George L. Wolff

Maternal epigenetics and methyl supplements affect agouti gene expression in Avy/a mice

‘Viable yellow’ (Avy/a) mice are larger, obese, hyperinsulinemic, more susceptible to cancer, and, on average, shorter lived than their non‐yellow siblings. They are epigenetic mosaics ranging from a yellow phenotype with maximum ectopic agouti overexpression, through a continuum of mottled agouti/yellow phenotypes with partial agouti overexpression, to a pseudoagouti phenotype with minimal ectopic expression. Pseudoagouti Avy/a mice are lean, healthy, and longer lived than their yellow siblings. Here we report that feeding pregnant black a/a dams methyl‐supplemented diets alters epigenetic regulation of agouti expression in their offspring, as indicated by increased agouti/black mottling in the direction of the pseudoagouti phenotype. We also present confirmatory evidence that epigenetic phenotypes are maternally heritable. Thus Avy expression, already known to be modulated by imprinting, strain‐specific modification, and maternal epigenetic inheritance, is also modulated by maternal diet. These observations suggest, at least in this special case, that maternal dietary supplementation may positively affect health and longevity of the offspring. Therefore, this experimental system should be useful for identifying maternal factors that modulate epigenetic mechanisms, especially DNA methylation, in developing embryos.—Wolff, G. L., Kodell, R. L., Moore, S. R., Cooney, C. A. Maternal epigenetics and methyl supplements affect agouti gene expression in Avy/a mice. FASEB J. 12, 949–957 (1998)

The case for strategic international alliances to harness nutritional genomics for public and personal health

Nutrigenomics is the study of how constituents of the diet interact with genes, and their products, to alter phenotype and, conversely, how genes and their products metabolise these constituents into nutrients, antinutrients, and bioactive compounds. Results from molecular and genetic epidemiological studies indicate that dietary unbalance can alter gene-nutrient interactions in ways that increase the risk of developing chronic disease. The interplay of human genetic variation and environmental factors will make identifying causative genes and nutrients a formidable, but not intractable, challenge. We provide specific recommendations for how to best meet this challenge and discuss the need for new methodologies and the use of comprehensive analyses of nutrient-genotype interactions involving large and diverse populations. The objective of the present paper is to stimulate discourse and collaboration among nutrigenomic researchers and stakeholders, a process that will lead to an increase in global health and wellness by reducing health disparities in developed and developing countries.

Soy Protein Isolate Reduces Hepatosteatosis in Yellow Avy/a Mice Without Altering Coat Color Phenotype

Agouti (A vy /a) mice fed an AIN-93G diet containing the soy isoflavone genistein (GEN) prior to and during pregnancy were reported to shift coat color and body composition phenotypes from obese-yellow towards lean pseudoagouti, suggesting epigenetic programming. Human consumption of purified GEN is rare and soy protein is the primary source of GEN. Virgin a/a female and Avy/a male mice were fed AIN-93G diets made with casein (CAS) or soy protein isolate (SPI) (the same approximate GEN levels as in the above mentioned study) for 2 wks prior to mating. A vy /a offspring were weaned to the same diets and studied at age 75 d. Coat color distribution did not differ among diets, but SPI-fed, obese A vy /a offspring had lower hepatosteatosis (P < 0.05) and increased (P < 0.05) expression of CYP4a 14, a PPARα-regulated gene compared to CAS controls. Similarly, weanling male Sprague-Dawley (SD) rats fed SPI had elevated hepatic Acyl Co-A Oxidase (ACO) mRNA levels and increased in vitro binding of PPARα to the PPRE promoter response element. In another hepatosteatosis model, adult SD rats fed a high fat/cholesterol diet, SPI reduced (P < 0.05) steatosis. Thus, 1) consumption of diets made with SPI partially protected against hepatosteatosis in yellow mice and in SD rats, and this may involve induction of PPARα-regulated genes; and 2) the lifetime (in utero, neonatal and adult) exposure to dietary soy protein did not result in a shift in coat color phenotype of A vy /a mice. These findings, when compared with those of previously published studies of A vy /a mice, lead us to conclude that: 1) the effects of purified GEN differ from those of SPI when GEN equivalents are closely matched; 2) SPI does not epigenetically regulate the agouti locus to shift the coat color phenotype in the same fashion as GEN alone; and 3) SPI may be beneficial in management of non-alcoholic fatty liver disease

Accelerated appearance of chemically induced mammary carcinomas in obese yellow (avy/a) (balb/c x vy)f1 hybrid mice.

