Continuous infusion of 20-hydroxyecdysone increased mass of triceps brachii in C57BL/6 mice.

Phytoecdysteroids have been attributed with numerous pharmacological properties in animals, including increasing muscle mass, and 20-hydroxyecdysone (20E) is one of the most abundant phytoecdysteroids produced by plants. In this study, the physiological and gene expression effects of 20E were analyzed in C57BL/6 mice given a continuous infusion of saline or 20E (5 mg/kg/day) for 5 or 15 days using subcutaneously implanted Alzet® osmotic pumps. The masses of the total body, muscle groups and organs were determined. There was a significant increase ( p = 0.01) in the mass of triceps brachii in mice treated with 20E for 5 days (115 ± 8 mg) compared with mice treated with saline for 5 days (88 ± 3 mg), however, there were no differences in the other measured parameters. To determine potential mechanisms of 20E in skeletal muscle, Illumina's Mouse Whole Genome-6 v2.0 Expression BeadChips were used to evaluate changes in gene expression of the triceps brachii after 20E infusion. Ingenuity Pathways Analysis was used to identify genes with the most evidence for differential expression, of which, 16 genes involved in the skeletal and muscular system were identified. Overall, the data suggest that 20E does not have potent anabolic properties, however, a muscle-specific increase was observed and genes were identified to provide an explanation for the muscle accretion.

Prion sequence polymorphisms and chronic wasting disease resistance in Illinois white-tailed deer (Odocoileus virginianus).

Nucleic acid sequences of the prion gene (PRNP) were examined and genotypes compiled for 76 white-tailed deer from northern Illinois, which previously tested positive for chronic wasting disease (CWD), and 120 negative animals selected to control for geographic location and age. Nine nucleotide polymorphisms, seven silent and two coding, were found in the sampled population. All observed polymorphisms except two of very low frequency were observed in both negative and positive animals, although five polymorphic loci had significantly different distributions of alleles between infected and non-infected individuals. Nucleotide base changes 60C/T, 285A/C, 286G/A and 555C/T were observed with higher than expected frequencies in CWD negative animals suggesting disease resistance, while 153C/T was observed more than expected in positive animals, suggesting susceptibility. The two coding polymorphisms, 285A/C (Q95H) and 286G/A (G96S), have been described in white-tailed deer populations sampled in Colorado and Wisconsin. Frequency distributions of coding polymorphisms in Wisconsin and Illinois deer populations were different, an unexpected result considering the sampled areas are less than 150 km apart. The total number of polymorphisms per animal, silent or coding, was negatively correlated to disease status. The potential importance of silent polymorphisms (60C/T, 153C/T, 555C/T), either individually or cumulatively, in CWD disease status has not been previously reported.

Characterization of calpastatin gene in fish: its potential role in muscle growth and fillet quality.

Calpastatin (CAST), the specific inhibitor of the calpain proteases, plays a role in muscle growth and meat quality. In rainbow trout (RBT), we identified cDNAs coding for two CAST isoforms, a long (CAST-L) and a short isoform (CAST-S), apparently derived from two different genes. Zebrafish and pufferfish CAST cDNA and genomic sequences were retrieved from GenBank and their exon/intron structures were characterized. Fish CASTs are novel in that they have fewer repetitive inhibitory domains as compared to their mammalian counterparts (one or two vs. four). The expressions of CAST mRNAs were measured in three RBT strains with different growth rates and fillet firmness that were fed either high energy or control diets. CAST-L and S expressions were significantly lower (p<0.01) in the strain that has the slowest growth rate and yielded the softest fillet. Strain or diet did not affect level of calpain mRNAs. However, the decrease in the CAST/calpain ratio at the mRNA level did not lead to a corresponding change in the calpain catalytic activity. Further investigation should reveal a potential use of the CAST gene as a tool to monitor fish muscle growth and fillet firmness.

Identification and molecular characterization of the rainbow trout calpains (Capn1 and Capn2): their expression in muscle wasting during starvation.

Calpains are calcium regulated proteases involved in cellular functions that include muscle proteolysis both ante- and postmortem. Here, we describe the molecular characterization of the rainbow trout catalytic subunits of the mu- and m-calpains, respectively. The cDNA sequence for Capn1 encodes a protein of 704 amino acids with a calculated molecular mass of 79.9 kDa. The amino acid sequence shows 66% and 86% identity with the mouse and zebrafish Capn1, respectively. The Capn2 cDNA codes for a protein consisting of 701 amino acid residues with a calculated molecular mass of 78.2 kDa. The protein shows 65% amino acid sequence identity with the mouse and chicken Capn2. The two isozymes of rainbow trout have the characteristic domains: I (propeptide), II (cysteine catalytic site), III (electrostatic switch), and IV (contains five EF-hands). Because starvation induces muscle wasting, the hypothesis of this study was that starvation could affect regulation of the calpain system in muscle. Starvation of rainbow trout fingerlings (15-20 g) for 35 days stimulated the expression of Capn1 (2.2-fold increase, P < 0.01), Capn2 (6.0-fold increase, P < 0.01), and calpastatins (1.6-fold increase, P < 0.05) as measured by quantitative real-time RT-PCR. The mRNA changes led to a 1.23-fold increase in the calpain catalytic activity. The results suggest a potential role of calpains in protein mobilization as a source of energy under fasting condition.

