Tohru Matsui

Activin A inhibits differentiation of 3T3-L1 preadipocyte

We investigated the effect of activin A on differentiation of 3T3-L1 preadipocyte. Activin A suppressed the induction of terminal differentiation markers such as glycerol-3-phosphate dehydrogenase (GPDH) activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein (aP2) mRNA when the cells were treated with activin A throughout the differentiation period. Activin A treatment during the early phase decreased GPDH activity and aP2 mRNA level, and also reduced the expression of peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer binding protein (C/EBP) alpha mRNAs without affecting the expressions of the active isoforms of C/EBPbeta and its mRNA. On the other hand, activin A treatment had no effect on the mitotic clonal expansion. These results indicate that activin A inhibits adipogenesis via affecting the transcriptional factor cascade upstream of PPARgamma expression.

Formaldehyde treatment suppresses ruminal degradation of phytate in soyabean meal and rapeseed meal

Most of the P in oilseed meal is in the form of phytate P, and phytate forms complexes with protein. Phytate P has been considered to be absorbed easily in ruminants because of phytate degradation in the rumen. Treatment of oilseed meals with formaldehyde improves the nutritional value of protein through suppressing its ruminal degradation. The present experiment was conducted to study the effects of formaldehyde treatment on phytate degradation in the rumen. The ruminal degradation of phytate in formaldehyde-treated soyabean meal or rapeseed meal was determined by a nylon-bag technique in sheep. Soyabean meal and rapeseed meal were treated with formaldehyde at levels of 3, 5 or 10 g/kg. Treatment with formaldehyde suppressed phytate and protein degradation in both the oilseed meals. Compared with the regular soyabean meal, the regular rapeseed meal showed lower degradability of phytate in the rumen. These results suggest that treatment with formaldehyde suppresses ruminal degradation of phytate in oilseed meal. Thus, the absorption of P from oilseed meal is probably decreased by this treatment in ruminants.

Development of intramuscular fat in Wagyu beef cattle depends on adipogenic or antiadipogenic substances present in serum

In blood, there are many kinds of adipogenic or antiadipogenic factors such as hormones and vitamins. In this study, adipogenic activity in sera of fattened beef cattle was evaluated using cultured mouse 3T3-L1 preadipocytes. After the preadipocytes were grown to reach confluence, serum of fattened beef cattle was added into the culture medium (10%, vol/vol)for 3 days, and thereafter cellular sn-glycerol-3-phosphate dehydrogenase (GPDH) activity was determined as an index ofadipocyte differentiation. Sera were collected from 19 beef cattle (Wagyu and Wagyu × Holstein cross cattle) from three different farms at slaughter. Cellular GPDH activity was significantly different among the farms, and was affected by sex difference (i.e. sera from fattened heifers induced higher GPDH activity than those from steers). There was a positive correlation between GPDH activity and beef marbling performance (T = 0·62, P < 0·02), suggesting that serum factor(s) play a role in development of intramuscular fat deposition. Adipogenic activity was negatively correlated with serum retinol concentration (r = −0·73, P < 0·001). Neither serum cholesterol, triacylglycerol nor non-esterified fatty acid was related to adipogenic activity.Furthermore, serum retinol concentration was negatively correlated with beef marbling performance. These data imply that retinol level in blood during the fattening period may influence intramuscular fat deposition of beef cattle through its antiadipogenic action on preadipocytes present in muscle tissues.

Induction of beige-like adipocytes in 3T3-L1 cells.

