Avy Mice Research Articles

Letrozole-Induced Polycystic Ovary Syndrome Attenuates Cystathionine-β Synthase Gene Expression in the Ovaries of Female Sprague Dawley Rats

Abstract Objectives Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects 10% of reproductive age women and leads to hyperandrogenism, abnormal menstrual cycles, and polycystic ovaries. Moreover, PCOS has been associated with elevated serum homocysteine; however, the characterization of one-carbon metabolism (OCM) in PCOS remains incomplete. The aim of our research was to examine OCM in a genetic and chemically-induced rodent model of PCOS: 1) viable yellow Agouti (Avy) mice; and 2) letrozole (Let)-induced Sprague Dawley (SD) rats. Methods Five wk old female Avy mice (N = 18), their lean controls (N = 18), and SD rats (N = 36) were acclimated for one wk. Following acclimation, the animals were placed on a modified standard AIN93G diet (energy, %: 50.4, carbohydrate; 17.3, protein; and 32.3, fat). Rats were randomly assigned to Let (1 g/kg BW) treatment or vehicle (carboxymethylcellulose) control that was administered via a subcutaneously implanted slow-release pellet every 30-d. For both models, 12 animals were randomly assigned to be euthanized during proestrus at one of the following ages: 8, 16 or 24 wk. Bodyweight and estrous cycles were measured daily. Ovaries were collected to assess gene expression of OCM. These data were analyzed using linear mixed models to determine the main effects of age and treatment at a significance level of P < 0.05. Results Letrozole significantly reduced the occurrence of proestrus and estrus stages (P = 0.0001 and P = 0.006, respectively). Additionally, Let-induced rats had increased BW compared to control rats, across all age groups (P < 0.0001). In contrast, Avy mice weighed less than their controls by 24 wk of age (P < 0.0001). Cystathionine-β synthase (CBS) mRNA expression was downregulated in the Let-induced vs. control rats at 16 (59%; P < 0.05) and 24 (77%; P < 0.01) wk of age. As expected, Cyp19A1, aromatase mRNA was downregulated in the Let-induced rats (P = 0.02). Interestingly, betaine-homocysteine s-methyltransferase (BHMT) mRNA increased as a function of age in Let-induced rats (P = 0.03). Conclusions These data demonstrate that Letrozole-induced PCOS temporally decreases ovarian CBS mRNA expression; whereas, BHMT mRNA is upregulated as a function of age. Funding Sources This work was supported by the National Institute of Child Health and Human Development.

Increased risk for T cell autoreactivity to \xdf-cell antigens in the mice expressing the Avy obesity-associated gene

There has been considerable debate as to whether obesity can act as an accelerator of type 1 diabetes (T1D). We assessed this possibility using transgenic mice (MIP-TF mice) whose ß-cells express enhanced green fluorescent protein (EGFP). Infecting these mice with EGFP-expressing murine herpes virus-68 (MHV68-EGFP) caused occasional transient elevation in their blood glucose, peri-insulitis, and Th1 responses to EGFP which did not spread to other ß-cell antigens. We hypothesized that obesity-related systemic inflammation and ß-cell stress could exacerbate the MHV68-EGFP-induced ß-cell autoreactivity. We crossed MIP-TF mice with Avy mice which develop obesity and provide models of metabolic disease alongside early stage T2D. Unlike their MIP-TF littermates, MHV68-EGFP–infected Avy/MIP-TF mice developed moderate intra-insulitis and transient hyperglycemia. MHV68-EGFP infection induced a more pronounced intra-insulitis in older, more obese, Avy/MIP-TF mice. Moreover, in MHV68-EGFP-infected Avy/MIP-TF mice, Th1 reactivity spread from EGFP to other ß-cell antigens. Thus, the spreading of autoreactivity among ß-cell antigens corresponded with the transition from peri-insulitis to intra-insulitis and occurred in obese Avy/MIP-TF mice but not lean MIP-TF mice. These observations are consistent with the notion that obesity-associated systemic inflammation and ß-cell stress lowers the threshold necessary for T cell autoreactivity to spread from EGFP to other ß-cell autoantigens.

