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Agouti is a gene responsible for the distribution of melanin pigment in mammals^[1]. The wild type agouti allele (A) presents a grey phenotype, however, many allele variants have been identified through genetic analyses, which result in a wide range of phenotypes distinct from the typical grey coat^[2]. The most widely studied allele variants are the lethal yellow mutation (A^y) and the viable yellow mutation (A^vy) which are caused by ectopic expression of agouti^[2]. These mutations are synonymous with the yellow obese syndrome which is characterized by early onset obesity, hyperinsulinemia and tumorigenesis^[2]^[3].

Pigment development

The murine agouti gene locus is found on chromosome 2 and encodes a 131 amino acid protein. This protein signals the distribution of melanin pigments in epithelial melanocytes located at the base of hair follicles^[4]. Expression is more sensitive on ventral hair than dorsal hair^[5]. Agouti is not directly secreted in the melanocyte as it works as a paracrine factor on dermal papillae cells to inhibit release of melanocortin^[6]. Melanocortin acts on follicular melanocytes to increase production of eumelanin, a melanin pigment responsible for brown and black hair. When agouti is expressed, production of phaeomelanin dominates, a melanin pigment that produces yellow or red colored hair^[7]. The dominance hierarchy of pigment expression explains the evolutionary persistence of the yellow phenotype of agouti, as pheomelanin expression always dominates over eumelanin expression^[4].

Mutations

The lethal yellow mutation (A^y) was the first embryonic mutation to be characterized in mice, as homozygous lethal yellow mice (A^y/ A^y) die early in development, due to an error in trophectoderm differentiation^[4]. Lethal yellow homozygotes are rare today, as lethal yellow and viable yellow heterozygotes (A^y/a and A^vy/a) persist more commonly. These phenotypes are caused by ectopic expression of the agouti gene and are associated with the yellow obese syndrome, characterized by early onset obesity, hyperinsulinemia and tumorigenesis^[4].

The lethal yellow (A^y) mutation is due to an upstream deletion at the start site of agouti transcription. This deletion causes the genomic sequence of agouti to be lost, except the promoter and the first non-encoding exon of Raly, a ubiquitously expressed gene in mammals^[5] The coding exons of agouti are placed under the control of the Raly promoter, initiating ubiquitous expression of agouti, increasing production of pheomelanin over eumelanin and resulting in the development of a yellow phenotype^[8].

The viable yellow (A^vy) mutation is due to a change in the mRNA length of agouti, as the expressed gene becomes longer than the normal gene length of agouti. This is caused by the insertion of a single intracisternal A particle (IAP) retrotransposon upstream to the start site of agouti transcription^[9]. In the proximal end of the gene, an unknown promoter then causes agouti to be constitutionally activated, and individuals to present with phenotypes consistent with the lethal yellow mutation. Although the mechanism for the activation of the promotor controlling the viable yellow mutation is unknown, the strength of coat color has been correlated with the degree of gene methylation, which is determined by maternal diet and environmental exposure^[9]. As agouti itself inhibits melanocortin receptors responsible for eumelanin production, the yellow phenotype is exacerbated in both lethal yellow and viable yellow mutations as agouti gene expression is increased.

Viable yellow (A^vy/a) and lethal yellow (A^y/a) heterozygotes have shortened life spans and increased risks for developing early onset obesity, type II diabetes mellitus and various tumors^[6]^[10]. The increased risk of developing obesity is due to the dysregulation of appetite, as agouti agonizes the agouti-related protein (AGRP), responsible for the stimulation of appetite via hypothalamic NPY/AGRP orexigenic neurons^[9]. Agouti also promotes obesity by antagonizing melanocyte-stimulating hormone (MSH) at the melanocortin receptor (MC4R), as MC4R is responsible for regulating food intake by inhibiting appetite signals^[11]. The increase in appetite is coupled to alterations in nutrient metabolism due to the paracrine actions of agouti on adipose tissue, increasing levels of hepatic lipogenesis, decreasing levels of lipolysis and increasing adipocyte hypertrophy^[12]. This increases body mass and leads to difficulties with weight loss as metabolic pathways become dysregulated. Hyperinsulinemia is caused by mutations to agouti, as the agouti protein functions in a calcium dependent manner to increase insulin secretion in pancreatic beta cells, increasing risks of insulin resistance^[13]. Increased tumor formation is due to the increased mitotic rates of agouti, which are localized to epithelial and mesenchymal tissues^[8].

Methylation and diet intervention

Correct functioning of agouti requires DNA methylation. Methylation occurs in six guanine-cytosine (GC) rich sequences in the 5’ long terminal repeat of the IAP element in the viable yellow mutation^[10]. When this area is unmethylated, ectopic expression of agouti occurs, and yellow phenotypes present. When the region is methylated, agouti is expressed normally, and grey phenotypes develop. The epigenetic state of the IAP element is determined by the level of methylation, as individuals show a wide range of phenotypes based on their degree of DNA methylation^[10]. Increased methylation is correlated with increased expression of the normal agouti gene. Low levels of methylation can induce gene imprinting which results in offspring displaying consistent phenotypes to their parents, as ectopic expression of agouti is inherited through non-genomic mechanisms^[9]^[14].

DNA methylation is determined in utero by maternal nutrition and environmental exposure^[10]. Methyl is synthesized de novo but attained through the diet by folic acid, methionine, betaine and choline, as these nutrients feed into a consistent metabolic pathway for methyl synthesis^[15]. Adequate zinc and vitamin B12 are required for methyl synthesis as they act as cofactors for transferring methyl groups^[16].

When inadequate methyl is available during early embryonic development, DNA methylation cannot occur, which increases ectopic expression of agouti and results in the presentation of the lethal yellow and viable yellow phenotypes which persist into adulthood. This leads to the development of the yellow obese syndrome, which impairs normal development and increases susceptibility to the development of chronic disease. Ensuring maternal diets are high in methyl equivalents is a key preventative measure for reducing ectopic expression of agouti in offspring. Diet intervention through methyl supplementation reduces imprinting at the agouti locus, as increased methyl consumption causes the IAP element to become completely methylated and ectopic expression of agouti to be reduced^[17]. This lowers the proportion of offspring that present with the yellow phenotype and increases the number offspring that resemble agouti wild type mice with grey coats^[9].

