Background/Aims: Dairy cows with ketosis are characterized by oxidative stress and hepatic damage. The aim of this study was to investigate hepatic oxidative stress and the apoptotic status of ketotic cows, as well as the underlying apoptosis pathway. Methods: The blood aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (GGT) activities and the haptoglobin (HP), serum amyloid A (SAA) and serum apoptotic cytokeratin 18 neo-epitope M30 (CK18 M30) concentrations were determined by commercially available kits and ELISA kits, respectively. Liver histology, TUNEL and Oil red O staining were performed in liver tissue samples. TG contents were measured using an enzymatic kit; Caspase 3 assays were carried out using the Caspase 3 activity assay kit; oxidation and antioxidant markers were measured using biochemical kits; apoptosis pathway were determined by qRT-PCR and western blot. Results: Ketotic cows displayed hepatic fat accumulation. The hepatic malondialdehyde (MDA) content was significantly increased, but the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were markedly decreased in ketotic cows compared with control cows, indicating that ketotic cows displayed severe oxidative stress. Significantly higher serum levels of the hepatic damage markers AST, ALT, GGT and GLDH were observed in ketotic cows than in control cows. The blood concentration of the apoptotic marker CK18 M30 and the number of TUNEL-positive cells in the liver of ketotic cows were 1.19- and 2.61-fold, respectively, higher than the values observed in control cows. Besides, Caspase 3 activity was significantly increased in the liver of ketosis cows. Importantly, the levels of phosphorylated c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) were significantly increased but the level of phosphorylated extracellular signal-regulated kinase1/2 (ERK1/2) was markedly decreased, which further promoted tumor protein 53 (p53) expression and inhibited nuclear factor E2-related factor 2 (Nrf2) expression. The apoptosis-related molecules p21, MDM2, Caspase 3, Caspase 9 and Bax were expressed at significantly higher levels in ketotic cows than in healthy cows, whereas the anti-apoptosis molecule Bcl-2 was expressed at significantly lower levels. Conclusions: Based on these results, ketotic cows display severe hepatic oxidative stress. The hepatic MAPK-p53-Nrf2 apoptotic pathway is over induced and partially mediated apoptotic damage in the liver.

The hepatic growth hormone (GH)-insulin-like growth factor (IGF)-I axis is essential for regulating intrahepatic lipid metabolism. Ketotic cows are characterized by high blood concentrations of fatty acids and β-hydroxybutyrate (BHB), which display lipotoxicity. The aim of this study was to investigate changes in the hepatic GH-IGF-I axis in ketotic cows and to determine the effects of fatty acids and BHB on the GH-IGF-I axis in calf hepatocytes. Liver and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) cows. Hepatocytes were isolated from calves and treated with various concentrations of GH, fatty acids, and BHB. The results showed that clinically ketotic cows displayed a high blood concentration of GH, a low blood concentration of IGF-I, and decreased hepatic GHR1A expression as well as impaired hepatic Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) signaling. In vitro, GH treatment induced activation of the JAK2-STAT5 pathway to increase the mRNA expression and secretion of IGF-I in calf hepatocytes. More importantly, treatment with fatty acids or BHB significantly inhibited GHR1A mRNA and JAK2 protein expression, as well as the STAT5 phosphorylation level and phospho-STAT5 nuclear translocation; these effects markedly reduced IGF1 mRNA expression and secretion in calf hepatocytes. In summary, these results indicate that high blood concentrations of fatty acids or BHB can impair the intrahepatic GH-mediated JAK2-STAT5 pathway and downregulate IGF-I expression and secretion in ketotic cows.

The aim of this study was to investigate the changes in hepatic oxidative phosphorylation (OXPHOS) complexes (COs) in patients and cows with non‐alcoholic steatohepatitis (NASH) and to investigate the mechanism that links mitochondrial dysfunction and hepatic insulin resistance induced by non‐esterified fatty acids (NEFAs). Patients and cows with NASH displayed high blood NEFAs, TNF‐α and IL‐6 concentrations, mitochondrial dysfunction and insulin resistance. The protein levels of peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α), mitofusin‐2 (Mfn‐2) and OXPHOS complexes (human: COI and COIII; cow: COI‐IV) were significantly decreased in patients and cows with NASH. NEFA treatment significantly impaired mitochondrial function and, increased reactive oxygen species (ROS) production, and excessive ROS overactivated the JNK and p38MAPK pathways and induced insulin resistance in cow hepatocytes. PGC‐1α and Mfn‐2 overexpression significantly decreased the NEFA‐induced ROS production and TNF‐α and IL‐6 mRNA expressions, reversed the inhibitory effect of NEFAs on mitochondrial function and attenuated the overactivation of the ROS‐JNK/p38MAPK pathway, alleviated insulin resistance induced by NEFAs in cow hepatocytes and HepG2 cells. These findings indicate that NEFAs induce mitochondrial dysfunction and insulin resistance mediated by the ROS‐JNK/p38MAPK pathway. PGC‐1α or Mfn‐2 overexpression reversed the lipotoxicity of NEFAs on mitochondrial dysfunction and insulin resistance. Our study clarified the mechanism that links hepatic mitochondrial dysfunction and insulin resistance in NASH.