Non-alcoholic fatty liver disease and non-alcoholic steatohepatitis

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of elevated liver enzymes and chronic liver disease in the Western countries.^1-3 Its incidence in adults and children is rising rapidly due to the ongoing epidemics of obesity and type2 diabetes.^{4, 5} It is a multifaceted metabolic disorder and is encountered in clinical practice by a variety of health care specialists ranging from primary care physicians and gastroenterologists to cardiologists, radiologists and gynecologists. It comprises of a spectrum of liver disease ranging from simple steatosis to full blown steatohepatitis that is characterized by steatosis, lobular inflammation, ballooning and fibrosis.⁶ Over the last several years, much progress has been made in terms of our understanding of its risk factors, pathogenesis, natural history, non-invasive markers and treatment. This review is tailored towards clinicians caring for patients with NAFLD and it discusses practical issues related to selected aspects of its evaluation and management.

Evaluation of newly suspected NAFLD

Suspected NAFLD represents one of the most common reasons why patients visit gastroenterologists and hepatologists in an ambulatory setting.^1-3 While some patients may have attributable abdominal symptoms and tender hepatomegaly, the majority are asymptomatic and their liver disease identified incidentally on routine blood tests and/or abdominal imaging. Its initial assessment consists of excluding competing and co-existing etiologies and identifying clinically important co-morbidities.

Competing etiologies

The diagnosis of NAFLD requires that there is no history of previous or ongoing significant alcohol consumption. There is no consistent agreement to the definition of significant alcohol consumption but it is generally believed that average alcohol consumption more than 2 drinks per day in women and more than 3 drinks per day in men is necessary to develop alcoholic fatty liver.^{7, 8} However, in individuals with metabolic risk factors such as obesity and diabetes, it is possible that alcohol consumed at lower quantities may promote hepatic steatosis. This notion is supported by a population-based study in which Ruhl and Everhart have shown that ≤ 1 alcohol drink per day causes elevated ALT only in the obese but not in normal weight individuals.⁹ This study is at odds with recent epidemiological data suggesting that modest wine consumption may reduce the prevalence of NAFLD.¹⁰

When the alcohol consumption history is insufficient, it becomes difficult to distinguish alcoholic and non-alcoholic forms of fatty liver, especially in those with obesity and associated metabolic risk factors. Conventional markers like GGT, MCV and AST/ALT ratio are not useful, and specific serum markers for chronic alcohol abuse are of limited utility.¹¹ The carbohydrate-deficient transferrin (CDT) is the most widely used and perhaps the most specific serum marker for detecting chronic alcohol abuse.¹² This test evaluates the ratio of desialylated transferrin to total transferrin and it has 81% sensitivity with 98% specificity in detecting chronic alcohol abuse. Unfortunately, this and other tests like the ratio of mitochondrial AST to total AST¹³ are not readily available in clinical practice.

More recently, investigators from the Mayo clinic have developed “ALD/NAFLD Index” (ANI) that consisted of five easily available variables: mean corpuscular volume, AST and ALT values, height and weight and gender (http://www.mayoclinic.org/gi-rst/mayomodel10.html).¹⁴ This weighted multivariate model uses logistic regression analysis to generate ANI score, and a score greater than zero incrementally favors ALD whereas a value less than zero favors NAFLD in decrement. For example, ANI of 8.95 would correspond to greater than 99% probability of alcoholic liver disease whereas a value of -5.04 would correspond to greater than 99% probability of NAFLD. As the majority with alcoholic liver disease in this study had high MELD scores, this tool is more likely applicable to patients with decompensated cirrhosis presenting for liver transplant evaluation rather than outpatients with fatty liver.

Elevated serum auto-antibodies are common in patients with NAFLD. Although low titer anti nuclear antibody (ANA) positivity can be seen in up to 33% of patients with NAFLD, ANA positivity in titers greater than 1:320 is generally rare.^15-17 Low titers of anti-smooth muscle and anti-mitochondrial antibodies may also be noted in patients with NAFLD.¹⁸ The presence of these auto-antibodies is generally thought to be an epiphenomenon, although in one study their presence correlated with more severe histological damage.^{19, 20} In patients with suspected NAFLD and ANA positivity at titers greater than 1:160 or ASMA positivity at titers greater 1:40, a liver biopsy may be considered to exclude the presence of autoimmune hepatitis.

