Physiol. Genomics 1: 151-163, 1999.
First published November 11, 1999;
1094-8341/99 $5.00
Physiological Genomics 1:151-163 (1999)
1094-8341/99 $5.00 © 1999 American Physiological Society
Review
Physiological consequences of ectopic agouti gene expression: the yellow obese mouse syndrome
GEORGE L. WOLFF1,2,3,
DEAN W. ROBERTS1,3 and
KATHLEEN G. MOUNTJOY4
1 Division of Biochemical Toxicology, National Center for Toxicological Research/Food and Drug Administration, Jefferson 72079
2 Department of Biochemistry/Molecular Biology
3 Department of Pharmacology/Interdisciplinary Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
4 Research Centre for Developmental Medicine and Biology and Department of Molecular Medicine, University of Auckland, Auckland 1, New Zealand
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ABSTRACT
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Wolff, George L., Dean W. Roberts, and Kathleen G. Mountjoy. Physiological consequences of ectopic agouti gene expression: the yellow obese mouse syndrome. Physiol. Genomics 1: 151–163, 1999.—This review summarizes primary and downstream phenotypic manifestations, with emphasis on altered responsiveness to environmental stimuli, of dominant yellow mutations at the mouse agouti locus. Obvious effects include hyperinsulinemia, obesity, stimulation of somatic growth and tumorigenesis, and coat color. Downstream influences of hyperinsulinemia and obesity on the individual's physiology determine important components of the obese yellow agouti mouse syndrome. Collectively, the phenotypic aberrations described support the concept that identical genomes are expressed in a spectrum of physiological phenotypes that reflect the complex interdependence of gene-regulated physiological pathways and processes in the organism throughout extended, but temporally ordered, periods of fetal and neonatal development and aging. This summary identifies important areas for additional research and provides integrated information required for a systematic approach to the development of interventions for common adult human health problems.
background genome; melanocortin; pseudoagouti mice; response to environmental stimuli; tumorigenesis
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INTRODUCTION
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PHYSIOLOGICAL GENOMICS WAS foreshadowed more than 65 years ago by Sewall Wright's statement (46) that "All characters are affected by many genes and each gene affects many characters." In those days, the study of the influence of genes on physiological parameters was called mammalian physiological genetics and emphasized the significance of interactions among genes, as well as the generally underappreciated influence of the background genome on individual gene expression (47).
Molecular genetics has revealed that the majority of these interactions actually occur at the physiological level rather than at the level of the gene. In this review, we shall use the terms "gene interactions" and "background genome effects" as shorthand for the following concepts. 1) Each gene specifies a protein or RNA that modulates a specific step in one or more physiological pathways or cellular processes. The apparent interactions among such genes actually represent the effect of each gene-specified protein or RNA on a different step in the same physiological pathway or cellular process. 2) The apparent effects of the background genome on expression of particular genes actually represent the effects of the qualitative and quantitative differences in the physiological pathways and cellular processes, resulting from the presence of different alleles at many loci and different patterns of expressed genes in each inbred strain and in each individual in noninbred populations.
The ontogeny of each individual mouse and human involves the development of functional physiological, immunologic, and cognitive systems through complex interactions, programmed sequential cell differentiation and migration, and subsequent organization of diverse cell types throughout an extended, but temporally ordered, period of fetal and neonatal development.
Today, the combination of physiological and molecular biological concepts and techniques facilitates the investigation of the reciprocal relations of gene function and physiology at all levels of biological organization. It is now possible to 1) define gene product (polypeptide) actions and interactions that, in many cases, modulate cellular signaling pathways and 2) trace in detail the intra- and intercellular molecular paths from gene to in vivo phenotype.
An illuminating example of the power of molecular techniques and concepts to address problems of physiological genetics is the "yellow agouti obese mouse syndrome." This syndrome, induced by alternative dominant alleles at the agouti locus on mouse chromosome 2, is characterized by yellowish hair, adult-onset obesity, hyperinsulinemia, somewhat increased lean body mass, and heightened susceptibility to endogenously and exogenously induced tumors (reviewed in Refs. 38, 44, 48). These phenotypic effects result, directly or indirectly, from the binding of agouti protein, specified by the agouti gene, to melanocortin receptors, the proteins specified by the melanocortin receptor genes. Thus the binding of agouti protein modulates the signaling pathways associated with the melanocortin receptors. The interaction occurs not between the agouti and melanocortin receptor genes per se but rather between the proteins specified by these genes. Therefore, the "interaction between the genes" occurs downstream at the level of the products of DNA transcription and translation. Each gene merely serves as a template for synthesis of a protein, which is a part of signaling pathways and/or cellular processes. In short, physiological genomics deals with the effects of the interactions of gene products in the complex network of homeostatic and cellular processes that constitute the living organism.
