Liver fat as risk factor of hepatic and cardiometabolic diseases

Non‐alcoholic fatty liver disease (NAFLD) is a disorder characterized by excessive accumulation of fat in the liver that can progress to liver inflammation (non‐alcoholic steatohepatitis [NASH]), liver fibrosis, and cirrhosis. Although most efforts for drug development are focusing on the treatment of the latest stages of NAFLD, where significant fibrosis and NASH are present, findings from studies suggest that the amount of liver fat may be an important independent risk factor and/or predictor of development and progression of NAFLD and metabolic diseases. In this review, we first describe the current tools available for quantification of liver fat in humans and then present the clinical and pathophysiological evidence that link liver fat with NAFLD progression as well as with cardiometabolic diseases. Finally, we discuss current pharmacological and non‐pharmacological approaches to reduce liver fat and present open questions that have to be addressed in future studies.

hyperlipidemia, and metabolic syndrome). 3,4 NAFLD is also increasingly recognized as an independent risk factor for cardiovascular diseases. 5 The gold standard for the diagnosis and staging of NAFLD remains liver biopsy, which is an invasive, time-and cost-consuming method, that is operator dependent showing moderate reproducibility. 6 These important limitations make liver biopsy a suboptimal screening method for NAFLD-or for monitoring disease progression and treatment response. 1,[6][7][8] Furthermore, no approved treatment for NAFLD exists to date, and several efforts for the development of drugs that can improve liver fibrosis have failed. 1,[6][7][8] Thus, there is an urgent need for noninvasive tools for diagnosing and staging NAFLD, for identifying patients at high risk for disease progression, and for monitoring treatment response. 6,[9][10][11] Additionally, therapeutic approaches targeting earlier stages of the disease may prove to be more fruitful.
For the diagnosis of NAFLD, the presence of ≥5% of fat in the liver is necessary. Accumulation of liver fat is thus the first and most crucial event for NAFLD development. In this review, we will first briefly present the current tools (non-invasive imaging modalities or blood-based tests) that are available for the quantitative or semiquantitative assessment of fat amount in the liver. Second, we will discuss how increased liver fat accumulation may promote liver inflammation and fibrosis, and we will summarize the evidence showing the value of non-invasive liver fat quantification for predicting histologic changes in NAFLD. Third, we will present the findings linking liver fat with metabolic diseases and its value as independent predictor of their development and progression. Finally, we will discuss the impact of lifestyle interventions, bariatric operations, and medications on reducing liver fat and improving NAFLD.

| METHODS OF DETECTION OF LIVER FAT
Hepatic steatosis can be quantified histologically by classifying liver fat into four grades, which represent the percentage of fatty hepatocytes: Steatosis (S) 0: 0-5%, S1: 5-33%, S2: 33-66%, S3: >66%. 12 However, because of its invasiveness, liver biopsy is not an appropriate tool for the diagnosis of liver steatosis. Non-invasive methods are more suitable for this purpose and can be divided into imaging methods and blood-based tests (see Table 1).

| Imaging methods
To assess hepatic steatosis, different imaging modalities are available, including ultrasound-based techniques, such as abdominal ultrasonography and controlled attenuation parameter (CAP), and magnetic resonance-based techniques such as MRI-estimated proton density fat fraction (MRI-PDFF).

| Ultrasonography
Transabdominal ultrasound is the most commonly used imaging method for the diagnosis of hepatic steatosis, as it is widely available and inexpensive. It is recommended as the first-line diagnosis and/or screening tool for liver fat 23,24 ; however, it can only detect steatosis with >10-20% liver fat content. 25 Taking liver biopsy as a reference, abdominal ultrasound has shown pooled sensitivities and specificities to distinguish moderate-to-severe steatosis from its absence of 85% (80-89%) and 93% (87-97%), respectively. 13 Limitations of the ultrasonography include that it is operator-dependent and that it cannot provide an absolute quantification of liver fat. Apart from establishing the diagnosis of steatosis, ultrasonography has been occasionally used to assess the severity of steatosis. 26 However, it demonstrates low sensitivity especially at detecting mild steatosis, and its accuracy is reduced in patients with obesity and renal disease. [25][26][27] Thus, it is considered a suboptimal method for monitoring disease progression or treatment response.