Epigenetic Responses to Low Dose Ionizing Radiation

Two epigenetically regulated subsets of genes that potentially link environmental exposures early in development to adult diseases are imprinted genes and those with metastable epialleles. Genes with metastable epialleles have highly variable expressions because of stochastic allelic modifications in the epigenome. Genomic imprinting is an unusual epigenetic form of gene regulation that results in monoallelic expression in a parent-of-origin dependent manner. The viable yellow agouti (Avy) mouse harbors a metastable Agouti gene because of an upstream insertion of a transposable element. We previously used this animal model to demonstrate that nutritional and chemical toxicant exposures during early development induce persistent epigenetic changes at the Avy locus that result in alterations in coat color and adult disease susceptibility. We also showed that low doses of ionizing radiation (

Maternal obesity heritably perturbs offspring metabolism for three generations without serial programming.

Maternal obesity can program offspring metabolism across multiple generations. It is not known whether multigenerational effects reflect true inheritance of the induced phenotype, or are due to serial propagation of the phenotype through repeated exposure to a compromised gestational milieu. Here we sought to distinguish these possibilities, using the Avy mouse model of maternal obesity. In this model, F1 sons of obese dams display a predisposition to hepatic insulin resistance, which remains latent unless the offspring are challenged with a Western diet. We find that F2 grandsons and F3 great grandsons of obese dams also carry the latent predisposition to metabolic dysfunction, but remain metabolically normal on a healthy diet. Given that the breeding animals giving rise to F2 and F3 were maintained on a healthy diet, the latency of the phenotype permits exclusion of serial programming; we also confirmed that F1 females remained metabolically healthy during pregnancy. Molecular analyses of male descendants identified upregulation of hepatic Apoa4 as a consistent signature of the latent phenotype across all generations. Our results exclude serial programming as a factor in transmission of the metabolic phenotype induced by ancestral maternal obesity, and indicate inheritance through the germline, probably via some form of epigenetic inheritance.

Age-specific locomotor response to nicotine in yellow and mottled yellow Avy/a mice

BackgroundMost Agouti viable yellow (Avy) mice display constitutive expression of agouti protein, which acts as an inverse agonist at the melanocortin receptor 4 (Mc4r), resulting in adult-onset obesity as well as an altered sensitivity to some drugs of abuse. We investigated the influence of excessive agouti expression on open-field locomotor response to daily 0.5 mg/kg (-)-freebase nicotine injections in 27 early adolescent and 27 young adult male Avy/a and a/a mice, and assessed the effects of nicotine administration (0.5 mg/kg) followed by open-field testing on serum corticosterone levels in a separate group of 25 young adult male Avy/a and a/a mice.FindingsYoung adult Avy/a mice displayed pronounced nicotine-induced hypolocomotion (a 24% reduction in distance traveled) compared to their a/a littermates. Early adolescent Avy/a mice did not differ from their a/a littermates or saline-matched controls in locomotion following nicotine administration. Young adult Avy/a mice also displayed increased thigmotaxis (a 5% increase in time spent outside the center of the apparatus) on the first day of nicotine administration as compared to saline-matched controls, while a/a mice did not. An increase in serum corticosterone levels 20 minutes after nicotine injection in a separate group of young adult male mice (n = 25) was proportionally similar between Avy/a and a/a mice.ConclusionsOverall, the results suggest an age- and epigenotype- or genotype-specific response to nicotine administration in young adult male Avy/a mice. It appears the Avy/a locomotor and thigmotaxic responses to acute nicotine administration are not mediated solely by hypothalamic-pituitary-adrenal (HPA) axis stimulation.

