Both pediatric and adult liver disease specialists are acutely aware of the growing problem of nonalcoholic fatty liver disease (NAFLD). The rise in the prevalence of NAFLD parallels the increase in global obesity rates, and the vital role of obesity and insulin resistance (IR) in NAFLD is undisputed. Not all obese individuals develop fatty NAFLD, however, and only a relative minority (5%-30%) meet strict histopathologic criteria for nonalcoholic steatohepatitis (NASH).1 Essential questions remain as to why some patients with NAFLD develop NASH and others do not. As those with NASH are at the greatest risk of progression to cirrhosis and liver cancer, it is important to understand the mechanisms that lead to necroinflammation and fibrosis from a substrate of hepatic steatosis.
The mediators of hepatic fibrosis have recently become a focal point for investigation as researchers attempt to gain a better understanding of the processes that trigger the cascade of activity that leads to collagen formation and deposition within the hepatic parenchyma. Zone 3 perisinusoidal fibrosis is commonly found in early adult disease with subsequent progression to portal fibrosis, whereas in pediatric NASH portal fibrosis is typically found early.2, 3
Hepatocyte regeneration in a normal liver occurs in the lobules by way of replication of hepatocytes. In the setting of liver injury, hepatic progenitor cells (HPCs), which are normally quiescent and are located in the canals of Hering, appear to play an essential role in the development of portal fibrosis. HPCs come into play as a secondary source of hepatocytes by way of hedgehog ligand-mediated epithelial to mesenchymal transformation (EMT).4 EMT also occurs during normal development as well as in tumor metastasis resulting in increased cellular proliferation. In the liver, EMT-derived hepatocytes are less vulnerable to apoptosis, although activation of HPCs also leads to the so-called ductular reaction (DR) with small biliary ductules composed of stromal and inflammatory cells producing proteins that activate hepatic stellate cells leading to fibrosis. This was elegantly shown by Richardson et al.5 in a study of 107 adult NAFLD patients and 11 controls where HPC expansion and DR was strongly associated with NASH and stage of fibrosis.
Further study in obese murine models has supported the role of EMT in hepatic fibrosis where exposure to the hepatotoxin ethionine leads to liver injury and subsequent expansion of hedgehog-responsive epithelial progenitor cells.6 Similarly, cirrhotic murine livers have been shown to demonstrate features of EMT compared to normal liver-derived hepatocytes after treatment with the apoptosis-inducing cytokine transforming-growth factor beta (TGF-β).7
In pediatric NAFLD, while the role of portal fibrosis has been established, the role of HPCs has not previously been investigated.8 In this issue of HEPATOLOGY, Nobili et al.9 assessed the role of HPCs in pediatric NAFLD comparing histologic specimens from normal controls, isolated fatty livers, and those meeting histopathologic criteria for NASH. This study confirmed an expanded HPC compartment in the setting of pediatric NASH that was correlated to the degree of fibrosis. This intuitively makes sense given the degree of portal-based fibrosis seen in pediatric populations and this study points to the role of DR in fibrosis in pediatric as well as adult NASH.
The importance and prominence of an intermediate hepatocyte (IH) pool was an additional novel finding of this study. IHs were defined as cells of intermediate size that showed faint cytokeratin-7 (CK7) immunoreactivity as found in cells of biliary epithelial lineage. In other chronic liver diseases such as hepatitis C, IHs have been shown to be associated with increased rates of hepatocellular carcinoma (HCC)10 and in this study, IHs were associated solely with NASH and were not seen in NAFLD without steatohepatitis. The number of IHs in pediatric NAFLD was directly associated with the number of HPCs (r = 0.685, P < 0.001) as well as the presence of hepatocyte ballooning (r = 0.534, P < 0.01) and NAFLD activity score (NAS) (r = 0.428, P < 0.05). This would suggest that stimulation of the HPC compartment with the production of IHs is an essential component of pediatric NASH, but requires further study in larger patient populations as well as in adult populations with NASH, particularly because IHs were not independently associated with fibrosis, steatosis, or inflammation.
The adipocytokines (or adipokines) produced by metabolically active visceral adipose tissue have been shown to be important regulators of insulin sensitivity as well as hepatic steatosis, necroinflammation, and fibrosis. This study is the first to investigate the expression of three adipokines by HPCs thought to be important in NAFLD. Adiponectin has been consistently shown to be deficient in NASH patients where it is associated with increased visceral fat and IR.11 Adiponectin expression in the HPCs of pediatric NASH was inversely associated with the NAS (r = −0.792, P < 0.001), hepatocyte ballooning (P = −0.408, P < 0.05), steatosis (−0.7609, P < 0.001), and lobular inflammation (−0.487, P < 0.01), but not fibrosis. Although this is consistent with our current knowledge of decreased adiponectin in NASH, the lack of association with HPC number and IR contradicts the findings of previous studies.5 Another difference from previous studies is that low adiponectin typically equates with increased IR, although this was not demonstrated here. One explanation for this difference is the pediatric population of this study, where IR is not always a prominent feature of disease and does not predict fibrosis.12 Further study in both adult and pediatric populations is required, ideally with an assessment of both hepatic as well as adipose tissue IR which may better reflect severity of NASH.13
Resistin is produced by adipocytes as well as macrophages and in animal models impairs insulin signaling by way of suppressor of cytokine signaling-3 (SOCS3), promotes the expression of the inflammatory cytokines tumor necrosis factor alpha (TNFα) and interleukin-12 (IL-12), and stimulates hepatic stellate cells, thus promoting fibrinogenesis.14 Resistin expression was found to be expanded in the HPCs of pediatric NAFLD patients and was correlated with the degree of fibrosis. This is entirely consistent with our current understanding of both pediatric and adult NASH and represents a potential mechanism of stellate cell activation and a possible novel target for NASH therapy. In fact, a recent study showed vitamin D deficiency in young obese rats led to steatohepatitis with increased resistin expression.15 Further study is required to determine if inhibition of resistin represents a novel pathway that may directly target hepatic fibrosis.
Glucagon-like peptide-1 (GLP-1) is an incretin hormone released by the intestine after meals that stimulates insulin and inhibits glucagon secretion. Incretin mimetics are prescribed in diabetes, where they improve glycemic control as well as promote modest weight loss. In this study, GLP-1 expression by HPCs was shown to be increased in pediatric NAFLD and was highest in those with definitive steatohepatitis. This is in contrast to a recent study evaluating the expression of GLP-1 receptor (GLP-1r) in adult NASH livers as well as obese rats with histopathology consistent with NASH, where hepatocyte expression of GLP-1r was reduced.16 In this and another recent animal study, treatment with the GLP-1 analog exenatide improved hepatic IR and fatty acid oxidation.17 Potentially, these disparate findings can be explained by the fact that the pediatric study was focused on HPCs, whereas the later study was hepatocytes of adult livers. Further study involving both cell types and assessing GLP expression would undoubtedly be of benefit.
Our understanding of the pathogenesis of NAFLD and NASH has come a long way from the original two-hit hypothesis first proposed in 1998. Differences between pediatric and adult NASH are becoming more evident and the work presented by Nobili et al. in this issue of HEPATOLOGY has expanded our knowledge base with regard to the role of HPCs and the DR in pediatric NASH fibrosis. The ever-complicated NAFLD jig-saw puzzle now has a few more pieces put in place but further work remains before we fully understand what the picture really looks like.