Medicine

Membrane Bound O-Acyltransferase 7 (MBOAT7) Shapes Lysosomal Lipid Homeostasis and Function to Control Alcohol-Associated Liver Injury

Department of Cancer Biology, Lerner Research Institute of the Cleveland Clinic, Cleveland, OH, USA
Center for Microbiome and Human Health, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA
Northern Ohio Alcohol Center (NOAC), Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA
Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA
Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute of the Cleveland Clinic, Cleveland, OH, USA
Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, Cleveland, OH, USA
Department of Pharmacology & Nutritional Sciences, Saha Cardiovascular Research Center, University of Kentucky College of Medicine, Lexington, KY, USA
Department of Family Medicine, Metro Health Medical Center, Case Western Reserve University, Cleveland, OH, USA
Lutheran Hospital, Cleveland Clinic, OH, USA
Department of Anatomical Pathology, Cleveland Clinic, Cleveland, OH, USA

https://doi.org/10.7554/eLife.92243.2

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Figures and data

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 chromatographytandem mass spectrometry (LC-MS/MS). n=10-16; ***p<0.001 and ****p<0.0001 in figure 1. ANOVA with Tukey’s post-hoc test.

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 model of ethanol-induced liver injury. (A) Hepatic Mboat7 expression was measured via 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, (G) Hepatic expression of Inflammatory gene measured by qPCR. n = 5-7; Data represent the mean ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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-axes, 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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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), 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 MBOAT7A-Huh7 hepatoma cells treated with or without 100 mM ethanol for 48 h was assessed by incubating cells with 10 pg/mL of lysosome indicator for 2 h and examined by flow cytometry. (n=5 from two experiments by normalizing to wild type group in each experiment; mean ± S.D. (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).

Demographic and clinical parameters for the entire cohort of healthy controls and heavy drinkers recruited for this study.

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 and E) Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST). (F) Hepatic triglycerides. (G) Hepatic expression of Inflammatory gene measured by qPCR. Data represent the mean ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

Hepatic Ceramide (Cer) 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 ceramides were quantified via liquid chromatography mass spectrometry (n=6/group). Data represent the mean ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

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 ± S.E.M. and groups not sharing a common letter superscript differ significantly (p≤0.05).

Genetic Deletion of MBOAT7 in Human Huh7 Cells is Associated with Diminished Lysosome Biogenesis and Ethanol-Induced Autophagy Dysregulation.
WT and MBOAT7A Huh7 hepatoma cells were treated with and without ethanol treatment (100mM) for 24 (A) and 48h (B). Total lysate were subjected to western blot analysis of LC3A/B, P62, Total mTOR, Total TFEB, LAMP-1, LAMP-2 and ATP6V1A.

Working Model.
MBOAT7 loss of function in either mouse or human hepatocytes is associated with decreased TFEB-driven lysosomal biogenesis and defective autophagy secondary to lysosomal dysfunction.

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