Abnormal lipid processing but normal long-term repopulation potential of myc−/− hepatocytes

Establishing c-Myc's (Myc) role in liver regeneration has proven difficult particularly since the traditional model of partial hepatectomy may provoke an insufficiently demanding proliferative stress. We used a model of hereditary tyrosinemia whereby the affected parenchyma can be gradually replaced by transplanted hepatocytes, which replicate 50-100-fold, over several months. Prior to transplantation, livers from myc−/− (KO) mice were smaller in young animals and larger in older animals relative to myc+/+ (WT) counterparts. KO mice also consumed more oxygen, produced more CO2 and generated more heat. Although WT and KO hepatocytes showed few mitochondrial structural differences, the latter demonstrated defective electron transport chain function. RNAseq revealed differences in transcripts encoding ribosomal subunits, cytochrome p450 members and enzymes for triglyceride and sterol biosynthesis. KO hepatocytes also accumulated neutral lipids. WT and KO hepatocytes repopulated recipient tyrosinemic livers equally well although the latter were associated with a pro-inflammatory hepatic environment that correlated with worsening lipid accumulation, its extracellular deposition and parenchymal oxidative damage. Our results show Myc to be dispensable for sustained in vivo hepatocyte proliferation but necessary for maintaining normal lipid homeostasis. myc−/− livers resemble those encountered in non-alcoholic fatty liver disease and, under sustained proliferative stress, gradually acquire the features of non-alcoholic steatohepatitis.

1 hour, then homogenized and subject to organic extraction as previously described [1]. Total protein content was determined using the BCA reagent.
To measure Acetyl CoA, a pulverized piece of flash frozen tissue was solubilized in ice cold lysis buffer (20 mM Tris, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 1% Triton X-100; 2.5 mM sodium pyrophosphate; 1 mM -glycerolphosphate; 1 mM Na3VO4; 1 μg/ml Leupeptin and 1 mM PMSF). The assay was then carried out according to manufacturer's instructions using an Acetyl-Coenzyme A Assay Kit (Sigma-Aldrich cat. # MAK039). ATP assays were performed by lysing tissues with 10% trichloroacetic acid to inactivate ATPases, and then diluting the samples 1:50 with PBS. 100 µl of this was mixed with 50 µl of ATPlite mammalian cell lysis solution in quadruplicate in 96 well plates. The remainder of the assay was carried out according to the manufacturer's instructions using the ATPlite TM Luminescence Assay System (Perkin Elmer, Waltham, MA). Results were normalized to total protein levels, which were determined on separate sets of identical wells.
Quantification of oxidative phosphorylation (Oxphos). Homogenates were prepared from fresh liver tissue as described in the protocol provided by the vendor (MiPNet17.02). Oxygen consumption was measured in MiR05 medium (0.5 mM EGTA, 3  ) and cytochrome C (10 µM) to assess outer mitochondrial membrane integrity. O 2 consumption rates were normalized to total protein content. Ten sets of livers were analyzed on different days and later assessed statistically by a Paired ttest.
In solution trypsin digestion for mass spectrometry. For each digestion, 20 µg of mitochondria were resuspended in 100 µl 50 mM NH4HCO3/0.02% ProteaseMAX™ Surfactant (Promega, Madison, WI). The mixture was then boiled for 10 min in the presence of 10 mM dithiothreitol and then alkylated by the addition of 45 mM iodoacetamide for 1 hr in the dark at room temperature). 0.5 µg of trypsin gold Supplemental Materials and Methods 3 (Promega, Madison, WI) was then added and digestion was performed overnight at 37°C. The resulting tryptic peptides were de-salted using PepClean C-18 Spin Columns (Pierce, Inc., Rockford, IL), vacuumdried, and re-suspended in 40 µl of 0.1% formic acid.
