Nonalcoholic Fatty Liver Disease Demonstrates a Pre-fibrotic and Premalignant Molecular Signature
Metabolic syndrome contributing to nonalcoholic fatty liver disease (NAFLD) can lead to hepatic dysfunction, steatohepatitis, cirrhosis, and hepatocellular carcinoma.
In this study, we tested whether diet-induced fatty liver in a mouse model physiologically mimicked human NAFLD, and whether transcriptional alterations in mouse fatty liver signified risk for the development of hepatitis, cirrhosis, and/or hepatocellular carcinoma.
SAMP6 strain mice were fed a low-fat diet or high-fat diet (HFD) for 6 months. Mouse livers were isolated and subjected to histology, immunohistochemistry, and whole transcriptome RNA sequencing. Sequences were aligned to the mouse reference genome, and gene expression signatures were analyzed using bioinformatics tools including Cufflinks, Pathview, Cytoscape, ClueGO, and GOstats.
Consistent with NAFLD, livers from HFD-fed mice demonstrated steatosis, high levels of inflammation, an up-regulation of genes encoding proteins associated with the complement pathway and immune responses, and down-regulation of those associated with metabolic processes. These livers also showed an up-regulation of genes associated with fibrosis and malignant transformation but no histological evidence of either pathobiology or DNA damage.
HFD-fed mice exhibited NAFLD that had incompletely transitioned from fatty liver to NASH. Importantly, bioinformatics approaches identified pre-fibrotic and premalignant signatures, suggesting that the pathogenesis of both fibrosis and cancer may initiate in fatty livers well before associated histological changes are evident.
KeywordsNAFLD Fibrosis Cancer Diet
Metabolic syndrome, a spectrum disorder defined by high blood pressure, high body mass index/abdominal circumference, glycemia, triglyceridemia, proinflammatory state, and high HDL cholesterol concentrations, has increased concurrent with obesity in the American population [1, 2, 3, 4]. In particular, nonalcoholic fatty liver disease (NAFLD), cirrhosis, and ultimately hepatocellular carcinoma can be consequent to many of the pathobiology associated with metabolic syndrome . NAFLD has become one of the most common liver diseases and an estimated 20–30% of individuals in the Western population are diagnosed with NAFLD and suffer from liver-related morbidity and mortality [1, 5]. Histologically, NAFLD is characterized by steatosis, e.g., fat accumulation. Fatty liver with demonstrable inflammation, DNA damage, and some degree of fibrosis is considered nonalcoholic steatohepatitis (NASH) . Quantification of the hepatocytes containing fat droplets in the liver can give an indication into the severity of the steatosis, and the recommended lower threshold has been set to 5% of the liver mass being composed of hepatocytes containing fat .
In order to better understand the relationship between obesity and downstream organ pathobiologies, we have developed a mouse model of diet-induced obesity that successfully mimics several aspects of metabolic syndrome. This model employs senescence-accelerated mouse prone 6 (SAMP6) mice, an inbred strain of AKR/J mice that exhibit an accelerated aging phenotype . SAMP6 and AKR/J mice fed a high-fat diet (HFD) for 6 months developed insulin resistance, hyperglycemia, obesity, urinary voiding dysfunction, and fatty livers . In the current study, we hypothesized that the fatty livers of the HFD-fed mice mimicked human NAFLD. Therefore, we determined whether these mice might provide a good model to study the chronology of transcriptional changes that potentially drive the development of NAFLD. These studies showed that livers from HFD-fed mice demonstrated steatosis, inflammation, an up-regulation of genes encoding proteins associated with the complement pathway and immune responses, down-regulation of those associated with metabolic processes, and little DNA damage. Therefore, the transcriptome signatures of the fatty livers from the HFD-fed SAMP6 mice were consistent with that of NAFLD that had incompletely transitioned from fatty liver to NASH. Surprisingly, the fatty livers also showed an up-regulation of genes associated with extracellular matrix deposition and malignant transformation, but no histological evidence of fibrosis or hepatocellular carcinoma. Therefore, the NAFLD livers expressed a pre-fibrotic and premalignant signature in the absence of any other indication of either fibrosis or cancer.
