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  Fatty Liver Disease

Nonalcoholic fatty liver disease.....liver enzymes-ALT/HIV/diet, exercise, metabolic syndrome, waist circumfrance, dysfunctional adipose tissue, diabetes, screening
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Nature Reviews Disease Primers - 17 December 2015
Nonalcoholic fatty liver disease (NAFLD) has been accepted as a bona fide disease entity in the late twentieth century. The disorder is characterized by excess accumulation of fat (in the form of triglycerides) in hepatocytes (>5% fat content in the liver; referred to as steatosis), leading to nonalcoholic fatty liver (NAFL). In the presence of additional factors, described below, hepatocyte injury and cell death occur along with lobular and portal inflammation, and the disease is referred to as nonalcoholic steatohepatitis (NASH). With progressive collagen deposition and subsequent vascular remodelling, cirrhosis may result.
The United States and Europe.
NAFLD is currently the most common cause of abnormal liver function tests in western countries. According to population studies using ultrasonography or CT imaging, the prevalence of NAFLD is in the range of 20-50%10, 11, 12, 13 (Table 1). The prevalence of NAFLD varies markedly between ethnic groups. An urban population study in the United States using proton magnetic resonance spectroscopy (1H-MRS) showed that the prevalence of hepatic steatosis was 45% in Hispanic, 33% in white and 24% in black populations14. These variations can be explained by differences in lifestyle, the prevalence of metabolic syndrome and genetics, such as a polymorphism of the patatin-like phospholipase domain-containing 3 (PNPLA3) gene, which encodes a lipase that mediates triacylglycerol hydrolysis in adipocytes (Fig. 2). NAFLD is strongly associated with metabolic disorders. Not surprisingly, fatty liver has been reported in 40-80% of patients with type 2 diabetes mellitus (T2DM) and 30-90% of patients who are obese15,16.
Asia, Africa and the Pacific Islands. The epidemiology of NAFLD in Asia-Pacific and African countries is shaped by vast differences in lifestyle and economic growth among countries and between urban and rural areas within the same country. For example, in China, the prevalence of NAFLD is higher in coastal and urban areas (up to 30%) than in inland and rural areas (around 1%)23. In urban areas, the prevalence of NAFLD is around 15-30% in Asia-Pacific countries and 10-20% in Africa.
Compared with western countries, the increase in NAFLD prevalence over time is more marked in Asia and the Pacific areas, probably reflecting rapid lifestyle changes. Studies from Japan and China recorded a twofold increase in the prevalence of NAFLD over a decade23,24. By contrast, the reported prevalence of NAFLD in the United States was already around 30-40% in 2004 and has not increased substantially thereafter14. The incidence of NAFLD is difficult to study because ultrasonography cannot reliably quantify liver fat. Nevertheless, according to a recent study from Hong Kong using paired 1H-MRS, 13.5% of community subjects developed NAFLD in 3-5 years25. Asians and Pacific Islanders develop obesity-related complications at a lower body mass index (BMI) than Europeans and Americans because of the tendency to have central fat accumulation. As a result, approximately 15-20% of Asian patients with NAFLD do not have obesity according to current definitions.
