Shyuichi Ohwada

Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle

Various cytokines are thought to play a role in muscle regeneration, however, the interaction and mechanisms of action of these cytokines remains largely unknown. In this study, we investigated the role of HGF, IGF-I, and IGF-II during myogenesis using the regeneration model of skeletal muscle as well as myoblast culture. RT-PCR analysis revealed that HGF and IGF-I expressions were markedly upregulated, in regenerating muscle. In contrast, there was no significant difference in IGF-II expression between normal and regenerating muscle. Immunohistochemical analysis demonstrated that HGF was expressed mostly by myocytes during the early stages of muscle regeneration. Additionally, HGF inhibited the formation of myotubes by myoblasts, but promoted cellular proliferation. Otherwise, IGF-I and IGF-II were expressed by myocytes through the early to middle stages of muscle regeneration. The addition of HGF to myoblast growing in vitro significantly increased the number of cells. These findings indicate that these three cytokines have pleiotropic effects in regenerating skeletal muscle.

Distribution of myofiber types in thigh muscles of chickens

The composition of myofiber types varies within thigh muscles of chickens. The present study was designed to determine whether or not myofiber types were distributed uniformly across the diameter of the thigh muscles of chickens. Cross sections from middle portions of muscles were used histochemically to examine differences in distribution and composition of myofiber types in the muscles. Myofibers that reacted moderately (M) or strongly (S) for myosin adenosine triphosphatase (ATPase) after preincubation at pH 4.3 were classified as type I. Type I myofibers reacted weakly (W), moderately (M), or strongly (S) for ATPase after preincubation at pH 10.6; these type I myofibers were subclassified into four types (ISW, ISM, ISS, and IMM). Myofibers that reacted negatively for acid‐stable ATPase and strongly for alkali‐stable ATPase were classified into two types: type IIA, with strong NADH tetrazolium reductase (NADH‐TR), and type IIB, with weak NADH‐TR activity. The M. pubo‐ischio‐femoralis pars lateralis had numerous type IIA myofibers and very few type ISM myofibers, whereas the pars medialis had many type IMM myofibers and few type ISS and IIA myofibers. The type I group of myofibers did not exceed about 50% in the other muscles, which had one to three types of type ISW, ISM, and ISS myofibers. The Mm. femorotibiales had more type ISW, and ISM myofibers in the deep regions near the femur than in the superficial regions. The M. iliotibialis cranialis, M. iliofibularis, and M. flexor cruris medialis had more type ISW, ISM, or ISS myofibers in the medial regions than in the lateral regions. A few type ISW myofibers were scattered in the cranial part of M. iliotibialis and in the M. ambiens. The M. flexor cruris lateralis pars pelvica had type IIA and IIB myofibers exclusively. All the muscles had type IIA myofibers. Type IIB myofibers existed in the muscles except the M. puboischio‐femoralis. Type IIA and IIB myofibers differed in proportion in different muscles and in their different regions. The type I group of myofibers was generally concentrated more in the deep regions near the femur and in the medial regions than in the superficial and lateral regions of the thigh muscles. The distribution of type IIA myofibers resembled that of type I group. Type IIB myofibers showed a distribution opposite to that of type I group and IIA myofibers. The spatial distribution of myofiber types within individual muscles can account for the various locomotory and postural requirements of the thigh.

Immunobiotic Lactobacillus jensenii Modulates the Toll-Like Receptor 4-Induced Inflammatory Response via Negative Regulation in Porcine Antigen-Presenting Cells

ABSTRACT Previously, we demonstrated that Lactobacillus jensenii TL2937 attenuates the inflammatory response triggered by activation of Toll-like receptor 4 (TLR-4) in porcine intestinal epithelial cells. In view of the critical importance of antigen-presenting cell (APC) polarization in immunoregulation, the objective of the present study was to examine the effect of strain TL2937 on the activation patterns of APCs from swine Peyers patches (PPs). We demonstrated that direct exposure of porcine APCs to L. jensenii in the absence of inflammatory signals increased expression of interleukin-10 (IL-10) and transforming growth factor β in CD172a+ APCs and caused them to display tolerogenic properties. In addition, pretreatment of CD172a+ APCs with L. jensenii resulted in differential modulation of the production of pro- and anti-inflammatory cytokines in response to TLR4 activation. The immunomodulatory effect of strain TL2937 was not related to a downregulation of TLR4 but was related to an upregulation of the expression of three negative regulators of TLRs: single immunoglobulin IL-1-related receptor (SIGIRR), A20, and interleukin-1 receptor-associated kinase M (IRAK-M). Our results also indicated that TLR2 has an important role in the anti-inflammatory activity of L. jensenii TL2937, since anti-TLR2 antibodies blocked the upregulation of SIGIRR and IRAK-M in CD172a+ APCs and the production of IL-10 in response to TLR4 activation. We performed, for the first time, a precise functional characterization of porcine APCs from PPs, and we demonstrated that CD172a+ cells were tolerogenic. Our findings demonstrate that adherent cells and isolated CD172a+ cells harvested from swine PPs were useful for in vitro study of the inflammatory responses in the porcine gut and the immunomodulatory effects of immunobiotic microorganisms.

