The health risks associated with obesity are widespread and

GASTROENTEROLOGY 2012;142:711 725 IN BASIC CLINICAL GASTROENTEROLOGY HEPATOLOGY Robert F. Schwabe and John W. Wiley, Section Editors Role of Obesity and Lipotoxicity in the Development of Nonalcoholic Steatohepatitis: Pathophysiology and Clinical Implications KENNETH CUSI Division of Endocrinology, Diabetes and Metabolism, University of Florida, Gainesville, Florida Podcast interview: www.gastro.org/gastropodcast. Also available on itunes. As obesity reaches epidemic proportions, nonalcoholic fatty liver disease (NAFLD) is becoming a frequent cause of patient referral to gastroenterologists. There is a close link between dysfunctional adipose tissue in NAFLD and common conditions such as metabolic syndrome, type 2 diabetes mellitus, and cardiovascular disease. This review focuses on the pathophysiology of interactions between adipose tissue and target organs in obesity and the resulting clinical implications for the management of nonalcoholic steatohepatitis. The release of fatty acids from dysfunctional and insulin-resistant adipocytes results in lipotoxicity, caused by the accumulation of triglyceride-derived toxic metabolites in ectopic tissues (liver, muscle, pancreatic beta cells) and subsequent activation of inflammatory pathways, cellular dysfunction, and lipoapoptosis. The cross talk between dysfunctional adipocytes and the liver involves multiple cell populations, including macrophages and other immune cells, that in concert promote the development of lipotoxic liver disease, a term that more accurately describes the pathophysiology of nonalcoholic steatohepatitis. At the clinical level, adipose tissue insulin resistance contributes to type 2 diabetes mellitus and cardiovascular disease. Treatments that rescue the liver from lipotoxicity by restoring adipose tissue insulin sensitivity (eg, significant weight loss, exercise, thiazolidinediones) or preventing activation of inflammatory pathways and oxidative stress (ie, vitamin E, thiazolidinediones) hold promise in the treatment of NAFLD, although their long-term safety and efficacy remain to be established. Better understanding of pathways that link dysregulated adipose tissue, metabolic dysfunction, and liver lipotoxicity will result in improvements in the clinical management of these challenging patients. Keywords: Insulin Resistance; NAFLD; Fatty Liver; Type 2 Diabetes Mellitus. The health risks associated with obesity are widespread and involve a variety of tissues, including the vascular bed, as illustrated in Figure 1. However, the impact of obesity varies widely among subjects even with a similar degree of body mass index (BMI). This is not only because BMI is a rather crude index of total adiposity, 1 but also because genetics and factors such as sex, age, ethnicity, cardiorespiratory fitness, and body fat distribution (visceral vs subcutaneous) also play a role. Recent studies indicate that the deleterious metabolic impact of obesity in nonalcoholic fatty liver disease (NAFLD) may also vary widely among patients with a similar BMI. 2,3 Female patients and older adults have more adiposity for any given BMI compared with younger male subjects. In any case, obesity early in life significantly shortens life expectancy and predicts the future development of metabolic syndrome, type 2 diabetes mellitus (T2DM), and cardiovascular disease (CVD). 4 In the United States, almost one-third of children aged 2 19 years and twothirds of adults are overweight or obese. 5 The increase in the prevalence of obesity is a worldwide problem, with an estimated 1.46 billion adults affected 6 and a reduction of life expectancy of up to 7 years compared with normal-weight individuals. 7 It is estimated that 170 million children (younger than 18 years of age) globally are overweight or obese, affecting more than 20% of all children in many countries (United States, Great Britain, Australia, Brazil, Chile). 6 Ethnic minorities in the United States, in particular the Hispanic population, 8 are much more vulnerable to the unfavorable conditions of modern lifestyles that promote obesity, affecting more immigrants undergoing acculturation and the economically disadvantaged. 9 Excess adiposity and poor cardiorespiratory fitness drive the epidemic of T2DM 10 and early CVD in population-based studies. 4,11 Abbreviations used in this paper: BMI, body mass index; CIMT, carotid-intima medial thickness; CV, cardiovascular; CVD, cardiovascular disease; FFA, free fatty acid; IL, interleukin; MRS, magnetic resonance imaging and spectroscopy; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PPAR, peroxisome proliferator-activated receptor; RCT, randomized controlled trial; T2DM, type 2 diabetes mellitus; TLR, Toll-like receptor; TNF, tumor necrosis factor; TZD, thiazolidinedione; VLDL, very-low-density lipoprotein. 2012 by the AGA Institute 0016-5085/$36.00 doi:10.1053/j.gastro.2012.02.003

712 KENNETH CUSI GASTROENTEROLOGY Vol. 142, No. 4 Figure 1. Obesity and triglyceride-derived toxic lipid metabolites accumulate in ectopic tissues and lead to multiorgan dysfunction and common chronic metabolic diseases such as NAFLD and to T2DM and CVD. The economic burden of obesity is also significant. For instance, the average annual medical costs are 1.6 times higher for obese patients with the metabolic syndrome compared with those without metabolic syndrome. 12 Obesity accounts for between 2% and 6% of total health care costs in many countries, 6 and in the United States health care expenses for obesity-related conditions were estimated to be approximately $117 billion or 10% of the total health care costs about a decade ago, with more recent estimates indicating that obesity-related conditions consume $168 billion yearly or 17% of total health care costs. 13 Recent reports on America s struggle with obesity suggest that if this trend continues, by 2030 an estimated 164 million Americans will be obese, with US health care spending rising by as much as $66 billion a year. 14 An estimated 1% reduction in BMI at the population level would decrease as many as 2.4 million cases of diabetes and 1.7 million cases of heart disease and stroke per year. Either we adopt strong proactive policies to reverse the current trend or it is unlikely that any health care system, beyond the specific model adopted, will be able to cope with the demands of obesity-related conditions in the near future. Adipose Tissue Dysfunction, Insulin Resistance, and Inflammation Adipose Tissue Adaptation in Obesity Normal adipocyte function depends on a number of factors, such as adipocyte number, size, the overall hormonal milieu, and interaction with other cell types within the adipose tissue bed. 15 Fat cells derive from multipotent mesenchymal stem cells that develop into adipoblasts and then to preadipose cells (lipid-depleted