ABSTRACT There are two types of brown adipocytes: classical brown adipocytes that form the brown fat depots and beige adipocytes that emerge in the white fat depots. Beige adipocytes have a low level of uncoupling protein 1 (Ucp1) expression in the basal state, but Ucp1 expression is increased in response to β adrenergic receptor activation. The present study explored the factors responsible for the differentiation of 3T3-L1 white preadipocytes to beige adipocytes. Significant expression of Ucp1 was not detected under any tested conditions in the absence of isoproterenol (Iso), an agonist of β adrenergic receptor. Iso-induced Ucp1 expression was significantly higher in the cells treated with a mixture of triiodothyronine (T3) and 3-isobutyl-1-methylxanthine (IBMX) for days 0–8 than in the control cells. Chronic IBMX treatment was indispensable for the enhanced Iso-induced Ucp1 expression, and treatment with additional rosiglitazone (Rosi) for days 0–8 further increased the Ucp1 expression. Recently, genes were identified that are predominantly expressed in beige adipocytes, which were induced from stromal vascular cells in white fat depots. However, the expression levels of the beige adipocyte-selective genes in the adipocytes induced by the mixture of T3, IBMX and Rosi did not differ from those in the control adipocytes. The present study indicates that 3T3-L1 cells can differentiate to beige-like adipocytes by prolonged treatment with the mixture of T3, IBMX and Rosi and that the gene expression profile of the adipocytes is distinct from those previously induced from white fat depots.

Diet-induced changes in Ucp1 expression in bovine adipose tissues.

Brown adipocytes, which regulate non-shivering thermogenesis, have been believed to exist in a limited number of mammalian species, and only under limited physiological conditions. Recent discoveries indicate that adult humans possess a significant number of functional brown adipocytes. This study explores the regulatory emergence of brown adipocytes in white adipose tissue (WAT) depots of fattening cattle. RT-PCR analyses indicated significant expression of Ucp1, a brown adipocyte-specific gene, in the WAT of 31-month-old Japanese Black steers. Immunohistochemical analysis revealed that Ucp1-positive small adipocytes were dispersed in the subcutaneous WAT. Next, we examined expression level of Ucp1 and other brown adipocyte-selective genes such as Pgc1α, Cidea, Dio2, Cox1, Cox7a1 and Cox8b in WAT of 30-month-old steers fed either diet with low protein/energy content (roughage diet) or that with high protein/energy content (concentrate diet) for 20months. Ucp1 expression in the subcutaneous WAT was significantly higher in the concentrate diet group than in the roughage diet group. Furthermore, the higher Ucp1 expression levels were limited to the subcutaneous WAT, and no differences between groups were detected in the mesenteric, perirenal, intermuscular or intramuscular WAT. Expression of Dio2, Cox1 and Cox8b was higher in the subcutaneous WAT but not in the mesenteric WAT of the concentrate diet group. Furthermore, expression of Prdm16, a positive regulator of differentiation toward brown adipocyte-lineage cells, and expression of leptin, a molecule that enhances activity of brown adipocytes, were significantly higher in the subcutaneous WAT of the concentrate diet group. This study demonstrates the presence of brown adipocytes in WAT depots of fattening cattle, and suggests the diet-related modulation of expression of genes predominantly expressed in brown adipocytes.

Fermentation of soybean meal with Aspergillus usamii improves zinc availability in rats.

Soybean meal was fermented withAspergillus usamii to improve zinc availability through the degradation of phytic acid. Rats fed a diet containing fermented soybean meal showed greater femoral zinc than did animals fed a diet containing regular soybean meal. Zinc solubility in the small intestine was higher in the rats fed fermented soybean meal than in the rats fed regular soybean meal. These results suggested that fermentation withAspergillus usamii improved zinc availability in dietary soybean meal, which was induced by the increase of zinc solubility in the small intestine. Adding the same amount of phytate that was contained in the regular soybean mealbased diet did not affect the amount of zinc present in rats fed a fermented soybean meal-based diet with sodium phytate. Phytase activity was found in fermented soybean meal, and this activity may degrade added phytate in fermented soybean meal-based diet.