Mildly increased serum ferritin is not uncommon in patients with NAFLD and it does not necessarily indicate co-existing iron overload.²¹ Metabolic syndrome and hyperinsulinemia are known to be associated with increased serum ferritin, and this association may be mediated by the presence of NAFLD.²² Nonetheless, elevated serum ferritin in a patient with suspect NAFLD should prompt testing for HFE gene mutations. The prevalence of HFE gene mutations in NAFLD patients has been variable depending on the population studied and their relevance remains largely unknown.^{23, 24} While the prevalence of HFE gene mutations and their clinical relevance remain unclear,^22-24 homozygote or heterozygote C282Y mutations along with elevated serum ferritin may justify a liver biopsy in patients with suspected NAFLD.

It is important to verify that chronic hepatitis B and hepatitis C have been convincingly excluded, and depending on the clinical scenario, rare disorders such as alpha-1 antitrypsin deficiency and Wilson’s disease should also be excluded. Thorough medication history is important as several commonly prescribed medications (e.g., tamoxifen, methotrexate, amiodarone) are noted for their ability to cause hepatic steatosis.¹

Evaluation for co-morbidities

The patients with NAFLD frequently have many clinically significant co-morbidities (Table 1). Obesity, type2 diabetes and hyperlipidemia are well known to co-exist in patients with NAFLD, and it is important to systematically characterize them. The body mass index and waist circumference should be measured to better characterize the degree (mild, moderate and severe) and the nature (central versus peripheral) of obesity. Type2 diabetes and glucose intolerance are very frequent in patients with NAFLD and they have prognostic significance.²⁵ In patients without preexisting type2 diabetes, the presence of glucose intolerance and insulin resistance should be evaluated by obtaining fasting blood glucose and insulin levels and hemoglobin A1c. In non-diabetic patients, insulin resistance should be assessed by calculating HOMA-IR (homeostasis model assessment-insulin resistance) or QUICKI (quantitative insulin-sensitivity check index).^{26, 27} Both HOMA-IR and QUICKI are mathematical transformation of fasting blood glucose and insulin levels. In our practice, we calculate HOMA-IR ([fasting insulin (μU/ml) * fasting glucose (mmol/L)]/22.5), and as a general guideline, we consider a HOMA-IR value > 3 to be clinically significant.²⁸ If not done recently, fasting lipid profile should be obtained as dyslipidemia is nearly universal. Typically, fasting serum triglyceride levels are high along with low HDL values. Recent data suggest that high-sensitivity CRP (hs-CRP) is frequently elevated in patients with NAFLD and should be measured along with fasting lipid profile to assess cardiovascular risk (vide infra).^{29, 30}

Table 1. Co-morbidities commonly associated with NAFLD.

Established conditions	Emerging conditions
Obesity	Obstructive sleep apnea
Type2 Diabetes/Glucose intolerance	Hypothyroidism
Dyslipidemia	Obstructive sleep apnea
Metabolic syndrome	Hypopituitarism

In addition, patients should be systematically explored for the presence of other co-morbidities such as obstructive sleep apnea,^{31, 32} hypopituitarism,³³ hypothyroidism,³⁴ and polycystic ovary syndrome^35-37. Obstructive sleep apnea (OSA) is commonly present in patients with NAFLD and may contribute to fatigue that sometimes these patients experience.³⁸ Some studies have suggested causal relationship between OSA and the pathogenesis of NAFLD but these are far from convincing.^{39, 40} Previous studies have shown that patients with hypothyroidism and hypopituitarism have increased prevalence of NAFLD.^{33, 34} Several studies have shown high prevalence of NAFLD in premenopausal women with polycystic ovary syndrome^{35, 37} and one study³⁶ suggested that such individuals are at higher risk for disease progression.

Identification of NASH in patients with NAFLD

The NAFLD can be categorized into simple steatosis and steatohepatitis (NASH). A well-defined case of NASH histologically exhibits macrovesicular steatosis, lobular inflammation, balloon degeneration of hepatocytes and zone-3 pericellular fibrosis.⁴¹ Undoubtedly, NASH is histologically progressive and can lead to cirrhosis and associated liver dysfunction. Simple steatosis has a relatively benign course but is not totally without histological consequences.⁴² For example, investigators from the Cleveland Clinic reported in their initial paper that 4% of patients with simple steatosis developed cirrhosis and 2% had liver-related mortality over a median follow-up of ∼ 8 years.⁶ The extended follow-up of the same cohort was reported in an abstract form recently and it confirmed that simple steatosis is not entirely benign.⁴³