The human homologue of agouti, the agouti signaling protein (ASP)

Agouti signaling protein (ASP) is the human homologue of murine agouti. It is encoded by the human agouti gene on chromosome 20 and is a 132 amino acid protein. It is expressed more broadly than murine agouti, as it is found in adipose tissue, pancreas, testes and ovaries^[16]. ASP has 85% similarity to the murine form of agouti^[18]. As ectopic expression of murine agouti leads to the development of the yellow obese syndrome, this is expected to be consistent in humans^[18]. The yellow obese syndrome increases the development of many chronic diseases, including obesity, type II diabetes mellitus and tumorigenesis^[2].

ASP has similar pharmacological activation to murine agouti, as melanocortin receptors are inhibited through competitive antagonism^[19]. Inhibition of melanocortin by ASP can also be through non-competitive methods, broadening its range of effects^[8]. The function of ASP differs to murine agouti. ASP effects the quality of hair pigmentation whereas murine agouti controls the distribution of pigments that determine coat color^[9]. ASP has neuroendocrine functions consistent with murine agouti, as it agonizes AGRP via AGRP neurons in the hypothalamus and antagonizes MSH at MC4Rs which reduce satiety signals. As appetite signals increase and satiety signals decrease, body mass is likely to increase and individuals are more susceptible to becoming obese. The mechanism underlying hyperinsulinemia in humans is consistent with murine agouti, as insulin secretion is heightened through calcium sensitive signaling in pancreatic beta cells. The mechanism for ASP induced tumorigenesis remains unknown in humans.

^ Silvers, W. K.; Russell, E. S. (1955). "An experimental approach to action of genes at the agouti locus in the mouse". Journal of Experimental Zoology. 130: 199–220.
^ ^a ^b ^c ^d Bultman, S. J.; Michaud, E. J.; Woychik, R. P. (1992). "Molecular Characterization of the Mouse Agouti Locus". Cell. 71: 1195–1204.
^ Wolff, G. L.; Roberts, D. W.; Mountjoy, K. G. (1999). "Physiological consequences of ectopic agouti gene expression: the yellow obese mouse syndrome". Physiological genomics. 1 (3): 151–163.
^ ^a ^b ^c ^d Mayer, T. C.; Fishbane, J. L. (1972). "). Mesoderm-Ectoderm Interaction In the Production of the Agouti Pigmentation Pattern in Mice". Genetics. 71: 297–303.
^ ^a ^b Melmed, S. (Ed) (2010). The Pituitary (3rd ed.). Cambridge: MA: Academic Press.
^ ^a ^b Miltenberger, R. J.; Mynatt, R. L.; Wilkinson, J. E.; Woychik, R. P. (1997). "The role of the agouti gene in the Yellow Obese Syndrome". Journal of Nutrition. 127 (9): 1902–1907.
^ Lu, D.; Willard, D.; Patel, I. R.; Kadwell, S.; Overton, L.; Kost, T.; Luther, M.; Wenbiao, C.; Woychik, R. P.; Wilkison, W. O.; Cone, R. R. (1994). "Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor". Nature. 371: 799–802.
^ ^a ^b ^c Tollefsbol, T. (Ed.) (2012). Epigenetics in Human Disease (6 ed.). Cambridge: MA: Academic Press.
^ ^a ^b ^c ^d ^e ^f Dolinoy, D. C. (2008). "The agouti mouse model: an epigenetic biosensor for nutritional and environmental alterations on the fetal epigenome". Nutrition Reviews. 66 (1): 7–11.
^ ^a ^b ^c ^d Spiegelman, B. M.; Flier, J. S. (1996). "Adipogenesis and obesity: rounding up the big picture". Cell. 87: 377–389.
^ Adan, R. A. H.; Tiesjema, B.; Hillebrand, J. J. G.; la Fleur, S. E.; Kas, M. J. H.; De Krom, M. (2006). "The MC4 Receptor and Control of Appetite". British Journal of Pharmacology. 149 (7): 815–827.
^ Johnson, P. R.; Hirsch, J. (1972). "Cellularity of adipose depots in six strains of genetically obese mice". Obesity Research. 7 (5): 506–514.
^ Moussa, N. M.; Claycombe, K. J. (1999). "The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity". Obesity Research. 13: 2–11.
^ Constância, M.; Pickard, B.; Kelsey, G.; Reik, W. (1998). "Imprinting Mechanisms". Genome Research. 8: 881–900.
^ Cooney, C. A.; Dave, A. A.; Wolff, D. L. (2002). "Maternal Methyl Supplements In Mice Affect Epigenetic Variation and DNA Methylation of Offspring". Journal of Nutrition. 132 (8): 2398–2400.
^ ^a ^b Wilson, B. D.; Ollman, M. M.; Kang, L.; Stoffel, M.; Bell, G. I.; Barsh, G. S. (1995). "Structure and Function of ASP, the human homolog of the mouse agouti gene". Human Molecular Genetics. 4 (2): 223–230.
^ Lopez-Calderero, I.; Sanchez Chavez, E.; Garcia-Carbonero, R. (2010). "The insulin-like growth fator pathway as a target for cancer therapy". Clinical and Translational Oncology. 12: 326–338.
^ ^a ^b Kwon, H. Y.; Bultman, S. J.; Loffler, C.; Chen, W. J.; Furdon, P. J.; Powell, J. G.; Usala, A. L.; Wilkison, W.; Hansmann, I.; Woychik, R. P. (1994). "Molecular structure and chromosomal mapping of the human homolog of the agouti gene". Proceedings of the National Academy of Sciences of the United States. 91 (21): 9760–9764.
^ Takeuchi, S. (2015). Handbook of Hormones. Cambridge: MA: Academic Press. pp. 66–67.

[1] Silvers, W. K.; Russell, E. S. (1955). "An experimental approach to action of genes at the agouti locus in the mouse". Journal of Experimental Zoology. 130: 199–220.

[Bultman-2] Bultman, S. J.; Michaud, E. J.; Woychik, R. P. (1992). "Molecular Characterization of the Mouse Agouti Locus". Cell. 71: 1195–1204.

