Schiff’s Diseases of the Liver
10th Edition

Chapter 39
Nonalcoholic Fatty Liver Disease
Stephen H. Caldwell
Abdullah M.S. Al-Osaimi
Curtis K. Argo
Nonalcoholic fatty liver disease (NAFLD), is an umbrella term that includes a range of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) (1,2). As apparent from the volume of literature published in just the last 3 years (well over 1,000 articles), it is increasingly recognized as a potentially serious condition, which can progress to cirrhosis, liver failure, and hepatocellular cancer (HCC) and has a worldwide distribution (3,4,5,6,7,8,9). The spectrum of clinical severity, in part, reflects the normal role that the liver plays in fat metabolism— a fat storage site where lipid peroxidation can lead to injury and activation of profibrotic cytokines in some individuals. The explanation for why some people develop steatosis and others do not and why some with steatosis develop injury is related to variable expression of obesity, type 2 diabetes, and the metabolic syndrome—conditions that represent a complex mixture of genetic predisposition and environmental factors (10). Several mechanisms appear to promote the accumulation of hepatic fat: De novo synthesis of triglycerides, impaired secretion of lipoprotein and, perhaps the most important in typical patients, increased delivery of fatty acids to the liver. These abnormalities correlate with central obesity and physical conditioning, providing the basis for conservative management of NAFLD with exercise, dietary changes, and weight loss. The prominent role of insulin resistance provides the basis for several of the most promising forms of pharmacologic intervention with insulin-sensitizing agents. Between these two broad categories of treatment, cytoprotective and antioxidant therapy remain under investigation as possible means of reducing oxidative injury.
Clinical and Histologic Criteria and Terminology
Although the exact histologic criteria continue to be debated, the term NASH is now widely regarded as the more severe form of “NAFLD.” Not as widely accepted is the distinction between “primary” NASH (usually associated with obesity and diabetes without other precipitating factors) and “secondary” NASH (associated with a specific disease or some medications) (Table 39.1). This distinction is limited because shared risk factors point to a common pathogenesis of “primary” NASH and some of the conditions that have been associated with “secondary” NASH. The presence or absence of insulin resistance may be a useful distinguishing feature. As such, steatohepatitis associated with toxin exposure may qualify as a distinct entity while many other “secondary forms” may actually represent an exacerbation of “primary” NASH (See “Epidemiology” and “Other Conditions Associated with Nonalcoholic Fatty Liver Disease/Nonalcoholic Steatohepatitis—“Secondary” Nonalcoholic Steatohepatitis”).
Clinical Criteria
By definition, the criteria for NASH require exclusion of alcohol as an etiology. The acceptable level of alcohol consumption is variable but the daily alcohol intake can be conservatively fixed as not exceeding 20 g/day in men and 10 g/day in women—levels below the risk level associated with increased risk of cirrhosis (30 g/day in men and 20 g/day in women) (11,12,13). However, these “cutoffs” leave an unresolved gray area in which a patient prone to NASH may consume alcohol

Sclose to the threshold for liver injury or may have consumed more significant alcohol (measured as lifetime exposure) in the past, leaving open the question of one chronic problem (NASH) superimposed on a past injury. These issues are further discussed in the subsequent text.
Table 39.1. Definitions and Terms
  1. NAFLD. Indicates the presence of fatty infiltration of the liver, defined as fat exceeding 5%–10% of liver weight and frequently taken as fat in >5%–10% macrosteatotic hepatocytes in biopsy specimens. Microsteatosis is an underappreciated aspect because of limitations of routine staining techniques. The term NAFLD includes the term NASH.
  2. Simple steatosis. A type of fatty infiltration (NAFLD) with no or minimal inflammation and no fibrosis. This is synonymous with type 1 disease, as classified by Matteoni (7) (see Table 39.2).
  3. NASH. A type of NAFLD with inflammation, ballooned hepatocytes, and/or fibrosis, usually beginning around the central vein, which may progress to cirrhosis. This is synonymous with type 3 or 4 disease, as classified by Matteoni (7) (see Table 39.2).
  4. “Primary” NAFLD or NASH. A term occasionally encountered in the literature but not uniformly accepted. It indicates typical NAFLD or NASH associated with central obesity and often type 2 diabetes mellitus but without a specific, additional etiology. The likelihood that many cases of “secondary” NAFLD or NASH represent unrecognized or exacerbated “primary” NAFLD or NASH makes the term less useful.
  5. “Secondary” NAFLD or NASH. NAFLD or NASH associated with a specific problem such as a toxin. Use of the term secondary NAFLD or NASH implies the absence of insulin resistance. Many patients previously classified as “secondary” may have exacerbation of underlying “primary” NASH, making this distinction less useful.
  6. “Presumed” NASH or NAFLD. Several epidemiologic and pediatric studies have utilized a presumptive diagnosis of NAFLD or NASH on the basis of abnormal liver enzyme levels, negative results of viral studies, and echogenic or “bright” liver on ultrasonography consistent with fatty infiltration. (See “Imaging in Nonalcoholic Fatty Liver Disease: Ultrasonography, Computed Tomography Scan, Magnetic Resonance Imaging and Magnetic Resonance Proton Spectroscopy”).
NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis.
Histologic Criteria
Steatosis, defined as hepatic fat exceeding 5% to 10% of total weight (14) and usually taken as fat identifiable in more than 5% to 10% of hepatocytes by light microscopy, is an essential feature of NAFLD. Within the spectrum of NAFLD, the term NASH indicates a more severe type of liver injury and worse prognosis compared to “simple steatosis,” which is distinguished by the absence of inflammation or fibrosis and appears to have a long-term stable course (Fig. 39.1) (15). Beyond this distinction, even accomplished pathologists debate the relative importance of specific histologic variables (16). Interestingly, more or less restrictive definitions of the term NASH appear to influence patient demographics, suggesting the presence of gender-based variation in disease expression (17).
Important histologic variables include fibrosis (usually sinusoidal in a pericentral vein or zone 3 distribution), ballooned hepatocytes sometimes containing Mallory hyaline and lobular inflammation (Figs. 39.2 and 39.3). Glycogenated nuclei are common (18,19). Other variables include portal injury, apoptotic bodies, microvesicular steatosis (much more evident using specialized fixation techniques such as osmium), and lipogranulomas (Figs. 39.3 and 39. 4) (20,21). Substantial concordance between observers has been reported for the extent of steatosis, location and severity of fibrosis, and balloon degeneration (22). The degree of fibrosis has been organized into a staging system developed by Brunt et al. (Table 39.2) (23). In addition, although not uniformly accepted, a useful histologic classification scheme (Table 39.2) for NAFLD has been proposed and is widely referenced. It ranges from simple steatosis to the more severe steatosis with balloon degeneration and Mallory bodies or fibrosis (7). Other scoring systems have also been proposed (24) but the most significant refinements in the histologic assessment has been the development of NASH activity

index (NAI) and NASH activity score (NAS), discussed further under “Scoring of the Biopsy (Nonalcoholic Steatohepatitis Activity Index, Nonalcoholic Steatohepatitis activity score).”
▪ Figure 39.1 Simple steatosis: The patient is a 47-year-old woman with mild obesity and an idiopathic, neurodegenerative disease and hepatomegaly. The biopsy specimen showed only minimal inflammation and no fibrosis. No inciting agents were identified to explain the liver condition (hematoxylin and eosin, 200×).
▪ Figure 39.2 A: Nonalcoholic steatohepatitis (NASH) with cirrhosis (stage 4). B: Early stage 3 (bridging fibrosis) with hematoxylin and eosin stain. C: The same biopsy accentuating the presence of bridging with a Masson trichrome stain. The specimen in (A) is from a 65-year-old women with moderate obesity and type 2 diabetes. She does not have complications of portal hypertension. The presence of macrovesicular steatosis, inflammation, and cirrhosis allows the diagnosis of NASH with cirrhosis (stage 4) (100×, hematoxylin and eosin). Specimens (B) (200×, hematoxylin and eosin) and (C) (200×, trichrome) are from her 40-year-old son who has mild liver enzyme abnormalities and mild (mostly truncal) obesity (body mass index = 30) without diabetes. NASH with fibrosis, mildly apparent on the hematoxylin and eosin stain (B), is accentuated with trichrome staining (C), which demonstrates bridging consistent with stage 3. Arrowheads in (B) and (C) define a fibrotic bridge bordering a regenerative nodule. These slides also illustrate a familial pattern seen in approximately 20% of patients.
“Presumed” Nonalcoholic Fatty Liver Disease
In several large epidemiologic studies, the diagnosis of “presumed NAFLD” has been made on the basis of noninvasive testing (See “Epidemiology”) (12). In general, such studies have utilized abnormal transaminases in the absence of other known liver disease and/or liver ultrasonography to make the diagnosis of fatty liver disease. However, the relationship between the diagnosis of “presumed” NAFLD and the histologic activity, stage, and prognosis is unreliable. Indeed, one of the major limitations of these studies is the inability to distinguish NASH from less severe forms of fatty liver such as simple steatosis.
▪ Figure 39.3 Balloon degeneration, Mallory hyaline, and glycogenated nucleus in nonalcoholic steatohepatitis. Long arrow indicates accumulation of perinuclear eosinophilic material (Mallory hyaline) in a ballooned hepatocyte (400×, hematoxylin and eosin). Agreement on what constitutes Mallory hyaline is sometimes hard to obtain. Ubiquitin stain, although infrequently used, can be employed to highlight Mallory hyaline (not shown). Also shown is a pale, glycogenated, nucleus (arrowhead), which is more typical of NASH compared to alcohol-related liver disease.
▪ Figure 39.4 Micro- and macrovesicular steatosis in nonalcoholic steatohepatitis. Arrowhead indicates a cell with small droplets of fat in addition to the more apparent and typically large droplet in macrovesicular steatosis (long arrow, 400x, hematoxylin and eosin). Special stains or fixation techniques, such as osmium tetroxide fixation (not shown), can be used to accentuate the often overlooked microvesicular component.

Cryptogenic Cirrhosis
Serial biopsy studies have established the potential progression of NASH to a stage of “bland” cirrhosis with loss of characteristic histology (Fig. 39.5) (25,26). The loss of fatty infiltration may be due to altered blood flow or decreased sinusoidal permeability and impaired lipoprotein delivery as the liver becomes fibrotic (27,28,29). A number of additional studies have strongly suggested that many cases of “cryptogenic” cirrhosis, a remarkably homogenous group (Fig. 39.6), are the result of such a process (30,31,32,33,34,35,36,37,38). Approximately two thirds of patients with this diagnosis, among the most common indications for liver transplantation, have major risk factors for NAFLD (e.g., obesity and diabetes). In a series of patients undergoing transplantation for “cryptogenic” cirrhosis, definitive features of NASH were evident in 17 of 30 and minor features were seen in an additional 10 patients (39). The significantly increased frequency of steatosis and steatohepatitis after transplantation for cryptogenic cirrhosis further support this relationship (40). On the basis of these associations and predominant histologic findings (and recognizing that other conditions are also involved with cryptogenic cirrhosis), a classification system of cryptogenic cirrhosis can be formulated, as shown in Table 39.3 (41).
Table 39.2. Classification and Stages of Nonalcoholic Fatty Liver Disease/Nonalcoholic Steatohepatitis
Fibrosis Stages of NASH (Brunt et al. (23))
Stage 1: Zone 3, pericentral vein, sinusoidal or pericellular fibrosis
Stage 2: Zone 3 sinusoidal fibrosis and zone 1 periportal fibrosis
Stage 3: Bridging between zone 3 and zone 1
Stage 4: Regenerating nodules, indicating cirrhosis
Types of NAFLD (Matteoni et al. (7))
Type 1: Simple steatosis (no inflammation or fibrosis)
Type 2: Steatosis with lobular inflammation but absent fibrosis or balloon cells
Type 3: Steatosis, inflammation, and fibrosis of varying degrees (NASH)
Type 4: Steatosis, inflammation, ballooned cells, and Mallory hyaline or fibrosis (NASH)
NASH, nonalcoholic steatohepatitis; NAFLD, nonalcoholic fatty liver disease.
Focal Steatosis and Focal Sparing
In a series of patients with various forms of fatty liver disease detected radiographically, focal steatosis was evident in approximately 15% and focal sparing (usually of the caudate lobe) was seen in 9% (42). Variation in blood flow (with resulting differences in

insulin exposure and nutrient delivery) is thought to explain both focal-sparing and focal steatosis (43,44). A relationship to insulin exposure has long been suspected as a factor because of the development of focal steatosis in continuous ambulatory peritoneal dialysis (CAPD) patients exposed to insulin in the peritoneal dialysis fluid (45,46). Histologically, the lesions vary from simple steatosis to steatohepatitis (45). Cirrhotic nodules have also been shown to occasionally have focal fatty change, possibly unrelated to fatty liver disease (47,48).
▪ Figure 39.5 Development of nonalcoholic fatty liver disease (NASH) after transplantation for cryptogenic cirrhosis. A: Explanted liver from an obese, diabetic man showing “bland” cirrhosis (40×, hematoxylin and eosin). B: Two years later (200×, hematoxylin and eosin), a repeat biopsy for abnormal liver enzymes revealed steatosis that was persistent and associated with mild inflammation at 3 years (C) (200×, hematoxylin and eosin). D: Four years after transplantation the patient developed ascites and repeat biopsy showed early cirrhosis with bridging and diminished fatty infiltration (100×, hematoxylin and eosin). Long arrow demonstrates a fibrous band.
Historic Perspective
Early Observations
A long-recognized association between the liver and fat storage is mirrored in a popular explanation of the origin of the Latin term for liver, ficatum, and the corresponding modern Greek term, sycoti, both of which were derived from the common name for fattened animal livers, iecur ficatum and hepar sykoton, respectively (D. Tniakou, personal,

