A novel leptin receptor variant with a conservative amino acid substitution (I359 V) in body weight selected and unselected mouse lines.

Recently published results revealed linkage of obesity traits to a chromosomal region near the leptin receptor gene (Lepr) locus in high body weight selected big and fat DU6i mice. The purpose of this study was the search for variants of the Lepr gene in selected and unselected mouse lines as a candidate for different body composition traits. The complete Lepr cDNA sequence was analysed in DU6i mice. In addition, body weight, abdominal fat weight and serum leptin levels were measured in 42 day old, male mice of the strains DU6i, DUKs, Him : OF1 and DBA/2. Sequence comparison to the published wild type sequence revealed three silent mutations and an amino acid exchange (I359 V) in the C2 domain of the putative leptin binding site in the line DU6i. Compared to the high body weight selected mice also unselected lean control DUKs mice and Him : OF1 mice harbour the amino acid substitution, although they show significantly lower values for body weight, abdominal fat weight and serum leptin levels. Therefore, we assume that the mutation in the C2 domain of Lepr alone might not result in impaired leptin binding or signaling.

Different isoforms of the soluble leptin receptor in non-pregnant and pregnant mice.

Leptin circulates in murine serum in a free and a bound form. As shown in humans, a soluble leptin receptor (sOB-R), which modulates the effects of its ligand, circulates in murine blood. The aim of our study was to determine abundance and biochemical nature of this protein. For the quantification of sOB-R we developed a ligand-immunofunctional assay (LIFA) which is based on both, leptin binding and immunological recognition. The use of this LIFA revealed that during late gestation sera of pregnant mice had a approximately 290-fold higher level of sOB-R than non-pregnant animals. As investigated by size exclusion chromatography these mice sera demonstrated a co-elution of their leptin binding activity with leptin immunoreactivity and levels of sOB-R measured by LIFA. Therefore, it has to be concluded that sOB-R represents the major leptin binding activity in murine circulation. The molecular analysis of sOB-R by Western blot and by cross-linking with 125I-leptin in sera of pregnant and non-pregnant mice demonstrated two different isoforms of sOB-R, which were capable of leptin binding. The sOB-R in serum migrated at a molecular weight of 150kDa in pregnant and only of 120kDa in non-pregnant animals. Deglycosylation of these isoforms led to sOB-R molecules which were found at the same molecular weight in SDS-PAGE. This finding indicates that both isoforms differ only in the degree of their glycosylation. In conclusion, the non-pregnant and the pregnant states are accompanied by differently glycosylated isoforms of sOB-R whose physiological relevance remains to be determined.

Intrinsic properties of muscle satellite cells are changed in response to long-term selection of mice for different growth traits.

Satellite cell cultures were derived from mice selected long-term over 70 generations for body weight (DU-6, growth), carcass protein amount (DU-6P, protein) and an index combining body weight and endurance treadmill performance (DU-6+LB, growth + fitness) at 42 days of age and from an unselected control line (DU-Ks). They were grown under identical environmental conditions to examine intrinsic cellular differences in proliferation, protein metabolism and responsiveness to growth factors. Growth kinetics (DNA and protein amounts) were determined over a 12-day period. During exponential growth, all growth-selected cultures grew faster than the control culture: (DU-6+LB=DU-6P)>DU-6>DU-Ks. The differences in DNA and protein levels were maintained until day 8. DU-Ks cultures reached similar levels as the growth (DU-6) and protein (DU-6P) cultures in terms of DNA at day 12 of cultivation. Thus, the cultures from the growth and protein lines, but not from the growth + fitness line, exhibited larger protein:DNA ratios (cell size) than the control cultures. Cell cultures from the selected lines were more responsive to serum and epidermal growth factor in terms of [(3)H] thymidine incorporation into DNA, whereas no stimulation by insulin or insulin-like growth factor-I was detectable in cultures from selected lines or controls. During differentiation, protein metabolism in cultures from selected lines was characterised by higher rates of protein synthesis (PS) and degradation (PD), as measured by [(3)H] phenylalanine incorporation or release, respectively, than in control cells. The ratios of the relative differences from the control in PS and PD were only >1.0 in the growth and protein lines. In conclusion, long-term selection for growth therefore modifies the intrinsic capability of satellite cells for proliferation and protein metabolism, with changes being dependent on the selection trait.