[Wolff-3] Wolff, G. L.; Roberts, D. W.; Mountjoy, K. G. (1999). "Physiological consequences of ectopic agouti gene expression: the yellow obese mouse syndrome". Physiological genomics. 1 (3): 151–163.

[Mayer-4] Mayer, T. C.; Fishbane, J. L. (1972). "). Mesoderm-Ectoderm Interaction In the Production of the Agouti Pigmentation Pattern in Mice". Genetics. 71: 297–303.

[Melmed-5] Melmed, S. (Ed) (2010). The Pituitary (3rd ed.). Cambridge: MA: Academic Press.

[Miltenberger-6] Miltenberger, R. J.; Mynatt, R. L.; Wilkinson, J. E.; Woychik, R. P. (1997). "The role of the agouti gene in the Yellow Obese Syndrome". Journal of Nutrition. 127 (9): 1902–1907.

[Lu-7] Lu, D.; Willard, D.; Patel, I. R.; Kadwell, S.; Overton, L.; Kost, T.; Luther, M.; Wenbiao, C.; Woychik, R. P.; Wilkison, W. O.; Cone, R. R. (1994). "Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor". Nature. 371: 799–802.

[Tollefsbol-8] Tollefsbol, T. (Ed.) (2012). Epigenetics in Human Disease (6 ed.). Cambridge: MA: Academic Press.

[Dolinoy-9] ^ ^a ^b ^c ^d ^e ^f Dolinoy, D. C. (2008). "The agouti mouse model: an epigenetic biosensor for nutritional and environmental alterations on the fetal epigenome". Nutrition Reviews. 66 (1): 7–11.

[Spiegelman-10] Spiegelman, B. M.; Flier, J. S. (1996). "Adipogenesis and obesity: rounding up the big picture". Cell. 87: 377–389.

[Adan-11] Adan, R. A. H.; Tiesjema, B.; Hillebrand, J. J. G.; la Fleur, S. E.; Kas, M. J. H.; De Krom, M. (2006). "The MC4 Receptor and Control of Appetite". British Journal of Pharmacology. 149 (7): 815–827.

[Johnson-12] Johnson, P. R.; Hirsch, J. (1972). "Cellularity of adipose depots in six strains of genetically obese mice". Obesity Research. 7 (5): 506–514.

[Moussa-13] Moussa, N. M.; Claycombe, K. J. (1999). "The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity". Obesity Research. 13: 2–11.

[Constância-14] Constância, M.; Pickard, B.; Kelsey, G.; Reik, W. (1998). "Imprinting Mechanisms". Genome Research. 8: 881–900.

[Cooney-15] Cooney, C. A.; Dave, A. A.; Wolff, D. L. (2002). "Maternal Methyl Supplements In Mice Affect Epigenetic Variation and DNA Methylation of Offspring". Journal of Nutrition. 132 (8): 2398–2400.

[Wilson-16] Wilson, B. D.; Ollman, M. M.; Kang, L.; Stoffel, M.; Bell, G. I.; Barsh, G. S. (1995). "Structure and Function of ASP, the human homolog of the mouse agouti gene". Human Molecular Genetics. 4 (2): 223–230.

[Lopez-Calderero-17] Lopez-Calderero, I.; Sanchez Chavez, E.; Garcia-Carbonero, R. (2010). "The insulin-like growth fator pathway as a target for cancer therapy". Clinical and Translational Oncology. 12: 326–338.

[Kwon-18] Kwon, H. Y.; Bultman, S. J.; Loffler, C.; Chen, W. J.; Furdon, P. J.; Powell, J. G.; Usala, A. L.; Wilkison, W.; Hansmann, I.; Woychik, R. P. (1994). "Molecular structure and chromosomal mapping of the human homolog of the agouti gene". Proceedings of the National Academy of Sciences of the United States. 91 (21): 9760–9764.

[19] Takeuchi, S. (2015). Handbook of Hormones. Cambridge: MA: Academic Press. pp. 66–67.