The first step in the management of NASH in most patients is the implementation of lifestyle modifications that focus on healthy eating habits and regular exercise....Fatty acids and the carbohydrates used to make fatty acids can also be diverted to oxidative pathways and this may explain the benefit of regular exercise in improving the metabolic syndrome....The mechanisms of lipotoxic injury remain uncertain, but oxidative stress has been proposed to have a role191. Oxidative stress with associated lipid peroxidation clearly occurs in NASH192, but whether it is causative or an epiphenomenon remains unknown.....More recently, in a large European observational cohort with NAFLD, statin use was associated with less steatosis, NASH and fibrosis in a dose-dependent manner; interestingly, the presence of the PNPLA3I148M polymorphism prevented the protective effect of statins199. In another recent study, statin use in patients with NAFLD was associated with a reduction in liver-related deaths, liver transplantations or other liver outcomes7......Most patients with NAFLD are overweight or obese, and even in those who are not, lifestyle changes that incorporate dietary modification and exercise are the cornerstone of therapy. Obesity has both health and psychosocial ramifications and can have a profound effect on an individual patient's QOL.....Another consequence of liver disease in patients with NASH is HCC. The incidence of HCC is increasing in both the United States and worldwide. In western countries, approximately 20% of HCCs are found in patients with NASH-related liver disease; and even more if HCCs occurring in the setting of cryptogenic cirrhosis are included, which often represents 'burned out' NASH that has lost its classic histological features (Fig. 1)215,216. A recent study in the United States based on Surveillance, Epidemiology, and End Results (SEER)-Medicare registries from 2004 to 2009 found NASH to be the underlying cause of HCC in 14.1% of nearly 5,000 cases, following alcoholic liver disease in 16% and hepatitis C virus infection in 54.9%. While this represented a 9% annual increase in NASH-related HCC, NASH-HCC cases represented only 5% of liver transplantations for HCC during the same time period, probably reflecting the burden of co-morbid illness in this population. Furthermore, patients with NASH and HCC were older and had shorter survival if they did not receive liver transplantation than other aetiologies of liver disease217.....Cirrhosis creates a milieu that is permissive for the development of HCC; thus, current screening recommendations focus primarily on patients with cirrhosis. However, reports of HCC occurring in the absence of cirrhosis are on the rise, with some even developing in the absence of profound fibrosis. By some estimates, 10-75% of cases of HCC may occur in non-cirrhotic NAFLD221,222. This is a serious problem because of the large number of patients affected by NAFLD and NASH at a population level. The individual risk of HCC in patients without cirrhosis who have NAFLD is even less well understood. Consequently, although some individuals at risk for HCC may not be identified at an early stage of malignancy, there is presently insufficient evidence to alter current screening guidelines. Optimal recommendations for HCC screening in patients with NAFLD may change in the future as more is learnt about individual risk in this challenging population......Until recently, the landscape of clinical trials for NASH was somewhat limited. Currently there are hundreds of clinical trials in NASH. The breadth of mechanisms being targeted reflects the complexity of the disease. As the field continues to advance and patients can be better classified by constellations of risk factors, or sub-classifications using genomics or metabolomics, we will be able to offer a more personalized, mechanistically derived treatment approach to each patient.
Current and Future Therapeutic Approaches to NAFLD/NASH - (08/05/17)
EASL: Emerging Treatments for ASH & NASH - AASLD 2016 - (07/03/17)
IAS: Fatty Liver in HIV+ at IAS - (08/05/17)
AASLD: NAFLD Debrief AASLD 2017 - (11/29/17)
Children and adolescents
The United States and Europe.
NAFLD is the most common liver disease in children and adolescents (Table 2). Similarly to adult patients, NAFLD is associated with obesity and metabolic syndrome27 and probably with increased levels of alanine aminotransferase (ALT) in the blood and PNPLA3 polymorphisms28,29 (Fig. 2). A recent analysis of the National Health and Nutrition Examination Survey, including data from >12,000 12-19-year-old Americans, showed that the prevalence of NAFLD (with NAFLD defined as a BMI in the >85th percentile and increased ALT levels of >22.1 U per litre for girls and >25.8 U per litre for boys) more than doubled over the past two decades27. The associated risk factors identified in this study confirm previously identified factors, such as age, increased BMI, male gender and Mexican-American race30. Modifiable factors were increased consumption of sugar-sweetened beverages and other lifestyle factors (such as a sedentary behaviour)27. Paediatric NAFLD prevalence has been predicted to increase even further31.
"Another important criterion is that NAFLD is tightly associated with metabolic syndrome and insulin resistance in the liver, muscle and adipose tissues126. NAFLD should be suspected in all individuals with one or more components of the metabolic syndrome, defined as the cluster of any three of the following five features: increased waist circumference(ethnicity adjusted), increased fasting glucose or T2DM, hypertriglyceridaemia, low HDL levels (sex adjusted) and increased blood pressure127. This association does not characterize NAFLD owing to the genetic variant in PNPLA3, but PNPLA3 mutation and metabolic syndrome may be associated; that is, frequently present in the same person80.