2005). A more scientific appreciation of fatty liver emerged in the 19th century when Virchow classified various types of fatty infiltration of the liver (49). The color, shape, and firmness of a fatty liver were described and fat-globules were proven to be within hepatic cells (50). Morgan, in the 1870s, described an association with obesity and overeating (51). This was extended many years later by Zelman, who reported the existence of liver damage with fibrosis and early cirrhosis in obese patients without a significant history of alcohol consumption (52). More recently, this concept resurfaced in patients who had undergone bypass surgery for morbid obesity (53,54,55,56,57,58). This was widely attributed at the time to postoperative, protein-calorie malnutrition or intestinal bacterial overgrowth, although a correlation between obesity and diabetes and potential liver damage was not strongly emphasized.
▪ Figure 39.6 Past series of patients with cryptogenic cirrhosis. Previous case series have shown a female predominance, onset in sixth or seventh decade, and mildly abnormal alanine transaminase (ALT) levels. [Series from references (30,31,32,33,34,35).]
Table 39.3. Cryptogenic Cirrhosis—Proposed Classification
Class 1—cirrhosis with features of steatohepatitis including scattered steatosis, ballooned hepatocytes, possibly with Mallory bodies, and glycogenated nuclei
Class 2—cirrhosis with features of autoimmune disease including plasma cells or granulomas
Class 3—cirrhosis with features of biliary obstruction including proliferation of bile ducts and cholestasis
Class 4—bland cirrhosis: Cirrhosis lacking other distinguishing features
From Contos MJ, Cales W, Sterling RK, et al. Development of nonalcoholic fatty liver disease after orthotopic liver transplantation for cryptogenic cirrhosis. Liver Transpl 2001;7:363–373 and Ayata G, Gordon FD, Lewis WD, et al. Cryptogenic cirrhosis: clinicopathologic findings at and after liver transplantation. Hum Pathol 2002;33:1098–1104.
The Ethanol Conundrum
As previously noted, the cutoff levels for classification of “nonalcoholic” versus “alcoholic” steatohepatitis (ASH) remain unresolved. Clearly, there are many patients with NASH and related cirrhosis without a history of current or past ethanol exposure (“teetotalers”). Before this recognition, a common experience was nicely summarized by Ludwig in his original description of NASH:
…we have encountered patients who did not drink, who had not been subject to bypass surgery, and who had not taken drugs that may produce steatohepatitis, yet had in their liver biopsy specimens changes that were thought to be characteristic of alcoholic liver disease. In these instances, the biopsy evidence sometimes caused clinicians to persevere unduly in their attempts to wrench from the patient an admission of excessive alcohol or to obtain a confirmation of such habits from relatives of the patients. Thus, the misinterpretation of the biopsy in this poorly understood and hitherto unnamed condition caused embarrassment to the patient and physician.”(2)
Nonetheless, it is widely suspected and recently documented that approximately 10% of patients classified as having NASH actually have had a significant lifetime exposure to ethanol when a more structured

history is obtained (59). Intuitively, synergy between ASH and NASH seems likely by the association of more severe alcohol-related liver disease with obesity (60,61,62,63). However, one study has indicated a lower risk of severe steatohepatitis among obese patients consuming moderate alcohol, possibly mediated by effects on insulin signaling (64,65). Because of the widely perceived health benefits of moderate ethanol ingestion (e.g., red wine), the difficulty in sorting out NASH from alcohol-related liver injury is likely to persist. Immunohistochemical stains for insulin receptors and regulators may provide a means of distinguishing the prominence of one pathway over another but they have not been validated and clinical utility not yet confirmed (66). With the possible exception of glycogenated nuclei (increased in NASH), other histologic features do not reliably distinguish between ASH and NASH (18,67).
A number of laboratory tests have been proposed to make this distinction including carbohydrate-deficient transferrin; however, none has proved satisfactory (68). Transaminase ratios may provide guidance: The aspartate transaminase (AST) to alanine transaminase (ALT) ratio is typically less than 1 in early or mild NASH, between 1 and 2 in more severe NASH, and more than 2 in more severe ASH (See “Clinical and Laboratory Findings”). The extent of elevation of γ-glutamyl transpeptidase (GGT) levels may also be useful (seldom >400 in NASH), but none of these relationships is entirely reliable and may be obscured by concomitant medication use. For these reasons, the history remains the most commonly used means of assessing alcohol consumption, especially when repeated by multiple health care providers over time.
“Hepatogenous” Diabetes
Complicating the relationships between insulin resistance, hyperinsulinemia, and NAFLD is the relationship between liver disease and diabetes, which has long been referred to as hepatogenous diabetes (a term coined by Naunyn in the early 1900s) (69). The issue is whether hyperinsulinemia coincides with the development of liver disease, as appears to be the case in NAFLD, or follows the development of liver disease, as in hepatogenous diabetes. A number of papers have established an increased prevalence of diabetes in cirrhosis of various etiologies (70). Impaired insulin sensitivity rather than decreased insulin metabolism from portosystemic shunting, are postulated to explain this condition (71,72). The role of cirrhosis-related abnormal skeletal muscle metabolism, a major target of insulin and one of earliest abnormalities detectable in type 2 diabetes, is yet to be investigated (73).
Epidemiology and Prevalence in High-Risk Groups
NAFLD is one of the most common of all liver disorders, especially in industrialized countries, and represents a significant source of disease (74,75,76,77,78,79,80). The estimated prevalence in the general population ranges from 2.8% to 20%, depending on the criteria used for estimation (81). In a series of 150 consecutive patients with abnormal liver enzymes for at least 6 months, 40% had steatosis, 15% had hepatitis C, and 2% had nonalcoholic steatohepatitis (82). In another series of 81 patients with abnormal liver enzymes and a negative serologic workup, 50% of the patients had steatosis and 32% had steatohepatitis (83). In the primary care setting, NAFLD accounts for approximately one third of cases of suspected chronic liver disease cases (84). Obesity, type 2 diabetes and hyperlipidemia have been the most constant conditions associated with steatosis and steatohepatitis and are predictors of more severe histologic disease (85). In a large study of risk factors for the presence of NAFLD at autopsy, Wanless and Lentz found mild to severe steatosis in approximately 70% of obese patients compared to 35% of lean patients and steatohepatitis in 18.5% of obese patients compared to just 2.7% of lean patients (86). Diabetes has also been identified as an independent risk factor for NASH. Bellentani et al. identified a 4.6-fold increased risk of fatty liver in obese patients compared to nonobese patients and also identified hypertriglyceridemia as a significant predictor of steatosis on liver ultrasonography (12). An overview of major conditions associated with NAFLD and NASH is shown in Table 39.4.
Biopsy studies in obese patients (body mass index [BMI] usually >30) showed steatosis in 85%, mild to moderate fibrosis in approximately 25% to 30%, and cirrhosis in 1% to 2% (87,88). Ratzui et al. found that approximately 30% of consecutive obese patients with abnormal liver enzymes had at least septal fibrosis and 10% had cirrhosis (89). In another series of obese patients undergoing gastroplasty, Garcia-Monzon et al. observed NASH in 69%, while 22% had simple steatosis and only 8% had a normal biopsy (90). Similar to other studies, one half of those with mild or severe steatohepatitis had normal liver enzymes (91). In another group of patients undergoing bariatric surgery, Dixon et al. reported that only 4% had normal results of biopsies; 71% had simple steatosis and 26% had steatohepatitis with variable degrees of fibrosis (64). Similar results were noted in a more recent study. A compilation of histology in these series of obese patients is shown in Figure 39.7 (92).
Table 39.4. Conditions Associated with Nonalcoholic Fatty Liver
Metabolic factors
   Obesity (especially truncal or central obesity)
   Type 2 diabetes mellitus
   Hyperlipidemia (especially hypertriglyceridemia)
   Systemic lipotoxicity
Specific conditions associated with fatty infiltration of the liver
   Metabolic syndrome (hyperinsulinemia, hypertension, obesity, polycystic ovary disease)
   Mitochondrial diseases
   Weber-Christian disease
Bariatric (weight loss) surgery
   Jejunoileal bypass (no longer performed)
   Gastric bypass or gastroplasty (less frequent compared to jejunoileal bypass)
   Nucleoside analogs
Parenteral nutrition and malnutrition
   Total parenteral nutrition
   Celiac disease
   Wilson disease
   Toxins (CCl4, perchloroethylene, phosphorous, ethyl bromide, petrochemicals)

Type 2 Diabetes Mellitus
Insulin resistance is common in patients with NASH, and hyperinsulinemia plays a pathogenic role in the progression of NASH even in the absence of overt diabetes (93,94). It is estimated that up to 75% of patients with type 2 diabetes have fatty infiltration (93,95,96), although ethnicity appears to influence the prevalence significantly (see subsequent text) (97). Fatty infiltration has been noted to commonly precede the development of overt diabetes in earlier studies (98). The progression to more overt diabetes in these patients depends on peripheral fat and skeletal muscle metabolism and pancreatic islet cell vitality. The severity of liver injury worsens with the degree of abnormal glucose metabolism among obese patients (99) and the impact on the overall clinical course is significant—the standardized mortality ratio in patients with type 2 diabetes is actually higher for cirrhosis than for cardiovascular disease (100,101). Younossi et al. further demonstrated that the coexistence of diabetes in patients with NAFLD more than doubled the prevalence of cirrhosis on diagnostic biopsy from 10% to 25% (102).
As with type 2 diabetes, large studies revealing the true prevalence of NAFLD and associated histology within different forms of hyperlipidemia are lacking. In one study using noninvasive imaging, it was shown that two thirds of patients with hypertriglyceridemia and one third of those with hypercholesterolemia have fatty liver (103). This is probably an underestimation because significant hyperlipidemia (i.e., triglyceride, total cholesterol, or high-density lipoprotein [HDL] cholesterol) was reported in 96% of patients with NASH in one large, well-characterized series (104), although a lower range (3% to 92%) was noted in a compilation of 13 series summarized by McCullough (105). It is likely that ethnic, presumably genetic, factors will influence the true prevalence (see “Genetic Variation”). However, the close relationship between hyperlipidemia and fatty liver disease raises further practical concerns about the effects of antihyperlipidemic medications. Although acute hepatitis appears to be rare

among patients with suspected NAFLD treated with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (See “Treatment of Nonalcoholic Fatty Liver Disease”), the long-term effects have not been adequately explored (106,107,108,109).
▪ Figure 39.7 Liver histology in series of obese patients. Only 10% of obese subjects have normal histology while 5% have previously unidentified cirrhosis. Approximately 85% have steatosis and one third of these have, in addition, nonalcoholic steatohepatitis (NASH) with varying degrees of fibrosis. (Adapted from findings in references 64,87,88,89,90,91,92.)
Metabolic Syndrome
Unexplained elevation of liver enzyme levels, attributed largely to NAFLD, are seen in approximately 7% of individuals meeting the criteria for the metabolic syndrome, defined by the Adult Treatment Panel-III criteria (Table 39.5) (110,111). However, this is probably an underestimation of the true prevalence, given the high prevalence of NAFLD in the general population, the high prevalence of features of the metabolic syndrome among patients with NAFLD, and the frequency of normal liver enzymes even in the setting of significant histologic disease (the last factor obscures the true prevalence if NAFLD is detected depending on abnormal aminotransferases) (112). It has been suggested that NAFLD can cause metabolic syndrome. However, current data indicate that both hepatic steatosis and central adiposity are independent risk factors for metabolic syndrome and all three conditions appear to be united by variable degrees of insulin resistance and systemic lipotoxicity (see subsequent text) (113,114,115).
Normal Body Mass Index
NAFLD has been well documented in patients with normal BMI (5,17,116). This group appears to contain relatively younger males with milder histology, visceral or central adiposity (without overt obesity by BMI), and hyperinsulinemia. Such individuals, possibly more common in Asian populations where visceral adiposity is seen with lower BMI, are thought to represent an initial stage of the insulin resistance syndrome (117,118,119,120). These findings are consistent with the major role of body fat distribution as opposed to simply the amount of body fat in the development of insulin resistance and NAFLD (121,122,123,124).
Table 39.5. Adult Treatment Panel-III Criteria for the Metabolic Syndrome (110)
Risk factor Definition
Abdominal obesity Waist circumference
   Men ≥102 cm (≥40 in.)
   Women ≥88 cm (≥35 in.)
Triglycerides ≥150 mg/dL
High-density lipoprotein cholesterol  
   Men <40 mg/dL
   Women <50 mg/dL
Blood pressure ≥130/85 mm Hg
Blood glucose ≥110 mg/dL
Pediatric Patients
In parallel to the increasing problems of obesity, a number of papers have now established NAFLD and NASH as potentially serious problems in children (125,126,127,128,129). As with adult fatty liver disease, the problem appears to exist worldwide. The histologic severity appears to vary substantially but fibrosis and cirrhosis have been described in 18 of 24 patients in one Canadian series (130). Recently acquired rather than long-standing obesity increases the risk of fatty infiltration in this group. Ethnic variation, male preponderance, and prominent portal tract injury standout in pediatric NASH (126,131). Ethnic influences appear similar to those in the adult disease (see subsequent text), with a greater risk among children, especially boys, of Hispanic or northern European descent compared to African American pediatric patients (132).
Nonalcoholic Steatohepatitis as a Factor in Other Liver Diseases
NAFLD is so common that it can be expected to coexist with virtually any other liver disease and to possibly influence the course of that disease (133). Several studies have indicated that steatosis (possibly mediated by core protein metabolism) (134) is associated with hepatitis C (especially genotype 3) and accelerates liver disease progression (135). The association with insulin resistance and type 2 diabetes has been sufficiently strong to dub hepatitis C as a metabolic disease (136), and a measure of insulin resistance in hepatitis C has been shown to predict fibrosis (137). Although viral metabolism may be important in this association (138), many patients with hepatitis C virus and features of NASH have coexisting risk factors for metabolic syndrome and, therefore, for NASH, suggesting a synergistic effect rather than a causal relationship (139,140). Iron loading of the liver has also been suggested as a factor in the progression of NASH, although this is controversial (141,142). As recently pointed out by Powell et al. hyperferritinemia in NAFLD correlates more closely with insulin resistance than with iron overload or HFE gene mutations and can be reversed with weight loss (143).
Genetic Factors in Nonalcoholic Fatty Liver Disease
Ethnic Variation
Several reports have described significant ethnic variation in the prevalence of NAFLD/NASH (144,145,146,147). This is explained, in part, by ethnic differences in the

distribution of body fat because central adiposity correlates better with fatty infiltration of the liver (and to insulin sensitivity) than does total body fat (148,149,150,151,152,153). The observation is consistent with the genetic associations of obesity and diabetes and the ethnic variation described in lipoprotein metabolism (154,155,156,157,158). Similar ethnic variation has been described in cryptogenic cirrhosis (144,159). Most of these studies, including the large epidemiologic study by Weston et al. have indicated that people primarily of African American descent seem to have a lower than expected prevalence of steatosis relative to the rates of obesity and diabetes (160). The most convincing data comes from Browning et al. who studied 2,287 subjects with magnetic resonance spectroscopy (MRS) (see subsequent text) to measure liver triglyceride content (161). The authors again noted that African Americans tended to have significantly less steatosis than Hispanic or non-Hispanic whites independent of the presence of obesity or diabetes, suggesting that additional factors influence the development of steatosis (Fig. 39.8). Similar variation in the prevalence of NAFLD among patients with diabetes has also recently been described. (Fig. 39.9).
Familial Factors
A high prevalence of afflicted first-degree relatives has been described in at least two studies of NASH (162,163). Both these studies reported an association with obesity and diabetes, and one showed a relationship with cryptogenic cirrhosis within kindreds, perhaps explaining prior reports of familial cirrhosis (164). However, it remains unclear whether these associations represent genetic mechanisms, common environmental exposures, and shared risks or perhaps some combination of both genetic risk and common health habits. The demonstration of impaired skeletal muscle mitochondrial metabolism associated with insulin resistance in the offspring of patients with type 2 diabetes further suggests a genetic risk closely related to handling and disposition of intracellular fat (165).
▪ Figure 39.8 Distribution of hepatic triglyceride content by ethnicity as assessed by 1H-magnetic resonance spectroscopy. Browning et al. (162) showed that the distribution of hepatic triglycerides content was skewed toward lower levels in blacks and slightly higher levels in Hispanics. (Reprinted with permission of Wiley-Liss Inc., a subsidiary of John Wiley & Sons, Inc.)
Genetic Variation
The search for genetic factors has involved seeking specific polymorphisms of candidate genes involved in fat metabolism, oxidative stress and cytokine activity, and genome-wide comparative surveys of single nucleotide polymorphisms (SNPs) (166,167). Experimentally, variation in hepatic lipogenic gene expression was seen in mouse strains in which, paradoxically, greater hepatic steatosis was associated with less insulin resistance (168). In humans, microarray analysis has shown that patients with NASH underexpress genes