| Controlled attenuation parameter (CAP)
CAP is a tool which is integrated into the vibration-controlled transient elastography (VCTE) device Fibroscan ® . CAP reflects the attenuation of the ultrasound signal in steatotic liver tissue quantitatively in dB/m and allows a non-invasive quantification of liver fat. 28  with CAP levels as well as with failures in CAP measurement, which are estimated to be about 8%. 29 Further studies are needed for precise differentiation of contiguous degrees of fatty liver. By comparing CAP and liver ultrasound head-to-head, it could be shown that the performance of CAP for detecting and grading liver steatosis was higher than that of liver ultrasound; however, the rate of overestimation was significantly higher for CAP than for ultrasonography (30.5% vs 12.4%; p < 0.05). 30 SmartExam is a recently developed software that allows continuous measurements of CAP during the entire examination and captures roughly 200 CAP values. Preliminary data suggest that continuous CAP has a lower measurement variability than the original method with using the median of 10 measurements. 31

| MRI-PDFF (proton density fat fraction)
MRI-PDFF is considered one of the most accurate methods to detect and quantify liver steatosis. It has the advantage to assess steatosis across the whole liver. In a head-to-head comparison with CAP, MRI-PDFF could show a higher accuracy in identifying all grades of liver steatosis (AUROC 0.99). 15 Furthermore, it has been shown that a reduction in MRI-PDFF values results in histologic improvement in NAFLD, including resolution of NASH, and fibrosis improvement. 32,33 However, because of its high costs and limited availability, MRI-PDFF is used almost exclusively in clinical trials but not in routine clinical practice.

| CT and magnetic resonance spectroscopy (MRS)
CT has been previously used for liver fat quantification by using the Hounsfield scale (HU). 34 Development of conversion equations based on very high linear correlations allows the conversion of the HU measurements to PDFF. 34 However, because of the risk of ionized radiation and the low accuracy of the method in mild steatosis, CT is considered not appropriate for diagnosing and monitoring liver fat.
MRS was the first method developed for liver fat quantification based on MRI. MRS has a sensitivity of 73-89% and specificity of 92-96% for accurately detecting different cut-off percentages of liver fat. 35 1 H-MRS is considered as the most accurate method to detect and quantify non-invasively liver steatosis. 36 Main disadvantage of the method is the limited reproducibility, because it demands focusing on single voxels instead of volumetric assessments followed in MRI.

| Blood-based tests
There are also various blood-based tests for the detection of liver fat, including fatty liver index (FLI), 16 SteatoTest™, 17 hepatic steatosis index (HSI), 19 index of NASH (ION), 20 NAFLD liver fat score (NAFLD-LFS), 21 and lipid accumulation product (LAP). 22 The features and performances of these tests are summarized in Table 1 In a between-tests comparison, MRI-PDFF outperformed CAP and all blood-based tests, whereas the performance of CAP was not significantly superior to the blood based-tests. 48 In another study, FLI score ≥60 demonstrated a sensitivity of 60% and specificity of 80% and NAFLD FLS ≥ À0.640 a sensitivity of 68% and specificity of 78% to detect steatosis diagnosed with MRI-PDFF, indicating that both tests are suboptimal for diagnosing steatosis. 49 The main limitation of the available blood-based tests is that they cannot provide an accurate assessment of liver fat % and consequently cannot be used for monitoring disease progression. Furthermore, they include gray zones, where steatosis can neither be confirmed nor be excluded. Moreover, they did not find their way into routine clinical practice as they do not add more information to the existing clinical standard with clinical, laboratory, and imaging examinations in suspected fatty liver.

