Low abundance of mitophagy markers is associated with reactive oxygen species overproduction in cows with fatty liver and causes reactive oxygen species overproduction and lipid accumulation in calf hepatocytes.

Mitochondria are the main site of fatty acid oxidation and reactive oxygen species (ROS) formation. Damaged or dysfunctional mitochondria induce oxidative stress and increase the risk of lipid accumulation. During the process of mitophagy, PTEN induced kinase 1 (PINK1) accumulates on damaged mitochondria and recruits cytoplasmic Parkin to mitochondria. As an autophagy receptor protein, sequestosome-1 (p62) binds Parkin-ubiquitinated outer mitochondrial membrane proteins and microtubule-associated protein 1 light chain 3 (LC3) to facilitate degradation of damaged mitochondria. In nonruminants, clearance of dysfunctional mitochondria through the PINK1/Parkin-mediated mitophagy pathway contributes to reducing ROS production and maintaining metabolic homeostasis. Whether PINK1/Parkin-mediated mitophagy plays a similar role in dairy cow liver is not well known. Thus, the objective of this study was to investigate mitophagy status in dairy cows with fatty liver and its role in free fatty acid (FFA)-induced oxidative stress and lipid accumulation. Liver and blood samples were collected from healthy dairy cows (n = 10) and cows with fatty liver (n = 10) that had a similar number of lactations (median = 3, range = 2 to 4) and days in milk (median = 6 d, range = 3 to 9 d). Calf hepatocytes were isolated from 5 healthy newborn female Holstein calves (1 d of age, 30-40 kg). Hepatocytes were transfected with small interfering RNA targeted against PRKN for 48 h or transfected with PRKN overexpression plasmid for 36 h, followed by treatment with FFA (0.3 or 1.2 mM) for 12 h. Mitochondria were isolated from fresh liver tissue or calf hepatocytes. Serum concentrations of β-hydroxybutyrate were higher in dairy cows with fatty liver. Hepatic malondialdehyde (MDA) and hydrogen peroxide (H2O2) were greater in cows with fatty liver. The lower protein abundance of PINK1, Parkin, p62, and LC3-II in hepatic mitochondrial fraction of dairy cows with fatty liver indicated the mitophagy was impaired. In hepatocytes, knockdown of PRKN decreased protein abundance of p62 and LC3-II in the mitochondrial fraction, and increased contents of triacylglycerol (TG), MDA, and H2O2. In addition, protein abundances of PINK1, Parkin, p62, and LC3-II were lower in the mitochondrial fraction from hepatocytes treated with 1.2 mM FFA than the hepatocytes treated with 0.3 mM FFA, whereas the content of TG, MDA, and H2O2 increased. In 1.2 mM FFA-treated hepatocytes, PRKN overexpression increased protein abundance of p62 and LC3-II in the mitochondrial fraction and decreased contents of TG, MDA, and H2O2. Together, our data demonstrate that low abundance of mitophagy markers is associated with ROS overproduction in dairy cows with fatty liver and impaired mitophagy induced by a high concentration of FFA promotes ROS production and lipid accumulation in female calf hepatocytes.

Fang Z ，Liu G ，Zhu M ，Wang S ，Jiang Q ，Loor JJ ，Yu H ，Hao X ，Chen M ，Gao W ，Lei L ，Song Y ，Wang Z ，Du X ，Li X ... -  《-》

Sirtuin 3 improves fatty acid metabolism in response to high nonesterified fatty acids in calf hepatocytes by modulating gene expression.

Sirtuin 3 (SIRT3), a mitochondrial deacetylase, is a key regulator of energy metabolism in the liver. In nonruminants, the hepatic abundance of SIRT3 is decreased in individuals with nonalcoholic fatty liver diseases, and recovery of SIRT3 alleviates hepatic triacylglycerol (TG) deposition. However, the level of SIRT3 expression and its effects on lipid metabolism in dairy cows have not been characterized. Here we studied the hepatic expression of SIRT3 in cows with fatty liver and the role of SIRT3 in fatty acid metabolism in bovine hepatocytes. This in vivo study involved 10 healthy cows and 10 cows with fatty liver, from which we collected samples of liver tissue and blood. Primary hepatocytes were isolated from Holstein calves and treated with 0, 0.5, or 1.0 mM nonesterified fatty acids (NEFA) for 24 h or transinfected with SIRT3 overexpression adenovirus (Ad-SIRT3)/SIRT3-short interfering (si)RNA for 48 h. Cows with fatty liver displayed lower serum glucose concentrations but higher serum NEFA and β-hydroxybutyrate concentrations relative to healthy cows. Cows with fatty liver also had significant lower mRNA and protein abundance of hepatic SIRT3. Incubation of primary hepatocytes with NEFA reduced SIRT3 abundance in primary hepatocytes in a dose-dependent manner. Fatty acid (1 mM) treatment also markedly increased the abundance of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) but significantly decreased the abundance of carnitine palmitoyltransferase I (CPT1A), carnitine palmitoyltransferase II (CPT2), and acyl-CoA oxidase (ACO). Knockdown of SIRT3 by SIRT3-siRNA downregulated the mRNA abundance of CPT1A, CPT2, and ACO. In contrast, overexpression of SIRT3 by Ad-SIRT3 upregulated the mRNA abundance of CPT1A, CPT2, and ACO; downregulated the mRNA abundance of ACC1 and FAS; and consequently, decreased intracellular TG concentrations. Overexpression of SIRT3 ameliorated exogenous NEFA-induced TG accumulation by downregulating the abundance of ACC1 and FAS and upregulating the abundance of CPT1A, CPT2, and ACO in calf hepatocytes. Our data demonstrated that cows with fatty liver had lower hepatic SIRT3 contents, and an increase in SIRT3 abundance by overexpression mitigated TG deposition by modulating the expression of lipid metabolism genes in bovine hepatocytes. These data suggest a possible role of SIRT3 as a therapeutic target for fatty liver disease prevention in periparturient dairy cattle.

