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HEP_24607_sm_SuppFig1.tif2747KSupporting Fig. 1. Deletion of MAT1A gene decreases TGH, Aada and Pemt mRNA levels in 3-month-old mice. Liver RNA was isolated from 3-month-old (3m) and 8-month-old (8m) wild type (WT) (□) and MAT1A-knockout (MAT1A-KO) (▪) mice. (A) Pemt, (B) Dgat2, triglyceride hydrolase (TGH), arylacetamide deacetylase (Aada), (C) Mttp and (D) Apob gene expression analysis were performed by qRT-PCR using TaqMan gene expression assaysTM (Applied Biosystems). The relative quantity of apoB mRNA was determined by the ΔΔCT method. Normalization was performed using the Visual Basic Application GeNorm. According to GeNorm, pyruvate carboxylase, glyceraldehyde-3-phosphate dehydrogenase and 18S rRNA were suitable genes for normalization. Values are means ± SEM of 4-8 animals per group.
HEP_24607_sm_SuppFig2.tif3301KSupporting Fig. 2. The mRNA expression levels of proteins required for normal VLDL assembly in mice are upregulated in hepatocytes exposed to AICAR or SAMe. Primary C57BL/6 mice hepatocytes isolated by collagenase digestion were seeded in 6 well dishes at 5 x 105 cell per well (n=6) in anfotericin/FBS containing medium and 1 hour later the medium was replaced for an anfotericin-free medium. The next day the medium was replaced by a FBS-free medium containing 0.2% (v/v) dexametasone (□, control) or dexametasone with 0.75 mM SAMe (▪) or 0.5 mM 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) (▪). For RNA extraction, the cells were collected in TRIzolTM 6 hours and 24 hours after adding the effectors. Gene expression was measured by qRT-PCR using TaqMan gene expression assaysTM (Applied Biosystems). The relative quantity of each gene was determined by the ΔΔCT method. Normalization was performed using the Visual Basic Application GeNorm. According to GeNorm, pyruvate carboxylase, glyceraldehyde-3-phosphate dehydrogenase and 18S rRNA were suitable genes for normalization.
HEP_24607_sm_SuppFig3.tif1575KSupporting Fig. 3. MTP triglyceride transfer activity correlates positively with the microsomal triglyceride content in both MAT1A-KO mice and in their controls. (A) 3-month-old and 8-month-old wild type (WT) and (B) MAT1A-knockout (MAT1A-KO) mice were fasted 2 hours before liver collection. Liver microsomes were isolated by differential centrifugation and MTP triglyceride (TG) transfer activity and microsomal TG content were measured as described in materials and methods. Correlation studies were carried out by using Pearson's correlation coefficient and significance was defined as p<0.05.
HEP_24607_sm_SuppFig4.tif2326KSupporting Fig. 4. The disrupted mobilization of triglycerides for VLDL secretion in 3-month-old MAT1A-KO mice is not repaired by treatment with the AMPK inhibitor compound cPrimary 3-month-old MAT1A-knockout (MAT1A-KO) mice hepatocytes were incubated 4 hours with 0.4 mM [3H]oleic acid (5 μCi/dish) complexed with 0.5% fatty acid free BSA and [14C]glycerol (0.5 μCi/dish) (pulse). Cells were washed for 1 hour in DMEM followed by a second wash with (▪) compound c (cc) dissolved in 2% DMSO or with 2% DMSO alone (control, ▪) and incubated for 4 hours in the same conditions. After both, the pulse and chase periods, cells and media were collected and the percentages of the total (A) [3H]-triglyceride (TG) and (B) [14C]-TG secreted to the media were calculated. (C) Inactivation grade of AMPK was established by inmunoblotting. Values are means ± SEM of n=5. Statistical differences versus control hepatocytes are denoted by *p<0.05 (Student's t test).
HEP_24607_sm_SuppFig5.tif3949KSupporting Fig. 5. MAT1A deletion alters apoB metabolism in mice. Liver microsomes were isolated from 3-month-old (3m) and 8-month-old (8m) wild type (WT) (□) and MAT1A-knockout (MAT1A-KO) (▪) mice by differential centrifugation and the microsomal lumen and membranes were separated, as indicated in Supporting Materials and Methods. The apoB protein mass in (A) lumen, (B) membranes and (C) total microsomes was determined by Western blotting. The luminal apoB was concentrated prior to Western blotting by immunoprecipitation. (D) Primary 3 month-old WT and MAT1A-KO mice hepatocytes were incubated overnight in DMEM, cells were washed and incubated in methionine/cysteine-free DMEM for 1 hour and radiolabeled for 2 hours with 50 μCi/ml [35S]methionine in the presence (▪) or absence (□) of the proteasome inhibitor MG132 (25 μM). Cells were scraped into the immunoprecipitation buffer and immunoprecipitation of apoB and transferrin were carried out in the cellular lysate. (E) Microsomes were incubated in the absence (□) or the presence of 50 μg/ml trypsin (▪) or of 50 μg/ml trypsin plus 1% (v/v) triton X-100 (▪) for 30 min on ice. Trypsin treatment was stopped by the addition of 50 μg of soybean trypsin inhibitor, microsomes were separated from trypsin by centrifugation, and the microsomal proteins were solubilised for SDS-PAGE and immunoblotting.
HEP_24607_sm_SuppFig6.tif9702KSupporting Fig. 6. ApoB localization in mice hepatocytes is altered by MAT1A deletion. Primary 3-month-old WT and MAT1A-KO mice hepatocytes were incubated for 4 hours in DMEM with (A) 0.5% fatty acid-free BSA or (B) 0.4 mM oleic acid in 0.5% fatty acid-free BSA. The coverslips were washed with PBS and the cells fixed with 3.7% formaldehyde. After permeabilization with 5% Triton X-100, cells were blocked using 10% foetal bovine serum in PBS and double labeled with anti-apoB antibody (red) and with BODIPY® 493/503 (green).
HEP_24607_sm_SuppFig7.tif4945KSupporting Fig. 7. Proposed models for the assembly of VLDL in MAT1A-KO mice. (A) The deletion of MAT1A gene in 3-month-old mice produces a rise in the secretion of small, TG-poor VLDL particles due to increased apoB availability. The lipolytic activity and gene expression of the main enzymes (TGH and AADA) implicated in the mobilization of TG from the cytoplasmic lipid droplets storage is repressed, decreasing the availability of TG to be channelled for VLDL secretion. (B) In 8-month-old MAT1A-KO mice, with established NASH, there is a more accentuated rise in the secretion of small, TG-poor VLDL particles, and the scenario concerning lipid secretion is different. The secretion of TG in VLDL is higher than in WT mice Abbreviations: SAMe, S-adenosylmethionine; TG, triglyceride; VLDL, very-low-density lipoprotein; DGAT, acyl-CoA:diacylglycerol acyltransferase; apo, apolipoprotein; PEMT, phosphatidylethanolamine N-methyltransferase; MTP, microsomal triglyceride transfer protein; ER, endoplasmic reticulum; PC, phosphatidylcholine; PE, phosphatidylethanolamine; TGH, triglyceride hydrolase; AADA, arylacetamide deacetylase. In white, decreased enzyme activity; in black, increased enzyme activity.
HEP_24607_sm_SuppTable1.doc34KSupporting Table 1. Primers used for qRT-PCR
HEP_24607_sm_SuppInfo.doc59KSupporting Information

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