Genomic scan for genes affecting body composition before and after training in Caucasians from HERITAGE.

An autosomal genomewide search for genes related to body composition and its changes after a 20-wk endurance-exercise training program has been completed in the HERITAGE Family Study. Phenotypes included body mass index (BMI), sum of eight skinfold thicknesses, fat mass (FM), fat-free mass, percent body fat (%Fat), and plasma leptin levels. A maximum of 364 sib-pairs from 99 Caucasian families was studied with the use of 344 markers with single-point and multipoint linkage analyses. Evidence of significant linkage was observed for changes in fat-free mass with the S100A and the insulin-like growth factor I genes (P = 0.0001). Suggestive evidence (2.0 < or = Lod < 3.0; 0.0001 < P < or = 0.001) was also observed for the changes in FM and %Fat at 1q31 and 18q21-q23, in %Fat with the uncoupling protein 2 and 3 genes, and in BMI at 5q14-q21. At baseline, suggestive evidence was observed for BMI at 8q23-q24, 10p15, and 14q11; for FM at 14q11; and for plasma leptin levels with the low-density lipoprotein receptor gene. This is the first genomic scan on genes involved in exercise-training-induced changes in body composition that could provide information on the determinants of weight loss.

A genome-wide scan for abdominal fat assessed by computed tomography in the Québec Family Study.

To identify chromosomal regions harboring genes influencing the propensity to store fat in the abdominal area, a genome-wide scan for abdominal fat was performed in the Quebec Family Study. Cross-sectional areas of the amount of abdominal total fat (ATF) and abdominal visceral fat (AVF) were assessed from a computed tomography scan taken at L4-L5 in 521 adult subjects. Abdominal subcutaneous fat (ASF) was obtained by computing the difference between ATF and AVF. The abdominal fat phenotypes were adjusted for age and sex effects as well as for total amount of body fat (kilogram of fat mass) measured by underwater weighing, and the adjusted phenotypes were used in linkage analyses. A total of 293 microsatellite markers spanning the 22 autosomal chromosomes were typed. The average intermarker distance was 11.9 cM. A maximum of 271 sib-pairs were available for single-point (SIBPAL) and 156 families for multipoint variance components (SEGPATH) linkage analyses. The strongest evidence of linkage was found on chromosome 12q24.3 between marker D12S2078 and ASF (logarithm of odds [LOD] = 2.88). Another marker (D12S1045) located within 2 cM of D12S2078 also provided evidence of sib-pair linkage with ASF (P = 0.019), ATF (P = 0.015), and AVF (P = 0.0007). Other regions with highly suggestive evidence (P < 0.0023 or LOD > or =1.75) of multipoint linkage and evidence (P < 0.05) of single-point linkage, all for ASF, included chromosomes 1p11.2, 4q32.1, 9q22.1, 12q22-q23, and 17q21.1. Three of these loci (1p11.2, 9q22.1, and 17q21.1) are close to genes involved in the regulation of sex steroid levels, whereas two others (4q32.1 and 17q21.1) are in the proximity of genes involved in the regulation of food intake. This first genome-wide scan for abdominal fat assessed by computed tomography indicates that there may be several loci determining the propensity to store fat in the abdominal depot and that some of these loci may influence the development of diabetes in obese subjects.

Genome-wide search for genes related to the fat-free body mass in the Québec family study.