The human homologue of this locus, agouti signaling protein (ASIP), located in a syntenic section on human chromosome 20q, is regularly expressed at a low level in several tissues. Because the murine agouti gene is normally transcribed only in the skin and then only transiently during formation of the yellow subapical band in the hair, the usual expression of ASIP in multiple tissues suggests that its transcription is regulated quite differently from that of agouti. The function of the ASIP protein in Homo sapiens is unknown (33).
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LETHAL (AY) AND VIABLE (AVY) YELLOW MUTATIONS AT THE MOUSE AGOUTI LOCUS
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Two of the dominant agouti mutations have been studied extensively, namely, "lethal yellow" (Ay), first described by Cuénot (5), and "viable yellow" (Avy), first described by Dickie (6). The Ay> mutation was propagated by the mouse fancy in Europe at least as early as the 1800s, whereas the Avy mutation was discovered at The Jackson Laboratory (Bar Harbor, ME) in 1960. In addition to the phenotypic traits common to the other dominant "yellow" mutants, the lethal yellow mutation is characterized by prenatal lethality of the homozygous Ay/Ay genotype. The viable yellow Avy/- phenotype differs from that of the clear yellow Ay/- phenotype by exhibiting eumelanic mottling, i.e., irregular areas or small spots of agouti/black hair on a yellow background.
Molecular definition of these mutations was made possible by identification and cloning of the agouti locus at the Oak Ridge National Laboratories (2). Briefly, Ay is characterized by a large deletion that includes the coding regions of the Raly (heterogeneous nuclear ribonucleo-protein associated with lethal yellow) gene, a member of the hnRNP gene family. The hnRNP proteins are involved in pre-mRNA packaging and processing. Although the precise function of Raly is unknown, the lethal yellow mutation suggests that it is important for early embryonic development.
Raly is constitutively expressed in all somatic cells. Therefore, fusion of the Raly promoter with the agouti gene results in ectopic overexpression of agouti (17). In short, the Raly promoter overrides the agouti gene's own promoter in regulating its transcription and thus induces uninterrupted formation of yellow pigment by the hair follicle melanocytes. The embryonic lethality, hitherto associated with the Ay/Ay genotype, may actually result from the absence of Raly expression in the developing embryo.
Ectopic overexpression of the agouti gene induced by a heterologous promoter is also basic to the syndrome expressed by Avy/- mice. In this case, however, it is due to insertion of an intracisternal A particle (IAP) into noncoding exon 1A (reviewed in Ref. 48).
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MATERNAL EFFECTS ON METHYLATION OF IAP LONG TERMINAL REPEATS
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When methylated, the long terminal repeats (LTRs) of the IAP inserted in the noncoding region of agouti cease to induce agouti transcription (18). When such methylation occurs in progenitor cells of some Avy/a or Aiapy (IAP yellow)/a melanocyte clones, the agouti gene's own promoter regulates its transcription in these clones so that pheomelanin is synthesized only during formation of the subapical yellow band in the hair. This results in eumelanic islands (mottling) on a yellow background.
Prenatal exposure to methyl-supplemented diets, maternally administered, increases such areas of eumelanic mottling, most likely due to increased IAP LTR methylation (42). In the most extreme case, with or without dietary methyl supplementation, a lean mouse, called "pseudoagouti," is produced with a coat color pattern that closely resembles that of the wild type.
Maternal factors strongly influence the genomic imprinting observed in the Avy mutants (reviewed in Ref. 42). The relatively low proportions of the pseudoagouti phenotype produced on most inbred and F1 hybrid backgrounds have made it economically impractical to elucidate the specific maternal factors involved in regulation of the genomic imprinting of the mutant agouti gene. An exception is strain AKR females mated with Avy-bearing males, inasmuch as their Avy/a offspring segregate into approximately equal proportions of mottled yellow and pseudoagouti mice (Ref. 35; K. Gärtner and G. L. Wolff, unpublished observations). Therefore, populations of mottled yellow, pseudoagouti, and black (AKR x C57BL/6-Avy)F1 hybrid mice would provide an economically justifiable experimental system for defining the maternal physiological and molecular factors involved in the regulation of genomic imprinting.