Liver biopsy is the current gold standard to identify steatohepatitis but it is not without controversies and practical difficulties. Several recent studies have highlighted its sampling variability and inter-observer discordance.^44-46 The precise histological definition of NASH is not entirely known and the experts believe that it should be based on pattern recognition rather a composite score of individual components such as steatosis, ballooning and fibrosis.⁴⁷ It remains controversial whether ballooning or pericellular fibrosis that should be considered as critical for the histological diagnosis of NASH. The recently described NAFLD Activity Score (NAS) is valuable for quantifying histological changes, especially in clinical trials,⁴⁷ but its generalizability and diagnostic utility are unknown.

Over the past several years, there has been significant interest to non-invasively predict liver histology in patients with NAFLD. However, it remains somewhat controversial which histological finding(s) should be targeted for non-invasive assessment. Most studies to date have attempted to predict advanced fibrosis (bridging fibrosis/cirrhosis),^{25, 48, 49} but it has been argued that one should attempt to predict steatohepatitis rather than advanced fibrosis.⁵⁰ It is our view that future studies should attempt to predict three histological states (simple steatosis, NASH without significant fibrosis and NASH with advanced fibrosis), rather than dichotomous descriptive states.

Aminotransferase values and common imaging tests such as liver ultrasound, computed tomography and magnetic resonance are of limited value in predicting liver histology. Numerous circulating biomarkers and prediction models have been investigated to noninvasively predict hepatic histology in patients with NAFLD (Table 2) and their full discussion is beyond the scope of this review. However, cytokeratin 18 (CK-18) fragments and serum dehydroepiandrosterone (DHEA), two hypothesis-driven circulating biomarkers, deserve further discussion. Based on experimental data that hepatocytes apoptosis may play an important role in the pathogenesis of NAFLD, Wieckowska et al. measured plasma CK-18 fragment levels in 44 consecutive individuals with suspected NAFLD undergoing liver biopsy.⁵¹ CK-18 is a major intermediate filament protein in hepatocytes and it is cleaved by the effector caspases (mainly caspase 3) upon the activation of the apoptosis cascade.^{52, 53} Compared to individuals normal histology and those without NASH, patients with definite NASH had significantly higher CK-18 fragment levels.⁵⁴ It was suggested that CK-18 fragment levels > 380.2 U/L can predict definite in a very precise fashion.⁵¹ A puzzling and unexplained aspect of this small cross-sectional study is that nearly 25% of patients with suspected NAFLD supposedly had normal hepatic histology on liver biopsy. More recently, Charlton et al. have shown that serum DHEA levels had a consistent and stepwise inverse relationship with the degree of hepatic fibrosis that persisted after adjusting for age.⁵⁵ This observation which was initially made on a derivation cohort consisting of 122 patients was subsequently reproduced on a validation cohort consisting of 361 NAFLD patients recruited from two separate academic centers.⁵⁵ These provocative preliminary data deserve further study but it may be too optimistic to assume that a single biomarker can reliably predict histology in NAFLD, a condition with relatively complex phenotype and multiple co-morbidities.

Table 2. Non-invasive biomarkers previously studied or currently under evaluation.

	Serum biomarkers	Imaging studies	Breath tests
Fibrosis	BARD score58 Fibrotest NAFLD fibrosis score56 ELF/OELF57, 59 Hyaluronic acid130	Transient elastography61, 131 MR elastography132 MR spectroscopy133, 134	[13C] methacetin135 [13C] ketoisocaproate135 Ethanol136 Acetone136
Oxidative stress	TBARS119 Oxidized-LDL119 Total antioxidant response137 Total lipid peroxide levels137
Hepatocyte apoptosis	CK-18 fragments51, 53
Inflammation	TNF-α/adiponectin ratio138, 139 hsCRP29 IL-6140 CC-chemokine ligand-2141