Fat-free mass (FFM) consists mostly of skeletal muscle and bone tissues, and identification of the genes and molecular mechanisms involved in the control of FFM would have implications for the understanding of sarcopenia and potentially osteoporesis associated with aging, as well as the response to starvation, refeeding, anorexia, and any other conditions in which lean body mass is important. A genome-wide search for genes related to body leanness has been completed in the Quebec Family Study (QFS). Microsatellite markers (N = 292) from the 22 autosomal chromosomes were typed. The mean spacing of the markers was 11.9 centimorgans (cM) (range, <0.1 to 41). FFM was calculated from percent body fat, derived from underwater weighing, and body weight and was adjusted by regression for age and sex effects before analysis. A maximum of 336 sib pairs or 609 pairs of extended relatives were analyzed using single-point Haseman-Elston regression (SIBPAL and RELPAL) and multipoint variance component (SEGPATH) linkage analyses. Significant linkages were observed on chromosomes 15q25-q26 for a CA repeat within the insulin-like growth factor 1 receptor (IGF1R) gene (Lod score = 3.56) and at 18q12 with D18S877 (Lod score = 3.53) and D18S535 (Lod score = 3.58), 2 markers located 10 cM apart. A moderately significant linkage was also observed on chromosome 7p15.3 with the marker D7S1808 (Lod score = 2.72). The most obvious candidate genes within the regions identified by these linkages include the IGF1R on 15q and neuropeptide Y (NPY) and growth hormone-releasing hormone (GHRH) receptor on 7p. On 18q, the melanocortin receptor 4 (MC4R) is not likely the candidate gene for the observed linkage. This study represents the first genome-wide search for genes that may be involved in the regulation of the lean component of body mass in humans.

Absence of linkage between VO2max and its response to training with markers spanning chromosome 22.

An extensive search for linkage between DNA markers and the response of VO2max to training has recently been launched in the HERITAGE Family Study. This is the first report on a genome-wide search strategy to locate chromosomal regions and positional candidate genes for cardiorespiratory endurance phenotypes. Linkage between seven markers spanning chromosome 22 spaced approximately 10 cM apart (D22S264, D22S274, D22S301, D22S304, D22S421, IL2RB, and PDGFB) and VO2max at baseline, as well as its response to endurance exercise training, was examined using the sib-pair linkage method. Markers were genotyped in at least 210 sib-pairs derived from 128 adult brothers (25 +/- 6 yr; mean +/- SD) and 138 sisters (24 +/- 6 yr) from 86 Caucasian families. VO2max, maximal heart rate, and maximal oxygen pulse were measured during stationary cycle tests before and after a standardized 20-wk endurance training program. On average, the initial VO2max was 2654 +/- 767 mL.min-1 while training increased VO2max significantly by 430 +/- 239 mL.min-1 or 16% (P < 0.0001). The VO2max response was adjusted for age and initial VO2max. No evidence of linkage was found between any of these markers on chromosome 22 and VO2max or its trainability.

Quantitative analysis of dystrophin in fast- and slow-twitch mammalian skeletal muscle.

The relative tissue content of dystrophin has been evaluated in the slow-twitch soleus (primarily type I fibers) and fast-twitch vastus lateralis (primarily type IIb fibers) muscles of the rat and mouse, as well as in human biopsy samples from the vastus lateralis and gluteus maximus muscles, using a sensitive immunochemical assay. The dystrophin content of the soleus muscle was approximately twofold higher than in the vastus lateralis muscle. This difference is not entirely explained by the higher total sarcolemmal surface of the smaller soleus muscle type I fibers, and is therefore attributed to a higher content of dystrophin in the type I fibers compared to type IIb fibers. PCR analysis of the dystrophin transcript levels in the two muscle types indicated no significant differences. Analysis of human muscle biopsies revealed a twofold higher dystrophin content in the vastus lateralis muscle compared to the gluteus maximum muscle. It is concluded that the tissue content of dystrophin may vary significantly among physiologically different skeletal muscle types.

Dystrophin: a sensitive and reliable immunochemical assay in tissue and cell culture homogenates.

A modified polyacrylamide gel electrophoresis system is described which provides excellent resolution of very high molecular weight proteins. This system has been successfully applied to the immunochemical detection of dystrophin in mouse and rat skeletal muscle, mouse myotubes in cell culture, and in human muscle-biopsy specimens. The mass of total homogenate protein (3-12 micrograms) and the relative quantity of dystrophin detected immunologically were found to be strongly correlated (r = 0.970 - 0.995). The method described here requires minute quantities of tissue or cells to accurately evaluate the relative amount of dystrophin present. The entire procedure for the detection of dystrophin is simple, rapid and cost efficient compared to other available techniques.

Characterization and expression of the prostatic arginine esterase gene, a canine glandular kallikrein.