| LIVER FAT AS RISK FACTOR FOR NAFLD PROGRESSION
3.1 | Pathophysiological mechanisms linking the amount of hepatic fat with NAFLD progression ( Figure 1) Liver fat is stored in the form of triacylglycerols (TGs) in patients with NAFLD. Around 60% of the hepatic TGs were derive from nonesterified free fatty acids (NEFAs) from adipose tissue after lipolysis, 25% from de novo hepatic lipogenesis by using 2-carbon precursors from glucose, fructose, and amino acids, and 15% from dietary fat that escapes storage in adipose tissue by spill over to NEFA pool or chylomicron remnant formation and uptake in the liver. 50 High caloric intake increases both the dietary fat available for uptake by the liver as well as the amount of carbohydrates that can be converted to fat through de novo lipogenesis. 1,7,8 Moreover, as body weight, body fat, and intracellular fat in muscle and adipose tissue increases, hepatic and peripheral insulin resistance develops. 1 F I G U R E 1 Role of liver fat accumulation in the development and progression of NAFLD and metabolic diseases. FFA in the liver derive from lipolysis of adipose tissue, dietary intake of lipids, and de novo lipogenesis (DNL) from carbohydrates. Increased caloric intake does not only directly provide the liver with higher amounts of lipids and carbohydrates, but it results in the long-term in obesity and insulin resistance (IR). IR in adipose tissue stimulates lipolysis and attenuates lipid uptake, whereas IR in muscle reduces glucose uptake, thus redirecting lipids and glucose to the liver. Furthermore, hepatic insulin resistance reduces glycogen synthesis and increases gluconeogenesis thus promoting hyperglycemia. FFAs in the liver either accumulate in the form of triglycerides or they return to circulation through VLDL. Increased secretion of VLDL and small-dense LDL may contribute to atherosclerosis in NAFLD. When the capacity of the liver for formation of triglycerides or secretion of VLDL is exceeded, toxic lipids accumulate in the hepatocytes promoting ER and oxidative stress as well as mitochondrial dysfunction. Apoptotic or necroptotic mechanisms are subsequently activated that result not only in hepatocyte damage or death but also in the release of pro-inflammatory and profibrotic signals. These signals will recruit macrophages from the periphery or activate the resident macrophages (Kupffer cells) and the fibrogenic hepatic stellate cells, thus leading to liver inflammation and fibrosis. Additionally, these signals (either damage associated molecular patterns, DAMPs, or hepatokines) are capable of inducing tissue-specific (e.g., in adipose tissue, pancreas, cardiovascular system) changes or of promoting chronic systemic inflammation that may aggravate insulin resistance, hyperglycemia, and hypertension. Gut dysbiosis as well as increased vasoconstriction observed in NAFLD may further promote arterial hypertension. Importantly, NAFLD is not always associated with elevated cardiovascular risk. Genetically driven NAFLD because of certain polymorphisms may even reduce cardiovascular risk by altering lipid metabolism (created with BioRender.com).
Important evidence supporting the causal relationship of liver fat accumulation with NAFLD progression is provided by genetic studies.
Specifically, the presence of several variants in certain genes, such as the rs738409 C > G single nucleotide polymorphism of PNPLA3, the TM6SF2 E167K variant, and the GCKR rs780094, has been associated with increased liver fat accumulation because of repression of lipase activity, 57 impairment of VLDL secretion, 58 and increased glucose uptake and de novo lipogenesis, respectively. 59 All these variants are associated at the same time with a profound increase of the risk for development of NASH and liver fibrosis as well as with liver-related events and all-cause mortality. 60-63

| Liver fat amount as marker of NAFLD status and progression in clinical studies
Because of accumulation of liver fat is the first step in a lengthy process that may lead to liver inflammation and fibrosis, it is reasonable to ask whether quantifying liver fat may serve as a prognostic marker of risk for development of NASH or liver fibrosis as well as whether treatments that aim to reduce liver fat can prevent disease progression. Specifically, identifying patients at high risk for NAFLD progression is extremely important because of the very high prevalence of the disease that makes general screening of the population and planning of regular follow ups in all patients with NAFLD challenging.
Additionally, no approved treatment for NAFLD exists to date, and most efforts that aimed to improve advanced liver fibrosis because of NAFLD have failed so far. This indicates that probably interventions at an earlier stage of NAFLD and especially in high-risk populations for developing advanced liver fibrosis in the future may be needed.
Furthermore, in contrast to assessment of liver inflammation, the accurate quantification of liver fat is possible with non-invasive imaging modalities, which supports the use of fat amount as a marker of disease state or treatment response; if indeed, it is strongly related to disease progression.

