Fatty liver and ketosis are common metabolic diseases for dairy cows in the transition period, a time when most dairy cows undergo negative energy balance (NEB) caused by increased requirement for energy to support milk synthesis (Fatima et al. Reference Fatima, Waters, O'Boyle, Seoighe and Morris2014; Schulz et al. Reference Schulz, Frahm, Meyer, Kersten, Reiche, Rehage and Danicke2014). This NEB initiates lipid mobilisation and subsequent high plasma concentrations of non-esterified fatty acids (NEFAs; McCabe et al. Reference McCabe, Waters, Morris, Kenny, Lynn and Creevey2012). Large quantities of NEFAs are metabolised into ketones, such as acetoacetic acid (AcAc), acetone and β-hydroxybutyrate (BHBA), or synthesised into triglycerides (TG) in hepatocytes, thereby inducing ketosis and/or fatty liver (Karcagi et al. Reference Karcagi, Gaal, Ribiczey, Huszenicza and Husveth2010; McArt et al. Reference McArt, Nydam, Oetzel, Overton and Ospina2013). NEFAs are first metabolised by hepatocytes via β-oxidation to produce AcAc, and subsequently to acetone and BHBA, which finally leads to ketosis (Hayirli, Reference Hayirli2006; Turk et al. Reference Turk, Podpečan, Mrkun, Kosec, Flegar-Meštrić, Perkov, Starič, Robić, Belić and Zrimšek2013). Unfortunately, clinical investigation has shown that ketosis can in turn further aggravate the development of fatty liver. The possible mechanism of this interaction is an interesting topic for further research.
It has been proposed that AcAc could act as a signalling molecule and be involved in the development of human diabetes, which is in some respects similar to ketosis and fatty liver in dairy cows (Abdelmegeed et al. Reference Abdelmegeed, Kim, Woodcroft and Novak2004; Kanikarla-Marie & Jain, Reference Kanikarla-Marie and Jain2015). Elevated levels of AcAc may increase lipid peroxidation in people with type 1 diabetes (Jain & McVie, Reference Jain and McVie1999), and high levels of AcAc constitute a risk factor for the oxidative modification of VLDL in diabetic patients (Jain et al. Reference Jain, McVie, Jaramillo and Chen1998). Similarly, levels of VLDL are significantly decreased in dairy cows with ketosis (Sun et al. Reference Sun, Zhang, Wu, Shu, Xia, Xu and Zheng2014). It is known that fatty liver occurs when the rate of hepatic TG synthesis exceeds the rate of TG disappearance through either hydrolysis or exported via VLDL (Gessner et al. Reference Gessner, Schlegel, Ringseis, Schwarz and Eder2014). The ability of dairy cows to secrete hepatic TG as VLDL is extremely low compared with nonruminant animals (McCabe et al. Reference McCabe, Waters, Morris, Kenny, Lynn and Creevey2012). The main structural proteins apolipoprotein B100 (ApoB100) and apolipoprotein E (ApoE), and the regulatory proteins microsomal triglyceride transfer protein (MTP) and low density lipoprotein receptor (LDLR), are all involved in the synthesis and assembly of VLDL in liver (Mason, Reference Mason1998; Larsson et al. Reference Larsson, Skogsberg and Björkegren2004; Sacks, Reference Sacks2015). It is unclear whether AcAc can regulate the expression of ApoB100, ApoE, MTP and LDLR, and hence influence the synthesis and assembly of VLDL in bovine hepatocytes.
We have previously shown that the ketone body, BHBA, can induce oxidative stress in bovine hepatocytes (Shi et al. Reference Shi, Li, Li, Li, Song, Deng, Wang, Zhang, Ding and Yin2014). In this study, we hypothesised that AcAc could also influence the synthesis and assembly of VLDL through a mechanism involving oxidative stress in bovine hepatocytes.
Materials and methods
Ethical approval of the study protocol
All the experiments were performed in accordance with the Guiding Principles in the Use of Animals adopted by the Chinese Association for Laboratory Animal Sciences. All the animal procedures were approved by the Institutional Animal Care and Use Committee of Jilin University (Changchun).
Materials
RPMI-1640 medium, heparin sodium, Collagenase IV and foetal bovine serum were purchased from Gibco (Grand Island, NY, USA). AcAc, HEPES, insulin, and NAC (N-acetylcysteine, an antioxidant) were provided by Sigma-Aldrich (St. Louis, MO, USA). Dexamethasone acetate, vitamin C, penicillin, streptomycin and other chemicals were purchased from Baoman Biotechnology (Shanghai, China). Six-well plates and filters were purchased from Corning Incorporated (Corning, NY, USA).