associated with the mitochondrial antioxidant system and overexpress acute-phase reactants that may play a role in insulin resistance (169,170). Variation in adiponectin and hormone-sensitive lipase polymorphisms (important in flux of fatty acids between the liver and periphery) has been described, as has variation in SNPs of microsomal triglyceride transfer protein (171,172). Other potential factors include the CD36 protein (e.g., fatty acid translocase—a scavenger-receptor expressed in adipose tissue where it functions as a transporter of fatty acids and oxidized low-density lipoprotein), lamin A mutations (associated with lipodystrophy), mitochondrial mutations (see subsequent text), iron overload, cytochrome P-450, peroxisome metabolism, and variation in the function of antioxidants (e.g., glutathione, vitamin E and superoxide dismutase) that are known to be depleted in conditions associated with hepatic lipid peroxidation (173,174,175,176,177,178,179,180,181,182). As previously described in patients with hepatitis C infection, a synergistic association between high expression of angiotensinogen and of transforming growth factor-β1 genotypes has also been described in obese patients with more severe NASH (183,184).
▪ Figure 39.9 Diabetes, ethnicity, and nonalcoholic fatty liver disease (NAFLD). Sundaram et al. demonstrated significantly increased odds ratios for NAFLD in patients with diabetes who were of predominantly European descent compared to patients of African American descent and in patients with metabolic syndrome (97).
Clinical and Laboratory Findings
Symptoms and Signs
Specific symptoms in NASH are infrequent. Associated signs and symptoms and laboratory findings are summarized in Table 39.6. In a compilation of several studies, Reid noted an absence of specific symptoms in 48% to 100% of patients (8). However, in another study, Sanyal noted fatigue in 45 of 62 patients (73%) and right upper quadrant pain in 30 of 62 (48%) (185). This discomfort is often mistaken for gallstone disease that can also be associated with obesity and hyperinsulinemia (186). Persistence of pain after cholecystectomy or an abnormal-appearing liver at the time of surgery often precipitates a referral to the liver clinic. Hepatomegaly is usually due to steatosis but may be due to hepatic glycogenosis in patients with diabetes (187). Acanthosis nigricans has been noted in some children with NASH. The presence of palmar erythema or spider angiomas suggests cirrhosis that may very subtle—we have seen a number of patients whose initial presentation of NASH with cirrhosis was the presence of rectal varices detected at screening colonoscopy. A family history of fatty liver, unexplained liver abnormalities, or cryptogenic cirrhosis is seen in approximately 20% to 25%. A subacute form of NASH with cirrhosis probably represents unrecognized chronic disease with unexplained sudden decompensation (188,189).
Table 39.6. Signs, Symptoms, and Laboratory Features
Asymptomatic (48%–100%)
Fatigue (∼70%)
Right upper quadrant pain (up to 50%)
Occasional neurological deficits (possibly part of systemic lipotoxicity)
Acanthosis nigricans in children
Palmar erythema and spider angiomas (if cirrhosis has developed)
Elevated aspartate transaminase and alanine transaminase levels (usually <2 × normal)
Mildly elevated γ-glutamyl transpeptidase level
Mildly elevated alkaline phosphatase level
antinuclear antibody positive in ∼30%
Increased immunoglobulin A
Abnormal iron indices in 20% to 60% (usually not with definite hemochromatosis)
See text references.
Not surprisingly, clinical findings associated with the metabolic syndrome, including obesity, type 2 diabetes, hyperlipidemia, hypertension, hyperuricemia, polycystic ovary syndrome (i.e., insulin resistance, diabetes, obesity, hirsutism, oligomenorrhea, or amenorrhea), and gallstone disease, are common (93,94,186,190,191,192,193,194,195,196,197). Changes in body composition due to aging and cirrhosis (both associated with loss of muscle and adipose tissue) may mask a history of prior, severe, and long-standing obesity (198,199). The relationships between the hypercoagulable component of metabolic syndrome (due to plasminogen activator inhibitor-1 increased with central obesity), systemic endothelial/vascular abnormalities, and steatohepatitis have not yet been fully explored (200,201,202).
Laboratory Features
Many patients present with only abnormal liver test results, especially ALT and/or AST often detected on routine screening or during institution of weight loss therapy, or antihypertensive, antidiabetic, or antihyperlipidemic therapy. Transaminase levels are usually less than two times the upper limit of normal; from a compilation of five earlier studies reviewed by Harrison and Neuschwander-Tetri, the average AST and ALT levels were 79 IU/L and 64 IU/L, respectively (203). AST, partly mitochondrial in origin (204), correlates imperfectly with the degree of inflammatory activity and injury as does the AST to ALT ratio, in that values less than 1 are consistent with mild disease while values greater than 1 often indicate fibrosis (205). Mild elevation of GGT and alkaline phosphatase levels may also

be present but are not as well studied. GGT was predictive of more severe histology in one study, although isolated GGT level elevation is of doubtful significance in this setting (89).
Nonalcoholic Fatty Liver Disease and Normal Aminotransferases
It is now well known that patients with significant histologic liver disease, including cirrhosis, may have normal aminotransferase levels (206,207). The lack of complete correlation between histology and aminotransferases is similar to that seen with hepatitis C—the relationship is present but imperfect. It is generally accepted that elevation of AST and ALT levels indicates hepatocyte injury or, possibly, the rate of cell turnover, which results from cell injury (208). Other explanations include the possibility that the “true normal” range in obese patients may be lower than that for lean individuals (209). Indeed, ALT levels are positively correlated with central obesity and hyperinsulinemia (leading to an ongoing effort to revise normal ranges) and negatively correlated with caffeine consumption (210,211,212). In addition, the use of a thiazolidinedione medication (troglitazone) was shown to normalize transaminase levels and improve parameters of inflammation, but all patients still met criteria for NASH on follow-up biopsy (213).
Other Laboratory Abnormalities
Abnormal sinusoidal deposition of immunoglobulin A (IgA), indistinguishable from that seen with alcohol-related liver disease, has previously been described in NASH (214). Serum IgA level elevation in alcohol-related liver disease results from neoantigen formation (215). Although not as well studied, isolated elevation of serum IgA level is seen in 25% of patients with NASH and a lower serum IgG to IgA ratio is associated with more severe fibrosis (216). Antinuclear antibody is seen in approximately 35% of patients with NASH (217,218). Abnormal iron indices, including ferritin and transferrin saturation, are seen in 20% to 60% of patients, but this is not usually associated with homozygous or heterozygous genetic hemochromatosis and the relationship to disease severity remains unsettled (219). Low platelets, often misdiagnosed previously as idiopathic thrombocytopenia, warrants an additional search for hypersplenism and cirrhosis (220).
Findings of Lipodystrophy
Fatty infiltration of the liver is a common feature of the lipodystrophies (221,222). These disorders, congenital or acquired, vary in the distribution of dystrophic fat. Common features include diabetes, hypertriglyceridemia, panniculitis, and focal or diffuse loss of subcutaneous fat. The mechanism is thought to involve failure of differentiation of preadipocytes, possibly due to lipin deficiency (223). The diagnosis depends on cross-sectional imaging of an involved area to demonstrate fat atrophy. Histologic NASH was observed in eight of ten patients in a recent small series that also demonstrated improvement with leptin replacement, supporting a role for leptin deficiency (224). Cirrhosis has been described among women with the acquired variety (225,226). A familial form of partial lipodystrophy has also been described in association with NAFLD (227). On the basis of the subtle presentation of lipodystrophy and lack of easily available tests, it seems likely that there is more overlap between NAFLD and lipodystrophy than is commonly appreciated. A potentially important distinguishing feature of the more severe forms of lipodystrophy is the characteristically low leptin level (228). Lipodystrophy related to antiretroviral therapy, perhaps now the most common form of this disorder, is discussed further in subsequent text.
Findings Suggestive of Mitochondrial Disease
The systemic nature of NAFLD and the presence of morphologic mitochondrial abnormalities in liver biopsy specimens (94,229) suggest that other mitochondria-related clinical manifestations may be prevalent in NAFLD (230). Features of systemic mitochondrial disease can be observed in patients with NASH including depression, ophthalmoplegia, neurodegenerative diseases, deafness, lipomatosis, and gut dysmotility (231). Few of these have been systematically investigated, although Al-Osaimi et al. recently reported an increased prevalence of a subtle form of gaze palsy in patients with NASH (232). Insulin resistance and dyslipidemia are features of symmetric lipomatosis or Madelung’s disease (associated with mitochondrial deoxyribonucleic acid [DNA] mutations) (233,234) and of maternally inherited diabetes and deafness (MIDD) syndrome (also associated with mitochondrial mutation) (235). Similar hepatic mitochondrial DNA mutations have been identified in some patients with NASH (236). In addition, cryptogenic cirrhosis has been described in certain mitochondrialopathies (237). Underlying these seemingly unrelated problems, mitochondrial heteroplasmy could play a role in the variable expression of a mitochondrial problem in different organs (238,239).
Weber-Christian Disease
Nodular panniculitis, especially over the lower extremities, is the most distinguishing feature of classical Weber-Christian disease (240,241). Steatohepatitis is a common finding, suggesting a systemic disorder of

fat metabolism. Liver chemistries may be only mildly increased or there may be an acute steatohepatitis with jaundice and Mallory bodies may be evident. The diagnosis can be established by demonstration of fat necrosis. In distinct contrast to many other forms of NAFLD, immunosuppression has been reported to be effective therapy, suggesting an autoimmune component (242).
Imaging in Nonalcoholic Fatty Liver Disease: Ultrasonography, Computed Tomography Scan, Magnetic Resonance Imaging, and Magnetic Resonance Proton Spectroscopy
Liver imaging plays an important role as an initial diagnostic tool, in epidemiologic studies, and in the evaluation of partial liver donors (Fig. 39.10) (243). Of particular relevance to epidemiologic studies, imaging is used to assess the distribution of body fat in comparison to anthropometric measurements (244,245). For example, computed tomographic (CT) measurement of abdominal visceral fat at the L4-5 intervertebral space, adjusted for age and sex (246), is the most established technique (247). Magnetic resonance imaging (MRI) (248,249) and ultrasonography (250) have also been utilized to quantitate body fat, especially central adiposity. However, none of the conventional imaging techniques can accurately grade or stage NASH (with the possible exception of advanced MRI), and the accuracy of even detecting steatosis is limited above levels of 20% to 30% (251,252).
Ultrasonography and Elasticity
Ultrasonography can detect the presence of hepatic steatosis through increased echogenicity and sound attenuation with defined criteria for fatty infiltration (253,254,255). However, its utility is limited because of difficulty in differentiating fibrosis from fatty infiltration (256,257), misinterpretation of focal fatty sparing as a hypoechoic mass (258), and poor detection if the degree of steatosis is less than 20% to 30%. Nonetheless, as initial testing in a suspected case and for large population screening, it is a reliable and economical means of assessment. Newer techniques including measurement of hepatic elasticity (decreased with fibrosis) are promising but have not been adequately evaluated in NAFLD in which fatty infiltration may present a problem by spuriously decreasing elasticity.
Computed Tomography
With CT, unenhanced scans remain the optimal technique to image hepatic fat when the diagnosis relies on attenuation differences between the liver and spleen (151,259,260). The sensitivity and specificity of detecting fatty liver (with spleen-minus-liver attenuation of 10 Hounsfield units) were 0.84 and 0.99, respectively, in one study (261). Recently, therapeutic trials have used the “liver-to-spleen ratio” (in Hounsfield units) in which values less than 1 are consistent with relatively greater steatosis. Contrast-enhanced CT scan has more limited utility because the optimal liver-minus-spleen attenuation differences are significantly influenced by the contrast injection rate and timing of measurement (262).
Magnetic Resonance Imaging and Spectroscopy
Conventional spin-echo MRI is insensitive in detecting fatty infiltration (263). This limitation is improved with refinements of the technique, including modified spin-echo and in-phase, out-of phase imaging (264,265). Proton (1H)-MRS is another means of very accurately assessing the degree of hepatic steatosis (266,267). In one study, the correlation between fat concentration

measured in the liver biopsies and 1H-spectroscopy was 0.9 (P < 0.001) (268). 31P spectroscopy (Fig. 39.11) offers the futuristic prospect of measuring cellular chemistry and metabolic parameters including adenosine triphosphate (ATP) homeostasis in the liver, lipid peroxidation, and phospholipid content of the liver (270,271,272,273,274,275).
▪ Figure 39.10 Radiologic imaging of nonalcoholic fatty liver disease (NAFLD). A: Sonographic appearance of NAFLD. B: Computed tomography (CT) appearance of NAFLD. (A) demonstrates a heterogeneous-appearing echotexture with findings of “bright liver” in the parenchyma; (B) shows a relatively hypodense liver compared to the spleen (liver-to-spleen ratio <1).
▪ Figure 39.11 Biopsy sampling error. Ratziu et al. (269) show evidence of the sampling error that can occur if inadequate liver biopsy specimens are obtained. The biopsy specimens of 15 of 51 patients showed at least a one-stage difference in fibrosis stage, and 2 showed a two-stage difference from the same right flank site with slightly different needle orientation, indicating the need for samples of at least 2 to 3 cm in length using a standard 15–gauge width needle to minimize sampling error. The symbols represent the results of an individual patient’s consecutive biopsies.
The Use of Liver Biopsy in Nonalcoholic Fatty Liver Disease
Liver biopsy remains the gold standard for confirming the diagnosis, staging the extent of injury, and grading the degree of activity. However, biopsy is often deferred, and a conservative course of exercise and diet is prescribed as initial steps unless the clinical evaluation indicates more advanced disease or when there is a question of medication-induced injury. Practically speaking, the importance of the biopsy is increasing as therapeutic interventions improve and with the recognition of occult cirrhosis as a significant consideration in the management of older patients with diabetes (see “Treatment of Nonalcoholic Fatty Liver Disease”). The use of surrogate tests such as serologic fibrosis markers, refined imaging techniques, or noninvasive measures of elasticity is promising but remains to be fully explored (276,277,278).
Predictors of Underlying Histology
Clinical predictors of findings on the initial biopsy specimen have been studied extensively (Table 39.7), although such noninvasive predictors should be regarded cautiously because of the high number of exceptions. Age (>40 to 50 years), the degree of obesity, the degree of diabetes or insulin resistance, hypertriglyceridemia, hypertension, family history of NASH or cryptogenic cirrhosis, complete abstinence from ethanol, transaminase level (AST and ALT) elevation (relatively weak marker), and an AST to ALT ratio more than 1 are often, but not invariably, predictive of more advanced histology, but conflicting studies exist (5,7,30,60,64,76,85,89,90,91,205,279,280,281,282,283,284,285). Other factors noted as predictive of more severe histology on the initial biopsy specimen include circulating antibodies to lipid peroxides (286) and the number of parameters positive from the Adult Treatment Panel-III criteria for metabolic syndrome (287). A number of these variables have been combined into composite scores, but these also have limited sensitivity and specificity and have not gained wide clinical acceptance. Incorporation of markers of collagen metabolism, such as serum hyaluronic acid, into a clinical score enhances the predictive value but still carries only a 76% accuracy rate in predicting significant injury on biopsy (288).
Table 39.7. Predictors of More Severe Histology in Nonalcoholic Steatohepatitis
Age >40–50 y or female gender
Degree of obesity or steatosis
Overt diabetes or increased insulin resistance
Elevated alanine transaminase level
Elevated aspartate transaminase level
Elevated γ-glutamyl transpeptidase level
Aspartate transaminase:alanine transaminase ratio >1
Elevated immunoglobulin A level
See text.