The prostatic arginine esterase gene was isolated from a genomic library prepared with dog liver DNA in lambda EMBL3. The selected clone contained an insert of approximately 17 kb which included the whole coding portion of arginine esterase mRNA (5 exons plus 4 introns), 2 kb upstream from the initiation site and 12 kb downstream from the polyadenylation site. The intron-exon boundaries were identical to all known mammalian kallikrein genes. The deduced amino acid sequence indicated a high degree of identity (51-61%) with other kallikreins expressed not only in the prostate but also in the pancreas of various animal species. The 5'-flanking sequences contained potential regulatory elements such as a variant TATA box (TTTAAA), a CCAAT box, a SP1 transcriptional factor binding site (GGGCGG), and two TGTCCT motifs resembling glucocorticoid response elements. Southern blot analysis with an amplified cDNA fragment of 487 bp corresponding to the 5' portion of the mRNA and with a DNA probe from a different portion of the arginine esterase gene indicated the presence of two to three homologous genes in the canine genome while in a previous study a single band was detected using a 400-bp arginine esterase cDNA corresponding to the 3' portion of the mRNA. These results suggest that the arginine esterase gene belongs to a small kallikrein gene family. Arginine esterase mRNA is expressed primarily in the prostate but also at an extremely low level (approximately a thousandfold less) in several other tissues including the liver, the gracilis thigh muscle, the kidney, and the pancreas.

Southern blot analysis with synthetic oligonucleotides. Application to prostatic protein genes.

1. An ethanol precipitation procedure was developed to purify radiolabeled DNA and oligonucleotide probes to be used in Southern blots. 2. The radiolabeled probes produced strong hybridization signals on a clear background on Southern blot analysis of single gene copies even after 5 days of exposure on X-ray films. 3. An oligonucleotide probe complementary to human glandular kallikrein-1 coding region (amino acids 161-167) detected a single DNA fragment after digestion with Bam H1, Hind III or Pst 1. 4. Another oligonucleotide probe coding for the same region of human prostate-specific antigen detected 3 DNA fragments on Southern blots by contrast to a 1.5 kb full length cDNA probe which detected the presence of only one strong hybridization signal. 5. Oligonucleotide probes appear to be excellent tools for gene mapping. Their sensitivity, specificity and limitations can be compared to the one of monoclonal antibodies used in epitope mapping of proteins.

Depressed translational activity in the androgen sensitive levator ani muscle of the rat.

The androgen-dependent levator ani (LA) muscle of the rat provides a suitable model to explore the molecular mechanism of steroid hormone action in target tissues. The objective of the present series of experiments was to study the effect of gonadectomy (GDX) and androgen replacement therapy on the in vitro protein synthetic capacity of the LA muscle. The incorporation of labeled methionine into the contractile protein fraction of the LA muscle maintained in organ culture decreases in a time-dependent manner following GDX. Translation of total polyadenylated mRNA in the rabbit reticulocyte translation system revealed that the decrease in protein synthetic capacity was not associated with differences in the template activity of the mRNA derived from GDX tissue. However, when polyribosomes were used to direct the same in vitro synthesis system, a significant time-dependent loss of translational activity was observed following GDX. The polyribosomes of the LA muscle of control and GDX rats were shown to contain equivalent amount of rRNA and mRNA of comparable translation efficiency. Collectively the results of these experiments indicate that the decrease in protein synthetic capacity of the LA muscle in androgen deficient rats is due, in part, to a repression of the translation process associated to the functional integrity of polyribosomes.

Nucleotide sequence of the androgen-dependent arginine esterase mRNA of canine prostate.

The nucleotide sequence of canine prostate arginine esterase mRNA was determined using a 400 bp cDNA clone and primer-extended cDNA transcripts for the 5'-coding and noncoding regions. The mRNA contains 864 nucleotides encoding a protein of 236 amino acids preceded by 24 amino acids which constitutes both the signal and the zymogen peptides. The sequence indicates the presence of one potential glycosylation site. A high degree of homology was found between the canine enzyme and other members of the kallikrein family including human prostate specific antigen. The protein appears to be specified by a single gene.

