Non-alcoholic fatty liver disease: an emerging pathological spectrum
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- Zafrani, E.S. Virchows Arch (2004) 444: 3. doi:10.1007/s00428-003-0943-7
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The spectrum of pathological lesions observed in non-alcoholic fatty liver disease (NAFLD) is wide and strongly resembles that of alcohol-induced liver disease. It ranges from fatty liver to steatohepatitis, progressive fibrosis and cirrhosis. Hepatocellular carcinoma is a possible complication of NAFLD, but whether it is related to frequently associated metabolic disorders (e.g., overweight, diabetes) or to underlying cirrhosis is unclear. This disease is the result of a multi-factorial process in which insulin resistance seems to play a major role in the initial accumulation of fat in the liver, whereas multiple causes of mitochondrial dysfunction and oxidative stress can induce the secondary occurrence of necroinflammatory lesions and fibrosis. Genetic factors might explain why only some patients with simple steatosis will develop steatohepatitis and fibrosis. Due to the increasing prevalence of obesity in Western countries, NAFLD will possibly be a public health problem and the liver disease of the future.
KeywordsNon-alcoholic fatty liver disease Steatohepatitis Liver fibrosis Insulin resistance Mitochondrial dysfunction
Non-alcoholic fatty liver disease (NAFLD) has probably become one of the most common causes of chronic liver disease in Western countries and might be, in the future, an important public health problem [4, 20, 53]. This condition, with an uncertain natural history, may be severe and is characterized by a wide spectrum of pathological lesions, ranging from simple steatosis to steatohepatitis, advanced fibrosis, cirrhosis and even hepatocellular carcinoma. These lesions closely resemble those induced by alcohol, but occur in patients who deny excessive alcohol consumption.
This disease raises numerous questions concerning its definition, epidemiology and associated conditions, its clinical presentation, natural history, pathophysiology and management. Addressing some of these questions will be the major purpose of the present review.
What is NAFLD?
Association of liver steatosis, inflammation and fibrosis has been known for several decades. It has been documented in non-alcoholic patients following surgical jejunoileal bypass for morbid obesity , in obese patients who had not undergone weight-loss surgery  and in diabetics . The term non-alcoholic steatohepatitis (NASH) was coined by Ludwig et al. to describe biopsy findings, including steatosis, necrosis, inflammation, Mallory bodies and fibrosis in patients, mainly obese women, who did not drink alcohol . This has brought interest to this entity and promoted further research, but NASH refers to necroinflammatory lesions associated with steatosis (i.e., steatohepatitis) and only corresponds to part of NAFLD, which is now the preferred term to more accurately designate the much wider spectrum, consisting of simple steatosis, steatohepatitis, cirrhosis and hepatocellular carcinoma. Recent conferences have also stressed the need for a consensus regarding classifying, grading and staging NAFLD [53, 65].
Although the value of liver biopsy in routine practice has been debated, essentially due to the risks and cost of this procedure and due to the lack of effective therapy, it is the only accurate diagnostic method, and it is essential for grading and staging the disease and for including patients in clinical trials [63, 65].
Hepatocellular steatosis (Fig. 1) is usually macrovesicular, made of large intracytoplasmic droplets displacing the nucleus at the cell periphery. Macrovesicular fatty change is occasionally associated with microvesicular steatosis, consisting of small intracytoplasmic lipid droplets leaving the nucleus at the center of the hepatocytes (Fig. 1b). Steatosis of varied degrees may either be diffuse within the hepatic lobule or predominate in zone 3, around terminal hepatic veins. Glycogen nuclei (Fig. 1b) are seen in at least one-third of the patients .
Steatohepatitis is defined by the association of steatosis with other hepatocellular lesions, essentially necroinflammatory changes. Ballooned hepatocytes are enlarged, with a clear cytoplasm, and apoptotic bodies can be noted. Mallory bodies (Fig. 1c), consisting of intracytoplasmic aggregates of cytokeratin intermediate filaments, are not constantly observed, and their presence is not necessary for the diagnosis of steatohepatitis. They may be small, sparse and inconspicuous . Eosinophilic inclusions, corresponding on electron microscopy to abnormal giant mitochondria with loss of cristae (Fig. 1d) and containing paracrystalline formations [15, 66], are occasionally observed in the cytoplasm of the hepatocytes.