Latent periods and cumulative incidence of mammary carcinomas (MT) up to 50 wk after initial gavage with 7.12-dimethylbenz[a]anthracene (DMBA) were determined in virgin yellow (Avy/A) and agouti (A/a) (BALB/cStCrlfC3Hf/Nctr X VY/WffC3Hf/Nctr-Avy) F1 hybrid female mice. When subcutaneous masses reached 5-10 mm in diameter, the mice were killed and necropsied, and the tissues examined histologically. No MT were found in control mice. Cumulative MT incidence in the 1.5-mg DMBA group (A) was 43% (41/95) among yellow mice, and 33% (32/96) among agoutis. In the 6.0-mg DMBA group (B), corresponding MT incidences were 86% (83/96) and 71% (67/95). In group A, the first percentile of MT detection was 13.0 wk after initial carcinogen treatment in yellow mice; it was 18.0 wk in agoutis. Corresponding latent periods for the 20th percentile were 34.3 and 47.0 wk. In group B, latencies for the first percentile were 8.3 and 9.0 wk. Corresponding latencies for the 20th percentile were 15.3 and 16.0 wk. Within genotypes and dose groups, rates of weight gain of mice that developed MT and those that did not were similar. We conclude that MT induced by low doses of DMBA arise more rapidly in yellow mice than in nonyellow littermates. The absence of spontaneous MTs, acceleration of chemically induced MT formation at a low dose level that does not induce general toxicity, and availability of genetically identical (except for one gene) normal control animals make this experimental system suitable for development of an assay to efficiently test the carcinogenic potential of low dose levels of chemical substances.

Genome-Linked Toxic Responses to Dietary Iron Overload

Genome-related differences to Fe overload between and within rodent species were evaluated in the present study. Male B6C3F, mice, yellow and black C5YSF1 mice, and Fischer 344 (F344) rats were fed AIN-76A diets containing 35 (control), 1,500, 3,500, 5,000, or 10,000 μg carbonyl Fe/g for 12 wk. No effects on body weight gain were observed in the B6C3F, and black C5YSF, mice, whereas at all doses of Fe above the control, weight gain was reduced in yellow C5YSF1 mice and F344 rats. At the 10,000 μg Fe/g dose, 9 of 12 rats died, but there was no mortality among the mice. In all animals, there was a dose-related increase in liver nonheme Fe, and the Fe was stored in hepatocytes predominantly in the periportal region. There was significant hypertrophy of the hepatocytes in both B6C3F, mice and F344 rats fed the 10,000 μg Fe/g diet. PCNA assays showed significant stimulatory effects of the high dose of Fe on hepatocyte proliferation in the F344 rats and the C5YSF, mice but not in the B6C3F, mice. In the rat, there was pancreatic atrophy with loss of both endocrine and exocrine tissue. Morphometric evaluation of pancreas showed fewer β cells in B6C3F, and yellow C5YSF1 mice but not in the black C5YSF 1 mice. There were fewer islets in the yellow C5YSF1 mice, and total and mean islet areas were smaller than in the control mice. Rats in the 10,000 μg Fe/g dose group had markedly exacerbated dose-dependent nephropathy and changes in glomerular and tubular epithelium associated with Fe accumulation. The rats also showed degeneration of the germinal epithelium of the testis, formation of multinucleated giant cells, and lack of mature sperm.

Potentiation of response to insulin and anti-insulin action by two human pituitary peptides in lean agouti A/a, obese yellow Avy/A, and C57BL/6J-ob/ob mice.

