Isolation of biliary lipid carrying vesicles, analysis of their biochemical properties and hepatic ABCG5 mRNA expression in diosgenin-fed rats

Abstract

Objective: An attempt was made to isolate vesicles, rich in biliary-type lipid from livers of ratsfed with diosgenin, which is reported to increase cholesterol carrying vesicles in hepatocytes, and to determine their lipid, alkaline phosphodiesterase I, protein status and hepatic ATP-binding cassette half-transporter genes 5/8 (ABCG5/ABCG8) expression. Material and Methods:The livers were removed, homogenized, subjected to centrifugation, and the microsomal fraction was obtained. This microsomal fraction was then loaded onto a 12% self generating gradients of OptiPrep TM in a Beckman Vti 65 vertical-tube rotor and centrifuge at 350000 xg for 90 min at 4°C. The gradient fractions were analysed for total phospholipid, cholesterol, protein and alkaline phosphodiesterase I (PDE). Results: There were no differences in total phospholipid, cholesterol and protein contents of the subcellular fractions of livers from control and diosgenin fed rats. However, PDE activity was significantly lower in some subcellular fractions in diosgenin-fed rats compared to controls. The gradient fractions with density 1.05-1.07 g/ml had increased amounts of cholesterol, PDE activity and some phospholipids, but contained very little protein. However, the gradient fractions with density 1.09-1.23 g/ml were significantly rich in cholesterol, phospholipids and protein, but not PDE activity, suggesting another population of vesicles. Hepatic ABCG5 mRNA expression was increased in diosgenin-fed rats compared with the control group, although, no significant difference was found in hepatic ABCG8 mRNA expression between control and diosgenin-fed rats. Conclusion: These findings suggested that ABCG5 is involved in diosgenin induced biliary cholesterol secretion and may provide an insight into mechanisms involved in biliary lipid transport. Key Words: ABCG5; ABCG8; biliary lipid; diosgenin; biliary cholesterol Conflict of interest:No conflicts of interest are declared by the authors. ÖZETAmaç: Hepatositlerde kolesterol taşıyıcı vezikülleri arttırdığı bildirilmiş olan diosgenin ile beslenen sıçan karaciğerlerinden, safra kökenli lipid yönünden zengin vezikülleri izole etmek ve onların lipid, alkalin fosfodiesteraz I, protein ile hepatik ATP-bağlayan kaset yarım taşıyıcı genleri 5/8 (ABCG5/ABCG8) ekspresyonlarının düzeylerinin saptanması amaçlandı. Gereç ve Yöntem: Karaciğerler alınarak, homojenize edildikten sonra santrifüjlenerek mikrozomal fraksiyonları elde edildi. Daha sonra bu mikrozomal fraksiyon, Beckman Vti 65 vertikal-tüp rotor içindeki, % 12'lik kendiliğinden gradiyent oluşturan OptiPrep TM' in üzerin konuldu ve 4°C'de, 350000 xg devirde, 90 dakika süreyle santrifüj edildi. Gradiyent fraksiyonlarının toplam fosfolipid, kolesterol, protein ve alkalin fosfodiesteraz I (PDE) için analizleri yapıldı. Bulgular: Kontrol ve diosgeninle beslenmiş sıçanlar arasında, subsellüler fraksiyonların toplam fosfolipid, kolesterol ve protein içerikleri yönünden farklılıklar bulunmadı. Bununla birlikte, diosgenin ile beslenen sıçanların, bazı subs...