Intensity of the inflammatory infiltration varies with the severity of steatohepatitis. Inflammation is usually mixed, made of lymphocytes and neutrophils located in the lobule (Fig. 1e), around altered hepatocytes or in the portal areas (Fig. 1f). The degree of stainable iron, when present, must be indicated.
Fibrosis and cirrhosis
Extent of fibrosis is of prognostic importance. Perisinusoidal fibrosis is initially mild and predominates in zone 3, around terminal hepatic veins (Fig. 1g). It is then associated with portal and periportal fibrosis with progressive formation of fibrous bridges between terminal hepatic veins and portal areas or between adjacent portal tracts (Fig. 1h). Cirrhosis is obtained when hepatocellular nodules are completely surrounded by annular fibrosis. Liver failure, portal hypertension and hepatocellular carcinoma may then complicate the disease [13, 34, 62]. Some degree of fibrosis is found in up to two-thirds of the patients at the time of diagnosis, whereas cirrhosis is noted in up to 16 percent [4, 63]. In cirrhotic patients, histological features suggestive of steatohepatitis (e.g., steatosis, predominance of neutrophils in the inflammatory infiltrate) may have regressed or are no longer present [4, 14, 60]. It is, thus, noteworthy that NAFLD is more frequently considered a major cause of cryptogenic cirrhosis [14, 58, 62, 63].
Grading and staging
Since prognosis of NAFLD depends on the severity of the pathological findings, grading and staging the lesions observed on liver biopsy appears necessary. Grading evaluates the degree of steatosis and necroinflammatory changes, whereas staging estimates the extent of fibrosis.
A system referring to NAFLD correlates certain histological features with long-term prognosis . Type 1 corresponds to simple steatosis, type 2 to steatosis with lobular inflammation, type 3 requires the additional presence of ballooned hepatocytes and type 4 the existence of Mallory bodies or fibrosis . Among 132 patients with a mean follow-up of 9 years, cirrhosis occurred in 3.4 percent of types 1 and 2 patients and in 24.7 percent of types 3 and 4 patients .
Another method, which is limited to the classification of NASH, has been proposed . Three grades of necroinflammatory activity, based on an overall impression of the intensity of hepatocellular and inflammatory changes, have been defined : mild (grade 1), moderate (grade 2) and severe (grade 3). Staging reflects the pattern and the extent of fibrosis as well as architectural remodeling : focal or extensive zone 3 perisinusoidal fibrosis is noted in stage 1; stage 2 adds focal or extensive periportal fibrosis; focal or extensive bridging fibrosis is observed in stage 3; stage 4 corresponds to cirrhosis. Such a grading and staging system is much like what is used for chronic hepatitis C [7, 72], but intra-observer and inter-observer variations in liver biopsy interpretation have not been studied.
Epidemiology and conditions associated with NAFLD
Conditions associated with non-alcoholic fatty liver disease
1. Insulin resistance
2. Disorders of lipid metabolism
3. Severe weight loss
Jejunoileal or gastric bypass
4. Total parenteral nutrition
5. Latrogenic agents
Drugs: amiodarone, tamoxifen, diltiazem
Toxic exposure: industrial solvents
Obesity, defined by a body mass index superior to 30 kg/m2, is clearly associated with NASH, but most patients are moderately overweight [53, 65]. The likelihood of developing NASH increases with the degree of obesity, and resolution of a fatty liver may occur following gradual weight loss . It must be noted that obesity is not invariably present in NASH patients, and many patients have a normal body weight .
Type 2 diabetes is a component of the metabolic syndrome and is associated with obesity and NAFLD [45, 60]. Both obesity and diabetes are associated with peripheral insulin resistance, hyperinsulinemia, increased free fatty acid levels and hypertriglyceridemia. Recently, the demonstration of peripheral insulin resistance, increased fatty acid β-oxidation and hepatic oxidative stress in patients, with either steatosis alone or NASH, suggests a link between obesity, insulin resistance and NAFLD .