April 2012 OBESITY LIPOTOXICITY IN DEVELOPMENT OF NASH 713 precursors of the future adult white adipocyte). Maturation depends on a complex signal system. Up-regulation by peroxisome proliferator-activated receptor (PPAR- ) is essential, in concert with other transcription factors such as sterol regulatory element binding protein 1c, CCAAT-enhancer-binding proteins, and bone morphogenetic proteins, among others. 15 These transcription factors give adipocytes great plasticity and a significant ability to adapt to overfeeding by means of hypertrophy and hyperplasia. Within this context, adipose tissue must be viewed primarily as a protective tissue that stores and prevents excessive exposure of other organs to fatty acids. The first adaptation in adults to avert systemic lipotoxicity from chronic overfeeding is enlargement of adipocytes (hypertrophy), followed by a longer-term compensatory mechanism involving fat cell replication (hyperplasia), the predominant mechanism in childhood obesity. Hypertrophic adipocytes develop a gene expression pattern that resembles that of macrophages and produce adipokines of the kind described in foam cells, the fat-loaded activated macrophages that are found in arterial plaques. 15,16 Protection from chronic energy supply and excess triglyceride accumulation in tissues such as the liver, muscle, and pancreatic beta cells calls for an extraordinary adaptation by adipocytes that involves activation of several inflammatory pathways but at the cost of adipose tissue insulin resistance. The most relevant of these pathways in obesity are the inhibitor B kinase/nuclear factor B pathway 17 in which free fatty acids (FFAs) activate Toll-like receptor (TLR)-4 receptors in macrophages and adipocytes 15,18 ; the c-jun N-terminal kinase/activator protein 1 pathway, where insulin signaling is inhibited in the presence of tumor necrosis factor (TNF)- 19,20 ; the cyclic adenosine monophosphate responsive element binding protein H (CREB-H) pathway, which promotes the secretion of acute-phase proteins such as C-reactive protein and generation of reactive oxygen species 21,22 ; and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. 21 All become activated by excess fatty acids, reactive oxygen species, endoplasmic reticulum stress, and lipid by-products such as diacylglycerol 23 and ceramide 24 and inhibited by adiponectin and thiazolidinediones (TZDs). Endocrine Role of Adipose Tissue As illustrated in Figure 2A, there is a close relationship among hypertrophic insulin-resistant adipocytes, dysregulated immunity both at the level of the adipose tissue and the liver, and lipotoxicity-induced steatohepatitis. To better understand this, it must be first recognized that adipocytes represent only 70% of the total adipose mass. The rest is a network collectively known as the stromal vascular fraction and composed of endothelial cells, pericytes, fibroblasts, early mesenchymal cells, preadipocytes, and macrophages, all playing a relevant role in autocrine-paracrine regulation of fat metabolism. Adipocytes and the stromal vascular fraction secrete a host of hormones, complement factors, cytokines (TNF-, interleukins [ILs], others), chemokines (monocyte chemoattractant protein 1, macrophage inhibitory factor, plasminogen activator inhibitor 1, and others), enzymes, and peptides known collectively as adipokines, many previously believed to be only secreted by macrophages. The complexity of adipokine functions has been reviewed in depth elsewhere. 15,16,25,26 Adipokines include growth factors such as insulin-like growth factor I, transforming growth factor, bone morphogenetic proteins, and potent angiogenic factors such as hypoxia-inducible factor 1 and vascular epithelial growth factor. Because adipose tissue must ensure a steady day-to-day energy supply to meet metabolic needs, adipocytes also secrete a host of hormones that regulate glucose and lipid metabolism such as angiotensin II, estrogens, glucocorticoids, PPARs, leptin, visfatin, resistin, retinol-binding protein 4, and others. Decreased secretion of adiponectin in obesity alters lipid metabolism and insulin sensitivity in the liver, and administration of recombinant adiponectin to adiponectin-deficient obese mice fed a high-fat diet dramatically alleviates hepatomegaly, steatosis, and inflammation. 27 In an interesting paradox, ob/ob mice overexpressing adiponectin are rescued from insulin resistance and diabetes despite a pathological expansion in adipose tissue mass. 28 In nonalcoholic steatohepatitis (NASH), decreased secretion of adiponectin by dysfunctional adipocytes contributes to steatohepatitis and fibrosis. 29 Reversal of adipose tissue insulin resistance 30 and an increase in plasma adiponectin concentration 31 by TZDs ameliorates lipotoxicity and steatohepatitis in patients with NASH. Role of Liver Macrophage Activation in the Development of Lipotoxic Liver Disease Adipose tissue macrophages are an important component of the stromal vascular fraction. 25,32,33 Both monocyte chemoattractant protein 1 and macrophage inhibitory factor, as well as necrotic fat cells, are powerful stimulants for adipose tissue macrophages recruitment, both locally and from bloodstream monocytes, forming the well-established crown -like structure. 34,35 As illustrated in Figure 2A, activation of adipose tissue macrophages plays a key role in adipocyte dysfunction, adipose tissue insulin resistance, release of excess FFAs into the circulation, and ectopic fat deposition in the liver. There are 2 types of macrophages: (1) M1 macrophages or classically activated macrophages that play a key role in humoral immunity and response to common pathogens; they secrete large amounts of proinflammatory cytokines (such as TNF-, inducible nitric oxide synthase, C-C chemokine receptor 2 or CCR2, and IL-12) and low levels of the anti-inflammatory cytokine IL-10 and (2) alternatively activated M2-type macrophages, with anti-inflammatory actions in adipose tissue. 36,37 An increase in number of M1 relative to M2 macrophages is characteristic of animals fed a high-fat diet 36 38 and human obesity. 16 Kupffer cells are the liver counterpart of adipose tissue macrophages. Bone marrow promonocytes and monoblasts differentiate into peripheral blood monocytes and