Isolation of cow primary hepatocytes and AcAc treatment
Primary bovine hepatocytes were isolated and cultured as previously described (Shi et al. Reference Shi, Li, Deng, Li, Sun, Yuan, Song, Wang, Li and Li2015). The concentrations of AcAc used in this study were based on the serum AcAc concentrations in dairy cows with ketosis or fatty liver. N-acetylcysteine (NAC) was an antioxidant in liver diseases. The hepatocytes were starved of serum overnight before AcAc treatment. Then, the hepatocytes were treated with 0 mM AcAc (control group, GC), 0·6 mM AcAc (low-dose group, GL), 2·4 mM AcAc (medial-dose group, GM), and 4·8 mM AcAc (high-dose group, GH), and 10 mM NAC + 4·8 mM AcAc (NAC + GH) for 24 h, respectively. Each treatment was replicated nine times.
The determination of the oxidative stress markers superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA)
After treatment with different concentrations of AcAc with or without NAC for 24 h, the hepatocytes were collected and washed twice in ice-cold phosphate-buffered saline (PBS). Then, the cells were lysed using P0013D (Beyotime, Jiangsu, China). Subsequently, the lysate was centrifuged at 12 000 g for 5 min at 4 °C. The supernatant was used to determine the content of MDA and the activities of GSH-Px, SOD and CAT using the appropriate biochemical kits (Beyotime, Jiangsu, China) according to the manufacturer's instructions.
Real-time PCR
Total RNA from hepatocytes was isolated with TRIzol reagent (TaKaRa Biotechnology Co., Ltd., Tokyo, Japan) according to the supplier's protocol. The concentration of RNA was determined using a K5500 Micro-Spectrophotometer (Beijing Kaiao Technology Development Co., Ltd., Beijing, China). Approximately 5 µg of total RNA was reverse-transcribed to cDNA in 20-μl reactions using PrimeScript Reverse Transcriptase (TaKaRa Biotechnology Co., Ltd., Tokyo, Japan) according to the manufacturer's instructions. All primers were designed using Primer Express software (PE Applied Biosystems, Inc., Foster City, CA, USA; sequences are given in online Supplementary Table S1) and synthesised from Sangon (Sangon Biotech Co., Ltd., Shanghai, China). The mRNA expression levels were evaluated by quantitative polymerase chain reaction (qRT-PCR) analysis using a SYBR Green QuantiTect RT-PCR Kit (Takara Biotechnology Co., Ltd.). qRT-PCR was performed on a 7500 Real-Time PCR System (Applied Biosystems/Life Technologies, Grand Island, NY, USA). The relative expression of each gene was calculated by the 2−ΔΔCT method and was normalised to β-actin levels.
Determination of VLDL content
After the hepatocytes were treated with AcAc, the medium from the 6-well plates was collected in 1·5 ml EP tubes. The VLDL content synthesised and secreted by hepatocytes was determined using ELISA kits (Shanghai Bluegene Biotech Co. Ltd., Shanghai, China; cat. no. E11V0006), according to the manufacturer's protocol.
Determination of TG content
After the cell medium was removed, hepatocytes were harvested and transferred into a centrifuge tube. Then, the cells were washed twice with ice-cold PBS and lysed using lysis buffer (Shanghai Bluegene Biotech Co. Ltd.) in ice bath conditions for 30 min. Finally, the lysate was centrifuged for 5 min at 12 000 g at 4 °C, and the supernatant was used to determine the content of triglycerides with a commercial kit (Applygen Technologies Inc., Beijing, China) using an automatic biochemical analyser (Shenyang EKSV Medical Equipment Co. Ltd., Shenyang, China).
Statistical analysis
The results are expressed as the mean ± standard error (sem). The data were analysed by a one-way ANOVA using the SPSS software program (SPSS Inc., Chicago, IL). The differences were considered significant at P < 0·05.
Results
The effect of AcAc on oxidative stress in bovine hepatocytes
Table 1 shows the effect of AcAc on different measures of oxidative stress in bovine hepatocytes. MDA content was increased by AcAC to a peak in the GH group which was partially reversed by the antioxidant, NAC. The antioxidant indicators SOD, CAT, and GSH-Px were detected by real-time PCR and enzyme activity. The mRNA expression of Mn SOD, Cu/Zn SOD, and GSH-Px were all decreased in the AcAc-treated groups compared to the GC group. The CAT mRNA expression level had no change in the GL group, but was significantly lower in the GM and GH groups when compared with the GC group. Enzyme activity data confirmed that all activities were significantly decreased in the GM and GH groups (data are presented in online Supplementary Table S2).
