Ketosis occurs most frequently in the peripartal period and is associated with liver injury and steatosis. Lysosomes serve as the terminal degradative station and contribute to liver homeostasis through their role in the digestion of dysfunctional organelles and lipid droplets.
Transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function. Thus, the objective of the present study was to investigate the status of lysosomal function and TFEB transcriptional activity and potential changes in abundance of upstream effectors of TFEB identified in nonruminants, including mechanistic target of
rapamycin kinase complex 1 (
mTORC1),
protein kinase B (Akt),
glycogen synthase kinase β (GSK3β), and extracellular signal-regulated kinase1/2 (ERK1/2), and to explore which factor induces the above changes. Liver and blood samples were collected from healthy cows (n = 10) and ketotic cows (n = 10) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9 d). Calf hepatocytes were isolated from Holstein calves and treated with 10 ng/mL
growth hormone (GH), 3.0 mM β-hydroxybutyrate (BHB), 1.5 ng/mL
interleukin-18 (IL-18), 0.15 ng/mL
tumor necrosis factor-α (TNF-α), or 1.2 mM
free fatty acid (FFA) for 12 h. Serum levels of FFA and activities of
alanine aminotransferase and
aspartate aminotransferase were greater in ketotic cows, whereas
glucose was lower. Additionally, ketotic dairy cows exhibited higher serum concentrations of GH,
IL-18, and TNF-α, and lower serum concentration of
insulin. The lower
protein abundance of
lysosome-associated membrane protein 1 (LAMP1) and
mRNA abundance of LAMP1 indicated that hepatic lysosomal mass was lower in ketotic cows. Furthermore, lower
protein abundance of
cathepsin D (CTSD) and
mRNA abundance of CTSD and V0 domain of the
vacuolar ATPase along with lower activity of β-N-
acetylglucosaminidase indicated impairment in hepatic lysosomal function due to
ketosis. The lower nuclear abundance, total
protein, and
mRNA abundance of TFEB and
peroxisome proliferator-activated receptor γ coactivator 1 α along with greater phosphorylated (p)-TFEB in the liver of ketotic cows indicated an impairment of hepatic TFEB transcriptional activity. The
protein abundances of phosphorylated mTOR (p-mTOR) and its downstream effectors
ribosomal protein S6 kinase B (RPS6KB) and eukaryotic factor 4E-binding
protein 1 (EIF4EBP1) were greater, whereas p-Akt, p-GSK3β, and p-ERK1/2 were lower in the liver of ketotic cows. Importantly, elevated phosphorylation of mTOR, RPS6KB, and EIF4EBP1 was observed in calf hepatocytes treated with GH, BHB,
IL-18, TNF-α, and FFA. Moreover, BHB, TNF-α, and FFA, not GH and
IL-18, reduced TFEB transcriptional activity and impaired lysosomal function in calf hepatocytes. Taken together, these data suggest that BHB, TNF-α, and FFA overactivate the hepatic
mTORC1 signaling pathway during
ketosis and further impaired TFEB transcriptional activity and lysosomal function, which may contribute to liver injury and steatosis.