Membrane-bound O-acyltransferase 7 (MBOAT7) shapes lysosomal lipid homeostasis and function to control alcohol-associated liver injury

Abstract

Recent genome-wide association studies (GWAS) have identified a link between single-nucleotide polymorphisms (SNPs) near the MBOAT7 gene and advanced liver diseases. Specifically, the common MBOAT7 variant (rs641738) associated with reduced MBOAT7 expression is implicated in non-alcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), and liver fibrosis. However, the precise mechanism underlying MBOAT7-driven liver disease progression remains elusive. Previously, we identified MBOAT7-driven acylation of lysophosphatidylinositol lipids as key mechanism suppressing the progression of NAFLD (Gwag et al., 2019). Here, we show that MBOAT7 loss of function promotes ALD via reorganization of lysosomal lipid homeostasis. Circulating levels of MBOAT7 metabolic products are significantly reduced in heavy drinkers compared to healthy controls. Hepatocyte- (Mboat7-HSKO), but not myeloid-specific (Mboat7-MSKO), deletion of Mboat7 exacerbates ethanol-induced liver injury. Lipidomic profiling reveals a reorganization of the hepatic lipidome in Mboat7-HSKO mice, characterized by increased endosomal/lysosomal lipids. Ethanol-exposed Mboat7-HSKO mice exhibit dysregulated autophagic flux and lysosomal biogenesis, associated with impaired transcription factor EB-mediated lysosomal biogenesis and autophagosome accumulation. This study provides mechanistic insights into how MBOAT7 influences ALD progression through dysregulation of lysosomal biogenesis and autophagic flux, highlighting hepatocyte-specific MBOAT7 loss as a key driver of ethanol-induced liver injury.

Keywords: MBOAT7; alcohol-associated liver disease; autophagy; medicine; mouse.

Figure 1.. MBOAT7 products are selectively reduced in heavy drinkers.

Plasma lysophosphatidylinositol (LPI – inset graph) and phosphatidylinositol (PI) species from both male and female healthy controls and heavy drinkers were measured by liquid chromatography–tandem mass spectrometry (LC–MS/MS). n = 10–16; ***p < 0.001 and ****p < 0.0001 in the figure. Analysis of variance (ANOVA) with Tukey’s post hoc test.

Figure 2.. Hepatocyte-specific deletion of Mboat7 promotes ethanol-induced liver injury.

Female control (Mboat7^fl/fl) or hepatocyte-specific Mboat7 knockout mice (Mboat7-HSKO) were fed with subjected the NIAAA (National Institute on Alcohol Abuse and Alcoholism) model of ethanol-induced liver injury. (A) Hepatic Mboat7 expression was measured via quantitative polymerase chain reaction (qPCR). (B) Western blot for hepatic microsomal MBOAT7 protein levels replicated in n = 3 mice. (C) Liver weight, (D) plasma alanine aminotransferase (ALT), (E) percent steatosis quantified by a blinded board-certified pathologist, (F) hepatic triglycerides, and (G) hepatic expression of inflammatory gene measured by qPCR. n = 5–7. Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 2—figure supplement 1.. Myeloid-specific deletion of Mboat7 does not promote ethanol-induced liver injury.

Female control (Mboat7^fl/fl) or myeloid-specific Mboat7 knockout mice (Mboat7-MSKO) were subjected to the NIAAA model of ethanol-induced liver injury. (A) Western blots from bone marrow derived macrophage (BMDM) or peritoneal macrophage (PM) collected from Mboat7^fl/fl or Mboat7-MSKO mice. (B) Initial and final body weight measured in Mboat7^fl/fl or Mboat7-MSKO mice. (C) Liver weight, (D, E) plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), (F) hepatic triglycerides, and (G) hepatic expression of inflammatory gene measured by qPCR. Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3.. Ethanol alters the liver lipidome in a MBOAT7-dependent manner.

Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure. Liver lysophosphatidylinositol (LPI) (A) and phosphatidylinositol (PI) species, including the MBOAT7 product PI 38:4 (B) and others (C), were quantified via liquid chromatography–tandem mass spectrometry (LC–MS/MS) in n = 5–7. (D) Principal component analysis for untargeted lipidomics analysis. The first and second principal components are plotted on the x- and y-axis, respectively, and sample treatment group is indicated by color. (E) Heatmap showing global lipidomic alterations in mouse liver. Total levels of endosomal/lysosomal lipids were measured by targeted and untargeted lipidomic approach using LC–MS/MS. (F) Total bis(monoacylglycero)phosphate (BMP) levels. (G) Total phosphatidylglycerol (PG) and (H) total cardiolipin (CL) from the liver of Mboat7^fl/fl or Mboat7-HSKO mice. Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 1.. Alterations in total hepatic lipid levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure, and untargeted lipidomics was performed to broadly examine major lipid classes in the liver. (A) Phosphatidylcholine (PC), (B) phosphatidylserine (PS), (C) phosphatidylethanolamine (PE), (D) phosphatidic acid (PA), (E) sphingomyelins (SM), (F) ceramides (Cer), (G) ether phosphatidylcholine, (H) ether phosphatidylethanolamine, (I) free fatty acids (FFA), (J) hexosylceramide (HexCer), (K) diacylglycerol (DAG), and (L) cholesteryl ester (CE) were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 2.. Hepatic phosphatidylcholine (PC) levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure, and untargeted lipidomics were performed to broadly examine major lipid classes in the liver. The molecular species of PC were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 3.. Hepatic bis(monoacylglycerol)phosphate (BMP) levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure, and targeted lipidomics was performed in the liver. The molecular species of BMP were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 4.. Hepatic phosphatidylglycerol (PG) levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure, and targeted lipidomics was performed in the liver. The molecular species of PG were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 5.. Hepatic cardiolipin (CL) levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure, and untargeted lipidomics was performed in the liver. The molecular species of CL were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 6.. Hepatic phosphatidylserine (PS) levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure, and untargeted lipidomics was performed in the liver. The molecular species of PS were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 7.. Hepatic phosphatidylethanolamine (PE) levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure, and untargeted lipidomics was performed in the liver. The molecular species of PE were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 8.. Hepatic phosphatidic acid (PA) levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure, and untargeted lipidomics was performed in the liver. The molecular species of PA were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 9.. Hepatic sphingomyelin (SM) levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure, and untargeted lipidomics was performed in the liver. The molecular species of SM were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 10.. Hepatic ceramide (Cer) levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat-HSKO mice were subjected to the NIAAA model of ethanol exposure, and untargeted lipidomics was performed in the liver. The molecular species of ceramides were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 3—figure supplement 11.. Hepatic ether-linked lipids levels in Mboat7-HSKO mice.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure, and untargeted lipidomics was performed in the liver. The molecular species of ether-linked lipids were quantified via liquid chromatography–mass spectrometry (n = 6/group). Data represent the mean ± standard error of the mean (SEM) and groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 4.. Mboat7-HSKO mice have dysregulated lysosome function in response to ethanol.

Age-matched female Mboat7^fl/fl or Mboat7-HSKO mice were subjected to the NIAAA model of ethanol exposure. (A) Total liver lysates were subjected to western blot analysis of major autophagy marker genes LC3A/B (P62), mammalian target of rapamycin (mTOR) and lysosome biogenesis genes (TFEB, LAMP-1, LAMP-2, and ATP6V1A). (B) Nuclear fractions from mouse livers of Mboat7^fl/fl and Mboat7-HSKO were subjected to western blot analysis of TFEB. (C) Lysosome protein degradation activity in wild-type and MBOAT7∆-Huh7 hepatoma cells treated with or without 100 mM ethanol for 48 hr was assessed by incubating cells with 10 µg/ml of lysosome indicator for 2 hr and examined by flow cytometry. n = 5 from two experiments by normalizing to wild-type group in each experiment; mean ± standard deviation (SD) (D) Expression levels of the genes encoding functions in lysosomal hydrolase and accessory, lysosomal m involved in lysosomal biogenesis in the liver of Mboat7^fl/fl and Mboat7-HSKO mice upon ethanol feeding. mRNA expression levels were determined by qPCR (n = 6/group). Groups not sharing a common letter superscript differ significantly (p ≤ 0.05).

Figure 4—figure supplement 1.. Genetic deletion of MBOAT7 in human Huh7 cells is associated with diminished lysosome biogenesis and ethanol-induced autophagy dysregulation.

WT and MBOAT7Δ Huh7 hepatoma cells were treated with and without ethanol treatment (100 mM) for 24 (A) and 48 hr (B). Total lysate were subjected to western blot analysis of LC3A/B, P62, Total mTOR, Total TFEB, LAMP-1, LAMP-2, and ATP6V1A.

Figure 4—figure supplement 2.. Working model.

MBOAT7 loss of function in either mouse or human hepatocytes is associated with decreased transcription factor EB (TFEB)-driven lysosomal biogenesis and defective autophagy secondary to lysosomal dysfunction.