The serial analysis of gene expression (SAGE) method is used to study global gene expression in cells or tissues in various experimental conditions. However, its reproducibility has not yet been definitively assessed. In this study, we have evaluated the reproducibility of the SAGE method and identified the factors that affect it. The determination coefficient (R2) for the reproducibility of SAGE is 0.96. However, there are some factors that can affect the reproducibility of SAGE, such as the replication of concatemers and ditags, the number of sequenced tags and double PCR amplification of ditags. Thus, corrections for these factors must be made to ensure the reproducibility and accuracy of SAGE results. A bioinformatic analysis of SAGE data is also presented in order to eliminate these artifacts. Finally, the current study shows that increasing the number of sequenced tags improves the power of the method to detect transcripts and their regulation by experimental conditions.
In order to characterize the action of androgen in skeletal muscle, we have investigated the effects of castration (GDX) and dihydrotestosterone (DHT) on global gene expression in mice. The serial analysis of gene expression method was performed in the muscle of male mice in six experimental groups: intact, GDX and GDX+DHT injection 1, 3, 6 or 24 h before they were killed. A total of 780 822 sequenced tags quantified the expression level of 80 142 tag species. Thirteen and seventy-nine transcripts were differentially expressed in GDX and DHT respectively (P,0·05), including eight partially characterized and 21 potential novel transcripts. The induced transcripts within 3 h after DHT injection were involved in the following functions: transcription, protein synthesis, modification and degradation, muscle contraction and relaxation, cell signaling, polyamine biosynthesis, cell cycle progression and arrest, angiogenesis, energy metabolism and immunity. However, the inductions of transcripts related to cell cycle arrest and angiogenesis were no longer significant 24 h after DHT injection. The current study might suggest that DHT promotes protein synthesis, cell signaling, cell proliferation and ATP production, as well as muscle contraction and relaxation at the transcriptional level in skeletal muscle in vivo.
Dietary red pepper suppresses energy intake and modifies macronutrient intake. We have investigated whether a stimulus in the mouth and the sensation of spiciness are necessary for red pepper-induced changes in energy and macronutrient intake in human volunteers. In a preliminary test, sixteen Japanese male volunteers tasted samples of a soup with graded doses of red pepper in order to define a moderate and a maximum tolerable (strong) dose of red pepper. On the day of the experiment, a standardised breakfast was given to the volunteers. At lunchtime, the subjects ingested one of four experimental soups containing either a placebo, a moderate or a strong dose of red pepper plus placebo capsules, or a placebo soup plus capsules delivering a strong dose of red pepper. The rest of the meal was given ad libitum to all subjects. The amount of food, protein and carbohydrate ingested was similar for all conditions. Energy and fat intake were similar after the ingestion of the moderate soup compared with placebo. However, the strong soup significantly lowered fat intake compared with placebo (P¼0·043), and ingestion of strong capsules also tended to suppress it (P¼ 0·080). Moreover, energy intake after strong soup and capsules tended to be lower than placebo (P¼0·089 and 0·076, respectively). The present results indicate that the maximum tolerable dose is necessary to have a suppressive effect of red pepper on fat intake. The main site of the action of red pepper is not in the mouth. Red pepper: Spiciness: Macronutrient intakeObesity is an important cause of morbidity and is having an increasingly negative impact on healthcare systems in both industrialised and developing countries (Taubes, 1998;Wickelgren, 1998). Obesity can be simply described as the outcome of a positive energy balance characterised by an excess of energy intake over expenditure. Consequently, the prevention and treatment of obesity is possible through an increase in energy expenditure or a decrease in energy intake. Capsaicin, the pungent principal of red pepper, is known to enhance energy metabolism via a stimulation of the sympathoadrenal system (Watanabe et al. 1991). As a result, an ingestion of capsaicin decreases body-fat mass in a dose-dependent manner (Watanabe et al. 1991). The ingestion of red pepper stimulates the postprandial increase in RMR and the thermic effect of feeding (TEF), suppresses energy intake and modifies macronutrient intake (Yoshioka et al. 1999(Yoshioka et al. , 2001). Dietary fat is an important factor in the development of obesity because of its high energy density (Warwick & Schiffman, 1992), its low potency to induce satiety (Lissner et al. 1987;Kendall et al. 1991;Thomas et al. 1992) and its little contribution to the TEF (Flatt et al. 1985;Schutz et al. 1989;Bennett et al. 1992). These studies led to the proposal that capsaicin (red pepper) could be useful to treat obesity or prevent its development (Doucet & Tremblay, 1997;Dulloo, 1998). Diamond et al. (1985) have demonstrated that the TEF consists of a cephalic p...
Sarcopenia is related to metabolic syndrome in postmenopausal women. Hormone replacement therapies with androgens improve muscle functions by molecular mechanisms that are still unknown, at least partly because the skeletal muscle transcriptome has been less characterized in females. We performed the serial analysis of gene expression method in six experimental groups, intact (male and female), ovariectomy (OVX), OVXCdihydrotestosterone (DHT) injection 1, 3, or 24 h before kill in mice. The 438 transcript species differentially expressed between gender showed that females had higher expression levels of mRNA related to cytoskeleton/contractile apparatus and mitochondrial processes as well as protein, lipid, and amino acid metabolisms. In females, OVX and DHT modulated 109 and 128 transcript species respectively. OVX repressed transcripts of fast/glycolytic fiber, glycolysis, and glucose transport, whereas all these effects were reversed 3 h after the DHT injection. Moreover, DHT treatment induced transcripts which reduce intracellular Ca 2C level at early time points. These results may suggest that DHT treatment in OVX mice increases muscle contractility by affecting fiber distribution and intracellular Ca 2C concentration as well as improving glucose metabolism. On the other hand, transcripts of fast/oxidative fiber, oxidative phosphorylation, and ATP production were repressed 24 h after DHT administration. In our previous study using male mice, transcripts in oxidative phosphorylation and ATP production were induced 24 h after DHT injection (Yoshioka M, Boivin A, Ye P, Labrie F & St-Amand J 2006 Effects of dihydrotestosterone on skeletal muscle transcriptome in mice measured by serial analysis of gene expression. Journal of Molecular Endocrinology 36 247-259). These results demonstrate gender differences in DHT actions on skeletal muscle, and contribute to a precise understanding of the molecular mechanisms of androgen actions in the female skeletal muscle.
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