CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation

Darren M. Gordon; Samuel O. Adeosun; Somtochukwu I. Ngwudike; Christopher D. Anderson; John E. Hall; Terry D. Hinds; David E. Stec

doi:10.1016/j.abb.2019.108072

CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation

Darren M. Gordon, Samuel O. Adeosun, Somtochukwu I. Ngwudike, Christopher D. Anderson, John E. Hall, Terry D. Hinds, David E. Stec

Research output: Contribution to journal › Article › peer-review

27 Scopus citations

Abstract

Obesity is the predominant cause of non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance and diabetes. NAFLD includes a spectrum of pathologies that starts with simple steatosis, which can progress to non-alcoholic steatohepatitis (NASH) with the commission of other factors such as the enhancement of reactive oxygen species (ROS). Biliverdin reductase A (BVRA) reduces biliverdin to the antioxidant bilirubin, which may serve to prevent NAFLD, and possibly the progression to NASH. To further understand the role of BVRA in hepatic function, we used CRISPR-Cas9 technology to target the Blvra gene in the murine hepa1c1c7 hepatocyte cell line (BVRA KO). BVRA activity and protein levels were significantly lower in BVRA KO vs. wild-type (WT) hepatocytes. Lipid accumulation under basal and serum-starved conditions was significantly (p < 0.05) higher in BVRA KO vs. WT cells. The loss of BVRA resulted in the reduction of mitochondria number, decreased expression of markers of mitochondrial biogenesis, uncoupling, oxidation, and fusion, which paralleled reduced mitochondrial oxygen consumption. BVRA KO cells exhibited increased levels of ROS generation and decreased levels of superoxide dismutase mRNA expression. In conclusion, our data demonstrate a critical role for BVRA in protecting against lipid accumulation and oxidative stress in hepatocytes, which may serve as a future therapeutic target for NAFLD and its progression to NASH.

Original language	English
Article number	108072
Journal	Archives of Biochemistry and Biophysics
Volume	672
DOIs	https://doi.org/10.1016/j.abb.2019.108072
State	Published - Sep 15 2019

Bibliographical note

Funding Information:
Flow cytometry analysis and FACS sorting was done through the UMMC Flow Cytometry Core which is supported in part by CEPR COBRE ( P20GM121334 ). This work was supported by grants from the National Heart, Lung and Blood Institute K01HL-125445 (T.D.H.), PO1HL-051971 (J.E.H.), and 1T32HL105324 (S.O.A) and the National Institute of General Medical Sciences P20GM104357-02 (J E H., D.E.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funding Information:
Flow cytometry analysis and FACS sorting was done through the UMMC Flow Cytometry Core which is supported in part by CEPR COBRE (P20GM121334). This work was supported by grants from the National Heart, Lung and Blood Institute K01HL-125445 (T.D.H.), PO1HL-051971 (J.E.H.), and 1T32HL105324 (S.O.A) and the National Institute of General Medical Sciences P20GM104357-02 (J?E?H. D.E.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2019 Elsevier Inc.

Keywords

Bilirubin
Hepatocytes
Lipid peroxidation
Mitochondria
Obesity
Reactive oxygen species
Steatosis

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1016/j.abb.2019.108072

Cite this

@article{f3caa054556c44599bf489557da5d7c6,

title = "CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation",

abstract = "Obesity is the predominant cause of non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance and diabetes. NAFLD includes a spectrum of pathologies that starts with simple steatosis, which can progress to non-alcoholic steatohepatitis (NASH) with the commission of other factors such as the enhancement of reactive oxygen species (ROS). Biliverdin reductase A (BVRA) reduces biliverdin to the antioxidant bilirubin, which may serve to prevent NAFLD, and possibly the progression to NASH. To further understand the role of BVRA in hepatic function, we used CRISPR-Cas9 technology to target the Blvra gene in the murine hepa1c1c7 hepatocyte cell line (BVRA KO). BVRA activity and protein levels were significantly lower in BVRA KO vs. wild-type (WT) hepatocytes. Lipid accumulation under basal and serum-starved conditions was significantly (p < 0.05) higher in BVRA KO vs. WT cells. The loss of BVRA resulted in the reduction of mitochondria number, decreased expression of markers of mitochondrial biogenesis, uncoupling, oxidation, and fusion, which paralleled reduced mitochondrial oxygen consumption. BVRA KO cells exhibited increased levels of ROS generation and decreased levels of superoxide dismutase mRNA expression. In conclusion, our data demonstrate a critical role for BVRA in protecting against lipid accumulation and oxidative stress in hepatocytes, which may serve as a future therapeutic target for NAFLD and its progression to NASH.",

keywords = "Bilirubin, Hepatocytes, Lipid peroxidation, Mitochondria, Obesity, Reactive oxygen species, Steatosis",

author = "Gordon, {Darren M.} and Adeosun, {Samuel O.} and Ngwudike, {Somtochukwu I.} and Anderson, {Christopher D.} and Hall, {John E.} and Hinds, {Terry D.} and Stec, {David E.}",

note = "Funding Information: Flow cytometry analysis and FACS sorting was done through the UMMC Flow Cytometry Core which is supported in part by CEPR COBRE ( P20GM121334 ). This work was supported by grants from the National Heart, Lung and Blood Institute K01HL-125445 (T.D.H.), PO1HL-051971 (J.E.H.), and 1T32HL105324 (S.O.A) and the National Institute of General Medical Sciences P20GM104357-02 (J E H., D.E.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding Information: Flow cytometry analysis and FACS sorting was done through the UMMC Flow Cytometry Core which is supported in part by CEPR COBRE (P20GM121334). This work was supported by grants from the National Heart, Lung and Blood Institute K01HL-125445 (T.D.H.), PO1HL-051971 (J.E.H.), and 1T32HL105324 (S.O.A) and the National Institute of General Medical Sciences P20GM104357-02 (J?E?H. D.E.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Publisher Copyright: {\textcopyright} 2019 Elsevier Inc.",

year = "2019",

month = sep,

day = "15",

doi = "10.1016/j.abb.2019.108072",

language = "English",

volume = "672",

}

TY - JOUR

T1 - CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation

AU - Gordon, Darren M.