Limitations of the Biopsy
The major limitations of biopsy include patient inconvenience, the potential for complications, performance in obese patients, and sampling error. In general, complication rates with liver biopsy are infrequent, and the variety of available techniques offer improved safety and applicability (289,290,291,292). Sampling error on percutaneous biopsy is well recognized but can be minimized by obtaining an adequate specimen more than 2 to 3 cm in length by 1.5 mm wide (15 to 16 gauge) (293,294,295). In the most convincing study, Ratziu et al. demonstrated a 10% to 15% risk of a two-stage error and roughly 40% risk of one-stage error in the fibrosis stage when specimens are less than about 2 to 3 cm in length (Fig. 39.11) (269).
Scoring of the Biopsy (Nonalcoholic Steatohepatitis Activity Index, Nonalcoholic Steatohepatitis Activity Score)
Although the Brunt score remains the most commonly used method of assessing the biopsy, recent introduction of composite scores have provided a useful means of assessing response to treatment particularly in clinical investigations (Table 39.8). Key parameters have been combined into the NAI and NAS (297,298). The NAI ranges from 0 to 12 and accounts for steatosis, necroinflammatory activity, and hepatocyte injury (ballooned cells), each of which is scored from 0 to 4. The more refined NAS uses a scale of 0 to 8

to score steatosis, lobular inflammation, and cellular ballooning. Fibrosis stage is scored separately in both composites.
Table 39.8. Histology Scoring Systems in Nonalcoholic Fatty Liver Disease
  Steatosis Inflammation (lobular) Hepatocyte injury (ballooning) Maximum score Fibrosis
NAS-I (NAFLD activity score) (Brunt et al., 1999) (23) 0–3 0–3 0–2 8 0–4
0—none 0—no foci 0—absent   0—none
1—<33% 1—1–2 foci/mpf 1—present-z3   1—sinusoidal
2—33%–66% 2—3–4 foci/mpf 2—marked-z3   2—sinusoidal and periportal
3—>66% 3—>4 foci/mpf     3—bridging fibrosis
NAI (NASH activity index) (Promrat et al. 2004) (296) 0–4 0–4 0–4 12 0–4
0—<5% 0—no foci 0—absent   0—none
1—5%–25% 1—<1 foci/mpf 1—only z3, <50% of CVs   1—perisinusoidal
2—>25%–50% 2—1 foci/2 mpf 2—only z3, >50% of CVs   2—perisinusoidal and periportal
3—>50%–75% 3—1–2 foci/mpf 3—both z2 and z3, (1/3–2/3)   3—bridging fibrosis
4—>75% 4—>2 foci/mpf 4—all zones (>2/3)   4—cirrhosis/regeneration
NAS-II (NASH clinical research network revision)
(Kleiner et al., 2005) (297)
0–3 0–3 0–2 8 0–4
0—<5% 0—no foci 0—absent   0—none
1—5%–33% 1—<2 foci/mpf 1—few ballooned cells   1—perisinusoidal or periportal
   1A—mild, z3, perisinusoidal
   1B—moderate, z3, perisinusoidal
2—>33%–66% 2—2–4 foci/mpf 2—many/prominent ballooning   2—perisinusoidal and portal/periportal
3—>66% 3—>4 foci/mpf     3—bridging fibrosis
NAS, NASH activity score, nonalcoholic stestohepatitis; NAI, NASH activity index, nonalcoholic steatohepatitis; NAFLD, nonalcoholic fatty liver disease; z2, zone 2; z3, zone 3; mpf, medium power field—200× magnification; CVs, central veins.
Natural History and Prognosis
Mortality Overview
Although patients with NAFLD frequently have substantial comorbid conditions that could influence survival, progressive liver disease often becomes the dominant problem. Consistent with this, obesity was recognized as a major risk factor for cirrhosis-related deaths in people who consume little or no alcohol (299). Among patients with type 2 diabetes in Japan, the cause of death was cirrhosis in 6.4% (compared to 19.5% from heart disease) (300). However, the observed versus expected deaths ratio (O/E ratio) was actually higher for cirrhosis than for heart disease (2.67 vs. 1.81). In another report, the five- and ten-year survival in NASH was estimated at 67% and 59%, respectively (301). These figures were lower than those for a matched population, but the difference did not reach statistical significance. In a 12-year follow-up, Cortez-Pinto et al. showed that patients with NASH had a liver-related death rate similar to that for ambulatory patients with ASH—both rates were significantly better than those for hospitalized patients with ASH (302). In one of the largest natural history studies published to date, Adams et al. showed that liver disease is the third leading cause of death in a group of 420 patients with NAFLD followed up for a mean of 8 years compared to liver disease as the thirteenth most common cause of death in the general population (303,304).
Variation Based on Initial Histology
In a retrospective study of adult patients in the United States, the overall mortality among those with fatty liver accompanied by inflammation, fibrosis, balloon cells, or Mallory hyaline was increased compared to crude death rates, and cirrhosis-related deaths were also increased when fatty infiltration was accompanied by the presence of these more severe histologic markers (7). In contrast, several prior studies have shown that simple steatosis or steatosis with minimal inflammation (Matteoni type 1 and 2) is a relatively stable condition (15,305). The relative stability of these milder forms of fatty infiltration indicates that NASH (fatty infiltration plus fibrosis and/or balloon cells) probably begins at the higher stage rather than progressing through stages from simple steatosis to more severe forms. However, this has not been established and such a transition can be seen with rapid weight loss (and perhaps with other forms of metabolic stress) (306,307).
Serial Biopsy Studies
A number of studies reporting serial biopsy in patients with NASH have now been published (5,25,26,89,308,309,310,311). Although the studies have a number of shortcomings, including variable biopsy techniques, different entry criteria, and incomplete data on confounding variables such as voluntary lifestyle changes and antidiabetic or antihyperlipidemic medications (312), there is sufficient data to draw some reliable observations. Compiling the studies, 177 patients have undergone a second biopsy after a mean of 4.5 years (Table 39.9). Cirrhosis developed in 10% of patients while fibrosis progressed in 33%, remained stable in 41%, and improved in 22%. In some of the patients, the progression to cirrhosis was surprisingly rapid over 1 to 2 years. Most papers show that as fibrosis progresses, aminotransferases, steatosis scores, and inflammation improve paradoxically. This is of some concern in the clinical setting in which normalization of the aminotransferases should be regarded with cautious optimism, especially in older patients. Normalization of these parameters is consistent with the progression of NASH to a “burned out” state, which is often recognized as “cryptogenic” cirrhosis (see preceding text).
Table 39.9. Serial Biopsies in Patients with Cryptogenic Cirrhosis
Author Number Years of follow-up Progressed to stage 4 Progressed to stage 2–3 No change Improved
Lee (1989) (308) 12 3.5 2 3 7 0
Powell (1990) (24) 13 4.5 3 3 6 1
Bacon (1994) (5) 2 5 1 0 1 0
Ratziu (2000) (89) 4 5 1 1 2 0
Harrison (2003) (308) 19 5.7 2 4 9 4
Fassio (2004) (309) 22 4.3 0 7 11 4
Adams (2005) (310) 98 3.2 9 24 35 30
Totals/Average 177 4.5 18 (10%) 59 (33%) 73 (41%) 39 (22%)

Nonalcoholic Steatohepatitis–Related Cirrhosis and Cryptogenic Cirrhosis
NASH-related cirrhosis appears, like other forms of cirrhosis, to progress through several stages. A small number of studies have addressed the natural history of this form of cirrhosis. Hui et al. compared the course of 23 patients with well-defined NASH cirrhosis to 46 matched patients with cirrhosis from hepatitis C (313). Nine of 23 patients with NASH–cirrhosis developed major complications of portal hypertension (e.g., ascites, encephalopathy, and variceal bleeding) during a mean follow-up of 7 years, with complication-free survival of 83%, 77% and 48% at 1, 3 and 10 years, respectively. Ratziu et al. compared the course of 27 overweight patients with cryptogenic cirrhosis to 10 lean patients with cryptogenic cirrhosis and 391 patients with hepatitis C–related cirrhosis in a retrospective follow-up cohort study (314). With a mean follow-up of 22 months, 2 of the 15 patients presenting only with abnormal liver test results developed major complications of portal hypertension and 5 developed HCC. The overall severity and risk for either a complication of portal hypertension or HCC were greater in obese patients with cryptogenic cirrhosis compared to the lean cryptogenic cirrhosis group but not different from patients with hepatitis C. The authors concluded that obesity-related cirrhosis behaves as aggressively as hepatitis C–related cirrhosis.
Hepatocellular Cancer
A number of studies have now shown an increased risk of HCC in obese patients and in those with diabetes (315,316,317,318,319). In addition to the natural history studies noted in the preceding text, there are also a number of well-documented NASH case reports and series indicating this progression (320,321,322). Although animal models of fatty liver exist in which HCC develops without cirrhosis, most human studies suggest that silent progression of NASH to cirrhosis is the more predominant pathway (323,324). The association of NASH with cryptogenic cirrhosis (see preceding text) and the risk of HCC in cryptogenic cirrhosis further strengthens the idea that for many patients with progressive NASH, HCC is an increasingly common late complication (325,326,327). Although the molecular events leading to HCC are yet to be fully defined, the proliferation of oval cells (hepatocyte progenitor cells) observed in human and experimental NAFLD may be a contributing factor (328) in addition to mutations in regulator genes such as PTEN that regulate certain aspects of both fat metabolism and cell proliferation (329).
Experimental and Animal Models of Nonalcoholic Fatty Liver Disease
Small Animal Models
Many advances in the understanding of NAFLD have resulted from the development of small animal models—usually mice or rats. This field has been reviewed extensively by Koteish and Diehl (330), Farrell (331), and Nanji (332). Several of the best known models include the hyperphagic ob/ob mouse, which has a congenital deficiency of leptin; the FA/FA rat, which has an impaired leptin receptor; and the methionine–choline–deficient (MCD) rodent. Studies in these animals, as well as the numerous transgenic models, have led to many seminal observations in NASH about hepatic fat metabolism and its regulation. Although providing insight into specific pathways, these models share the common problem of inadequately imitating the common form of human NAFLD. For example, the ob/ob mouse requires other provocative measures to produce significant injury in addition to simple steatosis, and the MCD model, although producing hepatic injury without further provocation, lacks insulin resistance (333).
Large Animal Steatosis and the Liver as a Normal Fat-Storing Organ
Fatty liver disease is a well-known problem in veterinary medicine. Variants of the disorder are seen in cows (334,335,336), hens (337), and cats (338,339) and can occur spontaneously or with phosphorous supplements in pigs (340,341,342). Hepatic “lipidosis” is reported to be one of the most common liver disorders in domestic cats and, similar to human steatohepatitis, has been associated with mitochondrial morphologic abnormalities (343). Seasonal variation in hepatic fat has been observed in deer (344). These observations suggest a close integration of the liver into the adipose system, which is evident experimentally in studies demonstrating the upregulation of genes governing adipocyte differentiation during liver regeneration (345,346).
Palmipedes (migratory geese) develop fatty liver before migration and utilize fat as a preferred source of energy for muscles through the expression of fatty acid–binding protein (347). This has been exploited in the production of foie gras in which geese are fed a corn-based diet, resulting in a 8- to 10-fold increase in liver size in as little as 2 weeks (from 100 to 800 g). The goose hepatocyte enlarges to three to four times the original diameter, with fat droplets observable as mixed micro- and macrosteatosis. Although early harvest limits the natural course, degenerative changes

is seen and cirrhosis is anecdotally noted (348). In addition, accidental exposure to moldy corn (mycotoxins) is associated with liver failure with ascites (349). Interestingly, significant subspecies variation exists in the lipogenic capacity (350). Indirect evidence suggests that the main mechanism involves increased lipid synthesis and altered very low density lipoprotein (VLDL) synthesis and secretion (351,352,353). These observations further illustrate the role of the liver within a broadened concept of the adipose system, with its inherent plasticity (transdifferentiation of fat stores) and its role in energy metabolism and thermoregulation (354,355,356,357). It has been hypothesized that skeletal muscle fat metabolism, which interacts with hepatic fat stores and strongly influences insulin sensitivity, confers a survival advantage under harsh conditions (358).
Pathogenesis of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis
Although many of the metabolic abnormalities noted in NAFLD have yet to be assimilated into a cohesive “story,” the importance of lipotoxicity as underlying insulin resistance, the metabolic syndrome and cellular injury in steatohepatitis, is emerging as the most likely primary pathway in typical cases of human NAFLD (359). In this light, NAFLD can be viewed as a potential adverse occurrence within the realm of the metabolic syndrome and more specifically as a possible adverse outcome of systemic lipotoxicity (360). Excessive fatty acids have a broad effect on many tissues and even influence gene expression and, hence, adipocytokine production in adipose tissue (361). However, it is very likely that there is substantial variation between groups or individuals in the relative importance of different mechanisms, as suggested in the wide ethnic variation in the development of steatosis—the first “hit” in the path to hepatic fibrosis. A plausible pathway for some of the most common abnormalities is shown in Figure 39.12.
The normal, healthy liver contains no more than 5% lipid by weight (362). The levels of both triglycerides (mostly unsaturated fatty acids) and free fatty acids (mostly saturated) are increased in the liver of obese patients (363). The development of cellular injury involves a cascade of events beginning with the development of steatosis (NAFLD) and the subsequent development of oxidative stress, lipid peroxidation, and cell injury, and activation of profibrotic cytokines, resulting in NASH (364). Increased hepatic fat stored in the form of triglyceride can be derived from the plasma fatty acid pool mostly released from peripheral adipose tissue by lipolysis, from dietary fat through chylomicron remnants, or from de novo synthesis within the liver. De novo lipid synthesis, largely from glucose, is governed by two main transcription factors that signal transcription of the enzyme systems responsible for fatty acid synthesis and subsequently for their esterification into triglyceride: Sterol regulatory element–binding protein (SREBP), which is governed by insulin, and carbohydrate response element–binding protein (CREBP), which is governed by glucose levels (365,366,367,368). SREBP-1 level appears to be increased in animal models of NAFLD, and it is suspected that a similar process is going on human NAFLD.
The disposition of fatty acid in the liver proceeds by one of several routes: Storage as triglyceride, export as VLDL, or oxidation. Regulation of the predominant form of disposition depends on a number of interacting factors based on energy homeostasis and influenced by peroxisome proliferators activated receptor (PPAR) activity and probably by the activity of the adrenergic nervous system (369,370,371). Although focal fat necrosis may occur from direct release of fat from swollen hepatocytes in NAFLD (372), toxicity, as noted by Bass and Merriman, results primarily from the indirect effects of lipid peroxidation and to a lesser extent from direct toxicity of fatty acids (369).
Insulin Resistance
Although epidemiologic work indicates that peripheral insulin resistance is neither sufficient nor essential for NAFLD, it is present in most patients with NAFLD and, therefore, is so closely intertwined with the disease as to be virtually inseparable in most cases. Insulin resistance is characterized by a reduced sensitivity to insulin in target tissues (e.g., muscle, adipose tissue, and liver), where it normally favors glucose transport into the cell, storage of glycogen, inhibition of lipolysis in adipose tissue, and inhibition of gluconeogenesis from the liver. The expected manifestations of insulin resistance include decreased peripheral (muscle) glucose utilization, enhanced lipolysis and mobilization of fatty acids from peripheral fat stores, and increased hepatic glucose output (normally suppressed by insulin). Insulin resistance, primarily mediated by excessive fatty acids (373,374,375,376,377,378,379), is observed in NAFLD using a variety of techniques (112,380,381,382). The relationship between fatty liver and insulin resistance represents a (sometimes precarious) balance between the three main target organs—skeletal muscle, adipose tissue, and the liver (383). Using modifications of the insulin “clamp” test, Sanyal et al. convincingly demonstrated that the predominant site of resistance in NAFLD is in the peripheral fat and skeletal muscle as opposed to the liver (94,384,385,386,387). Recent work using MRS of skeletal