There are numerous papers published in the literature describing non-invasive prediction models but the majority of them consisted of small sample size and lacked rigorous external validation. Three recently described models with relatively large sample size and some level of validation have shown encouraging results.^56-58 In a multicenter study consisted of 480 patients in the derivation and 253 in the validation cohorts, Angulo et al.⁵⁶ have shown that NAFLD fibrosis score consisting of 6 variables (age, BMI, AST/ALT ratio, hyperglycemia, platelet count and albumin) can reliably predict advanced fibrosis. The formula for the fibrosis score was “ - 1.675 + 0.037 × age (years) + 0.094 × BMI (kg/m2) + 1.13 × impaired glucose tolerance/diabetes (yes=1, no=0) + 0.99 × AST/ALT ratio - 0.013 × platelets (X10⁹/l) - 0.66 × albumin (g/dl)”, and a low cutoff point (score < -1.455) signified the absence of advanced fibrosis whereas a high cutoff point (score > 0.676) identified advanced fibrosis. It was concluded that NAFLD fibrosis score can be utilized as a triaging tool for optimizing liver biopsy yield in terms of identifying or excluding advanced fibrosis. More recently, Guha et al.⁵⁷ have compared the performance of “Enhanced Liver Fibrosis” (ELF) score to that of the “NAFLD Fibrosis Score” in predicting advanced fibrosis. The ELF is essentially the “Original European Liver Fibrosis” (OELF) test without “age” included in the algorithm. The OELF test, a model consisting of age and three serum markers of matrix turnover (TIMP 1, hyaluronic acid and P3NP), predicted advanced fibrosis in a variety of liver disorders.⁵⁹ In this report, the ELF score had excellent ability to predict different levels of fibrosis (any fibrosis, moderate fibrosis, or advanced fibrosis) and when it was combined with the “NAFLD fibrosis score”, its ability to predict different levels of fibrosis improved further.⁵⁷ Harrison et al. have recently developed BARD score to predict advanced fibrosis in patients with NAFLD⁵⁷. The BARD score is a weighted sum of three easily available variables [BMI > 28 kg/m² (1 point), AST/ALT ≥ 0.8 (2 points) and diabetes (1 point)] and authors have shown that a score of 2-4 was associated with an odds ratio 17 (95% CI: 9.2-31.9) for predicting advanced fibrosis⁵⁷. These promising models will need to be validated by external investigators before they are recommended for wide clinical use.

One French study tested “Fibrotest” for predicting advanced fibrosis in NAFLD and found encouraging results.⁶⁰ “Fibrotest” is a popular serum test for predicting advanced fibrosis in individuals with chronic hepatitis C, but more studies are needed to test its utility in NAFLD. Similarly, liver stiffness measured by transient elastography (FibroScan) may have some role in predicting the degree of fibrosis, but the data are sparse in terms of its utility in NAFLD.^{61, 62}

In summary, there has been significant research in developing biomarkers of liver histology in patients with NAFLD, and in fact one editorialist recently opined that the future is around the corner for non-invasive diagnosing progressive NASH.⁶³ While this may be the case for advanced fibrosis, insufficient attention has been paid to developing markers for non-invasively identifying steatohepatitis in patients with NAFLD. Until more definite data with external validation become available, we will continue to our current practice of recommending liver biopsy to selected patients with suspected NAFLD based on the presence of certain risk factors such as older age, diabetes, severe obesity and metabolic syndrome.

Management of patients with NAFLD

Advice regarding weight loss, exercise and specific diets

The NAFLD is largely a manifestation of obesity and metabolic syndrome and is characterized by excess calorie intake and lack of optimal health-related fitness or physical activity.⁶⁴ It is generally believed that weight loss is beneficial for patients with NAFLD, but data are sparse in terms of specifics such as how, how much and how rapidly to lose weight. Furthermore, precise hepatic and extrahepatic benefits of weight loss are not well defined. In a recent review, Bellentani et al.⁶⁵ pointed out that there are only four human studies consisting of less than 40 total patients that evaluated the effect of calorie restriction alone, and change in liver enzymes was the primary end point in all but one study. There are 10 published studies consisting of 626 total patients that evaluated the effect of calorie restriction combined with exercise, but liver histology was the primary end point in only four studies (123 patients).⁶⁵ This paucity of data makes it difficult to make evidence-based recommendations about dietary modification and exercise to treat NAFLD and NASH.