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@@ Line 1: / Line 1: @@
-''Agouti'' is a [[gene]] responsible for the distribution of [[melanin]] pigment in mammals<ref>{{cite journal |last1=Silvers |first1=W. K. |last2=Russell |first2=E. S. |title=An experimental approach to action of genes at the ''agouti'' locus in the mouse |journal=Journal of Experimental Zoology |date=1955 |volume=130 |pages=199-220}}</ref>. The wild type ''agouti'' allele (A) presents a grey phenotype, however, many allele variants have been identified through genetic analyses, which result in a wide range of phenotypes distinct from the typical grey coat<ref name="Bultman">{{cite journal |last1=Bultman |first1=S. J. |last2=Michaud |first2=E. J. |last3=Woychik |first3=R. P. |title=Molecular Characterization of the Mouse Agouti Locus. |journal=Cell |date=1992 |volume=71 |pages=1195-1204}}</ref>. The most widely studied allele variants are the ''lethal yellow'' mutation (A<sup>y</sup>) and ''viable yellow'' mutation (A<sup>vy</sup>), caused by ectopic expression of ''agouti''<ref name="Bultman"/>. These are synonymous with the ''yellow obese syndrome'' which is characterized by early onset [[obesity]], [[hyperinsulinemia]] and [[tumorigenesis]]<ref name="Bultman"/><ref name="Wolff">{{cite journal |last1=Wolff |first1=G. L. |last2=Roberts |first2=D. W. |last3=Mountjoy |first3=K. G. |title=Physiological consequences of ectopic agouti [[gene expression]]: the ''yellow obese mouse syndrome''. |journal=Physiological genomics |date=1999 |volume=1 |issue=3 |pages=151-163}}</ref>.
+''Agouti'' is a [[gene]] responsible for the distribution of [[melanin]] pigment in mammals<ref>{{cite journal |last1=Silvers |first1=W. K. |last2=Russell |first2=E. S. |title=An experimental approach to action of genes at the ''agouti'' locus in the mouse |journal=Journal of Experimental Zoology |date=1955 |volume=130 |pages=199-220}}</ref>. The wild type ''agouti'' allele (A) presents a grey phenotype, however, many allele variants have been identified through genetic analyses, which result in a wide range of phenotypes distinct from the typical grey coat<ref name="Bultman">{{cite journal |last1=Bultman |first1=S. J. |last2=Michaud |first2=E. J. |last3=Woychik |first3=R. P. |title=Molecular Characterization of the Mouse Agouti Locus. |journal=Cell |date=1992 |volume=71 |pages=1195-1204}}</ref>. The most widely studied allele variants are the ''lethal yellow'' mutation (A<sup>y</sup>) and the ''viable yellow'' mutation (A<sup>vy</sup>) which are caused by ectopic expression of ''agouti''<ref name="Bultman"/>. These mutations are synonymous with the ''yellow obese syndrome'' which is characterized by early onset [[obesity]], [[hyperinsulinemia]] and [[tumorigenesis]]<ref name="Bultman"/><ref name="Wolff">{{cite journal |last1=Wolff |first1=G. L. |last2=Roberts |first2=D. W. |last3=Mountjoy |first3=K. G. |title=Physiological consequences of ectopic agouti gene expression: the ''yellow obese mouse syndrome''. |journal=Physiological genomics |date=1999 |volume=1 |issue=3 |pages=151-163}}</ref>.
-[[File:Proposed mechanism for the relationship between ectopic agouti expression and the development of ‘yellow obese mouse syndrome’.jpg|thumb|'''Proposed mechanism for the relationship between ectopic ''agouti'' expression and the development of ''yellow obese mouse syndrome''''']]
+[[File:Proposed mechanism for the relationship between ectopic agouti expression and the development of ‘yellow obese mouse syndrome’.jpg|thumb|'''Proposed mechanism for the relationship between ectopic ''agouti'' expression and the development of ''yellow obese syndrome''''']]
 ===Pigment development===
-The murine ''agouti'' gene locus is found on chromosome 2 and encodes a 131 amino acid protein. This protein signals the distribution of [[melanin]] pigments in the epithelial melanocytes located at the base of hair follicles<ref name="Mayer">{{cite journal |last1=Mayer |first1=T. C. |last2=Fishbane |first2=J. L. |title=). Mesoderm-Ectoderm Interaction In the Production of the Agouti Pigmentation Pattern in Mice |journal=Genetics |date=1972 |volume=71 |pages=297-303}}</ref>. The type of pigment secreted is determined by mesoderm and ectoderm genotypes at the agouti locus. Expression is more sensitive on ventral hair than dorsal hair<ref name="Melmed">{{cite book |last1=Melmed |first1=S. (Ed) |title=The Pituitary |date=2010 |publisher=Academic Press |location=Cambridge: MA |edition=3rd}}</ref>. The mechanism of ''agouti'' expression differs to most genes that affect coat color, as the protein is not directly secreted in the melanocyte, instead, it is secreted as a [[paracrine]] factor from the dermal papillae cells to inhibit release of [[melanocortin]]<ref name="Miltenberger">{{cite journal |last1=Miltenberger |first1=R. J. |last2=Mynatt |first2=R. L. |last3=Wilkinson |first3=J. E. |last4=Woychik |first4=R. P. |title=The role of the agouti gene in the Yellow Obese Syndrome. |journal=Journal of Nutrition |date=1997 |volume=127 |issue=9 |pages=1902-1907}}</ref>. Melanocortin acts on follicular melanocytes to increase production of [[eumelanin]], a melanin pigment responsible for brown and black hair. However, when ''agouti'' is expressed, production of [[phaeomelanin]] dominates, a melanin pigment that produces yellow or red colored hair<ref name="Lu">{{cite journal |last1=Lu |first1=D. |last2=Willard |first2=D. |last3=Patel |first3=I. R. |last4=Kadwell |first4=S. |last5=Overton |first5=L. |last6=Kost |first6=T. |last7=Luther |first7=M. |last8=Wenbiao |first8=C. |last9=Woychik |first9=R. P. |last10=Wilkison |first10=W. O. |last11=Cone |first11=R. R. |title=Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor |journal=Nature |date=1994 |volume=371 |pages=799-802}}</ref>. The dominance hierarchy of pigment expression explains the evolutionary persistence of the yellow phenotype of ''agouti'', as [[pheomelanin]] expression always dominates over [[eumelanin]] expression<ref name="Mayer"></ref>.