Materials and Methods
Mouse Strains and Husbandry
The senescence-accelerated mouse prone 6 (SAMP6) mouse strain was used in this study (Harlan Laboratories, Indianapolis, IN) . Senescence-accelerated mouse (SAM) models have been developed by selective sister–brother breeding of the AKR/J strain and now comprise 12 lines: senescence-prone inbred (SAMP) strains 1, 2, 3, 6, 7, 8, 9, 10, and 11, and senescence-resistant (SAMR) strains 1, 4, and 5 . The pathobiologies of these mice differ widely, though the SAMP6 strain is characterized by amyloidosis, osteoporosis, and prostatic fibrosis [10, 11, 12]. Genetically, SAMP6 mice carry a mutant Abcb1a allele common to many of the SAMP strains, as well as a p.S540fs frameshift mutation in the Il4ra gene potentially associated with ulcerative colitis and osteoporosis [13, 14]. The rationale for using the SAMP6 mouse strain is based on our observation of the development of fatty liver in mice fed a high-fat diet, and lack of genetic predisposition for this pathobiology . Additionally, the accelerated aging phenotype facilitates response to a high-fat diet (which can be difficult to tolerate over extended periods of time) such that these mice demonstrate pronounced fatty liver and a metabolic syndrome phenotype, e.g., obesity, hyperglycemia, and insulin resistance, consistent with human type 2 diabetes, after 6 months on the diet . For these studies, mice were put on the following diets at 6–8 weeks of age: high-fat diet (HFD) containing 60% calories from fat, 20% protein, and 20% carbohydrates (58Y1, Test Diet, Richmond, IN) or low-fat diet (LFD) containing 10.2% calories from fat, 18.3% protein, and 71.5% carbohydrates (58Y2, Test Diet, Richmond, IN). These chows are considered “paired” diets by the manufacturer and are intended to be used in tandem for diet-induced obesity (DIO) studies . The mice were fed daily with fresh high- or low-fat chow (5 g/day) for 6 months . Mice were housed in the vivarium facility at the University of Massachusetts Boston under enriched conditions at a constant temperature (22–23 °C) with a 12/12-h light/dark cycle and optimal humidity and free access to tap water and food ad libitum. Upon completion of the studies, mice were killed and a portion of the liver tissues snap-frozen in liquid nitrogen and stored at − 80 °C, while another portion was fixed and paraffin-embedded. All animal procedures were performed using protocols approved by the Institutional Animal Care and Use Committee (IACUC).
Formalin-fixed paraffin-embedded tissue sections of 5 μM were deparaffinized using reagents and protocols from the manufacturer (HistoClear, Thermo Fisher Scientific, Waltham, MA). Antigen retrieval and immunohistochemistry were performed as described previously using HistoFine Simple Stain AEC Solution (Nichirei Biosciences, Tokyo, Japan) and Harris hematoxylin solution counterstain (Sigma-Aldrich)  and goat anti-mouse CD8 antibody (Abcam, ab203035). For positive control, mouse spleen sections were incubated with anti-CD8 primary and anti-rabbit secondary antibodies; for negative control, mouse spleen sections were incubated with anti-rabbit secondary antibody only. CD8 expression levels were assessed on whole tissue sections using ImmunoMembrane .
TUNEL staining for the detection of apoptotic cells was accomplished using reagents and protocols from the manufacturer (Roche In Situ Cell Detection Kit). The tissue sections were pre-incubated with DEPC to prevent proteinase K-mediated activation of DNAseI and consequent high numbers of false-positive apoptotic nuclei . As a positive control, sections of normal mouse liver were microwaved for 20 min to induce DNA damage; as a negative control, other sections were incubated, fixed, and permeabilized without exposure to proteinase K.
