Free fatty acids induce bile acids overproduction and oxidative damage of bovine hepatocytes via inhibiting FXR/SHP signaling.

Hepatic oxidative injury induced by free fatty acids (FFA) and metabolic disorders of bile acids (BA) increase the risk of metabolic diseases in dairy cows during perinatal period. However, the effects of FFA on BA metabolism remained poorly understood. In present study, high concentrations of FFA caused cell impairment, oxidative stress and BA overproduction. FFA treatment increased the expression of BA synthesis-related genes [cholesterol 7a-hydroxylase (CYP7A1), hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 7, sterol 12α-hydroxylase, sterol 27-hydroxylase and oxysterol 7α-hydroxylase], whereas reduced BA exportation gene (ATP binding cassette subfamily C member 1) and inhibited farnesoid X receptor/small heterodimer partner (FXR/SHP) pathway in bovine hepatocytes. Knockdown of nuclear receptor subfamily 1 group H member 4 (NR1H4) worsened FFA-caused oxidative damage and BA production, whereas overexpression NR1H4 ameliorated FFA-induced BA production and cell oxidative damage. Besides, reducing BA synthesis through knockdown of CYP7A1 can alleviate oxidative stress and hepatocytes impairment caused by FFA. In summary, these data demonstrated that regulation of FXR/SHP-mediated BA metabolism may be a promising target in improving hepatic oxidative injury of dairy cows during high levels of FFA challenges.

Expression of hepatic genes involved in bile acid metabolism in dairy cows with fatty liver.

Bile acids are cholesterol-derived molecules that are primarily produced in the liver. In nonruminants with fatty liver, overproduction of bile acids is associated with liver injury. During the transition period, fatty liver is a metabolic disorder that can affect up to 50% of high-producing dairy cows. The purpose of this study was to provide a comprehensive evaluation of hepatic bile acid metabolism in dairy cows with fatty liver by assessing the expression changes of genes involved in bile acid synthesis, export, and uptake. The serum activities of aspartate aminotransferase, alanine aminotransferase, and glutamate dehydrogenase and the concentration of total bile acids were all greater, whereas the serum concentration of total cholesterol was lower in cows with fatty liver than in healthy cows. The content of total bile acids was higher, but total cholesterol was slightly lower in liver tissues from fatty liver cows than from healthy cows. The hepatic mRNA abundance of cholesterol 7a-hydroxylase (CYP7A1); hydroxy-delta-5-steroid dehydrogenase, 3 ß- and steroid delta-isomerase 7 (HSD3B7); and sterol 12α-hydroxylase (CYP8B1), enzymes involved in the classic pathway of bile acid synthesis, was higher in fatty liver cows than in healthy cows. Compared with healthy cows, the hepatic mRNA abundance of alternative bile acid synthesis pathway-related genes sterol 27-hydroxylase (CYP27A1) and oxysterol 7α-hydroxylase (CYP7B1) did not differ in cows with fatty liver. The protein and mRNA abundances of bile acid transporter bile salt efflux pump (BSEP) were lower in the liver of dairy cow with fatty liver. Compared with healthy cows, the hepatic mRNA abundance of bile acid transporters solute carrier family 51 subunit α (SLC51A) and ATP binding cassette subfamily C member 1 (ABCC1) and 3 (ABCC3) was greater in cows with fatty liver, whereas the solute carrier family 51 subunit ß (SLC51B) did not differ. The expression of genes involved in bile acid uptake, including solute carrier family 10 member 1 (NTCP), solute carrier organic anion transporter family member 1A2 (SLCO1A2) and 2B1 (SLCO2B1) was upregulated in dairy cows with fatty liver. Furthermore, the hepatic protein and mRNA abundance of bile acid metabolism regulators farnesoid X receptor (FXR) and small heterodimer partner (SHP) were lower in cows with fatty liver than in healthy cows. Overall, these data suggest that inhibition of the FXR signaling pathway may lead to increased bile acid synthesis and uptake and decreased secretion of bile acids from hepatocytes to the bile, which elevates hepatic bile acid content in dairy cows with fatty liver. Because the hepatotoxicity of bile acids has been demonstrated on nonruminant hepatocytes, it is likely that liver injury is induced by increased hepatic bile acid content in dairy cows with fatty liver.

