Hepatic glutathione S-transferase activity in mice: effects of Avy/-genotype, obesity, lindane treatment, and sex

Phenotypically distinct but genetically identical obese mottled yellow Avy/a and lean pseudoagouti Avy/a sibling mice and their congeneic black a/a littermates provide an experimental system for distinguishing phenotypic effects from genotypic effects in the expression of the genotype at the organismic level. Hepatic glutathione S-transferase activity in obese yellow Avy/a (YS X VY) F-1 hybrid female mice was only about 66% of that found in their lean black a/a sisters. This decreased enzyme activity was not a direct effect of the Avy/a genotype but was associated with the obesity of the yellow mice since the enzyme activity in lean pseudoagouti Avy/a female siblings was similar to that found in the black a/a mice. Long-term feeding of 160 ppm lindane in the diet decreased the enzyme activity in all phenotypes but did not eliminate the difference between the obese yellow and lean pseudoagouti and black mice. Interpretation of the available data suggests that no direct relationship exists between the level of hepatic glutathione S-transferase activity and the enhancement of tumor formation in yellow Avy/a mice. Several inbred mouse strains and F-1 hybrids were also screened for this enzyme activity. No strain differences were found but sex differences within different inbred strains were not uniform. In the AE and YS strains and their F-1 hybrid enzyme activity was higher in female than in males. In contrast, BALB/c and VY strain males had higher enzyme activity than the corresponding females.     

Prenatal determination of obesity, tumor susceptibility, and coat color pattern in viable yellow (Avy/a) mice. The yellow mouse syndrome

Maturity-onset obesity and elevated circulating insulin levels are characteristic of some, but not all, mice bearing the viable yellow mutation (Avy) at the agouti locus. The expression of the Avy/a genotype in individual mice, which become obese and which remain lean is determined during prenatal development by as yet unidentified conditions in the dam's reproductive tract. One Avy/a phenotype is identified by a mottled yellow coat and characterized by adult obesity, elevated circulating insulin levels, and impaired glucose tolerance. These mice are notably more susceptible to hyperplasia and neoplasia. The alternative Avy/ a phenotype has a pseudoagouti coat, remains lean, is normoinsulinemic and normoglycemic, and in numerous other characteristics resembles congeneic lean black (a/a) littermates. Obese mottled yellow and lean pseudoagouti Avy/a mice differ in capacity to support the growth of ascites cells, in the growth response to castration, and in hepatic glutathione S-transferase activity, erythrocyte fragility, immune function, and susceptibility to Plasmodium yoelii pathogenesis. Our working hypothesis is that the constellation of characteristics, except coat color pattern, which differentiate the obese yellow mice from their lean littermates, is largely a consequence of the elevated circulating insulin levels that induce increased lipogenesis and decreased lipolysis, increased DNA and protein synthesis, increased mitosis in sensitive tissues, and increased proliferation of transformed cells.     

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

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 Avy/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 Avy/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 Avy/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.     

Diabetogenic response to streptozotocin varies among obese yellow and among lean agouti (BALB/c x VY)F1 hybrid mice

To test the hypothesis that the elevated insulin levels in obese neoplasia-susceptible yellow Avy/- mice might be a major factor stimulating tumor formation, it is necessary to use normoinsulinemic yellow mice. Although our attempt to obtain normoinsulinemic, euglycemic mice by streptozotocin treatment was unsuccessful, we did observe significant differences in the responsiveness to this treatment among mice of identical genotype. These differences were observed among female yellow Avy/A and agouti A/a (BALB/c x VY)F1 hybrid mice in the responses of body weight gain, plasma glucose, and plasma insulin levels to a single intraperitoneal injection of either 150 or 200 mg/kg streptozotocin (STZ) at 4 weeks of age followed by a 22-week observation period. Among animals treated with the high streptozotocin dose, 80% of the yellow mice gained almost no weight and became grossly hyperglycemic and hypoinsulinemic; however, only 55% of the agouti mice exhibited such a strong response. In the low dose group, 25% of the yellow mice responded with reduced body weight gain, decreased insulin, and elevated glucose levels whereas none of the agouti mice exhibited such responses. More pancreatic islet tissue mass was present in the untreated yellow control mice than among the comparable agouti mice by the end of the study. In both streptozotocin dose groups and in both genotypes, islet tissue mass was reduced to a much greater extent in the more responsive mice than in the less responsive mice. There appeared to be no correlation between islet tissue mass and insulin level. The phenotypic variation in responsiveness to an exogenous agent among test animals of a single inbred or F1 hybrid genotype reported here is not unique to this F1 hybrid since it is seen in most chronic bioassays when relatively low levels of agent are used.     