Liu L ，Xing D ，Du X ，Peng T ，McFadden JW ，Wen L ，Lei H ，Dong W ，Liu G ，Wang Z ，Su J ，He J ，Li X ... -  《-》

Low abundance of insulin-induced gene 1 contributes to SREBP-1c processing and hepatic steatosis in dairy cows with severe fatty liver.

Fatty liver is a major metabolic disorder of high-producing dairy cows during the transition period. In nonruminants, it is well established that insulin-induced gene 1 (INSIG1) plays a crucial role in regulating hepatic lipogenesis by controlling the anchoring of sterol regulatory element-binding protein 1 (SREBP-1) on the endoplasmic reticulum along with SREBP cleavage-activating protein (SCAP). Whether the INSIG1-SCAP-SREBP-1c transport axis is affected in cows experiencing fatty liver is unknown. Thus, the aim of this study was to investigate the potential role of INSIG1-SCAP-SREBP-1c axis in the progression of fatty liver in dairy cows. For in vivo experiments, 24 dairy cows at the start of their fourth lactation (median; range 3-5) and 8 d in milk (median; range 4-12 d) were selected into a healthy group [n = 12; triglyceride (TG) content <1%] and a severe fatty liver group (n = 12; TG content >10%) according to their hepatic TG content. Blood samples were collected for detecting serum concentrations of free fatty acids, β-hydroxybutyrate, and glucose. Compared with healthy cows, cows with severe fatty liver had higher serum concentrations of β-hydroxybutyrate and free fatty acids and lower concentration of glucose. Liver biopsies were used to detect the status of INSIG1-SCAP-SREBP-1c axis, and the mRNA expression of SREBP-1c-target lipogenic genes acetyl-CoA carboxylase α (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1). Cows with severe fatty liver had lower protein expression of INSIG1 in the hepatocyte endoplasmic reticulum fraction, greater protein expression of SCAP and precursor SREBP-1c in the hepatocyte Golgi fraction, and greater protein expression of mature SREBP-1c in the hepatocyte nuclear fraction. In addition, the mRNA expression of SREBP-1c-target lipogenic genes ACACA, FASN, and DGAT1 was greater in the liver of dairy cows with severe fatty liver. In vitro experiments were conducted on hepatocytes isolated from 5 healthy 1-d-old female Holstein calves, and hepatocytes from each calf were run independently. First, hepatocytes were treated with 0, 200, or 400 μM palmitic acid (PA) for 12 h. Exogenous PA treatment decreased INSIG1 protein abundance, enhanced the endoplasmic reticulum to Golgi export of SCAP-precursor SREBP-1c complex and the nuclear translocation of mature SREBP-1c, all of which was associated with increased transcriptional activation of lipogenic genes and TG synthesis. Second, hepatocytes were transfected with INSIG1-overexpressing adenovirus for 48 h and treated with 400 μM PA 12 h before the end of transfection. Overexpressing INSIG1 inhibited PA-induced SREBP-1c processing, upregulation of lipogenic genes, and TG synthesis in hepatocytes. Overall, the present in vivo and in vitro results indicated that the low abundance of INSIG1 contributed to SREBP-1c processing and hepatic steatosis in dairy cows. Thus, the INSIG1-SCAP-SREBP-1c axis may be a novel target for treatment of fatty liver in dairy cows.

Zhu Y ，Lei L ，Wang X ，Jiang Q ，Loor JJ ，Kong F ，Chen L ，Li J ，Zhao C ，Liu M ，Liu G ，Li X ... -  《-》

AdipoRon alleviates fatty acid-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes by promoting autophagy.

