Effect of peripheral 5-HT on glucose and lipid metabolism in wether sheep.

In mice, peripheral 5-HT induces an increase in the plasma concentrations of glucose, insulin and bile acids, and a decrease in plasma triglyceride, NEFA and cholesterol concentrations. However, given the unique characteristics of the metabolism of ruminants relative to monogastric animals, the physiological role of peripheral 5-HT on glucose and lipid metabolism in sheep remains to be established. Therefore, in this study, we investigated the effect of 5-HT on the circulating concentrations of metabolites and insulin using five 5-HT receptor (5HTR) antagonists in sheep. After fasting for 24 h, sheep were intravenously injected with 5-HT, following which-, plasma glucose, insulin, triglyceride and NEFA concentrations were significantly elevated. In contrast, 5-HT did not affect the plasma cholesterol concentration, and it induced a decrease in bile acid concentrations. Increases in plasma glucose and insulin concentrations induced by 5-HT were attenuated by pre-treatment with Methysergide, a 5HTR 1, 2 and 7 antagonist. Additionally, decreased plasma bile acid concentrations induced by 5-HT were blocked by pre-treatment with Ketanserin, a 5HTR 2A antagonist. However, none of the 5HTR antagonists inhibited the increase in plasma triglyceride and NEFA levels induced by 5-HT. On the other hand, mRNA expressions of 5HTR1D and 1E were observed in the liver, pancreas and skeletal muscle. These results suggest that there are a number of differences in the physiological functions of peripheral 5-HT with respect to lipid metabolism between mice and sheep, though its effect on glucose metabolism appears to be similar between these species.

Effect of myostatin on chemokine expression in regenerating skeletal muscle cells.

Transforming growth factor-ß (TGF-ß) is implicated in the regulatory expression of chemokines that control multiple steps in myogenesis. However, it remains to be established whether myostatin, a member of the TGF-ß superfamily, affects chemokine expression in skeletal muscle. We investigated the effects of myostatin on the expression of mRNAs and proteins for 4 chemokines (CXCL1, CXCL2, CXCL6, CCL2) in intact and regenerating musculus longissimus thoracis from normal-muscled (NM) and double-muscled (DM) cattle. These chemokines were expressed in regenerating muscle, and their expression was always lower in DM than in NM cattle. Immunohistochemistry revealed that CXCL1 and CXCL6 were detected in the regenerating areas of myoblasts and myotubes in both NM and DM cattle. In cultures of myoblasts isolated from the regenerating muscles, significantly less CXCL1, CXCL2 and CCL2 mRNA was expressed in DM myoblasts than in NM myoblasts during the proliferating stage (P-stage). The expression of CXCL1, CXCL2 and CCL2 mRNAs in NM myoblasts and CXCL1, CXCL2 and CXCL6 mRNAs in DM myoblasts decreased upon switching from P-stage to fusion stage (F-stage). Also, the expression of CXCL1, CXCL2 and CXCL6 mRNAs was significantly lower in DM than in NM myoblasts during the F-stage. The addition of 100 ng/ml myostatin during the F-stage attenuated the expression of CXCL1 and CXCL2 mRNAs and augmented that of CCL2. These results show for the first time that myostatin regulates the differential expression of chemokines in skeletal muscle cells.

Expression of myostatin in neural cells of the olfactory system.

Recent studies show that myostatin mRNA expression is found in some regions of the brain. However, the functional significance of this is currently unknown. We therefore investigated myostatin expression and function in the brain. In this study, we used immunohistochemistry, in situ hybridization, and RT-PCR analysis to reveal that myostatin is expressed in the mitral cells in the olfactory bulb (OB) and in neurons in the olfactory cortex (OC). Using 3D reconstruction, mitral cells positive for myostatin were positioned in the lateral and ventral regions of the OB. In contrast, myostatin-positive mitral cells were detected in mice at 2 weeks of age, but not on days 0 and 7 after birth. Activin receptor IIB, a myostatin receptor, was expressed in the OB, OC, hippocampus, and paraventricular thalamic nucleus. Moreover, c-Fos immunostaining in granule cells in the OB was augmented after intracerebroventricular injection of myostatin. These findings suggest that myostatin is localized in specific cells associated with the olfactory system of the brain and may act as a key inhibitor in cell and/or signal development of the olfactory system.

TIEG1 negatively controls the myoblast pool indispensable for fusion during myogenic differentiation of C2C12 cells.