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FUNCTIONS OF AGOUTI PROTEIN
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Agouti protein inhibits binding of 
-melanocyte-stimulating hormone (-MSH) to melanocortin receptor 1 (MC1-R) in hair follicle melanocytes. This prevents activation of adenylyl cyclase and a subsequent increase in intracellular cAMP concentration (reviewed in Ref. 4). As a result, eumelanin synthesis is prevented, and the default synthesis of pheomelanin proceeds, resulting in completely yellow hairs.
Ectopic agouti protein also binds to another melanocortin receptor, MC4-R, and inhibits its function in the hypothalamus (reviewed in Ref. 4). Disruption of the gene, Mc4r, results in obese mice that, however, do not have a yellow coat color (12). These mice are slightly longer than the normal control mice, indicating an effect on somatic growth; however, whether tumor formation is also increased has not been determined yet.
Besides inhibiting binding of -MSH to MC1-R and MC4-R, agouti protein also inhibits the activity of the promoter of microphthalmia-associated transcription factor (Mitf; chromosome 6) and "markedly reduces the expression of the protein" (1). Agouti protein antagonism of -MSH binding to MC1-R and MC4-R is likely to have numerous downstream effects, including effects on gene transcription. However, it is also not unreasonable to assume that agouti protein may interact directly with additional, as yet unidentified, gene loci, especially when ectopically expressed.
The only direct effect of agouti protein that has been demonstrated so far is binding to melanocortin receptors. The phenotypic expression of the agouti protein is altered by a protein specified by a recessive mutant, mahogany (mg), at the Mahogany locus. Because less pheomelanin is synthesized, the coat color of mg/mg Ay/- mice is darker than that of Mg/Mg Ay/- mice. Although the specific mode of action of the mahogany protein, either wild-type or mutant, is unknown, it appears, at the very least, to modulate the binding of agouti protein to MC1-R and MC4-R and may even bind agouti protein directly (20).
Agouti protein also enhances the influx of Ca2+ into skeletal muscle cells and thus increases their intracellular Ca2+ concentration ([Ca2+]i). The mode of action of the agouti protein in this case is unknown (47). Spider and snail venoms that attack Ca2+ channels (14) exhibit spatial homology with the agouti protein's COOH-terminal region in number and spacing of cysteine residues. This homology is particularly striking because this region of the agouti protein has functional activity similar to the intact protein (30); therefore, agouti may act on a specific Ca2+ channel.
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OBESITY AND HYPERINSULINEMIA
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A current working hypothesis postulates that hyperphagia in yellow agouti mice is induced by the inhibition, by ectopic agouti protein, of MC4-R, which are normally regulated by the agouti gene-related protein (Agrp) (22) in the hypothalamus. In addition, it is postulated that excess lipid synthesis and deposition results from the effects of agouti protein on Ca2+ influx and [Ca2+]i in adipocytes (reviewed in Ref. 49).
Yellow Avy/a mice were more sensitive than black a/a siblings to the neurochemical effects of early postnatal monosodium glutamate (MSG) administration. Food intake was decreased, as were the hypothalamic contents of dopamine, pro-opiomelanocortin (POMC), and ß-endorphin (3). In addition to decreasing POMC expression in the hypothalamus, it is likely that MSG treatment also decreases the expression of Mc3r and Agrp genes. Reduced expression of these genes in the arcuate nucleus may contribute to the delay in weight gain of the yellow agouti mice observed following neonatal MSG treatment.
Disruption, by adrenalectomy, of the ACTH feedback loop, which includes melanocortin receptors and POMC peptides, also slowed the weight gain of both yellow Avy/a and black a/a mice, with the reduction being greater in the yellow agouti mice. Corticosterone replacement in adrenalectomized yellow agouti mice resulted in a dose-dependent increase in body weight, including the adipose component (28).