Effects of hypophysectomy and the insulin-like and anti-insulin pituitary peptides on carbohydrate metabolism in yellow Avy/A (BALB/c x VY)F1 hybrid mice

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 less than 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 less than 0.001) than those in tissue from obese yellow mice, and the rates responded significantly (P less than 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 less than 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.     

Influence of Maternal Phenotype on Metabolic Differentiation of Agouti Locus Mutants in the Mouse

The results of extensive breeding experiments indicate that the phenotypic differentiation of embryos carrying the viable yellow, A ^vy, or mottled, a^m, mutations is influenced to a major extent by the agouti locus genotype and the strain genome of the dam. The A^vy/a and a^m/a genotypes are each expressed in a spectrum of coat color phenotypes. These can be grouped into two classes, mottled and pseudoagouti.—In a reciprocal cross of C57BL/6JNIcrWf and AM/Wf-a^m/a^m mice, 29.5% of the offspring of C57BL/6 dams were of the pseudoagouti phenotype, whereas no pseudoagouti offspring were produced by AM strain dams.—Mottled yellow A^vy/a mice become obese and tumor formation is enhanced in these mice in comparison with the lean pseudoagouti A^vy/a siblings.—In two different reciprocal crosses using four different inbred strains, the proportion of pseudoagouti A^vy/a offspring differed according to the strain of the dam. Regardless of strain, mottled yellow A ^vy/a dams produced significantly fewer pseudoagouti A ^vy/a offspring than did black a/a dams.—The data suggest that metabolic differentiation of A^vy/a zygotes into phenotypic classes with different susceptibilities to obesity and tumor formation is influenced to a considerable degree by the metabolic characteristics of the oviductal and uterine environment of the dam.     

Regulation of yellow pigment formation in mice: a historical perspective

Pigment synthesis by hair follicle melanocytes is modulated by a large number of environmental and genetic factors, many of which are discussed in this review. Eumelanic (non-yellow) pigment is produced by hair follicle melanocytes following the binding of alpha-melanocyte stimulating hormone to melanocortin receptor 1. Binding of this hormone to the melanocyte membrane is blocked by agouti signaling protein (ASP) which is encoded by the agouti locus and results in the synthesis of yellow pigment, instead of non-yellow (black/brown) pigment. The cyclical release of ASP by hair follicle cells results in a black/brown hair with a subapical yellow band. This is the wild-type coat color pattern of many mammals and is called agouti. Several dominant mutations at the agouti locus in mice, induced by retrotransposon-like intracisternal A particles, result in ectopic over-expression of ASP and animals with much higher proportions of all-yellow hairs. This abnormal presence of ASP in essentially all body cells results in the 'yellow agouti obese mouse syndrome.' The obesity has been associated with binding of ASP to melanocortin receptor 4 inactivating the latter. The syndrome also includes hyperinsulinemia, increased somatic growth, and increased susceptibility to hyperplasia and carcinogenesis. The physiologic and molecular bases for these syndrome components have not yet been elucidated. This historically orientated review is subdivided, where applicable, into pre- and post-1992 subsections to emphasize the impact of the cloning of the agouti and extension loci and their protein products on the identification of the molecular and physiological pathways modulating the manifold aspects of pheomelanogenesis.     

