Non-alcoholic fatty liver disease

Editor in Chief: Elliot Tapper, M.D., Beth Israel Deaconess Medical Center

Overview
Non-alcoholic fatty liver disease (NAFLD) is a spectrum of liver disease in the absence of excessive alcohol use that begins as fatty accumulation in the liver (hepatic steatosis). A fatty liver does not necessarily disturb the function of the liver, but by varying mechanisms and insults, it may progress to inflammation of the liver. When inflammation occurs in this setting, the condition is then called NASH - Non-alcoholic steatohepatitis. NASH, in turn, may progress to fibrosis and, later, cirrhosis. Studies of serial liver biopsies estimate a 26-37% rate of hepatic fibrosis and 2-15% rate of cirrhosis in less than 6 years. In 2001, NASH represented 2.9% of the indications of liver transplantation. The impact of NAFLD is manifest at each step along the spectrum of the diease. Studies in the United States and Sweden have revealed that both simple steatosis as well as steatohepatitis significantly reduce life expectancy, even when the diagnosis is made in children.

NAFLD/NASH was first described in a 1980 series of obese, non-alcoholic patients of the Mayo Clinic. Since that seminal description, our understanding of NAFLD has progressed minimally. The disease is most closely associated with the increasing obesity, insulin resistance, type two diabetes mellitus and hyperlipedmia endemic to the developed world. Roughly half of all patients with NAFLD, however, do not meet criteria for metabolic syndrome.

Epidemiology
Estimates are that between 30- 90 million Americans have some degree of NAFLD and 5-6% have NASH. A Japanese study estimates the prevalence of NAFLD in that country at 31 per 1000 and found an incidence of approximately 10% - 308 new cases of NAFLD in a group of 3,147 patients followed over 414 days. . In the third National Health and Nutrition Examination Survey (NHANES III), the peak prevalence of NAFLD in men occurred in the fourth decade and in the sixth decade for women.

Some have suggested a genetic or sociocultural component to NAFLD spectrum disease. As a part of the Dallas Heart Study, 2,240 patients - 1,105 african-americans, 401 hispanics and 734 caucasians - received abdominal MRI's from which we can infer the presence of steatosis. Hepatic steatosis was found in 45% of hispanics (both men and women), 33% of caucasians (42% of men, 24% of women) and 24% of african-american (23% of men, 24% of women). This pattern may hold true in children as well. In a San Diego study of 742 consecutive autopsies of children victims of trauma over 10 years, fatty liver was found in 9.6% of all children, 38% of the obese, 12% of hispanics, 10% of asians, 8.6% of caucasians and 1.5% of african-americans.

Overview
The exact cause is still unknown. However both obesity and insulin resistance likely play a strong role in this disease process. The exact reasons and mechanisms by which this disease progresses from steatosis to steatohepatitis and fibrosis is a subject of much research and debate. The prevailing wisdom comes from the so-called ‘two-hit hypothesis.’ The first hit is steatosis. The second hit is controversial and is likely numerous; likely any injury which causes a change that leads from hepatic steatosis to hepatic inflammation and fibrosis by way of lipid peroxidation. Oxidative stress, hormonal imbalances, endotoxemia and mitochondrial abnormalities may all be potential causes.

Insulin Resistance
Clinicopathologic studies in patients with NAFLD have confirmed that insulin resistance is a cause and not a result of liver disease. For example, 75% of NAFLD/NASH patients – lean or obese – having insulin resistance while only 7.5% of patients with Hepatitis C are insulin resistant. In another study, a fasting glucose >110 mg/dL was the most predictive feature of NAFLD.

Comorbid Liver Disease
The evidence for oxidative stress as a second-hit comes from many sources. For example, as in hemochromatosis liver disease, any degree of iron overload engenders enhanced free radical production in the liver. As many as 31% of patients with NASH have been discovered to have C282Y mutations of the HFE gene for hemochromatosis(compared to a prevalence of less than 12.5% in the general population). Similarly, while 2-3.6% of the population have the profibrotic MZ alpha-1 antitrypsin deficiency phenotype, it is present in as many as 16.7% of NASH patients requiring liver transplantation.