Diagnostic workup should include evaluation of family and personal history of components of the metabolic syndrome and assessment of liver tests, including platelet counts, albumin and coagulation, fasting blood glucose, triglycerides and HDL levels. The most common modes of presentation of NAFLD are the detection of unexplained abnormal liver enzymes and/or of bright liver at ultrasonography.
Blood parameters. Liver tests can show mild (twofold to fivefold) increases in the levels of ALT, alkaline phosphatase (ALP) and γ-glutamyltranspeptidase (GGT), but levels are normal in the majority of people with NAFLD (80%)128. The ratio of ALT to aspartate aminotransferase (AST) is greater than 1 unless advanced fibrotic NAFLD is present or the patient has covert AFLD. Increased levels of GGT do not discriminate between AFLD and NAFLD, as raised GGT levels are commonly associated with metabolic disease129. Ferritin may be increased in up to 60% of patients, but is mainly a marker of subclinical inflammation, given that iron overload is uncommon in NAFLD (4-6%)130. However, high ferritin levels (twofold to threefold) have been associated with more-advanced disease125. Autoantibodies (antinuclear antibody and α-smooth muscle actin-specific antibody) are present in approximately one-third of patients at low titres (1/80 and 1/160)131 as compared to absent in healthy individuals.
A test of insulin sensitivity should be considered in all suspected patients. The HOMA-IR is a good surrogate for insulin resistance in individuals who do not have diabetes132, although there is no universal agreement on threshold defining what is abnormal. Diagnostic workup should eventually include an oral glucose tolerance test according to standard criteria133 that provides additional information about the glucose tolerance status.
MRI. The leading quantitative MRI biomarker for hepatic steatosis is proton density fat fraction (PDFF)138. This biomarker measures the relative proportion of mobile protons that are attributable to fat and correlates closely with biochemically determined hepatic triglyceride concentration138. In numerous single-centre and multi-centre studies, PDFF has shown high accuracy for diagnosing, grading and longitudinally monitoring steatosis in adults and children, including individuals who are morbidly obese139, 140, 141, 142, 143, 144. Historically, measurement of PDFF required MRS, which is usually available only in academic centres and generally only assesses portions of the liver. In recent years, advanced MRI techniques have been developed that measure PDFF throughout the entire liver in a single breath-hold, thereby eliminating sampling variability. These techniques are becoming commercially available on the latest generation of magnetic resonance scanners made by the leading manufacturers. Importantly, PDFF measurements made on scanners of different field strength and manufacturer agree closely143, 144, 145. With appropriate quality control, these advanced PDFF-estimation techniques can be used in clinical trials (Fig. 5a).
Diagnosis of NASH
The diagnosis of NAFLD can be derived from classic risk factor assessment along with biochemical measures, and ultrasound-based and magnetic resonance-based detection of hepatic steatosis, yet the most relevant challenge to the clinician is the distinction between simple fatty liver and NASH because patients with NASH are at greater risk for developing progressive fibrosis. The presence of normal liver enzymes in the majority (80%) of individuals renders the identification of patients at risk particularly challenging128. Importantly, histological severity between patients with and those without abnormal liver aminotransferases does not differ.
The actual diagnosis of NASH requires microscopic evaluation of liver tissue, obtained by liver biopsy (Fig. 1). Histological analysis shows the constellation of steatosis, hepatocyte ballooning and lobular inflammation, typically in acinar zone 3 - the microcirculatory unit through which blood exits the liver146. Fibrosis better predicts outcomes and mortality7. Semi-quantitative grading and staging systems have been developed (Box 2). These systems are primarily used to the assess the severity of lesions. Grading involves the assessment of the activity of the disease: steatosis, inflammation and hepatocyte ballooning, whereas staging involves the assessment of fibrosis location and vascular remodelling. Although the grade might be reversible with appropriate intervention, the stage is probably less reversible.
As liver disease progression has been associated with persistence or worsening of metabolic risk factors147, most predictive indices of disease severity are based on components of the metabolic syndrome along with biochemical and imaging indicators of advanced liver disease. However, the large number of patients with NAFLD and the risk of complications associated with liver biopsy have led to the refinement of non-invasive techniques to predict the histological features of NAFLD and NASH.