Potentially contributing to the tendency toward obesity, 21-day-old Avy/A (BALB/c x VY)F1 mice have significantly (P < 0.001) more pancreatic ß-cells than their A/a siblings (30). If these extra ß-cells are secreting insulin, this may be sufficient to initiate increased lipid deposition and weight gain in yellow agouti mice, as suggested by the findings of Mynatt et al. (19). These workers found that the expression of agouti in adipose tissue of transgenic FVB/N mice, combined with an elevated circulating insulin level induced by daily subcutaneous insulin injections, caused more weight gain in the transgenic mice than in wild-type FVB/N mice. Peripheral insulin resistance would follow and result in the persistent hyperinsulinemia characteristic of obese mottled yellow agouti mice.
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EFFICIENCY OF FEED UTILIZATION
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In a study of the effects of 30% caloric restriction on (BALB/cStCrlfC3Hf/Nctr x VY/WffC3Hf/Nctr-Avy)F1 hybrid mottled yellow Avy/A and agouti A/a mice (41), the body weights of ad libitum-fed Avy/A mice increased considerably more relative to their feed consumption than did those of the ad libitum-fed A/a mice. Ad libitum-fed yellow agouti mice consumed only ~8.7 g feed per gram body weight gain during an active growth period between 16 and 44 wk. In contrast, the ad libitum-fed agouti mice consumed ~30.5 g feed per gram body weight gain, i.e., the ratio among agouti mice was 3.5-fold greater than among the yellow agouti mice. These data confirm the earlier conclusion that the Avy genotype affects the efficiency of food utilization (9).
Previously, we reported that Avy/A females gained ~14.0 g/kcal, whereas A/a females gained only ~3.5 g/kcal, a fourfold greater increase among yellow agouti mice than among agouti mice (9). This greater efficiency of calorie utilization by yellow agouti mice was abolished by caloric restriction, with a feed consumption-to-body weight gain ratio for both calorie-restricted Avy/a and calorie-restricted A/a mice of ~28.
Although the binding of ectopic agouti protein to MC4-R induces some hyperphagia, this does not appear to be the major cause of the obesity. In a study to determine possibly differential effects of the tumor promoter lindane (
-hexachlorocyclohexane) on yellow and pseudoagouti Avy/a mice (45), food consumption of the yellow agouti (VY x YS)F1 hybrid female mice was only ~10% greater than that of the pseudoagouti and black siblings. For three different diets (control, high fat, and high sucrose) in a different study (9), the weekly food intakes of yellow Avy/A (BALB/c x VY)F1 mice were 32%, 16%, and 4% greater, respectively, than those of the A/a siblings. Corresponding body weight gains of the yellow agouti mice were 432%, 238%, and 142% greater than those of the agouti mice. The greater body weight gain in the yellow agouti mice fed the high-fat and high-sucrose diets (~27 g) rather than the control diet (~21 g) was due to increased efficiency of feed utilization, since the absolute caloric intake per mouse with these diets was actually decreased.
Results of the above studies bolster the conclusion that ectopic agouti protein "alters the response to changes in dietary composition and affects the efficiency of food utilization more than the total caloric intake" (9). No published data appear to be available on energy expenditure during rest or activity, on core body temperature in yellow agouti mice, or on relative activities in yellow agouti mice of uncoupling protein (UCP) genes, which specify uncoupling proteins that may be involved in modulating metabolic rates (27). Accordingly, identification of the cellular and physiological processes that determine the efficiency of nutrient utilization and are modulated, directly or indirectly, by the ectopic agouti protein requires considerable further work.
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IMMUNITY AND HOST RESISTANCE TO MALARIAL PARASITE INFECTION
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Obese mottled yellow Avy/a (YS x VY)F1 hybrid mice differ from their lean pseudoagouti Avy/a and lean black a/a siblings in certain immune responses such as decreased antibody response to the T-dependent immunogen tetanus toxoid, enhanced antibody response to the T-independent immunogen type III pneumococcal polysaccharide, decreased rates of carbon clearance, and increased levels of IgA (26). The fact that the obese yellow agouti mice differ from both the lean pseudoagouti and lean black mice in these immune responses suggests that these alterations are probably associated, directly or indirectly, with the obese phenotype rather than with ectopic agouti protein per se; however, the possibility that ectopic agouti protein may directly affect immune responses cannot be excluded and requires further investigation.