Epigenetics for ecotoxicologists

Do genes define your destiny? The field of epigeneticsexamines how genes and the environment interact to formthe basis of heredity and comes up with some surprisingfindings. We often think of deoxyribonucleic acid (DNA) asthe sole physical basis for heredity, the genetic code thatdetermines everything from eye and hair color to specificpersonality traits. The growing field of epigenetics suggeststhat this traditional paradigm is an oversimplification. Epi-genetics is the study of factors that are heritable, but whichoccur by mechanisms other than changes in the DNA codeitself. C.H. Waddington first used the term epigenetics in theearly 1940s to describe ‘‘the interactions of genes with theirenvironment, which bring the phenotype into being.’’ Epi-genetic marks are susceptible to environmental influences—both chemical and nonchemical—and can be inherited inways that may seem counterintuitive (see sidebar, ExpandingIdeas About Biological Inheritance). Effects of early lifeexperiences, such as parental care or nutrition, can showup later in life and even be passed on to future generations.This emerging area of research has interesting and importantimplications for the field of ecotoxicology—from basicscience to international policy.In classic genetics, genetic material is in the form of DNAand encodes all of the information necessary for life. Thisinformation is copied faithfully and passed down as cellsdivide. Every cell contains a complete copy of the DNAcode, but the pattern of gene expression—that is, whichgenes are turned ‘‘on’’ (expressed) or ‘‘off’’ (nonex-pressed)—determines the cell type and function. A skin cellon your arm contains the same DNA code as a nerve cell inyour brain, but their extreme differences in structure andfunction are due to the particular set of genes that each isexpressing. Epigenetics refers to an annotation in the form ofchemical marks on top of the DNA code; the prefix ‘‘epi’’comesfromaGreekwordmeaning‘‘over’’or‘‘above.’’Thesechemical marks, which are discussed in detail below, affectwhich genes are expressed and at what levels. Epigeneticmarks are highly influenced by the environment and can beinherited along with the genetic code as cells divide mitoti-cally, and in some cases meiotically, from one generation tothe next.Epigenetics is receiving significant attention in the field ofbiomedicine. A PubMed search for ‘‘epigenetic’’ revealedthat more than 1,300 review articles have been written on thetopic, of which more than 50% were published in the lastthree years alone. Epigenetics provides a mechanism for theBarker hypothesis, which postulates that nutrition and otherenvironmentalfactorsearlyindevelopmentcanaltersuscept-ibility to chronic diseases in adulthood. A classic exampleof this is that individuals who were conceived during theDutchWinterHunger(1944–1945)havepersistentepigeneticalterations on a characteristic gestational marker, the IGF2gene, as adults. Furthermore, individuals who experiencedthe Dutch Winter Hunger have higher rates of metabolicdisorders and cardiovascular disease, the epigenetic mecha-nisms of which are the focus of ongoing studies. Nutritionaldeprivation may also have an impact on human longevity;indeed, lifespanisnegativelycorrelated withfoodabundance

EHP Paper of the Year, 2011

In 2008, Environmental Health Perspectives (EHP) established the Paper of the Year Award as a means of recognizing high-impact papers published in the journal (Tilson 2008). Originally, the Paper of the Year was selected on the basis of citations received over the preceding 60 months. Earlier this year EHP announced that it would be recognizing two papers each year (Tilson 2011). The EHP Classic Paper of the year will be the research article, commentary, or review article that is the most highly cited paper over the preceding 60 months. The winner of the 2011 EHP Classic Paper of the Year was “Maternal Genistein Alters Coat Color and Protects Avy Mouse Offspring from Obesity by Modifying the Fetal Epigenome” by Dana C. Dolinoy, Jennifer R. Weidman, Robert A. Waterland, and Randy Jirtle. The second paper to be recognized each year will be the EHP Paper of the Year. This award will recognize a highly cited paper published during the preceding year. Both awards are subject to the approval of the EHP Board of Associate Editors. In this issue, EHP announces that the Paper of the Year for 2011 is “Global Estimates of Ambient Fine Particulate Matter Concentrations from Satellite-Based Aerosol Optical Depth: Development and Application” by Aaron van Donkelaar, Randall V. Martin, Michael Brauer, Ralph Kahn, Robert Levy, Carolyn Verduzco, and Paul J. Villeneuve. This paper (van Donkelaar et al. 2010) was published in the June 2010 issue of the journal. At the time the paper was written, it was clear that chronic exposure to fine particulate matter (< 2.5 µm in diameter; PM2.5) could harm human health by producing morbidity and mortality. van Donkelaar et al. (2010) recognized that the sparseness of monitoring data, especially in developing countries, had hindered epidemiologic and health impact studies of PM2.5. They noted that satellite remote sensing offers unparalleled global coverage of aerosol optical depth (AOD), a measure of aerosol over the entire atmospheric column, and that the relationship between AOD and near-surface PM2.5 varies in both space and time. van Donkelaar et al. (2010) related AOD to near-surface PM2.5 by using a chemical transport model that predicts the state of the atmosphere from meteorologic data sets, emission inventories, and equations that represent the physical and chemical evolution of atmospheric constituents. van Donkelaar et al. (2010) combined AODs from two NASA (National Aeronautics and Space Administration) satellite instruments [Moderate Resolution Imaging Spectroradiometer (MODIS) and Multi-angle Imaging Spectroradiometer (MISR)] with the Goddard Earth Orbiting System chemical transport model to produce global long-term average (2001–2006) PM2.5 estimates at approximately 10 km × 10 km horizontal resolution. They found significant agreement with ground-based PM2.5 monitors, where present, both in North America and around the world. van Donkelaar et al. (2010) provided an observationally based data set to assess the impact of ambient PM2.5 exposure globally. The authors estimated that the World Health Organization (WHO) Air Quality Guideline of 10 μg/m3 is exceeded for 80% of the world’s population. Over eastern Asia, half the population has annual ambient mean PM2.5 exposures in excess of the WHO PM2.5 interim target of 35 μg/m3, and in eastern China annual mean levels exceed 80 μg/m3. EHP congratulates van Donkelaar and colleagues for their contribution to the environmental health science literature. Their findings have broad implications for the global environmental health and risk assessment communities.