The different measures of oxidative stress in bovine hepatocytes
Hepatocytes were treated with 0 mM AcAc (control group, GC), 0·6 mM AcAc (low-dose group, GC), 2·4 mM AcAc (medial-dose group, GM), and 4·8 mM AcAc (high-dose group, GH), and 10 mM NAC + 4·8 mM AcAc (NAC + GH), respectively. The data are shown as the mean ± sem. Asterisks denote significance (P < 0·05) from the GC (*) or from the GH (**).
The effect of AcAc on ApoB100, ApoE, MTP, and LDLR in bovine hepatocytes
The mRNA expression of ApoB100, ApoE, MTP, and LDLR (important molecules involved in the hepatic synthesis and assembly of VLDL) are shown in Table 2. With the exception of MTP, all were significantly lower in the GM and GH groups when compared to the GC group, but higher in the NAC + GH group when compared with the GH group. MTP mRNA expression was not altered by AcAc.
The levels of ApoB100, ApoE, MTP, LDLR, VLDL, and TG in bovine hepatocytes
Hepatocytes were treated as described in Table 1. The data are shown as the mean ± sem. Asterisks denote significance (P < 0·05) from the GC (*) or from the GH (**).
The effect of AcAc on the content of VLDL and TG in bovine hepatocytes
VLDL and TG contents were detected using ELISA kits (Table 2). VLDL content was gradually decreased in the GM and GH groups when compared to the GC group, but remarkably increased in the NAC + GH group when compared with the GH group. The content of TG changed in reverse to VLDL, increased in the GM and GH groups when compared with the GC group, but decreased in the NAC + GH group when compared with the GH group.
Discussion
Ketosis and fatty liver are closely related metabolic diseases in high-production dairy cows (Hayirli, Reference Hayirli2006; González et al. Reference González, Muiño, Pereira, Campos and Benedito2011). Dairy cows with ketosis or fatty liver are characterized by high blood levels of NEFA and AcAc (Gross et al. Reference Gross, Schwarz, Eder, van Dorland and Bruckmaier2013; Wang et al. Reference Wang, Zhu, Wang, Yao, Zhao and Liu2015). A previous study has shown that high concentrations of NEFAs could inhibit the synthesis and assembly of VLDL, leading to TG accumulation in bovine hepatocytes (Liu et al. Reference Liu, Li, Li, Guan, Song, Yin, Chen, Lei, Liu and Li2014). In this study, we showed that high concentrations of AcAc treatment could result in dramatically elevated TG content, and significantly decreased VLDL content in bovine hepatocytes. Therefore, we conclude that high levels of AcAc in the blood may be an important factor for the development of fatty liver. Furthermore, it also partly explains why dairy cows with ketosis are more susceptible to fatty liver during the transition period.
It is well known that VLDL serves as the main pathway to eliminate TG in liver (Mason, Reference Mason1998; Li et al. Reference Li, Li, Yang, Xiao, Fu, Deng, Ding, Wang, Liu and Li2014). Thus, abnormal assembly of VLDL is an important factor in the induction of fatty liver in dairy cows. ApoB100, the TG-binding protein, is the structural backbone for the formation and secretion of VLDL (Larsson et al. Reference Larsson, Skogsberg and Björkegren2004; Liu et al. Reference Liu, Li, Li, Guan, Song, Yin, Chen, Lei, Liu and Li2014). VLDL cannot be produced at sufficient amounts in dairy cows with fatty liver because of a low ApoB100 expression (Gessner et al. Reference Gessner, Schlegel, Ringseis, Schwarz and Eder2014). Our results showed that high levels of AcAc could significantly inhibit the expression of Apo B100 in bovine hepatocytes. Additionally, ApoE is also present as a structural constituent of VLDL, and plays a vital role in the regulation of hepatic VLDL-triglyceride secretion (Kuipers et al. Reference Kuipers, Jong, Lin, Eck, Havinga, Bloks, Verkade, Hofker, Moshage and Berkel1997). ApoE-deficient mice had impaired VLDL-triglyceride secretion, accumulated large amounts of TG, and developed fatty liver (Kuipers et al. Reference Kuipers, Jong, Lin, Eck, Havinga, Bloks, Verkade, Hofker, Moshage and Berkel1997; Tsukamoto et al. Reference Tsukamoto, Maugeais, Glick and Rader2000). Our results demonstrate that AcAc may reduce the structural constituents of VLDL by suppressing the expression of ApoB100 and ApoE genes in hepatocytes. Furthermore, some studies have indicated that MTP could regulate the expression of ApoB and ApoB-containing lipoprotein (Tietge et al. Reference Tietge, Bakillah, Maugeais, Tsukamoto, Hussain and Rader1999; Borradaile et al. Reference Borradaile, de Dreu, Barrett, Behrsin and Huff2003). MTP, an important regulatory molecule, is required for transport TG to be incorporated into lipoprotein (Mason, Reference Mason1998; Wang et al. Reference Wang, Chen, Lam, Han, Hassler, Finck, Davidson and Kaufman2012). However, AcAc had no effect on the expression of MTP in this study. Indeed, a clinical test reported that MTP expression was not significantly changed in cases of dairy cows with fatty liver, which further support our results (Bremmer et al. Reference Bremmer, Trower, Bertics, Besong, Bernabucci and Grummer2000). Importantly, another crucial regulatory molecule, LDLR, was down-regulated in AcAc-treated bovine hepatocytes. LDLR is a key receptor that is involved in the hepatic VLDL assembly, and ApoE is a high-affinity ligand for the LDLR (Larsson et al. Reference Larsson, Skogsberg and Björkegren2004; Sacks, Reference Sacks2015). Moreover, the secretion of ApoB100 was found to be inhibited by LDLR from hepatocytes (Twisk et al. Reference Twisk, Gillian-Daniel, Tebon, Wang, Barrett and Attie2000; Larsson et al. Reference Larsson, Skogsberg and Björkegren2004). These data provides evidence that AcAc may inhibit LDLR expression, which increases the secretion of ApoB100 from hepatocytes and affects ApoE combined with LDLR in the assembly of VLDL.
