Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
Phosphatidylcholine protects against steatosis in mice but not non-alcoholic steatohepatitis
Highlights
►Hepatic CTα knockout mice are susceptible to high fat diet-induced liver NASH. ►Hepatic CTα knockout mice have 25% lower hepatic PC resulting in steatosis. ►Diacylglycerol and ceramide are elevated by 50% in CTα-deficient livers. ►Normalization of hepatic PC prevents steatosis in hepatic CTα knockout mice. ►Hepatic level of PC is not the only determinant for the development of NASH.
Introduction
Non-alcoholic fatty liver disease (NAFLD) encompasses a wide spectrum of liver damage including steatosis, non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. The prevalence of NAFLD in the general population in North America is 10%–24%, and is 25–75% in the obese and diabetic population [1]. The progression from steatosis to NASH has been described as a “two-hit” model in which the “first hit” constitutes hepatic triacylglycerol (TG) accumulation and the “second hit” involves cellular stresses causing inflammation and apoptosis [2]. However, cellular determinants responsible for the pathogenesis of NASH are relatively unknown.
One of the most commonly used nutritional models for studying NASH is a diet deficient in both methionine and choline (MCD). The MCD diet induces histological characteristics of NASH including steatosis, inflammation, and fibrosis. In response to the MCD diet, steatosis is associated with reduced very-low-density lipoprotein (VLDL) secretion, as a result of reduced phosphatidylcholine (PC) biosynthesis [3], [4]. In addition to their role in PC biosynthesis, methionine and choline are important metabolites involved in methylation reactions, acetylcholine production and protein biosynthesis [5], [6]. Hence, many factors other than reduced PC biosynthesis might contribute to the pathogenesis of NASH in the MCD model, thereby limiting the MCD diet as a model to study the role of PC biosynthesis in the progression of NASH.
In all nucleated cells, PC is made via the CDP-choline pathway and flux through this pathway is regulated by CTP: phosphocholine cytidylyltransferase (CT) [7], [8]. Although two isoforms of CT exist (CTβ and CTα), CTα is believed to be the predominant isoform in the liver [9]. In the liver, PC can also be made via the phosphatidylethanolamine N-methyltransferase (PEMT) pathway in which PC is synthesized from sequential methylation reactions of phosphatidylethanolamine (PE) [10].
We generated mice deficient in either PEMT or hepatic CTα. Studies on Pemt−/− mice demonstrated that normal VLDL secretion requires PEMT, and that disruption of this pathway causes hepatic steatosis [11], [12]. CTα is also important for hepatic VLDL metabolism since liver-specific CTα knockout (LCTα−/−) mice have impaired apoB-100 secretion [13]. Furthermore, on a chow diet hepatic PC in LCTα−/− mice was 5–20% lower, and TG was 25–40% higher, than in LCTα+/+ mice [13]. These data demonstrate the importance of CTα in controlling hepatic VLDL secretion.
Patients with hepatic steatosis have 25% less hepatic PC than do patients without hepatic steatosis [14]. In addition, a functional polymorphism (V175M substitution) within the Pemt gene is associated with NAFLD in humans [15]. This polymorphism decreased the specific activity of PEMT when expressed in hepatoma cells and increased the susceptibility of patients to NAFLD [15]. Thus, impaired hepatic PC biosynthesis might be a determinant of NASH in humans. However, the direct role of impaired hepatic PC biosynthesis in the pathogenesis of NASH has not been investigated. Therefore, we challenged LCTα−/− mice with a high fat (HF) diet to determine whether or not impaired PC biosynthesis contributes to the pathogenesis of NASH. Our hypothesis predicted that LCTα−/− mice would develop hepatic steatosis when fed the HF diet due to impaired VLDL secretion. Surprisingly, within one week of being fed the HF diet, LCTα−/− mice developed NASH. To investigate potential treatments for NASH, LCTα−/− mice fed the HF diet were given a daily injection of CDP-choline (to bypass the CT deficiency), or were fed betaine in the HF diet (to supply methyl groups for the PEMT pathway). Furthermore, we hypothesized that adenoviral delivery of CTα would increase hepatic PC mass and prevent hepatic steatosis in LCTα−/− mice. The results show that impaired PC biosynthesis is not the sole determinant of NASH in LCTα−/− mice.
Section snippets
Animal handling and diets
All procedures were approved by the University of Alberta's Institutional Animal Care Committee in accordance with guidelines of the Canadian Council on Animal Care. Female C57BL/6:129P2 floxed (control) and LCTα−/− mice (2–4 months old) [13] were fed the HF diet (Bioserve, #F3282) for 7 days. Mice were fasted for 12 h before collection of blood by cardiac puncture. Plasma was stored at − 80 °C. Liver samples were freeze clamped and stored at − 80 °C.
In some experiments, the diet was supplemented with
The HF diet induces moderate steatohepatitis in livers of LCTα-deficient mice
Liver histology revealed markedly altered morphology of livers from LCTα-deficient mice fed the HF diet for 7 days (Fig. 1A). Plasma alanine aminotransferase (ALT) content was 7-fold higher in the LCTα−/− mice compared to floxed controls, indicating induced liver injury (Fig. 1B). To investigate the presence of NASH, liver histology was clinically assessed for the appearance of intra-acinar and portal inflammation, and ballooning degeneration of hepatocytes. Clinical assessment of liver
Discussion
We utilized LCTα−/− mice, without dietary reduction of PC precursors (choline and methionine), to investigate the relationship between impaired hepatic PC biosynthesis and NASH. This is the first study in which the direct role of only impaired PC biosynthesis in the pathogenesis of NASH has been investigated. LCTα−/− mice proved to be an attractive model for studies on NASH as liver disease developed within one week of feeding the HF diet as indicated by elevated levels of plasma ALT, increased
Acknowledgements
The authors thank Susanne Lingrell and Randy Nelson for excellent technical assistance and advice and Jean Vance for helpful comments. The research was supported by Canadian Institutes of Health Research Grant MOP5182. Dr. Vance is a Scientist of the Alberta Heritage Foundation for Medical Research.
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