It is generally recommended that overweight and obese patients with NAFLD lose 7-10% of their body weight by dietary modification and exercise over the course of 6-12 months. This is based on short-term studies showing that gradual weight loss of this magnitude improves insulin resistance and hepatic histology.^{3, 66} Our recommendations to enhance patient compliance with lifestyle modifications are shown in Table 3. Scientific evidence is lacking to make precise recommendations specific to modifying macronutrient composition, but it appears sensible to recommend low glycemic food with decreased saturated and trans-fat intake but increased mono and polyunsaturated fatty acid intake.⁶⁷ Evolving data suggest diets consisting of high fructose should be avoided by these patients.⁶⁸ Due to lack of safety and efficacy data, popular weight-loss diets such as Atkins, Ornish and South Beach diets should not be recommended.⁶⁷ In our clinical practice, we recommend diminished portions of balanced diet (consisting of low glycemic and low fat diet and increased portions of fruits and vegetables) and 5-7 sessions per week of moderate aerobic exercise with each session lasting for 30-45 minutes. However, such prescriptive recommendations made in a clinic setting are rarely effective both short term or long term.⁶⁵

Table 3. Strategies to enhance patient compliance in lifestyle modification.

Communicate with empathy Be sensitive to general stigma against obesity Discuss pros and cons of proposed changes to lifestyle Explore reasons for perpetual poor dietary and exercise choices Encourage self-efficacy Offer specific choices of food and exercise Design individualized program of eating and physical activity Explain treatment and its benefits

Orlistat, a reversible inhibitor of gastric and pancreatic lipase, may be effective in promoting limited weight loss in selected patients, but side effects such as diarrhea and bloating make it less desirable. In a randomized study, Harrison et al. have shown that orlistat does not cause weight loss or histological improvement above and beyond that is accomplished with calorie restriction alone.⁶⁹ Rimonabant (endocannabinoid receptor 1 antagonist) is approved in Europe for promoting weight loss, and it may have favorable anti-steatotic and anti-fibrotic properties.^70-74 Large clinical trials are underway with this and other similar compounds in NASH but their results will not become available for at least several years.

Advice regarding bariatric surgery

As many patients with NAFLD have severe obesity, it often comes up in clinical practice whether bariatric surgery in suitable in this patient population. Jejunoileal bypass was widely popular in mid-1950s to mid-1970s for the surgical treatment of obesity but due to disastrous hepatic and extrahepatic consequences it is now totally abandoned.^75-77 Over the last decade, an increasing number of foregut bariatric surgery procedures are being performed to treat obesity and its complications, and its short and long term benefits are becoming well established.^78-80 The commonly performed foregut bariatric surgery procedures include roux-en-Y gastric bypass (most common), adjustable gastric banding, gastroplasty and sleeve gastrectomy.^{81, 82} To date, there have been no studies that evaluated foregut bariatric surgery to specifically treat NAFLD but many published papers described its favorable effect of hepatic histology when performed for other indications and thus introducing selection bias .^78-80 In general, liver histology improves significantly following foregut bariatric surgery with very minimal risk of worsening.^{83, 84} In a recent meta-analysis consisting of 15 studies and 766 paired liver biopsies, Mummadi et al.⁸⁴ have shown that all components of NAFLD show significant improvement following foregut bariatric surgery. Pooled proportion of patients with improvement or resolution in steatosis was 93% (95% CI: 84-98%), with improvement or resolution of steatohepatitis was 82% (95% CI: 64 - 95%), with improvement in fibrosis when assessed using needle biopsies was 73% (95% CI: 65 - 81%). We speculate that liver disease is unlikely to worsen in association with rapid and profound weight loss unless there are additional risks such as bacterial overgrowth (e.g., jejunoileal bypass) or nutrient depletion (e.g., kwashiorkor). This forms the basis for our view that very long roux limb (i.e., > 150 cms) should be avoided in patients with advanced fibrosis. Compensated cirrhosis is not a contraindication for foregut bariatric surgery provided it is performed by an experienced surgeon and clinically evident portal hypertension is absent (no esophageal or abdominal varices). There are reports that cirrhosis may reverse following bariatric surgery.^{85, 86}

In our practice, we recommend foregut bariatric surgery as a therapeutic possibility for the severely obese NAFLD patients with advanced fibrosis who failed to lose weight despite repeated nutritional counseling. In those with cirrhosis, we exclude clinical portal hypertension by performing abdominal imaging and upper endoscopy. Bariatric surgery may be particular attractive for carefully selected patients with Child’s A cirrhosis not only because it may stabilize or improve the liver disease but it also may enhance their future suitability for liver transplantation.

Role of insulin sensitizers

As insulin resistance is nearly universal in patients with NASH, it is not surprising that many studies tested insulin sensitizers as its treatment. However, a large number of them are proof-of concept studies with small number of patients without rigorous study design, making it difficult to make definite recommendations. Biguanides (metformin) and thiazolidinediones (pioglitazone and rosiglitazone) are the two classes of insulin sensitizers studied in humans.