muscle indicates that insulin resistance in metabolic syndrome results from skeletal muscle lipotoxicity and secondary changes in mitochondrial metabolism (388). Excessive skeletal muscle fatty acid also leads to inhibition of insulin-stimulated glucose transport through the effects on insulin receptor substrate-1 (IRS-1) (389). Teleologically, insulin resistance in this setting can be seen as a normal response to excessive energy substrate availability (diet and obesity) and underutilization (activity).
Lipid Peroxidation and Hepatic Lipotoxicity
Lipid peroxidation reflects an imbalance between pro- and antioxidant substances (oxidative stress) (390). It is a branching, chain reaction stimulated by a free radical attack on unsaturated fatty acids (Fig. 39.12) (391). Free radicals, which initiate the process, may be derived from mitochondrial, peroxisomal, or cytochrome P-450 fat metabolism, with the formation of superoxide, hydrogen peroxide, and hydroxyl radicals. The products of the reaction are another free radical and a lipid hydroperoxide, which, in a reaction catalyzed by iron, forms a second (lipid) free radical and, therefore, amplifies the process. Damage involves chemical bonding with other cellular constituents including membrane lipids, proteins, and DNA (392). Although difficult to measure directly, lipid peroxidation is the main process leading to inflammation, activation of cytokines, stimulation of stellate cells, and fibrosis (393,394). Levels of the markers of lipid peroxidation (e.g., nitrotyrosine, 4-hydroxynonenal [4-HNE], and malonic dialdehyde [MDA]) are increased in human NASH and are associated with mitochondrial abnormalities (94,395,396). The latter may be an injury response or due to increased activity of uncoupling protein (UCP), which decreases oxidative stress and is associated with abnormal oxidative phosphorylation and an adenosine triphosphate deficit (94,270,397,398,399,400). Cytokine level elevation activates stellate cells with secondary fibrosis. Interestingly, a major site of oxidative injury appears to be the border area of small fat droplets that consist of a unique phospholipid monolayer (401,402).
Cytokine Activation and Fibrosis
Cytokine level elevation, especially tumor necrosis factor-α (TNF-α), has been well described in NAFLD (403). In animal models, obesity itself appears to sensitize the liver to cytokine-mediated injury (404,405), TNF-α has been shown to induce mitochondrial UCP in regenerating liver in animal models of nonalcoholic fatty liver, and transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) have also been implicated as mediators of fibrosis in NASH (403,406,407). Cell culture experiments indicate that stimulation of TNF-α results from fatty acid–mediated destabilization of lysosomes (408). Indirect evidence suggests that lipoperoxide-induced expression of inflammatory cytokines is mediated by the transcription factor nuclear factor-κB (409). A number of additional transcription factors (e.g., PTEN) and cytokines (e.g., osteopontin) are being recognized that, at least in experimental conditions, appear to interact with each other and influence insulin signaling and fibrosis pathways (410,411,412).
Adiponectin and Leptin (Adipocytokines)
Adiponectin is the most abundant protein in the adipocyte and participates in glucose homeostasis and insulin signaling through receptors in the muscle (adipoR1) and liver (adipoR2) (413,414,415,416,417). Its level is decreased in both obese/diabetic mice and humans and in patients with NASH compared to body fat–matched controls (416,418,419). Unlike leptin (see subsequent text) or TNF-α, adiponectin levels appear to be significantly different in patients with simple steatosis versus those with NASH, suggesting a more significant role for it in disease pathogenesis including a possible anti-inflammatory effect (420,421,422,423). Leptin is a circulating protein coded for by the obesity gene (chromosome 7q31 in humans) and produced primarily in white adipose tissue and its level is increased in cirrhosis (424,425,426,427). Its primary role is to govern satiety through action at the hypothalamus; however, human obesity is usually associated with elevated leptin levels (428). It has been variably implicated in the development of histologic injury in human and experimental NAFLD (429,430,431,432,433). In a recent study from Angulo et al. elevated leptin levels in progressive NASH were attributed to factors involved in production; no difference in leptin was seen between patients with worsening injury or those without on serial biopsy (434).
Other Cellular Injuries (Ballooning and Apoptosis)
Although the ballooned hepatocyte constitutes a marker for more progressive injury, a consensus definition remains elusive. The normal diameter of the hepatocyte varies from 13 to 30 μm (435). On light microscopy, ballooned cells are described as 1.5 to 2 times the normal size, located predominantly in zone 3 compared to other zones, and have rarefied cytoplasm. Ballooning in viral hepatitis involves hydropic changes and irregular dilatation of the smooth endoplasmic reticulum (436,437). However, electron microscopy using osmium fixation, which highlights fat droplets, suggests that most such cells in NASH


are microsteatotic (438). Coupled with observations of localization of lipid peroxidation (see preceding text) and localization of Mallory bodies (439), these observations suggest that handling of small-droplet storage fat is a significant source of injury in NASH. Apoptotic bodies are only occasionally seen in NASH specimens, although signal pathways for apoptosis are significantly activated in human NASH (440). This paradoxical situation is the result of rapid “cleanup” of apoptotic bodies such that they are infrequently seen or it may represent the result of a (sometime precarious) balance of pro- and antiapoptotic factors involving adaptive changes in the mitochondrion that play a central role in the regulation of apoptosis (441,442,443). Such adaptive (or maladaptive) changes may make the fatty liver more resilient in terms of localized cell death but more prone to necrosis because these processes are closely interrelated and coregulated (444).
▪ Figure 39.12 Mechanism of steatohepatitis—increased hepatic fat stores result from increased delivery of fatty acids, increased de novo synthesis, and decreased export of fat. The excessive free fatty acids both result from and promote extrahepatic insulin resistance. Fatty acid oxidation leads to the formation of free radicals, which derive from mitochondrial, peroxisomal, and cytochrome P-450 oxidation. Free radicals (e.g., super oxide, hydrogen peroxide, and hydroxyl radical) can directly activate transcription factors, resulting in overexpression of cytokines and, if not neutralized by the antioxidant system (superoxide dismutase and glutathione peroxidase), the free radicals (X) can trigger the chain reaction of lipid peroxidation (rancidification). Unsaturated, polyenoic fatty acids are especially susceptible. This produces a carbon-centered lipid radical, which reacts with oxygen to form an oxygen-centered lipid peroxyl radical. This substance subsequently reacts with a second fatty acid to form another lipid free radical (propagation) and a lipid hydroperoxide. The latter is unstable and, in the presence of iron and another fatty acid, reacts to form yet another lipid radical (amplification). Alternatively, the lipid hydroperoxide may degrade to malonic dialdehyde (MDA) (detected by the thiobarbituric acid reactant [TBAR] reaction) or to ethane or pentane (detected in breath tests), react with other radicals to form a stable pigment (e.g., ceroids and lipofuscins), or may cross-link with deoxyribonucleic acid (DNA) or other cellular proteins (neoantigen formation). This oxidative stress induces cytochrome P-450 fatty acid oxidation and causes mitochondria changes with enlargement and formation of crystalline bodies possibly a result of abnormal expression of uncoupling protein. This leads to an adenosine triphosphate (ATP) deficit that increases the risk of necrosis and probably stimulates apoptosis, resulting in sporadic cell death and increased cell turnover. Lipid peroxidation also causes transcription of profibrotic cytokines that activate stellate cells, producing fibrosis. Countering the process of lipid peroxidation is the antioxidant system, which neutralizes lipid radicals by combination with vitamin E. The latter is then restored by shuffling the radical groups to glutathione through selenium. GSH, glutathione; NADPH, nicotinamide adenosine dinucleotide phosphate, reduced form; GSSG, oxidized glutathione; IgA, immunoglobulin A.
Mitochondrial Changes and Adenosine Triphosphate Homeostasis
The mitochondrion may be both an especially important source of reactive oxygen species and a target for injury resulting from lipid peroxidation (196,445,446). Its unique evolutionary history places it in a central position of several major metabolic pathways, including fatty acid synthesis and β-oxidation, oxidative phosphorylation (ATP generation), and signaling pathways for the process of apoptosis (447). Mitochondrial morphologic abnormalities (Fig. 39.13) have been observed in both ASH and NASH (94,229,448,449,450). Commonly noted intramitochondrial inclusions are thought to be either a protein or phospholipid precipitate (451). It is thought that these morphologic abnormalities correlate with functional abnormalities, including respiratory chain dysfunction, and by their distribution may be part of the adaptive process to oxidative stress that make the liver more tolerant to reactive oxygen species but more susceptible to ischemic injury (398,452,453). For example, the poor function of steatotic livers in transplantation has been attributed in part to abnormal ATP homeostasis with depletion of electron transport components and increased susceptibility to ischemic injury (454,455,456,457).
Impaired function of the mitochondrial electron transport chain (ETC) in NAFLD has been described in several studies (458,459), attributable in part the to overexpression of UCP induced by increased fatty acids (460). Perez-Carreras et al. noted reduction in the ETC activity to 40% to 70% of normal in all the major complexes (I to V) in human NASH. In vivo impairment of ATP synthesis was observed by Cortez-Pinto et al. using 31P MRS of the liver in controls compared to patients

with NAFLD (Fig. 39.14) (270). In experimental conditions, complex I and V (ATP synthase) dysfunction have been implicated in reperfusion injury, and these results open inquiry as to what the optimal protective mechanism is for the ETC during liver transplantation. (461,462). In a recent exhaustive review of the subject, Pessayre and Fromenty proposed that NASH is primarily a “mitochondrial disease.” Although this may initially seem to be an overstatement, a cogent argument is presented on the fundamental role of the mitochondrion in hepatic fat metabolism, skeletal muscle physiology, insulin resistance, and pancreatic islet cell vitality, placing the mitochondrion at a central point in the overall pathophysiology of NAFLD (463).
▪ Figure 39.13 Deformed mitochondria with crystalline inclusions in nonalcoholic steatohepatitis: A: An elongated mitochondrion with two bundles of parallel, crystalline structures (arrows) near a large fat droplet. B: Two mitochondria cut tangentially showing crystalline inclusions in an area of cytoplasm between two large fat droplets (arrows). Note the paucity of normal cristae.
▪ Figure 39.14 Abnormal adenosine triphosphate (ATP) homeostasis in nonalcoholic fatty liver disease. Initial fructose metabolism requires the expenditure of energy in the form of ATP. Intravenous injection of fructose normally produces a significant drop in hepatic ATP stores, as measured by magnetic resonance spectroscopy, followed by return to baseline. The baseline in patients with fatty liver is similar to that in healthy controls, but there is a blunted recovery indicating the presence of a relative energy deficit in the fatty liver. NS, not significant. (Adapted from
Cortez-Pinto, Chatham J, Chacko VP, et al. Alterations in liver ATP homeostasis in human nonalcoholic steatohepatitis: a pilot study. JAMA 1999;282:1659–1664.
Cytochrome P-450
Similar to alcohol-related liver disease, induction of cytochrome CYP 2E1 in NAFLD has been described as a possible source of oxidative stress and activation of cytokines (464,465,466) through enhanced microsomal ω-oxidation of fatty acids (normally a minor pathway of fatty acid metabolism). Expression of CYP 2E1 is influenced by a high-fat, low-carbohydrate diet and colocalizes in immunohistochemical stains to fatty cells and markers of lipid peroxidation (467,468). It is associated with increased activity of mitogen-activated protein kinases (MAPKs) through the activity of extracellular signal–regulated kinases 1 and 2 (ERK1/2), which participate in the regulation of cell death (apoptosis) pathways (469). On the basis of animal studies, it is unlikely that P-450 2E1 plays a singular role in NASH because CYP 2E1 nullizygous, transgenic mice also develop a steatohepatitis-like picture (470). Indeed, Schattenberg et al. (469) showed experimentally that overexpression of CYP 2E1 is protective against oxidative injury and decreases apoptosis but increases the risk of necrosis induced by fatty acid exposure. Consistent with this adaptive process, inhibition of CYP 2E1 in experimental conditions increases formation of Mallory bodies (471).
Abnormal Lipoprotein Metabolism
Consistent with its close association with hyperlipidemia and metabolic syndrome, NASH has been associated with abnormal apolipoprotein (apo) metabolism (472). Two groups have reported decreased apoB-100 secretion in NASH, indicating possible impairment of VLDL synthesis (a major component of which is apoB-100) (473,474), and another group has described differences in apoA-I (a component of HDL) in patients with NAFLD compared to controls (475). Isolation of small lipid droplets from hepatocytes of rats in experimental studies has demonstrated that the lipid droplets

