized a, a* and A^ at the molecular level. Here, we report that insertions of 11, 6, and 0.6 kb are present at precisely the same position in the first intron of the Agouti gene in a, fl', and A^, respectively. The a insertion consists of a 5.5-kb VL30 element that has incorporated 5.5 kb of additional sequence internally; this internal sequence is flanked by 526-bp direct repeats. The a* allele contains only the VL30 element and a single, internal 526-bp repeat. The A^ allele has only a solo VL30 LTR. Based on the comparison of the structure of the a* and A^ insertions, we propose that reverse mutations occur by excision of inserted sequences in a through homologous recombination, utilizing either the 526-bp direct repeats to generate a* or the VL30 LTRs to generate A^. Moreover, the analysis of these three alleles has allowed us to identify additional exons of the Agouti gene that give rise to alternatively processed forms of Agouti mRNA. We demonstrate that the distinct insertions in a, a* and A^ cause pigmentation differences by selectively inactivating the expression of different forms of Agouti transcripts.
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Molecular characterization of the mouse Agouti locus
Cell, 1992Co-Authors: Scott J. Bultman, Edward J Michaud, Richard P. WoychikAbstract:Summary The Agouti ( a ) locus acts within the microenvironment of the hair follicle to regulate coat color pigmentation in the mouse. We have characterized a gene encoding a novel 131 amino acid protein that we propose is the one gene associated with the Agouti locus. This gene is normally expressed in a manner consistent with a locus function, and, more importantly, its structure and expression are affected by a number of representative alleles in the Agouti dominance hierarchy. In addition, we found that the pleiotropic effects associated with the lethal yellow ( A y ) mutation, which include pronounced obesity, diabetes, and the development of neoplasms, are accompanied by deregulated overexpression of the Agouti gene in numerous tissues of the ault animal.
Mitchell L. Klebig - One of the best experts on this subject based on the ideXlab platform.
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Combined effects of insulin treatment and adipose tissue-specific Agouti expression on the development of obesity
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: R. L. Mynatt, R. J. Miltenberger, Mitchell L. Klebig, Michael B. Zemel, John E. Wilkinson, William O. Wilkison, Richard P. WoychikAbstract:The Agouti gene product is a secreted protein that acts in a paracrine manner to regulate coat color in mammals. Several dominant mutations at the Agouti locus in mice cause the ectopic, ubiquitous expression of Agouti, resulting in a condition similar to adult-onset obesity and non-insulin-dependent diabetes mellitus. The human Agouti protein is 85% homologous to mouse Agouti; however, unlike the mouse Agouti gene, human Agouti is normally expressed in adipose tissue. To address whether expression of Agouti in human adipose tissue is physiologically relevant, transgenic mice were generated that express Agouti in adipose tissue. Similar to most humans, these mice do not become obese or diabetic. However, we found that daily insulin injections significantly increased weight gain in the transgenic lines expressing Agouti in adipose tissue, but not in nontransgenic mice. These results suggest that insulin triggers the onset of obesity and that Agouti expression in adipose tissue potentiates this effect. Accordingly, the investigation of Agouti’s role in obesity and non-insulin-dependent diabetes mellitus in mice holds significant promise for understanding the pathophysiology of human obesity.
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Analysis of the function of the Agouti gene in obesity and diabetes
1996Co-Authors: Randall L. Mynatt, R. J. Miltenberger, Mitchell L. KlebigAbstract:This chapter discusses the Agouti gene and dominant mutations in that gene that lead to Agouti-induced obesity, and recent work with transgenic mice to elucidate the role of Agouti in obesity. Agouti was cloned in 1992 by the lab of Rick Woychik at Oak Ridge National Laboratory, making it the first of many recently cloned mouse obesity genes. Sequence analysis predicted that mouse Agouti is a secreted protein of 131 amino acids. The mature protein has a basic central region (lys57-arg85), a proline-rich domain (pro86-pro91) and a C-terminal region (cys 92-cys 13 1) containing 10 cysteine residues which form 5 disulfide bonds. The human homologue of Agouti has also been cloned by the Woychik lab and maps to human chromosome 20q 11.2. Human Agouti is 132 amino acids long and is 85% similar to the mouse Agouti protein and is normally expressed in adipose tissue. The researchers have been able to recapitulate obesity, hyperinsulinemia, and hyperglycemia with the ubiquitous expression of Agouti. Agouti expression in either liver and adipose tissue alone does not cause obesity, and there`s a dose-dependent effect of Agouti on body weight, food efficiency, body temperature, and insulin and glucose levels.