Use of Embryo Transfer to Determine the Effects of a Maternal Diet Enriched in Methyl Components on Body Weight and Serum Glucose Concentrations in Viable Yellow Offspring.

Epigenetics might account for various disease states, including obesity and diabetes, which are not explained by classic genetics. The best characterized animal model in which an epigenetic change in a gene is inherited and leads to well defined pathological outcomes is the viable yellow (Avy) mouse, which possess coat colors ranging from fully yellow to mottled, to completely brown, depending on degree of methylation of an intracisternal A particle (IAP) inserted in the promoter of their agouti gene. Yellow Avy mice with a demethylated IAP develop “yellow obese syndrome”, typified by obesity and diabetes; whereas, brown Avy, siblings, with a methylated IAP site, and black (a/a) siblings remain healthy. Addition of nutrients, likely to enrich the pool of methyl donors and vitamin co-factors required for methylation, to the diet of dams from the time of conception to term has been reported to increase DNA methylation of the IAP promoter site, such that more offspring are brown and less prone to “yellow-obese syndrome”. However, no previous study has determined when offspring are most sensitive to components in maternal diet. Herein, we used an embryo transfer approach to determine when during this time period, offspring are sensitive to methyl components. C57Bl6J females, maintained on either AIN93G control (C) or methyl-enriched (M) diet, were bred to Avy/a males. Zygotes from these matings were transferred into CD1 recipient females, also on either C or M diet. Thus, there were four groups (C to C, 20 litters, 84 pups; C to M, 22 litters, 108 pups; M to C, 14 litters, 79 pups; M to M, 9 litters, 50 pups). Coat color of the pups was assessed at 21 days of age, and all pups were maintained on the C diet. Each week, a subset of each group was weighed and saphenous vein blood serum collected and assayed for glucose by using a Bayer Contour blood glucose monitoring system. Our initial statistical assessment of coat color differences was by Chi-Squared analysis, and, based on this analysis, it appeared that there was a significant shift in offspring coat color based on donor and recipient diet combinations with M to M unexpectantly yielding more yellow coat color offspring (P values ranging from 0.001 to 0.04). However, when data were analyzed by GLIMMOX, which treats the litter rather than individual pups as a unit and is the more appropriate statistical test, there was no difference in coat color between the four groups (P = 0.81). At 40 days of age, offspring born to C to C and M to C groups weighed less than C to M and M to M offspring (C to C = 23.3 ± 1.4 g, M to C = 23.3 ± 1.1 g, C to M = 27.4 ± 1.4 g, M to M = 29.2 ± 1.5 g, P < 0.05). At this age, offspring in C to C group demonstrated lower serum glucose concentrations than mice in the other three groups (C to C = 126.6 ± 11.6 mg/dl, C to M = 160.0 ± 12.0 mg/dl, M to C = 155.6 ± 9.3 mg/dl, and M to M = 177.1 ± 12.1 mg/dl, P < 0.05). These studies demonstrated that when litter is treated as the experimental unit, there were no differences in coat color brought about by increased methyl donors in maternal diet. However, offspring exposed to M diet during gestation were heavier and exhibited higher serum glucose concentrations than those exposed to a control diet throughout gestation. These results suggest that methyl supplements or other components in the methyl-enriched diet influence the embryonic developmental outcome from periconception to term and possibly even increase the risk for adult-onset diseases, including obesity and diabetes. (poster)

Does In Utero Bisphenol A Exposure Alter Anxiety Behaviors in Yellow and Brown Coat Color Avy Mice?

Does In Utero Bisphenol A Exposure Alter Anxiety Behaviors in Yellow and Brown Coat Color Avy Mice?