Targeted mass spectrometry assays for selected peptides. Selective/multiple reaction monitoring (SRM/MRM)-based targeted mass spectrometry was performed on a TSQ Quantum Ultra instrument (Thermo Fisher Scientific) coupled to a Thermo Fisher Nanoflow Dionex Ultimate 3000 liquid chromatography system. Nano-LC separations, each performed in duplicate, used an analytical C18 PicoChip™ columns packed with 10.5 cm Reprosil C18 3µm 120Å chromatography media with a 75 µm ID column and a 15 µm tip (New Objective, Inc., Woburn, MA). Mobile phase A consisted of 0.1% formic acid in HPLC water and mobile phase B consisted of 0.1% formic acid in 100% acetonitrile. 2 g of tryptic peptides were loaded onto the column and washed with mobile phase A for 4min at 6 l/min. The peptides were then eluted into the analytical C18 column at a flow rate of 300 nl/min with a gradient comprised of 0-5% solvent B for 4.5 min, 5-40% solvent B for 31.5 min, 40-95% solvent B for 4 min and 95% solvent B for 8 min. The collision energies were calculated using a linear equation CE =0.034 x m/z + 3.314. The full width at half maximum was set to be 0.7 Da for Q1 and Q3. The instrument was operated using scheduled SRM mode with 1 sec cycling time and 5 min retention time window. The majority of peptides had base peak widths of ~20 sec base and ~12 data points were acquired per chromatogram peak. Skyline software [2] was used to facilitate targeted SRM assay method development and data analyses. The transitions of the targeted peptides were originally selected based on mouse tandem spectrum library downloaded from PeptideAtlas (http://www.peptideatlas.org/) and subsequently optimized on the TSQ. All selected peptides and their corresponding SRM assay parameters were listed in Supplemental Table S1. Peak areas of all transitions for the same peptide were summed and the total peak area was used as metric for relative quantitation. Student's t test was applied to log-transformed total peak area to determine the significance of the differences between groups.
Unbiased label free mass spectrometry assays. 2 µg of tryptic peptides per sample were analyzed with reverse-phased LC-MS/MS using a nanoflow LC (EASY-nLC II, Thermo Fisher) coupled online to Supplemental Materials and Methods 4 LTQ/Orbitrap Velos Elite hybrid mass spectrometer (Thermo-Fisher). Mobile phases contained 0.1% formic acid in HPLC grade water for solvent A and 0.1% formic acid in 100% acetonitrile for solvent B.
Peptides were first loaded onto a C-18 trap column (Thermo Fisher) and desalted on line for 6 l solvent A. Peptides were then eluted onto a capillary column (75 m inner diameter x 360 µm outer diameter x 15 cm long (Polymicro Technologies, Phoenix, AZ) slurry-packed-in-house with 5 µm particle size, 125 Ǻ pore size C-18 silica-bonded stationary phase (Phenomenex, Torrance, CA) and resolved using a 100 min gradient at the flow rate of 0. Mass tolerance was set to 20 ppm for initial search and 4.5 ppm for the main search for precursor ions, and 0.5 Da for fragment ions. Minimum peptide length was set to 7 amino acids and the false discovery rate (FDR) based on a target-decoy approach was set to 1%. The match between runs options was enabled with 2 min match time window and 20 min alignment window. The intensity values from MaxQuant output for identified peptides were used for relative quantitation across samples. Only peptides with non-zero intensity values in all samples were included for statistical test. Student's t test was applied to log-transformed intensities to identify peptides with significant difference between WT and KO. The linear step-up (LSU) [3]   Cambridge, UK.). Adapter sequences, primers, Ns, and reads with quality score below 28 were trimmed using fastq-mcf of ea-utils and PRINSEQ (http://prinseq.sourceforge. net/manual.html). Reads with a remaining length of less than 20 bp after trimming were discarded. Paired end reads were mapped to the mouse genome (m10) using TopHat (http://ccb.jhu.edu/software/tophat/index.shtml) in a strand specific manner. Read coverage on forward and reverse strands for genome browser visualization was computed using SAMtools, BEDtools, and UCSC Genome Browser utilities. Pairwise differential expression was quantified using Cuffdiff and DESeq [4,5]. Cufflinks was used to determine FPKM levels for each gene from the TopHat alignment and was used as input for Cuffdiff. Significant differentially expressed genes were determined by adjusted P-value with a threshold of 0.05. DESeq w a s utilized Supplemental Figure S1. Deletion of myc coding exons 2 and 3 from KO hepatocytes. Complex II could not be measured in situ and was instead measured separately on isolated mitochondrial lysates as described previously [1,8]. In situ enzyme activities for each of the indicated complexes are depicted here graphically after adjusting for differences in the protein content of each complex based on densitometric scanning of BNGE profiles [3].  Supplemental Table S1. The resulting MS data were analyzed using the differential mass spectrometry tools (InfoClinika, Seattle WA) that are included in the CHORUS data analysis environment (www.chorusproject.org). Total peak areas from 2439 peptides were collected from 5 individual liver mitochondrial preparations from each group and matched to 377 mitochondrial proteins using the PeptideAtlas. All results were compared using both a Students t-test and Wilcoxon signed-rank test.
Supplemental Table S3. Transcripts identified by Ingenuity Pathway Analysis from the top 10 deregulated pathways in transplanted livers. Differential gene expression profiling of genes identified as described for Figure 3C, performed on isolated hepatocytes from 2 WT mice and 4 KO mice.