Endotoxins
One of the original theories of NASH pathogenesis derived from clinical experience involving obese patients who developed cirrhosis after a jejuno-ileal bypass. This sort of intestinal deformity may increase the concentration of bacterial endotoxins in the portal circulation, which in turn may cause an elevation of intrahepatic levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha. One study found the rate of small bowel bacterial overgrowth to be present in twice as many patients with NASH as control. Furthermore, some degree of steatohepatitis can even be reversed after treatment with metronidazole.

Adiponectin
Many groups have implicated variations in different metabolic pathways. One of the principle pathways under investigation is that which is affected by adiponectin. Adiponectin is an anti-atherogenic, insulin sensitizing cytokine whose secretion is decreased in obesity. One study found an inverse relationship between circulating concentrations of adiponectin and tumor necrosis factor. . Another implication of the research on adiponectin is that different dietary fats have variable effects on adiponectin levels, with polyunsaturated fatty acids leading to decreased levels and more hepatic inflammation.

Adenosine
Another pathway under investigation is purinergic metabolism. CD39 is the dominant vascular (and immune cell) ectonucleotidase in the liver that hydrolyzes extracellular ATP and ADP to AMP which can then be converted to adenosine via ecto-5’-nucleotidase/CD73. Alterations in purinergic signaling induced by altered CD39 expression have major impacts upon hepatic metabolism, repair mechanisms, regeneration and associated immune responses. Varying levels of CD39 and adenosine have thus been implicated in the spectrum of NAFLD/NASH phenotypes.

Based on knockout studies, the experimental evidence is mounting in support of a major role for both CD39 and adenosine in the development of steatosis, inflammation and, later, fibrosis. Firstly, the deletion of CD39 and thus the local reduction of adenosine results in hepatic insulin resistance and increased serum levels of several inflammatory cytokines.

Secondly, adenosine appears to be a critical supportive link in the cell’s cascade of responses to inflammation; Adenosine suppresses inflammation and, as inflammation, tissue repair and scarring are closely linked events, it enhances fibrosis by increasing matrix formation in healing insulted tissues and facilitating regeneration. CD39 deletion shifts the local population of cytokines toward the pro-inflammatory and non-fibrinogenic (e.g. interferon gamma).

Thirdly, in CD39 knockout models of hepatitis and pancreatitis, there is a marked decrease in fibrogenesis. And adenosine receptor A2A is a major factor in the pathogenesis of cirrhosis.

Fibroblast Growth Factor 21
Fibroblast growth factor 21 (FGF21) has emerged as an important metabolic regulator of glucose and lipid metabolism. Essentially, FGF21 moderates or induces the hepatic response to a fasting state: gluconeogenesis, fatty acid oxidation, and ketogenesis. Moreover, it is a crucial component of the hepatic lipid oxidation machinery. This probably occurs as a function of proliferator-activated receptor activation. .

While the present evidence is contradictory for FGF21's role in the setting of fatty liver, it is evolving. In one study, supplemental, recombinant FGF21 was given to mice and resulted in reduced blood glucose, insulin, and lipid levels and reversed hepatic steatosis. FGF21 also dramatically improved hepatic and peripheral insulin sensitivity. At the same time, studies in humans have shown that circulating FGF21 concentrations were increased in subjects who were either overweight or had type 2 diabetes or impaired glucose tolerance. The most recent study has shown that while FGF21 levels are associated with BMI in humans, they are not nutritionally regulated. It may only be a marker of - not causally linked to - NAFLD.

Uric Acid
Another candidate in the pathophysiology of NAFLD is uric acid. While it remains to be seen whether uricemia is causal or a marker of disease, a hypothesis generating paper from China implicates uric acid in NAFLD. A population-based prospective study in China to found that 11.80% (813/6890) subjects developed NAFLD over 3 years of follow-up. Interestingly, the incidence of NAFLD increased with progressively higher baseline serum uric acid levels (7.2%, 9.5%, 11.5%, 13.8%, and 17.2% in quintile 1, quintile 2, 3, 4 and 5, respectively). In animal studies conducted by the same group, they were able to show that hypouricemic medications reduced hepatic steatosis and hyperlipidemia.

