1. Human Biology and Medicine
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Obesity-linked suppression of membrane-bound O-Acyltransferase 7 (MBOAT7) drives non-alcoholic fatty liver disease

  1. Robert N Helsley
  2. Varadharajan Venkateshwari
  3. Amanda L Brown
  4. Anthony D Gromovsky
  5. Rebecca C Schugar
  6. Iyappan Ramachandiran
  7. Kevin Fung
  8. Mohammad Nasser Kabbany
  9. Rakhee Banerjee
  10. Chase K Neumann
  11. Chelsea Finney
  12. Preeti Pathak
  13. Orabi Danny
  14. Lucas J Osborn
  15. William Massey
  16. Renliang Zhang
  17. Anagha Kadam
  18. Brian E Sansbury
  19. Calvin Pan
  20. Jessica Sacks
  21. Richard G Lee
  22. Rosanne M Crooke
  23. Mark J Graham
  24. Madeleine E Lemieux
  25. Valentin Gogonea
  26. John P Kirwan
  27. Daniela S Allende
  28. Mete Civelek
  29. Paul L Fox
  30. Lawrence L Rudel
  31. Aldons J Lusis
  32. Matthew Spite
  33. Jonathan Mark Brown  Is a corresponding author
  1. Cleveland Clinic, United States
  2. Brigham's and Womens Hospital, Harvard Medical School, United States
  3. University of California, Los Angeles, United States
  4. Ionis Pharmaceuticals Inc, United States
  5. Bioinfo, Canada
  6. Cleveland State University, United States
  7. University of Virginia, United States
  8. Wake Forest University School of Medicine, United States
  9. Brigham's and Women's Hospital, Harvard Medical School, United States
Research Article
  • Cited 4
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Cite this article as: eLife 2019;8:e49882 doi: 10.7554/eLife.49882

Abstract

Recent studies have identified a genetic variant rs641738 near two genes encoding membrane bound O-acyltransferase domain-containing 7 (MBOAT7) and transmembrane channel-like 4 (TMC4) that associate with increased risk of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), alcohol-related cirrhosis, and liver fibrosis in those infected with viral hepatitis1-7. Based on hepatic expression quantitative trait loci analysis it has been suggested that MBOAT7 loss of function promotes liver disease progression1-7, but this has never been formally tested. Here we show that Mboat7 loss, but not Tmc4, in mice is sufficient to promote the progression of NAFLD in the setting of high fat diet. Mboat7 loss of function is associated with accumulation of its substrate lysophosphatidylinositol (LPI) lipids, and direct administration of LPI promotes hepatic inflammatory and fibrotic transcriptional changes in an Mboat7-dependent manner. These studies reveal a novel role for MBOAT7-driven acylation of LPI lipids in suppressing the progression of NAFLD.

Article and author information

Author details

  1. Robert N Helsley

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5000-3187
  2. Varadharajan Venkateshwari

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  3. Amanda L Brown

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  4. Anthony D Gromovsky

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  5. Rebecca C Schugar

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  6. Iyappan Ramachandiran

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  7. Kevin Fung

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  8. Mohammad Nasser Kabbany

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  9. Rakhee Banerjee

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  10. Chase K Neumann

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  11. Chelsea Finney

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  12. Preeti Pathak

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  13. Orabi Danny

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  14. Lucas J Osborn

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  15. William Massey

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  16. Renliang Zhang

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  17. Anagha Kadam

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  18. Brian E Sansbury

    Center for Experimental Therapeutics and Reperfusion Injury, Brigham's and Womens Hospital, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  19. Calvin Pan

    Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.
  20. Jessica Sacks

    Department of Pathobiology, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  21. Richard G Lee

    Cardiovascular Group, Ionis Pharmaceuticals Inc, Carlsbad, United States
    Competing interests
    Richard G Lee, employee at Ionis Pharmaceuticals, Inc.
  22. Rosanne M Crooke