EHP Classic Paper of the Year, 2011

Environmental Health Perspectives (EHP) established the Paper of the Year in 2008 (Tilson 2008) as a way of highlighting high-quality articles published in the journal. Until now, the Paper of the Year for any given year was selected on the basis of citations received over the preceding 60 months. Starting this year, this award will be known as the EHP Classic Paper of the Year. This award will be given to the most highly cited Research Article, Commentary, or Review Article over the preceding 60 months. A new award, the EHP Paper of the Year, will honor a Research Article published in the preceding year. Final selection of both awards will be subject to approval by the EHP Board of Associate Editors. The winner of the EHP Paper of the Year will be announced later in the calendar year. We are proud to announce that the 2011 EHP Classic Paper of the Year is “Maternal Genistein Alters Coat Color and Protects Avy Mouse Offspring from Obesity by Modifying the Fetal Epigenome” by Dana C. Dolinoy, Jennifer R. Weidman, Robert A. Waterland, and Randy L. Jirtle. This article was published in the April 2006 issue of EHP (Dolinoy et al. 2006) and has been cited more than 35 times per year since it was published. At the time the paper was written, it was increasingly recognized that exposure to nutritional, chemical, and behavioral factors could alter gene expression and affect health and disease not only by mutating promoter and coding regions of genes but also by modifying the epigenome. The epigenome comprises the heritable changes in gene expression that occur in the absence of changes to the DNA sequence itself, including DNA methylation and chromatin packaging. If the genome is compared to the hardware in a computer, the epigenome is the software that directs the computer’s operation. Dolinoy et al. (2006) argued that identifying epigenetic targets and defining how they are influenced by nutrition and the environment might lead to the development of innovative diagnostic, treatment, and prevention strategies that target the “epigenomic software” rather than the “genomic hardware.” Dolinoy et al. (2006) used the viable yellow agouti (Avy) mouse as an epigenetic biosensor to demonstrate that genistein, the major phytoestrogen in soy, increases DNA methylation of the Agouti gene, resulting in population-level decreased incidence of adult-onset obesity, diabetes, and cancer. At the inception of this study, epidemiological data suggested that increased dietary genistein plays a role in decreased incidence of cancer in Asians compared with Westerners. Thus, they hypothesized that early dietary exposures, including genistein, might be linked to adult health status via epigenetic mechanisms. These authors demonstrated that maternal dietary genistein supplementation of mice during gestation at levels comparable to humans consuming high soy diets shifted the coat color of heterozygous viable yellow agouti (Avy/a) offspring toward pseudoagouti. This phenotypic change was significantly associated with increased methylation of six cytosine–guanine sites in a retrotransposon upstream of the transcription start site of the Agouti gene. A significant finding was that genistein-induced hypermethylation persisted into adulthood, decreasing ectopic Agouti expression and protecting offspring from adult-onset obesity. Dolinoy et al. (2006) provided the first evidence that in utero dietary genistein affects gene expression and alters susceptibility to obesity in adulthood by permanently altering the epigenome. They also established the framework for future studies by showing that both genistein and methyl donors, such as folic acid, betaine, and choline, counteract DNA hypomethylation caused by bisphenol A, an endocrine-active agent used to make polycarbonate plastic. Their results showed that simple dietary changes can protect against the deleterious effects of environmental toxicants on the fetal epigenome (Dolinoy et al. 2007). EHP congratulates Dolinoy and colleagues for their contribution to the environmental health science literature. In addition to demonstrating that single-nucleotide polymorphisms can affect environmentally responsive genes, they demonstrated that early nutritionally and environmentally induced epigenetic modifications may be an alternative mechanism underlying individual susceptibilities to environmental agents.

