Nonalcoholic Fatty Liver Disease -- New Insight Into a Major Cause of Obesity-Re

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Nonalcoholic Fatty Liver Disease -- New Insight Into a Major Cause of

Obesity-Related Morbidity and Mortality

Disclosures

F. Balistreri, MD

Introduction

Research presented during this year's meeting of the American

Association for the Study of Liver Diseases (AASLD) addressed

important advances in our understanding of the hepatic consequences

of obesity -- nonalcoholic fatty liver disease (NAFLD), a spectrum of

disorders characterized by macrovesicular steatosis that occur in the

absence of consumption of harmful amounts of alcohol. This spectrum

ranges from fatty liver alone to nonalcoholic steatohepatitis (NASH);

the latter represents a shift from simple steatosis to an

inflammatory/fibrosing disease. Sustained liver injury may lead to

progressive fibrosis and cirrhosis.

The Increasing Prevalence of NAFLD and NASH

NAFLD is the hepatic component of the metabolic syndrome, which is

characterized by obesity, type 2 diabetes mellitus, insulin

resistance, dyslipidemia, and hypertension. Zelber-Sagi and colleagues

[1] screened 326 individuals to explore the utility of NAFLD in

predicting the metabolic syndrome in the general population. The

prevalence of NAFLD was 29% and the prevalence of the metabolic

syndrome was 13%; 71% of individuals with the metabolic syndrome had

NAFLD. Including NAFLD as one of the features of the metabolic

syndrome increased the prevalence to 21%. NAFLD-related morbidity is

highly significant and rivals the costs and resource utilization

associated with the management of hepatitis C virus (HCV) infection.

The number of patients hospitalized for the principal diagnosis of

NAFLD in the United States in 2002 was approximately 70% of the

number of hospitalizations for HCV-related disease.[2]

The prevalence of NAFLD differs among racial groups. Whereas the risk

factors for the metabolic syndrome are highly prevalent in black

patients, there is an unexpected relative paucity of documented NAFLD

in this population. Troy and colleagues[3] reported that Hispanics

and whites were overrepresented and that blacks were underrepresented

in biopsy-proven NAFLD. In addition, in patients with type 2 diabetes

mellitus, the prevalence of NAFLD was higher among whites than among

blacks.[4] The low prevalence of NAFLD in the black population may

result from a difference in genetic susceptibility.

In another study presented during this year's meeting, Xanthakos and

colleagues[5] determined the prevalence of hepatic steatosis in a

population-based cohort of 281 young adult women by measuring hepatic

fat fraction with magnetic resonance imaging (MRI). Only 3% of

subjects had an elevated hepatic fat fraction; the prevalence of

hepatic steatosis was lower in blacks (2%) than whites (4%). Subjects

with an abnormal hepatic fat fraction had higher mean alanine

aminotransferase (ALT) levels, body mass index (BMI), and waist

circumference. Only 4% of all subjects had abnormal serum ALT (> 35

U/L), with no differences among the races. Thus, significant hepatic

steatosis was infrequent in young adult women, despite a high

prevalence of obesity (42%), central adiposity (34%), and elevated

fasting insulin (41%). Prospective studies should further evaluate

the role of age, duration of obesity, and race in determining the

prevalence and severity of NAFLD.

Clinical Features

NAFLD and the Metabolic Syndrome

Individuals with NAFLD have significantly higher levels of glucose,

insulin, triglycerides, serum ALT, and higher BMI and waist

circumference (central adiposity) than controls.[1] The strongest

independent predictors of NAFLD are waist circumference, BMI, and

triglyceride content. NAFLD predicts the metabolic syndrome

independently of insulin and BMI. In addition, NAFLD is a stronger

predictor for the combination of hyperglycemia, low levels of high-

density lipoprotein (HDL), hypertriglyceridemia, and hypertension

than is central obesity.