Obese mottled yellow Avy/a (YS x VY)F1 hybrid mice also differ from their lean pseudoagouti Avy/a and lean black a/a siblings in their response to infection with the murine malarial parasite Plasmodium yoelii. These differences include lower peak parasitemia and an altered survival pattern (24). Because erythrocytes are the targets of malaria, differences in hematological responses (31) may contribute to the atypical response to P. yoelii infection observed in yellow agouti obese mice (44). Notably, iron overload, induced by feeding AIN-76A diet fortified with 1,500, 3,500, 5,000, or 10,000 mg/kg carbonyl iron for 12 wk to mottled yellow Avy/a and black a/a (C57BL/6NNctr x YS/WffC3Hf/Nctr-Avy)F1 hybrid male mice (31), significantly decreased (P < 0.001) the number of erythrocytes in the yellow agouti mice but not in the black mice (unpublished observations). Erythrocytes from the yellow agouti mice were also more fragile, exhibiting increased susceptibility to osmotic stress (P < 0.01) (44), which may be associated with hyperinsulinemia as postulated by Engström et al. (7) with respect to similar observations in obese Lepob/Lepob mice.
Altered resistance of yellow agouti mice to P. yoelii may also be related to decreased T cell competence. Infection of agouti A/a (BALB/c x VY)F1 hybrid mice and their mottled yellow Avy/A siblings with P. yoelii resulted in an increase in relative spleen-to-body weight ratio that was significantly greater in agouti mice compared with their yellow agouti siblings (P < 0.001). This is consistent with the report that splenomegaly is a T cell-dependent, protective response to P. yoelii infection (25) and the observation that yellow agouti mice have a decreased capacity (P < 0.005) to mount a T cell-dependent antibody response to tetanus toxoid (26).
Although leptin, agouti protein, and melanocortin receptors are all associated with immune function, their precise interrelationships require additional definition. Compared with their lean littermates, obese yellow agouti mice have increased leptin levels (14). Leptin differentially regulates the proliferation of naive and memory T cells, directly linking immune function with nutritional status (13). MC1-R, antagonized by agouti protein, is expressed on macrophages (29). Mahogany associates agouti protein with immune functions, since it is a homologue of attractin, produced by activated T cells (10), and also necessary for agouti antagonism of melanocortin receptors (20).
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DIFFERENTIAL MORTALITY
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Differential mortality, unrelated to treatment, was observed between the mottled yellow and pseudoagouti Avy/a phenotypes between 17 and 24 mo of age (Table 1) in the lindane study (45). The mortality rate for yellow agouti mice was approximately double (P < 0.001) that of the pseudoagouti and black phenotypes. Whether this was a consequence of the obesity and its effects on the physiology of the animal or was due to more direct interference with homeostatic processes by the ectopic agouti protein is unknown. The body weight of yellow agouti mice normally begins to decline, for unknown reasons, toward that of their nonyellow siblings beginning around 18 mo of age; thus this decline in body weight seems to coincide with the increased mortality.
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Table 1. Differential mortality among mottled yellow Avy/a, pseudoagouti Avy/a, and black a/a female (YSxVY)F1 hybrid mice between about 17 and 24 mo of age
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STIMULATION OF SOMATIC GROWTH AND TUMORIGENESIS
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Probably the most significant and challenging "yellow agouti mouse" problem remaining to be addressed is the physiological/metabolic basis of the stimulatory effect on somatic growth and tumorigenesis associated with ectopic expression of the agouti protein. The lean body mass of Avy/a and Ay/a mice has long been known to be somewhat greater than that of their non-Avy or non-Ay littermates. Likewise, lean pseudoagouti Avy/a mice are slightly (~5%), but significantly (P < 0.01), heavier than their lean black a/a siblings (37). This strongly suggests that even a low level of ectopic agouti protein affects one or more cellular processes involved in somatic growth.
It has been known, at least since the 1940s, that yellow agouti mice exhibit a higher frequency of tumor formation whether induced endogenously or exogenously (reviewed in Ref. 36). The most obvious factor to explain this growth effect has been the hyperinsulinemia associated with obesity. This possibility seemed to be supported by the observation that allogeneic ascites tumor cells formed larger subcutaneous solid tumors when implanted in yellow Avy/a and Ay/a mice than in black a/a mice (reviewed in Ref. 44).
