Objective Both non-alcoholic fatty liver disease (NAFLD) and the multitarget complexity of microRNA (miR) suppression have recently raised much interest, but the in vivo impact and context-dependence of hepatic miR-target interactions are incompletely understood. Assessing the relative in vivo contributions of specific targets to miR-mediated phenotypes is pivotal for investigating metabolic processes.
Design We quantified fatty liver parameters and the levels of miR-132 and its targets in novel transgenic mice overexpressing miR-132, in liver tissues from patients with NAFLD, and in diverse mouse models of hepatic steatosis. We tested the causal nature of miR-132 excess in these phenotypes by injecting diet-induced obese mice with antisense oligonucleotide suppressors of miR-132 or its target genes, and measured changes in metabolic parameters and transcripts.
Results Transgenic mice overexpressing miR-132 showed a severe fatty liver phenotype and increased body weight, serum low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) and liver triglycerides, accompanied by decreases in validated miR-132 targets and increases in lipogenesis and lipid accumulation-related transcripts. Likewise, liver samples from both patients with NAFLD and mouse models of hepatic steatosis or non-alcoholic steatohepatitis (NASH) displayed dramatic increases in miR-132 and varying decreases in miR-132 targets compared with controls. Furthermore, injecting diet-induced obese mice with anti-miR-132 oligonucleotides, but not suppressing its individual targets, reversed the hepatic miR-132 excess and hyperlipidemic phenotype.
Conclusions Our findings identify miR-132 as a key regulator of hepatic lipid homeostasis, functioning in a context-dependent fashion via suppression of multiple targets and with cumulative synergistic effects. This indicates reduction of miR-132 levels as a possible treatment of hepatic steatosis.
- FATTY LIVER
- NONALCOHOLIC STEATOHEPATITIS
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Contributors GH planned and performed all of the experiments, analysed and interpreted data and wrote the manuscript; NY constructed the model and performed the partial least square and principal component analysis (PCA) analysis; YT performed in vivo experiments and contributed to the biochemical analysis; RH performed quantitative PCR, planned and performed the Fluidigm analyses and contributed to the in vivo experiments and biochemical analysis; ERB performed western blots, planned some of the experiments and edited the manuscript; SU and JT planned, performed and analysed metabolic cage data; RZ measured miR-132 levels; RZ and BE contributed to some of the in vivo experiments; YRK contributed methionine-deficient and choline-deficient diet, high fat and high sucrose diet and choline-deficient and ethionine-supplemented diet mouse tissues and histology; EK contributed choline-deficient high fat diet (CDHFD) mouse tissues and histology; OP performed pathological analysis; ES contributed to the clinical aspects; EP advised and contributed mouse non-alcoholic steatohepatitis (NASH) samples; MH contributed murine CDHFD liver NASH samples; DSG planned the experiments, interpreted data and edited the manuscript; HS guided the project, planned the experiments, interpreted data and edited the manuscript; all coauthors read and commented on the manuscript contents.
Funding This study was supported by the European Research Council Advanced Award CholinomiRs 321501, the European Commission FP-7 Health-2013-Innovation Grant #602133, the European Union's Horizon 2020 research and innovation programme 639314, the Israel Science Foundation grant 817/13 and The Hebrew University's Translational Research programme (to HS). MH received an European Research Council (ERC) consolidator grant HepatoMetaboPath.
Competing interests None declared.
Patient consent Obtained.
Ethics approval ProteoGenex, Culver City, California, USA.
Provenance and peer review Not commissioned; externally peer reviewed.
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