Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin

  1. Rima S Chakrabarti
  2. Sally A Ingham
  3. Julia Kozlitina
  4. Austin Gay
  5. Jonathan C Cohen
  6. Arun Radhakrishnan
  7. Helen H Hobbs  Is a corresponding author
  1. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
  2. University of Texas Southwestern Medical Center, United States

Abstract

Cholesterol partitions into accessible and sequestered pools in cell membranes. Here we describe a new assay using fluorescently-tagged anthrolysin O, a cholesterol-binding bacterial toxin, to measure accessible cholesterol in human red blood cells (RBCs). Accessible cholesterol levels were stable within individuals but varied >10-fold among individuals. Significant variation was observed among ethnic groups (Blacks>Hispanics>Whites). Variation in accessibility of RBC cholesterol was unrelated to the cholesterol content of RBCs or plasma, but was associated with the phospholipid composition of the RBC membranes and with plasma triglyceride levels. Pronase treatment of RBCs only modestly altered cholesterol accessibility. Individuals on hemodialysis, who have an unexplained increase in atherosclerotic risk, had significantly higher RBC cholesterol accessibility. Our data indicate that RBC accessible cholesterol is a stable phenotype with significant inter-individual variability. Factors both intrinsic and extrinsic to the RBC contribute to variation in its accessible. This assay provides a new tool to assess cholesterol homeostasis among tissues in humans.

Article and author information

Author details

  1. Rima S Chakrabarti

    Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  2. Sally A Ingham

    Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  3. Julia Kozlitina

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  4. Austin Gay

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  5. Jonathan C Cohen

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
  6. Arun Radhakrishnan

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7266-7336
  7. Helen H Hobbs

    Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, United States
    For correspondence
    helen.hobbs@utsouthwestern.edu
    Competing interests
    Helen H Hobbs, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8700-9897

Funding

Howard Hughes Medical Institute

  • Rima S Chakrabarti
  • Sally A Ingham
  • Helen H Hobbs

National Institutes of Health (PO1 HL20948)

  • Austin Gay
  • Jonathan C Cohen
  • Arun Radhakrishnan
  • Helen H Hobbs

Welch Foundation (I-1793)

  • Arun Radhakrishnan

American Heart Association (12SDG12040267)

  • Arun Radhakrishnan

National Institutes of Health (5T32-GM008203)

  • Austin Gay

National Institutes of Health (UL1TR001105)

  • Julia Kozlitina
  • Helen H Hobbs

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Human subjects: All study protocols were approved by the Institutional Review Board (IRB) of the University of Texas Southwestern Medical Center, and all subjects provided written informed consent. Each participant completed a detailed staff-administered survey, including questions about socioeconomic status, medical history and medication use. Ancestry was self-reported.

Reviewing Editor

  1. Stephen G Young, University of California, Los Angeles, United States

Publication history

  1. Received: November 16, 2016
  2. Accepted: January 28, 2017
  3. Accepted Manuscript published: February 7, 2017 (version 1)
  4. Accepted Manuscript updated: February 8, 2017 (version 2)
  5. Version of Record published: February 23, 2017 (version 3)

Copyright

© 2017, Chakrabarti 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.

Metrics

  • 2,479
    Page views
  • 475
    Downloads
  • 29
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Rima S Chakrabarti
  2. Sally A Ingham
  3. Julia Kozlitina
  4. Austin Gay
  5. Jonathan C Cohen
  6. Arun Radhakrishnan
  7. Helen H Hobbs
(2017)
Variability of cholesterol accessibility in human red blood cells measured using a bacterial cholesterol-binding toxin
eLife 6:e23355.
https://doi.org/10.7554/eLife.23355

Further reading

    1. Biochemistry and Chemical Biology
    Meiling Wu, Anda Zhao ... Dongyun Shi
    Research Article

    Antioxidant intervention is considered to inhibit reactive oxygen species (ROS) and alleviates hyperglycemia. Paradoxically, moderate exercise can produce ROS to improve diabetes. The exact redox mechanism of these two different approaches remains largely unclear. Here, by comparing exercise and antioxidants intervention on type 2 diabetic rats, we found moderate exercise upregulated compensatory antioxidant capability and reached a higher level of redox balance in the liver. In contrast, antioxidant intervention achieved a low-level redox balance by inhibiting oxidative stress. Both of these two interventions could promote glucose catabolism and inhibit gluconeogenesis through activation of hepatic AMPK signaling, therefore ameliorating diabetes. During exercise, different levels of ROS generated by exercise have differential regulations on the activity and expression of hepatic AMPK. Moderate exercise-derived ROS promoted hepatic AMPK glutathionylation activation. However, excessive exercise increased oxidative damage and inhibited the activity and expression of AMPK. Overall, our results illustrate that both exercise and antioxidant intervention improve blood glucose in diabetes by promoting redox balance, despite different levels of redox balance. These results indicate that the AMPK signaling activation, combined with oxidative damage markers, could act as a sensitive biomarker, reflecting the threshold of redox balance defining effective treatment in diabetes. These findings provide theoretical evidence for the precise treatment of diabetes by antioxidants and exercise.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Edmundo G Vides, Ayan Adhikari ... Suzanne R Pfeffer
    Research Advance

    Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed 'Site #1', can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed 'Site #2', that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.