714 KENNETH CUSI GASTROENTEROLOGY Vol. 142, No. 4 Figure 2. (A) Adipose tissue is composed of adipocytes and the stromal vascular fraction that includes macrophages and other immune cells that have a relevant role in the autocrine-paracrine regulation of adipocytes. In obesity, activation of macrophages/immune system contributes to the development of dysfunctional, insulin-resistant adipocytes that release excessive amounts of FFA and cause insulin resistance and lipoapoptosis in distant tissues (liver, muscle, pancreatic beta cells, vascular bed, other). Accumulation of triglyceride-derived toxic lipid metabolites activates intracellular inflammatory pathways within hepatocytes and Kupffer and other immune cells, in resemblance to defects within adipocytes. Activation of hepatic stellate cells leads to collagen deposition and the potential for cirrhosis. (B) PPAR- are widely distributed among adipocytes, hepatocytes, and hepatic stellate cells, as well as macrophages and immune cells infiltrating adipose and liver tissue that may be targeted by PPAR- agonists during TZD therapy in patients with NASH. line the walls of the liver sinusoids as specialized macrophages of the reticuloendothelial system. As shown in Figure 2A, they are a major source of inflammatory cytokines and in their interaction with B and T cells play a similar role to M1 polarized macrophages in adipose tissue in the development of insulin resistance, inflammation, and apoptosis. 38 40 Experimental depletion of Kupffer cells prevents high-fat diet induced 41 and alco-

April 2012 OBESITY LIPOTOXICITY IN DEVELOPMENT OF NASH 715 hol-induced 42 hepatic steatosis and inflammation in rodents. Alternative M2 activation of Kupffer cells by PPAR- agonists reverses obesity-induced hepatic insulin resistance. 38 Adipose tissue macrophage activation precedes Kupffer cell activation. When C57BL/6 mice are fed a high-fat diet, macrophage activation and gene expression of a number of proinflammatory adipokines (such as IL-1, IL-1R, TNF-, transforming growth factor, and others) occur rapidly in adipose tissue and animals eventually develop steatohepatitis. 43,44 However, M1 macrophage liver infiltration and steatohepatitis occur weeks after adipose tissue dysregulation. Taken together, M1/M2 Kupffer cell imbalance (M1 M2) and the release of inflammatory mediators by Kupffer cells are critical to the pathogenesis of NASH and an attractive treatment target. Cross Talk Between Adipose Tissue and the Liver The importance of PPAR- signaling is highlighted in Figure 2B, where it can be appreciated that PPAR- are widely distributed among adipocytes (most abundant), macrophages, and several stromal vascular fraction cells types as well as at the level of the liver. Their widespread distribution explains the multiple metabolic and antiinflammatory effects of PPAR- agonists and provides a rationale for their clinical use in NAFLD. TZDs improve adipocyte and whole-body insulin sensitivity by multiple mechanisms that include up-regulation of the PPAR- coactivator 1 (PGC-1 ), enhancement of adipocyte mitochondrial biogenesis and oxidative capacity, as well as an increase in adiponectin gene expression. 45,46 PPAR- agonists exert anti-inflammatory effects in NASH by targeting Kupffer cells, 47,48 and both PPAR- 49 and adiponectin 26 regulate hepatic stellate cell function. TZDs have been reported to have significant antifibrotic effects in animal studies. 50 53 Although reversal of fibrosis has yielded mixed results in human trials, 54 56 the discrepant results may be due to species differences or because clinical studies have been of relative short duration. More potent/specific PPAR- ligands may have greater potential to prevent hepatic stellate cell lipotoxicity, a possibility that remains to be explored. Clinical Impact of Adipose Tissue Dysfunction and Lipotoxicity The prior section has set the stage for understanding the clinical impact of excess fatty acids across a variety of tissues and the role of obesity in NAFLD. Role of Excess FFAs on Skeletal Muscle Insulin Resistance in Humans The potential for FFAs to alter skeletal muscle glucose metabolism was first proposed more than 50 years ago 57 and has been extensively investigated ever since. Work performed in our laboratory 58 61 and many others, as reviewed in Samuel et al, 23 Kelley and Mandarino, 62 and Boden 63 confirmed and expanded the observation that an increase in plasma FFA concentration impairs insulin signaling and causes skeletal muscle insulin resistance in healthy subjects. FFA-induced insulin resistance in healthy insulin-sensitive subjects develops well within the range of plasma FFA concentrations typically observed in obesity and T2DM ( 600 700 mol/l or 2- fold greater than the levels of lean healthy insulin-sensitive subjects). 61 On the other hand, a decrease in plasma FFA concentration by acipimox, a nicotinic acid analogue inhibitor of adipose tissue lipolysis, rapidly improves muscle insulin sensitivity. 64 Excess fatty acid supply promotes skeletal muscle insulin resistance together with an increase in intramyocellular lipids. However, rather than the accumulation of intramyocellular lipids per se, skeletal muscle insulin resistance correlates more closely with a variety of lipidderived toxic metabolites from incomplete oxidation of fatty acids, such as acylcarnitines and long-chain fatty acyl CoAs, 65 ceramides, 66,67 and/or diacylglycerols. 23,67 Complicating the matter is that intramyocellular lipids and certain intramyocellular diacylglycerols have been reported to be markedly higher in insulin-sensitive trained athletes, although ceramides were tightly linked to decreased insulin action in obese insulin-resistant subjects. 68 Although there is considerable controversy as to which metabolite is of greater metabolic significance to muscle insulin resistance, these studies show that lipid-derived metabolites impair insulin signaling and activate inflammatory pathways, including certain protein kinase C isoforms and I /nuclear factor B. One must also bear in mind that skeletal muscle is the target of circulating inflammatory cytokines, such as TNF- and IL-6, and that activated M1 inflammatory macrophages originating from adipose tissue in between muscle fibers may infiltrate skeletal muscle and cause insulin resistance in a similar manner as described earlier for adipose tissue. 69 Skeletal Muscle Insulin Resistance in Patients With NASH The clinical implication from the data reviewed is that even a mild increase in adiposity and plasma FFA level may cause muscle lipotoxicity, so it comes as no surprise that muscle insulin resistance is fully established in obese patients with NAFLD, 3,54,70 even in nonobese individuals with NASH if they have adipose tissue insulin resistance. 71 73 Obese subjects without adipose tissue insulin resistance behave metabolically similarly to lean subjects with near-normal muscle insulin sensitivity and usually do not develop NAFLD. 2,3,71 An abrupt deterioration in muscle insulin sensitivity occurs in obese patients with NAFLD that is proportional to the degree of adipose tissue insulin resistance, indicating a primary defect in adipose tissue rather than in skeletal muscle. 3 However, because metabolic abnormalities are fully established when adult patients with NAFLD are studied, the initial/ intrinsic defect(s) leading to insulin resistance in muscle, liver, and adipose tissue remains to be established.

