Non-alcoholic fatty liver disease: causes, diagnosis, cardiometabolic consequences, and treatment strategies

Review

Non-alcoholic fatty liver disease: causes, diagnosis, cardiometabolic consequences, and treatment strategies Norbert Stefan, Hans-Ulrich Häring, Kenneth Cusi

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing worldwide. In some patients with NAFLD, isolated steatosis can progress to advanced stages with non-alcoholic steatohepatitis (NASH) and fibrosis, increasing the risk of cirrhosis and hepatocellular carcinoma. Furthermore, NAFLD is believed to be involved in the pathogenesis of common disorders such as type 2 diabetes and cardiovascular disease. In this Review, we highlight novel concepts related to diagnosis, risk prediction, and treatment of NAFLD. First, because NAFLD is a heterogeneous disease, the advanced stages of which seem to be strongly affected by comorbidities such as insulin resistance and type 2 diabetes, early use of reliable, non-invasive diagnostic tools is needed, particularly in patients with insulin resistance or diabetes, to allow the identification of patients at different disease stages. Second, although the strongest genetic risk alleles for NAFLD (ie, the 148Met allele in PNPLA3 and the 167Lys allele in TM6SF2) are associated with increased liver fat content and progression to NASH and cirrhosis, these alleles are also unexpectedly associated with an apparent protection from cardiovascular disease. If consistent across diverse populations, this discordance in NAFLDrelated risk prediction between hepatic and extrahepatic disease might need to be accounted for in the management of NAFLD. Third, drug treatments assessed in NAFLD seem to differ with respect to cardiometabolic and antifibrotic efficacy, suggesting the need to better identify and tailor the most appropriate treatment approach, or to use a combination of approaches. These emerging concepts could contribute to the development of a multidisciplinary approach for endocrinologists and hepatologists working together in the management of NAFLD.

Introduction The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing worldwide, and 25% of the global adult population is potentially affected by the disease.1 A total of 3% to 10% of all children and about 34% of children who are obese in developed countries are thought to have NAFLD.2,3 The increasing prevalence of NAFLD is accompanying the increasing prevalence of other non-communicable diseases, including type 2 diabetes, cardiovascular disease, obesity-associated and type 2 diabetes-associated cancer, and advanced liver diseases such as hepatic cirrhosis and hepatic cancer.4−7 The increasing prevalence of these diseases is related to unhealthy lifestyles, particularly unhealthy diet, which drives the increase in cardiometabolic diseases, cancers, and NAFLD.8−11 Cardiovascular disease is the leading cause of death in people with NAFLD.12,13 However, because people with NAFLD are often obese (or have features of poor metabolic health even at normal weight) and can have impaired glucose and lipid metabolism, insulin resistance, prediabetes, or type 2 diabetes,14−22 the extent to which specifically the prevention and treatment of NAFLD could reduce morbidity and mortality in these individuals remains unclear. Importantly, NAFLD is a heterogeneous disease, and understanding the extent to which the liver phenotype contributes to cardiometabolic risk in NAFLD is crucial to provide clarity in this regard. NAFLD can be categorised histologically into non-alcoholic fatty liver and nonalcoholic steatohepatitis (NASH). Non-alcoholic fatty liver is defined as the presence of at least 5% hepatic steatosis without evidence of hepatocellular injury in the form of hepatocyte ballooning. NASH is defined as the presence of a least 5% hepatic steatosis and inflammation with

hepatocyte injury (eg, ballooning), with or without fibrosis. Because from an epidemiological perspective the increased cardiometabolic risk in NAFLD seems to depend strongly on the presence of advanced stages of NAFLD, such as NASH with moderate-to-advanced fibrosis,12,13 improved implementation of existing, and further development of novel, non-invasive tools to diagnose these different stages of the disease, is necessary. Furthermore, the extent to which the prediction of hepatic and extrahepatic risk differs in NAFLD, and the mechanism explaining this phen­omenon, needs to be investigated. Finally, accumulating evidence from the most recent pharma­ cological clinical trials in patients with NAFLD suggests that each drug tested will have a broad spectrum of effects with respect to their antiinflammatory, antifibrotic, and cardiometabolic potential, making tailored and combin­ation therapeutic approaches the logical path in future management.