Although the deleterious effects of central adiposity on glucose and

lipid metabolism are well recognized, independent favorable effects

of peripheral adiposity on features of the metabolic syndrome have

been suggested. Perlemuter and colleagues[6] studied 1155 obese women

to test the hypothesis that trunk fat mass and leg fat mass have

independent and opposite effects on liver injury. In univariate

analyses, serum ALT was positively correlated with BMI and trunk fat

mass, aspartate aminotransferase (AST) was negatively correlated with

height and leg fat mass, and gamma-glutamyltranspeptidase was

positively correlated with total and trunk fat mass. Leg fat mass may

confer independent protective effects against obesity-associated

liver dysfunction in contrast to the deleterious effects of trunk fat

mass. Regional body composition must be taken into account in the

assessment of the relationship between body weight and shape, as well

as in NAFLD.

NAFLD is associated with an increased risk for mortality in patients

with type 2 diabetes mellitus. In this context, and colleagues

[7] compared 116 patients with newly diagnosed type 2 diabetes

mellitus who subsequently developed NAFLD vs 221 patients with type 2

diabetes mellitus who did not develop NAFLD. Overall, 29% of subjects

died. In a multivariable analysis adjusted for differences in age,

sex, and date of diabetes diagnosis, death was significantly

associated with a diagnosis of NAFLD. NAFLD remained an independent

risk factor for mortality when controlling for smoking, hypertension,

obesity, or hyperlipidemia. Only ischemic heart disease was

independently associated with higher mortality among patients with

NAFLD.

Is NAFLD an Early Marker of Coronary Heart Disease?

The prevalence of NAFLD among 100 patients with acute myocardial

infarction was found to be 66% and 50% for women and men,

respectively -- a prevalence significantly higher than that found in

the general population.[8] Moreover, NAFLD was associated with

greater severity of coronary disease. Both of these findings were

independent of BMI. Fracanzani and colleagues[9] reported early

atherosclerotic damage in 125 patients with NAFLD; the independent

predictors of vascular damage were steatosis, age, and systolic blood

pressure. In another study presented during this year's AASLD

meeting, Schwimmer and colleagues[10] hypothesized that fatty liver

is a risk factor for early onset atherosclerosis and that

atherosclerosis begins in childhood and progresses through adulthood

to form the lesions that cause cardiovascular disease. Therefore,

they reviewed the autopsy findings from 817 children. Fatty liver was

present in 15%, mild atherosclerosis was present in 21%, and more

severe atherosclerosis was present in 2% of subjects. Atherosclerosis

was significantly more common in individuals with fatty liver (30%)

than in those without fatty liver (19%). The investigators concluded

that fatty liver may not only be a marker of atherosclerosis but also

a mediator.

Novel NAFLD-Associated Clinical Features

Hepatocellular Carcinoma. Primary liver cancer is a major cause of

mortality in NASH. The combination of obesity, NASH, and

hepatocellular carcinoma (HCC) is a new, emerging triad that was

essentially nonexistent before 1999.[11] Yatsuji and colleagues[12]

documented that older age and advanced fibrosis were significant risk

factors for HCC in NASH. Thus, regular screening for HCC is important

in patients with NASH who have these risk factors. Obesity is also a

prognostic factor in patients with HCV-related HCC. In a related

study, multivariate analysis indicated that BMI > 28 was the only

host-related factor associated with a poor outcome for early-stage

HCC in patients with HCV infection; BMI also influenced age of onset

of HCC.[13]

Obstructive Sleep Apnea. This condition, a common comorbidity in

patients with morbid obesity, is an independent risk factor for NASH.

[14]

Increased Sensitivity of Steatotic Livers to Drug- and Toxin-Induced

Toxicity[15]. Obese mice with NASH are more susceptible to

acetaminophen-induced liver injury, most likely as a result of

augmented oxidative stress in hepatocytes.[16]

Screening and Diagnosis

There are no established standards for screening for fatty liver.