| Liver fat amount as marker of biopsy-proven steatosis, inflammation, and fibrosis
Several clinical studies have demonstrated so far that changes in liver fat content assessed by MRI-PDFF correlate strongly with histologic changes of NAFLD. Specifically, a decline or increase of liver fat in MRI-PDFF of approximately 5-6% identified an improvement or worsening in steatosis grade in liver histology with 90% specificity and almost 60% sensitivity. 64 In another study, the patients with improvement in steatosis grade in liver histology had a mean reduction of liver fat in MRI-PDFF of approximately 20% compared to 0.8% reduction in patients without improvement of steatosis grade. 65 Similar findings have been reported also in children with NAFLD, where improvement or worsening of steatosis grade was detected with 90% specificity when fat percent in MRI PDFF was reduced by 11% or increased by 5.5% from the baseline, respectively. 66 Importantly, apart from liver steatosis grade, changes in liver fat content correlate also strongly with alterations in NAFLD activity score and fibrosis stage in histology. Specifically, a meta-analysis including seven studies showed that reduction of more than 30% in liver fat was associated with 7 times higher probability for histologic response, defined as a 2-point improvement in NAFLD activity score with at least 1-point improvement in lobular inflammation or ballooning and 5.45 times higher probability for NASH resolution. 32 In another study, more than 30% decline in liver fat assessed by MRI-PDFF was associated in 40% of the cases with fibrosis regression, 25% with no changes and 13% with fibrosis progression. The adjusted OR for fibrosis regression by more than 30% reduction in liver fat was 6.46. 33  fibrosis progression compared to subjects in the lower liver fat group (38.1% vs 11.8%). After adjusting for age, sex, ethnicity, and BMI, the higher liver fat group had a 6.7 times higher risk of fibrosis progression, suggesting that indeed higher liver fat content can predict progression of liver fibrosis. 69 Similarly, liver fat was positively associated with overall mortality in another study including 129 patients with biopsy-proven NAFLD that were followed prospectively for more than 20 years. 70 In contrast, several cross-sectional as well as longitudinal studies that assessed liver fat either histologically or with CAP reported that fat amount was either not associated with liver-related events and mortality or was even lower in patients with HCC, decompensated liver cirrhosis, or portal hypertension. [71][72][73][74][75] This paradox is explained by the fact that the importance of liver fat for NAFLD progression may vary between the different stages of the disease.
Initially, liver fat is an important contributor for development of hepatic inflammation and activation of fibrotic mechanisms. As disease progresses though to advanced fibrosis and cirrhosis, liver fat is reduced. This phenomenon has been described as "burned-out" NASH, and the mechanisms leading to it remain still unclear. It is suspected that an increase in adiponectin, which downregulates fatty acid synthesis and increases β-oxidation may lead to a reduction of liver fat in later stages of NAFLD. 76 However, robust evidence that can explain this phenomenon is currently lacking. Nevertheless, this observation indicates that liver fat amount and its changes can be used as marker of NAFLD status and treatment response in the early or middle stages of the disease and not in advanced fibrosis or cirrhosis.

| LIVER FAT AMOUNT AS RISK FACTOR OF CARDIOMETABOLIC DISEASES
Several studies have suggested that not only the presence of liver fat but also the amount of liver fat is associated with the development and progression of metabolic diseases and their complications.

| Diabetes
Evidence from studies investigating NAFLD pathophysiology support the presence of an association between liver fat and diabetes. relative risk ratio to be in the impaired fasting glucose (IFG) group, in the impaired glucose tolerance group (IGT), in the combined IFG + IGT group, or to have undiagnosed T2DM, respectively. 95 In a large meta-analysis including 19 observational studies with almost 300,000 individuals, the presence of NAFLD was associated with 1.8-fold higher risk of incident diabetes, which was further increased up to 2.6-fold in patients with more severe steatosis according to ultrasonography. 96 Similarly, an FLI ≥ 60 was associated with twofold higher risk of prediabetes and ninefold to tenfold higher risk for T2DM in a population of individuals with overweight/obesity. 97 Nasr et al.
showed that each steatosis grade increase in liver biopsy was associated with a HR of 1.6 for development of T2DM after adjusting for age, BMI, and fibrosis stage. 70 People with grade 3 steatosis demonstrated additionally higher mortality risk. Importantly, the elevated risk for development of diabetes was decreased in subjects in whom the liver fat amount was reduced in subsequent biopsies. 70 In another recent study, liver fat amount assessed with MRI-PDFF (and not fibrosis markers) was associated with hepatic insulin sensitivity among patients with T2DM and obesity. 98 An important question though is still whether the association between liver fat and development of diabetes is a causal one or whether liver fat is simply reflecting the overall metabolic health. In a recently published study that used over Specifically, the prevalence of hypertriglyceridemia or dyslipidemia (high LDL-C with low HDL-C) is estimated to be approximately 70% in patients with NAFLD. 103 Lipoprotein subfraction analyses suggested that NAFLD is characterized by high ratios of total cholesterol and triglyceride to HDL-cholesterol, by high ratio of apolipoprotein B to apolipoprotein A1, and by large VLDL particle size and decreased LDL and HDL particle size. [104][105][106] Importantly, these changes are associated with increased risk of atherogenesis; they are observed regardless of BMI and they are mainly driven by higher liver fat content and not by the presence of liver inflammation. 105 Finally, the relation of liver fat with hyperlipidemia/dyslipidemia has been also observed in children and adolescents. 107 Specifically, higher liver fat, even within the normal range and after adjusting for BMI, was associated with higher insulin resistance, total cholesterol, and triglycerides. 107 Similarly, liver fat was independently associated with triglycerides and insulin resistance in adolescents with obesity. 108