THIAZOLIDINEDIONE INDUCES THE ADIPOSE DIFFERENTIATION OF FIBROBLAST‐LIKE CELLS RESIDENT WITHIN BOVINE SKELETAL MUSCLE

To investigate the role of peroxisome proliferator‐activated receptor γ (PPARγ) in adipocyte formation within the skeletal muscle of beef cattle, fibroblast‐like cells were isolated from the longissimus muscle of cattle and cultured with activators of murine PPARγA thiazolidinedione T‐174, which is a specific ligand for PPARγ, stimulated adipose differentiation (evaluated by counting differentiated adipocytes under microscopic observation) in a dose‐dependent fashion. A peroxisome proliferator Wy14,643 which strongly activates the α isoform of murine PPAR also stimulated differentiation but its potency was weaker than that of T‐174. Unexpectedly, 15‐deoxy‐Δ12,14‐prostaglandin J2, which is believed to be an endogenous ligand for PPARγ, could not induce adipose differentiation in doses which have been found to be effective on rodent cells. Immunoblotting analysis confirmed the significant expression of PPARγ protein in fibroblast‐like cell cultures prepared from bovine skeletal muscle. In conclusion, bovine skeletal muscle contains adipose precursor cells expressing functionally active PPARγ.

Effect of exercise on iron metabolism in horses

We investigated the effect of exercise on iron metabolism in horses. Four horses were walked on a mechanical walker for 1 wk (pre-exercise). They then performed moderate exercise on a high-speed treadmill in the first week of the exercise and relative high in the second week and high in the third week. Serum iron was significantly lower in the third week of exercise than in the pre-exercise. Transferrin saturation (TS) was significantly lower in the first and third weeks of exercise than in the pre-exercise. Serum haptoglobin was significantly lower in the first week of exercise than in the pre-exercise and further significantly lower in the second and third weeks than in the first. The packed cell volume did not change during the experiment. The exercise significantly increased the apparent absorption of iron. Urinary iron excretion did not change throughout the experiment. Sweat iron loss did not change during the exercise. The exercise significantly increased iron balance. We considered that hemolysis is induced by moderate exercise and is further enhanced by heavy exercise, which decreases serum iron and TS. However, the increase in iron absorption compensates for the adverse effect of exercise on iron status. Therefore, exercise does not induce anemia in horses.

PCR detection of bovine mitochondrial DNA derived from meat and bone meal in feed

Because bovine meat and bone meal (MBM) is thought to be a major source of bovine spongiform encephalopathy, we developed a PCR-based method for detection of bovine MBM in animal feed. We isolated bone particles from feed containing bovine MBM using a separation technique based on specific gravity and then washed bone particles with sodium hypochlorite solution and an EDTA-proteinase K solution. The mitochondrial DNA was extracted from bone particles and amplified using PCR with cattle-specific primers. Bovine DNA was not detected in a milk replacer containing dried skim milk and dried whey, but bovine DNA was detected in the milk replacer that was mixed with bovine MBM. Other cattle-derived materials in feeds did not interfere with the selective detection of bovine MBM. This method allowed detection of bovine mitochondrial DNA in feed with 0.1% added bovine MBM. When the treatment with sodium hypochlorite was excluded, bovine DNA derived from MBM could not be distinguished from bovine DNA derived from other bovine materials. However, the exclusion of this treatment improved the detection limit of bovine MBM in feed. This method appears suitable for the selective detection of bovine MBM in feed.

Role of endogenous TGF‐β family in myogenic differentiation of C2C12 cells

The present study evaluated endogenous activities and the role of BMP and transforming growth factor‐β (TGF‐β), representative members of the TGF‐β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C‐terminal serines was monitored, since TGF‐β family members signal via the phosphorylation of Smads in a ligand‐dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation‐related changes were consistent with those in the expression of Ids, well‐known BMP‐responsive genes. Treatment with inhibitors of BMP type I receptors or noggin in C2C12 myoblasts down‐regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP‐2 during the myoblast phase also inhibited myotube differentiation through the down‐regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF‐β‐responsive Smad, was higher 8–16 days after differentiation stimulation. A‐83‐01, an inhibitor of TGF‐β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF‐β family are changed during myogenesis in a pathway‐specific manner, and that the activities are required for myogenesis. J. Cell. Biochem. 112: 614–624, 2011.