In situ localization of agouti signal protein in murine skin using immunohistochemistry with an ASP-specific antibody

Switching between production of eumelanin or pheomelanin in follicular melanocytes is responsible for hair color in mammals; in mice, this switch is controlled by the agouti locus, which encodes agouti signal protein (ASP) through the action of melanocortin receptor 1. To study expression and processing patterns of ASP in the skin and its regulation of pigment production in hair follicles, we have generated a rabbit antibody (termed alphaPEP16) against a synthetic peptide that corresponds to the carboxyl terminus of ASP. The specificity of that antibody was measured by ELISA and was confirmed by Western blot analysis. Using immunohistochemistry, we characterized the expression of ASP in the skin of newborn mice at 3, 6, and 9 days postnatally. Expression in nonagouti (a/a) black mouse skin was negative at all times examined, as expected, and high expression of ASP was observed in 6 day newborn agouti (A/+) and in 6 and 9 day newborn lethal yellow (A(y)/a) mouse skin. In lethal yellow (pheomelanogenic) mice, ASP expression increased day by day as the hair color became more yellow. These expression patterns suggest that ASP is delivered quickly and efficiently to melanocytes and to hair matrix cells in the hair bulbs where it regulates melanin production.     

Female Fertility and Mating Type Effects on Effective Population Size and Evolution in Filamentous Fungi

The murine agouti locus regulates a switch in pigment synthesis between eumelanin (black/brown pigment) and phaeomelanin (yellow/red pigment) by hair bulb melanocytes. We recently described a spontaneous mutation, hypervariable yellow (A(hvy)) and demonstrated that A(hvy) is responsible for the largest range of phenotypes yet identified at the agouti locus, producing mice that are obese with yellow coats to mice that are of normal weight with black coats. Here, we show that agouti expression is altered both temporally and spatially in A(hvy) mutants. Agouti expression levels are positively correlated with the degree of yellow pigmentation in individual A(hvy) mice, consistent with results from other dominant yellow agouti mutations. Sequencing of 5' RACE and genomic PCR products revealed that A(hvy) resulted from the integration of an intracisternal A particle (IAP) in an antisense orientation within the 5' untranslated agouti exon 1C. This retrovirus-like element is responsible for deregulating agouti expression in A(hvy) mice; agouti expression is correlated with the methylation state of CpG residues in the IAP long terminal repeat as well as in host genomic DNA. In addition, the data suggest that the variable phenotype of A(hvy) offspring is influenced in part by the phenotype of their A(hvy) female parent.     

A Transgenic Mouse Assay for Agouti Protein Activity

The mouse agouti gene encodes an 131 amino acid paracrine signaling molecule that instructs hair follicle melanocytes to switch from making black to yellow pigment. Expression of agouti during the middle part of the hair growth cycle in wild-type mice produces a yellow band on an otherwise black hair. The ubiquitous unregulated expression of agouti in mice carrying dominant yellow alleles is associated with pleiotropic effects including increased yellow pigment in the coat, obesity, diabetes and increased tumor susceptibility. Agouti shows no significant homology to known genes, and the molecular analysis of agouti alleles has shed little new light on the important functional elements of the agouti protein. In this paper, we show that agouti expression driven by the human β-ACTIN promoter produces obese yellow transgenic mice and that this can be used as an assay for agouti activity. We used this assay to evaluate a point mutation associated with the a(16H) allele within the region encoding agouti's putative signal sequence and our results suggest that this mutation is sufficient to cause the a(16H) phenotype. Thus, in vitro mutagenesis followed by the generation of transgenic mice should allow us to identify important functional elements of the agouti protein.     