Lysine alpha-ketoglutarate reductase and lysine oxidation are distributed in the extrahepatic tissues of chickens.

In animals, lysine oxidation is thought to occur primarily via the activity of lysine alpha-ketoglutarate reductase (LKR). This activity was reported previously in chicken liver, but no work on the tissue distribution of the enzyme in chickens has been reported. Therefore, LKR activity was assayed in liver, kidney, pancreas, heart, brain, lung, spleen, muscle, and intestinal tissues in chickens as was the in vitro ability of tissue homogenates to oxidize lysine. Additionally, the expression of LKR mRNA was assessed by RT-PCR. We found LKR to be present in all tissues studied by both enzymatic analysis and mRNA abundance. Additionally, all tissues assayed oxidized lysine. The extent of lysine oxidation differed among the tissues, consistent with the different pathways of lysine oxidation in the different tissues. These studies demonstrate that LKR is widely distributed in chicken tissues and that tissues other than liver can contribute to whole-body lysine oxidation.

The effects of in ovo rhIGF-I administration on expression of the growth hormone secretagogue receptor (GHSR) during chicken embryonic development.

Growth hormone secretion is under the control of a pair of hypothalamic factors, growth hormone releasing hormone and somatostatin. The growth hormone secretagogue receptor (GHSR) and its endogenous ligand represent a novel third method regulating the release of growth hormone. Early chicken embryonic development has been proposed to be independent of GH. However, recent evidence shows that peripheral GH secretion has paracrine/autocrine functions during embryonic development. In the current study, we used the reverse-transcriptase polymerase chain reaction to determine the expression pattern of the GHSR during embryonic development and the effects of in ovo recombinant human (rh) IGF-I administration on its expression pattern. Eggs were injected once with 100 ng rhIGF-I in 10 mM acetic acid, and 0.1% BSA per embryo on embryonic day 3. Total RNA was isolated from whole embryos on embryonic day (E) 0-6 (n=6 per day), thoracic/abdominal halves of the embryos on E7- E8 (n= 6 per day) and Pectoralis muscle on E9-E20 (n= 4 per day). We found that GHSR expression was low during E0-E4, followed by an increase on E5 and remained constant through E17. GHSR expression then increased on E18 before reducing on E20. A similar pattern was found in the rhIGF-I treated embryos with the exception of a significant increase in GHSR expression on E8. These data indicate that the GHSR may be active in regulating GH secretion during early embryonic development, and upregulation of the GHSR gene following IGF-I administration may have an important role in the determination of postnatal muscle growth.

Myostatin and TGF-beta2 gene expression patterns in response to in ovo administration of rhIGF-I during chicken embryonic development.

The objective of the study was to evaluate the impact of in ovo administration of recombinant human insulin-like growth factor-I (rhIGF-I) on myostatin and transforming growth factor-beta2 (TGF-beta2) gene expression during chicken embryogenesis with emphasis on skeletal muscle development. Eggs were injected once with 100 ng rh IGF-I in 10 mM acetic acid, 0.1% BSA per embryo on day 3 of embryonic development. Total RNA was isolated from whole embryos on each of embryonic days (E) 0 to 6 (n = 6 per day/per treatment), from thoracic/abdominal halves of the embryo at E 7 to 8 (n = 6 per day/per treatment), and from pectoralis muscle tissues at E 9 to 20 (n = 4 per day/per treatment). Reverse-transcription polymerase chain reaction (RT-PCR) was used to synthesize cDNAs. Myostatin mRNA isolated from pectoralis muscles of the rhIGF-I treated group increased on E 10 (approximately 2.5 fold) and remained high through E 13, whereas myostatin mRNA from control pectoralis muscles increased at E 9 and remained high until E 12. TGF-beta2 gene expression from in ovo rhIGF-I treated pectoralis muscles dramatically increased at E 13 (approximately 2.5 fold), in contrast to E 14 from control pectoralis muscle, and gradually declined through E 16. Our results demonstrate that in ovo administration of rhIGF-I on E 3 may alter developmental expression patterns of myostatin and TGF-beta2 genes.

IGF-I, IGF-II, and IGF-receptor-1 transcript and IGF-II protein expression in myostatin knockout mice tissues.

Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to demonstrate whether a correlation exists between insulin-like growth factors (IGFs)-positive regulators of growth-and myostatin, a negative regulator of muscle growth. IGF-I, -II, and IGF-receptor-1 (IGF-R1) mRNA and IGF-II protein expressions were determined in control and myostatin knockout mice tissues. IGF-I gene expressions were similar between control and knockout mice tissues, whereas IGF-II mRNA levels were significantly higher in myostatin knockout mice kidney and soleus muscles than those of control mice (P <.01). IGF-R1 mRNA levels from control mice heart (P <.05) and kidney (P <.01) were significantly higher than in myostatin knockout mice, whereas levels were lower in pectoralis muscle of control mice than knockout mice (P <.01). The strongly IGF-II-positive cells in soleus muscle were more common in myostatin knockout mice and were seen in a few foci in control mice. IGF-II immunoreactivity in both control and myostatin knockout mice kidneys was localized to the epithelium of renal tubules and collecting ducts. Reciprocal changes in the expression of myostatin and IGF-II and IGF-R1 may underlie normal growth of skeletal muscle and other organs in mammals, and the changes in these tissues associated with disease.