In rare disorders of lipid metabolism (e.g., abetalipoproteinemia) as well as in rare syndromes characterized by severe insulin resistance (e.g., limb lipodystrophy), NAFLD with possible advanced fibrosis and cirrhosis may be observed [53, 59]. Other metabolic, surgical and genetic conditions are also associated with NALFD: jejunoileal bypass  or gastroplasty [31, 50] as treatments for morbid obesity, biliopancreatic diversion, extensive small bowel resection and prolonged total parenteral nutrition , small intestinal diverticulosis with bacterial overgrowth , Wilson’s disease  and Weber-Christian syndrome .
Several drugs (e.g., amiodarone, tamoxifen, antiretroviral nucleoside analogues) and environmental factors (e.g., industrial solvents) may be responsible for hepatic lesions identical to those observed in NAFLD and alcohol-induced liver disease [23, 53, 65]. It is of interest that many of these conditions have in common either abnormal fat metabolism or mitochondrial dysfunction. Since NAFLD does not constantly occur in every patient having one of the above mentioned disorders or being exposed to iatrogenic agents, it is reasonable to postulate that this liver disease results from the conjunction or the potentiation of deleterious effects of several factors.
NAFLD and viral hepatitis C
Although steatosis and fibrosis are two of the histological hallmarks of chronic viral hepatitis C , it is generally not admitted that viral C infection should be included in the list of causes of NAFLD. However, it is of interest that, in chronic viral hepatitis C, steatosis seems to be more prevalent in patients infected with the genotype 3a virus  and that its degree is related to body mass index and to progressive fibrosis . Steatosis might be due to a direct cytopathogenic effect of hepatitis C virus, since it has been shown in transgenic mice and in cell cultures that structural and non-structural viral proteins modify triglyceride metabolism and induce mitochondrial alterations, increasing oxidative stress [42, 54]. Steatosis might also interfere with response to antiviral therapy in chronic hepatitis C. Absence of steatosis is, indeed, associated with a better response , whereas high body mass index is an independent risk factor for non-response .
Viral hepatitis C and NAFLD are common causes of liver disease. It is, therefore, not surprising that they can coexist in the same patients. A recent report has shown that steatohepatitis was observed in 5.5 percent of all cases of chronic viral hepatitis C, and that among 85 patients with histological features of steatohepatitis, 54 also had hepatitis C viral infection without history of alcohol abuse .
Clinical presentation, laboratory abnormalities and radiological findings
Most patients with NAFLD are asymptomatic, and the liver disease may be incidentally discovered during evaluation of hypertension, diabetes or morbid obesity [53, 65]. Hepatomegaly is the only physical finding in most patients [4, 6, 45]. Stigmata of signs of chronic liver disease, such as spider nevi, palmar erythema or signs of portal hypertension, should remind that cirrhosis can already be constituted at the time of diagnosis in up to 16 percent of the patients [4, 63] and that NAFLD is probably a major cause of cryptogenic cirrhosis [14, 62, 63].
Mild to moderate elevation of serum aspartate or alanine aminotransferase activities is usually the only laboratory abnormality, and NAFLD is the most usual cause of unexplained persistent elevation of these enzymatic activities, once hepatitis C and other known etiologies of chronic liver disease have been ruled out . The ratio of aspartate aminotransferase to alanine aminotransferase is usually less than 1, but it increases with progression of fibrosis, leading to a loss of diagnostic value in patients with cirrhosis [4, 6]. Serum alkaline phosphatase or γ-glutamyltransferase activities may be mildly elevated. Presence of antinuclear antibodies in the serum, the significance of which is unknown, has been reported in 10 to 25 percent of patients . It is not surprising that 30 to 50 percent of patients with NASH have diabetes or glucose intolerance [26, 40, 45, 75] and that 20 to 80 percent of patients have hypertriglyceridemia [26, 40, 45, 60]. Other abnormalities, including hypoalbuminemia, prolonged prothrombin time and hyperbilirubinemia, may be found in patients with cirrhosis. Changes in blood iron store parameters have been noted in approximately 10 percent of patients [5, 6, 61], but hepatic iron levels are usually within the normal ranges [5, 6, 49, 61]. It has been suggested that iron overload could be associated with increased fibrosis . However, these findings have not been confirmed by other studies [5, 19, 76]. The significance of hemochromatosis gene mutations in the pathogenesis and progression of NAFLD remains controversial [19, 30].