Inbred lines of mice derived from long-term growth selected lines: unique resources for mapping growth genes.

Lines of mice selected for many generations for high or low growth in several laboratories around the world have been collected, and from these, inbred lines are being developed by recurrent full-sib mating in Edinburgh. There are seven high selected lines and four low lines (each low line is from the same base population as one of the high lines), and the histories of each are summarized. Mean body weight of males at 70 days of age in the Edinburgh laboratory in the heaviest inbred line (77 g) is 4.8-fold higher than in the lightest line (16 g), and 1.9-fold higher than in the least extreme high line (41 g). Litter size, food intake, and fat content also differ substantially. These inbred extreme selected lines are a uniquely valuable resource for QTL or gene mapping, candidate gene identification, and elucidation of epistatic effects.

Genome-wide search for loci controlling serum IGF binding protein levels of mice.

A segregating F(2) pedigree based on two mouse lines (DU6i and DBA/2) with extremely different growth characteristics was generated to search for loci affecting serum levels of insulin-like growth factor (IGF) binding proteins (IGFBPs) and to estimate their effects on growth and body composition. DU6i is characterized by high body mass and obesity associated with hyperinsulinemia, hyperleptinemia, and elevated serum IGF-I concentrations. Furthermore, significantly elevated serum levels of IGFBP-2, IGFBP-3, and IGFBP-4 were found in DU6i vs. DBA/2 mice. Linkage analysis identified loci with major effects on the serum level of IGFBP-3 on Chromosome 5 at 58 cM (Igfbp3q1; F = 9.9) and on Chromosome 10 at 46 cM (Igfbp3q2; F = 33.8). A locus significantly influencing serum IGFBP-2 levels in males was found on Chromosome 7. Additional linkage was detected in males and females for IGFBP-2 on Chromosomes 8, 11, 14, 17, and X, and for IGFBP-4 on Chromosome 4. Additional loci affecting IGFBPs acted in a sex-specific manner. The identified loci coincide in part with chromosomal regions controlling growth and obesity. Thus, multiple genes or pleiotropic gene effects may be assumed for these chromosomal regions. The identification of quantitative trait loci for IGFBPs as subcomponents of growth regulation and differentiation will further improve the understanding of complex trait regulation.

Single QTL effects, epistasis, and pleiotropy account for two-thirds of the phenotypic F(2) variance of growth and obesity in DU6i x DBA/2 mice.

Genes influencing body weight and composition and serum concentrations of leptin, insulin, and insulin-like growth factor I (IGF-I) in nonfasting animals were mapped in an intercross of the extreme high-growth mouse line DU6i and the inbred line DBA/2. Significant loci with major effects (F > 7.07) for body weight, obesity, and muscle weight were found on chromosomes 1, 4, 5, 7, 11, 12, 13, and 17, for leptin on chromosome 14, for insulin on chromosome 4, and for IGF-I on chromosome 10 at the Igf1 gene locus itself and on chromosome 18. Significant interaction between different quantitative trait loci (QTL) positions was observed (P < 0.01). Evidence was found that loci having small direct effect on growth or obesity contribute to the obese phenotype by gene-gene interaction. The effects of QTLs, epistasis, and pleiotropy account for 64% and 63% of the phenotypic variance of body weight and fat accumulation and for over 32% of muscle weight and serum concentrations of leptin, and IGF-I in the F(2) population of DU6i x DBA/2 mice. [The quantitative trait loci described in this paper have been submitted to the Mouse Genome Database.]

Thiazolidinedione-induced activation of the transcription factor peroxisome proliferator-activated receptor gamma in cells adjacent to the murine skeletal muscle: implications for fibroblast functions.