Astrocyte Leptin Receptor (ObR) and Leptin Transport in Adult-Onset Obese Mice

The agouti viable yellow (A vy) spontaneous mutation generates an unusual mouse phenotype of agouti-colored coat and adult-onset obesity with metabolic syndrome. Persistent production of agouti signaling protein in A vy mice antagonizes melanocortin receptors in the hypothalamus. To determine how this disruption of neuroendocrine circuits affects leptin transport across the blood-brain barrier (BBB), we measured leptin influx in A vy and B6 control mice after the development of obesity, hyperleptinemia, and increased adiposity. After iv bolus injection, (125)I-leptin crossed the BBB significantly faster in young (2 month old) B6 mice than in young A vy mice or in older (8 month old) mice of either strain. This difference was not observed by in situ brain perfusion studies, indicating the cause being circulating factors, such as elevated leptin levels or soluble receptors. Thus, A vy mice showed peripheral leptin resistance. ObRa, the main transporting receptor for leptin at the BBB, showed no change in mRNA expression in the cerebral microvessels between the age-matched (2 month old) A vy and B6 mice. Higher ObRb mRNA was seen in the A vy microvasculature with unknown significance. Immunofluorescent staining unexpectedly revealed that many of the ObR(+) cells were astrocytes and that the A vy mice showed significantly more ObR(+) astrocytes in the hypothalamus than the B6 mice. Although leptin permeation from the circulation was slower in the A vy mice, the increased ObR expression in astrocytes and increased ObRb mRNA in microvessels suggest the possibility of heightened central nervous system sensitivity to circulating leptin.

Mahogany, blood–brain barrier, and fat mass surge in AVY mice

Avy/agouti (Avy) mice have late onset obesity related to overexpression of agouti signaling protein (ASP) in the hypothalamus. As mahogany modulates the actions of ASP, we tested the transport of mahogany peptide across the blood-brain barrier (BBB). The brain uptake of mahogany peptide was significantly higher in young Avy mice, and it preceded the surge of fat mass quantified by nuclear magnetic resonance. The results suggest a role of accelerated BBB transport in the epigenetics of Avy mice.

Assessing the Effects of High Methionine Intake on DNA Methylation

Methylation of DNA occurs at cytosines within CpG (cytosine-guanine) dinucleotides and is 1 of several epigenetic mechanisms that serve to establish and maintain tissue-specific patterns of gene expression. The methyl groups transferred in mammalian DNA methylation reactions are ultimately derived from methionine. High dietary methionine intake might therefore be expected to increase DNA methylation. Because of the circular nature of the methionine cycle, however, methionine excess may actually impair DNA methylation by inhibiting remethylation of homocysteine. Although little is known regarding the effect of dietary methionine supplementation on mammalian DNA methylation, the available data suggest that methionine supplementation can induce hypermethylation of DNA in specific genomic regions. Because locus-specific DNA hypomethylation is implicated in the etiology of various cancers and developmental syndromes, clinical trials of "promethylation" dietary supplements are already under way. However, aberrant hypermethylation of DNA could be deleterious. It is therefore important to determine whether dietary supplementation with methionine can effectively support therapeutic maintenance of DNA methylation without causing excessive and potentially adverse locus-specific hypermethylation. In the viable yellow agouti (Avy) mouse, maternal diet affects the coat color distribution of offspring by perturbing the establishment of methylation at the Avy metastable epiallele. Hence, the Avy mouse can be employed as a sensitive epigenetic biosensor to assess the effects of dietary methionine supplementation on locus-specific DNA methylation. Recent developments in epigenomic approaches that survey locus-specific DNA methylation on a genome-wide scale offer broader opportunities to assess the effects of high methionine intake on mammalian epigenomes.

The hypothalamus and the regulation of energy homeostasis: lifting the lid on a black box.

The hypothalamus is the focus of many peripheral signals and neural pathways that control energy homeostasis and body weight. Emphasis has moved away from anatomical concepts of 'feeding' and 'satiety' centres to the specific neurotransmitters that modulate feeding behaviour and energy expenditure. We have chosen three examples to illustrate the physiological roles of hypothalamic neurotransmitters and their potential as targets for the development of new drugs to treat obesity and other nutritional disorders. Neuropeptide Y (NPY) is expressed by neurones of the hypothalamic arcuate nucleus (ARC) that project to important appetite-regulating nuclei, including the paraventricular nucleus (PVN). NPY injected into the PVN is the most potent central appetite stimulant known, and also inhibits thermogenesis; repeated administration rapidly induces obesity. The ARC NPY neurones are stimulated by starvation, probably mediated by falls in circulating leptin and insulin (which both inhibit these neurones), and contribute to the increased hunger in this and other conditions of energy deficit. They therefore act homeostatically to correct negative energy balance. ARC NPY neurones also mediate hyperphagia and obesity in the ob/ob and db/db mice and fa/fa rat, in which leptin inhibition is lost through mutations affecting leptin or its receptor. Antagonists of the Y5 receptor (currently thought to be the NPY 'feeding' receptor) have anti-obesity effects. Melanocortin-4 receptors (MC4-R) are expressed in various hypothalamic regions, including the ventromedial nucleus and ARC. Activation of MC4-R by agonists such as alpha-melanocyte-stimulating hormone (a cleavage product of pro-opiomelanocortin which is expressed in ARC neurones) inhibits feeding and causes weight loss. Conversely, MC4-R antagonists such as 'agouti' protein and agouti gene-related peptide (AGRP) stimulate feeding and cause obesity. Ectopic expression of agouti in the hypothalamus leads to obesity in the AVY mouse, while AGRP is co-expressed by NPY neurones in the ARC. Synthetic MC4-R agonists may ultimately find use as anti-obesity drugs in human subjects Orexins-A and -B, derived from prepro-orexin, are expressed in specific neurones of the lateral hypothalamic area (LHA). Orexin-A injected centrally stimulates eating and prepro-orexin mRNA is up regulated by fasting and hypoglycaemia. The LHA is important in receiving sensory signals from the gut and liver, and in sensing glucose, and orexin neurones may be involved in stimulating feeding in response to falls in plasma glucose.

