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Cited by 145Cholesterol and Lipid MetabolismMicroRNA in disease regulationLipid metabolism and disorders

The DOI matches its Crossref record; 32 of the 32 references it cites were found in a registry or an index.

Long-term therapeutic silencing of miR-33 increases circulating triglyceride levels and hepatic lipid accumulation in mice

Open access
Goedeke, Leigh, Salerno, Alessandro, Ramírez, Cristina M, Guo, Liang, Allen, Ryan M, Yin, Xiaoke, Langley, Sarah R, Esau, Christine, Wanschel, Amarylis, Fisher, Edward A, Suárez, Yajaira, Baldán, Angel, Mayr, Manuel, Fernández‐Hernando, Carlos·2014·EMBO Molecular Medicine
DOI 10.15252/emmm.201404046PMC PMC4197861
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Abstract

Plasma high‐density lipoprotein (HDL) levels show a strong inverse correlation with atherosclerotic vascular disease. Previous studies have demonstrated that antagonism of miR‐33 in vivo increases circulating HDL and reverse cholesterol transport (RCT), thereby reducing the progression and enhancing the regression of atherosclerosis. While the efficacy of short‐term anti‐miR‐33 treatment has been previously studied, the long‐term effect of miR‐33 antagonism in vivo remains to be elucidated. Here, we show that long‐term therapeutic silencing of miR‐33 increases circulating triglyceride (TG) levels and lipid accumulation in the liver. These adverse effects were only found when mice were fed a high‐fat diet (HFD). Mechanistically, we demonstrate that chronic inhibition of miR‐33 increases the expression of genes involved in fatty acid synthesis such as acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS) in the livers of mice treated with miR‐33 antisense oligonucleotides. We also report that anti‐miR‐33 therapy enhances the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP‐responsive genes, including ACC and FAS. Taken together, these results suggest that persistent inhibition of miR‐33 when mice are fed a high‐fat diet (HFD) might cause deleterious effects such as moderate hepatic steatosis and hypertriglyceridemia. These unexpected findings highlight the importance of assessing the effect of chronic inhibition of miR‐33 in non‐human primates before we can translate this therapy to humans.