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

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 ß-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 ß-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 ß-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.

Overactivation of hepatic mechanistic target of rapamycin kinase complex 1 (mTORC1) is associated with low transcriptional activity of transcription factor EB and lysosomal dysfunction in dairy cows with clinical ketosis.

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 ß (GSK3ß), 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 ß-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 ß-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-GSK3ß, 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.

Free fatty acids impair autophagic activity and activate nuclear factor kappa B signaling and NLR family pyrin domain containing 3 inflammasome in calf hepatocytes.

Free fatty acids (FFA)-induced hepatic inflammation agravates liver injury and metabolic dysfunction in dairy cows with ketosis or fatty liver. Under stressful conditions, autophagy is generally considered as a cell protection mechanism, but whether the FFA-induced inflammatory and stress effect on hepatocytes involves an autophagy response is not well known. Thus, the objective of this study was to investigate the effects of FFA on autophagy and the role of autophagy in the activation of NF-κB (nuclear factor kappa B) signaling and NLRP3 (NLR family pyrin domain containing 3) inflammasome in calf hepatocytes. Calf hepatocytes were isolated from 3 healthy Holstein female new-born calves (1 d of age, 30-40 kg) and exposed to various concentrations of FFA (0, 0.3, 0.6, or 1.2 mM) after treatment with or without the autophagy inhibitor chloroquine (CQ) or the autophagy activator rapamycin. Expression of autophagy markers, LC3 (microtubule-associated protein 1 light chain 3) and p62 (sequestosome 1), NF-κB signaling, and NLRP3 inflammasome-related molecules were analyzed via western blot and quantitative real-time PCR. Results revealed that 0.6 and 1.2 mM FFA activated NF-κB signaling and NLRP3 inflammasome as indicated by an elevated ratio of p-NF-κB/NF-κB, protein abundance of NLRP3 and CASP1 (caspase 1), activity of CASP1, and mRNA abundance of IL1B and IL18. In addition, hepatocyte treated with 0.6 and 1.2 mM FFA or autophagy inhibitor CQ displayed increased protein abundance of p62 and LC3-II. Moreover, there was no difference in protein abundance of p62 and LC3-II between calf hepatocytes treated with 1.2 mM FFA and 1.2 mM FFA plus CQ, indicating that FFA inhibits autophagic activity in calf hepatocytes. Treatment with CQ led to overactivation of NF-κB signaling and NLRP3 inflammasome. Furthermore, CQ plus 1.2 mM FFA aggravated FFA-induced inflammation. In contrast, induction of autophagy by rapamycin ameliorated the FFA-activated NF-κB signaling and NLRP3 inflammasome as demonstrated by a lower ratio of p-NF-κB/NF-κB, protein abundance of NLRP3 and CASP1, activity of CASP1, and mRNA abundance of IL1B and IL18. Overall, inhibition of autophagy exacerbated, whereas induction of autophagy alleviated, FFA-induced inflammatory processes in calf hepatocytes, suggesting that impairment of autophagy might be partly responsible for hepatic inflammation and subsequent liver injury in dairy cows with ketosis or fatty liver. As such, regulation of autophagy may be an effective therapeutic strategy for controlling overt inflammatory responses in vivo.

Propionate alleviates palmitic acid-induced endoplasmic reticulum stress by enhancing autophagy in calf hepatic cells.