Lancet Diabetes Endocrinol 2018 Published Online August 30, 2018 http://dx.doi.org/10.1016/ S2213-8587(18)30154-2 Department of Internal Medicine IV, University Hospital Tübingen, Tübingen, Germany (Prof N Stefan MD, Prof H-U Häring MD); Institute of Diabetes Research and Metabolic Diseases, Helmholtz Centre Munich, University of Tübingen, Tübingen, Germany (Prof N Stefan, Prof H-U Häring); German Centre for Diabetes Research, Tübingen, Germany (Prof N Stefan, Prof H-U Häring); Division of Endocrinology, Diabetes and Metabolism, University of Florida, Gainesville, FL, USA (Prof K Cusi MD); and Division of Endocrinology, Malcom Randall Veterans Administration, Medical Center, Gainesville, FL, USA (Prof K Cusi) Correspondence to: Prof Norbert Stefan, Department of Internal Medicine IV, University Hospital Tübingen, Tübingen 72076, Germany [email protected]

Unhealthy lifestyle and NAFLD Which parameters of an unhealthy lifestyle are the most important risk factors driving the epidemic of NAFLD? Overnutrition and sedentarism often result in obesity and hepatic steatosis; however, these factors might not necessarily result in hepatocyte necrosis, inflammation, and fibrosis. Furthermore, a subset individuals with obesity (about 25–30%) might have metabolically healthy obesity.23 Key lifestyle parameters, such as an increased intake of glucose, fructose, and saturated fat, induce hepatic de-novo lipogenesis, subclinical inflammation in adipose tissue and liver, and insulin resistance in adipose tissue, the liver, and skeletal muscle. These lifestyle parameters are also accompanied by an increased risk of type 2 diabetes, in which β-cell dysfunction-mediated

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Flour

both aerobic and resistance exercise reduce liver fat content by similar amounts. However, the beneficial effects of exercise seem to depend substantially on weight loss,28 although in short-term studies steatosis improved without a substantial decrease in bodyweight.29

Soda

Lifestyle-independent causes of NAFLD Unhealthy lifestyle Glucose, fructose, saturated fat, and positive energy balance Ageing

Visceral obesity and lipodystrophy-like phenotype

Fatty acids, ceramides, cytokines, and dysregulated adipokines

De-novo lipogenesis, triglyceride synthesis

Diabetes Insulin Insuliin

4.5 4.5 M

Inflammation, fibrosis Insulin resistance

Gut G t dysbiosis dysbiosis bi i

Glucose

Genetics: PNPLA3, TM6SF2, MBOAT7, GCKR, and HSD17B13

DAMPs PAMPs SCFA, and ceramides

Figure 1: Causes of NAFLD An unhealthy lifestyle involving a positive energy balance with increased caloric intake, particularly of glucose, fructose, and saturated fat, and sedentary behaviour results in an increased total and visceral fat mass, type 2 diabetes, gut dysbiosis, insulin resistance, and increased hepatic de-novo lipogenesis. Under these conditions, diabetes-associated hyperinsulinaemia and hyperglycaemia exacerbate hepatic de-novo lipogenesis. Gut dysbiosis via increased release of DAMPs and PAMPs, such as lipopolysaccharides, and dysregulated release of SCFAs and ceramides induce hepatic inflammation and fibrosis. This process is amplified by a lipodystrophy-like phenotype with expansion of visceral adipose tissue resulting in increased release of fatty acids and ceramides and a dysregulated pattern of cytokines and adipokines. A genetic predisposition for hepatic lipid accumulation, inflammation, and fibrosis also contributes to the pathogenesis of NAFLD. DAMPs=damage-associated molecular patterns. GCKR=glucokinase regulator. HSD17B13=hydroxysteroid 17-beta dehydrogenase 13. MBOAT7=membrane-bound O-acyltransferase domain-containing 7. NAFLD=non-alcoholic fatty liver disease. PAMPs=pathogen-associated molecular patterns. PNPLA3=patatin-like phospholipase domain-containing protein 3. SCFAs=short-chain fatty acids. TM6SF2=transmembrane-6 superfamily member 2.

hyperglycaemia promotes hepatic de-novo lipogenesis.24,25 Increased proinflammatory cytokine and dysregulated adipokine secretion from adipose tissue contributes to the process of increased lipid storage in the liver.24,25 Increased ceramide signalling also has an important role in the pathogenesis of NAFLD.26,27 Additionally, gut dys­ biosis, induced by an unhealthy diet, is believed to contribute to the accumulation of fat in the liver and the pathogenesis of NASH. In the presence of dysbiosis and a leaky gut, bacteria-derived products can induce adipose tissue inflammation, hepatic steatosis, and hepatic inflammation (figure 1).27 Finally, a sedentary lifestyle can also promote the development of NAFLD. In this respect, increases in 2

