Nonalcoholic Fatty Liver Disease: From Pathogenesis to Emerging TreatmentThe multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD)
Introduction
Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome and is defined as the accumulation of fat in the liver in patients who do not consume excessive alcohol. The prevalence of NAFLD is 20–30% in adults and is higher in industrialized countries [1]. NAFLD is asymptomatic in most affected patients and is associated with obesity and features of the metabolic syndrome, namely hypertension, dyslipidemia, central adiposity and insulin resistance (IR) or diabetes [2], [3]. The term NAFLD encompasses a wide spectrum of conditions, from simple accumulation of fat (‘fatty liver’ or steatosis) to steatohepatitis (NASH), fibrosis and cirrhosis with its clinical consequences [4].
Despite its high prevalence, only a small proportion of subjects with NAFLD have NASH with consequent higher risk of liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) (Fig. 1). While patients with simple fatty liver have similar life expectancy to the general population, those with NASH have an impaired survival, due primarily to cardiovascular and liver-related causes. This concept was recently challenged by two studies with longitudinal follow-up, which showed that advanced fibrosis, but not the presence of NASH (as diagnosed by the NAS score) predicted overall mortality in patients with NAFLD [5], [6].
This could be due to shortcomings of the NAS score, as the presence and grading of steatosis have a disproportionate impact compared to lobular inflammation and ballooning, while portal inflammation is not part of the score [7]. Alternatively, lobular inflammation and ballooning could be epiphenomena similarly to simple steatosis, not related to the activation of fibrogenic pathways.
The diagnosis of NAFLD remains one of exclusion and liver biopsy is still the gold standard to differentiate fatty liver from NASH and to stage fibrosis, although several noninvasive markers have been recently introduced for the latter [8].
The underlying mechanism for the development and progression of NAFLD is complex and multifactorial. Different theories have been formulated, leading initially to the ‘two hits hypothesis’. According to this, hepatic accumulation of lipids secondary to sedentary lifestyle, high fat diet, obesity and insulin resistance, acts as the first hit, sensitizing the liver to further insults acting as a ‘second hit’. The ‘second hit’ activates inflammatory cascades and fibrogenesis [9]. This has been supported by animal models of obesity, such as the leptin deficient ob/ob mice, characterized by increased hepatic lipid accumulation, where a second insult is necessary to initiate inflammation and fibrosis [10].
However, it became rapidly evident that this view is too simplistic to recapitulate the complexity of the human NAFLD where multiple parallel factors, acting synergistically in genetically predisposed individuals, are implicated in the development and progression of disease.
Consequently, a multiple-hit hypothesis has now substituted the outdated two-hit hypothesis for the progression of NAFLD. In this review, we explore the potential mechanisms that are implicated in the pathogenesis and progression of NAFLD and that delineate the multiple-hit hypothesis.
Dietary habits, environmental and genetic factors can lead to the development of insulin resistance, obesity with adipocyte proliferation and changes in the intestinal microbiome.
Insulin resistance is one of the key factors in the development of steatosis/NASH and results in increased hepatic de novo lipogenesis (DNL) and impaired inhibition of adipose tissue lipolysis, with consequent increased flux of fatty acids to the liver [11]. Insulin resistance also promotes adipose tissue dysfunction with consequent altered production and secretion of adipokines and inflammatory cytokines [12].
Fat accumulates in the liver in the form of triglycerides, and this happens contemporarily with increased lipotoxicity from high levels of free fatty acids, free cholesterol and other lipid metabolites: as a consequence, mitochondrial dysfunction with oxidative stress and production of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress associated mechanisms, are activated [13].
Also, altered gut flora leads to further production of fatty acids in the bowel, increased small bowel permeability and thus increased fatty acid absorption and raised circulating levels of molecules which contribute to the activation of inflammatory pathways and release of proinflammatory cytokines such as IL-6 and TNF-α [14].
In subjects predisposed by genetic factors or epigenetic modifications, all these factors affect hepatocyte fat content and liver inflammatory environment, thus leading to a state of chronic hepatic inflammation (Fig. 2) through heterogeneous hepatocellular damage pathways, with possible progression to hepatocellular death (for both direct toxicity and apoptosis activating mechanisms), activation of hepatic stellate cells and deposition of fibrous matrix.
Although the dictum was that steatosis always precedes inflammation, it is now recognized that NASH can be the initial liver lesion: the timing and combination of genetic, external and intracellular events rather than the simple sum of hepatic insults result in different pathways which lead to steatosis or NASH respectively [15].
Section snippets
Fat Metabolism, Lipotoxicity and Insulin Resistance
Fat accumulates in the liver of patients with NAFLD mainly in the form of triglycerides [16], [17]. Triglycerides derive from esterification of glycerol and free fatty acids (FFAs). Once synthesized, triglycerides enter storage or secretory pools, which have distinct rates of turnover. FFAs derive either from diet or from the adipose tissue via lipolysis and/or from hepatic DNL. Once in the hepatocytes, FFAs undergo acyl-CoA synthases activity and form fatty acyl-CoAs, which may enter either
Adipose Tissue Dysfunction
Adipose tissue is not inert as traditionally thought but has an endocrine function and secretes hormones (adipokines) such as leptin and adiponectin [67].
Obesity-related adipocyte hypertrophy and/or insulin resistance result in an imbalance of adipokines that may profoundly affect not only the adipose tissue itself but also the liver [68].
Adipose tissue contributes to the maintenance of low-grade inflammatory states by producing pro-inflammatory cytokines: serum levels of IL-6 and adipocytes
Genetic Determinants
Genetic variants, especially in the form of single nucleotide polymorphisms, influence hepatic FFAs flux, oxidative stress, response to endotoxins and cytokine production and activity, and are determinants of NAFLD development and progression [88].
Genome-wide association studies have established the role of patatin-like phospholipase 3 (PNPLA3) single nucleotide polymorphisms in the development and progression of NAFLD, in particular the I148 M (rs738409 C/G) variant. PNPLA3 gene encodes for a
Epigenetic Factors
Clinical studies investigating epigenetic reprogramming in NASH are just beginning to emerge.
Epigenetic modifications are stable changes at transcriptional level, such as DNA methylation, histones modifications and activity of microRNAs (miRNAs), which do not alter the basic DNA sequences and contribute to the cell homeostasis exhibiting a high degree of developmental and environmentally driven plasticity [100]. It has been hypothesized that the disruption of this balance may determine an
Dietary Factors
Dietary factors, both in terms of quantity and caloric intake but also of specific nutrients, contribute to the development of NAFLD and NASH. In a study of 18 healthy individuals, doubling the regular caloric intake with fast-food based meals resulted in profound ALT elevations and increased steatosis in less than 4 weeks [117].
Fructose is a lipogenic, pro-inflammatory dietary factor that results in oxidative stress and upregulation of TNF-α [118]. It is cleared by the liver within first-pass
Conclusions
The pathogenesis of NAFLD and its progression is a complex process, which cannot be completely explained by the ‘two-hit’ hypotheses. It is becoming increasingly evident that the different histological patterns of NAFLD, namely simple steatosis and NASH not only have different risk of progression but may also reflect different disease entities in terms of pathogenesis. A number of diverse parallel processes contribute to the development of steatosis and liver inflammation. The gut microbiome
Conflicts of Interest
None.
Acknowledgments
Dr. Buzzetti was supported by an educational grant from AIGO (Associazione Italiana Gastroenterologi Ospedalieri) and MIMI (Montecatini Interactive Medicine International).
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