+The murine ''agouti'' gene locus is found on chromosome 2 and encodes a 131 amino acid protein. This protein signals the distribution of [[melanin]] pigments in epithelial melanocytes located at the base of hair follicles<ref name="Mayer">{{cite journal |last1=Mayer |first1=T. C. |last2=Fishbane |first2=J. L. |title=). Mesoderm-Ectoderm Interaction In the Production of the Agouti Pigmentation Pattern in Mice |journal=Genetics |date=1972 |volume=71 |pages=297-303}}</ref>. Expression is more sensitive on ventral hair than dorsal hair<ref name="Melmed">{{cite book |last1=Melmed |first1=S. (Ed) |title=The Pituitary |date=2010 |publisher=Academic Press |location=Cambridge: MA |edition=3rd}}</ref>. ''Agouti'' is not directly secreted in the melanocyte as it works as a [[paracrine]] factor on dermal papillae cells to inhibit release of [[melanocortin]]<ref name="Miltenberger">{{cite journal |last1=Miltenberger |first1=R. J. |last2=Mynatt |first2=R. L. |last3=Wilkinson |first3=J. E. |last4=Woychik |first4=R. P. |title=The role of the agouti gene in the Yellow Obese Syndrome. |journal=Journal of Nutrition |date=1997 |volume=127 |issue=9 |pages=1902-1907}}</ref>. Melanocortin acts on follicular melanocytes to increase production of [[eumelanin]], a melanin pigment responsible for brown and black hair. When ''agouti'' is expressed, production of [[phaeomelanin]] dominates, a melanin pigment that produces yellow or red colored hair<ref name="Lu">{{cite journal |last1=Lu |first1=D. |last2=Willard |first2=D. |last3=Patel |first3=I. R. |last4=Kadwell |first4=S. |last5=Overton |first5=L. |last6=Kost |first6=T. |last7=Luther |first7=M. |last8=Wenbiao |first8=C. |last9=Woychik |first9=R. P. |last10=Wilkison |first10=W. O. |last11=Cone |first11=R. R. |title=Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor |journal=Nature |date=1994 |volume=371 |pages=799-802}}</ref>. The dominance hierarchy of pigment expression explains the evolutionary persistence of the yellow phenotype of ''agouti'', as [[pheomelanin]] expression always dominates over [[eumelanin]] expression<ref name="Mayer"></ref>.
 ===Mutations===
-The ''lethal yellow'' mutation (A<sup>y</sup>) was the first embryonic mutation to be characterized in mice, as homozygous ''lethal yellow'' mice (A<sup>y</sup>/ A<sup>y</sup>) die early in development, due to an error in trophectoderm differentiation<ref name="Mayer"></ref>. However, ''lethal yellow'' homozygotes are rare today, as ''lethal yellow'' and ''viable yellow'' heterozygotes (A<sup>y</sup>/a and A<sup>vy</sup>/a) persist more commonly. These phenotypes are caused by [[ectopic expression]] of the ''agouti'' gene and are associated with the ''yellow obese syndrome'', characterized by early onset [[obesity]], [[hyperinsulinemia]] and [[tumorigenesis]]<ref name="Mayer">{{cite journal |last1=Mayer |first1=T. C. |last2=Fishbane |first2=J. L. |title=). Mesoderm-Ectoderm Interaction In the Production of the Agouti Pigmentation Pattern in Mice |journal=Genetics |date=1972 |volume=71 |pages=297-303}}</ref>.
+The ''lethal yellow'' mutation (A<sup>y</sup>) was the first embryonic mutation to be characterized in mice, as homozygous ''lethal yellow'' mice (A<sup>y</sup>/ A<sup>y</sup>) die early in development, due to an error in trophectoderm differentiation<ref name="Mayer"></ref>. ''Lethal yellow'' homozygotes are rare today, as ''lethal yellow'' and ''viable yellow'' heterozygotes (A<sup>y</sup>/a and A<sup>vy</sup>/a) persist more commonly. These phenotypes are caused by [[ectopic expression]] of the ''agouti'' gene and are associated with the ''yellow obese syndrome'', characterized by early onset [[obesity]], [[hyperinsulinemia]] and [[tumorigenesis]]<ref name="Mayer">{{cite journal |last1=Mayer |first1=T. C. |last2=Fishbane |first2=J. L. |title=). Mesoderm-Ectoderm Interaction In the Production of the Agouti Pigmentation Pattern in Mice |journal=Genetics |date=1972 |volume=71 |pages=297-303}}</ref>.
-The ''lethal yellow'' (A<sup>y</sup>) mutation is due to a deletion upstream to the start site of ''agouti'' transcription. The deletion causes the genomic sequence of ''agouti'' to be lost, except for the promoter and the first non-encoding exon of ''Raly'', a mammalian ubiquitously expressed gene<ref name="Melmed"/> The coding exons of ''agouti'' are placed under the control of the ''Raly'' promoter, initiating ubiquitous expression of ''agouti'', increasing production of [[pheomelanin]] over [[eumelanin]] and resulting in the development of a yellow phenotype<ref name="Tollefsbol">{{cite book |last1=Tollefsbol |first1=T. (Ed.) |title=Epigenetics in Human Disease |date=2012 |publisher=Academic Press |location=Cambridge: MA |edition=6}}</ref>. The ''viable yellow'' (A<sup>vy</sup>) mutation is due to a change in the mRNA length of ''agouti'', as the expressed gene becomes longer than the normal gene length of agouti. This is caused by the insertion of a single intracisternal A particle (IAP) retrotransposon upstream to the start site of ''agouti'' transcription<ref name="Dolinoy">{{cite journal |last1=Dolinoy |first1=D. C. |title=The agouti mouse model: an epigenetic biosensor for nutritional and environmental alterations on the fetal epigenome |journal=Nutrition Reviews |date=2008 |volume=66 |issue=1 |pages=7-11}}</ref>. In the proximal end of the gene, an unknown promoter then causes ''agouti'' to be constitutionally activated, and individuals to present with phenotypes consistent with the ''lethal yellow'' mutation. Although the mechanism underlying the activation of the promotor controlling the ''viable yellow'' mutation is unknown, the strength of coat color has been correlated with the degree of gene [[methylation]], which is determined by maternal diet and environmental exposure<ref name="Dolinoy" />. As ''agouti'' itself acts as an inhibitor of melanocortin receptors responsible for eumelanin production, the yellow phenotype is exacerbated in both ''lethal yellow'' and ''viable yellow'' mutations.
+The ''lethal yellow'' (A<sup>y</sup>) mutation is due to an upstream deletion at the start site of ''agouti'' transcription. This deletion causes the genomic sequence of ''agouti'' to be lost, except the promoter and the first non-encoding exon of ''Raly'', a ubiquitously expressed gene in mammals<ref name="Melmed"/> The coding exons of ''agouti'' are placed under the control of the ''Raly'' promoter, initiating ubiquitous expression of ''agouti'', increasing production of [[pheomelanin]] over [[eumelanin]] and resulting in the development of a yellow phenotype<ref name="Tollefsbol">{{cite book |last1=Tollefsbol |first1=T. (Ed.) |title=Epigenetics in Human Disease |date=2012 |publisher=Academic Press |location=Cambridge: MA |edition=6}}</ref>.