Nuclear peroxisome proliferator-activated receptor gamma (PPARgamma) is the target of antidiabetogenic thiazolidinediones (TZD). However, recent studies failed to show that TZD has an effect in vitro on insulin-regulated glucose uptake in skeletal muscles, the major site of glucose disposal. The potential effects of TZD on cells adjacent to skeletal muscles are not well characterized but may be involved in TZD's actions. Hence, we studied these cells from mice treated with the carrier and with the TZD ciglitazone (9 nmol/g body weight). The cells were typified by lipid enrichment (floating adipocytes and macrophages), by the ectopic expression of cellular fibronectin (fibroblasts), fibronectin and PPARgamma (preadipocytes), PPARgamma and CD11b/Mac-1 (active macrophages) as revealed by flow cytometry and immunoblotting. The glucose transporter 4 proteins (GLUT4) and the uptake of glucose and long-chain fatty acids (LCFA) were determined flow cytometrically using fluorescent derivatives of glucose (NBDG) and LCFA (C16-Bodipy). The expression of tumor necrosis factor alpha (TNFalpha) in CD11b/Mac-1-positive and CD11b/Mac-1-negative cells separated by magnetic immunobeads was analyzed. The results showed that TZD treatment upregulated GLUT4 expression, and increased insulin-regulated NBDG uptake and C16-Bodipy binding and influx, at the same time as increasing the quantity of PPARgamma-expressing fibroblasts; this indicates the development of the preadipocyte phenotype. In contrast, TZD lowered the number of adipocytes (0.6-fold compared to the carrier-treated control) perhaps through an action of TNFalpha from CD11b- and PPARgamma-expressing macrophages. The data suggest that the regulatory effects of TZD on energy homeostasis involve two major targets: the PPARgamma-positive fibroblasts whose adipogenic program is promoted, and CD11b-PPARgamma-expressing macrophages which become cytotoxic and fibrogenic because of the effects of TNFalpha on neighboring adipocytes and fibroblasts, respectively.

Quantitative trait loci affecting body weight and fatness from a mouse line selected for extreme high growth.

Quantitative trait loci (QTL) influencing body weight were mapped by linkage analysis in crosses between a high body weight selected line (DU6) and a control line (DUKs). The two mouse lines differ in body weight by 106% and in abdominal fat weight by 100% at 42 days. They were generated from the same base population and maintained as outbred colonies. Determination of line-specific allele frequencies at microsatellite markers spanning the genome indicated significant changes between the lines on 15 autosomes and the X chromosome. To confirm these effects, a QTL analysis was performed using structured F2 pedigrees derived from crosses of a single male from DU6 with a female from DUKs. QTL significant at the genome-wide level were mapped for body weight on chromosome 11; for abdominal fat weight on chromosomes 4, 11, and 13; for abdominal fat percentage on chromosomes 3 and 4; and for the weights of liver on chromosomes 4 and 11, of kidney on chromosomes 2 and 9, and of spleen on chromosome 11. The strong effect on body weight of the QTL on chromosome 11 was confirmed in three independent pedigrees. The effect was additive and independent of sex, accounting for 21-35% of the phenotypic variance of body weight within the corresponding F2 populations. The test for multiple QTL on chromosome 11 with combined data from all pedigrees indicated the segregation of two loci separated by 36 cM influencing body weight.

Long-term selection for protein amount over 70 generations in mice.

Based on the outbred mouse strain Fzt: Du, which has been obtained by systematic crossing of four inbred and four outbred lines, a long-term selection experiment was carried out for total protein amount (PA) in the carcass, starting in 1975. An unselected control line (CO) was kept under the same management but without continuous protein analysis. The protein amount of male carcasses at 42 days of age (P42) increased from 2.9 g in generation 0 to 5.2 g at generation 70, representing 97% of a theoretical selection limit. The total selection response amounts to 2.3 g, which is about 80% above the initial value and corresponds to 9 sigma p or 12 sigma A. The estimated realized heritability of protein amount decreased from 0.56 to 0.03 at generation 70, which was due to an increase in phenotypic variance from 0.065 to 0.24 g2 and a reduction in genetic variance from 0.04 to 0.01 g2. Half the selection response was obtained after about 18 to 23 generations, a half-life of 0.25 to 0.3 Ne. The maximum selection response was 0.094 g/generation and the response was 0.01 g/generation at generation 70. The measurements of body weights at 0, 10, 21, 42 and 63 days throughout the experiment showed a strong correlated effect for all weights. The PA mice are one of the heaviest lines of mice ever reported, and do not differ significantly in their body composition from control mice at 42 days. The direct selection response was due primarily to increased general growth. Body weight and protein amount are phenotypically and genetically highly correlated (rp = 0.82, rA approximately 1); however, selection for body weight led to fatter animals, whereas selection for protein opposed increased fatness (at least until selection age). This may be of general importance in animal breeding. The comparatively high selection response in this experiment seems due to the heterogeneity of the base population, the relatively high effective population size, and the duration of the experiment.