    Cardiovascular Group, Ionis Pharmaceuticals Inc, Carlsbad, United States
    Competing interests
    Rosanne M Crooke, employee at Ionis Pharmaceuticals, Inc.
  23. Mark J Graham

    Cardiovasular Group, Ionis Pharmaceuticals Inc, Carlsbad, United States
    Competing interests
    Mark J Graham, employee at Ionis Pharmaceuticals, Inc.
  24. Madeleine E Lemieux

    Bioinfo, Ontario, Canada
    Competing interests
    No competing interests declared.
  25. Valentin Gogonea

    Department of Chemistry, Cleveland State University, Cleveland, United States
    Competing interests
    No competing interests declared.
  26. John P Kirwan

    Department of Pathobiology, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  27. Daniela S Allende

    Department of Anatomical Pathology, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  28. Mete Civelek

    Department of Biomedical Engineering, University of Virginia, Charlottesville, United States
    Competing interests
    No competing interests declared.
  29. Paul L Fox

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    Competing interests
    No competing interests declared.
  30. Lawrence L Rudel

    Department of Pathology, Section on Lipid Sciences, Wake Forest University School of Medicine, Winston-Salem, United States
    Competing interests
    No competing interests declared.
  31. Aldons J Lusis

    Department of Medicine, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    No competing interests declared.
  32. Matthew Spite

    Center for Experimental Therapeutics and Reperfusion Injury, Brigham's and Women's Hospital, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  33. Jonathan Mark Brown

    Department of Cardiovascular and Metabolic Sciences, Cleveland Clinic, Cleveland, United States
    For correspondence
    brownm5@ccf.org
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2708-7487

Funding

National Heart, Lung, and Blood Institute (R01-HL122283)

  • Jonathan Mark Brown

National Heart, Lung, and Blood Institute (R00-HL12172)

  • Mete Civelek

National Heart, Lung, and Blood Institute (R01-HL106173)

  • Matthew Spite

National Heart, Lung, and Blood Institute (F32-HL136044)

  • Brian E Sansbury

National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK120679)

  • Jonathan Mark Brown

National Institute on Alcohol Abuse and Alcoholism (P50-AA-024333)

  • Daniela S Allende
  • Jonathan Mark Brown

National Heart, Lung, and Blood Institute (P01 HL029582)

  • Paul L Fox

National Institute on Alcohol Abuse and Alcoholism (U01-AA021893)

  • Daniela S Allende

National Institute of Diabetes and Digestive and Kidney Diseases (U01-DK061732)

  • Daniela S Allende

National Institute of Diabetes and Digestive and Kidney Diseases (R01-DK103637)

  • Daniela S Allende

National Heart, Lung, and Blood Institute (P01-HL49373)

  • Lawrence L Rudel

National Heart, Lung, and Blood Institute (P01-HL30568)

  • Aldons J Lusis

All coauthors are responsible for the content of this work, and different aspects of this work was funded by the National Institutes of Health (NIH) and the American Heart Association (AHA).