are composed of neutral fats consisting mainly of esters of linoleic, oleic, and palmitic acid (24). Similar studies in human liver biopsy samples from patients with ASH have shown that the lipid composition of accumulating lipid droplets is similar regardless of size but that the smallest droplets, based on size, density, and lipid composition, resemble a precursor of plasma VLDL (476,477). Taken together with observations of cellular ballooning (see preceding text), these observations again point to the abnormal handling of the small storage lipid droplets as being significant in NASH progression.
Peroxisomal Metabolism
The peroxisome is involved in numerous metabolic pathways including synthesis of plasmalogens, bile acids, and cholesterol, and oxidation of very long chain fatty acids, branched-chain fatty acids, dicarboxylic acids, polyunsaturated fatty acids (PUFA), L-pipecolic acid, and phytanic acid (478,479). Steatohepatitis develops in mice lacking peroxisomal fatty acyl-CoA oxidase (480), and morphologic abnormalities with diminished size but increased number of microsomes have been described in human fatty liver (481). Peroxisomal fatty acid oxidation represents another potential source of reactive oxygen species including superoxide and hydrogen peroxide that form during peroxisomal oxidation of very long chain fatty acids and metabolism of dicarboxylic acids (derived from cytochrome P-450 ω-oxidation of very long chain fatty acids) (482). Multiple inherited disorders of peroxisomal metabolism have been described including Zellweger’s syndrome, adrenoleukodystrophy, and Refsum’s disease. Although disturbed peroxisomal metabolism does not appear to be a primary factor in most patients with NASH (based on normal levels of dicarboxylic acid (94) and normal very long chain fatty acid profiles in patients with NASH—Caldwell et al.), it is possible that genetic abnormalities, nutritional abnormalities, or adaptive changes in the peroxisome may contribute to the condition in some patients.
Other Conditions Associated with Nonalcoholic Fatty Liver Disease/Nonalcoholic Steatohepatitis—“Secondary” Nonalcoholic Steatohepatitis
The foregoing discussion has largely centered on what some refer to as “primary” NASH or NAFLD typically associated with insulin resistance and the metabolic syndrome. The presence of other factors may suggest a separate disease with overlapping features of NASH (Tables 39.1 and 39.4) distinguished primarily by the lack of insulin resistance. However, this does not exclude the likelihood that many such patients have underlying “primary” NASH exacerbated by some other insult.
Bariatric Surgery
Historically, weight-reduction therapy played an important role in the recognition of NAFLD/NASH because of the unexpected exacerbation that was noted in some patients after early jejunoileal bypass (483). Stimulation of TNF by bacterial endotoxin has been postulated as an etiologic factor, but this was not supported by efforts to prevent injury using oral antibiotics (484) or by reports of a similar process after gastroplasty, in which bacterial overgrowth is less of a problem (485). Micronutrient deficiency has also been proposed. The rate of weight loss may be a key factor by increasing the rate at which intra-abdominal fat is mobilized. In spite of its historical association with exacerbation of NASH, weight loss surgery (particularly gastric bypass) remains a viable option in some patients (see subsequent text).
Medication Induced
A number of medications have been implicated as causes of steatohepatitis. In several cases, particularly with nifedipine and diltiazem (486,487), the association may be one of common drug use in patients at high risk for NASH. Similarly, Chitturi and Farrell pointed out in a recent paper that the risks for methotrexate-induced steatohepatitis are almost identical to those of NASH, suggesting a high degree of overlap between these entities (466,488). Tamoxifen-induced steatohepatitis presents a particularly difficult balance of risk–benefit in patients on therapy for prior breast cancer (489,490). Although liver injury was not considered a side effect in a recent review of adjuvant breast cancer therapy (491), the authors and others have observed severe steatohepatitis with cirrhosis in this setting (492,493,494). The risk factors for tamoxifen-induced steatohepatitis are, as with methotrexate, similar to those of NASH, suggesting a possible synergistic effect of the drug in a patient prone to NASH. Amiodarone, which has long been associated with phospholipidosis and steatohepatitis (495,496,497), has become an extremely common medication in cardiology. As with tamoxifen, its use should not be undertaken without some consideration of the high likelihood that many recipients will have preexisting fatty liver because of shared risks between NASH and heart disease. The issue again becomes one of risk versus benefit. We have observed advanced cirrhosis with death due to

liver failure in this setting and associated litigation. To our knowledge, an adequate risk–benefit analysis has not been done.
Human Immunodeficiency Virus Therapy
An acquired lipodystrophy, associated with insulin resistance and steatosis and sharing features with multiple symmetrical lipomatosis, has been described with nucleoside analog therapy for human immunodeficiency virus (HIV) infection (on highly-active antiretroviral therapy [HAART]) (498,499,500,501). The syndrome may be increased in women, may present acutely, and can be associated with a Reye’s syndrome–like picture, neuropathy, myopathy, and pancreatitis (502). There are increased concerns that patients with this syndrome are at increased risk of developing NAFLD and the possibility of progression to NASH and cirrhosis. The pathogenesis of NAFLD from HAART therapy is related to insulin resistance (383), mitochondrial DNA damage (503), and the development of lactic acidosis (504).
Parenteral Nutrition, Malnutrition, and Celiac Disease
Liver disease, often with macro- and microvesicular steatosis, is one of the most common and potentially severe side effects of total parenteral nutrition (TPN) (505). In one series of patients on extended TPN, macrovesicular steatosis was seen in 63% of patients with cholestasis and 100% of those without cholestasis (506). Microvesicular steatosis and phospholipidosis (fat-laden cells in the sinusoidal space or portal tract) are also common features. Both the amount of lipid infusion and its composition appear to affect the expression of liver disease in this setting (507). Choline deficiency may play a role in some patients. At the opposite end of the nutrition spectrum, fatty liver is a common finding in kwashiorkor, in which export of lipid from the liver because of protein deficiency (diminished apoprotein B) is thought be the primary mechanism (508). In both types of nutritional fatty liver, zone 1 (periportal) involvement may predominate. There have been reports suggesting an association between NASH/NAFLD and celiac disease (509,510,511). Bardella et al. examined 59 consecutive patients with elevated transaminase levels and NAFLD and found that 6 patients had positive tissue transglutaminase antibodies and 2 (3.4%) were positive for antiendomysial antibodies. Overall, two patients (3.4%) were positive for both antibodies and had positive histology (512). In another report, Nehra et al. observed that 1 out of 47 patients with NASH was positive for antiendomysial antibodies (514).
Solvents and Industrial Agents
A variety of toxins have been implicated in the development of fatty liver diseases (514). Better described agents include carbon tetrachloride (now rarely used), dimethylformamide, perchloroethylene, and petrochemical derivatives (515,516,517,518). Other compounds and elements that have been implicated include phosphorous (See “Experimental and Animal Models of Nonalcoholic Fatty Liver Disease”), ethyl bromide, ethyl chloride, and rare earths. Synergy between exposure to these agents and disease progression in an obese patient and/or a patient with diabetes is suspected but not established. Cotrim et al. have demonstrated a potentially progressive form of NAFLD that results from petrochemical exposure and occurs in the absence of insulin resistance (519).
Wilson Disease
Macro- and microvesicular steatosis are well-known features of Wilson disease (520,521). Consideration of Wilson disease should be made especially with steatohepatitis in a younger individual. It is not known how often the carrier state for mutations in the nuclear-encoded gene for copper-transporting ATPase (522) could play a role in more typical cases of NASH. We have noted borderline values of ceruloplasmin occasionally in patients with some features of obesity-related steatohepatitis (S. Caldwell, unreported clinical observation, 2004). Mitochondrial injury, mutations, and premature oxidative aging were recently described in patients with Wilson disease, suggesting a possible overlap (through mitochondrial dysfunction) with more typical NAFLD and NASH (523).
Inherited Metabolic Diseases
Macrovesicular steatosis can be seen in a variety of inherited metabolic diseases, most, but not all, of which present in childhood. Disorders include glycogen storage diseases (524), galactosemia (525), tyrosinemia (526), heterozygous hypobetalipoproteinemia (527,528), and abetalipoproteinemia. Both of the latter disorders are characterized by impaired formation of VLDL due to decreased synthesis of apolipoprotein B. A number of lipid storage diseases, (e.g., cholesterol ester storage, Niemann-Pick disease, Tay-Sachs disease, and Gaucher’s disease) can have excessive fatty infiltration of the liver with cholesterol esters, sphingolipids, phospholipids, sphingomyelin, gangliosides, or glucocerebrosides. Presentation as systemic diseases in infancy (although not exclusively so) and the distribution (predominantly in the reticuloendothelial cells) distinguish the lipid storage disorders from typical NAFLD/NASH (21,529).

Managing the Obese Patient with Diabetes and Cirrhosis
Silent progression of NASH to cirrhosis, often in association with normalization of the liver enzymes, has led to the common situation in which otherwise stable patients with type 2 diabetes are found incidentally to have cirrhosis (299,312). Although early data indicates that therapy aimed at diabetes may ameliorate steatohepatitis (See “Antidiabetic Agents”), there is little knowledge on the effects of other commonly employed medications such as antidepressants, sulfonylureas, HMG-CoA reductase inhibitors, or even insulin itself. Moreover, other adjunct treatments that are commonly used in these patients (e.g., aspirin for coronary disease or angiotensin-converting enzyme inhibitors for prevention of diabetic kidney disease) may have adverse effects if cirrhosis has developed (530). This situation arises because of the insidious development of the hyperdynamic state of cirrhosis as a result of portosystemic shunting. One of the hallmarks of this striking change in physiology is systemic vasodilatation with associated changes in renal hemodynamics. Therefore, in addition to considering disease-specific conditions such as the possibility of varices or HCC, broad treatment considerations need to include a reconsideration of certain tenets of diabetes management. Unfortunately, this aspect of NAFLD has not been adequately investigated.
Treatment of Nonalcoholic Fatty Liver Disease
Who Should be Treated?
Patient selection and the relative risk–benefit of different interventions remain one of the most challenging aspects of treating NAFLD. Although less severe forms of NAFLD, such as simple steatosis or steatosis with only inflammation (types 1 and 2 NAFLD), may progress to cirrhosis, most studies support an increased risk of progression, mainly in the presence of more severe histology at baseline such as ballooned cells and fibrosis (NASH or types 3 and 4 NAFLD) (7,15,305,309,531,532). These data indicate the need for careful patient selection in studying the effects of as yet unapproved pharmacologic interventions (Table 39.10). In general, there is a consensus that dietary changes and increasing activity are cornerstones and that these lifestyle changes are typically part of standard recommendations in spite of limited data and variable acceptance by the patient (see subsequent text). Voluntary adoption of these recommendations complicate the interpretation of pharmacologic therapy—most of the existing publications have lacked controls and, although most have included anthropometric indices such as weight and some have accounted for dietary changes, none has accounted for the degree of conditioning that could influence steatosis and may not be reflected in anthropometric measurements.
Endpoints of Therapy
The primary endpoint of therapy remains changes in the histology (See “Scoring of the Biopsy (Nonalcoholic Steatohepatitis Activity Index, Nonalcoholic Steatohepatitis activity score)”). However, sampling error, especially with cores less than 2 cm, is a potential problem and few of the studies reviewed in the subsequent text have provided sufficient details of the biopsy to account for this confounding variable (269,293,294,295). Novel markers of histologic injury especially applicable to the research setting include stains for lipid peroxide by-products, electron microscopy to assess mitochondrial morphology, and markers of stellate cell activation (557). The major surrogate markers for liver injury include the serum aminotransferase levels (approximation of inflammatory activity), imaging to assess hepatic fat content (e.g., ultrasonography, CT scan, MRI, MRS), and serologic fibrosis panels that are in development. Other important measures include anthropometric indices (e.g., weight, BMI), the degree of physical conditioning (e.g., lactate threshold), measures of insulin signaling (e.g., glucose tolerance testing, HOMA or Quicki, insulin clamp tests), serologic or urinary markers of lipid peroxidation (e.g., malondialdehyde or hydroxynonenal), and cytokine levels such as TNF-α, TGF-β, and adiponectin.
Initial Intervention
Lifestyle changes remain a cornerstone of initial management (558,559,560). Optimistically, diet modification and exercise can be accomplished in obese patients, with as many as 80% of patients achieving dietary goals and 36% achieving exercise goals (561). Pessimistically, even intensive counselling to reduce fat intake (<30% of daily calories) and engage in regular physical activity produces only a modest 5% sustained weight loss (562,563,564). In NAFLD, surprisingly little is known about the effects of specific diet types or how much exercise should be recommended or how best to convey this advice.
Exercise Alone
Visceral adiposity and steatosis have been shown to correlate inversely with the degree of cardiorespiratory fitness (114,565). However, the relative benefit of