It is well documented that elevated oxidative stress is implicated in the secretion of TG-VLDL in some metabolic diseases (Anderson et al. Reference Anderson, Evans, Ellis, Graham, Morris, Jackson, Lewis, Rees and Frenneaux2001; Pan et al. Reference Pan, Cederbaum, Zhang, Ginsberg, Williams and Fisher2004; Bloomer et al. Reference Bloomer, Harvey, Lee and Stockton2015). Moreover, cows with ketosis or fatty liver displayed oxidative stress. MDA is considered the classical oxidative stress marker in cells (Rybka et al. Reference Rybka, Kędziora-Kornatowska, Banaś-Leżańska, Majsterek, Carvalho, Cattaneo, Anacker and Kędziora2013; Zhang et al. Reference Zhang, Jin, Zhang, Yu, Sun and Lu2015). In this study, we found that the content of the oxidative stress marker MDA was significantly increased in hepatocytes treated with AcAc, and that this was reversible by the antioxidant molecule NAC. Previous studies showed that decreasing MDA production mainly depended on three important antioxidant enzymes SOD, CAT, and GSH-Px in cells (Rybka et al. Reference Rybka, Kędziora-Kornatowska, Banaś-Leżańska, Majsterek, Carvalho, Cattaneo, Anacker and Kędziora2013; Sangwan et al. Reference Sangwan, Tomar, Ali, Singh and Singh2015; Shi et al. Reference Shi, Li, Deng, Li, Sun, Yuan, Song, Wang, Li and Li2015), all of which were decreased by AcAc treatment. Therefore, we propose that high concentrations of AcAc can induce oxidative stress in bovine hepatocytes. This agrees with previous observations of AcAc-induced oxidative stress in rat hepatocytes, human erythrocytes and vascular endothelial cells (Jain et al. Reference Jain, McVie, Jaramillo and Chen1998; Jain & McVie, Reference Jain and McVie1999; Abdelmegeed et al. Reference Abdelmegeed, Kim, Woodcroft and Novak2004; Kanikarla-Marie & Jain, Reference Kanikarla-Marie and Jain2015). Our previous studies have demonstrated that oxidative stress was a common mechanism for inflammatory injury of liver in dairy cows (Shi et al. Reference Shi, Li, Li, Li, Song, Deng, Wang, Zhang, Ding and Yin2014, Reference Shi, Li, Deng, Li, Sun, Yuan, Song, Wang, Li and Li2015). Here, we can conlude that high levels of AcAc can induce oxidative stress and influence the synthesis and assembly of VLDL, thereby resulting in TG accumulation in bovine hepatocytes. This may explain why ketosis can further aggravate the development of fatty liver, due to the possible mechanism of high blood levels of AcAc inhibiting the hepatic VLDL assembly through induction of oxidative stress.
Supplementary material
The supplementary material for this article can be found at http://dx.doi.org/10.1017/S0022029916000546
This work was supported by the National Key Technology R&D Program (grant no. 2012BAD12B03 and 2013BAD21B01), the National High Technology Research and Development Program 863 (2013AA102806), the National Natural Science Foundation of China (Beijing, China; grant nos. 31360630, 31372494, 31402265, 31472247, 31460681, and 31572581), and the Science Fund for Distinguished Young Scholars of Jilin University (grant no. 201100009)