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ectopic expression of the Agouti gene in transgenic mice causes obesity features of type ii diabetes and yellow fur
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Mitchell L. Klebig, John E. Wilkinson, J G Geisler, Richard P. WoychikAbstract:Abstract Mice that carry the lethal yellow (Ay) or viable yellow (Avy) mutation, two dominant mutations of the Agouti (a) gene in mouse chromosome 2, exhibit a phenotype that includes yellow fur, marked obesity, a form of type II diabetes associated with insulin resistance, and an increased susceptibility to tumor development. Molecular analyses of these and several other dominant "obese yellow" a-locus mutations suggested that ectopic expression of the normal Agouti protein gives rise to this complex pleiotropic phenotype. We have now tested this hypothesis directly by generating transgenic mice that ectopically express an Agouti cDNA clone encoding the normal Agouti protein in all tissues examined. Transgenic mice of both sexes have yellow fur, become obese, and develop hyperinsulinemia. In addition, male transgenic mice develop hyperglycemia by 12-20 weeks of age. These results demonstrate conclusively that the ectopic Agouti expression is responsible for most, if not all, of the phenotypic traits of the dominant, obese yellow mutants.
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a molecular model for the genetic and phenotypic characteristics of the mouse lethal yellow ay mutation
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Edward J Michaud, Liane B Russell, Mitchell L. Klebig, Scott J. Bultman, M J Van Vugt, Lisa Stubbs, Richard P. WoychikAbstract:Abstract Lethal yellow (Ay) is a mutation at the mouse Agouti locus in chromosome 2 that causes a number of dominant pleiotropic effects, including a completely yellow coat color, obesity, an insulin-resistant type II diabetic condition, and an increased propensity to develop a variety of spontaneous and induced tumors. Additionally, homozygosity for Ay results in preimplantation lethality, which terminates development by the blastocyst stage. The Ay mutation is the result of a 170-kb deletion that removes all but the promoter and noncoding first exon of another gene called Raly, which lies in the same transcriptional orientation as Agouti and maps 280 kb proximal to the 3' end of the Agouti gene. We present a model for the structure of the Ay allele that can explain the dominant pleiotropic effects associated with this mutation, as well as the recessive lethality, which is unrelated to the Agouti gene.
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Molecular analysis of reverse mutations from nonAgouti (a) to black-and-tan (a(t)) and white-bellied Agouti (Aw) reveals alternative forms of Agouti transcripts
Genes & Development, 1994Co-Authors: Scott J. Bultman, Mitchell L. Klebig, Edward J Michaud, Hope O. Sweet, Muriel T. Davisson, Richard P. WoychikAbstract:The Agouti gene regulates the differential production of eumelanin (black or brown) and phaeomelanin (yellow) pigment granules by melanocytes in the hair follicles of mice. The original nonAgouti [a] allele, which confers a predominantly black coat color, has been shown to revert to two other more dominant Agouti alleles, black-and-tan {a*) and white-bellied Agouti (A**^, with an exceptionally high frequency. The a' and A^ alleles confer phenotypes in which the pigmentation is not uniformly distributed over the dorsal and ventral surfaces of the animal; in both cases the ventral surface of the animal is markedly lighter than the dorsal surface due to an increase in phaeomelanin production. To understand the unusually high reversion rate of a to a' or A^, and to decipher the molecular events associated with the different pigmentation patterns associated with these three Agouti alleles, we have characterized a, a* and A^ at the molecular level. Here, we report that insertions of 11, 6, and 0.6 kb are present at precisely the same position in the first intron of the Agouti gene in a, fl', and A^, respectively. The a insertion consists of a 5.5-kb VL30 element that has incorporated 5.5 kb of additional sequence internally; this internal sequence is flanked by 526-bp direct repeats. The a* allele contains only the VL30 element and a single, internal 526-bp repeat. The A^ allele has only a solo VL30 LTR. Based on the comparison of the structure of the a* and A^ insertions, we propose that reverse mutations occur by excision of inserted sequences in a through homologous recombination, utilizing either the 526-bp direct repeats to generate a* or the VL30 LTRs to generate A^. Moreover, the analysis of these three alleles has allowed us to identify additional exons of the Agouti gene that give rise to alternatively processed forms of Agouti mRNA. We demonstrate that the distinct insertions in a, a* and A^ cause pigmentation differences by selectively inactivating the expression of different forms of Agouti transcripts.