Doubt regarding hyperinsulinemia as the primary promoter of tumorigenesis in yellow agouti mutant mice was generated by the appearance of benign lung tumors in lindane-treated lean pseudoagouti Avy/a and obese mottled yellow Avy/a (YS x VY)F1 female mice with the same frequency, whereas no such tumors developed in the sibling black a/a mice (45). Because the pseudoagouti mice are normoinsulinemic, hyperinsulinemia could not have played a role in the growth of these tumors.
That the somatic and neoplastic growth effects associated with ectopic agouti protein are probably related is suggested by the enhancement of hyperplastic mammary, bladder, and lung lesions in yellow agouti mice (reviewed in Ref. 44). Hyperplasia results in a larger population of cells at risk for neoplastic transformation and therefore, on a stochastic basis, would be the source of more tumors than the analogous normal cell population. Thus the earlier and greater mammary tumor prevalence among virgin yellow Avy/A (C3H/HeNIcrWf x VY/Wf-Avy)F1 hybrid female mice was, most likely, a consequence of the greater number of hyperplastic alveolar nodules (HAN) that appeared earlier in yellow agouti females than in the agouti females. Thus, in yellow agouti mice, the pool of cells at risk was earlier and larger than that of the agouti mice.
Consistent with this tendency toward increased relative hyperplasia, the grade of bladder hyperplasia induced by feeding N-acetylaminofluorene in the diet was higher in yellow Avy/A (BALB/cStCrlfC3Hf/Nctr x VY/WffC3Hf/Nctr-Avy)F1 hybrid mice at each dose level than in their agouti A/a siblings (reviewed in Ref. 44). Similarly, after 6 mo of lindane feeding, pulmonary Clara cell hyperplasia was present in 77% of the yellow agouti (YS/ChWffC3Hf/Nctr x VY/WffC3Hf/Nctr-Avy>)F1 hybrid mice but in only 50% of the pseudoagouti and 56% of the black mice (45). After 24 mo of treatment, the prevalence of this lesion had equalized among the three phenotypes. Here again, lesion formation occurred earlier and more rapidly among the yellow agouti mice.
Lindane feeding to another set of female yellow agouti and black mice was stopped at 6 mo of age to determine whether continuous treatment was necessary to maintain hyperplasia or tumorigenesis (45). At 13 mo of age, the prevalence of Clara cell hyperplasia among these yellow agouti mice had regressed by only 18% in comparison with the continuously fed mice; in contrast, among black mice the prevalence had decreased by 48%. Apparently, the presence of ectopic agouti protein was conducive to maintenance of Clara cell hyperplasia.
Formation of benign lung tumors was promoted by lindane treatment in yellow and pseudoagouti females but not in black females. Among yellow agouti females, tumor prevalence was 15%, among pseudoagouti mice it was 7%, but among black mice it was only 1% above the respective background prevalences. Among the yellow agouti mice fed the lindane-supplemented diet to 25 mo of age, terminal lung tumor prevalence was 19% compared with 10% among the mice fed the diet to only 6 mo of age but held to 25 mo of age.
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HEPATOCELLULAR ADENOMAS
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The incidence (12%) of hepatocellular adenomas in lean normoinsulinemic pseudoagouti Avy/a mice fed lindane for 24 mo was greater (P = 0.03) than that observed in normoinsulinemic black a/a mice (3%) but less (P = 0.0001) than the incidence (35%) in hyperinsulinemic yellow agouti mice. Presumably, agouti protein is synthesized in fewer hepatocytes in pseudoagouti mice because of increased IAP LTR methylation in the hepatocyte population compared with mottled yellow Avy/a mice. If the expression of agouti protein in hepatocytes is a factor in hepatic tumorigenesis, this could account for the lower frequency of hepatocellular adenomas in this phenotype compared with the mottled yellow phenotype. These observations imply that the ectopic expression of agouti protein per se in at least some hepatocytes in the pseudoagouti mice may either facilitate initiation or itself exert an initiating effect whereby some cells are transformed and subsequently promoted by the lindane (45).
The carcinoma incidence among the yellow agouti mice fed the control diet was 13% (12/93) and in the lindane-fed group it was 17% (16/94); in contrast, carcinoma prevalence among pseudoagouti mice was only 2% in the control group and 5% in the treated group, whereas among the black mice it was only 3% in the control group and 1% in the treated group. Thus the progression of some adenomas to carcinomas was facilitated or stimulated by some endogenous factor in the yellow agouti mice that was not present in either the pseudoagouti or black mice. Hyperinsulinemia and DNA fragility are two possible candidates. This suggestion is supported by the finding of E. J. Michaud and A. Kuklin (personal communication) in which, following initiation with diethylnitrosamine, transgenic FVB/N mice bearing an expression vector, containing the agouti gene, targeted to hepatocytes develop significantly more hepatocellular tumors than nontransgenic littermates.