Abstract Insulin-like and anti-insulin effects of human growth hormone (hGH) were examined by determining the effects of two peptides representing portions of the hGH molecule in lean agouti A/a and obese yellow A vy /A and ob/ob mice. The peptides were the amino terminal segment, residue 1–43 (hGH1–43), which has been shown to potentiate the response to insulin and another peptide, hyperglycemic peptide (HP), with unknown structure, which has anti-insulin activity. The anti-insulin component is an acidic low molecular weight peptide which co-purifies with hGH but was not recognized by antibodies to intact hGH and did not cross-react with anti-hGH1–43 antiserum. The purpose of these studies was to further understand the multiple actions of hGH and its acute and chronic effects on response to insulin. Injections of hGH1–43 dramatically enhanced the effect of insulin on glucose clearance of obese yellow A vy /A and ob/ob mice and increased the insulin-stimulated glucose oxidation in adipose tissue of yellow mice, but had no direct effect on blood glucose or insulin levels of either genotype. Administration of HP to obese yellow mice produced hyperglycemia and suppressed serum insulin concentrations. Tissues from lean agouti and obese yellow mice treated with HP in vitro showed decreased basal and insulin-stimulated glucose oxidation as well as decreased 14C incorporation into lipids. Chronic treatment of obese yellow and ob/ob mice with HP increased fasting blood glucose and impaired glucose tolerance. The effect of HP was more pronounced in obese yellow mice and the ob/ob mice were more sensitive to the diabetogenic actions of intact hGH. These data provide further evidence for the existence of two opposing biologic activities derived from disparate amino acid sequences in hGH. Additionally, the data indicate that assays using obese yellow A vy /A mice can distinguish the effects of hGH from those of the individual peptides to a greater degree than assays using obese ob/ob mice.

Variability in gene expression and tumor formation within genetically homogeneous animal populations in bioassays

Considerable variation in susceptibility to tissue-specific tumor formation in response to chronic treatment with low or intermediate dose levels of putative carcinogens is observed within populations of genetically homogeneous test animals under controlled environmental conditions. Experimental evidence from National Toxicology Program studies is reviewed, as are studies of differing degrees of carcinogenic response and tumor promotion among iso-and congenic mice carrying the Avy (viable yellow) mutation. The data suggest that individual variations in carcinogenic response among genetically homogeneous animals may derive primarily from differences in regulation of gene transcription. Differences in posttranscriptional and posttranslational processing of gene products are probably also contributing factors. The viable yellow Avy/a mouse model system is uniquely suited for investigating the developmental and molecular bases of this phenotypic variability in genetically homogeneous populations since various degrees of carcinogenic response and promotion of tumor formation can be predicted, a priori, at least as early as 7 days of age by correlation with coat color patterns. Ectopic expression of the agouti protein results in enhanced susceptibility to tumor formation in tissues which are already sensitized to neoplastic transformation by their strain genome. The differences in tumorigenic response and coat color pattern among Avy/- mice appear to be associated with different DNA methylation states of the promoter of an intracisternal A particle inserted into exon 1A of the agouti gene.

Neonatal MSG reduces hypothalamic DA, β-endorphin, and delays weight gain in genetically obese (Aviable yellow/a) mice

Neonatal treatment with monosodium glutamate (MSG) decreases proopiomelanocortin (POMC) peptides and results in obesity. The yellow mouse is a model of obesity induced by the viable yellow (Avy) gene at the agouti locus on Chromosome 2, which results in overproduction of a POMC receptor antagonist. Thus we hypothesized that MSG, when imposed on the genetically susceptible model, would alter the development of obesity. Both yellow obese (Avy) and black lean (alpha/alpha) males were injected on Postnatal Days 1, 3, 5, 7, and 9 with 2.0 mg/g body weight MSG or saline SC. Their food intake, growth parameters, and neurochemical status were examined. Paradoxically, MSG interacted with the yellow phenotype to delay the rapid rate of weight gain characteristic of this model (p < 0.05). Food intake was decreased (p < 0.05) in both phenotypes treated with MSG, as was hypothalamic content of dopamine (p < 0.05) and of the POMC peptide, beta-endorphin (p < 0.001). The yellow obese phenotype was more sensitive than the black lean phenotype to the neurochemical effect of early postnatal MSG administration. Recent reports suggest the agouti locus protein is an antagonist of the receptor for another POMC peptide, melanocyte-stimulating hormone (MSH). Therefore, the balance of functional activity between various POMC peptides appears to be an important factor in the development of both acquired and genetic obesity.