Collagen content was determined using Masson’s trichrome-stained tissue sections as described previously . The stained tissue sections were digitally imaged using a PathScan Enabler IV and color segmented using a subprogram within MATLAB (R2010a; MathWorks, Natick, Mass) which separates and quantifies color elements from trichrome images, permitting quantitation of blue-stained areas corresponding to extracellular collagen. Using the area of a single tissue section as the denominator, this approach provides a means to calculate the portion of this area that is made up by mature extracellular collagen I (the numerator), hence the percentage of extracellular collagen type I (numerator/denominator × 100) in that tissue section. By extension, this method also permits calculation of the percentage of the entire piece of tissue that is made up of mature extracellular collagen I .
Tissue Homogenization and RNA Preparation
One hundred milligrams of frozen tissue was disrupted in 1 mL TRIZOL reagent (Invitrogen, Carlsbad, CA) using an electric homogenizer until the tissue was completely dissolved in solution. Once homogenized, aliquots of the solution were transferred to Eppendorf tubes and left in TRIZOL at room temperature for 5 min. Phase separation was then performed using 200 µl chloroform for 15 s and left at room temperature for 5 min. Samples were then centrifuged at 12000g for 15 min at 2–8 °C. The aqueous phase (top) was transferred to a fresh RNAse-free tube and RNA precipitated with isopropanol. RNA pellets were collected via centrifugation, washed, resuspended, reprecipitated, suspended in 50 μl DEPC water, and stored at − 80 °C until use.
Library Preparation and Sequencing
Libraries were prepared from RNA isolated from three HFD-fed and three LFD-fed SAMP6 mice. Total RNA samples were DNAse-treated, and 300 ng was used as input to create single-indexed (six base pairs) RNASeq libraries using the TruSeq RNA preparation kit according to the manufacturer’s protocol (Illumina, Carlsbad, CA). Samples were pooled together and sequenced on a two-lane flow cell using onboard cluster generation with the Illumina HiSeq™ 2500 instrument. A 51-cycle paired-end rapid run of the single-indexed RNASeq libraries was performed. Raw data of the Bcl base call files were then demultiplexed upon completion.
Bcl base call files were converted to FASTQ format using Illumina’s bcl2fastq conversion tool v1.8.4. Stringent conversion parameters were set by not allowing a mismatch in the index during the demultiplexing process. Additionally, FASTQ files were created overwriting the default parameter to split the files after 4 million reads, generating a single FASTQ file for each sample replicate. Alignment of reads was carried out using Tophat v2.0.14 post-FASTQ file conversion using Illumina’s bcl2fastq tool v1.8.4 [19, 20]. Alignment parameters for Tophat consisted of utilizing the default parameters, which were sufficient for downstream analysis of data. The reference genome file used for the alignment was the mm10 Genome Reference Consortium GRCm38.p4, and the gene transcript file was provided by iGenomes (Illumina, Carlsbad, CA) . Cufflinks v2.2.1 was used to estimate the relative abundance of transcripts aligned for all the samples . Cuffdiff v2.2.1 was used to perform the differential gene expression analysis post-quantification of the transcripts for the samples . EdgeR analysis of gene transcripts differentially expressed by the livers of HFD-fed and LFD-fed mice was performed using the empirical analysis of digital gene expression data in R (edgeR) using a statistical cutoff and was set to p value ≤ 0.001 . The edgeR table for genes demonstrating ≥ 2 or ≤ 2 fold difference between HFD-fed and LFD-fed mice is provided in Supplementary Table I.
Gene set enrichment analysis using Pathview v1.10.1 was used to elucidate the top cellular pathways that are overall up-regulated, or down-regulated by HFD compared to LFD . In addition, selected pathways of interest were also picked for visualization of the overall regulation of the pathways. The statistical cutoff was set to a q value ≤ 0.05. Network analysis of the differential expression analysis was accomplished using Cytoscape v3.3.0 . Gene sets of twofold or higher up-regulated genes in HFD mice, and 0.1–0.5-fold lower down-regulated genes in the HFD mice with a p value cutoff of 0.05 was used as the gene set entry for the network analysis of the mouse interactome. Interactomes used for this analysis were provided by the Cytoscape program, loaded with the mouse interactome from BioGRID database release 3.4.129.