Peripheral Serotonin Enhances Lipid Metabolism by Accelerating Bile Acid Turnover

Serotonin is synthesized by two distinct tryptophan hydroxylases, one in the brain and one in the periphery. The latter is known to be unable to cross the blood-brain barrier. These two serotonin systems have apparently independent functions, although the functions of peripheral serotonin have yet to be fully elucidated. In this study, we have investigated the physiological effect of peripheral serotonin on the concentrations of metabolites in the circulation and in the liver. After fasting, mice were ip injected with 1 mg serotonin. The plasma glucose concentration was significantly elevated between 60 and 270 min after the injection. In contrast, plasma triglyceride, cholesterol, and nonesterified fatty acid concentrations were decreased. The hepatic glycogen synthesis and concentrations were significantly higher at 240 min. At the same time, the hepatic triglyceride content was significantly lower than the basal levels noted before the serotonin injection, whereas the hepatic cholesterol content was significantly higher by 60 min after the injection. Furthermore, serotonin stimulated the contraction of the gallbladder and the excretion of bile. After the serotonin injection, there was a significant induction of apical sodium-dependent bile acid transporter expression, resulting in a decrease in the concentration of bile acids in the feces. Additionally, data are presented to show that the functions of serotonin are mediated through diverse serotonin receptor subtypes. These data indicate that peripheral serotonin accelerates the metabolism of lipid by increasing the concentration of bile acids in circulation.

Orally Administered Prion Protein Is Incorporated by M Cells and Spreads into Lymphoid Tissues with Macrophages in Prion Protein Knockout Mice

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases. Infection by the oral route is assumed to be important, although its pathogenesis is not understood. Using prion protein (PrP) knockout mice, we investigated the sequence of events during the invasion of orally administered PrPs through the intestinal mucosa and the spread into lymphoid tissues and the peripheral nervous system. Orally administered PrPs were incorporated by intestinal epitheliocytes in the follicle-associated epithelium and villi within 1 hour. PrP-positive cells accumulated in the subfollicle region of Peyers patches a few hours thereafter. PrP-positive cells spread toward the mesenteric lymph nodes and spleen after the accumulation of PrPs in the Peyers patches. The number of PrP molecules in the mesenteric lymph nodes and spleen peaked at 2 days and 6 days after inoculation, respectively. The epitheliocytes in the follicle-associated epithelium incorporating PrPs were annexin V-positive microfold cells and PrP-positive cells in Peyers patches and spleen were CD11b-positive and CD14-positive macrophages. Additionally, PrP-positive cells in Peyers patches and spleen were detected in the vicinity of peripheral nerve fibers in the early stages of infection. These results indicate that orally delivered PrPs were incorporated by microfold cells promptly after challenge and that macrophages might act as a transporter of incorporated PrPs from the Peyers patches to other lymphoid tissues and the peripheral nervous system.

Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice

Insulin‐like growth factor‐1 (IGF‐1) is a positive regulator in proliferation and differentiation of skeletal muscle cells, while myostatin (MSTN) is a member of transforming growth factor β superfamily that acts as a negative regulator of skeletal muscle mass. The present study was performed to detail whether a correlation exists between MSTN and IGF‐1 in skeletal muscle of IGF‐1 knockout mice (IGF‐1−/−) and their wild type (WT; i.e., IGF‐1+/+) littermates. The body weight of IGF‐1−/− animals was 32% that of WT littermates. The fiber cross‐sectional areas (CSA) and number of fibers in M. rectus femoris of IGF‐1−/− animals were 49 and 59% those of WT animals, respectively. Thus, muscle hypoplasia of IGF‐1−/− undoubtedly was confirmed. Myostatin mRNA levels and protein levels were similar between M. gastrocnemius of IGF‐1−/− and WT animals. Myostatin immunoreactivity was similarly localized in muscle fibers of both IGF‐1−/− and WT M. rectus femoris. The mRNA levels of MyoD family (Myf5, MyoD, MRF4, myogenin) were differentially expressed in IGF‐1−/− M. gastrocnemius, in which the mRNA expression of MRF4 and myogenin was significantly lower, whereas there were no changes in the mRNA expression of Myf5 and MyoD. These findings first describe that myostatin expression is not influenced by intrinsic failure of IGF‐1, although MRF4 and myogenin are downregulated.

