These authors contributed equally to this study; M.L.M.C. and J.M.M. share senior authorship.
Steatohepatitis/Metabolic Liver Disease
Impaired liver regeneration in mice lacking glycine N-methyltransferase†
Version of Record online: 17 APR 2009
Copyright © 2009 American Association for the Study of Liver Diseases
Volume 50, Issue 2, pages 443–452, August 2009
How to Cite
Varela-Rey, M., Fernández-Ramos, D., Martínez-López, N., Embade, N., Gómez-Santos, L., Beraza, N., Vázquez-Chantada, M., Rodríguez, J., Luka, Z., Wagner, C., Lu, S. C., Martínez-Chantar, M. L. and Mato, J. M. (2009), Impaired liver regeneration in mice lacking glycine N-methyltransferase. Hepatology, 50: 443–452. doi: 10.1002/hep.23033
Potential conflict of interest: Nothing to report.
- Issue online: 29 JUL 2009
- Version of Record online: 17 APR 2009
- Accepted manuscript online: 17 APR 2009 12:00AM EST
- Manuscript Accepted: 1 APR 2009
- Manuscript Received: 25 NOV 2008
- National Institutes of Health (NIH). Grant Numbers: AT-1576, DK15289, SAF 2008-04800, HEPADIP-EULSHM-CT-205, ETORTEK-2008
- Fundación “La Caixa”
- Instituto de Salud Carlos III
Additional Supporting Information may be found in the online version of this article.
|HEP_23033_sm_SupFig1.tif||48K||Supporting Figure 1. HGF has no effect on LKB1 and AMPK phosphorylation in hepatocytes isolated from GNMT-KO mice. We have previously shown, that in WT mouse hepatocytes HGF treatment (25 ng/ml) induces LKB1 and AMPK phosphorylation (). In this experiment, LKB1 and AMPK phosphorylation was analyzed (30 μg/lane), via Western blotting with the indicated antibodies, in GNMT-KO hepatocytes before and 1 hour after the addition of HGF (25 ng/ml). Results are representative of three independent experiments.|
|HEP_23033_sm_SupFig2.tif||50K||Supporting Figure 2. Knockdown of GNMT with RNAi reduces HGF-induced cyclin D1 expression. Freshly isolated rat hepatocytes (15) were transfected with 1.5 μg/106 cells of GNMT specific RNAi (Qiagen, SI00258377) or control RNAi (Qiagen, 1022076) by electroporation using the rat hepatocyte Nucleofector® Kit from Amaxa (following the instructions provided by the supplier) and cultured in minimum essential medium (MEM) supplemented with 5% FBS. Two hours after attachment, the culture medium was replaced by MEM supplemented with 5% FBS and triamcinolone (100 nM) (30). Twenty-four hours after electroporation, the medium was replaced with serum-free MEM, and treated with HGF (25 ng/mL) for another 24 hours. mRNA was then isolated. (A) Real-time PCR analysis of GNMT and MAT1A mRNA expression in hepatocytes transfected with GNMT specific RNAi or control RNAi, 24 hours after electroporation. (B) Real-time PCR analysis of cyclin D1 mRNA expression of hepatocytes transfected with GNMT specific RNAi or control RNAi and treated with HGF during 24 hours. Each bar represents the mean ± SD of at least quadruplicate experiments (*p < 0.05 vs. control). Values were normalized with 18S ribosomal RNA expression. PCR was executed with the following primers: MAT1A-F 5′-GCTATGCCACTGACGAGACA -3′, MAT1A-R 5′-CAGAGATGACGATGGTGTGG-3′; GNMT-F 5′-AACAACAAAGCCCACATGGT-3′, GNMT-R 5′-TCTTCTTGAGCACGTGGATG -3′; 18S-F 5′-ACGGACCAGAGCGAAAGCAT -3′, 18S-R 5′-TGTCAATCCTGTCCGTGTCC-3′; Cyclin D1-F 5′-AGATGTGAAGTTCATTTCCAACC-3′, Cyclin D1-R 5′-TCACACTTGATGACTCTGGAAAG -3′.|
|HEP_23033_sm_SupFig3.tif||123K||Supporting Figure 3. Knockdown of GNMT with RNAi reduces cyclin A and D1 expression and cytoplasmic HuR accumulation in livers after partial hepatectomy (PH). Three month-old male WT mice were injected intravenously in the tail vein (200 μl of a 60 μM solution) with GNMT specific RNAi (Sense 5′-AUAUGCGCUUAAGGAGCGC-3′) or control RNAi (Sense 5′-AAUCGCAUAGCGUAUGCCGUU-3′) (Eurofins mwg/operon) per mice at 24 hours and 2 hours before PH. Livers were then removed during PH (time 0 hours) and 48 hours after PH, and mRNA and protein extracted. (A) Real-time PCR analysis of GNMT mRNA expression and (B) Western blot analysis of GNMT protein levels, in livers from mice injected with GNMT specific RNAi or control RNAi, 24 hours after injection (time 0 hours). (C) Real-time PCR analysis of cyclin D1 and A2 mRNA expression, and (D) Western blot analysis of cyclin D1 and A and cytoplasmic HuR protein levels in livers from mice injected with GNMT specific RNAi or control RNAi, 24 hours after the injection (time 0 hours) and 48 hours after the PH. Each bar represents the mean ± SD of quadruplicate experiments (*p < 0.05 vs. control). Values of mRNA expression were normalized with 18S ribosomal RNA expression. PCR was executed with the following primers: GNMT-F 5′-AGTACAAGGCGTGGTTGCTT-3′, GNMT-R 5′-ATCTTTGTCCAGCGTCAACC -3′; 18S-F 5′-GCACCACCACCCACGGAATCG -3′, 18S-R 5′-TTGACGGAAGGGCACCACCAG-3′; Cyclin D1-F 5′-GCCTCTAAGATGAAGGAGACCAT-3′, Cyclin D1-R 5′-ATTTTGGAGAGGAAGTGTTCGAT -3′, Cyclin A2-F 5′-ATAGATTCCTCTCCTCCATGTCTG-3′, Cyclin A2-R 5′-AAAGCAAGGACTTTCAATACAAGG-3′.|
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