During this meeting, Bedogni and colleagues[17] presented the results

of a study in which they screened for NAFLD in a representative

sample (n = 496) of the Italian population using a predictive model

based on sex, age, BMI, and waist circumference. Using this

predictive model they were able to detect 82% of cases of fatty

liver. Hyperinsulinemia was also identified as a strong predictor of

increased serum ALT levels, independent of NAFLD.[18]

The presence of fat in the liver can also be suggested by the use of

several imaging modalities; however, no current noninvasive method

can distinguish NASH from NAFLD. Because liver biopsy remains the

gold standard for grading and staging (ie, recognition of

inflammation and fibrosis), a critical issue in management is whether

individual patients should undergo biopsy to detect progressive

disease. During this year's meeting, 2 studies reported a clear

relationship between serum hyaluronic acid and fibrosis; inclusion of

this marker into a composite score predicted NASH and improved

selection of patients with NAFLD for liver biopsy.[19,20]

Other Noninvasive Diagnostic Approaches

MRI is an accurate tool for quantifying liver fat. MRI measurements

of liver fat content correlate with image analysis of histologic

sections and with a pathologist's estimate of liver fat (both r =

0.92, P < .001).[21] " Dynamic " breath tests can detect specific

alterations in metabolic pathways. The 13C-methacetin breath test, a

microsomal liver function test, enables quantitative evaluation of

cytochrome P450-dependent liver function, whereas the 13C-octanoate

breath test evaluates hepatic mitochondrial beta-oxidation. Both

mechanisms increase oxidative stress, which is implicated in the

pathogenesis of NASH, as discussed below. A unique breath test system

incorporating 13C-methacetin breath test and 13C-octanoate breath

test assesses the extent of hepatic injury and reliably distinguishes

NASH from steatosis.[22] 13C-octanoate breath test findings

correlated with insulin resistance and hyperinsulinemia, and 13C-

methacetin breath test predicted the extent of hepatic fibrosis.

Decreased production of adiponectin, an anti-inflammatory cytokine

secreted by adipocytes, may be the link between the metabolic

syndrome, type 2 diabetes mellitus, and NAFLD. Shimada and colleagues

[23] reported that serum levels of adiponectin were significantly

lower, and serum levels of type IV collagen, iron, and ferritin were

all significantly higher in NASH; 90% of patients with NASH were

diagnosed by combined evaluation of serum adiponectin and collagen

levels. Subclinical glucose intolerance is common in NAFLD; a simple

model incorporating C-peptide levels, glucose level at 60 minutes

during oral glucose tolerance test, and serum ALT also distinguishes

NASH from steatosis.[24] Takahashi and colleagues[25] validated the

usefulness of xenon-computed tomography measures, corresponding to

the fat content in the liver, as a surrogate marker of hepatic

steatosis. Finally, in another study presented during this year's

AASLD meeting, transient elastography, a novel method for the

noninvasive measurement of liver stiffness, was applied to detect

cirrhosis in patients with NASH.[26]

The Multifactorial Pathogenesis of NASH

NASH Is a Generalized Proinflammatory State

Obesity in and of itself represents a low-grade systemic

inflammation; this is reflected by elevated serum markers, such as C-

reactive protein and tumor necrosis factor (TNF)-alpha, in patients

with NAFLD.[27,28]

The " 2-Hit Theory "

Obesity and insulin resistance are key correlates of hepatic

inflammation and fibrogenesis; insulin resistance is an antecedent in

the accumulation of hepatocellular fat, whereas excess intracellular

fatty acids, oxidative stress, energy depletion, and mitochondrial

dysfunction cause cellular injury in the steatotic liver. Retention

of lipid in the hepatocyte triggers oxidative stress, generating

reactive oxygen species and cytokine release, culminating in NASH.

High serum free fatty acid concentrations allow greater hepatic fatty

acid uptake. Increased fatty acid delivery to the liver affects

hepatocytes, interfering with insulin function and mitochondrial beta-

oxidation. Although oxidative stress is a key factor, angiogenesis

also plays an important role in liver fibrogenesis. In addition,

leptin exerts potent proangiogenic activity.[29] Because

neovascularization mediated by leptin is essential for development of

liver fibrosis, antiangiogenic therapy may forestall fibrosis in NASH.

Kakajima and colleagues[30] demonstrated that insulin resistance,

severe hepatic steatosis, and overproduction of reactive oxygen

species impair regenerative responses. Therefore, the steatotic liver

consists of senescent hepatocytes with insufficient regenerative

capacity and is vulnerable to a " second-hit " insult. Alterations in

hepatic mitochondrial proteins (the proteome) adversely affect the

rate and efficiency of energy synthesis.[31] The loss of proteins in

the respiratory chain will increase formation of reactive oxygen

species that will contribute to hepatocellular injury.