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Figure 1 Long-term anti-miR-33 therapy results in hypertriglyceridemia in mice fed a HFD A qRT-PCR analysis of hepatic miR-33 expression levels in the livers of mice treated with PBS, control ASO, or miR-33 ASO, and fed a chow diet (CD).B, C Plasma cholesterol (B) and HDL-C (C) levels in the livers of mice treated with PBS, control ASO, or miR-33 ASO for 4 and 12 weeks and fed a CD.D Lipoprotein profile analysis obtained from pooled plasma of mice administered PBS, control ASO, or miR-33 ASO.E, F Circulating triglyceride (TG) levels (E) and body weight (F) of mice injected with PBS, control ASO, or miR-33 ASO, and fed a CD.G qRT-PCR analysis of hepatic miR-33 expression levels of mice treated with PBS, control ASO, or miR-33 ASO, and fed a high-fat diet (HFD).H-J Plasma cholesterol (H), HDL-C (I) and triglyceride (J) levels of mice treated with PBS, control ASO, or miR-33 ASO for 4 and 12 weeks and fed a HFD.K Representative Western blot of plasma ApoB-100 expression of mice treated with PBS, control ASO, or miR-33 ASO and fed a HFD for 20 weeks.L, M Cholesterol (L) and triglyceride (M) distribution in different lipoprotein fractions isolated from mice treated with PBS, control ASO, or miR-33 ASO, and fed a HFD.N Body weight of mice treated with PBS, control ASO, or miR-33 ASO for 20 weeks and fed HFD. Data information: All the data represent the mean ± SEM; (PBS n = 10, control ASO n = 12 and miR-33 ASO n = 12) and *P < 0.05 comparing miR-33 ASO group with PBS and control ASO groups. Lipoprotein fractionation analyses were performed using pooled plasma from five mice in each group. Source data is available online for this figure.
Figure 1 Long-term anti-miR-33 therapy results in hypertriglyceridemia in mice fed a HFD A qRT-PCR analysis of hepatic miR-33 expression levels in the livers of mice treated with PBS, control ASO, or miR-33 ASO, and fed a chow diet (CD).B, C Plasma cholesterol (B) and HDL-C (C) levels in the livers of mice treated with PBS, control ASO, or miR-33 ASO for 4 and 12 weeks and fed a CD.D Lipoprotein profile analysis obtained from pooled plasma of mice administered PBS, control ASO, or miR-33 ASO.E, F Circulating triglyceride (TG) levels (E) and body weight (F) of mice injected with PBS, control ASO, or miR-33 ASO, and fed a CD.G qRT-PCR analysis of hepatic miR-33 expression levels of mice treated with PBS, control ASO, or miR-33 ASO, and fed a high-fat diet (HFD).H-J Plasma cholesterol (H), HDL-C (I) and triglyceride (J) levels of mice treated with PBS, control ASO, or miR-33 ASO for 4 and 12 weeks and fed a HFD.K Representative Western blot of plasma ApoB-100 expression of mice treated with PBS, control ASO, or miR-33 ASO and fed a HFD for 20 weeks.L, M Cholesterol (L) and triglyceride (M) distribution in different lipoprotein fractions isolated from mice treated with PBS, control ASO, or miR-33 ASO, and fed a HFD.N Body weight of mice treated with PBS, control ASO, or miR-33 ASO for 20 weeks and fed HFD. Data information: All the data represent the mean ± SEM; (PBS n = 10, control ASO n = 12 and miR-33 ASO n = 12) and *P < 0.05 comparing miR-33 ASO group with PBS and control ASO groups. Lipoprotein fractionation analyses were performed using pooled plasma from five mice in each group. Source data is available online for this figure.CC BY · PubMed Central
Figure 2 Antagonism miR-33 in mice fed a HFD results in moderate hepatic steatosis A–D Hepatic content of triglycerides (A), diglycerides (B), free fatty acids (C), and cholesterol esters (D) quantified from liver tissue of mice treated with PBS, control ASO, or miR-33 ASO for 20 weeks and fed a chow diet (CD) or high-fat diet (HFD). Data represent the mean ± SEM; (PBS n = 3, control ASO n = 6 and miR-33 ASO n = 6) and *P < 0.05 comparing PBS and miR-33 ASO group with control ASO group.E Representative liver sections isolated from mice treated with PBS, control ASO, or miR-33 ASO stained with H&E, picrossirius red, and Oil Red O. Scale bar = 70 μm.F–J qRT-PCR analysis of genes involved in fatty acid synthesis (F), cholesterol metabolism (G), fatty acid oxidation and lipolysis (H), glucose metabolism (I) and lipoprotein metabolism (J) in liver tissues from mice treated with PBS, control ASO or miR-33 ASO. The mRNA fold change from each gene from mice treated with miR-33 ASO or control ASO-treated mice compared to PBS-treated mice was calculated. Data represent the mean ± SEM; (PBS n = 3, control ASO n = 6 and miR-33 ASO n = 6) and *P < 0.05 comparing miR-33 ASO group with PBS and control ASO group.K Representative Western blot analysis of ABCA1, NPC1, SRC1, RIP140, CROT, and NFYC from liver lysates of mice treated with PBS, control ASO, or miR-33 ASO. Tubulin was used as a loading control. Source data is available online for this figure.
Figure 2 Antagonism miR-33 in mice fed a HFD results in moderate hepatic steatosis A–D Hepatic content of triglycerides (A), diglycerides (B), free fatty acids (C), and cholesterol esters (D) quantified from liver tissue of mice treated with PBS, control ASO, or miR-33 ASO for 20 weeks and fed a chow diet (CD) or high-fat diet (HFD). Data represent the mean ± SEM; (PBS n = 3, control ASO n = 6 and miR-33 ASO n = 6) and *P < 0.05 comparing PBS and miR-33 ASO group with control ASO group.E Representative liver sections isolated from mice treated with PBS, control ASO, or miR-33 ASO stained with H&E, picrossirius red, and Oil Red O. Scale bar = 70 μm.F–J qRT-PCR analysis of genes involved in fatty acid synthesis (F), cholesterol metabolism (G), fatty acid oxidation and lipolysis (H), glucose metabolism (I) and lipoprotein metabolism (J) in liver tissues from mice treated with PBS, control ASO or miR-33 ASO. The mRNA fold change from each gene from mice treated with miR-33 ASO or control ASO-treated mice compared to PBS-treated mice was calculated. Data represent the mean ± SEM; (PBS n = 3, control ASO n = 6 and miR-33 ASO n = 6) and *P < 0.05 comparing miR-33 ASO group with PBS and control ASO group.K Representative Western blot analysis of ABCA1, NPC1, SRC1, RIP140, CROT, and NFYC from liver lysates of mice treated with PBS, control ASO, or miR-33 ASO. Tubulin was used as a loading control. Source data is available online for this figure.CC BY · PubMed Central
Figure 3 Anti-miR-33 therapy causes a profound alteration in the liver proteome A, B Liver protein extracts from control ASO (green color) and miR33 ASO-treated (red color) mice were quantified using difference gel electrophoresis (DIGE, n = 4 per group) (A). Among the differentially expressed spots was major urinary protein (MUP). Note the pronounced upregulation of MUP (white box) in response to miR-33 ASO. Results were reproduced by using a dye-swap (B): control ASO (red color), miR-33 ASO (green color).
Figure 3 Anti-miR-33 therapy causes a profound alteration in the liver proteome A, B Liver protein extracts from control ASO (green color) and miR33 ASO-treated (red color) mice were quantified using difference gel electrophoresis (DIGE, n = 4 per group) (A). Among the differentially expressed spots was major urinary protein (MUP). Note the pronounced upregulation of MUP (white box) in response to miR-33 ASO. Results were reproduced by using a dye-swap (B): control ASO (red color), miR-33 ASO (green color).CC BY · PubMed Central

