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Skeletal muscle miR-34a/SIRT1:AMPK axis is activated in experimental and human non-alcoholic steatohepatitis

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Abstract

Non-alcoholic fatty liver disease (NAFLD) pathogenesis associates with intramyocellular lipid deposition and mitochondrial dysfunction. microRNAs (miRs), including pro-apoptotic miR-34a, are modulated during disease progression in liver tissue and plasma. We aimed to investigate the functional role of the miR-34a/SIRT1:AMP-activated protein kinase (AMPK) pathway in modulating local mitochondrial dysfunction in the skeletal muscle of human and experimental non-alcoholic steatohepatitis. Muscle biopsies were obtained from morbid obese NAFLD patients undergoing bariatric surgery. C57BL/6N mice were fed different NAFLD-inducing diets and C2C12 muscle cells incubated with palmitic acid (PA) in the presence or absence of an AMPK activator, or upon miR-34a functional modulation. Several muscle miRNAs, including miR-34a, were found increased with human NAFLD progression. Activation of the miR-34a/SIRT1:AMPK pathway, concomitant with impairment in insulin signalling mediators and deregulation of mitochondrial-shaping proteins, was evident in C2C12 cells incubated with PA, as well as in the skeletal muscle of all three diet-induced NAFLD mice models. Functional studies established the association between miR-34a- and PA-induced muscle cell deregulation. Of note, activation of AMPK almost completely prevented miR-34a- and PA-induced cellular stress. In addition, the miR-34a/SIRT1:AMPK pathway and mitochondrial dynamics dysfunction were also found amplified in muscle of human NAFLD. Finally, muscle miR-34a expression and mitofusin 2 (Mfn2) protein levels correlated with hallmarks of NAFLD and disease progression. Our results indicate that activation of the miR-34a/SIRT1:AMPK pathway leads to mitochondrial dynamics dysfunction in skeletal muscle of human and experimental NAFLD, representing an appealing prospective target in metabolic syndrome.

Key messages

  • Skeletal muscle microRNAs are modulated during NAFLD progression.

  • Palmitic acid-induced muscle cell dysfunction occurs, at least in part, through activation of the miR-34a/SIRT1:AMPK pathway.

  • miR-34a/SIRT1:AMPK activation associates with mitochondria dynamics dysfunction in human NAFLD.

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Abbreviations

ALT:

Alanine aminotransferase

AMPK:

AMP-activated protein kinase

BSA:

Bovine serum albumin

CDAA:

Choline-deficient amino acid-defined

Drp1:

Dynamin-related protein 1

FF:

Fast food

FFA:

Free fatty acid

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

HFCD:

High-fat choline-deficient

INSR:

Insulin receptor

IR:

Insulin resistance

IRS1:

Insulin receptor substrate 1

LKB1:

Liver Kinase B1

miRNAs/miRs:

MicroRNAs

Mfn2:

Mitofusin 2

NAFLD:

Non-alcoholic fatty liver disease

NASH:

Non-alcoholic steatohepatitis

PA:

Palmitic acid

PGC-1α:

Peroxisome proliferator-activated receptor-γ coactivator-1α

SIRT1:

Sirtuin 1

UDCA:

Ursodeoxycholic acid

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Acknowledgements

We thank Tânia Carvalho, Histology and Comparative Pathology Laboratory, Instituto de Medicina Molecular, for the histological and pathology analyses and Drª Elisa Alves, Clinical Analysis Core Laboratory, Faculty of Pharmacy, University of Lisbon, for serum analyses. The study was supported in part by Gilead Sciences Research Scholars Program in International Liver Disease and Fundação para a Ciência e a Tecnologia (FCT) through grants PTDC/BIM-MEC/0895/2014 and UID/DTP/04138/2013, and fellowships SFRH/BD/91119/2012 (MBA), SFRH/BD/88212/2012 (PMR), and SFRH/BD/104160/2014 (ALS). Work in MGC lab is supported by a centre grant to BioISI, reference UID/MULTI/04046/2013, from FCT/MCTES/PIDDAC, Portugal.

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Author notes

  1. Cecília M. P. Rodrigues and Rui E. Castro share senior authorship

Authors and Affiliations

  1. Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, Lisbon, 1649-003, Portugal

    André L. Simão, Marta B. Afonso, Pedro M. Rodrigues, Cecília M. P. Rodrigues & Rui E. Castro

  2. BioISI – Biosystems and Integrative Sciences Institute, Faculty of Sciences, Universidade de Lisboa, Lisbon, Portugal

    Margarida Gama-Carvalho

  3. Gastroenterology, Hospital Santa Maria, Lisbon, Portugal

    Mariana V. Machado & Helena Cortez-Pinto

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  1. André L. Simão
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Contributions

RC and CR were responsible for the study concept and design, obtained funding and interpreted the data. AS was involved in performing the experiments, analysis and data interpretation and writing. MA and PR helped in performing the experiments. MGC performed the miRNA array analysis. MM and HCP were responsible for the collection and selection of human specimens. All authors critically revised the manuscript and approved its final version.

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Correspondence to Rui E. Castro.

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Simão, A.L., Afonso, M.B., Rodrigues, P.M. et al. Skeletal muscle miR-34a/SIRT1:AMPK axis is activated in experimental and human non-alcoholic steatohepatitis. J Mol Med 97, 1113–1126 (2019). https://doi.org/10.1007/s00109-019-01796-8

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