Gene ontology analysis of the gene sets, both twofold or higher up-regulated genes in HFD mice and 0.1–0.5-fold lower down-regulated genes in the HFD mice, was performed using ClueGO and GOstats. ClueGO v2.2.3 was used for the analysis of the two sets of genes, both up-regulated and down-regulated genes in the HFD mice . GOstats v2.36.0 was used to assess biological processes associated with genes differentially in the livers of the HFD-fed and LFD-fed mice . Statistical cutoff for both computational programs was set to a p value of ≤ 0.05.
NanoString Quantitation of RNA Transcripts
NanoString nCounter™ Mouse Inflammation v2 panel consisting of 248 inflammation-related mouse genes and six internal reference genes was interrogated using the same total RNA isolated from the liver tissues. Hundred nanograms of input total RNA was used in the hybridization reactions as recommended in the NanoString’s nCounter™ Gene Expression Assay Manual (NanoString Technologies, Seattle, WA). After hybridization, data were collected by the nCounter Prep Station v126.96.36.199 and nCounter Digital Analyzer v188.8.131.52. Raw nCounter data were analyzed using the nSolver analysis software v2.5.34. Normalization of the data was carried out in two different steps. First using the geometric mean of the positive controls probes provided by NanoString, the data were normalized to this prior to normalization using the housekeeping probes. Normalized data, and genes with counts higher than 10 were then used for the rest of the analysis. All statistical significance was set to a p value of ≤ 0.05.
Liver Histology and Phenotyping
Given these observations, we assessed that the HFD-fed mouse fatty livers were consistent with NAFLD that exhibited inflammation, but little DNA damage, therefore, had incompletely transitioned from fatty liver to NASH .
Validation of RNASeq Data
Premalignant and Pre-cirrhotic Gene Signatures Associated with Diet-Induced NAFLD
The previous research from our group showed that HFD-fed SAMP6 mice become obese and develop type 2 diabetes, consistent with the onset of metabolic syndrome . The HFD-fed mice also developed markedly enlarged fatty livers, suggesting that they might serve as a good murine model for NAFLD. Livers from HFD-fed mice demonstrated simple steatosis and inflammation, but little DNA damage, suggesting pathological changes consistent with NAFLD fatty liver that had not completely transitioned to NASH . RNASeq studies were largely confirmatory, showing an up-regulation of genes encoding proteins associated with the complement pathway and immune responses, and down-regulation of those associated with metabolic processes [32, 33, 34, 43]. Although histological examination of HFD-fed mouse fatty livers did not exhibit evidence of fibrosis or cancer, bioinformatic approaches applied to transcriptional analysis revealed pre-fibrotic and premalignant transcriptional signatures that were absent in the RNA of LFD-fed mice. These findings suggest that the pathogenesis of both fibrosis and cancer may initiate in fatty livers transitioning to NASH well before associated histological changes are evident. These data suggest that genomic changes driving disease progression toward cirrhosis and hepatocellular carcinoma may be initiated at a very early stage in fatty livers that have not completely transitioned to NASH.