During the transition period in dairy cows, high circulating concentrations of nonesterified fatty acids (NEFA) increase hepatic lipid deposits and are considered a major pathological factor for liver damage. We investigated whether AdipoRon, a synthetic small-molecule agonist of adiponectin receptors 1 and 2 shown to prevent liver lipid accumulation in nonruminants, could alleviate NEFA-induced lipid accumulation and mitochondrial dysfunction. Bovine hepatocytes were isolated from 5 healthy Holstein female newborn calves (1 d of age, 30-40 kg, fasting), and independently isolated hepatocytes from at least 3 different calves were used for each subsequent experiment. The composition and concentration of NEFA used in this study were selected according to hematological criteria of dairy cows with fatty liver or ketosis. First, hepatocytes were cultured with various concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 12 h. In a second experiment, hepatocytes were treated with AdipoRon at different concentrations (0, 5, 25, or 50 μM for 12 h) and times (25 μM for 0, 6, 12, or 24 h) with or without NEFA (1.2 mM) treatment. In the last experiment, hepatocytes were treated with AdipoRon (25 μM), NEFA (1.2 mM), or both for 12 h after treatment with or without the autophagy inhibitor chloroquine. Hepatocytes treated with NEFA had increased protein abundance of sterol regulatory element-binding protein 1c (SREBP-1c) and mRNA abundance of acetyl-CoA carboxylase 1 (ACACA), and decreased protein abundance of peroxisome proliferator-activated receptor α (PPARA), proliferator-activated receptor gamma coactivator-1 α (PGC-1α), mitofusin 2 (MFN2), cytochrome c oxidase subunit IV (COX IV), and mRNA abundance of carnitine palmitoyltransferase 1A (CPT1A), along with lower ATP concentrations. AdipoRon treatment reversed these effects, suggesting this compound had a positive effect on lipid metabolism and mitochondrial dysfunction during the NEFA challenge. In addition, upregulated expression of microtubule-associated protein 1 light chain 3-II (LC3-II, encoded by MAP1LC3) and downregulated expression of sequestosome-1 (SQSTM1, also called p62) indicated that AdipoRon enhanced autophagic activity in hepatocytes. The fact that chloroquine impeded the beneficial effects of AdipoRon on lipid accumulation and mitochondrial dysfunction suggested a direct role for autophagy during NEFA challenge. Our results suggest that autophagy is an important cellular mechanism to prevent NEFA-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes, which is consistent with other studies. Overall, AdipoRon may represent a promising therapeutic agent to maintain hepatic lipid homeostasis and mitochondrial function in dairy cows during the transition period.

Zhao C ，Wu B ，Li J ，Jiang Q ，Loor JJ ，Liu M ，Chen L ，Zhu Y ，Gao W ，Du X ，Song Y ，Liu G ，Lei L ，Li X ... -  《-》

Taraxasterol alleviates fatty acid-induced lipid deposition in calf hepatocytes by decreasing ROS production and endoplasmic reticulum stress.

Increased concentrations of free fatty acids (FFAs) induce reactive oxygen species (ROSs) generation and endoplasmic reticulum (ER) stress, thus, increasing the risk of fatty liver in dairy cows during the periparturient period. In non-ruminants, Taraxasterol (Tara; a pentacyclic triterpenoid found in medicinal plants) plays an important role in anti-inflammatory and antioxidant reactions. Whether Tara can alleviate or prevent fatty liver in ruminants is unknown. We addressed whether Tara supply could dampen lipid accumulation, ROSs production, and ER stress caused by FFAs in calf hepatocytes. Primary calf hepatocytes were isolated from five healthy calves (1 d old, female, 30-40 kg, fasting, rectal temperature 38.7-39.7 °C). In the first experiment, hepatocytes were incubated with various concentrations of Tara (2.5, 5, and 10 μg/mL) for 12 h prior to the 1.2-mM FFAs challenge. Results indicated that the level of ROSs was lowest with 5 μg/mL Tara. Thus, to further characterize the molecular mechanisms whereby Tara protects from FFAs-induced lipid deposition in calf hepatocytes, we performed incubations with 5 μg/mL Tara for 12 h prior to a 1.2-mM FFAs challenge for an additional 12 h. Results indicated that 1.2-mM FFAs challenge increased mitochondrial membrane potential (MMP), enhanced expression of proteins and mRNA associated with ER stress (PERK, IRE1, GRP78, ATF6, and CHOP) and fatty acid synthesis (FASN, ACC1, and SREBP-1c), and ultimately led to increased lipid droplet synthesis. In contrast, Tara treatment alleviated these negative effects after 1.2-mM FFAs challenge. To determine whether Tara protects against FFAs-induced lipid droplet synthesis by alleviating oxidative stress, hepatocytes were treated with 5 μg/mL Tara for 22 h prior to H2O2 (440 μM) challenge for 2 h. Compared with H2O2 treatment alone, results revealed a marked decrease in ROSs, MMP, and protein abundance of ER stress (GRP78, ATF6, and CHOP) and lipid droplet synthesis in response to Tara prior to H2O2 challenge. Data suggested that the increase in mitochondrial ROSs production contributes to lipid accumulation in calf hepatocytes. Collectively, our in vitro data indicate that Tara alleviates fatty acid-induced lipid deposition. Further research is warranted to ascertain that Tara can be helpful in the therapeutic management of early lactating cows to control or alleviate excessive hepatic lipid deposition.

Li M ，He Y ，Zhang W ，Yin Y ，Jiang Q ，Loor JJ ，Wang J ，Wen J ，Yang W ，Xu C ，Zhang B ... -  《-》