Ethics

Animal experimentation: Rat Studies of Diet-Induced ObesitySprague Dawley Rats were received at 12 weeks of age and were housed in individual cages, kept at a constant temperature and ambient humidity in a 12-hlight/dark cycle. Animals were then randomly assigned to either a standard chow diet or a high-fat diet (D12492, 60% fat, Research Diets, New Brunswick, NJ, USA) ad libitum to establish diet-induced obesity as previously described20. After 6 months of HFD-feeding, livers were excised for standard qPCR analysis of Mboat7 expression.Hybrid Mouse Diversity Panel92 inbred strains of 8-week-old male mice (180 individual mice) were fed a high fat, high sucrose diet (D12266B, Research Diets, New Brunswick, NJ) for 8 weeks before tissue collection44. Gene expression of Mboat7 in white adipose tissue and liver were measured and correlated with obesity related traits using biweight midcorrelation analysis as previously described4.Mouse Studies of Mboat7 Loss of FunctionTo explore the role of Mboat7 in diet-induced obesity, NAFLD progression, and insulin resistance, we utilized an in vivo knockdown approach in 8-week old adult mice. Selective knockdown of Mboat7 was accomplished using 2'-O-ethyl (cET) modified antisense oligonucleotides (ASO). All ASOs used in this work were synthesized, screened, and purified as described previously45 by Ionis Pharmaceuticals, Inc. (Carlsbad, CA). For Mboat7 knockdown studies, adult (8 week old) male C57BL/6 mice were purchased from Jackson Labs (Bar Harbor, ME USA), and maintained on either a standard rodent chow diet or a high fat diet (HFD, D12492 from Research Diets Inc.) and injected intraperitoneally biweekly with 12.5 mg/kg of either non-targeting control ASO or one of two independent ASOs directed against murine Mboat7 for a period of 20 weeks. Similar results were seen with two independent ASOs targeting different regions of the Mboat7 mRNA, hence key data using one Mboat7 ASO are shown. All rodents were maintained in an Association for the Assessment and Accreditation of Laboratory Animal Care, International-approved animal facility, and all experimental protocols were approved by the Institutional Animal Care and use Committee of the Cleveland Clinic (IACUC protocols # 2015-1519 and # 2018-2053).

Human subjects: Human MBOAT7 Expression Levels in Lean and Obese SubjectsThe majority of subjects recruited to examine MBOAT7 expression levels were morbidly obese bariatric surgery patients, but we were able to obtain liver biopsies from 10 subjects with a BMI under 30 as normal weight controls. For recruitment, adult patients undergoing gastric bypass surgery at Wake Forest School of Medicine were consented via written consent and enrolled by a member of the study staff following institutionally approved IRB protocols as previously described41. Exclusion criteria included: positive hepatitis C antibody, positive hepatitis B surface antigen, history of liver disease other than NAFLD, Childs A, B, or C cirrhosis, past or present diagnosis/treatment of malignancy other than non-melanocytic skin cancer, INR greater than 1.8 at baseline or need for chronic anticoagulation with warfarin or heparin products, use of immunomodulation for or history of inflammatory diseases including but not limited to malignancy, rheumatoid arthritis, psoriasis, lupus, sarcoidosis and inflammatory bowel disease, and greater or equal to 7 alcohol drinks per week or 3 alcoholic drinks in a given day each week. In addition to bariatric surgery patients, a small number of non-obese subjects (body mass index <30.0) consented to liver biopsy during elective gall bladder removal surgery (n=10). Each subject was assigned a unique identifier which was used throughout the study and did not include any identifiable information about the patient such as name, address, telephone number, social security number, medical record number or any of the identifiers outlined in the HIPAA Privacy Rule regulations. Only the principal investigator had access to the code linking the unique identifier to the study subject. Basic clinical information was obtained via self-reporting and a 15 ml baseline blood sample was obtained at the time of enrollment. A subset of this cohort has been previously described20,41. At the time of surgery, the surgeon collected a roughly 1-gram sample from the lateral left lobe. Wedge biopsies were rinsed with saline and immediately snap frozen in liquid nitrogen in the operating room before subsequent storage at -80{degree sign}C. For data shown in Figure 7 showing levels of MBOAT7 substrate and product lipids, de-identified patient samples from the Cleveland Clinic hepatology clinic (IRB # 10-947) were analyzed. These patients had biopsy proven Ishak fibrosis scores42 of 0 (normal) or 4 (advanced fibrosis). For analysis of hepatic MBOAT7 expression, RNA isolated from liver biopsies were used for quantitative real time PCR (qPCR) as described below.

Reviewing Editor

  1. Peter Tontonoz, University of California, Los Angeles, United States

Publication history

  1. Received: July 3, 2019
  2. Accepted: October 11, 2019
  3. Accepted Manuscript published: October 17, 2019 (version 1)
  4. Version of Record published: November 12, 2019 (version 2)

Copyright

© 2019, Helsley et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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