716 KENNETH CUSI GASTROENTEROLOGY Vol. 142, No. 4 FFA and Pancreatic Beta Cell Lipotoxicity in Humans Studies in humans. During fasting, fatty acids play an essential role as the main energy substrate for pancreatic beta cell function and short-term elevations in plasma FFA levels stimulate insulin secretion in humans. However, a chronic (24- to 72-hour) increase in plasma FFA levels is harmful, and since first described, 74 the role of pancreatic beta cell lipotoxicity has been the subject of intense investigation. Our group reported for the first time in humans that a sustained elevation of plasma FFA level impairs insulin secretion in lean, nondiabetic subjects genetically predisposed to develop T2DM. 59 FFAinduced pancreatic beta cell dysfunction can be rapidly reversed in these subjects by decreasing the release of FFAs from adipose tissue by experimentally inhibiting hormone-sensitive lipase with the nicotinic acid derivative acipimox. 64 These results are consistent with epidemiological observations that link chronically elevated plasma FFA levels with T2DM. 75,76 This also offers an attractive hypothesis to explain why impaired glucose tolerance and T2DM in obese insulin-resistant patients with NAFLD 73 affect both insulin secretion and insulin action. 77 Prediabetes and T2DM in Patients With NAFLD Although the etiology of pancreatic beta cell failure in T2DM remains unclear, there is consensus that obesity is directly related to the diabetes epidemic worldwide. The magnitude of the problem is apparent when it is estimated that in the United States more than one-third of adults aged 60 years or older ( 57 million) have prediabetes (defined as impaired fasting glucose, impaired glucose tolerance, or a plasma A 1c concentration between 5.7% and 6.4%) and diabetes mellitus affects 25 million Americans. 78 By 2030, it will exceed 38 million people in the United States. The presence of diabetes is associated with worse liver disease, although the underlying mechanisms are poorly understood. 79,80 For instance, in epidemiological studies, T2DM is associated with a 2- to 4-fold increase in serious liver disease, 81 83 cirrhosis, and hepatocellular carcinoma. 79,84 87 In patients with NAFLD, T2DM is associated with more severe hepatic and adipose tissue insulin resistance 70,73 and worse NAFLD activity scores and liver fibrosis. 70,73,81,82,88 90 However, well-controlled long-term prospective studies on the natural history of NAFLD in T2DM are lacking and estimates of T2DM in NAFLD have been based largely on medical history or the less sensitive plasma fasting glucose or A 1c levels. When screening for diabetes is performed using not only fasting plasma glucose but the gold standard oral glucose tolerance test, prediabetes (defined as both impaired fasting glucose and impaired glucose tolerance) and T2DM may be 3-fold higher in obese patients with NAFLD compared with patients without NAFLD. 73 Similar high prevalence rates of impaired glucose tolerance and T2DM (ranging from 33% to 62%) have been recently reported in studies from China, 91 Japan, 92 and a predominantly European population from Australia. 93 The clinical implication is that patients with NAFLD may benefit from early screening for T2DM to allow timely intervention to prevent liver and diabetes-related complications. FFAs, Hepatic Insulin Resistance, and NASH Clinical observations on the development of lipotoxic liver disease in humans. There is a close relationship between adipose tissue and liver metabolism in humans because adipocytes supply more than two-thirds of fatty acids used for hepatic triglyceride synthesis. 94 Hepatic insulin resistance in lean subjects is rapidly induced experimentally by a lipid infusion (ie, within 2 4 hours), 61 affecting both hepatic gluconeogenesis 95 and glycogenolysis. 96 Increased hepatic gluconeogenesis has been documented in NAFLD. 97,98 As observed for muscle and pancreatic beta cells, 64 pharmacologically decreasing plasma FFA levels restores hepatic insulin sensitivity. 99 Excess fatty acids not only induce hepatic insulin resistance but also impair insulin clearance in vitro and in vivo 100 102 and in humans. 59,103,104 This leads to the typical hyperinsulinemia of insulin-resistant states and of patients with NA- FLD. 54,70,72,105,106 In NAFLD, reduction of insulin clearance is proportional to the amount of liver fat. 107 Hyperinsulinemia, in the setting of elevated hepatic fatty acid flux as observed in obesity and NAFLD, stimulates hepatic sterol regulatory element binding protein 1c (SREBP-1c) activity and is associated with increased hepatic de novo lipogenesis 108 and oversecretion of very-low-density lipoprotein (VLDL). 109 However, it must be kept in mind that although de novo lipogenesis is increased in NAFLD, it contributes less to the total hepatic triglyceride pool in comparison to fatty acids derived from adipose tissue that account for the majority ( 60%) of hepatic triglyceride accumulation in NAFLD. 108 Figure 3 summarizes for the clinician a large body of basic and clinical work about the natural history of NAFLD. The linear model outlined should be viewed just as a schematic representation of a complex process, keeping in mind that only a minority of subjects with bland steatosis will develop cirrhosis, at least within the follow-up window of currently available studies. 82,110 115 Adipose tissue insulin resistance is present in the majority of patients with NAFLD, whether patients are obese or not, 72 with the liver behaving as the metabolic sensor of dysfunctional adipose tissue and a main target of this lipotoxic state. In our experience, in obese patients with NAFLD, there is a progressive deterioration in plasma triglyceride and high-density lipoprotein cholesterol levels, hepatic insulin resistance, and liver fibrosis as adipose tissue insulin resistance worsens. 3 Obesity, T2DM, and metabolic syndrome are characterized by oversecretion of VLDL driven by the high plasma FFA concentration and hepatic flux of fatty acids. Hepatic assembly and secretion of VLDL are controlled by complex posttranscriptional pathways that control apolipoprotein B metabolism. 116 Under normal physiological conditions, apolipoprotein B

