Phosphatidylcholine transfer protein/StarD2 promotes microvesicular steatosis and liver injury in murine experimental steatohepatitis

Abstract

Mice fed a methionine- and choline-deficient (MCD) diet develop steatohepatitis that recapitulates key features of nonalcoholic steatohepatitis (NASH) in humans. Phosphatidylcholine is the most abundant phospholipid in the surfactant monolayer that coats and stabilizes lipid droplets within cells, and choline is required for its major biosynthetic pathway. Phosphatidylcholine-transfer protein (PC-TP), which exchanges phosphatidylcholines among membranes, is enriched in hepatocytes. PC-TP also regulates fatty acid metabolism through interactions with thioesterase superfamily member 2. We investigated the contribution of PC-TP to steatohepatitis induced by the MCD diet. Pctp^-/- and wild-type control mice were fed the MCD diet for 5 wk and were then euthanized for histopathologic and biochemical analyses, as well as determinations of mRNA and protein expression. Whereas all mice developed steatohepatitis, plasma alanine aminotransferase and aspartate aminotransferase activities were only elevated in wild-type mice, indicating that Pctp^-/- mice were protected from MCD diet-induced hepatocellular injury. Reduced hepatotoxicity due to the MCD diet in the absence of PC-TP expression was further evidenced by decreased activation of c-Jun and reduced plasma concentrations of fibroblast growth factor 21. Despite similar total hepatic concentrations of phosphatidylcholines and other lipids, the relative abundance of microvesicular lipid droplets within hepatocytes was reduced in Pctp^-/- mice. Considering that the formation of larger lipid droplets may serve to protect against lipotoxicity in NASH, our findings suggest a pathogenic role for PC-TP that could be targeted in the management of this condition.NEW & NOTEWORTHY Phosphatidylcholine-transfer protein (PC-TP) is a highly specific phosphatidylcholine-binding protein that we previously showed to regulate hepatocellular nutrient metabolism through its interacting partner thioesterase superfamily member 2 (Them2). This study identifies a pathogenic role for PC-TP, independent of Them2, in the methionine- and choline-deficient diet model of experimental steatohepatitis. Our current observations suggest that PC-TP promotes liver injury by mediating the intermembrane transfer of phosphatidylcholines, thus stabilizing more pathogenic microvesicular lipid droplets.

Keywords: lipid droplet; methionine- and choline-deficient diet; nonalcoholic steatohepatitis; phospholipid; triglyceride.

Fig. 1.

PC-TP contributes to hepatocellular injury and microvesicular steatosis in mice fed an MCD diet. WT and Pctp^−/− mice were fed a chow or the MCD diet for 5 wk. A: body weight plotted as a function of time in mice fed the MCD diet. Influence of MCD diet on hepatic Pctp mRNA expression (B) and protein abundance (immunoblot and quantification; C) in WT mice. Representative hematoxylin and eosin- (D) and Sirius red (E)-stained liver images (×40 magnification; white bar = 50 µm). Arrows indicate areas of macrovesicular and microvesicular steatosis. NAFLD activity score (NAS; F) and components that comprise this score (G) in MCD diet-fed mice. NAFLD fibrosis score (H) and hepatic hydroxyproline concentrations (I) in MCD diet-fed mice. Plasma activities of alanine aminotransferase (ALT; J) and aspartate aminotransferase (AST; K). L: relative abundance of microvesicular and macrovesicular lipid droplets in the liver of MCD diet-fed mice. M: hepatic perilipin 2 (Plin2) protein abundance (immunoblot and relative quantification). Values represent means ± SE. A: n = 22–24 per group. B, C, and F–M: chow-fed mice, n = 4–5; MCD diet-fed mice, n = 13–15. *P < 0.05, Pctp^−/− vs. WT; §P < 0.05, MCD vs. chow diet.

Fig. 2.

PC-TP does not alter hepatic lipid concentrations or compositions in MCD diet-fed mice. WT and Pctp^−/− mice were fed a chow or MCD diet for 5 wk. Hepatic concentrations of total (A) and molecular species of (B) phosphatidylcholine, total (C) and molecular species of (D) diglyceride, total (E) and molecular species of (F) triglyceride, and total (G) and molecular species of (H) ceramide were quantified by mass spectrometry and are displayed as arbitrary units (AU). For each lipid molecular species, the number to the left of the colon indicates the total number of carbons, and the number to the right of the colon denotes the total number of unsaturations. Hepatic gene expression of transcription factors and enzymes that regulate lipid metabolism (I) and lipid biogenesis (J). Concentrations of plasma triglycerides (K) and cholesterol (L) as well as hepatic cholesterol (M) were assessed enzymatically. Values represent means ± SE. A–H: n = 4–5. I–M: n = 13–15. *P < 0.05, Pctp^−/− vs. WT; §P < 0.05, MCD vs. chow diet.

Fig. 3.

PC-TP alters hepatocellular stress pathways during MCD diet. WT and Pctp^−/− mice were fed a chow or MCD diet for 5 wk before measurements of fibroblast growth factor 21 (FGF21) mRNA expression in liver (A) and FGF21 protein concentration in plasma (B). Plasma concentration of β-hydroxybutyrate (C), hepatic concentration of free fatty acids (D), hepatic malondialdehyde (MDA) concentrations (E), and hepatic expression of genes central to cellular stress, inflammation, and fibrogenesis (F). Immunoblot of liver homogenate (G) and relative quantification of phospho-JNK over total JNK (H) and phospho-c-Jun Ser63 (I) and Ser73 (J) over total c-Jun. K: hepatic expression of genes that reflect endoplasmic reticulum (ER) stress. L: hepatic mRNA expression of liver receptor homolog-1 (LRH-1), along with downstream transcriptional targets. M: tolerance tests to glucose (GTT), insulin (ITT), and pyruvate (PTT). Values represent means ± SE. Chow-fed mice, n = 4–5; MCD diet-fed mice, n = 13–15. *P < 0.05, Pctp^−/− vs. WT; §P < 0.05, MCD vs. chow diet.

Fig. 4.

PC-TP-interacting protein Them2 does not promote hepatic injury in response to the MCD diet. A: hepatic Them2 mRNA expression in WT and Pctp^−/− mice fed a MCD diet for 5 wk. WT and Them2^−/− mice were fed a chow or the MCD diet for 5 wk. B: representative images of hematoxylin and eosin-stained liver sections (×40 magnification; white bar = 50 µm). NAFLD activity score (NAS; C) and NAS components (D). Plasma activities of alanine aminotransferase (ALT; E) and aspartate aminotransferase (AST; F). G: relative abundance of macrovesicular and microvesicular lipid droplets in livers of MCD diet-fed mice. Plasma concentration of β-hydroxybutyrate (H) and hepatic malondialdehyde (MDA; I). Values represent means ± SE. A: n = 7–15. C–I: n = 3–7. *P < 0.05, Pctp^−/− vs. WT; §P < 0.05, MCD vs. chow diet.

Fig. 5.

Schematic diagram: MCD diet reduces hepatic phosphatidylcholine concentrations. Under these limiting conditions, PC-TP is upregulated and functions to redistribute phosphatidylcholine molecules between the endoplasmic reticulum (ER) and lipid droplets (LDs) with sufficient or excess phosphatidylcholine, to preserve the stability of small LDs that promote liver injury. In the absence of PC-TP, smaller lipid droplets with insufficient phosphatidylcholine on their surfaces coalesce into larger lipid droplets that are less metabolically active and hepatotoxic.