Agouti signaling protein stimulates islet amyloid polypeptide (amylin) secretion in pancreatic beta-cells

Ectopic overexpression of the murine agouti gene results in yellow coat color, obesity, hyperinsulinemia, and type II diabetes. We have shown the human homologue of agouti (agouti signaling protein; ASP) to regulate human adipocyte metabolism and lipid storage via a Ca(2+)-dependent mechanism. We have also demonstrated agouti expression in human pancreas, and that ASP stimulates insulin release via a similar Ca(2+)-dependent mechanism. Plasma amylin is also elevated in agouti mutant mice. Amylin is cosecreted with insulin from beta-cells, and overexpression of human amylin in beta-cells in yellow agouti mutant mice resulted in accelerated pancreatic amyloid deposition, severely impaired beta-cell function, and a diabetic phenotype. We report here that ASP stimulates amylin release in both the HIT-T15 beta-cell line and human pancreatic islets in the presence of a wide range of glucose concentrations (0-16.7 mmol/L), similar to its effect on insulin release; this effect was blocked by 30 mumol/L nitrendipine, confirming a Ca(2+)-dependent mechanism. Accordingly, ASP stimulation of amylin release may serve as a compensatory system to regulate blood glucose in yellow agouti mutants.     

Agouti: from mouse to man, from skin to fat

The agouti protein regulates pigmentation in the mouse hair follicle producing a black hair with a subapical yellow band. Its effect on pigmentation is achieved by antagonizing the binding of alpha-melanocyte stimulating hormone (alpha-MSH) to melanocortin 1 receptor (Mc1r), switching melanin synthesis from eumelanin (black/brown) to phaeomelanin (red/yellow). Dominant mutations in the non-coding region of mouse agouti cause yellow coat colour and ectopic expression also results in obesity, type 11 diabetes, increased somatic growth and tumourigenesis. At least some of these pleiotropic effects can be explained by antagonism of other members of the melanocortin receptor family by agouti protein. The yellow coat colour is the result of agouti chronically antagonizing the binding of alpha-MSH to Mc1r and the obese phenotype results from agouti protein antagonizing the binding of alpha-MSH to Mc3r and/or Mc4r. Despite the existence of a highly homologous agouti protein in humans, agouti signal protein (ASIP), its role has yet to be defined. However it is known that human ASIP is expressed at highest levels in adipose tissue where it may antagonize one of the melanocortin receptors. The conserved nature of the agouti protein combined with the diverse phenotypic effects of agouti mutations in mouse and the different expression patterns of human and mouse agouti, suggest ASIP may play a role in human energy homeostasis and possibly human pigmentation.     

Spontaneous tumors in nude mice: effect of the viable yellow gene.

The incidence and type of spontaneous tumors in athymic nude (nu/nu) mice (partially inbred in CBA/H background) which were also carrying the viable yellow gene (Avy, derived from C57BL/6JAvy mice), were comparable to those observed in the phenotypically normal nu/+ and +/+ control crosses carrying the Avy gene. The Avy gene increases the incidence of spontaneous and induced tumors in most mouse strains. These results would argue against the thymus dependency of the putative immunological surveillance mechanisms.     

The mouse pink-eyed dilution allele of the P-gene greatly inhibits eumelanin but not pheomelanin synthesis

The mouse pink-eyed dilution (p) locus is known to control eumelanin synthesis, melanosome morphology, and tyrosinase activity in melanocytes. However, it has not been fully determined whether the mutant allele, p affects pheomelanin synthesis. Effects of the p allele on eumelanin and phemelanin synthesis were investigated by chemical analysis of dorsal hairs of 5-week-old mice obtained from the F(2) generations (black, pink-eyed black, recessive yellow, pink-eyed recessive yellow, agouti, and pink-eyed agouti) between C57BL/10JHir (B10)-congenic pink-eyed black mice (B10-p/p) and recessive yellow (B10-Mc1r(e)/Mc1r(e)) or agouti (B10-A/A) mice. The eumelanin content was dramatically (>20-fold) decreased in pink-eyed black and pink-eyed agouti mice, whereas the pheomelanin content did not decrease in pink-eyed black, pink-eyed recessive yellow, or pink-eyed agouti mice compared to the corresponding P/- mice. These results suggest that the pink-eyed dilution allele greatly inhibits eumelanin synthesis, but not pheomelanin synthesis.     