April 2012 OBESITY LIPOTOXICITY IN DEVELOPMENT OF NASH 717 Figure 3. Schematic representation of the pathophysiology of NASH (see text for details). is degraded within hepatocytes. However, in the presence of excess hepatic triglyceride (and to a lesser degree cholesterol) concentration, apolipoprotein B secretion is markedly increased. 117 In patients with high liver fat content, there is also a failure of insulin to suppress VLDL secretion in contrast to a normal suppression in subjects with low (normal) liver fat content. 118 In humans, VLDL secretion rate increases linearly with increasing intrahepatic triglyceride accumulation but hepatic VLDL-TG export is inadequate to normalize hepatic triglyceride content in NAFLD. 119,120 Increased hepatic VLDL secretion lowers high-density lipoprotein cholesterol levels and leads to small, dense, low-density lipoprotein cholesterol molecules, the typical triad of NAFLD and other insulinresistant states. Low plasma adiponectin levels may contribute to hepatic steatosis and VLDL dysregulation in NAFLD as well as necroinflammation in NASH 117,121 because the hormone is essential to the control of hepatic lipogenesis and an increase in plasma adiponectin concentration during TZD treatment is closely associated with liver metabolic and histologic improvement. 31 Genetic factors likely play a significant role in the development of hepatic steatosis and NASH. Candidate genes include those influencing oxidative stress, triglyceride and fatty acid metabolism, extracellular matrix synthesis/degradation (ie, liver fibrosis), endotoxin pathways (ie, TLR-4), and cytokine production by macrophages and adipose tissue. 122 The most reproducible genome-wide association study in NAFLD involves an allele of patatinlike phospholipase 3 (PNPLA3; rs738409). It is most common in the Hispanic population 123 but has been also found in other populations. 124 126 However, the role of PNPLA3 in the pathogenesis of NAFLD remains to be fully established. Another study reported that variants in the gene encoding apolipoprotein C3 (APOC3) (T-455C at rs2854116 and C-482T at rs2854117) contributed to liver fat and plasma triglyceride concentration in Indian subjects with NAFLD. 127 However, in a Finnish population, although carriers of the PNPLA3 allele variant had a higher liver fat content as compared with the noncarrier wild-type homozygotes (median, 11.3% vs 4.2%), no such association was observed with variants in APOC3. 128 The lack of a role for APOC3 is consistent with results from the Dallas Heart Study. 129 Ethnicity may be another important aspect to the development of NAFLD and NASH but may be less of a factor in the Hispanic population than previously believed. In early studies, worse steatosis in Hispanic subjects was also associated with confounders that conferred a greater risk for developing NAFLD, such as insulin resistance and obesity. 100 However, when Hispanic and white subjects are carefully matched for the degree of obesity, there are no significant differences in the severity of insulin resistance or steatohepatitis. 70 This observation has been recently confirmed in a large data-

718 KENNETH CUSI GASTROENTEROLOGY Vol. 142, No. 4 base of 1026 adults with biopsy-confirmed NASH from the NASH Clinical Research Network. 130 The prevalence of steatohepatitis (NASH) among patients with NAFLD varies, depending on the population and geographical location of the study, but is generally accepted in obese individuals to range from 15% to as high as 40% and higher in the presence of features of the metabolic syndrome. 81,87,88,90,131 136 Disease progression from bland steatosis to hepatic lipotoxic liver disease and steatohepatitis (Figure 3) is associated with mitochondrial dysfunction, endoplasmic reticulum stress, reactive oxygen species formation, and activation of inflammatory pathways (ie, c-jun N-terminal kinase, I /nuclear factor B, TLR4) by toxic lipid metabolites such as diacylglycerols, ceramides, and others that exceed the scope of this review. 137,138 Abnormal mitochondrial function has been reported in a number of studies using diverse techniques in NAFLD. 98,139 142 Many intracellular lipid abnormalities have been reported such as increased ratio of stored saturated-to-unsaturated fatty acids, dysregulated fatty acid desaturases and lipid partitioning, increased intracellular cholesterol levels, 143 145 and the observation that metabolic and histologic abnormalities are more closely related to fatty acid saturation and intrahepatic metabolic fate than the overall triglyceride concentration. Saturated fatty acids, such as palmitate, readily accumulate toxic triglyceride-derived metabolites and induce mitochondrial dysfunction, caspase activation, and lipoapoptosis, whereas hepatocytes exposed to unsaturated fatty acids accumulate triglycerides without harm and channel saturated fatty acids into less toxic lipid pools. 138,143,146,147 Therefore, hepatic steatosis per se may not be deleterious (indeed, most patients with steatosis do not have steatohepatitis) and should not become the primary target of treatment or drug development. As proof of concept, reduction of triglyceride synthesis in diabetic db/db mice fed a methionine and choline-deficient diet by an antisense oligonucleotide to acyl coenzyme A/diacylglycerol acyltransferase 2 (the final enzyme that catalyzes triglyceride synthesis) reduces hepatic steatosis but exacerbates oxidative stress, liver injury, and fibrosis. 137 On the other hand, inhibition of VLDL secretion in mice lacking microsomal triglyceride transfer protein (Mttp / ) promotes hepatic steatosis but does not trigger activation of the I /nuclear factor B, insulin resistance, or necroinflammation. 148 Consistent with this observation, patients with familial hypobetalipoproteinemia have severe hepatic steatosis due to a genetic defect in the assembly of apolipoprotein B that impairs hepatic VLDL triglyceride secretion but maintains normal insulin sensitivity. 