+The ''viable yellow'' (A<sup>vy</sup>) mutation is due to a change in the mRNA length of ''agouti'', as the expressed gene becomes longer than the normal gene length of agouti. This is caused by the insertion of a single intracisternal A particle (IAP) retrotransposon upstream to the start site of ''agouti'' transcription<ref name="Dolinoy">{{cite journal |last1=Dolinoy |first1=D. C. |title=The agouti mouse model: an epigenetic biosensor for nutritional and environmental alterations on the fetal epigenome |journal=Nutrition Reviews |date=2008 |volume=66 |issue=1 |pages=7-11}}</ref>. In the proximal end of the gene, an unknown promoter then causes ''agouti'' to be constitutionally activated, and individuals to present with phenotypes consistent with the ''lethal yellow'' mutation. Although the mechanism for the activation of the promotor controlling the ''viable yellow'' mutation is unknown, the strength of coat color has been correlated with the degree of gene [[methylation]], which is determined by maternal diet and environmental exposure<ref name="Dolinoy" />. As ''agouti'' itself inhibits melanocortin receptors responsible for eumelanin production, the yellow phenotype is exacerbated in both ''lethal yellow'' and ''viable yellow'' mutations as ''agouti'' gene expression is increased.
-''Viable yellow'' (A<sup>vy</sup>/a) and ''lethal yellow'' (A<sup>y</sup>/a) heterozygotes have shortened life spans and increased risks for developing early onset obesity, type II diabetes mellitus and various tumors<ref name="Miltenberger" /><ref name="Spiegelman">{{cite journal |last1=Spiegelman |first1=B. M. |last2=Flier |first2=J. S. |title=Adipogenesis and obesity: rounding up the big picture |journal=Cell |date=1996 |volume=87 |pages=377-389}}</ref>. The mechanisms underlying the increased risks of developing obesity are due to the dysregulation of appetite, as ''agouti'' agonizes the [[agouti-related protein]] (AGRP), responsible for the stimulation of appetite via hypothalamic NPY/AGRP orexigenic neurons<ref name="Dolinoy" />. Agouti also promotes obesity by antagonizing [[melanocyte-stimulating hormone]] (MSH) at the melanocortin receptor (MC4R), as [[MC4R]] is responsible for regulating food intake by inhibiting appetite signals<ref name="Adan">{{cite journal |last1=Adan |first1=R. A. H. |last2=Tiesjema |first2=B. |last3=Hillebrand |first3=J. J. G. |last4=la Fleur |first4=S. E. |last5=Kas |first5=M. J. H. |last6=De Krom |first6=M. |title=The MC4 Receptor and Control of Appetite |journal=British Journal of Pharmacology |date=2006 |volume=149 |issue=7 |pages=815-827}}</ref>. The increase in appetite is coupled to alterations in nutrient metabolism due to the [[paracrine]] actions of agouti on adipose tissue, increasing levels of hepatic [[lipogenesis]], decreasing levels of [[lipolysis]] and increasing adipocyte hypertrophy<ref name="Johnson">{{cite journal |last1=Johnson |first1=P. R. |last2=Hirsch |first2=J. |title=Cellularity of adipose depots in six strains of genetically obese mice. |journal=Obesity Research |date=1972 |volume=7 |issue=5 |pages=506-514}}</ref>. This increases body mass and leads to difficulties with weight loss as metabolic pathways are dysregulated. Hyperinsulinemia is caused by mutations to ''agouti'', as the agouti protein functions in a calcium dependent manner to increase insulin secretion in pancreatic beta cells, increasing risks of [[insulin resistance]]<ref name="Moussa">{{cite journal |last1=Moussa |first1=N. M. |last2=Claycombe |first2=K. J. |title=The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity. |journal=Obesity Research |date=1999 |volume=13 |pages=2-11}}</ref>. Increased tumor formation is due to the increased mitotic rates of ''agouti'', which are localized to epithelial and mesenchymal tissues<ref name="Tollefsbol"/>.
+''Viable yellow'' (A<sup>vy</sup>/a) and ''lethal yellow'' (A<sup>y</sup>/a) heterozygotes have shortened life spans and increased risks for developing early onset obesity, [[type II diabetes mellitus]] and various tumors<ref name="Miltenberger" /><ref name="Spiegelman">{{cite journal |last1=Spiegelman |first1=B. M. |last2=Flier |first2=J. S. |title=Adipogenesis and obesity: rounding up the big picture |journal=Cell |date=1996 |volume=87 |pages=377-389}}</ref>. The increased risk of developing obesity is due to the dysregulation of appetite, as ''agouti'' agonizes the [[agouti-related protein]] (AGRP), responsible for the stimulation of appetite via hypothalamic NPY/AGRP orexigenic neurons<ref name="Dolinoy" />. Agouti also promotes obesity by antagonizing [[melanocyte-stimulating hormone]] (MSH) at the melanocortin receptor (MC4R), as [[MC4R]] is responsible for regulating food intake by inhibiting appetite signals<ref name="Adan">{{cite journal |last1=Adan |first1=R. A. H. |last2=Tiesjema |first2=B. |last3=Hillebrand |first3=J. J. G. |last4=la Fleur |first4=S. E. |last5=Kas |first5=M. J. H. |last6=De Krom |first6=M. |title=The MC4 Receptor and Control of Appetite |journal=British Journal of Pharmacology |date=2006 |volume=149 |issue=7 |pages=815-827}}</ref>. The increase in appetite is coupled to alterations in nutrient metabolism due to the [[paracrine]] actions of agouti on adipose tissue, increasing levels of hepatic [[lipogenesis]], decreasing levels of [[lipolysis]] and increasing adipocyte hypertrophy<ref name="Johnson">{{cite journal |last1=Johnson |first1=P. R. |last2=Hirsch |first2=J. |title=Cellularity of adipose depots in six strains of genetically obese mice. |journal=Obesity Research |date=1972 |volume=7 |issue=5 |pages=506-514}}</ref>. This increases body mass and leads to difficulties with weight loss as metabolic pathways become dysregulated. Hyperinsulinemia is caused by mutations to ''agouti'', as the agouti protein functions in a calcium dependent manner to increase insulin secretion in pancreatic beta cells, increasing risks of [[insulin resistance]]<ref name="Moussa">{{cite journal |last1=Moussa |first1=N. M. |last2=Claycombe |first2=K. J. |title=The Yellow Mouse Obesity Syndrome and Mechanisms of Agouti-Induced Obesity. |journal=Obesity Research |date=1999 |volume=13 |pages=2-11}}</ref>. Increased tumor formation is due to the increased mitotic rates of ''agouti'', which are localized to epithelial and mesenchymal tissues<ref name="Tollefsbol"/>.
