González-Valero L, Rodríguez-López J M, Lachica M, Fernández-Fígares I
Department of Physiology and Biochemistry of Animal Nutrition,Estación Experimental del Zaidín,Consejo Superior de Investigaciones Científicas,Camino del Jueves s/n,18100 Armilla,Granada,Spain.
Animal. 2014 Nov;8(11):1873-80. doi: 10.1017/S1751731114001748. Epub 2014 Jul 15.
There are important differences in terms of metabolic activity, energy utilization and capacity of protein and fat deposition when Iberian and modern pigs are compared. Primary culture of hepatocytes was used to evaluate hepatic function and sensitivity to hormones between breeds without the interference of circulating blood factors. Hepatocytes were isolated from pure Iberian (n=10) and Landrace (n=8) pigs of similar BW (24.5±12.1 and 32.9±6.1 kg BW, respectively), by collagenase perfusion. Monolayers were established in medium containing fetal bovine serum for 1 day and switched to serum-free medium for the remainder of the culture period. Hepatocytes were maintained in William's E supplemented with β-mercaptoethanol (0.1 mM), glutamine (2 mM), antibiotics (gentamicin, penicillin, streptomycin and amphotericin B), dimethyl sulfoxide (1 µg/ml), dexamethasone (10-8 M), insulin (0.173 and 17.3 nM) and glucagon (0.287, 2.87 and 28.7 nM) for 24 to 48 h. Gluconeogenesis (GNG), glycogen degradation, triglycerides (TG) content and esterification, β-hydroxybutyrate (BHB) synthesis, IGF-1 synthesis, albumin and urea synthesis were determined. Iberian pigs had greater capacity of GNG than Landrace (24%, P<0.05), although no difference in glycogen degradation was found (P>0.10). TG content and esterification tended to be lower in hepatocytes from Iberian compared with Landrace pigs (12% and 31%, respectively; 0.10<P<0.05). Furthermore, addition of free fatty acids (CLA or linoleic acid, 0.2 mM) increased TG content (64%, P<0.001) although no difference between fatty acids was found. When free fatty acids were compared, a trend toward increased esterification (41%, P=0.078) was found for CLA. Although glucagon stimulated and insulin inhibited BHB synthesis, no difference between breeds was found (P>0.10). IGF-1 synthesis was diminished in hepatocytes from Iberian compared with Landrace pigs (16%, P<0.05). On the contrary, rate of albumin synthesis was greater in Iberian compared with Landrace pigs (58%, P<0.05). Finally, the capacity of urea synthesis was lower in hepatocytes of Iberian compared with Landrace pigs (37%, P<0.05). When ammonia was added to the media, urea concentration increased (648%, 1108% and 2791% when 0 mM was compared with 2.5, 5 and 10 mM, respectively). Urea synthesis increased on increasing ammonia content (55% and 325% when 0 mM was compared with 5 and 10 mM, respectively; P<0.0001). In conclusion, the genetic background accounts for important differences in protein and energy metabolism pathways found in primary culture of hepatocytes from lean and obese pigs.
比较伊比利亚猪和现代猪时，在代谢活性、能量利用以及蛋白质和脂肪沉积能力方面存在重要差异。利用肝细胞原代培养来评估不同品种间的肝功能及对激素的敏感性，且不受循环血液因子的干扰。通过胶原酶灌注法，从体重相似的纯种伊比利亚猪（n = 10）和长白猪（n = 8）中分离肝细胞（体重分别为24.5±12.1千克和32.9±6.1千克）。将单层细胞接种于含胎牛血清的培养基中培养1天，之后在无血清培养基中继续培养。肝细胞在添加了β-巯基乙醇（0.1 mM）、谷氨酰胺（2 mM）、抗生素（庆大霉素、青霉素、链霉素和两性霉素B）、二甲基亚砜（1 µg/ml）、地塞米松（10 - 8 M）、胰岛素（0.173和17.3 nM）以及胰高血糖素（0.287、2.87和28.7 nM）的威廉姆斯E培养基中维持培养24至48小时。测定糖异生（GNG）、糖原降解、甘油三酯（TG）含量及酯化、β-羟基丁酸（BHB）合成、IGF - 1合成、白蛋白和尿素合成情况。伊比利亚猪的糖异生能力比长白猪更强（24%，P < 0.05），不过在糖原降解方面未发现差异（P > 0.10）。与长白猪相比，伊比利亚猪肝细胞中的TG含量及酯化水平往往较低（分别为12%和31%；0.10 < P < 0.05）。此外，添加游离脂肪酸（共轭亚油酸或亚油酸，0.2 mM）可使TG含量增加（64%，P < 0.001），尽管未发现不同脂肪酸之间存在差异。比较游离脂肪酸时，发现共轭亚油酸存在酯化增加的趋势（41%，P = 0.078）。尽管胰高血糖素刺激而胰岛素抑制BHB合成，但未发现品种间存在差异（P > 0.10）。与长白猪相比，伊比利亚猪肝细胞中的IGF - 1合成减少（16%，P < 0.05）。相反，伊比利亚猪的白蛋白合成速率比长白猪更高（58%，P < 0.05）。最后，与长白猪相比，伊比利亚猪肝细胞中的尿素合成能力较低（37%，P < 0.05）。向培养基中添加氨后，尿素浓度升高（与0 mM相比，2.5 mM、5 mM和10 mM时分别升高648%、1108%和2791%）。随着氨含量增加，尿素合成增加（与0 mM相比，5 mM和10 mM时分别增加55%和325%；P < 0.0001）。总之，遗传背景导致了瘦猪和肥猪肝细胞原代培养中蛋白质和能量代谢途径的重要差异。