149 Still, there is an association between the magnitude of hepatic triglyceride accumulation and lipotoxicity-induced mitochondrial dysfunction in NAFLD. For instance, the combination of chronically elevated plasma FFA and insulin levels causes more severe hepatic steatosis and mitochondrial dysfunction than either factor alone in vivo 150 and likely plays a similar role in humans. 151 In middle-aged, obese patients with T2DM stratified based on liver triglyceride content measured by magnetic resonance imaging and spectroscopy (MRS), steatohepatitis worsens as hepatic fat increases from normal up to between 20% and 30% with no additional harm beyond this degree of triglyceride accumulation. 152 Finally, other mechanisms in addition to lipotoxicity, or overlapping as parallel hits, may play a role in the development of steatohepatitis in NASH and activate inflammation and ER stress, 153 including a broad spectrum of signals that range from dysfunctional gut microbiota 153 to increased hepatocyte free cholesterol concentration. 154,155 All must be kept in mind and point to the complexity and challenges of any given working hypothesis to explain the pathogenesis of NAFLD/NASH. Development of severe fibrosis is believed to occur in 5% to 10% of patients with NASH, and advanced fibrosis at baseline is a prominent histologic feature for disease progression. 89,135,136,156 This finding was recently confirmed in 2 long-term observational studies with 7 to 12 years of follow-up. 115,157 Development of cirrhosis (Figure 3) depends on poorly understood mechanisms that involve a cross talk among lipoapoptotic hepatocytes, Kupffer cells, and activated hepatic stellate cells. 137,158 Obesity is a major clinical risk factor for cirrhosis. 88,89,136,156,159,160 Hepatic stellate cells in vitro are very susceptible to palmitate and other long-chain fatty acids. 161,162 Recent metabolic studies in patients with NAFLD suggest that liver fibrosis correlates closely with severe adipose tissue insulin resistance, 3 rendering further support to the link between obesity and fibrosis and making adipose tissue a potential target for the prevention of disease progression. Cardiovascular Disease in NAFLD There is significant interest in understanding the role of NAFLD and NASH in the development of CVD. 156,163,164 The association of NAFLD with the metabolic syndrome and T2DM led to the connection between fatty liver and the development of CVD. 165 167 Epidemiological studies report that patients with elevated plasma aminotransferase concentrations have a higher Framingham risk score and/or number of cardiovascular (CV) events compared with those with normal levels. 168 171 As summarized in Figure 4, there are numerous mechanisms that may accelerate atherosclerosis and premature CVD in patients with NAFLD. It is known that individuals with NAFLD are characterized by abnormal endothelial function. 172 Increased fatty acids impair endothelial cell insulin signaling and nitric oxide production in a dose-dependent manner through the activation of the inhibitor B kinase /nuclear factor B pathway, 173 and experimentally induced plasma FFA elevation in humans alters endothelial function. 174,175 Increased hepatic gluconeogenesis occurs in NAFLD, 97,98 and hepatic insulin resistance is associated with increased insulin secretion to maintain a normal rate of hepatic glucose production and fasting plasma glucose. 3,54,76,92,93,106 This chronic demand on the pancreatic beta cell is believed to be a predisposing factor

April 2012 OBESITY LIPOTOXICITY IN DEVELOPMENT OF NASH 719 Figure 4. CV risk factors that coexist in patients with NAFLD (see text for details). for the development of T2DM in genetically predisposed subjects. 77 Hepatic insulin resistance and steatosis frequently precede the development of T2DM. 76 Hepatic steatosis is an independent risk factor for elevated C-reactive protein and additively associated with obesity and the metabolic syndrome in the development of systemic inflammation. 176 As previously discussed, hyperinsulinemia from increased insulin secretion and decreased insulin clearance correlates with the severity of hepatic steatosis 3,107,152 and chronically elevated plasma insulin levels may promote atherogenesis. Hyperglycemia per se is an established factor for CVD, as well as the typical atherogenic dyslipidemia in NAFLD driven by VLDL oversecretion, as discussed previously. Increased myocardial triglyceride accumulation is observed in obesity and T2DM 177 and is associated with myocardial insulin resistance, impaired ventricular energy metabolism, and coronary dysfunction. 178,179 Hepatic triglyceride content correlates inversely with myocardial glucose uptake and metabolism as measured by positron emission tomography as the phosphocreatine/adenosine triphosphate ratio. 180 There is a close correlation between NAFLD and myocardial steatosis and early diastolic dysfunction. 181 Interventions that mitigate hepatic steatosis have beneficial effects on myocardial function. Pioglitazone ameliorates liver and myocardial steatosis, 182 although improvement of cardiac function by the TZD may involve mechanisms beyond myocardial accumulation of toxic triglyceride-derived metabolites. 183 Whether NAFLD and CVD are mechanistically related or merely an association within a state of lipotoxicity remains to be established. Conceptually, studies attempting to link CVD with NAFLD can be divided into 3 major categories: (1) small longitudinal studies about the natural history of NAFLD comparing outcomes with the general population or subjects without NAFLD, 82,112,171,184 186 (2) cross-sectional studies measuring atherosclerotic burden in patients with NAFLD (Table 1), and (3) larger epidemiological studies examining the prevalence of CV events in patients with and without NAFLD (Table 1). Most of these studies are weak because the diagnosis of fatty liver was based on liver aminotransferase levels or ultrasonography, both rather insensitive tests for the diagnosis of NAFLD. In some studies, patients with NAFLD lacked proper controls or the positive association with CVD disappeared after adjusting for traditional risk factors. Interpretation of the role of