Imaging findings (i.e., increased echogenicity, low density hepatic parenchyma on computed tomography) reflect the presence of steatosis and are useful to detect a space-occupying lesion that could correspond to hepatocellular carcinoma, occurring either on NAFLD-associated cirrhosis or on non-cirrhotic liver.
Natural history of NAFLD: from steatosis to hepatocellular carcinoma?
As already emphasized, NAFLD is characterized by a spectrum of pathological lesions ranging from simple steatosis to cirrhosis and its complications, including hepatocellular carcinoma. It is usually considered that these lesions represent different stages in the natural history of the disorder.
In patients with only fatty liver at the time of diagnosis, progression to steatohepatitis with fibrosis seems rare. In one longitudinal series of 40 patients with only fatty liver, no instance of evolution toward fibrosis or cirrhosis was observed after a median follow-up of 11 years . Other reports also consider NAFLD a low-grade severity disorder [40, 60]. Matteoni et al.’s findings, which stress the importance of histologically grading necroinflammatory activity for predicting progression toward cirrhosis, have already been detailed . As a matter of fact, it must be pointed out that there is no correlation between liver biopsy findings and biochemical abnormalities. It has, indeed, been recently shown that a low normal alanine aminotransferase value is not a guarantee against steatohepatitis with advanced fibrosis, since the entire spectrum of NAFLD could be seen in individuals with normal values, and the histological lesions did not differ whether alanine aminotransferase levels were elevated or not .
In patients with NASH, multivariate analyses show that older age, obesity and diabetes are independent predictors of marked liver fibrosis . In severely obese patients, a raised index of insulin resistance, systemic hypertension and elevated serum alanine aminotransferase activity have been demonstrated to be independent predictors of NASH, whereas moderate alcohol consumption seemed to reduce the risk of NAFLD, possibly by reducing insulin resistance . In obese patients with minimal fibrosis, histological improvement may occur following slowly achieved weight loss . If weight loss is rapid, progression of the disease may be accelerated .
The precise risk of mortality in patients with NAFLD is not known. Many factors might be implicated, including complications and comorbidities of obesity and diabetes and liver disease, itself. Cirrhosis independently increases mortality , probably due to liver failure, portal hypertension and hepatocellular carcinoma. Growing evidence indicates that hepatocellular carcinoma should be considered in the natural history of progressive NAFLD [13, 48, 52, 60], since clinical and biological features suggestive of NASH are more frequently observed in patients with hepatocellular carcinoma arising on cryptogenic cirrhosis than in age- and sex-matched patients with hepatocellular carcinoma arising on viral or alcoholic cirrhosis . Whether tumor development is secondary to the metabolic abnormalities associated with NASH or to underlying cirrhosis is not clear. Interestingly, hepatocellular carcinoma may be observed in the absence of cirrhosis in NASH patients with minimal portal and periportal fibrosis . In addition, the recently published results of a large prospectively studied cohort of American adults has demonstrated that increased body weight, with possible NAFLD, was associated with increased death rates for all cancers, including liver cancers, especially in men .
The pathological lesions and the natural history of NAFLD probably reflect a complex multi-factorial process in which the genetic environment is of importance , since it has yet to be understood why only steatosis occurs in some patients whereas others develop steatohepatitis, cirrhosis or hepatocellular carcinoma. Insulin resistance, abnormal metabolism of fatty acids, mitochondrial dysfunction and oxidative stress, dysregulation of cytokines and production of various mediators are the main interacting factors implicated in the pathophysiology of NAFLD. A recent study has shown that 4 genes contributing to impaired insulin sensitivity were overexpressed in the livers of patients with NAFLD, whereas 12 genes important for maintaining mitochondrial function were underexpressed .