Comparison of the results of the current study with other studies in the literature is complicated by the use of different diets and different mouse strains. In particular, the balance between fat and carbohydrate contents of different mouse chows may account for some of the similarities and differences in liver pathology and RNA signatures observed between studies. For example, Kristiansen et al.  detailed that C57Bl/6J mice fed the high-fat, high-carbohydrate (40% kcal from each) AMylin Liver Nash Model (AMLN) diet for 26 weeks developed liver disease consistent with nonalcoholic steatohepatitis (NASH), e.g., the mouse liver tissues demonstrated high levels of steatosis (score 3) and stage 1–3 fibrosis. RNASeq analysis revealed differential gene expression signatures consistent with elevated inflammatory response pathways, expression of multiple collagen types, and decreased metabolic pathways, similar to the transcript profiles observed in the study reported here. However, Kristiansen et al.  did not report transcript signatures suggestive of association with growth factor pathways or with the transformed phenotype in the AMLN diet-fed mice. The Kristiansen et al. study determined that AMLN mice evinced an elevation in plasma insulin levels of about threefold compared to lean chow-fed mice, but this difference was not statistically significant and was not accompanied by differences in fasting blood glucose levels . Thus, unlike the HFD-fed mice utilized in the current study, the AMLN-fed mice did not develop a phenotype consistent with type 2 diabetes. Another study exclusively examined the role(s) of type 2 diabetes in the development of liver pathobiology. This study, by Zhang et al. , examined the RNA signature of liver tissues from db/db mice, who developed a syndrome similar to type 2 diabetes. Liver tissues from these mice exhibited down-regulation of genes that encode proteins associated with the immune response (including many of the complement genes) and an up-regulation of genes associated with metabolic processes . Some genes associated with hepatocellular carcinoma were up-regulated, though a pro-fibrotic signature was not detected . Notably, the db/db mice were not fed a high-fat diet and did not develop fatty liver. In contrast, the SAMP6 HFD-fed mice developed both type 2 diabetes  and fatty liver. Studies by Kristiansen et al.  and Zhang et al.  suggest that the up-regulation of immune response transcripts and down-regulation of metabolic function transcripts is likely associated more with the intake of high-fat rather than high-carbohydrate diets, though the use of different mouse strains (C57Bl/6, db/db, and SAMP6) in those and the current study likely contribute to the observed differences in liver pathobiologies as well.
Unlike either the Kristiansen et al.  or Zhang et al.  studies, the current study detected a premalignant RNA signature in HFD-fed mouse fatty livers. Studies by Shen et al.  showed that induction of hepatocellular carcinoma with the carcinogen diethylnitrosamine (DEN) was accelerated in C57BL/6 mice fed a high-fat diet compared to those fed with regular mouse chow. These studies further identified activating HRAS mutations in liver tissues of DEN-treated obese mice, which are known to stimulate kinase-mediated signaling pathways. Interestingly, the current study identified significantly elevated levels of transcripts encoding P13K/AKT signaling pathway proteins in the livers of HFD-fed mice, a finding consistent with the identification of amplification and activating mutations of PI3K and Akt genes in human NAFLD-related liver cancers  which has been translated to therapeutic approaches targeting PI3K/Akt signaling to treat human hepatocellular carcinoma . These data suggest that the up-regulation of growth factor receptor tyrosine kinase signaling pathways in fatty liver may signal cellular changes consistent with incipient tumorigenesis. The current study also showed that HFD-fed mice demonstrated up-regulation of genes encoding proteins in the Wnt/β-catenin signaling pathway which is known to be subject to mutation and up-regulation in ~ 50% of human hepatocellular carcinomas . Genes encoding G-protein-coupled receptors (GPCRs), which promote cell survival, proliferation, invasion, metastasis, neovascularization, and cell–cell signaling contributing to hepatocellular carcinoma, often through cross-talk with PI3K and Wnt signaling pathways , were up-regulated in hepatic tissues from HFD-fed mice. Notably, many of the genes that were up-regulated in hepatic tissues from HFD-fed mice encode proteins that “drive” expression of the hallmarks of cancer across diverse tumor types, including (as noted above) cell proliferation, angiogenesis, PI3K/Akt signaling, and cell–cell signaling .
Transcriptomic data reported here identified a pre-fibrotic signature that was evident at the transcriptional, but not protein or histological, levels. Hepatic fibrosis is associated with increased levels of ECM, which include collagen, laminin, hyaluron, elastin, and fibronectin . Genes encoding collagen and laminin, but not fibronectin or elastin, were up-regulated in hepatic tissues from HFD-fed mice. Taken together, these data suggest that the gene expression profile of liver tissues from HFD-fed mice displays an incipient and early, though incomplete, fibrotic signature.