| Arterial hypertension
Multiple mechanisms have been suggested to be involved in the promotion of hypertension by NAFLD. These include the induction of vs ≥ 60 = 1 vs 1.83 vs 2.09, respectively). 111 In another study that included 1051 subjects that were followed for 6.2 years, one SD increase of liver fat assessed by liver computed tomography was associated with 42% increased odds of incident hypertension. 114 This increased risk was maintained after adjusting for different factors including visceral adipose tissue mass and BMI. 114 Thus, not only the presence of NAFLD but also the amount of liver fat is related to the risk of arterial hypertension. Nevertheless, it is important here to mention that studies that have evaluated prospectively and in large cohorts the relationship between liver fat amount assessed by MRI-PDFF or liver histology and incident arterial hypertension are currently lacking.

| Cardiovascular risk
Because there is a close pathophysiologic and epidemiologic link between components of metabolic syndrome and NAFLD, multiple studies have also assessed whether NAFLD increases cardiovascular risk. A recent meta-analysis of 36 longitudinal studies that included data on almost 6 million middle-aged individuals and almost 100 thousand cases of fatal and non-fatal CVD with a median follow up of 6.5 years have shown that NAFLD was associated with a 45% increased risk of CVD events after adjustment for multiple factors, such as BMI, age, sex, adiposity measures, hypertension, dyslipidemia, and pre-existing diabetes. This risk was especially high for patients with higher fibrosis stage. 115 Other studies have suggested that the ratios of aspartate/alanine transaminase 116 representing a marker of liver inflammation or FLI score, representing liver fat content, are predictors of cardiovascular events. 117,118 In this context, a large prospective analysis based on UK Biobank has reported that FLI between 30 and 59 is associated with 16% higher risk and FLI ≥ 60 with 25% increased risk for major cardiovascular events at a median follow-up of 11.62 years after adjusting for multiple factors including transaminases and presence of diabetes, hypertension, and statin therapy. 117 FLI seems also to have an independent prognostic value for cardiovascular events in newly diagnosed, treatment-naïve hypertensive patients. 119 Furthermore, in an analysis including more than 5 million young adults aged 20 to 39 years, FLI between 30 and 59 was associated with 28% increased risk of myocardial infarction and 18% of stroke, whereas FLI above 60 was associated with 73% and 41% increased risk, respectively. 120 Apart from the association of liver fat content with components of the metabolic syndrome, several studies have linked liver fat with other surrogate markers of cardiovascular health. Specifically, liver fat has been associated with the calcification in the thoracic aorta and celiac trunk, 121 with the descending and infrarenal aortic diameter, 122 with plaque presence, 122 as well as with the echogenicity and thickness of carotid intima-media. [122][123][124] Additionally, liver fat assessed by FLI has been associated with 10-year Framingham risk score beyond other cardiovascular risk factors. 125,126 Moreover, larger amount of liver fat is correlated with larger volumes of epicardial fat and coronary artery calcification. 127 Additionally, in another study, a decrease in liver fat was associated with a reduction in carotid intima-media progression. 122

| Dissociation between fatty liver and cardiometabolic diseases
Although the majority of epidemiologic and experimental evidence supports the presence of a detrimental relationship between liver fat and cardiometabolic diseases, there is a significant heterogeneity in the findings as well as reports for inverse associations, most probably reflecting the complex pathophysiology of NAFLD. 80,128 There are many reasons explaining the discrepancies between studies. First of all, quantification of liver fat relies primarily on triglyceride concentra-