Although the epidemic of NAFLD is believed to be driven largely by unhealthy lifestyles, ageing and genetics (which to date have not been shown to be considerably influenced by lifestyle factors) might also have an important role. Numerous inherited and acquired genomic and epigenomic changes have a cumulative effect on the ageing phenotype.30 Additionally, ageassociated decline in skeletal muscle mass and functional deterioration is believed to contribute to the pathogenesis of many non-communicable chronic diseases, including NAFLD.31 Finally, the age-related decline in sex hormones and sex-hormone receptor expression, both in men and women, not only results in a redistribution of adipose tissue from the lower body to the upper body, and from subcutaneous adipose depots to visceral depots, but also in increased ectopic storage of lipids in the liver.16,32,33 Genome-wide association and exome-sequencing studies have provided robust evidence that genetic variability in PNPLA3, TM6SF2, MBOAT7, GCKR, HSD17B13, and other genes is associated with suscep­ tibility to and progression of NAFLD. These genes are strongly involved in regulating the mobilisation of triglycerides from lipid droplets (PNPLA3), secretion of VLDL (TM6SF2), remodelling of hepatic phosphati­dyl­ inositol acyl chain (MBOAT7), de-novo lipogenesis (GCKR), or bioactive lipid and oestradiol signalling (HSD17B13; figure 1).34−36

Diagnosis and staging of NAFLD For the diagnosis of hepatic steatosis, liver ultrasound is easily available in clinical practice, and with an overall sensitivity of 85% and specificity of 94%, this method is fairly accurate.37 However, the ability of liver ultrasound to detect hepatic steatosis at the ¹H-magnetic-resonance spectroscopy (¹H-MRS) cutoff of 5·6%38 is poor, and the optimum sensitivity for liver ultrasound is achieved at a liver fat content of at least 12·5% (sensitivity of about 80–85%).39 Among the proposed indices for the diagnosis of hepatic steatosis, the widely used fatty liver index40 has a low sensitivity (73%) and specificity (74%) compared with ¹H-MRS-diagnosed hepatic steatosis.41 CT-based diagnosis of hepatic steatosis is regarded as quite accurate; however, because of the use of radiation and the fact that this method of diagnosis was outperformed by dual-gradient echo MRI and ¹H-MRS,42 it cannot be recommended for the diagnosis of hepatic steatosis. The controlled attenuation-parameter (CAP) feature, which has been developed to quantify ultrasound attenuation during measurement of liver-stiffness vibration-con­ trolled elastography, has a sensitivity of 69% and a

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specificity of 82% to detect biopsy-diagnosed steatosis.43 CAP (sensitivity 72%, specificity 86%) was outperformed by MRI-based proton-density fat fraction (PDFF; sen­ sitivity 96%, specificity 100%) for the diagnosis of biopsyproven steatosis.44 Once hepatic steatosis is diagnosed by non-invasive methods, the true challenge is to measure disease severity by establishing the presence of steatohepatitis, and especially of moderate-to-severe fibrosis (fibrosis stage of at least F2). Increased plasma alanine amino­ transferase (ALT) concentrations are not accurately predictive45 of disease severity, and even a plasma ALT concentration of two times the upper limit of normal (>70 U/L) predicts NASH with a sensitivity of only 50% and a specificity of 61%.46 Thus, a liver biopsy remains the most specific test to assess the nature and severity of liver diseases. However, the cost and potential complications of biopsy have led to considerable interest in the development of novel, non-invasive methods for use in clinical practice.47 Many non-invasive biomarkers and radiological modalities have been proposed for diagnosis of NASH and fibrosis.47,48 Among other bio­ markers of NASH, circulating concentrations of cytokeratin-18 fragments were proposed to be the most reliable predictors of NASH in patients with NAFLD. However, the clinical utility of cytokeratin 18 is limited by several issues, such as the relatively low power to distinguish NAFLD from NASH or to determine the severity of NASH fibrosis.45,49 Blood lipidomics and several scores based on anthropometrics, transaminases, lipidomic parameters, cytokeratin-18 fragments, adipo­ nectin, and resistin have been proposed;45 however, these methods are not widely used in routine clinical practice. Because the stage of fibrosis is considered to be the most important determinant of mortality from liver disease and cardiovascular disease in patients with NAFLD,12,13 much interest surrounds the establishment of reliable, non-invasive tests for the diagnosis of the stage of fibrosis. In this respect, clinical parameters such as age, BMI, platelet count, liver transaminase concentrations, or the diagnosis of impaired fasting glucose or diabetes are being used to generate noninvasive fibrosis scores.17,47,48 Among these scores, both the NAFLD fibrosis score (NFS)50 and the fibrosis-4 (FIB-4) index51 are based on routine clinical parameters and inexpensive biochemical measurements, and have a fairly high sensitivity and specificity for the diagnosis of advanced fibrosis (fibrosis stages F3–F4), with an overall positive predictive value of about 80% and negative predictive value of about 90%.52,53 The most extensively studied methods for the prediction of liver-related mortality, as per European guidelines,52 are the NFS,50 FIB-4 index,51 enhanced liver fibrosis test,54 and FibroTest55 (known as FibroSure in the USA; table 1). However, these methods might be more useful to guide disease management in hepatology clinics in which patients usually have more advanced disease than in

