Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE (-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis

Narjes Nasiri-Ansari; Chrysa Nikolopoulou; Katerina Papoutsi; Ioannis Kyrou; Christos S. Mantzoros; Georgios Kyriakopoulos; Antonios Chatzigeorgiou; Vassiliki Kalotychou; Manpal S. Randeva; Kamaljit Chatha; Konstantinos Kontzoglou; Gregory Kaltsas; Athanasios G. Papavassiliou; Harpal S. Randeva; Eva Kassi

doi:10.3390/ijms22020818

Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE (-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis

Narjes Nasiri-Ansari, Chrysa Nikolopoulou, Katerina Papoutsi, Ioannis Kyrou, Christos S. Mantzoros, Georgios Kyriakopoulos, Antonios Chatzigeorgiou, Vassiliki Kalotychou, Manpal S. Randeva, Kamaljit Chatha, Konstantinos Kontzoglou, Gregory Kaltsas, Athanasios G. Papavassiliou, Harpal S. Randeva^*, Eva Kassi^*

^*Corresponding author for this work

Aston Medical School

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

Abstract

Aims/hypothesis: SGLT-2 inhibitors (SGLT-2i) have been studied as potential treatments against NAFLD, showing varying beneficial effects. The molecular mechanisms mediating these effects have not been fully clarified. Herein, we investigated the impact of empagliflozin on NAFLD, focusing particularly on ER stress, autophagy and apoptosis. Methods: Five-week old ApoE(-/-) mice were switched from normal to a high-fat diet (HFD). After five weeks, mice were randomly allocated into a control group (HFD + vehicle) and Empa group (HFD + empagliflozin 10 mg/kg/day) for five weeks. At the end of treatment, histomorphometric analysis was performed in liver, mRNA levels of Fasn, Screbp-1, Scd-1, Ppar-γ, Pck-1, Mcp-1, Tnf-α, Il-6, F4/80, Atf4, Elf2α, Chop, Grp78, Grp94, Χbp1, Ire1α, Atf6, mTor, Lc3b, Beclin-1, P62, Bcl-2 and Bax were measured by qRT-PCR, and protein levels of p-EIF2α, EIF2a, CHOP, LC3II, P62, BECLIN-1 and cleaved CASPASE-8 were assessed by immunoblotting. Results: Empagliflozin-treated mice exhibited reduced fasting glucose, total cholesterol and triglyceride serum levels, as well as decreased NAFLD activity score, decreased expression of lipogenic enzymes (Fasn, Screbp-1c and Pck-1) and inflammatory molecules (Mcp-1 and F4/80), compared to the Control group. Empagliflozin significantly decreased the expression of ER stress molecules Grp78, Ire1α, Xbp1, Elf2α, Atf4, Atf6, Chop, P62(Sqstm1) and Grp94; whilst activating autophagy via increased AMPK phosphorylation, decreased mTOR and increased LC3B expression. Finally, empagliflozin increased the Bcl2/Bax ratio and inhibited CASPASE-8 cleavage, reducing liver cell apoptosis. Immunoblotting analysis confirmed the qPCR results. Conclusion: These novel findings indicate that empagliflozin treatment for five weeks attenuates NAFLD progression in ApoE(-/-) mice by promoting autophagy, reducing ER stress and inhibiting hepatic apoptosis.

Original language	English
Article number	818
Number of pages	21
Journal	International Journal of Molecular Sciences
Volume	22
Issue number	2
DOIs	https://doi.org/10.3390/ijms22020818
Publication status	Published - 15 Jan 2021

Bibliographical note

© 2021 by the authors. Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Keywords

Apoptosis
Autophagy
ER stress
Inflammation
NAFLD
SGLT-2 inhibitors

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Nasiri-Ansari, N., Nikolopoulou, C., Papoutsi, K., Kyrou, I., Mantzoros, C. S., Kyriakopoulos, G., Chatzigeorgiou, A., Kalotychou, V., Randeva, M. S., Chatha, K., Kontzoglou, K., Kaltsas, G., Papavassiliou, A. G., Randeva, H. S., & Kassi, E. (2021). Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE (-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis. International Journal of Molecular Sciences, 22(2), Article 818. https://doi.org/10.3390/ijms22020818

