Comparative Proteomic Analysis Of Livers From Ketotic Cows

Differential expression proteins of livers between ketotic and healthy cows were displayed through 2D-E, and were identified through MALDI-TOF MS/MS technique. Altogether thirty-eight difference proteins were identified. Two proteins of the total, 3-hydroxyacyl-CoA dehydrogenase type-2 (HCDH) and acetyl-coenzyme A acetyltransferase 2 (ACAT) were confirmed by Weston-blot. Differential expression proteins of livers from ketotic cows involved in pathways of energy metabolism, carbohydrate metabolism, fatty acid metabolism, amino acid metabolism, antioxidation metabolism, constitutive protein and nucleic acid metabolism. The above results provide systematic data on elucidating liver metabolic conditions and characteristics of ketosis, and on constructing liver metabolic profile of ketotic cows.The expression levels of 3-hydroxyacyl-CoA dehydrogenase type-2 (HCDH), acetyl-Coenzyme A acetyltransferase 2 (ACAT), elongation factor Tu (EF-Tu), alpha-enolase (α-Enolase) and creatine kinase (CK) in liver of ketotic cows were 1.5 times more than those of healthy cows(P<0.05). HCDH, ACAT and EF-Tu were down-regulated,α-enolase and CK were up-regulated. Based on further analysis of liver difference expression proteins of ketotic cows and the published documents, liver metabolic profile of bovine ketosis was established. High level of ketone body in blood and liver could inhibit ketogenesis in liver of ketotic cows through negative feedback, which results in an accumulation of acetyl coenzyme A in hepatic cells. Accumulative acetyl coenzyme A in hepatic cells depresses the level of ACAT, and weakens ketogenesis. Accumulative acetyl coenzyme A in hepatic cells also reduces fatty acidβ-oxidation. EF-Tu was down regulated in liver of ketotic cows, indicating that protein synthesis is weakened, because most glucogenic amino acids may take part in TAC and glyconeogenesis, which produces mass ATP and stores as the form of creatine phosphate, in consequence CK is up-regulated. But the result is contrary with previous study. Theα-enolase was up-regulated, indicating that glyconeogenesis is enhanced by glucogenic amino acids. Based on the above results, the primary metabolic characteristics in livers of ketotic cows is that the pathways of fatty acids oxidation, ketogenesis and protein synthesis in liver of ketotic cows are weakened and gluconeogenesis is enhanced. But, ketotic cows remaines under the condition of hypoglycin, hyperketonemia, ATP surplus stored as creatine phosphate and acetyl coenzyme A accumulation in hepatocyte of ketotic cows.To identify liver fatty acids oxidation characteristics of ketotic cows, serum glucose, BHBA and NEFA, and liver CPT II, HAD and OAA were detected. The results indicated that the high concentrations of serum BHBA and NEFA, the low concentrations of serum glucose, and the low activities of liver OAA, CPT II, HAD were observed in ketotic cows. There were inverse correlation among the concentration of serum NEFA and the activities of liver OAA, CPT II and HAD. It is considered that high concentration of serum NEFA may be a primary cause that induces that decrease of fatty acids oxidation ability in liver of ketotic cows.