In clinical practice, interpretation of non-invasive markers should be performed by hepatologists according to the clinical context and considering all the other clinical and laboratory findings. The combination of two unrelated markers is recommended, as no single test has an advantage over the others in the prediction of the severity of liver disease148. Among the different strategies, algorithms that combine transient elastography and serum biomarkers are the most attractive and validated148. However, in case of unexplained discordance of non-invasive tests, a liver biopsy should be performed.
In clinical practice, interpretation of non-invasive markers should be performed by hepatologists according to the clinical context and considering all the other clinical and laboratory findings. The combination of two unrelated markers is recommended, as no single test has an advantage over the others in the prediction of the severity of liver disease148. Among the different strategies, algorithms that combine transient elastography and serum biomarkers are the most attractive and validated148. However, in case of unexplained discordance of non-invasive tests, a liver biopsy should be performed.
Biomarkers and scores. Screening of steatosis is usually based on ultrasonography in individual patients, but indices or biomarkers are preferred in larger-scale screening studies. The best-validated biomarkers are the Fatty Liver Index, the SteatoTest and the NAFLD liver fat score. Simple steatosis does not increase liver-related mortality, but these biomarkers can variably predict metabolic and cardiovascular outcomes or mortality149. Serum markers of fibrosis seem to have a better performance, particularly the NAFLD Fibrosis Score (NFS), Fibrosis-4 (FIB-4), BARD and commercially available panels, such as FibroTest, FibroMeter and the Enhanced Liver Fibrosis (ELF) test150 (Table 3). All of these tools have acceptable diagnostic accuracy, but only NFS and FIB-4 have been extensively validated19. All of these tests perform best at excluding severe fibrosis-cirrhosis, with negative predictive values of >90%, but are typically less accurate in the determination of less-severe fibrosis150. NFS, FIB-4, ELF and FibroTest can also predict overall, cardiovascular and liver-related mortality149.
Elastography. Imaging techniques that are collectively known as elastography have been developed to indirectly measure tissue stiffness non-invasively. The measurement involves the assessment of the propagation of shear waves within the liver, from which the hepatic stiffness is inferred. Depending on the device, results can be reported as shear wave speed in metres per second or as one of several elastic moduli (for example, Young modulus or complex shear modulus) in kPa154. These various elastography-derived parameters have emerged as the leading imaging biomarkers of hepatic fibrosis, but like serum markers, they perform best only at detecting advanced (stage 3 or stage 4) fibrosis.
Ultrasound-based elastography. The first ultrasound-based method to measure liver stiffness was VCTE155. This technology uses a non-imaging device to track shear waves that are transmitted transiently into the liver by an external vibration source; an anatomical image is not acquired and the location from which the measurements are made is not recorded.
More recently, elastography capabilities have been added to clinical ultrasound scanners154. Elastography-enabled scanners use ultrasound to track shear waves that are generated transiently in situ within the liver by an ultrasound push pulse (also known as an acoustic radiation force impulse); measurement locations are recorded on an anatomical image154,156,157 (Fig. 5b). As each manufacturer uses proprietary hardware and software, measurements made with different devices and even with different transducers (probes) on the same device do not agree157,158. Thus, any given patient should be monitored serially with a single device and using the same probe. VCTE and elastography-enabled scanners have been shown to provide high accuracy for the diagnosis of advanced fibrosis in patients with viral hepatitis infection159,160. Single-centre studies suggest these devices also accurately diagnose advanced fibrosis in patients with NAFLD160, although multi-centre validation studies have not yet been performed. None of these devices can diagnose early fibrosis accurately. The technical success rate, reliability, robustness and precision of these devices for measuring stiffness parameters in NAFLD has not been extensively studied, and their performance in detecting changes in fibrosis in the context of treatment trials is unknown157. Until such data become available, caution is advised for use of these devices in NAFLD clinical trials.
Magnetic resonance-based elastography. Magnetic resonance elastography uses a specialized MRI sequence to visualize shear waves that are delivered continuously into the liver by an external acoustic source161. The wave images are processed by a so-called inversion algorithm to generate 'elastograms' (Ref. 162) (Fig. 5c). These computer-generated images depict the spatial distribution of a stiffness parameter known as the magnitude of the complex shear modulus (usually referred to as 'shear stiffness' in the medical literature). The hepatic stiffness is recorded from representative portions of the liver. Magnetic resonance elastography is now commercially available on commercial MRI scanners, with the same hardware to generate the shear waves and the same inversion algorithm to process the wave images. Small single-centre studies in healthy volunteers suggest that measurements made with different scanners agree163. Large studies in patients with NAFLD are needed to confirm cross-platform reproducibility.