Androgen regulation of canine prostatic arginine esterase mRNA using cloned cDNA.

Canine prostatic arginine esterase complementary DNA has been cloned in pPBS27, a new cloning vector. The relative abundance of androgen-regulated mRNA in intact dog prostate was reflected by the finding that a high proportion of the clones in the cDNA library hybridized strongly by plaque or colony hybridization with a poly(A)+ RNA probe from intact dog prostate but not with a poly(A)+ RNA probe from castrated dog prostate. One clone carrying a 400 base pairs cDNA insert was selected for further studies. Translation of the hybrid-selected RNA in a cell-free system resulted in the production of a 31 kDa peptide immunoprecipitable by antibodies against arginine esterase. This identification was confirmed by partial sequence analysis of the cDNA revealing an encoding protein with high homology to known kallikreins. Northern blot analysis of poly(A)+ and total RNA showed that arginine esterase mRNA had an approximate size of 1.0 kb which corresponded to a major androgen-regulated RNA species that could be observed after denaturing agarose gel electrophoresis of prostatic poly(A)+ RNA from intact dogs. Dot-blot analysis showed that dogs which had been castrated 3 weeks before had more than 100-fold lower arginine esterase mRNA level than intact dogs or castrated dogs treated with Depo-testosterone.

Lack of effect of anabolic steroids on specific mRNAs of skeletal muscle undergoing compensatory hypertrophy.

Compensatory hypertrophy of the fast-twitch plantaris muscle (HP) was induced in male rats to determine whether the resulting translational activity of isolated polyribosomes could be modified in this process and by the androgen status. HP induced a significant increase in free androgen binding sites and a typical protein synthesis pattern characterized by a slow myosin light chain isozyme (LC-1S), an increase in fast isozymes (LC-1F,2F) and of beta-tropomyosin/alpha-tropomyosin ratio. The variations in receptor occupancy following castration and treatments with four anabolic steroids (AS) did not result in modification of the template activity of major HP mRNAs. These data suggest that the slight increase of steroid receptors found in HP remains insufficient to trigger an androgenic response in skeletal muscle.

The influence of immobilization stress on cardiac protein synthesis. A possible regulatory role for glucocorticoids.

High serum corticosterone levels and transient depletion of cytosolic glucocorticoid receptor binding capacity were observed in rat cardiac muscle following immobilization stress. To evaluate the effect of this treatment on the protein synthetic capacity, biologically active polyribosomes were used to direct the in vitro synthesis of polypeptides in the rabbit reticulocyte lysate. The results of these experiments indicate that the template activity of several messenger RNAs coding for major myofibrillar proteins was increased. This change in protein synthetic activity was not observed in adrenalectomized animals. Collectively, these results suggest that glucocorticoids are involved in the regulation of cardiac protein biosynthesis.

Hydrophobic behavior of sciatin and transferrin on a size-exclusion column.

Sciatin and transferrin are very similar glycoproteins which differ slightly in their carbohydrate content. In two-dimensional gel electrophoresis, they have one different isoform at pI lower than 5.77. However, highly different elution profiles have been recorded following size-exclusion HPLC. The use of a I-125 silica gel column has thus provided convincing evidence that both proteins do not show exactly the same hydrophobic three-dimensional structure.

Variations of glucocorticoid receptors in intact or denervated muscles: lack of cause-effect relationship with muscle atrophy in the rat.

The process of muscular atrophy following denervation has been tentatively ascribed to the influence of glucocorticoids (G) because of the rapid increase of cytosolic G receptors (RG) after sciatic nerve section. It appears however that the level of muscular atrophy is similar: in slow or fast-twitch muscles in spite of huge variations in RG; in intact or adrenalectomized (ADR-X) rats. Moreover, the protein muscle profile of intact of ADR-X rats after gel electrophoresis is similar but drastically decreased after 3 weeks of denervation. We conclude that there is no cause-effect relationship between muscle atrophy and RG elevation after nerve section.

Modulation of a major 30-kDa skeletal muscle protein by thyroid hormone.