@article{90846da0d1d84595bd9dc34070487043,

title = "Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE (-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis",

abstract = "Aims/hypothesis: SGLT-2 inhibitors (SGLT-2i) have been studied as potential treatments against NAFLD, showing varying beneficial effects. The molecular mechanisms mediating these effects have not been fully clarified. Herein, we investigated the impact of empagliflozin on NAFLD, focusing particularly on ER stress, autophagy and apoptosis. Methods: Five-week old ApoE(-/-) mice were switched from normal to a high-fat diet (HFD). After five weeks, mice were randomly allocated into a control group (HFD + vehicle) and Empa group (HFD + empagliflozin 10 mg/kg/day) for five weeks. At the end of treatment, histomorphometric analysis was performed in liver, mRNA levels of Fasn, Screbp-1, Scd-1, Ppar-γ, Pck-1, Mcp-1, Tnf-α, Il-6, F4/80, Atf4, Elf2α, Chop, Grp78, Grp94, Χbp1, Ire1α, Atf6, mTor, Lc3b, Beclin-1, P62, Bcl-2 and Bax were measured by qRT-PCR, and protein levels of p-EIF2α, EIF2a, CHOP, LC3II, P62, BECLIN-1 and cleaved CASPASE-8 were assessed by immunoblotting. Results: Empagliflozin-treated mice exhibited reduced fasting glucose, total cholesterol and triglyceride serum levels, as well as decreased NAFLD activity score, decreased expression of lipogenic enzymes (Fasn, Screbp-1c and Pck-1) and inflammatory molecules (Mcp-1 and F4/80), compared to the Control group. Empagliflozin significantly decreased the expression of ER stress molecules Grp78, Ire1α, Xbp1, Elf2α, Atf4, Atf6, Chop, P62(Sqstm1) and Grp94; whilst activating autophagy via increased AMPK phosphorylation, decreased mTOR and increased LC3B expression. Finally, empagliflozin increased the Bcl2/Bax ratio and inhibited CASPASE-8 cleavage, reducing liver cell apoptosis. Immunoblotting analysis confirmed the qPCR results. Conclusion: These novel findings indicate that empagliflozin treatment for five weeks attenuates NAFLD progression in ApoE(-/-) mice by promoting autophagy, reducing ER stress and inhibiting hepatic apoptosis.",

keywords = "Apoptosis, Autophagy, ER stress, Inflammation, NAFLD, SGLT-2 inhibitors",

author = "Narjes Nasiri-Ansari and Chrysa Nikolopoulou and Katerina Papoutsi and Ioannis Kyrou and Mantzoros, {Christos S.} and Georgios Kyriakopoulos and Antonios Chatzigeorgiou and Vassiliki Kalotychou and Randeva, {Manpal S.} and Kamaljit Chatha and Konstantinos Kontzoglou and Gregory Kaltsas and Papavassiliou, {Athanasios G.} and Randeva, {Harpal S.} and Eva Kassi",

note = "{\textcopyright} 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). ",

year = "2021",

month = jan,

day = "15",

doi = "10.3390/ijms22020818",

language = "English",

volume = "22",

journal = "International Journal of Molecular Sciences",

issn = "1661-6596",

publisher = "MDPI AG",

number = "2",

}

Nasiri-Ansari, N, Nikolopoulou, C, Papoutsi, K, Kyrou, I, Mantzoros, CS, Kyriakopoulos, G, Chatzigeorgiou, A, Kalotychou, V, Randeva, MS, Chatha, K, Kontzoglou, K, Kaltsas, G, Papavassiliou, AG, Randeva, HS & Kassi, E 2021, 'Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE (-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis', International Journal of Molecular Sciences, vol. 22, no. 2, 818. https://doi.org/10.3390/ijms22020818

Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE (-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis. / Nasiri-Ansari, Narjes; Nikolopoulou, Chrysa; Papoutsi, Katerina et al.
In: International Journal of Molecular Sciences, Vol. 22, No. 2, 818, 15.01.2021.

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

TY - JOUR

T1 - Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE (-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis

AU - Nasiri-Ansari, Narjes

AU - Nikolopoulou, Chrysa

AU - Papoutsi, Katerina

AU - Kyrou, Ioannis

AU - Mantzoros, Christos S.

AU - Kyriakopoulos, Georgios

AU - Chatzigeorgiou, Antonios

AU - Kalotychou, Vassiliki

AU - Randeva, Manpal S.

AU - Chatha, Kamaljit

AU - Kontzoglou, Konstantinos

AU - Kaltsas, Gregory

AU - Papavassiliou, Athanasios G.

AU - Randeva, Harpal S.