In single-centre studies, magnetic resonance elastography has been shown to diagnose advanced fibrosis in adult NAFLD with high accuracy164, 165, 166. The accuracy of magnetic resonance elastography is probably higher than that of ultrasound-based elastography167.
As with ultrasound-based techniques, accuracy for the detection of early fibrosis is modest. The precision of magnetic resonance elastography in the NAFLD population has not been studied extensively. Multi-centre validation studies are needed before magnetic resonance elastography can replace biopsy in NAFLD clinical trials.
Follow-up monitoring
Patient monitoring should include routine biochemistry, non-invasive monitoring of fibrosis and the assessment of co-morbidities. Given that the risk of developing T2DM and cardiovascular complications is increased in patients with NAFLD122, oral glucose tolerance tests should be performed whenever glucose metabolism is abnormal (that is, a fasting glucose of 100-126 mg per dl; glycated haemoglobin (HbA1c) of 5.7-6.4%)133 and carotid doppler ultrasonography122; referral to other specialists might be required. The timing of follow-up testing is still debated, and a definite evaluation for risks of progression and costs associated with investigations has not yet been performed. In patients with simple fatty liver, a reasonable approach is a follow up in primary care setting, unless worsening of metabolic risk factors occurs. In patients with NASH and/or fibrosis, a yearly monitoring policy is advisable. If indicated, on a case-by-case basis a repeat biopsy could be performed after 5-7 years."
Pathophysiology of NAFLD

Fatty liver disease is characterized by hepatic steatosis; there may also be varying degrees of hepatocyte injury, inflammation and, in some cases, fibrosis (Fig. 1).
The evolution of hepatic steatosis in obesity. Several factors contribute to the accumulation of hepatic lipids in the setting of obesity (Fig. 3). First, weight gain is associated with marked expansion of adipose tissue, which leads to dysfunction and eventual death of adipocytes. Dysfunction of adipose tissue results in local inflammation and the upregulation of cytokines that promote insulin resistance. Insulin resistance, in turn, compromises the ability of adipocytes to store fat, resulting in the release of free fatty acids into the circulation, which then become available for uptake by ectopic organs such as the liver37. As a consequence of obesity-related adipose tissue dysfunction, the liver is exposed to high levels of circulating free fatty acids as well as high levels of insulin, which is produced to compensate for systemic insulin resistance. Hepatocytes take up these fatty acids via the transporters fatty acid transport protein 5 (FATP5; also known as bile acyl-CoA synthetase) and CD36, which are also upregulated in obesity38,39. Fatty acid accumulation in hepatocytes prompts the synthesis of triglycerides; during this process, diacylglycerol intermediates accumulate, which impair hepatic insulin signalling by activating protein kinase Cε (PKCε)40. Hepatocyte insulin resistance fuels gluconeogenesis, promoting hyperglycaemia and prompting even more compensatory insulin production.
The liver itself can contribute to hepatic steatosis by producing lipid from carbohydrate in a process called de novolipogenesis (DNL). The enzymes responsible for DNL are upregulated by insulin and glucose through the action of two transcription factors, sterol regulatory element-binding protein 1 (SREBP1) and carbohydrate-responsive element-binding protein (ChREBP)41. In the normal liver, DNL is not a main source of hepatic lipid, but in the setting of obesity and hyperinsulinaemia, DNL can contribute as much as 25% of total hepatic lipid stores, and is considered an important factor in the development of NAFLD42,43. Why an insulin-dependent process such as DNL would be upregulated in hepatocytes if uptake of excess circulating fatty acids has rendered them insulin resistant seems paradoxical. One explanation is that hepatocyte insulin resistance manifests downstream from the insulin receptor44; another is that DNL can also be induced by insulin-independent pathways, such as endoplasmic reticulum stress-mediated activation of SREBP1 (Ref. 45).