Molecular Markers for the agouti Coat Color Locus of the Mouse

The agouti (a) coat color locus of the mouse acts within the microenvironment of the hair follicle to control the relative amount and distribution of yellow and black pigment in the coat hairs. Over 18 different mutations with complex dominance relationships have been described at this locus. The lethal yellow (Ay) mutation is the top dominant of this series and is uniquely associated with an endogenous provirus, Emv-15, in three highly inbred strains. However, we report here that it is unlikely that the provirus itself causes the Ay-associated alteration in coat color, since one strain of mice (YBR-Ay/a) lacks the provirus but still retains a yellow coat color. Using single-copy mouse DNA sequences from the regions flanking Emv-15 we have detected three patterns of restriction fragment length polymorphisms (RFLPs) within this region that can be used as molecular markers for different agouti locus alleles: a wild-type agouti (A) pattern, a pattern which generally cosegregates with the nonagouti (a) mutation, and a pattern which is specific to Emv-15. We have used these RFLPs and a panel of 28 recombinant inbred mouse strains to determine the genetic linkage of these sequences with the agouti locus and have found complete concordance between the two (95% confidence limit of 0.00 to 3.79 centimorgans). We have also physically mapped these sequences by in situ hybridization to band H1 of chromosome 2, thus directly confirming previous assignments of the location of the agouti locus.     

Genetic controls over melanocyte differentiation: interaction of agouti-locus and albino-locus genetic defects

Tyrosinase activities and dopachrome conversion activity were evaluated in extracts made from skins of 6-day-old mice that were mutant at the agouti and albino loci. Dopa oxidase (DO) activity of tyrosinase in fully pigmented (C/C) mice is reduced in extracts made from skins of yellow 6-day-old mice as compared to those of black mice. Dopachrome conversion (DC) activity is absent from skin extracts of normal yellow mice and is present in normal black mice. DC activity is a characteristic of a separate enzyme which has been called dopachrome conversion factor or dopachrome oxidoreductase. We measured the dopa oxidase activity and dopachrome conversion activity in skin extracts of yellow mice and black mice that were mutant at the albino (C) locus. Extracts made from extreme-dilution (ce/ce) mice do not have DO activity. Those from yellow extreme-dilution mice do not have DC activity, while those from black, extreme-dilution mice do. The DO and DC activities that characterize skin extracts made from platinum (cp/cp) yellow mice are similar to those of platinum black mice. These observations suggest possible mechanisms by which the functions controlled by the agouti and albino loci interact to control melanogenesis.     

Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin

Melanocytes produce two chemically distinct types of melanin pigments, eumelanin and pheomelanin. These pigments can be quantitatively analyzed by acidic permanganate oxidation or reductive hydrolysis with hydriodic acid to form pyrrole-2,3,5-tricarboxylic acid or aminohydroxyphenylalanine, respectively. About 30 coat color genes in mice have been cloned, and functions of many of those genes have been elucidated. However, little is known about the interacting functions of these loci. In this study, we used congenic mice to eliminate genetic variability, and analyzed eumelanin and pheomelanin contents of hairs from mice mutant at one or more of the major pigment loci, i.e., the albino (C) locus that encodes tyrosinase, the slaty (Slt) locus that encodes tyrosinase-related protein 2 (TRP2 also known as dopachrome tautomerase, DCT), the brown (B) locus that encodes TRP1, the silver (Si) locus that encodes a melanosomal silver protein, the agouti (A) locus that encodes agouti signaling protein (ASP), the extension (E) locus that encodes melanocortin-1 receptor, and the mahogany (Mg) locus that encodes attractin. We also measured total melanin contents after solubilization of hairs in hot Soluene-350 plus water. Hairs were shaved from 2-3-month-old congenic C57BL/6J mice. The chinchilla (c(ch)) allele is known to encode tyrosinase, whose activity is about one third that of wild type (C). Phenotypes of chinchilla (c(ch)/c(ch)) mice that are wild type or mutant at the brown and/or slaty, loci indicate that functioning TRP2 and TRP1 are necessary, in addition to high levels of tyrosinase, for a full production of eumelanin. The chinchilla allele was found to reduce the amount of pheomelanin in lethal yellow and recessive yellow mice to less than one fifth of that in congenic yellow mice that were wild type at the albino locus. This indicates that reduction in tyrosinase activity affects pheomelanogenesis more profoundly compared with eumelanogenesis. Hairs homozygous for mutation at the slaty locus contain 5,6-dihydroxyindole-2-carboxylic acid (DHICA)-poor melanin, and this chemical phenotype was retained in hairs that were mutant at both the brown locus and the slaty locus. Hair from mice mutant at the brown locus, but not at the slaty locus, do not contain DHICA-poor melanin. This indicates that the proportion of DHICA in eumelanin is determined by TRP2, but not by TRP1. Mutation at the slaty locus (Slt(lt)) was found to have no effect on pheomelanogenesis, supporting a role of TRP2 only in eumelanogenesis. The mutation at silver (si) locus showed an effect similar to brown, a partial suppression of eumelanogenesis. The mutation at mahogany (mg) locus partially suppressed the effect of lethal yellow (Ay) on pheomelanogenesis, supporting a role of mahogany in interfering with agouti signaling. These results show that combination of double mutation study of congenic mice with chemical analysis of melanins is useful in evaluating the interaction of pigment gene functions.     