Effects of Hypophysectomy and the Insulin-Like and Anti-Insulin Pituitary Peptides on Carbohydrate Metabolism in Yellow Avy/A (BALB/c × VY)F1 Hybrid Mice

Abstract The amino-terminal portion of human growth hormone, residues 1–43 (hGH1–43), has insulin-potentiating action, while a hyperglycemic pituitary peptide (HP), which co-purifies with human growth hormone (hGH), is antagonistic to the action of insulin. The effects of hGH, hGH1–43, and HP on glucose metabolism were assessed in young (4–5 weeks) and adult (6–8 months) hypophysectomized yellow Avy/A mice which lacked any interfering endogenous pituitary hormones, and compared with age-matched intact obese yellow Avy/A and lean agouti A/a mice. Treatment with hGH1–43 or HP did not promote body growth in hypophysectomized yellow mice; but after 2 weeks of treatment with hGH, there was a significant increase in body weight (P < 0.05). Treatment with HP raised blood glucose and lowered insulin concentrations in obese yellow mice, but not in agouti or hypophysectomized yellow mice. The severely impaired glucose tolerance of the hypophysectomized yellow mice was improved by acute (60 min) and chronic (3 days) treatment with hGH1–43 as well as by 2 weeks of treatment with hGH; in contrast, HP had no effect. Glucose oxidation in adipose tissue from obese yellow mice was low and showed essentially no response to stimulation by insulin at doses lower than 1000 microunits/ml. Basal glucose oxidation rates in adipose tissue taken from agouti and hypophysectomized yellow mice were significantly higher (P < 0.001) than those in tissue from obese yellow mice, and the rates responded significantly (P < 0.05) to 100 microunits/ml insulin. The insulin binding affinities in liver membranes from agouti mice were higher than those from either obese or hypophysectomized yellow mice. The insulin receptor densities were similar in both agouti and obese yellow mice, but higher in hypophysectomized yellow mice (P < 0.05). Treatment with hGH1–43 slightly increased, although not significantly, the insulin receptor density in yellow obese mice while hGH showed essentially no change. Therefore, hypophysectomy appeared to increase tissue response and decrease insulin resistance by increasing receptor numbers and lowering the circulating insulin levels. Furthermore, the insulin-like action of hGH was elicited directly in vivo by hGH1–43 in hypophysectomized yellow mice.

Serum and pituitary concentrations of growth hormone and prolactin in pygmy mice.

Summary Basal levels of GH and PRL in the serum and pituitary gland of normal (Pg/ Pg) and pygmy (pg/pg) (VY X C3H) F-l hybrid mice were measured by homologous radioimmunoassays. The serum concentrations of GH and PRL in pygmy mice at approximately 90 days of age were found to be no different from those in corresponding normal controls. The pituitary concentrations of the two hormones, however, were substantially subnormal in pygmy mice. Perphena-zine injection failed to stimulate PRL release by pygmy (pg/pg) pituitaries, in contrast to the marked increase in serum PRL levels of the normal Pg/Pg mice. Daily administration of 100 μg of ovine PRL for 8 weeks produced none or very little increase in the body weight of pygmy mice. These results suggest that the pg mutation induces a multifaceted change affecting the synthesis, release, and function of growth hormone and prolactin. The ovine PRL used in these studies was supplied by the Pituitary Hormone Distribution Program of the National Institute of Arthritis, Metabolic, and Digestive Diseases.