Secondary causes
NAFLD can also be caused by the following medications (termed secondary NAFLD):
 * Amiodarone
 * Antiviral drugs (nucleoside analogues)
 * Aspirin / NSAIDS
 * Corticosteroids
 * Methotrexate
 * Nifedipine
 * Perhexiline
 * Tamoxifen
 * Tetracycline
 * Valproic acid

Signs, Symptoms and Associations
Most patients with NAFLD have no or few symptoms. Infrequently patients may complain of fatigue, malaise and dull right upper quadrant abdominal discomfort. Mild jaundice can rarely be noticed. More commonly it is diagnosed as a result of abnormal liver function tests during routine blood tests. Often following an asymptomatic course, the disease may present first with cirrhosis and/or the complication of portal hypertension. In that respect, NASH is becoming an increasingly common indication for liver transplantation. As awareness of this condition spreads, it has been regarded as a major cause of cryptogenic cirrhosis of the liver. The diagnosis of cryptogenic cirrhosis is usually made in patients with similar clinical characteristics to those with NAFLD spectrum disease. Cryptogenic cirrhotics tend to be women, aged 63 (+/- 11) years who are obese and type 2 diabetics. Moreover, there are case reports of patients with NASH who received serial liver biopsies where there was a progression to cirrhosis with a dissapearance of the histologcal stigmatia of NASH. Without the index biopsy, these patients' cirrhosis would have been classified as cryptogenic.

Diagnosis
Disturbed liver enzymes are common. Typically, one finds a 2-4 fold elevation of the ALT above normal limit and an ALT/AST ratio of greater than 1. This ratio is imperfect, as AST tends to rise with the degree of fibrosis. Furthermore,high ALT values within the reference range (less than 40 IU) are still predictive of NAFLD/NASH. Another blood test that can be elevated is the ferritin. Typically, and except in very advanced disease, the liver's synthetic function is intact with normal albumin and INR.

Imaging is often ordered in the workup of suspected NAFLD. Ultrasound and computed tomography have sensitivities between 93-100%, but 62-76% positive predictive values. Problematically, ultrasound of fatty liver reveals a hyperechoic echotexture - a so-called 'bright liver' - that can often be indistinguishable from fibrosis and generally cannot reliably delineate NAFLD from NASH. Computed tomography is less sensitive, rarely detecting steatosis when fatty infiltration is less than 33%, but is potentially more specific. Statistics are similar for MRI, however using advanced MR techniques, some groups have been able to both quantify steatosis and differentiate steatohepatitis from steatosis.

A biopsy of the liver is still considered the gold standard in diagnosis. This is especially true for those patients with elevated liver enzymes for whom a non-invasive workup is inconclusive; 34% of these patients, in one series, were found to have NASH. Classically, biopsy reveals macrovesicular steatosis, inflammation, ballooning degeneration, zone 3 perivenular/periportal/perisinusoidal fibrosis and, finally, mallory bodies. Unfortunately, however, a standard biopsy is only able to sample a volume that is 1/50,000th of the liver, underscoring substantial room for sampling error.

When considering NAFLD, other tests are generally performed, including those for associated conditions (e.g. glucose, hemoglobin A1C) and those to distinguish this disease from viral hepatitis. Additionally, autoimmune causes are ruled out with serology. TSH is warranted, as hypothyroidism is more prevalent in NASH patients.

Treatment
Trials are presently being conducted to optimize treatment of NASH. No standard treatment has yet emerged as the "gold standard". General recommendations include improving metabolic risk factors - weight loss, treating diabetes, managing lipids - and reducing alcohol intake. One of the largest trials of NAFLD/NASH therapy evaluated rosiglitazone, an insulin-sensitizing thiazolidenedione. This study found, after one year, no significant improvement in markers of liver injury. In May of 2010, Sanyal and colleagues published a randomized, placebo controlled trial of Vitamin E (800 IU daily) and pioglitazone (30 mg daily) with paired-biopsies. They found that Vitamin E was best able to achieve improvement in NAFLD/NASH, vis-a-vis improvements in the histological stigmata of NASH (steatosis, lobular inflammation, ballooning degeneration and fibrosis). Pioglitazone was able to achieve significant improvements in some of the individual markers. Steatohepatitis resolved in 47% of patients taking Vitamin E and 36% taking pioglitazone. Neither medication could achieve significant improvements in fibrosis or portal inflammation.