Metformin

Although its exact mechanism of action is not entirely clear, its therapeutic benefit as an anti-diabetic agent and insulin sensitizer is well recognized. Its anti-diabetic action is likely related to decreased hepatic gluconeogenesis, decreased glucose absorption and increased insulin sensitivity by facilitating glucose uptake and utilization.^{86, 87} In addition, its stimulatory effect on AMP activated protein kinase or modulation of hepatic TNFα expression may be benefit favorably.^{88 89} A summary of studies evaluating metformin to treat NASH is shown in Table 4. A recent meta-analysis published in Cochrane database showed that metformin leads to normalization of serum aminotransferases in a significantly greater proportion of patients compared to dietary modification (OR: 2.83, 95% CI: 1.27 - 6.31and improved steatosis by imaging (OR: 5.25, 95% CI: 1.09 - 25.21).⁹⁰ The total number of patients treated with metformin in controlled studies is admittedly small and its favorable effect on hepatic histology may not be robust, but we favor its use in non-diabetic patients with NASH because of its safety profile. As most non-diabetic patients with NASH have glucose intolerance, it has the added benefit of lowering the risk of developing frank diabetes.⁹¹ As metformin has not been studied to treat NASH in diabetic individuals, its role in diabetic population is not known. An ongoing, multicenter study comparing metformin to vitamin E or placebo in pediatric patients with NASH (TONIC; NCT00063635) should provide more insight into metformin’s role in treating NASH.

Table 4. Selected studies of metformin in patients with NAFLD.

Author (ref)	N	Design	Comparator	Population	Duration	Liver enzymes	Histology
Marchesini et al142	14	Open label Single arm	None	Adults Mostly non-diabetic	4 mo	Improved	Not evaluated
Nair et al143	15	Open label Single arm	None	Non-diabetics	12 mo	Improved	Improved inflammation
Uygun et al144	36	Open label	Calorie restricted diet	Non-diabetics	6 mo	Improved	Improved inflammation
Bugianesi et al145	55	Randomized clinical trial	Calorie restricted diet	Non-diabetics	12 mo	Improved	Improved steatosis, inflammation and fibrosis
Schwimmer et al146	10	Open label Single arm	None	Non-diabetics	6 mo	Improved	Not evaluated
Loomba et al147	14	Open label Single arm	None	Non-diabetics	48 wks	Improved	Improved steatosis and inflammation
Nobili et al148	57	Open label	Antioxidant	Non-diabetics	24 months	No difference	No difference

Thiazolidinediones (TZD)

TZDs are a novel class of oral anti-diabetic medications that improve insulin resistance by acting as selective PPAR-γ agonists.^{92, 93} Troglitazone, the first generation TZD, has been withdrawn from the market due to its hepatotoxicity⁹⁴ whereas rosiglitazone and pioglitazone are the second generation TZDs that are currently available for clinical use.^{93, 95} They redistribute fat from muscle and liver to adipose tissue and thereby improving peripheral (skeletal muscle) and hepatic insulin sensitivity.⁹³ In addition, they increase circulating levels of adiponectin which is produced exclusively by the adipose tissue and has insulin sensitizing properties.⁹⁶

There has been significant interest in evaluating TZDs to treat NASH, and to our knowledge, eight studies have been published either as full length papers or solely as an abstract to date.^97-104 Troglitazone was tested in one study⁹⁷ whereas rosiglitazone in 2^{98, 103} and pioglitazone in 5 studies.^{99-102, 104} Four were randomized controlled studies with 213 total enrolled patients with histologically proven NASH.^101-104 Selected characteristics and outcomes of three studies that randomized at least 50 patients are shown in Table 5. In general, TZDs improve hepatic histology in patients with NASH although their favorable effect on steatosis is more striking than on other histological variables such as inflammation, ballooning or fibrosis. Their favorable effect on liver histology and liver biochemistries disappears upon their discontinuation, suggesting that long term treatment is needed to maintain their therapeutic benefits¹⁰⁵. This is potentially a significant issue as recent studies questioned the long term safety of TZDs (especially rosiglitazone)¹⁰⁶. As majority of the participants in these studies were non-diabetic, it is not clear if TZDs are equally effective in diabetics with NASH. In fact, the presence of diabetes was a negative predictor of response to rosiglitazone in one study.¹⁰³ Furthermore, Ratziu et al. recently raised the possibility that TZDs alone without lifestyle modification may not be as effective.¹⁰³ Overall, there are more questions than answers about the role of TZDs in patients with NASH and the ongoing large US multicenter study (PIVENS; NCT00063622) may provide some additional insight.