Insulin resistance, fatty acids and steatosis
Whereas steatosis remains the only liver lesion in many patients with NAFLD, some silently develop necroinflammation and progressive fibrosis. The reasons for the possible deleterious effects of steatosis are not completely understood, but the presence of oxidizable lipids in hepatocytes could trigger lipid peroxidation, mitochondrial dysfunction, oxidative stress and cytokine production .
Mitochondrial dysfunction, oxidative stress and cytokine production
Do pathophysiological mechanisms explain the histopathological lesions of NAFLD?
TNF-α and Fas ligand trigger apoptosis by permeabilizing mitochondrial membranes
TGF-β stimulates transglutaminase, which allows polymerization of cytokeratins to form Mallory bodies
Interleukin-8 and 4-hydroxynonenal are chemoattractive for neutrophils
TGF-β activates perisinusoidal stellate cells, and lipid peroxidation products increase their production of collagen, thus, leading to perisinusoidal fibrosis and cirrhosis; in addition, hyperleptinemia, a common finding in obese patients, might promote profibrogenic responses in the liver, in part by upregulating TGF-β [33, 35, 67]
As far as hepatocellular carcinoma is concerned, several clinical studies strongly suggest that it should be considered within the NAFLD spectrum [13, 48, 52, 60], but mechanisms of carcinogenesis in this context have not been studied, and it is, indeed, unclear whether this tumor is related to NASH-related cirrhosis or to metabolic disturbances associated with NAFLD.
Management of NAFLD should take into account the pathophysiological mechanisms that are implicated, the natural history of the disease and the safety and cost of the therapeutic options [53, 65]. Resolution of histological abnormalities, as determined by liver biopsy, should remain the gold standard for treatment outcomes. Until now, there are no published controlled trials of treatment modalities in NAFLD. Management is, therefore, directed toward correction of risk factors, and treatments try to correct putative pathophysiological abnormalities. Exercise and diet are essential, and weight must be controlled. Insulin resistance must be improved by antidiabetic/insulin sensitizing agents, and hyperlipidemia must be treated. Drugs with potential hepatoprotective effects, antioxydants agents and iron reduction therapy may be used [53, 65].
Whether alcohol intake should be prohibited is not clearly established. In the absence of formal data, the pragmatic recommendation is abstinence if significant fibrosis is present . The concomitant use of drugs that may induce steatohepatitis (e.g., amiodarone, tamoxifen) requires weighing of risks and benefits . An interesting unresolved issue is the problem of occupational exposure to industrial solvents and their harmful effects on the liver [23, 53].
Since NAFLD is probably a major cause of cryptogenic cirrhosis, many patients have likely been transplanted for such an indication. Both recurrence and de novo occurrence of NASH have been reported after surgery [21, 37]. Fatty liver constantly recurs within 5 years, but progression to steatohepatitis with fibrosis is very rare, and clinical outcome is similar to that of other groups of patients undergoing transplantation . NAFLD may also develop in patients receiving a graft for indications other than cryptogenic cirrhosis, i.e., alcoholic cirrhosis, primary biliary cirrhosis and primary sclerosing cholangitis . Fatty liver is noted in 25 percent of such patients within 5 years, and there is no instance of progression to fibrosis . The occurrence of NAFLD following transplantation may be related, in part, to weight gain and also to immunosuppression with corticosteroids and cyclosporine [17, 21, 55].
NAFLD is a major cause of liver-related morbidity in Western countries, with possible progression to cirrhosis, liver failure and hepatocellular carcinoma. Liver biopsy remains the best procedure to provide diagnostic and prognostic information. Since insulin resistance and oxidative stress play a major role in its pathogenesis, NAFLD should be searched for in overweight and diabetic patients. Management is usually aimed at reducing weight and treating associated disorders, but the role of specific pharmacological agents remains to be established. Considering the surge of obesity in developed areas of the world, is NAFLD the liver disease of the future?