Fernández-Fígares I, Shannon A E, Wray-Cahen D, Caperna T J
Growth Biology Laboratory, Animal and Natural Resources Institute, Beltsville Agricultural Research Center-East, USDA/ARS, Building 200, Room no. 202, USDA, MD 20705, USA.
Domest Anim Endocrinol. 2004 Aug;27(2):125-40. doi: 10.1016/j.domaniend.2004.02.003.
A study was conducted to elucidate hormonal control of ketogenesis and glycogen deposition in primary cultures of porcine hepatocytes. Hepatocytes were isolated from pigs (54-68 kg) by collagenase perfusion and seeded into collagen-coated T-25 flasks. Monolayers were established in medium containing fetal bovine serum for 1 day and switched to a serum-free medium for the remainder of the culture period. Hepatocytes were maintained in DMEM/M199 containing 1% DMSO, dexamethasone (10(-6) or 10(-7) M), linoleic acid (3.4 x 10(-5) M), and carnitine (10(-3) M) for 3 days. On the first day of serum-free culture, insulin was added at 1 or 100 ng/ml and glucagon was added at 0, 1, or 100 ng/ml. Recombinant human leptin (200 ng/ml) was added during the final 24 h; medium and all cells were harvested on the third day. Concentrations of acetoacetate and beta-hydroxybutyrate (ketone bodies) in media and glycogen deposition in the cellular compartment were determined. Ketogenesis was highly stimulated by glucagon (1 and 100 ng/ml) and inhibited by insulin. In contrast, glycogen deposition was stimulated by insulin and attenuated by glucagon; high insulin was also associated with a reduction in the ketone body ratio (acetoacetate:beta-hydroxybutyrate). High levels of dexamethasone stimulated ketogenesis, but inhibited glycogen deposition at low insulin. Culture of cells with leptin for 24 h, over the range of insulin, glucagon, and dexamethasone concentrations had no effect on either glycogen deposition or ketogenesis. These data suggest that while adult porcine hepatocytes are indeed sensitive to hormonal manipulation, leptin has no direct influence on hepatic energy metabolism in swine.
进行了一项研究以阐明猪肝细胞原代培养中酮体生成和糖原沉积的激素调控。通过胶原酶灌注从猪（54 - 68千克）中分离肝细胞，并接种到胶原包被的T - 25培养瓶中。单层细胞在含有胎牛血清的培养基中培养1天，然后在剩余培养期切换到无血清培养基。肝细胞在含有1%二甲基亚砜、地塞米松（10⁻⁶或10⁻⁷M）、亚油酸（3.4×10⁻⁵M）和肉碱（10⁻³M）的DMEM/M199中维持3天。在无血清培养的第一天，加入1或100 ng/ml的胰岛素，并加入0、1或100 ng/ml的胰高血糖素。在最后24小时加入重组人瘦素（200 ng/ml）；在第三天收获培养基和所有细胞。测定培养基中乙酰乙酸和β-羟基丁酸（酮体）的浓度以及细胞区室中的糖原沉积。胰高血糖素（1和100 ng/ml）高度刺激酮体生成，而胰岛素抑制酮体生成。相反，胰岛素刺激糖原沉积，胰高血糖素使其减弱；高胰岛素水平还与酮体比率（乙酰乙酸：β-羟基丁酸）降低有关。高浓度地塞米松刺激酮体生成，但在低胰岛素水平时抑制糖原沉积。在胰岛素、胰高血糖素和地塞米松浓度范围内，用瘦素培养细胞24小时对糖原沉积或酮体生成均无影响。这些数据表明，虽然成年猪肝细胞确实对激素操作敏感，但瘦素对猪的肝脏能量代谢没有直接影响。