The Role of Gastrointestinal Flora

Lipopolysaccharide derived from intestinal bacteria may contribute to

inflammation in NASH. Horie and colleagues[32] reported that mice

with fatty livers are more sensitive to lipopolysaccharide.

Continuous stimulation of intestinal lipopolysaccharide induces

further inflammation via activation of Kupffer cells, which produce

inflammatory cytokines such as TNF-alpha. Enhancement of the TNF-

alpha-mediated signaling pathway causes liver fibrosis in an animal

model of NASH.[33] Activation of stellate cells is also involved in

fibrogenesis; the latter occurs in parallel to expression of other

profibrotic cytokines.[34] Bigorgne and colleagues[35] reported that

lipopolysaccharide also directs lymphocyte homing to the liver. Thus,

liver inflammation, which may be triggered by endogenous

lipopolysaccharide, is due to an intrinsic capacity of lymphocyte

subpopulations to migrate specifically to the liver and to an

increased sensitivity of a steatotic liver to inflammation. Precise

identification and inhibition of factors responsible for lymphocyte

homing will open new therapeutic approaches. In humans, small-bowel

bacterial overgrowth has been postulated to increase intestinal

permeability, which allows entry of lipopolysaccharide into the

portal circulation, stimulating proinflammatory and suppressing anti-

inflammatory cytokines, leading to hepatic injury. Portal

inflammation marks the occurrence of this " second hit. " [36] However,

Pham and colleagues[37] were unable to demonstrate a clear

association between bacterial overgrowth and intestinal permeability

and severity of NAFLD.

Genetic and Environmental Factors Play a Role in Progression From

NAFLD to NASH

Familial clustering of insulin resistance and liver disease supports

the postulate of a genetic predisposition to NAFLD.[38] In this

context, and colleagues[39] searched for hemochromatosis (HFE)

mutations in 127 patients with biopsy-proven NASH. The prevalence of

C282Y and H63D HFE mutations was found to be 14% and 21%,

respectively -- suggesting that HFE mutations contribute to

progression of liver disease in NASH.

What Can We Learn From Models of Fatty Liver?

Methionine- and choline-deficient (MCD) diets rapidly induce severe

steatohepatitis in mice (inflammatory infiltrate, hepatocellular

necrosis, and pericellular fibrosis) that mimics the steatohepatitis

found in humans with NASH. The diet is 10% fat -- twice the fat

content of rodent chow. The MCD diet induces adaptive changes in

hepatic lipid metabolism to address such a dietary fat load.[40] The

liver still suppresses fatty acid synthesis and enhances fatty acid

oxidation, but these changes are insufficient to prevent lipid

accumulation, possibly because of impaired egress of triglyceride as

very-low-density lipoprotein. Thus, defective regulation of

triglyceride metabolism may contribute to the pathogenesis of NAFLD.

[41]

Treatment of NAFLD -- Potential Approaches

Treatment of Risk Factors

Triglycerides, low-density lipoproteins, and BMI are all increased in

patients with type 2 diabetes and NAFLD, suggesting that greater

control of these variables may prevent NAFLD in diabetics.[4]

Improved liver histology occurs after weight reduction (ie, diet and

exercise or, in extreme cases, surgery). Features of the metabolic

syndrome were reduced in a cohort of 177 obese children after

participation in a weight-management program; serum ALT levels also

improved significantly.[42]

Diet composition may also be important. Kang and colleagues[43]

determined that patients with metabolic syndrome consumed a diet

containing more carbohydrate and less fat than those without

metabolic syndrome; there was no difference in physical activity or

total caloric consumption between the groups. Replacing long-chain

triglycerides with medium-chain triglycerides reduces alcohol-induced

liver injury. However, when corn oil was isocalorically replaced with

medium-chain triglycerides in an experimental model of NASH, striking

hepatic inflammation developed along with an increase in TNF-alpha.

[44] Thus, contrary to expectations, medium-chain triglycerides

exacerbated liver injury in NASH. Accordingly, the practice of

dispensing medium-chain triglycerides to patients with NASH should be

reassessed.

