Evaluation of hepatic AMPK, mTORC1, and autophagy-lysosomal pathway in cows with mild or moderate fatty liver

Abstract

ABSTRACT: The aim of the present study was to investigate the activity of AMPK and mTORC1 as well as TFEB transcriptional activity and autophagy-lysosomal function in the liver of dairy cows with mild fatty liver (FL) and cows with moderate FL. Liver and blood samples were collected from healthy dairy cows (n = 10; hepatic triglyceride content <1% wet weight) and cows with mild FL (n = 10; 1% ≤ hepatic triglyceride content < 5% wet weight) or moderate FL (n = 10; 5% ≤ hepatic triglyceride content < 10% wet weight) that had a similar number of lactations (median = 3, range = 2–4) and days in milk (median = 6 d, range = 3–9). Blood parameters were determined using a Hitachi 3130 autoanalyzer with commercially available kits. Protein and mRNA abundances were determined using western blotting and quantitative real-time PCR, respectively. Activities of calcineurin and β-N-acetylglucosaminidase were measured with commercial assay kits. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Blood concentrations of glucose were lower in moderate FL cows (3.03 ± 0.21 mM) than in healthy (3.71 ± 0.14 mM) and mild FL cows (3.76 ± 0.14 mM). Blood concentrations of β-hydroxybutyrate (BHB, 1.37 ± 0.15 mM in mild FL, 1.88 ± 0.17 mM in moderate FL) and free fatty acids (FFA, 0.69 ± 0.05 mM in mild FL, 0.96 ± 0.09 mM in moderate FL) were greater in FL cows than in healthy cows (BHB, 0.76 ± 0.12 mM; FFA, 0.42 ± 0.04 mM). Compared with healthy cows, phosphorylation of AMPK was greater and phosphorylation of its downstream target acetyl-CoA carboxylase 1 was lower in cows with mild and moderate FL. Phosphorylation of mTOR was lower in cows with mild FL compared with healthy cows. In cows with moderate FL, phosphorylation of mTOR and its downstream effectors was greater than in healthy cows and cows with mild FL. The mRNA abundance of TFEB was downregulated in cows with moderate FL compared with healthy cows and mild FL cows. In mild FL cows, the mRNA and protein abundances of TFEB were greater than in healthy cows. Compared with healthy cows, the mRNA abundances of autophagy markers sequestosome-1 and microtubule-associated protein 1 light chain 3-II, and the protein and mRNA abundances of lysosome-associated membrane protein 1 and cathepsin D were increased in mild FL cows but decreased in moderate FL cows. Compared with healthy cows, the mRNA abundance of mucolipin 1 and activities of β-N-acetylglucosaminidase and calcineurin were higher in cows with mild FL but lower in cows with moderate FL. These data demonstrate that hepatic AMPK signaling pathway, TFEB transcriptional activity, and autophagy-lysosomal function are increased in dairy cows with mild FL; the hepatic mTORC1 signaling pathway is inhibited in mild FL cows but activated in moderate FL cows; and activities of AMPK and TFEB as well as autophagy-lysosomal function are impaired in moderate FL cows.

Keywords

• fatty liver
• energy metabolism
• TFEB
• autophagy