Effects of MSH on food intake, body weight and coat color of the yellow obese mouse

Viable yellow obese mice (Avy/a) were treated for 10 days with 5, 15, 50 or 150 micrograms/d of either alpha-MSH or desacetyl-MSH. The half-maximal effect on weight gain occurred with a dose of 5 micrograms/d for desacetyl-MSH and at a 30 fold higher level of 150 micrograms/d for alpha-MSH. In contrast, the half-maximal stimulation of eumelanin production by alpha-MSH occurred at 15 micrograms/d and with desacetyl-MSH at 150 micrograms/d, a 10-fold increase. Desacetyl-MSH produced a dose-related increase in the weight of muscle, as well as white and brown adipose tissue. Desacetyl-MSH and alpha-MSH both increased plasma corticosterone concentrations, but desacetyl-MSH was more potent. In a 2 x 2 factorial designed study, body weight was significantly increased in viable yellow mice only by treatment with desacetyl-MSH but in the lean animals, both alpha-MSH and desacetyl-MSH increased body weight. Food intake was significantly different between genotypes, and was stimulated by desacetyl-MSH. These studies demonstrate potent differences in biological actions on food intake, body weight, and a variety of organ weights between acetylated and desacetylated forms of MSH.     

Altered Circadian Period of Locomotor Activity in Carbonic Anhydrase II-Deficient Mice

This study finds lengthened circadian period in a congenic strain of mice homozygous for a null mutation in carbonic anhydrase isoenzyme-II gene on proximal Chromosome 3. Carbonic anhydrase II has the highest turnover rate of any constitutive enzyme. It catalyzes the reversible hydration of carbon dioxide to control intercellular acid/base balance. A strain of congenic mice has a carbonic anhydrase II null mutation within a DBA/2J inbred strain insert on a C57BL/6J inbred strain background. The locomotor activity levels and period of circadian rhythms were examined in the homozygous null mutants and their progenitors, mice heterozygous for the region around the carbonic anhydrase gene. The heterozygous mice siblings and the wild-type siblings served as the controls. During behavioral studies, male and female offspring and parents were housed singly in constant darkness. Locomotor activity was monitored using an infrared photobeam array. Mice homozygous for the carbonic anhydrase null mutation had a longer circadian period than either heterozygote or wild type littermates. Carbonic anhydrase null mutants also had low locomotor activity compared to either heterozygous or wild-type litter mates. This implies that either the physiological changes resulting from absence of carbonic anhydrase II isozyme or the presence of DBA/2J alleles around the carbonic anhydrase locus influence the circadian period and level of locomotor activity in laboratory mice.