Table 5.

Randomized controlled trials of TZDs consisting at least 50 randomized patients

First author (Ref)	n	Study design	Duration	Histological Improvement- Pre & Post				Mean weight gain	Comments
				Histologic variable	TZD	Placebo	TZD vs. Placebo
Belfort102	55	Hypocaloric diet + Pioglitazone versus Hypocaloric diet + Placebo *Diabetics + Non-diabetics	6 mo	Steatosis	Yes	No	Yes	2.5 kg	TZD group had significant increase in serum adiponectin levels and this inversely correlated with improved hepatic steatosis
				Inflmm	Yes	Yes	Yes
				Ballooning	Yes	No	Yes
				Fibrosis	Yes	No	Borderline
				NAS	Yes	No	Yes
Ratziu103	63	Rosiglitazone versus Placebo * Diabetics + Non-diabetics	12 mo	Steatosis	Yes	No	Yes	1.5 kg	- Primary end point was >30% reduction in hepatic steatosis and predictors of response included rosiglitazone treatment, lack of diabetes at entry and greater steatosis at baseline - No active lifestyle modification in either groups - Ranked assessment of pre-and post-treatment biopsies showed improved steatosis, ballooning and fibrosis in the rosiglitazone group - Relatively lower NAS at entry may at least in part explain why there was no change in NAS was seen in this study
				Inflamm	No	No	No
				Ballooning	No	No	No
				Fibrosis	No	No	No
				NAS	No	No	No
Gurupras ad 104	74	Diet + exercise + Pioglitazone versus Diet + exercise + Pioglitazone * Only non-diabetics	12 mo	Steatosis	Yes	Yes	No	2.6 kg	NAS score reported. Placebo group had mean weight loss of 3.5 kg but TZD group had mean weight gain of 2.6 kg
				Inflamm	Yes	No	No
				Ballooning	Yes	Worse	Yes
				Fibrosis	Yes	No	No
				NAS	N/A	N/A	N/A

Promising agents

There is intense research into developing suitable treatment for NASH and a list of compounds that are being tested to treat NASH in humans is shown in Table 6. Oral endocannabinoid receptor (CB1 receptor) antagonists are of potential benefit due to multiple potential favorable effects (on body weight, fibrogenesis and de novo lipogenesis) and large multicenter studies are underway. Neuropsychiatric side-effects are concern, but if proven effective, they may have a role at least in a select group of patients without underlying neuropsychiatric co-morbidities. The CB-1 receptor antagonist studies in humans have just begun and their results will not be available for few years. Based on promising animal data¹⁰⁷ and its ability to promote weight loss¹⁰⁸, we have initiated an open-label study of exenatide in 2006, but its recruitment has been hampered due to its injectable route of administration and potential GI side effects.

Table 6. Various agents in evaluation to treat NASH in Humans (www. Clinicaltrials.gov).

Agent	Nature of the Study
Metformin vs. Vit E vs. Placebo (TONIC)	Phase III, multicenter, RCT
Pioglitazone vs. Metformin vs. placebo (PIVENS)	Phase III, multicenter, RCT
Omega-3 fatty acids	Phase II, pilot study
Polyunsaturated fatty acids	Phase II, pilot study
Orlistat	Phase III, multicenter study
Silophus	Phase II, pilot study
Silymarin	Phase II, multicenter RCT
Rimonabant	Phase IIb, multicenter RCT
Recombinant leptin	Phase II, pilot study
Pentoxiphylline	Phase II, pilot study
Pentoxiphylline vs. Pioglitazone	Phase II, pilot study
Exendatide	Phase II, pilot study
Rosi vs.Rosi + metformin vs. rosi + losartan	Phase III, single center
SAMe	Phase II, pilot study
Iron depletion	Phase II, pilot study
CP 945598	Phase IIb, multicenter RCT
TRO 19622†	Phase II, pilot study
ASP9831Ψ	Phase II, pilot study
L-Alanine	Phase II, pilot study

^†Cholesterol like small molecule and is a potent neuroprotective agent in-vitro. May have mitochondrial protective effects