-===Methylation and diet intervention===
-Correct functioning of ''agouti'' requires DNA methylation. The methylation mechanism for the ''viable yellow'' mutation occurs in six guanine-cytosine (GC) rich sequences in the 5’ long terminal repeat of the IAP element<ref name="Spiegelman" />. When this area is unmethylated, ectopic expression of ''agouti'' occurs, and yellow phenotypes present. When the region is methylated, ''agouti'' is expressed normally, and grey phenotypes develop. The epigenetic state of the IAP element varies through different methylation densities, as individuals show a wide range of phenotypes based on their degree of DNA methylation<ref name="Spiegelman" />. Increased methylation is correlated with increased expression of the normal ''agouti'' gene. Low levels of methylation can also induce [[gene imprinting]]. This results in offspring with consistent phenotypes to their parents, as ectopic expression of ''agouti'' is inherited through non-genomic mechanisms<ref name="Dolinoy" /><ref name="Constância">{{cite journal |last1=Constância |first1=M. |last2=Pickard |first2=B. |last3=Kelsey |first3=G. |last4=Reik |first4=W. |title=Imprinting Mechanisms |journal=Genome Research |date=1998 |volume=8 |pages=881-900}}</ref>.
+===Methylation and diet intervention===
-DNA methylation is determined ''in utero'', by maternal nutrition and environmental exposures<ref name="Spiegelman" />. Methyl is synthesized ''de novo'' or attained through the diet by folic acid, methionine, betaine and choline, as these nutrients feed into a consistent metabolic pathway for methyl synthesis<ref name="Cooney">{{cite journal |last1=Cooney |first1=C. A. |last2=Dave |first2=A. A. |last3=Wolff |first3=D. L. |title=Maternal Methyl Supplements In Mice Affect Epigenetic Variation and DNA Methylation of Offspring. |journal=Journal of Nutrition |date=2002 |volume=132 |issue=8 |pages=2398-2400}}</ref>. Adequate [[zinc]] and [[vitamin B12]] are also required for methyl synthesis as these are required cofactors for transferring methyl groups<ref name="Wilson">{{cite journal |last1=Wilson |first1=B. D. |last2=Ollman |first2=M. M. |last3=Kang |first3=L. |last4=Stoffel |first4=M. |last5=Bell |first5=G. I. |last6=Barsh |first6=G. S. |title=Structure and Function of ASP, the human homolog of the mouse agouti gene. |journal=Human Molecular Genetics |date=1995 |volume=4 |issue=2 |pages=223-230}}</ref>.
+Correct functioning of ''agouti'' requires DNA methylation. Methylation occurs in six guanine-cytosine (GC) rich sequences in the 5’ long terminal repeat of the IAP element in the ''viable yellow'' mutation<ref name="Spiegelman" />. When this area is unmethylated, ectopic expression of ''agouti'' occurs, and yellow phenotypes present. When the region is methylated, ''agouti'' is expressed normally, and grey phenotypes develop. The epigenetic state of the IAP element is determined by the level of methylation, as individuals show a wide range of phenotypes based on their degree of DNA methylation<ref name="Spiegelman" />. Increased methylation is correlated with increased expression of the normal ''agouti'' gene. Low levels of methylation can induce [[gene imprinting]] which results in offspring displaying consistent phenotypes to their parents, as ectopic expression of ''agouti'' is inherited through non-genomic mechanisms<ref name="Dolinoy" /><ref name="Constância">{{cite journal |last1=Constância |first1=M. |last2=Pickard |first2=B. |last3=Kelsey |first3=G. |last4=Reik |first4=W. |title=Imprinting Mechanisms |journal=Genome Research |date=1998 |volume=8 |pages=881-900}}</ref>.
+DNA methylation is determined ''in utero'' by maternal nutrition and environmental exposure<ref name="Spiegelman" />. Methyl is synthesized ''de novo'' but attained through the diet by folic acid, methionine, betaine and choline, as these nutrients feed into a consistent metabolic pathway for methyl synthesis<ref name="Cooney">{{cite journal |last1=Cooney |first1=C. A. |last2=Dave |first2=A. A. |last3=Wolff |first3=D. L. |title=Maternal Methyl Supplements In Mice Affect Epigenetic Variation and DNA Methylation of Offspring. |journal=Journal of Nutrition |date=2002 |volume=132 |issue=8 |pages=2398-2400}}</ref>. Adequate [[zinc]] and [[vitamin B12]] are required for methyl synthesis as they act as cofactors for transferring methyl groups<ref name="Wilson">{{cite journal |last1=Wilson |first1=B. D. |last2=Ollman |first2=M. M. |last3=Kang |first3=L. |last4=Stoffel |first4=M. |last5=Bell |first5=G. I. |last6=Barsh |first6=G. S. |title=Structure and Function of ASP, the human homolog of the mouse agouti gene. |journal=Human Molecular Genetics |date=1995 |volume=4 |issue=2 |pages=223-230}}</ref>.
-When there is inadequate methyl during early embryonic development, DNA methylation cannot occur, which increases ectopic expression of ''agouti'' and results in the presentation of the ''lethal yellow'' and ''viable yellow''phenotypes which persist into adulthood. This leads to the development of the ''yellow obese syndrome'', which impairs normal development and increases risks for disease development later in life. Ensuring maternal diets are high in methyl equivalents is a key preventative measure for reducing ectopic expression of ''agouti'' in offspring.
-Diet intervention through methyl supplementation has decreased levels of imprinting at the ''agouti'' locus, as increased consumption of methyl equivalents causes the IAP element to become completely methylated and reduced ectopic expression of ''agouti''<ref name="Lopez-Calderero">{{cite journal |last1=Lopez-Calderero |first1=I. |last2=Sanchez Chavez |first2=E. |last3=Garcia-Carbonero |first3=R. |title=The insulin-like growth fator pathway as a target for cancer therapy. |journal=Clinical and Translational Oncology |date=2010 |volume=12 |pages=326-338}}</ref>. This lowers the proportion of heterozygotes in offspring which present with the yellow phenotype, and increase offspring that resemble the ''agouti'' wild type mice with a grey coat<ref name="Dolinoy" />.