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Metadata

Authors
Goedeke, L., Salerno, A., Ramírez, C. M., Guo, L., Allen, R. M., Yin, X., Langley, S. R., Esau, C., Wanschel, A., Fisher, E. A., Suárez, Y., Baldán, A., Mayr, M., & Fernández‐Hernando, C.
Affiliations
  • Yale University (US)
  • New York University (US)
  • Saint Louis University (US)
  • King's College London (GB)
  • Regulus Therapeutics (United States) (US)
ORCID
Venue
EMBO Molecular Medicine
Publisher
Springer Science and Business Media LLC
Published
2014-07-18
Volume
6
Issue
9
Pages
1133-1141
ISSN
  • 1757-4676 (ISSN-L)
  • 1757-4684
DOI
10.15252/emmm.201404046
PMC
PMC4197861
OpenAlex
W2142082511
Cited by
145
References
32
Citations by year
2026: 2 · 2025: 12 · 2024: 6 · 2023: 8 · 2022: 10 · 2021: 10 · 2020: 11 · 2019: 12 · 2018: 11 · 2017: 17 · 2016: 23 · 2015: 21 · 2014: 2
FWCI
20.84
Type
journal article
Language
en
Open access
Licence
Locations
  • EMBO Molecular Medicine (publishedVersion, other-oa)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • National University of Singapore (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • ORCA Online Research @Cardiff (Cardiff University) (submittedVersion)
  • Research Portal (King's College London) (submittedVersion, other-oa)
Funders
  • National Institutes of Health (R01HL107953, R01HL107953‐04S1, R01HL106063, 1F31AG043318, R01HL107794)
  • Foundation Leducq Transatlantic Network of Excellence in Cardiovascular Research
  • Heart Research UK
  • Department of Health via the National Institute for Health Research
  • Biomedical Research Centre
  • NHS Foundation Trust
  • King's College London
  • King's College Hospital NHS Foundation Trust
  • British Heart Foundation
  • AHA Predoctoral Fellowship (11PRE7240026)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Cholesterol and Lipid Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Lipid metabolism and disorders
Keywords
miR-33, antisense oligonucleotides, hypertriglyceridemia, hepatic steatosis, fatty acid synthesis, acetyl-CoA carboxylase, fatty acid synthase, SREBP, high-fat diet, HDL cholesterol, reverse cholesterol transport, atherosclerosis
MeSH terms
Animals, Fatty Liver, Liver, Male, Mice, Inbred C57BL, Triglycerides, Oligonucleotides, Antisense, Gene Silencing, MicroRNAs, Lipid Metabolism, Diet, High-Fat

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