The transforming growth factor (TGF)-ß inducible early gene (TIEG)-1 is implicated in the control of cell proliferation, differentiation, and apoptosis in some cell types. Since TIEG1 functioning may be associated with TGF-ß, a suppressor of myogenesis, TIEG1 is also likely to be involved in myogenesis. Therefore, we investigated the function of TIEG1 during myogenic differentiation in vitro using the murine myoblasts cell line, C2C12. TIEG1 expression increased during differentiation of C2C12 cells. Constitutive expression of TIEG1 reduced survival and decreased myotube formation. Conversely, knocking down TIEG1 expression increased the number of viable cells during differentiation, and accelerated myoblast fusion into multinucleated myotubes. However, expression of the myogenic differentiation marker, myogenin, remained unaffected by TIEG1 knockdown. The mechanism underlying these events was investigated by focusing on the regulation of myoblast numbers after induction of differentiation. The knockdown of TIEG1 led to changes in cell cycle status and inhibition of apoptosis during the initial stages of differentiation. Microarray and real-time PCR analyses showed that the regulators of cell cycle progression were highly expressed in TIEG1 knockdown cells. Therefore, TIEG1 is a negative regulator of the myoblast pool that causes inhibition of myotube formation during myogenic differentiation.

Myostatin down-regulates the IGF-2 expression via ALK-Smad signaling during myogenesis in cattle.

Myostatin (MSTN) is a negative regulator during muscle differentiation, whereas insulin-like growth factors (IGFs) are essential for muscle development. MSTN and IGFs act oppositely during myogenesis, but there is little information on the mutual relationship of MSTN and IGFs. The present study was conducted to examine whether MSTN affects IGF expression during early myogenesis in cattle. IGF-1 mRNA was similarly expressed in M. longissimus thoracis of double-muscled (DM) and normal (NM) Japanese shorthorn cattle. IGF-2 mRNA expression was consistently higher in the normal and regenerating muscle of DM cattle than those of NM cattle. When myoblasts were isolated from regenerating M. longissimus thoracis, IGF-2 mRNA expression showed a significant increase in differentiating DM derived myoblasts (DM-myoblasts) as compared with differentiating NM derived myoblasts (NM-myoblasts). An addition of recombinant mouse myostatin (rMSTN) to myoblast cultures attenuated IGF-2 mRNA expression and decreased myotube formation, but did not effect IGF-1 mRNA expression. An activin-like kinase (ALK) inhibitor, SB431542, mediates MSTN action, suppressed the translocation of Smad2/3 into the nucleus in DM-myoblasts, and restored the attenuated IGF-2 mRNA expression and the decreased myotube formation induced by rMSTN in myoblast cultures. The findings indicate that MSTN may negatively regulate myoblast differentiation by suppressing IGF-2 expression via ALK-Smad signaling.

Possible role of TIEG1 as a feedback regulator of myostatin and TGF-beta in myoblasts.

Myostatin and TGF-beta negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-beta signaling remains unclear. TGF-beta inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-beta signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-beta signaling using C2C12 myoblasts. Myostatin and TGF-beta induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage of differentiation. In contrast, TGF-beta enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-beta in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-beta. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-beta that prevents excess action in myoblasts.

The modulation of collagen fibril assembly and its structure by decorin: an electron microscopic study.

The present study was carried out to determine the effect of decorin in the process of collagen assembly. Collagen fibrils were obtained in vitro by aggregation from commercialized acid-soluble type I collagen with the addition of different concentrations of decorin (0-25 microg/ml). All specimens were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The distribution of collagen fibril diameters was also analyzed by TEM. In samples without or with low concentrations of decorin, highly porous collagen fiber networks were formed. On the other hand, dense networks were observed in samples treated with high concentrations of decorin. The influence of decorin secreted by cells on collagen fibrils was observed by SEM, and the fiber network elasticity was measured using a rheometer. SEM images showed that collagen fiber networks without fibroblasts were much looser than those cultured with normal fibroblasts. The networks cultured with the fibroblasts were composed of straight fibers with large diameters. On the other hand, collagen fiber networks cultured with siRNA-decorin-transfected (siDT) fibroblasts had loose, meandering fibers with small diameters. The elasticity of collagen fiber networks cultured with untransfected fibroblasts showed no significant difference over the 7-day incubation period. However, significantly lower elastic values were obtained for collagen fiber networks treated with siDT cells on days 3 and 7. In addition, after treatment with 5.0 or 25 microg/ml decorin, the l collagen fiber networks cultured with siDT cells exhibited an altered structure that showed a dense structure similar to that of the fiber networks cultured with untransfected fibroblasts. In conclusion, this in vitro study showed that decorin is a regulatory and architecturally small leucine-rich repeat proteoglycan in the process of collagen fibril assembly.