Epigenetic inheritance at the agouti locus in the mouse.

Epigenetic modifications have effects on phenotype, but they are generally considered to be cleared on passage through the germ line in mammals, so that only genetic traits are inherited. Here we describe the inheritance of an epigenetic modification at the agouti locus in mice. In viable yellow ( A(vy)/a) mice, transcription originating in an intra-cisternal A particle (IAP) retrotransposon inserted upstream of the agouti gene (A) causes ectopic expression of agouti protein, resulting in yellow fur, obesity, diabetes and increased susceptibility to tumours. The pleiotropic effects of ectopic agouti expression are presumably due to effects of the paracrine signal on other tissues. Avy mice display variable expressivity because they are epigenetic mosaics for activity of the retrotransposon: isogenic Avy mice have coats that vary in a continuous spectrum from full yellow, through variegated yellow/agouti, to full agouti (pseudoagouti). The distribution of phenotypes among offspring is related to the phenotype of the dam; when an A(vy) dam has the agouti phenotype, her offspring are more likely to be agouti. We demonstrate here that this maternal epigenetic effect is not the result of a maternally contributed environment. Rather, our data show that it results from incomplete erasure of an epigenetic modification when a silenced Avy allele is passed through the female germ line, with consequent inheritance of the epigenetic modification. Because retrotransposons are abundant in mammalian genomes, this type of inheritance may be common.

Angiotensinogen gene expression in adipose tissue: analysis of obese models and hormonal and nutritional control.

Synthesis of angiotensin II (ANG II) has recently been described in adipose cells and has been linked to regulation of adiposity. Angiotensinogen (AGT), the substrate from which ANG II is formed, was previously shown to be elevated in adipose tissue of obese (ob/ob and db/db) mice and regulated by nutritional manipulation. It is unknown, however, whether overexpression of adipose AGT can be extended to other models of obesity and whether hormonal and/or nutritional factors directly regulate AGT expression in adipocytes. We investigated these possibilities by analyzing AGT mRNA levels in adipose tissue of obese Zucker rats, viable yellow (Avy) mice, and humans and by treating 3T3-L1 adipocytes with insulin, glucose, and a beta-adrenergic agonist. We demonstrate that AGT mRNA is decreased by approximately 50 and 80%, respectively, in adipose tissue of obese vs. lean Zucker rats and Avy mice. We also report that AGT is expressed at variable levels in human adipose tissue. Finally, we show that AGT mRNA is upregulated by insulin and downregulated by beta-adrenergic stimulation in adipocytes.

Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity.

The brown fat-specific mitochondrial uncoupling protein (UCP) provides a mechanism for generating heat by uncoupling respiration and oxidative phosphorylation. It has been suggested that this system of thermogenesis can provide a defense against obesity. To test this idea, we created a transgenic mouse in which the fat-specific aP2 gene promoter directed Ucp expression in white fat and provided for the constitutive expression of Ucp in brown fat. Transgenic mice showed both Ucp mRNA and immunoreactive UCP in white fat at 2-10% the level normally measured in brown fat. A reduction in subcutaneous fat of aP2-Ucp C57BL/6J mice was observed at 3 mo of age. When the transgene was expressed in Avy genetically obese mice reductions in total body weight and subcutaneous fat stores were observed. Female transgenic Avy mice at 13 mo of age weighed 35 grams, a weight indistinguishable from nontransgenic C57BL/6J mice. Gonadal fat showed an increase in a novel adipocyte derivative that did not accumulate lipids and that constituted approximately 80% of the mass of the tissue in Avy transgenic. A major effect of aP2-Ucp in brown fat was to reduce endogenous gene expression by as much as 95%. The results suggest that UCP synthesized from the aP2 gene promoter is thermogenically active and capable of reducing fat stores.

