Introduction
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‐
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 [
4]. 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) [
6]. 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 [
7].
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 [
8]. 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 [
9]. 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.
Discussion
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 [
9]. 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 [
31]. 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‐
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. [
44] 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. [
44] 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 [
44]. 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. [
45], 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 [
45]. Some genes associated with hepatocellular carcinoma were up-regulated, though a pro-fibrotic signature was not detected [
45]. 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 [
9] and fatty liver. Studies by Kristiansen et al. [
44] and Zhang et al. [
45] 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. [
44] or Zhang et al. [
45] studies, the current study detected a premalignant RNA signature in HFD-fed mouse fatty livers. Studies by Shen et al. [
46] 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 [
47] which has been translated to therapeutic approaches targeting PI3K/Akt signaling to treat human hepatocellular carcinoma [
48]. 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 [
49]. 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 [
50], 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 [
51].
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 [
52]. 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 [
9].
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 [
53]. NAFLD patients can benefit from treatment options including exercise- and dietary intervention-based lifestyle changes [
54] 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 [
55] 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.