Toshihiko Kamada

Tissue distribution of lipase genes related to triglyceride metabolism in laying hens (Gallus gallus).

The triglyceride lipase gene family, including lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL), carboxyl ester lipase (CEL), endothelial lipase (EL), Lipase H, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), plays a critical role in lipid metabolism in mammals. In this study, we have identified and characterized the expression profile of these genes in the chicken, Gallus gallus. Chicken LPL and ATGL have been cloned, and HTGL, EL, Lipase H, and CEL sequences were found in the chicken genome database. The deduced amino acid sequences of HTGL, EL, Lipase H, and CEL were 66, 75, 63, and 65% identical with their respective human genes, suggesting conservation of important enzymatic functions. In contrast, a homologue of the HSL gene was not identified in the chicken genome. We performed RT-PCR using chicken liver, muscle, abdominal adipose tissue, or pancreas mRNA as the template, and all partial products were completely matched to the corresponding predicted sequences of triglyceride lipase gene members. Quantitation by qPCR of the transcript levels of these genes in 13 tissues indicates that the expression patterns diverge greatly between species. A particularly interesting pattern was observed in the distribution of EL and HTGL mRNA, which were highly expressed in kidney and ovary. This is the first report of HTGL, EL, Lipase H, and CEL in a pre-mammalian species and reveals novel details about specific features of the expression of these important molecules in lipid metabolism.

Regulation of bile acid, cholesterol, and fatty acid synthesis in chicken primary hepatocytes by different concentrations of T0901317, an agonist of liver X receptors.

Liver X receptors (LXRs) are members of the nuclear receptor family of transcription factors. They play a crucial role in lipid metabolism processes such as bile acid and fatty acid synthesis, as well as minor or limited roles in the regulation of cholesterol synthesis and uptake in mammals. In avian species, however, little is known about the role of LXRs except for the fact that they are involved in the stimulation of fatty acid synthesis. In this study, we characterize the expression profile of genes related to bile acid, cholesterol, and fatty acid synthesis and VLDL secretion in chicken primary hepatocytes treated with T0901317, a synthetic agonist of LXR. The activity of chicken cholesterol 7α hydroxylase (CYP7A1), a key enzyme in bile acid synthesis, mRNA expression, and bile acid excretion, was stimulated by supplementation of the culture medium with a low concentration (0.01 μM) of T0901317. In contrast, the levels of sterol regulatory element binding protein (SREBP)-1, fatty acid synthase mRNA, and VLDL-triacylglycerol in cells cultured in the presence of a high concentration (10 μM) of T0901317 were higher than those cultured in zero or low concentrations of T0901317. These results suggest that cellular responses to this LXR agonist were similar to those present in mammals. A novel finding of this study concerned changes to the regulation of cholesterol synthesis and uptake in chicken hepatocytes treated with T0901317. Levels of SREBP-2,3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and low-density lipoprotein receptor (LDLr) mRNA expression increased as a function of increasing T0901317 (up to 1.0 μM), but remained similar to those in cells cultured under control conditions when the concentration of T0901317 was increased to 10 μM. These results suggest that LXRs play an important role in cholesterol synthesis and uptake in chicken hepatocytes and, as such, differ to findings in mammals where the effect of LXR agonists on cholesterol synthesis plays only a minor role in the regulation of cellular sterol homeostasis.

Changes in peroxisome proliferator‐activated receptor gamma gene expression of chicken abdominal adipose tissue with different age, sex and genotype

Peroxisome proliferatior-activated receptor gamma (PPARgamma) is a transcription factor that regulates adipocyte differentiation, and the activation of PPARgamma increases fat deposition in growing chickens. The aim of the present study was to investigate whether the levels of PPARgamma gene expression were related to fat pad weight in abdominal adipose tissue in growing chickens with different genotype and sex. Body weight and abdominal adipose tissue weight in broiler chickens (Ross strain) were higher than the other genotypes (Road Island Red, White Leghorn, and Japanese native poultry (Tsushima)) at 3 and 5 weeks of age. PPARgamma mRNA expression in abdominal adipose tissue tended to increase with age, as evidenced by higher expression levels at 5 weeks than at 1 week of age in all sex and genotype of chickens. In broiler chickens, the PPARgamma expressions were significantly higher than the other genotypes. PPARgamma mRNA expression levels in abdominal adipose tissue of female chickens rapidly increased at 3 weeks, and were unchanged until 5 weeks, while those in male chickens gradually increased until 5 weeks. In addition, abdominal adipose tissue weight was correlated with PPARgamma expression levels. These results demonstrated that PPARgamma gene expression is a useful marker of fat deposition in chickens, suggesting that PPARgamma is a key factor of fat accumulation in chicken abdominal fat pad.