Localization of interleukin-18 and its receptor in somatotrophs of the bovine anterior pituitary gland

A pro-inflammatory cytokine, interleukin 18 (IL-18), induces intracellular expression of IL-1 and the release of IL-6. IL-1 and IL-6 has been detected in anterior pituitary cells, suggesting that IL-18 is produced in anterior pituitary cells and may serve to aid immuno-endocrine regulation. In the present study, we addressed this hypothesis by investigating the intracellular localization of IL-18 and its receptor in bovine anterior pituitary gland. IL-18 mRNA and its protein were detected in the anterior pituitary gland by RT-PCR and Western blotting. In situ hybridization showed that IL-18 mRNA was expressed in the anterior pituitary cells. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs. Furthermore, the expression of GH mRNA in IL-18 immunoreactive cells was confirmed by immuno-laser microdissection. These results first demonstrated that somatotrophs produced IL-18. Subsequently, the distribution of the IL-18 receptor alpha (IL-18Rα) was investigated in order to understand IL-18 signaling among the anterior pituitary cells. Bovine IL-18Rα cDNA was partially sequenced and detected in the anterior pituitary gland by RT-PCR. Immunohistochemistry of IL-18Rα, IL-18 and GH showed that IL-18Rα was co-localized in IL-18 immunoreactive cells or somatotrophs. These data suggest that IL-18 acts on somatotrophs as an immuno-endocrine mediator through the autocrine pathway.

Effects of Na-butyrate supplementation in milk formula on plasma concentrations of GH and insulin, and on rumen papilla development in calves

Although the growth-promoting action of sodium-butyrate (Na-butyrate) used as a feed additive has been observed in calves and pigs, the precise mechanisms involved remain to be clarified. In this study, pre-weaning calves were given milk formula (MF) supplemented with butyrate for 6 weeks to investigate its effects on postprandial changes in the plasma concentrations of metabolic hormones, and, simultaneously, on growth performance, the weight of the digestive organs and rumen papilla development. Ingestion of MF increased (P<0.05) the plasma concentrations of GH and insulin as well as the glucose level, but decreased the non-esterified fatty acid concentration. Na-butyrate supplementation in MF or in lactose solution (with the same quantity of lactose contained in the MF, 5%) suppressed the increase in plasma insulin and GH concentrations, and the plasma IGF1 level was not changed. The length of the rumen papilla and the weight of the perirenal fat tended to increase in the calves fed with Na-butyrate-supplemented MF, but the weight of the liver, spleen, and stomach were not changed. In addition, there was no difference in the expression of mRNA for sodium-dependent glucose transporter-1 in the small intestinal epithelial tissues. We conclude that the accelerated growth performance related to the intake of Na-butyrate used as a feed additive reported previously in several species is partly due to improved insulin sensitivity and a better digestive functional development. These data could be applicable to animal and human nutrition.

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

The transforming growth factor (TGF)‐β 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‐β, 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. J. Cell. Physiol. 226: 1128–1136, 2011.

Serotonin Improves High Fat Diet Induced Obesity in Mice.

There are two independent serotonin (5-HT) systems of organization: one in the central nervous system and the other in the periphery. 5-HT affects feeding behavior and obesity in the central nervous system. On the other hand, peripheral 5-HT also may play an important role in obesity, as it has been reported that 5-HT regulates glucose and lipid metabolism. Here we show that the intraperitoneal injection of 5-HT to mice inhibits weight gain, hyperglycemia and insulin resistance and completely prevented the enlargement of intra-abdominal adipocytes without having any effect on food intake when on a high fat diet, but not on a chow diet. 5-HT increased energy expenditure, O2 consumption and CO2 production. This novel metabolic effect of peripheral 5-HT is critically related to a shift in the profile of muscle fiber type from fast/glycolytic to slow/oxidative in soleus muscle. Additionally, 5-HT dramatically induced an increase in the mRNA expression of peroxisome proliferator-activated receptor coactivator 1α (PGC-1α)-b and PGC-1α-c in soleus muscle. The elevation of these gene mRNA expressions by 5-HT injection was inhibited by treatment with 5-HT receptor (5HTR) 2A or 7 antagonists. Our results demonstrate that peripheral 5-HT may play an important role in the relief of obesity and other metabolic disorders by accelerating energy consumption in skeletal muscle.