Nutrients from the diet also serve as substrates for hepatic triglyceride synthesis and thus participate in the development of hepatic steatosis. Dietary sugars are converted to fatty acids via DNL, and dietary fats are taken up by the liver along with adipose tissue-derived fatty acids. Roughly 40% of the lipid that accumulates in a fatty liver derives from dietary sugars and fats. Lipolysis of dysfunctional adipose tissue contributes the remaining 60%43.
The gut-liver axis in NAFLD. Interactions between the intestine and the liver are emerging as important determinants of NAFLD. Obesity, which is a key factor contributing to the development of NAFLD, is associated with alterations in the intestinal microbiota that enhance nutrient extraction from the diet46. To date, NAFLD has not yet been linked to any specific microbiotic signature, but people with NAFLD have intestinal microbiota that is clearly different from that of healthy individuals47. Intestinal dysbiosis can contribute to liver disease by weakening the intestinal barrier and enabling the translocation of bacteria or bacterial products, such as endotoxin or ethanol, into the portal circulation. Dysbiosis can also lead to the luminal degradation of beneficial nutrients, such as choline, which are important for the maintenance of hepatic lipid homeostasis11.
Mechanisms of cell death in a fatty liver. One of the hallmarks that differentiates NASH from NAFL is the presence of hepatocyte injury. Hepatocytes can be damaged by several mechanisms in the setting of NAFLD (Fig. 3). First, the demand for metabolism of excess fatty acids places strain on hepatocyte mitochondria; over time this leads to mitochondrial uncoupling, the production of reactive oxygen species (ROS) and the activation of Jun N-terminal kinase (JNK)48. Once initiated, ROS production and JNK activation continue in a feed-forward loop, which results in mitochondrial damage, impaired ATP production and cell death49,50. Excess fatty acids can also injure hepatocytes by inducing endoplasmic reticulum stress, which leads to mitochondrial dysfunction and cell death through caspase-2-mediated cleavage of BH3-interacting domain death agonist (BID)51, 52, 53. However, another pathway via which fatty acids can kill hepatocytes is through the activation of death receptors. Several death receptors (such as FAS, death receptor 5 (DR5; also known as TNFRSF10B and TRAIL-R2) and tumour necrosis factor receptor superfamily member 1A (TNFRSF1A; also known as TNFR1) are upregulated on hepatocytes in the setting of steatosis; the activation of death receptors has been implicated as an important stimulus for hepatocyte apoptosis and necroptosis in NASH54, 55, 56, 57.
Inflammation and fibrosis in fatty livers. Activation of the immune system is a key feature of NASH (Fig. 3). The classic effectors of hepatic inflammation in NASH are Kupffer cells and recruited macrophages, but natural killer T cells play an important part in macrophage recruitment58, and both natural killer T cells and T cells are emerging as contributors to progressive liver disease59, 60, 61, 62, 63. Several compounds can trigger inflammation in fatty livers - including fatty acids, bacterial endotoxin reaching the liver from the intestine and damage-associated molecular patterns (DAMPs) released from dying hepatocytes - and induce inflammation by activating Toll-like receptors (TLRs) and inflammasomes in target immune cells64,65, stimulating the production of an array of cytokines and chemokines58,66,67.
The development of hepatic fibrosis portends a poor outcome in NASH7. The central event in hepatic fibrogenesis is the activation of hepatic stellate cells68; these cells are susceptible to stimulation by various compounds that are present in a diseased fatty liver. Like inflammatory cells, stellate cells can be activated by DAMPs from dying hepatocytes or other sources that engage TLRs on the cell surface69. Other compounds such as free cholesterol can amplify hepatic fibrosis by upregulating the expression of TLRs on stellate cells70. Oxidative stress can also activate stellate cells; advanced glycation end products, which are abundant in individuals with diabetes, induce ROS production by stellate cells by inducing the enzyme NADPH oxidase 2 (NOX2; encoded by CYBB)71. One unique consideration in NASH is the role of ballooned hepatocytes in hepatic fibrosis. Ballooned cells produce Sonic Hedgehog72, a protein that promotes tissue fibrosis73. Whether Sonic Hedgehog stimulates fibrosis through direct effects on stellate cells or indirect effects on fatty liver injury is currently under study74.