Despite the use of different mouse models and dietary approaches, common themes emerge from a consideration of the studies discussed above and those reported here. Clearly, both high-fat and high-carbohydrate diets contribute to the development and progression of NAFLD. However, the combined data reported above suggest that the transition from NAFLD to NASH and to hepatocellular carcinoma may be more strongly promoted by a high-fat than high-carbohydrate diet. However, the current study suggests that high-fat diet alone may be insufficient for a complete transition from NAFLD fatty liver to NAFLD NASH. Clearly, the development and analysis of additional mouse models would be useful to better delineate these pathobiological mechanisms.
Although the present study was limited by the relatively small number of mice examined, the data show a clear distinction between HFD- and LFD-fed mice, suggesting that the major results and conclusions would not have changed significantly by including a higher number of mice in the study. Another potential limitation is the inability to carry out the study to its logical conclusion, e.g., keeping the mice on their respective diets for 12–24 months. This limitation is unavoidable, however, as the high-fat diet is intolerable over extended periods of time and can lead to acute lower urinary tract dysfunction, including acute urinary retention, which can be fatal .
The studies described above imply that perhaps more aggressive clinical attention and intervention may be warranted for patients with NAFLD. One recent study reached this conclusion and proposed increased clinical awareness and improved screening strategies to translate recent treatment progress into early treatment and improved quality of life for patients with type 2 diabetes and fatty liver . NAFLD patients can benefit from treatment options including exercise- and dietary intervention-based lifestyle changes  to improve the disease biochemically and histologically. Evidence that NAFLD is now the most common cause of liver disease in children from the developed world  provides additional rationale for early intervention to prevent liver damage and progression to cirrhosis or carcinoma among NAFLD patients. Fortunately, the use of mouse models such as those presented here and in the studies cited above may identify molecular DNA, RNA, and protein markers that can determine the extent of liver pathobiology even in the absence of tissue histopathology. Such markers could be useful for identifying patients who may benefit from lifestyle changes to reduce liver tissue injury and help repair organ function at an early stage before irreparable damage occurs.
High-fat diet-fed SAMP6 mice develop “diabesity,” characterized by concurrent obesity and metabolic syndrome, as well as markedly enlarged fatty livers, suggesting that they might serve as a good murine model for NAFLD. High-fat diet-fed SAMP6 mouse fatty livers physiologically mimic human NAFLD in that they demonstrate simple steatosis and an up-regulation of genes encoding proteins associated with the complement pathway and immune responses, and down-regulation of those associated with metabolic processes. These livers also showed pre-fibrotic and premalignant transcriptional signatures, suggesting that the pathogenesis of both fibrosis and cancer may initiate in fatty livers well before associated histological changes are evident. The use of molecular makers identified from mouse studies could be useful for identifying patients who may benefit from lifestyle changes to reduce liver tissue injury and help repair organ function at an early stage before irreparable damage occurs.
Thank you to the members of the CPCT laboratory for assistance and support. Funding for this research was provided in part by the Sanofi Genzyme grant to the College of Science and Mathematics, the Massachusetts Life Science Center, and NIH/NIGMS R25 GM076321 (D.A). We thank Dana-Farber/Harvard Cancer Center in Boston, MA, for the use of the Rodent Histopathology Core, and Dr. Roderick Bronson, who provided histodiagnostic service and is supported in part by an NCI Cancer Center Support Grant # NIH 5 P30 CA06516.
DA and MG-K performed mouse husbandry and molecular analyses. DA, JAR-N, CC, and TR performed RNA sequencing data alignment and analyses. JAM designed and supervised the studies. Funding was provided by the Sanofi Genzyme grant to the College of Science and Mathematics, the Massachusetts Life Science Center (J.A.M.), and NIH/NIGMS R25 GM076321 (D.A). The authors have no competing interests.
Compliance with ethical standards
Conflict of interest
Other than the financial support cited above, there are no further financial or other conflicts of interest to disclose.
This study is fully compliant with publication and institutional ethical standards.
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