tions (in MRI and MRS) or percentage of cells containing intracellular
lipid droplets (histology). 67,129 However, it is now accepted that triglyceride accumulation in the liver acts most probably protectively and not detrimentally. 8,80,128,130 Specifically, it may protect the liver from increased availability of fatty acyl-CoAs and formation of toxic lipids (lipotoxicity) that will stimulate inflammatory processes both locally (in the liver) and systemically. 128 Furthermore, it seems that not only the amount but also the type of accumulated lipid species plays a role in liver inflammation, insulin resistance, and cardiometabolic risk. For example mono-or poly-unsaturated fatty acids act beneficially by reducing oxidative stress and inflammation, whereas saturated fatty acids, lysophosphatidylcholines, and ceramides act detrimentally by promoting cellular damage. 131 Diacylglycerols (DAGs) and ceramides affect also insulin signaling leading to increased glucose production. According to other studies, not the abundance but the compartmentation especially of diacylglycerols in the membrane is an important contributing factor to hepatic insulin resistance. 117 Similarly, not only the concentrations of lipid droplets but their protein and lipid composition, their intracellular localization, and their distribution-zonation in the liver affect their functional properties. 132 Consequently, it may not be triglyceride accumulation per se, but rather the exhaustion of "detoxification" and fat storage capacities in the liver that contributes to inflammation, insulin resistance, and elevated cardiovascular risk. In that case, liver fat assessment serves only as a proxy marker of the on-going pathophysiologic processes. This may explain why many but not all patients with NAFLD demonstrate insulin resistance and increased cardiovascular risk. only in obese and not in normal-weight populations. 136 Moreover, the polymorphism was not causally associated to ischemic heart disease in a mendelian randomization study 137 or it was even linked to lower risk for coronary artery disease in another large genetic study. 138 These heterogeneous findings have been attributed to different pathophysiologic mechanisms, such as to less de novo lipogenesis and ceramide accumulation and higher concentrations of polyunsaturated triglycerides in people with this polymorphism. 139,140 Similarly, TM6SF2 protein is involved in triglyceride and lipoprotein secretion.
The rs58542926 TM6SF2 C > T variant is associated with fatty liver because of increased intracellular lipid retention as well as with insulin resistance and diabetes. 136,138,141 However, the increased retention of the lipids in the liver results in lower circulating total cholesterol levels and reduced risk for cardiovascular complications. 138,142 Finally, the rs1260326 of glucokinase regulator (GCKR) leads to increased glucose uptake in the liver and consequently to increased hepatic de novo lipogenesis. However, this polymorphism is associated with lower insulin resistance and reduced risk of type 2 diabetes, especially in European and Asian populations. 143 Another example showing the dissociation between liver fat and cardiometabolic risk is the burned-out NASH. Specifically, as NAFLD progresses to its late stages of advanced fibrosis or cirrhosis, liver fat content decreases. However, these patients with advanced fibrosis or cirrhosis seem to demonstrate the highest cardiovascular risk. 76,115 This increased risk may though reflect the accumulated negative impact of chronic long-term exposure of the cardiovascular system to hepatic lipotoxicity and inflammation. Liver fat quantification in these advanced stages of NAFLD will thus not capture disease temporal dynamics and may not serve as a reliable proxy marker linking fatty liver with cardiovascular risk.
Apart from genetically related NAFLD and cases of advanced liver fibrosis/cirrhosis, significant variation in the association between NAFLD and cardiometabolic state further exists. This has been attributed to different mechanisms involved in NAFLD development. Specifically, some recent approaches focus on dissecting fatty liver from visceral obesity-associated insulin resistance. 80 In a recent analysis, fetuin-A and adiponectin levels have been suggested to serve as main distinguishers between these two conditions. 80 In other approaches, anthropometric and routine biochemical parameters have been used to identify clusters among patients with NAFLD that differ in cardiometabolic risk. [144][145][146] Sex, age, obesity, dyslipidemia, and insulin resistance seem to be the most important factors for clustering, but it still remains unclear whether the parameters themselves and not liver phenotype are the main drivers of altered cardiovascular risk in these patients.

