Hideo Yano

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.

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.

Nitric oxide suppresses preadipocyte differentiation in 3T3-L1 culture

Nitric oxide (NO) is an important chemical messenger controlling many physiological functions, involving cell proliferation, and differentiation. The purpose of this study was to investigate the effect of NO on adipocyte differentiation using a murine preadipocyte cell line, 3T3-L1. The treatment with a NO donor, 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), reduced some markers of adipocyte differentiation such as glycerol-3-phosphate dehydrogenase activity, and intracellular lipid accumulation. To examine whether these effects of NOC18 on adipocyte differentiation markers are due to its cytotoxity, lactate dehydrogenase (LDH) release from the cells were measured. NOC18 did not affect LDH release into the culture medium. Thus, the suppressive actions of NO donor were unlikely to result from its cytotoxicity. Peroxisome proliferator-activated receptor (PPAR) γ is a critical transcription factor for adipocyte differentiation and adipocyte fatty acid binding protein (aP2) gene is one of its targets. Protein expression of PPARγ was not diminished by NOC18 treatment, although mRNA expression of aP2 was reduced. Electrophoretic mobility shift assay showed that NOC18 interfered with the DNA binding activity of PPARγ. Therefore, the present experiment suggest that NO suppresses adipocyte differentiation through suppressing the transcriptional activity of PPARγ, without suppressing its expression level.

The changes in body weight and plasma metabolite levels during leptin injection are caused by the reduction of food intake in sheep.

Leptin is thought to act on the central nervous system as an important regulator of food intake and body weight in mice. To examine the actions of leptin in sheep, we injected recombinant mouse leptin into the lateral cerebral ventricle of ewes. Six ewes were intracerebroventricularly cannulated and given free access to forage and water. The ewes were injected with phosphate-buffered saline (PBS) as a vehicle for 5 days through the cannula and then with PBS + leptin for 7 days consecutively. Although food intake and body weight did not change during PBS injection, the administration of leptin decreased food intake and body weight. Plasma glucose levels decreased and plasma non-esterified fatty acid levels increased on the last few days of leptin administration. Approximately 2 weeks after normal food intake was recovered, the amount of diet was individually reduced in accordance with the recorded food intake during the leptin administration in each sheep. During food restriction, body weight and plasma metabolite concentrations were similarly changed as during the leptin-administration period. These results indicate that leptin decreases food intake in sheep. Moreover, it is likely that the changes in body weight and plasma metabolites were mainly induced by the suppression of food intake during leptin injection.

Milk calcium taken with cheese increases bone mineral density and bone strength in growing rats.

We investigated the calcium bioavailability of milk calcium, taken with or without cheese. Twenty-four 6-week-old male rats for a meal-feeding experiment were trained to consume an AIN-76 diet within 2 h (2 times per day) for 2 weeks. The rats were then divided into three experimental groups, each fed 2 types of experimental diets: Control group, Cheese group, and Ca-Cheese group. The rats were each alternately given 2 types of experimental diets at 2-h meal-feeding for 31 days. The breaking force and energy of the femur in the Ca-Cheese group were significantly higher than in the control group. The bone mineral density (BMD) of the lumbar spine and the femur in the Ca-Cheese group was also significantly higher than in the other two groups. These results indicate that milk calcium taken with cheese increases bone strength and BMD efficiently, results that may be useful for the prevention of osteoporosis.