AU - Kassi, Eva

N1 - © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

PY - 2021/1/15

Y1 - 2021/1/15

N2 - Aims/hypothesis: SGLT-2 inhibitors (SGLT-2i) have been studied as potential treatments against NAFLD, showing varying beneficial effects. The molecular mechanisms mediating these effects have not been fully clarified. Herein, we investigated the impact of empagliflozin on NAFLD, focusing particularly on ER stress, autophagy and apoptosis. Methods: Five-week old ApoE(-/-) mice were switched from normal to a high-fat diet (HFD). After five weeks, mice were randomly allocated into a control group (HFD + vehicle) and Empa group (HFD + empagliflozin 10 mg/kg/day) for five weeks. At the end of treatment, histomorphometric analysis was performed in liver, mRNA levels of Fasn, Screbp-1, Scd-1, Ppar-γ, Pck-1, Mcp-1, Tnf-α, Il-6, F4/80, Atf4, Elf2α, Chop, Grp78, Grp94, Χbp1, Ire1α, Atf6, mTor, Lc3b, Beclin-1, P62, Bcl-2 and Bax were measured by qRT-PCR, and protein levels of p-EIF2α, EIF2a, CHOP, LC3II, P62, BECLIN-1 and cleaved CASPASE-8 were assessed by immunoblotting. Results: Empagliflozin-treated mice exhibited reduced fasting glucose, total cholesterol and triglyceride serum levels, as well as decreased NAFLD activity score, decreased expression of lipogenic enzymes (Fasn, Screbp-1c and Pck-1) and inflammatory molecules (Mcp-1 and F4/80), compared to the Control group. Empagliflozin significantly decreased the expression of ER stress molecules Grp78, Ire1α, Xbp1, Elf2α, Atf4, Atf6, Chop, P62(Sqstm1) and Grp94; whilst activating autophagy via increased AMPK phosphorylation, decreased mTOR and increased LC3B expression. Finally, empagliflozin increased the Bcl2/Bax ratio and inhibited CASPASE-8 cleavage, reducing liver cell apoptosis. Immunoblotting analysis confirmed the qPCR results. Conclusion: These novel findings indicate that empagliflozin treatment for five weeks attenuates NAFLD progression in ApoE(-/-) mice by promoting autophagy, reducing ER stress and inhibiting hepatic apoptosis.

AB - Aims/hypothesis: SGLT-2 inhibitors (SGLT-2i) have been studied as potential treatments against NAFLD, showing varying beneficial effects. The molecular mechanisms mediating these effects have not been fully clarified. Herein, we investigated the impact of empagliflozin on NAFLD, focusing particularly on ER stress, autophagy and apoptosis. Methods: Five-week old ApoE(-/-) mice were switched from normal to a high-fat diet (HFD). After five weeks, mice were randomly allocated into a control group (HFD + vehicle) and Empa group (HFD + empagliflozin 10 mg/kg/day) for five weeks. At the end of treatment, histomorphometric analysis was performed in liver, mRNA levels of Fasn, Screbp-1, Scd-1, Ppar-γ, Pck-1, Mcp-1, Tnf-α, Il-6, F4/80, Atf4, Elf2α, Chop, Grp78, Grp94, Χbp1, Ire1α, Atf6, mTor, Lc3b, Beclin-1, P62, Bcl-2 and Bax were measured by qRT-PCR, and protein levels of p-EIF2α, EIF2a, CHOP, LC3II, P62, BECLIN-1 and cleaved CASPASE-8 were assessed by immunoblotting. Results: Empagliflozin-treated mice exhibited reduced fasting glucose, total cholesterol and triglyceride serum levels, as well as decreased NAFLD activity score, decreased expression of lipogenic enzymes (Fasn, Screbp-1c and Pck-1) and inflammatory molecules (Mcp-1 and F4/80), compared to the Control group. Empagliflozin significantly decreased the expression of ER stress molecules Grp78, Ire1α, Xbp1, Elf2α, Atf4, Atf6, Chop, P62(Sqstm1) and Grp94; whilst activating autophagy via increased AMPK phosphorylation, decreased mTOR and increased LC3B expression. Finally, empagliflozin increased the Bcl2/Bax ratio and inhibited CASPASE-8 cleavage, reducing liver cell apoptosis. Immunoblotting analysis confirmed the qPCR results. Conclusion: These novel findings indicate that empagliflozin treatment for five weeks attenuates NAFLD progression in ApoE(-/-) mice by promoting autophagy, reducing ER stress and inhibiting hepatic apoptosis.

KW - Apoptosis

KW - Autophagy

KW - ER stress

KW - Inflammation

KW - NAFLD

KW - SGLT-2 inhibitors

UR - https://www.mdpi.com/1422-0067/22/2/818

UR - http://www.scopus.com/inward/record.url?scp=85099544365&partnerID=8YFLogxK

U2 - 10.3390/ijms22020818

DO - 10.3390/ijms22020818

M3 - Article

SN - 1661-6596

VL - 22

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

IS - 2

M1 - 818

ER -

Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE (-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis

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