exercise without weight loss (i.e., the “fit fat” individual) versus aggressive dieting has not been extensively explored in NAFLD (566). Exercise affects insulin signaling primarily through its effect on skeletal muscle substrate utilization and mitochondrial oxidative phosphorylation (567,568). The most effective “conditioning” exercise is the one that just passes beyond the lactate threshold—a level usually associated with some degree of discomfort (569). A number of studies have shown that increasing activity reduces expression of the metabolic syndrome, suggesting that a similar effect might be found with NAFLD (570,571). However, even trained athletes who are obese and with high calorie intake (e.g., Sumo wrestlers) have increased features of metabolic syndrome, indicating a limited benefit of exercise without concomitant weight loss (572).
Table 39.10. Pharmacologic Treatment of Nonalcoholic Fatty Liver Disease/Nonalcoholic Steatohepatitis
Authors N Design Agent Daily dose Duration Transaminases Histology Hepatic fat by imaging
Caldwell et al. (2001) (213) 10 Open label Troglitazone 400 mg 3–6 mo Improved Improved mild inflammation Not evaluated
Acosta et al. (2001) (533) 8 Case series Pioglitazone Variable 2–12 mo Improved Improved Not evaluated
Sanyal et al. (2004) (534) 10 RCT Pioglitazone 30 mg 6 mo Improved Improved steatosis, ballooning and fibrosis (with vitamin E) Not evaluated
Shadid et al. (2003) (535) 5 Open label Pioglitazone 30 mg 4–5 mo Improved Not evaluated Not evaluated
Neuschwander-Tetri (2003) (536) 30 Open label Rosiglitazone 8 mg 48 wk Improved Improved steatosis, inflammation, and fibrosis CT scan: Improved
Promrat et al. (2004) (296) 18 Open label Pioglitazone 30 mg 48 wk Improved Improved steatosis, inflammation, and fibrosis MRI: Improved
Tiikkainen et al. (2004) (537) 9 Open label Rosiglitazone 8 mg 16 wk Improved Not evaluated MRI: Improved
Coyle et al. (1999) (538) 2 Open label Metformin 500 mg 4 mo Improved Improved inflammation Not evaluated
Marchesini et al. (2001) (539) 14 Open label Metformin 1.5 g 4 mo Improved Not evaluated U/S: Decreased hepatomegaly
Lavine et al. (2004) (540) 10 Open label Metformin 1 g 6 mo Improved Not evaluated MRI: Improved
Nair et al. (2004) (541) 15 Open label Metformin 20 mg/kg 12 mo Improved Improved inflammation Not evaluated
Uygun et al. (2004) (542) 36 Open label Metformin 1.5 g 6 mo Improved Improved inflammation U/S: Improved
Tiikkainen et al. (2004) (537) 11 Open label Metformin 2 g 16 wk Improved Not evaluated MRI: No improvement
Buigianesi et al. (2005) (543) 55 Open label Metformin 2 g 12 mo Improved Improved steatosis, inflammation, and fibrosis in limited sample Not evaluated
Guma (1997) (544) 24 RCT UDCA 10 mg/kg 6 mo Improved Not evaluated Not evaluated
Ceriani (1998) (545) 31 RCT UDCA 10 mg/kg 6 mo Improved Not evaluated Not evaluated
Laurin (2002) (546) 24 Open label UDCA 13–15 mg/kg 12 mo Improved Improved steatosis Not evaluated
Mendez-Sanchez (2002) (547) 33 RCT UDCA 1,200 mg 6 wk Improved Not evaluated Not evaluated
Santos (2003) (548) 30 RCT UDCA 10 mg/kg 3 mo Improved Not evaluated Not evaluated
Bauditz (2004) (549) 12 Open label UDCA 7–10 mg/kg 6 mo Improved Not evaluated Not evaluated
Lindor (2004) (550) 166 RCT UDCA 13–15 mg/kg 24 mo Improved Improved steatosis Not evaluated
Lavine (2000) (551) 11 Open label Vitamin E 400–1,200 IU 4–10 mo Improved Not evaluated Not evaluated
Hasegawa (2001) (552) 12 Open label Vitamin E 300 mg 12 mo Improved Improved steatosis, inflammation, and fibrosis Not evaluated
Sanyal (2004) (534) 10 Open label Vitamin E 400 IU 6 mo Improved Mild improved steatosis Not evaluated
Harrison (2003) (553) 45 RCT Vitamin E 1,000 IU 6 mo Improved Improved fibrosis (with vitamin C) Not evaluated
Kugelmas (2003) (554) 16 RCT Vitamin E 800 IU 3 mo Improved Not evaluated Not evaluated
Vajro (2004) (555) 28 RCT Vitamin E 400–1,000 IU 5 mo Improved Not evaluated Not evaluated
Kawanaka (2004) (556) 10 Open label Vitamin E 300 mg 6 mo Improved Not evaluated Not evaluated
RCT, randomized controlled trial; CT, computerized tomography; MRI, magnetic resonance imaging; U/S, ultrasonography; UDCA, ursodeoxycholic acid.
Dietary Weight Loss and Exercise
Diet- and exercise-induced weight loss has shown promise in treating NAFLD but may also be associated with progression of liver disease if the rate of loss is greater than 1.6 kg/week and especially with drastic calorie reduction (306,573,574,575,576,577). In addition, weight loss may not restore normal insulin secretory pulses, suggesting persistent islet cell dysfunction in some patients (578). However, improvement in liver enzymes and histology has been shown with nutritional counselling and weight reduction (579,580). Ueno et al. reported significant improvement in liver enzyme levels and degree of steatosis in 15 obese patients treated with diet (25 kcal/kg ideal body weight per day) and exercise (walking and jogging) for 3 months compared to a control group, but fibrosis was not significantly altered (581). Not surprisingly, clinical improvement has been correlated with improving insulin signaling (582). Nonetheless, the paucity of more definitive data on this practical form of intervention is striking (583).
Weight Loss in Children
A number of small studies support the use of weight loss therapy in children. In one study, 33 obese children underwent a moderate hypocaloric diet (35 kcal/kg per day) and aerobic activities (6 hour/week or more) to achieve weight loss of approximately 500 g/week over a period of 6 months (584). All patients who lost at least 10% body weight had normalized liver enzyme levels and improved ultrasonographic findings. In a similar study, seven patients were treated with hypocaloric diet and exercise to obtain a weight loss approximately 500 g/week. All had significant reduction of aminotransferase levels; four out of five patients who had previous ultrasonography showed decreased evidence of steatosis. One patient who had a follow-up liver biopsy showed improvement in steatohepatitis (585).
Dietary Composition
Dietary lipid composition may be especially important for both insulin signaling and risk of lipid peroxidation (586). Dietary fat predominantly (98%) consists of triglyceride (glycerol and three fatty acids). Fatty acids affect the phospholipid composition of cell and organelle membranes and influence insulin sensitivity, gene regulation (PPARs), differentiation in adipocytes, and prostaglandin physiology (361,587,588,589). Fatty acids reach the liver from albumin-bound fatty acids released by adipose tissue and from chylomicron remnants directly from dietary fat (361,590). Diets enriched in PUFA (fish oil or ω-3 fatty acids) have not been adequately investigated for their potentially beneficial or detrimental effects in NAFLD, but some encouraging data (see subsequent text) has begun to emerge in spite of concerns for the role of ω-3 fatty acids in alcohol-related liver disease (591,592,593).
A study in MCD mice demonstrated that hepatic fatty acid content influences PPARα activity, which subsequently influences the activity of cytochrome P-450; this appears to promote lipid turnover in vivo and improves steatohepatitis (594). Supplementation of the diet with ω-3 fatty acids reduced collagen content in mice exposed to thioacetamide, whereas a mixture of ω-3 and ω-6 fatty acids had no effect (595). Lipid composition has also been shown to influence the expression of UCP-2 in the rat liver (596), and supplementing of ω-3 fatty acids in both the ob/ob mouse model and in the Fisher 344 leptin-resistant rat increased fat degradation factors (PPARα) and decreased hepatic fat synthetic factors (SREBP-1) (597,598). Conjugated dienoic derivatives of linoleic acid (conjugated linoleic acid [CLA]), have variable effects on hepatic and peripheral fat stores, depending on the content of other dietary fats (599). In humans, ongoing trials using indirect indices of NASH (e.g., aminotransferases, echotexture, Doppler blood flow, TNF) indicate improvement with ω-3 supplements (e.g., eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) (600,601,602).
Weight Loss Supplements
Orlistat (tetrahydrolipostatin) decreases fat absorption by inhibiting lipase. Harrison et al. have reported benefit in weight loss (mean of 10 kg) and histologic parameters (i.e., steatosis and fibrosis) in two pilot studies (603,604). Other existing studies are of relatively short duration and uncontrolled but have shown general improvement in surrogate markers of injury (605). Malabsorption of fat-soluble vitamins (including vitamin E) is of theoretic concern. There is presently too little data on sibutramine (a serotonin and norepinephrine reuptake inhibitor that is approved as a weight loss agent), although one uncontrolled study

has suggested improved aminotransferases with associated weight loss (575,605).
Weight-Reduction Surgery
Variations of gastric bypass or gastric restriction are becoming increasingly popular, although these interventions are not without the risk of hepatic decompensation (606,607). Early studies showed a reduction in the levels of markers of metabolic syndrome (e.g., glucose, insulin, fibrinogen, triglycerides, and uric acid) and ALT levels and a reduction in steatosis but a slight overall increase in inflammation and no significant change in fibrosis. However, reduced perisinusoidal fibrosis was reported in another study by Silverman et al. (99). Moreover, resolution of histologic NASH was observed in one study using adjustable gastric banding, and an even more substantial reduction in fibrosis was recently reported in one large series, although caution is warranted, given the potential for deterioration with too rapid weight loss (608,609,610). Prebypass treatment with ursodeoxycholic acid (UDCA) and vitamin E has been advocated in patients with active NASH but has not been studied in a controlled trial.
Ursodeoxycholic Acid and Cytoprotective Agents
The potential benefits of UDCA derive from its effects on mitochondrial membrane stability, improved blood flow, and/or immunomodulation (611,612,613,614,615,616). Several studies, some published only as abstracts, suggested a potential benefit of UDCA (544,545,546,547,548,549). In a fully published paper, Laurin treated 24 patients with NASH using UDCA (13 to 15 mg/kg) and observed improvement in liver enzyme levels and amelioration of steatosis (without change in fibrosis or inflammation) on biopsy at 12 months. Similarly, Ceriani et al. demonstrated normalization of liver enzymes in 14 of 16 patients treated with UDCA (10 mg/kg) compared to 4 of 15 on placebo and Guma et al. demonstrated liver enzyme normalization in 10 of 13 treated patients (10 mg/kg per day) versus 3 of 11 patients in a placebo group. However, in the largest study, Lindor et al. conducted a randomized trial in 166 patients (550). All patients were encouraged to lose weight and randomized to UDCA (13 to 15 mg/kg per day) or placebo for 2 years. Both groups had similar improvement in aminotransferases, and follow-up biopsy, available in two thirds of the patients, revealed similar levels of improvement in both groups. Whether negative results from this controlled trial reflect a true lack of response from UDCA is debatable (617). The surprising improvement in the placebo group in this study has pointed out the need for controlling for voluntary lifestyle changes when assessing pharmacologic intervention. Another recent randomized controlled trial of 48 patients (available only as abstract), in which patients were treated with the combination of UDCA (12 to 15 mg/kg) and vitamin E (800 IU/day) for 2 years versus single or double placebo controls, showed significant improvement in NAS only in the group treated with combination therapy (618).
Other agents with potential cytoprotective properties include taurine, an amino acid that alters bile acid physiology and that has been reported to normalize liver enzymes in children with NAFLD (619). Triacetyl uridine is another potentially important cytoprotective agent in experimental choline-deficient rats but has not been tested in humans (620).
Antioxidants and Nutritional Supplements
Vitamin E supplementation has been studied in both experimental and clinical settings (621). “Vitamin E” refers to a family of tocopherols and tocotrienols (each with different forms named α, β, γ, and δ) that exhibit antioxidant activity. Commercially available vitamin E supplements usually contain only α-tocopherol—the relative efficacy of mixed tocopherols compared to α-tocopherol alone is not established (622,623,624,625). Experimentally, a number of encouraging studies have been published (626,627). Similarly, human studies have shown reduced lipid peroxidation and cytokine levels with vitamin E therapy (552,556). However, pilot studies and two randomized controlled studies have produced varying results (551,553,554,555). A placebo-controlled trial in 14 children showed liver enzyme improvement with vitamin E (400 IU daily for 2 months followed by 100 IU daily for 3 months), but the placebo group also demonstrated a significant improvement in liver test results in parallel with weight loss. In adults, Harrison et al. randomized 45 patients with biopsy-proven NASH to vitamin E (1,000 IU daily) plus vitamin C (1,000 mg daily) versus placebo. Repeat liver biopsy after 6 months showed a small improvement in fibrosis score in the vitamin E group; however, there was no significant improvement in inflammation or necrosis score on biopsy. A second controlled study in 16 adults showed no benefit of vitamin E over diet and exercise alone. Although caution is warranted in patients with prior coronary artery disease where vitamin E is associated with blunted efficacy of statin drugs (628), significant toxicity has not been observed in these studies, but concerns over doses exceeding 400 IU/day (629) and the limited efficacy have tempered enthusiasm (630). On the other hand, its use in combination with other agents (e.g., UDCA and pioglitazone) has been more encouraging.

Betaine (trimethylglycine), a methyl donor in an alternative pathway for remethylation of homocysteine to methionine and S-adenosyl-methionine (SAMe), promotes the conversion of phosphotidylethanolamine to phosphotidylcholine (lecithin), which, in turn, promotes the export of fat from the liver as VLDL (631). Substantial benefit has been reported by Abdelmalek et al. in ten patients with NASH treated with betaine solution (20 g/day for 10 to 12 months) in terms of biochemical and multiple histologic parameters including steatosis, inflammation, and fibrosis (632). These agents may also serve to replenish glutathione and improve mitochondrial membrane fluidity (633,634,635). Silymarin, the active component of milk thistle extract, is an extraordinarily popular over-the-counter supplement that has been observed to decrease the expression of CYP 3A4 and decrease mitochondrial respiration in hepatocyte culture but has not, to our knowledge, been carefully studied in NASH (636). N-Acetylcysteine, which is converted to glutathione in the liver, has produced improvement in liver enzymes (histology not done) in a small pilot study at a dosage of 1 g/day for 3 months (637). Lecithin increases plasma free choline and decreases hepatic steatosis in long-term TPN patients (638,639). Experimentally, probiotics have shown some encouraging results, possibly through their effects on secondary mediators of inflammation (640). Lazaroids, or 21-aminosteroids, are additional antioxidants that have not been studied in NASH but warrant consideration (641).
Antidiabetic Agents
Because insulin resistance appears to be the crucial problem in most patients with NASH, insulin-sensitizing agents have emerged as the most promising form of pharmacologic therapy, especially in those with progressive fibrosis (Figs. 39.15, 39.16 and 39.17) (642). Other antidiabetic strategies agents have been less well studied (643). For example, neither insulin nor sulfonylureas have been carefully studied for effects on fatty liver. The major adverse effects of insulin is weight gain with truncal obesity that could exacerbate NAFLD (644). On the other hand, control of blood sugar level in patients with overt diabetes will likely decrease de novo fat synthesis in the liver through effects on CREBP. Acipimox is an inhibitor of lipolysis that is thought to improve insulin sensitivity by lowering free fatty acid levels but its effect on NAFLD has not been studied (645). Similarly, experimental antidiabetic agents such as D-chiro-inositol and newer agents such as the incretins have not, to our knowledge, been studied in NAFLD (646,647).
TZDs are a group of agents that act as ligands for the peroxisome-proliferator–activated receptor-γ (PPAR-γ) (expressed in adipose tissue, intestines, macrophages, and muscle and activated by fatty acids),

which is a member of a nuclear receptor superfamily that regulates gene expression of enzymes involved in lipid and glucose metabolism. The PPAR-γ receptor, the ligand, and a coactivator form a heterodimer with retinoid X receptor, which binds to the PPAR-γ response element of specific genes (648). The most profound effect of TZDs is in adipocyte differentiation (649). As a result, TZDs are often associated with increased peripheral but decreased central adiposity (650,651). Other effects include increased expression of glucose transporters (652), increased mitochondrial mass (653) and altered thermogenesis (through effects on UCP) (654), decreased cytokine expression, and inhibited inducible nitric oxide synthase, as well as decreased ceramide-induced apoptosis in pancreatic islet cells (655,656). Because PPAR-γ receptors are not