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LIVER WEIGHT
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An increase in liver weight (P < 0.05) in response to lindane, detectable after 6 mo of treatment in the yellow and pseudoagouti Avy/a mice, was another indication of the presence of ectopic agouti protein in the livers of pseudoagouti mice, since such an increase was not detected in black a/a mice until after 24 mo of treatment (45). As shown in Table 2, liver weights of both phenotypes increased in response to lindane treatment; however, the increase was greater among the livers from yellow agouti mice than among those from pseudoagouti mice. This suggests that the difference in response may have been due to the lower concentration of ectopic agouti protein in the livers and/or the lower insulin levels in the pseudoagouti mice.
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Table 2. Proportional increases in liver weights of female mottled yellow and pseudoagouti Avy/a (YSxVY)F1 hybrid mice after lindane treatment of different durations
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HEPATIC BENZO(A)PYRENE MONOOXYGENASE ACTIVITY
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After 12 mo of lindane treatment, hepatic benzo(a)pyrene monooxygenase activity exhibited significant differences between treated and control pseudoagouti as well as black mice; however, such differences were not observed between treated and control obese yellow agouti mice. Treated pseudoagouti and black mice exhibited greater enzyme activity than their respective controls. In contrast, treated and control yellow agouti mice had lower enzyme activities than either treated pseudoagouti or black mice (45).
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TUMOR LATENCY
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The first sarcomas induced by the subcutaneous implantation of plastic films were detected at 16 mo postimplantation in mottled yellow Avy/a YS/ChWffC3Hf/Nctr-Avy mice compared with 22 mo postimplantation in their black a/a siblings. Among Avy/a VY/WffC3Hf/Nctr-Avy mice, however, the first sarcomas were already detected at 11 mo postimplantation compared with 14 mo postimplantation in the black a/a VY/Wf mice (44). Thus, although the tumors appeared 21–27% earlier in yellow agouti mice than in their black siblings regardless of strain, the background genome determined the basic length of time required from implantation to tumor detection. This is a vivid example of the importance of the interaction of a mutant gene with different background genomes in determination of a physiological endpoint.
Another example of the shorter latency/more rapid growth of tumors in yellow agouti mice are mammary carcinomas, induced by 7,12-dimethylbenz(a)anthracene (DMBA) in yellow Avy/A (BALB/c x VY)F1 hybrid female mice. These appeared about 28% earlier and with a 29% greater frequency compared with their agouti A/a sisters (reviewed in Ref. 44).
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DNA STRAND BREAKAGE
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A clue to at least one aspect of the basis for the liver tumorigenesis effect may be provided by a recent observation on hepatocytes in a chronic iron overload study. Frequencies of DNA strand breaks in hepatocytes from mottled yellow Avy/a, pseudoagouti Avy/a, and black a/a male (C57BL/6N x YS/WffC3H/Nctr-Avy)F1 hybrid mice were determined by a previously described method (23) (B. Miller and S. J. James, personal communication). The mice had been fed iron-free AIN-93M diet fortified with 35 mg/kg, 1,500 mg/kg, 3,500 mg/kg, or 5,000 mg/kg carbonyl iron for 12 mo.
Iron overload had no effect on either DNA strand breakage or hepatocellular tumor incidence at any dose level in any of the mouse phenotypes. However, obese yellow Avy/a mice exhibited a significantly higher (P < 0.01, adjusted for multiple comparisons) frequency of DNA strand breakage [3,392 ± 74 strand breaks (n = 11)] than lean pseudoagouti Avy/a mice [2,911 ± 104 strand breaks (n = 6)] or black a/a mice [2,790 ± 74 strand breaks (n = 12)].
These data suggest that ectopic agouti protein increases chromosomal fragility, at least in hepatocytes, although it is difficult to imagine the mechanism involved. However, if confirmed, such fragility might constitute a mutational event facilitating initiation of neoplastic transformation and thus could contribute to the increased liver tumorigenesis characteristic of yellow Avy/- mice.