Negative energy balance-induced high blood concentrations of free fatty acids during the early postpartum period in dairy cows is a major cause of liver injury. Cows in severe negative energy balance often have suboptimal intakes of feed, which contributes to shortfalls in production of ruminal propionate and circulating glucose. Although increasing propionate production by the rumen through feed additives such as propylene glycol is effective in helping cows alleviate the shortfall in dietary energy supply, mechanisms whereby propionate affects liver function beyond gluconeogenesis are unknown. Therefore, the objective of this study was to investigate whether propionate could protect calf hepatic cells from palmitic acid (PA)-induced lipotoxicity and the underlying mechanisms. Calf hepatic cells were isolated from 5 healthy calves (1 d old, female, 30-40 kg, fasting) and treated with various concentrations of PA (0, 100, 200, or 400 µM) and propionate (0, 1, 2, or 4 mM) after being administered with or without autophagic inhibitor. Propionate enhanced autophagic activity in calf hepatic cells, as indicated by elevated expression of autophagy markers LC3-II (microtubule-associated protein 1 light chain 3-II, encoded by MAP1LC3) and decreased expression of SQSTM1 (sequestosome-1, also called p62). Conversely, PA suppressed autophagic activity and decreased cell viability, which was improved by propionate in calf hepatic cells. In addition, propionate decreased the phosphorylation of proteins EIF2AK3 (kinase R/PKR like ER kinase) and ERN1 (inositol-requiring enzyme 1α) and cleaved ATF6 (activating transcription factor 6) in PA-treated calf hepatic cells, indicating the suppression effect of propionate on endoplasmic reticulum (ER) stress. However, inhibition of autophagic activity by chloroquine or bafilomycin A1 impede the beneficial effects of propionate on ER stress and cell viability. These results demonstrated that propionate alleviates ER stress and elevates cell viability in PA-treated calf hepatic cells by enhancing autophagy, which implies that autophagy may be a promising target in improving liver injury of dairy cows during transition period.

Hepatic autophagy and mitophagy status in dairy cows with subclinical and clinical ketosis.

Severe negative energy balance around parturition is an important contributor to ketosis, a metabolic disorder that occurs most frequently in the peripartal period. Autophagy and mitophagy are important processes responsible for breaking down useless or toxic cellular material, and in particular damaged mitochondria. However, the role of autophagy and mitophagy during the occurrence and development of ketosis is unclear. The objective of this study was to investigate autophagy and mitophagy in the livers of cows with subclinical ketosis (SCK) and clinical ketosis (CK). We assessed autophagy by measuring the protein abundance of microtubule-associated protein 1 light chain 3-II (LC3-II; encoded by MAP1LC3) and sequestosome-1 (p62, encoded by SQSTM1), as well as the mRNA abundance of autophagy-related genes 5 (ATG5), 7 (ATG7), and 12 (ATG12), beclin1 (BECN1), and phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3). Mitophagy was evaluated by measuring the protein abundance of the mitophagy upstream regulators PTEN-induced putative kinase 1 (PINK1) and Parkin. Liver and blood samples were collected from healthy cows [n = 15; blood ß-hydroxybutyrate (BHB) concentration <1.2 mM], cows with SCK (n = 15; blood BHB concentration 1.2 to 3.0 mM) and cows with CK (n = 15; blood BHB concentration >3.0 mM with clinical signs) with similar lactation numbers (median = 3, range = 2 to 4) and days in milk (median = 6, range = 3 to 9). The serum activity of aspartate aminotransferase and alanine aminotransferase was greater in cows with CK than in healthy cows. Levels of oxidative stress biomarkers malondialdehyde and hydrogen peroxide were also higher in liver tissue from ketotic cows (SCK and CK) than from healthy cows. Compared with cows with CK and healthy cows, the hepatic mRNA abundance of MAP1LC3, SQSTM1, ATG5, ATG7, ATG12, and PIK3C3 was upregulated in cows with SCK. Compared with healthy cows, cows with SCK had a lower abundance of p62 and a greater abundance of LC3-II, but levels of both were higher in cows with CK. The mRNA abundance of ATG12 was lower in cows with CK than in healthy cows. Furthermore, the hepatic protein abundance of PINK1 and Parkin was greater in cows with SCK and slightly lower in cows with CK than in healthy cows. These data demonstrated differences in the hepatic activities of autophagy and mitophagy in cows with SCK compared with cows with CK. Although the precise mechanisms for these differences could not be discerned, autophagy and mitophagy seem to be involved in ketosis.