NAFLD is also challenging if these patients already have a much worse CV risk profile compared with controls without NAFLD, even after adjustment for this by statistical analysis. Taking into account the preceding limitations, there is some indication that NAFLD carries a higher risk of CVD. For instance, Matteoni et al 185 was among the first to report increased CV events in a group of 132 patients with biopsy-proven NASH followed up for up to 18 years. Coronary artery disease was the second cause of mortality after neoplasm, although the number of events was rather small (11 total). Dam-Larsen et al 186 arrived at a similar conclusion in a cohort of 417 patients with NAFLD followed up for up to 20.4 years, with the main causes of death being CVD and cancer. In another study in 420 patients from Minnesota, overall mortality and liver-related mortality after 7.6 years was significantly higher in subjects with NAFLD compared with the general population, with coronary heart disease among the leading causes of death. 112 Recent reports appear to coincide with the observation that NAFLD increases several fold the risk of CVD. 184,187

720 KENNETH CUSI GASTROENTEROLOGY Vol. 142, No. 4 Table 1. Studies Assessing CV Burden in Patients With and Without NAFLD Author (year) Method used for the diagnosis of NAFLD Primary end point Increased CVD risk? (after adjustment for confounders) a Villanova et al (2005) Liver biopsy Endothelial function b Yes Brea et al (2005) Liver US CIMT/carotid US No Volzke et al (2005) Liver US CIMT No Targher et al (2005) Liver US CV events Yes Targher et al (2006) Liver biopsy CIMT Yes Mirbagheri et al (2007) Liver US Coronary angiography Yes Hamaguchi et al (2007) Liver US CV events Yes Schindhelm et al (2007) ALT CV events Yes McKimmie et al (2008) c CT CIMT No Fracanzani et al (2008) Liver US or biopsy CIMT Yes Goessling et al (2008) AST, ALT CV events No Haring et al (2009) US CV events Yes Poanta et al (2011) c US CIMT No ALT, alanine aminotransferase; AST, aspartate aminotransferase; CT, computed tomography; US, ultrasonography. a CVD risk after adjustment for traditional risk factors (age, sex, body mass index, smoking, T2DM, metabolic syndrome, others). b Measured by brachial artery flow-mediated vasodilation. c All patients had T2DM. Several cross-sectional studies have compared patients with NAFLD to healthy controls in studies using surrogate markers of CVD. A study by Villanova et al 188 showed an association between fatty liver and endothelial dysfunction measured by brachial artery flow-mediated vasodilation, a marker of early atherosclerosis, in 52 patients with NAFLD (39 with biopsy-proven NASH) compared with patients without NAFLD. Mixed results have been reported in studies that have used carotid-intima medial thickness (CIMT) as a marker of atherosclerotic burden (Table 1). Although some studies showed a significant increase in atherosclerosis, 166,189 191 in several studies the association of NAFLD with CVD did not hold when adjusted for traditional CV risk factors. 166,189,192,193 Two studies in patients with T2DM also reported negative results for the association of CIMT with increased CVD in NAFLD. 192,193 The risk of CV events in NAFLD may be higher than in the general population (Table 1). One study reported that among 2103 patients with T2DM, 5-year nonfatal coronary heart disease, ischemic stroke, and CV death occurred more frequently in patients with NAFLD than those without a fatty liver measured by ultrasonography. 194 Adjustment for traditional CV risk factors attenuated but did not completely abolish the association. Several studies have used plasma aminotransferases as a surrogate marker of NAFLD. 169 171,195 In a cross-sectional study, Ioannou et al 170 found a strong correlation between elevated alanine aminotransferase level and 10-year risk of coronary heart disease estimated by the Framingham risk score, with a threshold for increased CV risk with a plasma alanine aminotransferase level greater than 43 IU/L in men and 30 IU/L in women. In 1439 patients from the Hoorn study, 195 the predictive value of elevated alanine aminotransferase level for coronary heart disease persisted independent of the presence of metabolic syndrome or other traditional risk factors. In the Framingham Offspring Heart Study, 169 the development of metabolic syndrome and diabetes was closely related to the presence of elevated ALT level over a follow-up period of 20 years. However, although CVD was increased in age/ sex-adjusted models in patients with NAFLD, this association was no longer significant in multivariate-adjusted models that included classic CV risk factors. In summary, the debate about CVD in NAFLD revolves on whether hepatic steatosis per se (or steatohepatitis) confers a CV risk above and beyond that of the clustering of deleterious metabolic risk factors characteristic of this population. Current evidence has been overall inconsistent due to a number of factors ranging from inadequate diagnosis of NAFLD (usually not relying on MRS or a liver biopsy but liver aminotransferase levels or ultrasonography), inadequate adjustment for confounders, short-term follow-up, frequent use of surrogate end points of CVD (ie, endothelial dysfunction, CIMT), and few CV events when used as the primary end point. Thus, although metabolic and clinical factors would support the notion that NAFLD carries an increased risk of CVD, larger, long-term prospective studies are needed to fully establish the nature of this association. Treatment of Lipotoxic Liver Disease (NASH) Lifestyle Intervention It is well established that lifestyle interventions that result in weight loss decrease CVD 196 and delay the development of T2DM. 197 However, large long-term trials are yet unavailable in NAFLD, so the most effective lifestyle intervention remains unclear because studies have been usually small and uncontrolled and included diverse diets and exercise programs. 198 201 Weight loss improves liver aminotransferase levels and hepatic steatosis when measured either by ultrasonography or MRS in proportion to the total amount of weight loss, but few studies have used paired liver histology to