+When inadequate methyl is available during early embryonic development, DNA methylation cannot occur, which increases ectopic expression of ''agouti'' and results in the presentation of the ''lethal yellow'' and ''viable yellow'' phenotypes which persist into adulthood. This leads to the development of the ''yellow obese syndrome'', which impairs normal development and increases susceptibility to the development of chronic disease. Ensuring maternal diets are high in methyl equivalents is a key preventative measure for reducing ectopic expression of ''agouti'' in offspring. Diet intervention through methyl supplementation reduces imprinting at the ''agouti'' locus, as increased methyl consumption causes the IAP element to become completely methylated and ectopic expression of ''agouti'' to be reduced<ref name="Lopez-Calderero">{{cite journal |last1=Lopez-Calderero |first1=I. |last2=Sanchez Chavez |first2=E. |last3=Garcia-Carbonero |first3=R. |title=The insulin-like growth fator pathway as a target for cancer therapy. |journal=Clinical and Translational Oncology |date=2010 |volume=12 |pages=326-338}}</ref>. This lowers the proportion of offspring that present with the yellow phenotype and increases the number offspring that resemble ''agouti'' wild type mice with grey coats<ref name="Dolinoy" />.
 ===The human homologue of ''agouti'', the ''agouti signaling protein'' (ASP)===
-''Agouti signaling protein'' (ASP) is the human homologue of murine ''agouti''. It is encoded by the human agouti gene on chromosome 20 and is a 132 amino acid protein. It is expressed more broadly than murine ''agouti'', as it is found in adipose tissue, pancreas, testis and ovary<ref name="Wilson" />. ASP has 85% similarity to the murine form of ''agouti''<ref name="Kwon">{{cite journal |last1=Kwon |first1=H. Y. |last2=Bultman |first2=S. J. |last3=Loffler |first3=C. |last4=Chen |first4=W. J. |last5=Furdon |first5=P. J. |last6=Powell |first6=J. G. |last7=Usala |first7=A. L. |last8=Wilkison |first8=W. |last9=Hansmann |first9=I. |last10=Woychik |first10=R. P. |title=Molecular structure and chromosomal mapping of the human homolog of the agouti gene |journal=Proceedings of the National Academy of Sciences of the United States |date=1994 |volume=91 |issue=21 |pages=9760-9764}}</ref>. As ectopic expression of ''agouti'' leads to the development of ‘yellow obese syndrome’ in mice, this is expected in humans<ref name="Kwon" />. The ''yellow obese syndrome'' increases the development of chronic diseases, including obesity, type II diabetes mellitus and tumorigenesis<ref name="Bultman" />.
+''Agouti signaling protein'' (ASP) is the human homologue of murine ''agouti''. It is encoded by the human agouti gene on chromosome 20 and is a 132 amino acid protein. It is expressed more broadly than murine ''agouti'', as it is found in adipose tissue, pancreas, testes and ovaries<ref name="Wilson" />. ASP has 85% similarity to the murine form of ''agouti''<ref name="Kwon">{{cite journal |last1=Kwon |first1=H. Y. |last2=Bultman |first2=S. J. |last3=Loffler |first3=C. |last4=Chen |first4=W. J. |last5=Furdon |first5=P. J. |last6=Powell |first6=J. G. |last7=Usala |first7=A. L. |last8=Wilkison |first8=W. |last9=Hansmann |first9=I. |last10=Woychik |first10=R. P. |title=Molecular structure and chromosomal mapping of the human homolog of the agouti gene |journal=Proceedings of the National Academy of Sciences of the United States |date=1994 |volume=91 |issue=21 |pages=9760-9764}}</ref>. As ectopic expression of murine ''agouti'' leads to the development of the ''yellow obese syndrome'', this is expected to be consistent in humans<ref name="Kwon" />. The ''yellow obese syndrome'' increases the development of many chronic diseases, including obesity, type II diabetes mellitus and tumorigenesis<ref name="Bultman" />.
-ASP has similar pharmacological activation to murine ''agouti'', as melanocortin receptors are inhibited through competitive antagonism<ref>{{cite book |last1=Takeuchi |first1=S. |title=Handbook of Hormones |date=2015 |publisher=Academic Press |location=Cambridge: MA |pages=66-67}}</ref>. Inhibition of melanocortin by ASP can also be through non-competitive methods, broadening its range of effects<ref name="Tollefsbol"/>. ASP does not have the same function as murine ''agouti''. ASP effects the quality of hair pigmentation whereas murine ''agouti'' controls the distribution of pigments for the determination of coat color<ref name="Dolinoy"/>. ASP also has neuroendocrine functions consistent with murine ''agouti'', as it agonizes AGRP via AGRP neurons in the hypothalamus and antagonizes MSH at MC4Rs decreasing satiety signals. As appetite signals increase and satiety signals decrease, body mass increases and individuals are more susceptible to becoming obese. The mechanism underlying hyperinsulinemia in humans is consistent with murine ''agouti'', as insulin secretion is heightened through calcium sensitive signaling in pancreatic beta cells. The mechanism for ASP induced tumorigenesis remains unknown in humans.
+ASP has similar pharmacological activation to murine ''agouti'', as melanocortin receptors are inhibited through competitive antagonism<ref>{{cite book |last1=Takeuchi |first1=S. |title=Handbook of Hormones |date=2015 |publisher=Academic Press |location=Cambridge: MA |pages=66-67}}</ref>. Inhibition of melanocortin by ASP can also be through non-competitive methods, broadening its range of effects<ref name="Tollefsbol"/>. The function of ASP differs to murine ''agouti''. ASP effects the quality of hair pigmentation whereas murine ''agouti'' controls the distribution of pigments that determine coat color<ref name="Dolinoy"/>. ASP has neuroendocrine functions consistent with murine ''agouti'', as it agonizes AGRP via AGRP neurons in the hypothalamus and antagonizes MSH at MC4Rs which reduce satiety signals. As appetite signals increase and satiety signals decrease, body mass is likely to increase and individuals are more susceptible to becoming obese. The mechanism underlying hyperinsulinemia in humans is consistent with murine ''agouti'', as insulin secretion is heightened through calcium sensitive signaling in pancreatic beta cells. The mechanism for ASP induced tumorigenesis remains unknown in humans.