Effects of stress-related signal molecules on cells associated with muscle tissue.

OBJECTIVE: To elucidate the physiologic background that makes muscle hypertrophy, especially that due to strenuous exercise, often parallel to stress sensitivity and signs of acute phase immune response. STUDY DESIGN: We used an animal model: lines of mice with hypertrophied (H) and normally developed (N) hind leg muscles, six in each case. Functional and receptor tests on cells from digested muscle tissue were made and analyzed by microplate cytofluorimetry and flow cytometry. RESULTS: Higher percentages of cells with a phagocyte marker (> 2-fold) and with opioid (about 1.3-fold) receptors were found, whereas the portion of glucocorticoid receptor-bearing cells tended to differ only among H and N. Naloxone, an opioid receptor antagonist, failed only in H to exert a suppressive effect on dihydrorhodamine 123 oxidation. CONCLUSION: These results and differences in responses of lipid trafficking and proteolytic activities to cortisol, naloxone and adrenergic receptor agonists suggest that not only the cell population associated with muscle tissue but also receptor-mediated responses that are known to be related to stress coping are different between H and N.

[The effect of cage type and population density on the body weight development of laboratory mice]. / Einfluss von Käfigtypen und Besatzdichten auf die Körpermasseentwicklung von Labormäusen.

The effect of different keeping conditions on body weight gain was studied in 200 male Fzt:DU mice between days 21 and 70 of life. The animals were divided in four different housing groups: DU-B (housing in stress cages), DU-B+HI (housing in stress cages and afterwards in standard cages type HI), DU-HI (housing in standard cages type HI) and DU-S+AP (housing in standard group cages). Housing in stress cages resulted in lower body weight gain at every age; subsequent housing in standard cages type HI for one week failed to compensate this effects. Likewise, keeping of four mice in standard cages type I (DU-HI) between days 42 and 70 of life also resulted in delayed body weight gain. It is possible that the chosen population density was not an optimum for normal weight gain during the adult life. Animals kept in standard stocking cages (DU-S+AP) had the highest body weight gain.

[Selection of laboratory rats for fertility components in synchronized estrus using Suisychron-Praemix. 1. Effects of selection and synchronization]. / Selektion auf Komponenten der Fruchtbarkeit bei der Laboratoriumsratte unter den Bedingungen der Brunstsynchronisation mit Suisynchron-Prämix. 1. Mitteilung: Wirkung von Selektion und Synchronisation.

The following results were obtained from experimental studies into effects of selection for fertility and into synchronised oestrus though eight generations of laboratory rats: -- The result of selection recordable from the population with selection only was better than that recorded from the population with selection and synchronised oestrus. This has been attributed to higher correlation between phenotype and genotype in the selection variant. -- Direct negative impact of synchronised oestrus on selection index was recorded from a population with random mating. -- The effects of synchronised oestrus regarding selection success were offset but not entirely ruled out by selection. -- Through the generations of both populations with synchronised oestrus the effects of synchronisation were lowered due to both physiological adaptation to Suisynchron application and contraselection with regard to suitability for synchronisation.

[Selection of laboratory rats for fertility components in synchronized estrus using Suisynchron-Praemix. 2. Results of litter development]. / Selektion auf Komponenten der Fruchtbarkeit bei der Laboratoriumsratte unter den Bedingungen der Brunstsynchronisation mit Suisynchron-Prämix. 2. Mitteilung: Ergebnisse der Wurfentwicklung.

The following results were obtained from experimental studies into selection of laboratory rats for fertility and random mating with oestrus being synchronised by means of Suisynchron-Prämix, through eight generations: --Success of selection without synchronised oestrus was higher than that with synchronised oestrus by selection according to the basic index and with one and the same selection intensity. --Litter size and weight were higher in the selected variants, as a result of increased potential fertility (rate of ovulation). --In the population with selection and synchronised oestrus a negative relationship was found to exist between the directly correlated selection results regarding fertility parameters, on the one hand, and the effect of synchronisation, on the other. --Both the rates of ovulation and litter parameters were lower in the population with selection and synchronised oestrus, as a result of synchronisation. --The effect of synchronised oestrus on the population with selection and synchronised oestrus was partially eliminated owing to selection for fertility.