| Lifestyle modification
The cornerstone of NAFLD therapy is lifestyle modification with weight loss and enhanced physical activity. A biopsy-based randomized controlled clinical trial over 52 weeks has shown that calorie reduction and physical activity improved steatosis in 65% of patients after 1 year with a weight loss of 5% to 7%. 147 A weight loss of 7% to 9%, resulted in NASH resolution in 64%, and a weight loss of ≥10% led to a regression of fibrosis by at least one stage in 45% and stabilization of fibrosis in the remaining 55%. 147 Clinically significant weight loss usually requires a reduction of 500-1000 kcal/d from the baseline or a hypocaloric diet with a target of 1200 kcal/d for women and 1400-1500 kcal/d for men. Several hypocaloric diets may be appropriate for weight loss in subjects with liver fat and/or NAFLD. 148 The Mediterranean diet is the best studied diet in NAFLD. In contrast to the Western diet, which is rich in highly processed foods with a high content of saturated fatty acids and carbohydrates, the Mediterranean diet is rich in dietary fibers, monounsaturated and omega-3 fatty acids, and phytosterols. 149 It is thought to reduce the risk and progression of NAFLD through an antioxidant and anti-inflammatory effect. Even in the absence of weight loss, a Mediterranean diet reduced hepatic steatosis compared with a low-fat, high-carbohydrate diet. 150 In contrast to the Mediterranean diet, other hypocaloric diets, such as low-carbohydrate and high-protein diets, meal replacement protocols, or intermittent fasting, are not specifically recommended in NAFLD guidelines, as sufficient data on the effects on histologic NAFLD/NASH endpoints are still lacking. 40 The beneficial effect of a hypocaloric diet on liver steatosis and NAFLD may be enhanced by physical activity. Furthermore, it may improve NAFLD independently from achieving weight loss by reducing hepatic fat content and leading to a reduction of lipolysis in adipocytes, free fatty acid delivery to the liver, hepatic de novo lipogenesis, and an improvement in peripheral insulin sensitivity. 151,152 A systematic review and meta-analysis demonstrated that predominantly aerobic exercise resulted in a significant reduction in liver fat content even in the absence of dietary interventions. 153 This has been challenged in a recent study which aimed to assess whether alternate day fasting and exercise alone or in combination are more efficient at reducing intrahepatic triglyceride content. Exercise alone provided rather modest reduction in liver fat (À1.3%), compared to alternateday fasting alone (À2.25%) or to their combination (À5.48%). 154 Nevertheless, it is recommended to perform 150-300 min of moderateintensity exercise or 75-150 min of high-intensity exercise per week. 152 However, lifestyle modifications aiming at weight loss remain challenging in clinical practice. Data from the noninterventional FLAG registry could show that less than 50% of patients with a mean BMI of 30 kg/m 2 who received lifestyle advice achieved any weight loss after 12 months, and only 17.1% achieved more than 5%. 155

| Bariatric procedures
In patients with grade III obesity (BMI ≥ 40 kg/m 2 ) or grade II obesity (BMI ≥ 35 kg/m 2 ) with metabolic comorbidities where lifestyle modifications fail, bariatric surgery should be considered. 156 The most commonly performed procedures are laparoscopic Roux-Y gastric bypass surgery and laparoscopic sleeve gastrectomy. 156  respectively. 160 To support weight reduction and improvement of steatosis in the short-term, endoscopic bariatric therapies seem promising; however, studies on the long-term effect and safety are currently lacking.

| Pharmacologic intervention
Currently, there are no approved pharmacologic interventions for the reduction of liver fat and/or the treatment of NAFLD. However, numerous drug therapy approaches are in advanced phases of clinical trials. 161 A selection of current phase III trials can be found in Table 2. Consequently, these medications are also tested regarding their data on compensated liver cirrhosis and primary as well as key secondary endpoints were measured non-invasively. If the latter will be accepted by regulatory authorities for future phase III approval studies, performing of NASH clinical trials will be greatly simplified, as liver biopsy will no longer be needed.

| Aramchol
Aramchol, a partial inhibitor of hepatic stearoyl-CoA desaturase (SCD1), improved steatohepatitis and fibrosis in rodents and reduced steatosis in an early clinical trial. It has been tested in a phase IIb trial over 52 weeks including 247 patients with NASH. Aramchol 600 mg/d demonstrated a placebo-corrected decrease in liver triglycerides measured by MR spectroscopy of À3.1 (95% CI À6.4 to 0.2, p = 0.066); however, it did not meet the prespecified significance level. 193 Nevertheless, the observed safety and changes in liver histology and enzymes provided a rationale for evaluating aramchol in an ongoing phase 3 program.