Thyroidectomy results in the transformation of type II fibres to type I in rat soleus muscle. In vitro translations containing polyribosomes indicate that the template activity of mRNA coding for a 30-kDa protein is increased in hypothyroid (6 months) rats. The cellular content of this protein is also increased in hypothyroid rats. The in vitro synthesis of the 30-kDa protein is not observed in thyroidectomized (10 weeks) rats that have been treated with triiodothyronine. The synthesis and accumulation of this protein are directly related to the proportion of type I fibres in rat skeletal muscle and appear to be modulated by thyroid hormone.

Determination of occupied cytoplasmic glucocorticoid receptor sites by an exchange assay in rat muscles.

We have established an assay to determine total cytoplasmic glucocorticoid receptor sites in heart, vastus and levator-ani-bulbocavernosus muscles from rats. We have tested the exchange of endogenously bound hormone with labeled triamcinolone acetonide by performing long incubation at 0-4 degrees C. This procedure appeared to produce an adequate exchange of exogenously added unlabeled corticosterone in the range of 16 nM (normal unstressed concentration of corticosterone in plasma) with vastus and levator ani-bulbocavernosus muscles. Beyond that level, the displacement of unlabeled corticosterone by labeled triamcinolone acetonide was less efficient. By contrast, in heart and especially in liver cytosols, the exchange was adequate even in the presence of high concentrations of corticosterone (64 and 460 nM respectively). Using the exchange assay conditions (20 h of incubation at 0-4 degrees C with 16 nM of labeled triamcinolone acetonide), we found no significant difference between glucocorticoid receptor levels in intact male and female rats. In both sexes, these glucocorticoid receptor values were not affected by gonadectomy, but increased more than 2-fold after adrenalectomy. No seasonal variations were recorded in intact or adrenalectomized rats.

[Partial identification and variations of the cytoplasmic androgen receptor in the rat kidney]. / Caractérisation partielle et variations du récepteur cytoplasmique des androgènes dans le rein de rat.

In the course of our studies on the fate of cytoplasmic receptors for androgens in various target organs, such a receptor has been partially characterized in rat kidney cytosol: the high affinity constant, the specificity for testosterone and the formation of a hormone-receptor complex with a 8-10S sedimentation coefficient on sucrose density gradient led us to estimate that this macromolecule was similar to the rat prostate androgen receptor. This receptor is partially occupied by circulating androgens because the number of binding sites increases following castration or hypophysectomy. Constant levels of receptors have been recorded for several months in absence of testosterone. The age and the sex of animals, as well as the functional state of thyroid gland, did not affect the number of binding sites. These receptor characteristics may explain the slight renotrophic action of androgens in rat kidney.

[Measurement of cytoplasmic androgen receptor in compensatory renal hypertrophy in the rat]. / Mesure du récepteur cytoplasmique des androgènes au cours de l'hypertrophie rénale compensatrice chez le rat.

In order to determine whether kidney hypertrophy secondary to unilateral nephrectomy was an appropriate model to study cytoplasmic androgen receptor modulation, this receptor was measured three weeks after surgery in intact or castrated animals. Our results demonstrate that the process of kidney hypertrophy is not accompanied by a proportional elevation of the number of androgen receptors and that these receptors are not significantly influenced by the gonadal activity in the rat.

Binding of methyltrienolone to glucocorticoid receptors in rat muscle cytosol.

Androgen receptor measurements with [3H]-methyltrienolone [17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881)] or [3H]testosterone in vastus lateralis muscle cytosol of intact male rats yielded similar numbers of binding sites. Gonadectomy significantly increased the androgen receptor values, and both steroids gave similar results. The binding of [3H]-testosterone was not affected by adrenalectomy. In contrast, [3H]R1881 binding increased 2-fold after adrenalectomy. Competition experiments as well as sucrose density gradient analysis indicated that R1881, in addition to its binding of the androgen receptor, was also bound to glucocorticoid receptor. These results show that the synthetic androgen R1881 must be used with caution in tissues suspected to contain glucocorticoid receptors.