Pharmacologic Treatment of Insulin Resistance -- Improving Insulin

Sensitivity

In humans with NASH, the insulin sensitizer metformin* improves

biochemical indices of hepatocellular injury and insulin resistance.

Conde de la and colleagues[45] investigated the protective

effect of metformin in a model of oxidative stress-induced apoptosis.

Metformin protected hepatocytes against reactive oxygen species-

induced apoptosis. This novel protective mechanism suggests that

metformin is a candidate for treatment of oxidative stress-related

liver disease.

Thiazolidinediones (peroxisome proliferator-activated receptor gamma

[PPAR-gamma] agonists) reverse insulin resistance and reduce hepatic

fat stores in type 2 diabetes mellitus. PPAR-alpha belongs to the

superfamily of ligand-activated nuclear hormone receptors that

regulate lipid metabolism. Harano and colleagues[46] demonstrated

that a PPAR-alpha ligand, fenofibrate,* improved steatosis in mice

via activation of lipid oxidation. PPAR-alpha agonists exert

antioxidative effects by inducing antioxidant enzymes, in addition to

diminishing steatosis.

Beta-glucosylceramide,* a naturally occurring glycolipid, has immune

modulatory effects. Administration of beta-glucosylceramide

ameliorated hepatic steatosis, hyperinsulinemia, hyperglycemia, serum

triglyceride levels, and intraperitoneal fat accumulation in a rat

model of type 2 diabetes mellitus.[47] Thus, beta-glucosylceramide is

a promising agent for the treatment of both NASH and the metabolic

syndrome.

Use of Lipid-Lowering Agents

Fernandez-Checa and colleagues[48] reported that free cholesterol

accumulation in the liver plays a role in the pathogenesis of NAFLD;

hepatic free cholesterol sensitizes fatty livers to TNF-mediated

steatohepatitis. This study provides a rationale for the use of

statins* in the treatment of this disease.

Drugs That Protect Hepatocytes/Antioxidants

Silymarin,* the seed extract of milk thistle, is a mixture of

flavonolignans and is a hepatoprotective agent. Liu and colleagues

[49] reported that silymarin improved hepatic steatosis in mice by

stimulating secretion of adiponectin. Silymarin may thus offer an

alternative option for treating NAFLD/NASH. Vitamin E,* considered to

be an antioxidant agent, reduces liver inflammation and necrosis.

Lydataki and colleagues[50] normalized serum ALT levels in patients

with NASH by treatment with vitamin E. A-Kader and colleagues[51]

treated 20 children with NAFLD with ursodeoxycholate* and vitamin E

for 12 months. They found significant improvement in serum ALT but no

improvement in histology. Finally, Mukherjee and colleagues[52]

assessed the effect of betaine,* a naturally occurring choline

metabolite, on liver function tests and hepatic fibrosis in a

prospective cohort of 35 patients with NASH. Betaine was well

tolerated and appeared to improve both biochemical parameters and

liver histology in this setting.

Novel Approaches

Hepatic iron overload is frequent in NAFLD, and iron depletion

improves liver function tests and insulin resistance. The addition of

iron depletion therapy to standard diet/exercise reduces serum ALT

levels in NASH independently from iron stores, thus representing a

promising effective, safe, and low-cost therapy.[53]

Glucagon-like peptide-1 (GLP-1),* produced by the small intestine, is

a known incretin hormone that reverses hepatic steatosis and features

of metabolic syndrome via a direct effect on hepatocyte fat

metabolism. GLP-1 has beneficial effects in the setting of NAFLD,

including improved insulin resistance, enhanced adiponectin

production, and induction of weight loss.[54]

The Future -- The NAFLD Research Agenda

Future research should further define the role of insulin resistance

and abnormal lipoprotein metabolism in the pathogenesis of

hepatocellular injury. The link between obesity and inflammation must

also be explored. A clear understanding of genetic factors and

environmental influences that predispose to NAFLD/NASH is needed.

Thus, insights provided by studies presented during this year's AASLD

meeting clearly inform future studies and will stimulate the

development of noninvasive predictors of disease and more effective

preventive and therapeutic strategies.