April 2012 OBESITY LIPOTOXICITY IN DEVELOPMENT OF NASH 721 assess response. Orlistat may enhance weight loss but does not per se improve histologic response. 202,203 Weight loss of 9%, either assisted by orlistat use or not, improves hepatic steatosis and necroinflammation. 203 Promrat et al 204 randomized 31 obese subjects to 48 weeks of lifestyle intervention (200 minutes a week of moderate physical activity) versus standard dietary counseling alone. The lifestyle arm lost 9.3% of weight compared with only 0.2% in the dietary counseling arm. Participants who lost 7% compared with those who lost 7% had significant improvements in steatosis, lobular inflammation, and ballooning injury, although fibrosis was unchanged. In a number of studies using MRS (not liver histology) as the primary end point, both diet alone 205 208 or combined with exercise 207,209 212 led to a significant reduction of liver steatosis in the order of 40% (range, 20% 81%). Reduction of steatosis was proportional to the intensity of the lifestyle intervention, but its impact on other aspects of liver histology was not determined. In the only study using both liver MRS and liver biopsies to assess response to treatment in patients with NASH, 54 diet plus pioglitazone led to a 52% reduction in liver steatosis measured by MRS and a significant 50% mean decrease in steatosis, ballooning necrosis, and inflammation on histology. Whether this may apply to lifestyle intervention studies alone remains to be established. Although hepatic fat reduction usually requires a minimum total body weight loss of 5%, a 3% decrease may still be associated with a significant decrease in hepatic steatosis. 207,209,211 This is because hepatic triglyceride reduction depends not only on the magnitude of weight loss but also the dietary composition and intensity of physical activity prescribed. Both low-carbohydrate 208,213 215 and low saturated fat 206 diets reduce hepatic steatosis. Both approaches were equally effective in a 6-month study in 170 patients with NAFLD. 216 Moderate exercise 2 to 3 times a week (without dietary modification or a change in body weight) has been reported to decrease hepatic steatosis in NAFLD over a period of 6 to 12 weeks. 217,218 Because weight loss is difficult to achieve and even more to maintain, bariatric surgery has emerged as a valid alternative. 200,219 221 Both Roux-en-Y gastric bypass or laparoscopic adjustable gastric banding have resulted in marked improvement of comorbidities (diabetes, hypertension, and dyslipidemia) as well as in liver histology such as reduced steatosis, necroinflammation, and ballooning. Changes in fibrosis have been more inconsistent, with even some studies reporting worsening of fibrosis following bariatric surgery. 222,223 Procedures with a malabsorptive component, such as Roux-en-Y gastric bypass, lead to a greater weight loss and metabolic benefit than laparoscopic adjustable gastric banding, although the popularity of the latter has increased because it is less invasive. The best bariatric surgery approach for NAFLD is unknown because there have been no prospective randomized controlled trials (RCTs) comparing both procedures. Moreover, results of currently available studies should be interpreted with caution because they are usually retrospective, limited by patient selection bias, poorly standardized in terms of presurgical/postsurgical dietary procedures, and lack of in-depth metabolic testing. In summary, dietary intervention (with or without exercise) improves hepatic steatosis and the metabolic profile of patients with NAFLD. In short-term studies, a 5% weight loss appears to be the minimal threshold to decrease triglyceride accumulation, whereas a 7% to 10% weight loss appears to be needed to reduce necroinflammation. The most effective lifestyle or surgical intervention to alter the natural history of NASH remains to be established. This calls for a multicenter long-term controlled trial comparing different lifestyle and surgical approaches in this population. Pharmacologic Interventions in NASH Many agents have been tested with disappointment in the management of NASH, and only vitamin E and pioglitazone have yielded significant promise in the treatment of patients with NASH. 80,160,201,224 Vitamin E The mechanism of action of vitamin E in patients with NASH remains unclear but is believed to be related to amelioration of intracellular oxidative stress. Early pilot studies showed mixed results on liver histology, 225 227 but its efficacy in adults was shown in a 2-year RCT in 247 subjects with NASH. 56 For unclear reasons, results in children and adolescents aged 8 17 years of age have instead been somewhat disappointing when they have been given vitamin E or metformin. 228 Neither agent was superior to placebo in terms of a reduction of plasma alanine aminotransferase level 40 U/L or 50% of baseline levels. This primary end point was achieved with vitamin E in only 25.9% of patients, compared with 15.8% receiving metformin and 17.2% receiving placebo. In the subset of children that underwent liver biopsies before and after treatment, neither vitamin E nor metformin had significant effects on the individual parameters of steatosis, fibrosis, or lobular inflammation. However, vitamin E significantly decreased hepatocellular ballooning (44% vs 21% placebo; P.02), which together with a trend for a reduction in steatosis improved the overall NAFLD activity score and the rate of NASH resolution (58% vs 28% placebo; P.006). Of note, metformin also improved hepatocellular ballooning to the same degree as vitamin E (44% vs 21% placebo; P.02) without improving any other histologic parameter, raising the question about the significance of hepatocellular ballooning in the context of other histologic parameters being unchanged. Therapeutically, vitamin E is attractive because it likely targets intracellular pathways different from insulin sensitizers and offers an opportunity for combined therapy in NASH. Metformin Improvement of insulin resistance by metformin appears to occur primarily at the level of the liver and,