^ΨNovel PDE4 inhibitor which may be improve hepatic inflammation and fibrosis

Cardiovascular disease in NAFLD

The cardiovascular morbidity and mortality is perhaps one of the most important aspects of NAFLD and NASH, and our knowledge of their association is evolving rapidly.^109-112 The patients with NAFLD have very high prevalence of cardiovascular risk factors and atherosclerosis and high incidence of cardiovascular morbidity and mortality.^{43, 113, 114} Over the last decade, numerous studies have demonstrated that patients with NAFLD are enriched with classic cardiovascular risk factors such as obesity, insulin resistance and type2 diabetes, dyslipidemia, and the metabolic syndrome.^115-117 Cross-sectional studies consisting of control groups have shown increased prevalence of endothelial dysfunction, ¹¹⁸ elevated levels of ox-LDL,¹¹⁹ Framingham coronary risk scores ^{109, 119} in NAFLD patients. Cross-sectional studies have also shown increased prevalence of premature atheroma formation,¹²⁰ carotid artery intima media thickness (surrogate for atherosclerosis),^{121, 122} vulnerable coronary plaques, ¹²³ increased mediastinal fat and abnormal left ventricular energy metabolism.¹²⁴ Recently it has been suggested that NAFLD poses cardiovascular risk above and beyond that is conferred by the presence of the metabolic syndrome.^{43, 113, 114, 125} In a nested case-control, prospective study, Targher et al. have shown that NAFLD in diabetic individuals is associated with moderately increased risk of incident cardiovascular disease even after adjusting for classic risk factors, glycemic control and the metabolic syndrome.¹²⁵ Most importantly, several longitudinal studies have shown that cardiovascular disease is much more common than liver disease as a cause of death in patients with NAFLD.^{43, 113, 114} In a longitudinal study consisting of 420 Olmsted county residents with NAFLD, Adams et al.¹¹³ have shown that ischemic heart disease accounts of 25% of deaths, compared to liver disease accounting for 13% of deaths. By linking the NHANES III to Linked Mortality Files, Ong et al.¹¹⁴ have shown that cardiovascular disease is the most common cause of death and it exceeds that of liver disease among 817 individuals with suspected NAFLD in comparison to 10,468 persons without liver disease. More recently, Rafiq et al.⁴³ have reported extended follow-up of an expanded NAFLD cohort that has been described previously. The mortality rate over an 11.1 year median follow-up (longest f/u 28.5 years) was 45% and the most common cause of death was coronary artery disease. All these data provide unequivocal evidence that coronary artery disease is a serious threat to patients with NAFLD. Therefore, it has become our practice to emphasize the significance of cardiovascular disease to patients with NAFLD and their primary care providers.

Statins remain a cornerstone for managing dyslipidemia and coronary artery disease. Despite initial concerns, several recent studies have shown that statins can be safely used in patients with underlying liver disease.^126-129 Studies have not been conducted to specifically show that statins diminish cardiovascular morbidity and mortality in patients with NAFLD but there are no suspected reasons why they would be any less effective. Minor fluctuations in aminotransferases upon initiating statin therapy are not uncommon but serious hepatotoxicity is quite rare¹²⁶ and even when happens, it is almost universally reversible upon prompt recognition and withdrawal of the offending agent.¹²⁷

In summary, when a patient with suspected NAFLD seen in the clinic it is important to carefully evaluate for competing etiologies and clinically important co-morbidities. Many advances have been made in terms of non-invasive biomarkers for predicting advanced fibrosis but insufficient attention has been paid to predicting steatohepatitis. Sustained weight loss can be effective to treat NASH but it is difficult to achieve. Foregut bariatric surgery can be quite effective in improving hepatic histology in selected patients without liver failure or significant portal hypertension. TZDs have shown promise but recent studies raised doubts about their long term safety. Large multicenter studies of CB1 receptor antagonists are underway but their results will not be available for several years. Several recommendations made in this review are not entirely evidence-based and thus should be cautiously accepted while we await more data and practice guidelines by the consensus of the experts. There is an ongoing effort to develop a multi-society (AASLD ACG, ACP and AGA) consensus practice guideline for managing patients with NAFLD and this would clearly identify the level of evidence available for different recommendations.

Abbreviations

NAFLD

Non-alcoholic fatty liver disease

NASH

Nonalcoholic steatohepatitis

NAS

NAFLD Activity Score

HOMA-IR

Homeostatic Model Assessment - Insulin Resistance

TZD

Thiazolidinediones

ELF

Enhanced Liver Fibrosis Score