The association of agouti protein with increased DNA strand breakage may not be limited to hepatocytes. Fibroblast cell lines from yellow Avy/a mice exhibited a significant degree of spontaneous transformation (11). In contrast, no fibroblast cell lines established from black a/a mice exhibited spontaneous transformation. When the transformed cell lines were analyzed for a restriction fragment length variant (RFLV), which is informative for Avy-bearing and a-bearing chromosomes, most of the transformed cells had lost the a-associated RFLV, and one transformant exhibited amplification of the Avy-associated RFLV. The increase in gene dosage of the Avy allele and loss of the a allele may have involved increased chromosomal fragility initiated by agouti protein.
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GROWTH HORMONE, INSULIN, AND INSULIN-LIKE GROWTH FACTOR I
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Because yellow agouti mice exhibit increased somatic growth, it seems paradoxical that growth hormone levels in yellow Avy/a mice "were low throughout most of the day with little indication of a diurnal rhythm" (16). In contrast, the black a/a siblings showed a large number of diurnal fluctuations in growth hormone concentration.
Insulin levels increased with age in yellow agouti mice but not in black mice, whereas growth hormone levels decreased in both genotypes. Thus the insulin-to-growth hormone ratio increased ~3.5 times as fast in yellow agouti mice compared with that in black mice between 30 and 90 days of age.
A marked biphasic increase in serum insulin-like growth factor I (IGF-I) levels in both black a/a and yellow Avy/a VY/WffC3Hf/Nctr-Avy mice of both sexes has been observed (41). The first increase, between 2–3 wk and 3–4 wk of age, was about twice as great in the yellow agouti mice (312% in females, 322% in males) compared with that in the black mice (167% in females, 154% in males). Between 3–4 wk and 4–5 wk of age, serum IGF-I concentration decreased 33% among the yellow agouti females and 42% among the black females. The second increase in IGF-I levels occurred between 4–5 and 6–7 wk of age, namely 121% among the yellow agouti females and 288% among the black females. The serum IGF-I concentrations then remained similar in both genotypes to at least 12–13 wk of age.
The first phase of the increase was twice as great among the yellow agouti mice compared with that in the black mice. Conversely, the second increase was greater among the black than among the yellow agouti mice. Thus the total increase in circulating IGF-I between the ages of 2–3 and 6–7 wk, 553% among yellow agouti females and 504% among the black females, was similar in both genotypes. However, the first, presumptively mitogenic, stimulus was stronger and occurred about 2 wk earlier in yellow agouti mice than in black mice.
Transiently higher circulating IGF-I levels in yellow agouti mice may alter their normal metabolic programming and contribute to the greater lean body mass observed in Avy/- mice; it may also be a factor in the greater metabolic efficiency observed in yellow agouti mice. This difference in timing of a major increase in serum IGF-I concentration between yellow Avy/a and black a/a strain VY mice reflects altered developmental programming induced, directly or indirectly, by ectopic agouti protein with implied effects on growth.
The background strain genome plays a major role in determining the response of plasma insulin and glucose concentrations to a glucose load. This is indicated by the markedly different responses to glucose tolerance tests of lean black a/a mice of the inbred YS and VY strains (Fig. 1). These data suggest that the YS mice have an inherent genome-associated insulin resistance on which, in the obese yellow Avy/a YS mice, the insulin resistance associated with their obesity is superimposed.
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Fig. 1. Concentrations of plasma insulin (A) and plasma glucose (B) during glucose tolerance tests of lean black a/a female mice of the inbred YS and VY strains and their F1 hybrid offspring. Each time point represents mean ± SE of the indicated number of mice. Each mouse was injected with 0.2 ml saline sc for 3 consecutive days. On day 4, 2 µg of dexamethasone were given intraperitoneally at 8:30 AM and food was removed for 6 h. At 2:30 PM, 1 mg glucose/g body wt in saline was injected intraperitoneally. Blood samples at 0, 30, 60, 120 min were obtained by orbital sinus puncture. Time 0 sample was collected immediately before glucose injection. These glucose tolerance tests were performed by Dr. L. G. Frigeri while at The Whittier Institute for Diabetes and Endocrinology (La Jolla, CA).
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CASTRATION: RESPONSE OF CIRCULATING PLASMA CORTICOSTERONE
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