highly expressed in liver, the benefit to patients with NASH is likely to primarily involve indirect mechanisms.
▪ Figure 39.15 Improvement in insulin resistance and histology with metformin in patients with nonalcoholic steatohepatitis. Since 1999, several studies have demonstrated metformin’s beneficial effects on biochemical, histologic, and metabolic parameters associated with NASH. The histologic parameter was standardized across all four studies by calculating the nonalcoholic fatty liver disease activity score (NAS) from data presented in the studies. ALT, alanine transaminase. (Adapted from references 537,541,542,543.)
▪ Figure 39.16 Comparison of thiazolidinediones (TZDs) with metformin shows greater improvement in biochemical, metabolic, and hepatic steatosis in the former. Equivalent doses of TZDs show similar effects on alanine transaminase (ALT), insulin resistance, and hepatic steatosis, which are thought to be due to their effects on mobilization of central fat to peripheral areas. Although these data are not conclusive, metformin appears to have a more modest effect compared with the TZDs. (Adapted from references 296, 536, 537.)
▪ Figure 39.17 Thiazolidinediones appear to improve fibrosis after treatment. In the studies by Neuschwander-Tetri et al. and Promrat et al., fibrosis scores on posttreatment (tx) liver biopsies had significantly decreased after 6 months of treatment with either rosiglitazone or pioglitazone. (Adapted from
Neuschwander-Tetri BA, Brunt EM, Wehmeier KR, et al. Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR-γ ligand rosiglitazone. Hepatology 2003;38:1008–1017.
Promrat K, Lutchman G, Uwaifo GI, et al. A pilot study of pioglitazone treatment for nonalcoholic steatohepatitis. Hepatology 2004;39:188–196.
A 4- to 6-month course of troglitazone normalized liver enzymes in seven of ten patients with NASH and improved inflammation in four of seven with follow-up biopsies (most with loss of polymorphonuclear cells) with no change in fibrosis scores (213). Long-term histologic follow-up 4 to 5 years after completing the study in these patients has shown near resolution in one patient who had subsequent success with weight loss and diet and progression to uncomplicated cirrhosis in another patient who was unable to achieve lifestyle changes (657). Rosiglitazone has been evaluated in two publications by Neuschwander-Tetri et al. (536,658). These studies showed significant improvement in the posttreatment biopsy and almost one half of the patients no longer met the histologic criteria for NASH. These changes were paralleled by improvement in indices of insulin activity and decreased hepatic fat content. Consistent with the effect of TZD on peripheral fat stores, 67% gained weight during the study (median increase of 7.3%). Laboratory follow-up on this group of patients also showed a relapse of the aminotransferase levels to baseline levels, indicating that the changes obtained were not sustained.
A similar effect of pioglitazone in both aminotransferase levels and histologic parameters has also been observed (533,535,659). Sanyal et al. performed a controlled trial of pioglitazone versus vitamin E, showing superiority of TZD in terms of histologic remission (534). The most detailed report available is that by Promrat et al. who treated 18 patients with pioglitazone, 30 mg daily, for 48 weeks with endpoints of histologic activity, insulin activity, body composition (dual-energy x-ray absorptiometry), and hepatic fat content by MRI (296). All patients received recommendations for increased physical activity, reduced calorie diet, and a daily multivitamin. Therapy was associated with normalization of the aminotransferase levels, a significant reduction in a composite histologic score (the NAI—see preceding text), a reduction in steatosis confirmed by MRI, and improved insulin sensitivity. Sustained weight gain was seen in 72% of patients (average of 3.5 kg). Preliminary results from a placebo-controlled trial indicate a correlation between higher adiponectin levels, reduced steatosis, and improved fibrosis in the treated group (660).
In the existing pilot studies (including troglitazone), elevation of liver enzyme levels during treatment has been reported in approximately 3 of 79 patients (one each for the major TZDs studied). No cases of severe hepatitis or exacerbation of NASH has been noted. Although troglitazone was well tolerated even in the presence of stable cirrhosis, a rare but potentially severe idiopathic toxicity led to its removal from the market (661,662,663). Even more rare toxicity has been reported with rosiglitazone and pioglitazone (664,665,666). The mechanism of idiosyncratic toxicity with TZDs is uncertain, but several lines of evidence (including a combined toxicity of troglitazone with an HMG-CoA reductase inhibitor) (667) point to rare but potentially significant mitochondrial dysfunction. Weight gain is the most common side effect noted in the existing studies. It is suspected that successful dietary and exercise intervention can prevent this development, but predictive factors remain to be defined. The effect of shifting fat stores on myocyte function, exercise tolerance, and endurance in these patients has not been adequately addressed. Edema, sometimes associated with congestive heart failure, has been reported, but the relationship between the medication and cardiac dysfunction is debated (668,669).
Metformin is an insulin-sensitizing, oral biguanide approved for use in type 2 diabetes in the United States since 1995 (670). The biguanides exert substantial changes in cellular bioenergetics without inducing weight gain, by reducing hepatic glucose production and increasing peripheral glucose utilization (671). Its major site of action is the mitochondria and causes a reduction of ATP in isolated rat hepatocytes, reduced activity of complex I in the mitochondrial respiratory chain, reduced fatty acid oxidation, and increased lactate production in preadipocytes under experimental conditions (653,672,673). Signaling is through adenosine monophosphate–activated protein kinase—this pathway is similarly activated with exercise, suggesting that in some ways metformin simulates the effects of exercise on glucose transport (674).
Favorable studies of metformin in the fatty liver of ob/ob mouse (675) along with a favorable side effect profile, especially the absence of weight gain seen with the TZDs, have led to sustained interest in this agent in treating NASH. Coyle et al. noted that metformin improved overall histology in two patients in one of the first reports of histologic changes (538). Less favorably, Tiikkainen et al. in a human study in patients with type 2 diabetes examining the relative effects of metformin (2 g/day) and rosiglitazone (8 mg/day), for 16 weeks, on steatosis and serum adiponectin levels, revealed that both drugs similarly increased insulin sensitivity but only rosiglitazone reduced hepatic steatosis (537). However, Marchesini et al. demonstrated improved liver enzymes, reduced hepatomegaly, and improved hepatic perfusion in patients treated with metformin (1,500 mg/day) for up to 6 months, and in a follow-up study, Bugianesi et al. reported improvement in several histologic parameters (e.g., steatosis, necroinflammation and fibrosis) independent of dietary changes (539,543,676).

Lavine and Schwimmer reported similar histologic improvement in ten children along with reduced steatosis by MRS (540). However, less definitive improvement in histologic parameters were observed in two other studies by Uygun et al. and Nair et al. (541). The overall safety profile of metformin is favorable and lactic acidosis appears to be rare, but caution is warranted in renal insufficiency (677).
Antihyperlipidemic Agents
Fibrates are lipid-lowering agents that act on PPAR-α receptors located primarily in the liver, heart, muscle, and kidney (normally activated by adipose tissue–derived fatty acids) that increase fatty acid uptake, mitochondrial β-oxidation, peroxisomal oxidation of fatty acids, and ω-oxidation of fatty acids in the P-450 system (648). However, histology-based studies of fibrates have not shown significant beneficial effects, although the available data is limited and some studies have reported improved transaminase levels (546,678,679).
With statin drugs (HMG-CoA reductase inhibitors), some type of interaction with NAFLD seems likely because the drugs are acting in part on hepatic fat metabolism and two thirds to three fourths (noninvasive testing) of hyperlipidemic patients have NAFLD. Not much is known about the long-term effects of these agents on NAFLD; however, the incidence of acute hepatitis, judged by the aminotransferase levels, appears to be quite low (108,680). On the other hand, induction of SREBP (a transcription factor governing hepatic fat synthesis), diminished secretion of VLDL and perhaps depleted ubiquinone raise concern about long-term effects (106). The anti-inflammatory effects of the statins may cause decreased cellular injury although there is reason to suspect that these agents will increase liver fat stores. However, limited available studies suggest an overall beneficial effect in NAFLD (107,601,681,682). It seems likely that this complex interaction represents the interaction between a number of covariables including the degree of physical conditioning (insulin signaling), ethanol use, dietary fatty acid composition, and genetic variables in fatty acid metabolism. Additional data is needed, especially in light of efforts to make these agents available “over-the-counter” (683).
Other Agents
Recent experimental data from Diehl et al. indicate that modulation of the adrenergic system may significantly influence NAFLD (370,684). A close association between the sympathetic nervous system (SNS) and the fatty liver seems likely on the basis of the close association between obesity and the SNS (685). Whether pharmacologic manipulation of the system in humans might be beneficial in select cases remains to be investigated. Other agents, also related more classically to the cardiovascular system, include angiotensin receptor blockers (ARBs). One small uncontrolled study in human patients with NASH and hypertension treated with losartan has shown histologic benefit after 48 weeks (686).
Liver Transplantation, Disease Recurrence, and Donor Organs
Transplantation for patients with advanced NASH is often complicated because of the presence of comorbid conditions related to obesity, diabetes, and hyperlipidemia (687,688). Although guidelines do not exist, these conditions warrant careful consideration of the long-term benefits of transplantation in this setting. Recurrence of disease is an additional concern. A number of reports have now documented the recurrence of NAFLD and NASH after transplantation (689,690,691,692). In addition, two series have shown a high risk for the development of NAFLD in patients with cryptogenic cirrhosis after liver transplantation (see “Cryptogenic Cirrhosis”). Immunosuppression regimens likely play a role: Steroid therapy promotes fatty change and cyclosporine interacts with the mitochondrial transition pore that regulates the electrochemical gradient across the mitochondrial membrane (693). The relative risks and benefits of different immunosuppression regimens in these patients are not established but the course may be severe. Steatosis in donor livers is associated with poor graft function likely due to abnormal mitochondrial function and disturbed ATP homeostasis (see “Pathogenesis of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis”) (694). The problem has become magnified with efforts to develop living-related donor programs in which prediction of steatosis in a partial donor can be difficult (695). The role of preoperative dietary changes in prospective living donors remains to be fully investigated (696).
Annotated References
Adams LA, Sanderson S, Lindor KD, et al. The histological course of nonalcoholic fatty liver disease: a longitudinal study of 103 patients with sequential liver biopsies. J Hepatol 2005;42:132–138.
Angulo P, Keach JC, Batts KP, et al. Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis. Hepatology 1999;30:1356–1362.
Dixon JB, Bathal PS, O’Brien PE. Nonalcoholic fatty liver disease: predictors of nonalcoholic steatohepatitis and liver fibrosis in the severely obese. Gastroenterology 2001;121:91–100.
Garcia-Monzon C, Martin-Perez E, Lo Iacono O, et al. Characterization of pathogenic and prognostic factors of nonalcoholic steatohepatitis associated with obesity. J Hepatol 2000;33:716–724.
Hui JM, Kench JG, Chitturi S, et al. Long-term outcomes of cirrhosis in nonalcoholic steatohepatitis compared with hepatitis C. Hepatology 2003;38:420–427.

Ratzui V, Bonyhay L, Di Martino V, et al. Survival, liver failure, and hepatocellular carcinoma in obesity-related cryptogenic cirrhosis. Hepatology 2002;35:1485–1493.
Ratziu V, Giral P, Charlotte F, et al. Liver fibrosis in overweight patients. Gastroenterology 2000;118:1117–1123.
These papers have provided important observations about the prediction of severity of underlying liver disease in consecutive obese patients or, in the Angulo paper, among consecutive patients with NASH. Among the salient findings were associations of more severe histology with increased obesity, more severe insulin resistance, hypertension, hypertriglyceridemia, elevation of transaminases (not invariably present) and/or AST:ALT ratio more than 1, age over 40 to 50 years, and female gender. From serial biopsy studies in patients with NASH, cirrhosis develops in about 10% while fibrosis progresses in 33%, remains stable in 41%, and improves in 22% over about 5 years. Once cirrhosis develops, there is a significant risk of decompensated portal hypertension and hepatocellular carcinoma.
Brunt EM, Janney CG, Di Bisceglie AM, et al. Nonalcoholic steatohepatitis: a proposal for grading and staging the histologic lesions. Am J Gastroenterol 1999;94:2467–2474.
Kleiner DE, Brunt EM, Van Natta ML, et al. Nonalcoholic steatohepatitis clinical research network. Design and validation of a histologic scoring system for NAFLD. Hepatology 2005;41:1313–1321.
Matteoni CA, Younossi ZM, Gramlich T, et al. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999;116:1413–1419.
The paper by Matteoni et al. elucidated the relationships between types of NAFLD, including simple steatosis or steatosis with mild inflammation (types 1 and 2), and more severe histology associated with fibrosis (types 3 and 4). Types 3 and 4 are associated with greater likelihood of progression. The paper by Brunt et al. provided a widely referenced scoring scheme for grading and staging NASH. Many of the key histologic parameters have subsequently been combined by Kleiner et al. into a composite score (the NASH activity score or NAS) that will facilitate assessment of therapeutic interventions and comparison between different treatments.
Caldwell SH, Oelsner DH, Iezzoni JC, et al. Cryptogenic cirrhosis: clinical characterization and risk factors for underlying disease. Hepatology 1999;29:664–669.
Contos MJ, Cales W, Sterling RK, et al. Development of nonalcoholic fatty liver disease after orthotopic liver transplantation for cryptogenic cirrhosis. Liver Transpl 2001;7:363–373.
Ong J, Younossi ZM, Reddy V, et al. Cryptogenic cirrhosis and post-transplantation non-alcoholic fatty liver disease. Liver Transpl 2001;7:797–801.
Powell EE, Cooksley WG, Hanson R, et al. The natural history of nonalcoholic steatohepatitis: a follow-up study of forty-two patients for up to 21 years. Hepatology 1990;11:74–80.
The paper by Powell et al. demonstrated a key observation that NASH may progress to a “bland” cirrhosis with loss of histologic features. This relationship was extended by Caldwell et al. to show that many cases of cryptogenic cirrhosis likely result from prior, unrecognized NASH in obese patients—especially women. An age difference of approximately 10 years (mean age 50 in NASH and 60 in cirrhosis) suggests that the sixth decade is commonly associated with progression to cirrhosis in patients with NASH. The high occurrence (recurrence) of NASH after liver transplantation shown in studies by Contos et al. and Ong et al. indicates the potential severity of the disease and further strengthened the suspected relationship between NASH and cryptogenic cirrhosis.
Cortez-Pinto H, Chatham J, Chacko VP, et al. Alterations in liver ATP homeostasis in human nonalcoholic steatohepatitis: a pilot study. JAMA 1999;282:1659–1664.
Day CP, James OFW. Steatohepatitis: a tale of two hits. Gastroenterology 1998;114:842–845.
Donnelly KL, Smith CI, Schwarzenberg SJ, et al. Sources of fatty acids in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest 2005;115:1343–1351.
Feldstein AE, Werneburg NW, Canbay A, et al. Free fatty acids promote hepatic lipotoxicity by stimulating TNF-α expression via a lysosomal pathway. Hepatology 2004;40:185–194.
Hui JM, Hodge A, Farrell GC, et al. Beyond insulin resistance in NASH: TNF-α or adiponectin? Hepatology 2004;40:46–54.
Pessayre D, Fromenty B. NASH: a mitochondrial disease. J Hepatol 2005;42:928–940.
Accumulation of hepatic lipid, the first step in the “two-hit” hypothesis (steatosis and oxidative stress) of NASH results from both increased uptake of fatty acids from the periphery and from de novo lipogenesis within the liver. Resulting lipid peroxidation underlies many of the changes that ultimately develop into progressive NASH including mitochondrial dysfunction (in both liver and other organs), activation of apoptosis pathways, cytokine synthesis, and activation of stellate cells with resulting fibrosis. The “communications,” through cytokines and adipocytokines, between fat stores and targets of insulin activity appear to play a significant role in the ultimate stability of the problem or the development of cellular injury.
Huang MA, Greenson JK, Chao C, et al. One-year intense nutritional counseling results in histological improvement in patients with nonalcoholic steatohepatitis: a pilot study. Am J Gastroenterol 2005;100:1072–1081.
Neuschwander-Tetri BA, Brunt EM, Wehmeier KR, et al. Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR-γ ligand rosiglitazone. Hepatology 2003;38:1008–1017.
Promrat K, Lutchman G, Uwaifo GI, et al. A pilot study of pioglitazone treatment for nonalcoholic steatohepatitis. Hepatology 2004;39:188–196.
Ratziu V, Charlotte F, Heurtier A, et al. LIDO Study Group. Sampling variability of liver biopsy in nonalcoholic fatty liver disease. Gastroenterology 2005;125:1898–1906.
Although there is yet surprisingly limited data, diet and exercise, most likely through their effect on insulin signaling and energy regulation, are the cornerstones of therapy, as demonstrated in the report by Huang et al. These voluntary lifestyle changes (diet and exercise) and sampling variability on liver biopsy noted in the study by Ratziu et al. are two of the most significant confounding variables that must be considered in assessing clinical trials of pharmacologic therapy (aimed especially at patients with evidence of ongoing histological injury). Using a variety of clinical endpoints in addition to histology—such as global measures of hepatic steatosis, anthropometric measures, insulin signaling, and cytokine levels—the insulin-sensitizing agents known as thiazolidinediones (e.g., rosiglitazone and pioglitazone) are emerging as the most promising agents, although side effects and durability of the response are yet to be fully defined in controlled trials.
Marchesini G, Brizi M, Morselli-Labate AM, et al. Association of nonalcoholic fatty liver disease with insulin resistance. Am J Med 1999;107:450–455.
Sanyal AJ, Campbell-Sargent C, Mirshahi F, et al. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 2001;120:1183–1192.
Using different methods to assess insulin activity, these two papers demonstrated insulin resistance as a key feature of NAFLD. Salient observations including demonstration of insulin resistance in obese and lean patients with fatty liver, shown by Marchesini et al. and extensive analysis of fat metabolism in NASH, by Sanyal et al. indicated diminished

suppression of lipolysis with insulin infusion and increased rates of fatty acid oxidation associated with oxidative stress and mitochondrial injury in NASH
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