Role of peroxisome proliferator-activated receptor β/δ in chicken adipogenesis

The adipocyte differentiation process involves a cascade of transcriptional events that culminates in the expression of peroxisome proliferator-activated receptor (PPAR)gamma. The present study was undertaken to identify the role of PPARbeta/delta in chicken adipocyte differentiation, with experiments performed using a PPARgamma agonist, a PPARbeta/delta agonist, a PPARgamma antagonist and fatty acid (oleate). Preadipocyte cells cultured in a differentiation medium (DMEM containing 500nM dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine, 20microg/mL bovine insulin and 10% fetal bovine serum) supplemented with 200microM oleate resulted in a significant increase in adipocyte fatty acid binding protein (aP2) mRNA expression after 24h and 7d of culture compared to cells cultured in a differentiation medium alone, while supplementation of the differentiation medium with GW501516 (a PPARbeta/delta agonist) did not affect aP2 mRNA expression levels. Supplementation of the differentiation medium with troglitazone (a PPARgamma agonist) and GW501516 induced preadipocyte differentiation; a significant increase of aP2 mRNA expression was observed in cells after incubation for 7d, but not after 24h of incubation. These results suggest that PPARbeta/delta does not play a key role in adipocyte differentiation, but it does enhance the transformation of immature into mature adipocytes in chickens. In addition, oleate functions not only as an activator of PPARs but also induces PPARgamma gene expression via alternative pathway of PPARs activation. These results establish the importance of exogenous fatty acid in the processes of adipogenesis and fat accumulation in chickens.

Kinematic gait analysis and lactation performance in dairy cows fed a diet supplemented with zinc, manganese, copper and cobalt.

This study investigated how supplementation of the diet of dairy cows with trace minerals (zinc, manganese, copper and cobalt) affected kinematic gait parameters and lactation performance. Eight Holstein cows were divided into two groups, with each group receiving a different dietary treatment (control diet, or control diet supplemented with trace minerals) in a two-period crossover design. Kinematic gait parameters were calculated by using image analysis software. Compared to cows fed the control diet, cows that received the trace mineral-supplemented diet exhibited significantly increased walking and stepping rates, and had a shorter stance duration. Feed intake and milk production increased in cows fed the trace mineral-supplemented diet compared with control groups. The plasma manganese concentration was not different in control and experimental cows. In contrast, cobalt was only detected in the plasma of cows fed the supplemented diet. These results provide the first evidence that trace mineral supplementation of the diet of dairy cows affects locomotion, and that the associated gait changes can be detected by using kinematic gait analysis. Moreover, trace mineral supplementation improved milk production and only minimally altered blood and physiological parameters in dairy cows.

Comparison of Behavior of Pigs in Individual and Group Penning.

肥育豚における同腹以外の個体との群編成はストレスの原因になるとも考えられている。未知の個体が群を形成した時の行動と, 他個体の干渉の無い単飼の時の行動とを比較して, 群飼によりどのような影響を受けるかの検討を行った。異なった4ヶ所の農場から肥育末期の豚を1頭ずつ導入し, 単飼での行動を観察した後, 群を編成して行動を7日間観察した。群を編成した後, 行動割合が一定になるのに4日以上を要したが, 行動が安定した群編成後6～7日の平均と単飼時とを比較すると, いずれの条件でも休息行動が1日の80%以上を占め, 次いで採食行動が10%程度出現するといった概況は類似していたが, 休息行動の総時間はやや群飼で多く, 採食行動において群飼が総時間で26%, 回数で42%少なく, 持続時間は33%長かった。群飼では長時間連続で休み, 集中的な採食行動を示すと言えた。単飼条件で揃っていなかった豚間の行動リズムが群飼条件では揃うようになった。群編成初日の特にはじめの3時間は敵対行動が多発し, 休息行動があまり見られないなど異なった様相が見られたが, その後は安定化へ向かった。これらの結果から, 群編成は一時的にストレスとなるがすぐに安定し, むしろ単飼時より落ちついていると見られる状況が出現するので大きなストレスとなっていないのではないかと考えられた。