AU - Adeosun, Samuel O.

AU - Ngwudike, Somtochukwu I.

AU - Anderson, Christopher D.

AU - Hall, John E.

AU - Hinds, Terry D.

AU - Stec, David E.

N1 - Funding Information: Flow cytometry analysis and FACS sorting was done through the UMMC Flow Cytometry Core which is supported in part by CEPR COBRE ( P20GM121334 ). This work was supported by grants from the National Heart, Lung and Blood Institute K01HL-125445 (T.D.H.), PO1HL-051971 (J.E.H.), and 1T32HL105324 (S.O.A) and the National Institute of General Medical Sciences P20GM104357-02 (J E H., D.E.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding Information: Flow cytometry analysis and FACS sorting was done through the UMMC Flow Cytometry Core which is supported in part by CEPR COBRE (P20GM121334). This work was supported by grants from the National Heart, Lung and Blood Institute K01HL-125445 (T.D.H.), PO1HL-051971 (J.E.H.), and 1T32HL105324 (S.O.A) and the National Institute of General Medical Sciences P20GM104357-02 (J?E?H. D.E.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Publisher Copyright: © 2019 Elsevier Inc.

PY - 2019/9/15

Y1 - 2019/9/15

N2 - Obesity is the predominant cause of non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance and diabetes. NAFLD includes a spectrum of pathologies that starts with simple steatosis, which can progress to non-alcoholic steatohepatitis (NASH) with the commission of other factors such as the enhancement of reactive oxygen species (ROS). Biliverdin reductase A (BVRA) reduces biliverdin to the antioxidant bilirubin, which may serve to prevent NAFLD, and possibly the progression to NASH. To further understand the role of BVRA in hepatic function, we used CRISPR-Cas9 technology to target the Blvra gene in the murine hepa1c1c7 hepatocyte cell line (BVRA KO). BVRA activity and protein levels were significantly lower in BVRA KO vs. wild-type (WT) hepatocytes. Lipid accumulation under basal and serum-starved conditions was significantly (p < 0.05) higher in BVRA KO vs. WT cells. The loss of BVRA resulted in the reduction of mitochondria number, decreased expression of markers of mitochondrial biogenesis, uncoupling, oxidation, and fusion, which paralleled reduced mitochondrial oxygen consumption. BVRA KO cells exhibited increased levels of ROS generation and decreased levels of superoxide dismutase mRNA expression. In conclusion, our data demonstrate a critical role for BVRA in protecting against lipid accumulation and oxidative stress in hepatocytes, which may serve as a future therapeutic target for NAFLD and its progression to NASH.

AB - Obesity is the predominant cause of non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance and diabetes. NAFLD includes a spectrum of pathologies that starts with simple steatosis, which can progress to non-alcoholic steatohepatitis (NASH) with the commission of other factors such as the enhancement of reactive oxygen species (ROS). Biliverdin reductase A (BVRA) reduces biliverdin to the antioxidant bilirubin, which may serve to prevent NAFLD, and possibly the progression to NASH. To further understand the role of BVRA in hepatic function, we used CRISPR-Cas9 technology to target the Blvra gene in the murine hepa1c1c7 hepatocyte cell line (BVRA KO). BVRA activity and protein levels were significantly lower in BVRA KO vs. wild-type (WT) hepatocytes. Lipid accumulation under basal and serum-starved conditions was significantly (p < 0.05) higher in BVRA KO vs. WT cells. The loss of BVRA resulted in the reduction of mitochondria number, decreased expression of markers of mitochondrial biogenesis, uncoupling, oxidation, and fusion, which paralleled reduced mitochondrial oxygen consumption. BVRA KO cells exhibited increased levels of ROS generation and decreased levels of superoxide dismutase mRNA expression. In conclusion, our data demonstrate a critical role for BVRA in protecting against lipid accumulation and oxidative stress in hepatocytes, which may serve as a future therapeutic target for NAFLD and its progression to NASH.

KW - Bilirubin

KW - Hepatocytes

KW - Lipid peroxidation

KW - Mitochondria

KW - Obesity

KW - Reactive oxygen species

KW - Steatosis

UR - http://www.scopus.com/inward/record.url?scp=85071001562&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85071001562&partnerID=8YFLogxK

U2 - 10.1016/j.abb.2019.108072

DO - 10.1016/j.abb.2019.108072

M3 - Article

C2 - 31422074

AN - SCOPUS:85071001562

SN - 0003-9861

VL - 672

JO - Archives of Biochemistry and Biophysics

JF - Archives of Biochemistry and Biophysics

M1 - 108072

ER -

CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

Other files and links

Fingerprint

Cite this