Insulin resistance vs. hyperinsulinemia in hypertension: insulin regulation of Ca2+ transport and Ca(2+)-regulation of insulin sensitivity.

Hypertension in obesity and insulin resistance has been attributed to insulin stimulation of sympathetic neural output and renal sodium retention. However, recent data demonstrates a significant vasodilatory effect of insulin and suggests that vascular smooth muscle resistance to this action may instead be the cause of hypertension in insulin resistance. This concept is supported by the observation that pharmacological amplification of peripheral insulin sensitivity results in reduced arterial pressure. Insulin attenuates vasoconstrictor responses to pressor agonists and accelerates vascular smooth muscle relaxation, while these effects are blunted in obesity and insulin resistance. Insulin regulation of vasoconstriction and vascular relaxation appears to be secondary to regulation of intracellular Ca2+ ([Ca2+]i), as insulin attenuates both voltage- and receptor-mediated Ca2+ influx and stimulates both the transcription and activity of Ca(2+)-ATPase in vascular smooth muscle cells. Further, these effects are also blunted in insulin resistance. Although [Ca2+]i plays a poorly understood role in insulin signalling, increases beyond an optimal range results in impaired insulin sensitivity, possibly by Ca(2+)-inhibition of insulin-induced dephosphorylation of insulin-sensitive substrates. Consistent with this concept, ectopic overexpression of the agouti gene in the viable yellow (Avy) mouse results in increased skeletal myocyte [Ca2+]i. Accordingly, increased [Ca2+]i in primary insulin target tissues appears to result in peripheral insulin resistance which then results in aberrant regulation of vascular smooth muscle [Ca2+]i and increases in arterial pressure.

Coat Color in Intact and Neurointermediate Lobe-Grafted Agouti Mice: Effect of Dopamine Agonists and Antagonists

The natural variation in the coat color of agouti C3H Avy mice was observed and reflectometrically recorded during the first 6 months of life. Three well-defined color stages were detected: (a) yellow coat from birth to the 6th week; (b) dark-gray coat from the 8th week to the 6th month, and (c) from the 6th month onward a yellow coat prevailed. To clarify the role played by the pituitary pars intermedia on these coat color variations, pimozide or d-butaclamol were chronically administered to newborn and 6-month-old mice. These drugs produced an intense darkening in the color of the growing hairs in newborn mice and in the color of the newly grown hair of adult preplucked mice. Pituitary pars intermedia grafted in adult yellow mice also produced an intense coat darkening of newly grown hair. Dopaminergic agonists, administered either subcutaneously or close to the grafts, prevented this darkening effect. It is suggested that pars intermedia peptides might be responsible for the hair color cycle in this mouse strain and that peptide secretion is controlled by a tonic inhibitory dopaminergic mechanism which operates from the first days of life.

Alteration of the Agouti mouse coat color pattern by bromoergocryptine. Possible involvement of MSH.

In the Agouti mice C3H Avy the coat color of the dorsum changes from birth to maturity. In young animals the dorsal tegument is yellow whereas in adult mice it shifts to dark gray. The administration of bromoergocryptine, a dopamine agonist, as a single injection in a beeswax pellet prevented the color change resulting in the persistence of the immature pattern. After plucking an area of the dorsum of an adult animal, the regrown hair was dark; however, when bromoergocryptine was administered at the time of plucking the new hair was not dark but yellow. When MSH was injected in adult animals previously treated with the drug, the 'bleaching' effect of bromoergocryptine was abolished, suggesting that this substance did not act directly upon melanin synthesis. In bromoergocryptine-treated mice the MSH content of the pars intermedia was decreased and the ultrastructure of this lobe contained cells with signs of hypoactivity. These two observations suggested that the effect of the drug on the coat color might be ascribed to inhibition of the secretion of MSH. The results may indicate that in the Agouti C3H Avy mice MSH plays a physiological role in determining the coat color. On the other hand, in CH57BL mice bromoergocryptine did not elicit color changes suggesting that in this strain the normal color of the hair is not MSH-dependent.