Parameters and biomarkers

Cutoffs for advanced fibrosis*

Non-invasive biomarker detection methods NAFLD fibrosis score50

Age, BMI, IFG and diabetes, AST-to-ALT ratio, platelets, and albumin

≤−1·455 >0·676

FIB-4 index51

Age, AST, ALT, and platelet

<1·3 >2·67

Enhanced liver fibrosis test54

Age, hyaluronic acid, aminoterminal propeptide of type III collagen, and tissue inhibitor of matrix metalloproteinase 1

≥9·8

FibroTest (FibroSure)55

Total bilirubin, γ-glutamyltransferase, α2-macroglobulin, apolipoprotein A1, and haptoglobin, corrected for age and sex

>0·30 >0·70

Non-invasive imaging VCTE56

Ultrasound-based measurement of low-frequency (50 Hz) elastic shear-wave velocity

>9·6

MRE57

MRI-based imaging of low-frequency mechanical waves

>3·64

*Some indices have two cutoffs (to maximise sensitivity or specificity), which create grey zones of indeterminate values. For example, for the NAFLD fibrosis score, when applying the low cutoff score (−1·455) advanced fibrosis could be excluded with high accuracy (negative predictive value of 93% in the estimation groups, and 88% in the validation groups). By applying the high cutoff score (0·676), the presence of advanced fibrosis could be diagnosed with high accuracy.50 NAFLD=non-alcoholic fatty liver disease. IFG=impaired fasting glucose. AST=aspartate aminotransferase. ALT=alanine aminotransferase. FIB-4=fibrosis-4. VCTE=vibration-controlled transient elastography. MRE=magnetic resonance elastography.

Table 1: Non-invasive estimation of fibrosis

endocrinology or primary care settings, because the sensitivity and specificity of these tests are lower for earlier and more moderate stages of fibrosis than for advanced fibrosis, and they have broad diagnostic grey zones (in which up to a third of patients cannot be classified).17 Vibration-controlled transient elastography (VCTE) and magnetic resonance elastography (MRE) have higher sensitivity and specificity for diagnosis of advanced fibrosis than do the NFS and the FIB-4 index (table 1).47 Notably, VCTE is the best validated and most commonly used type of elastography worldwide.56 MRE is an MRI-based imaging method that involves low-frequency mechanical waves in the liver. MRE is the most expensive technique for non-invasive measurement of fibrosis; however, MRE is better than VCTE for the detection of early fibrosis and of fibrosis stages F3–F4. Furthermore, the higher the BMI of the patient, the better the performance of MRE compared with VCTE.57 Other methods such as multiparametric MRI, which uses T1, T2*, and PDFF to quantify iron deposition, hepatic fibroinflammation, and hepatic steatosis, also provides information about liver anatomy58 and might in the future also prove to be effective for the diagnosis of advanced stages of liver disease.

Heterogeneity and dynamics of NAFLD In their 2016 global meta-analysis, Younossi and colleagues1 reported that the pooled overall prevalence of NASH in patients with NAFLD amounted to 59·10%. Although this proportion is very high, the investigators1 also found that the prevalence of NASH in NAFLD strongly depended on a clinically based indication for biopsy. For example, in North America the prevalence of

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NASH in patients with NAFLD was 60·64% in patients with an indication for a biopsy, whereas the prevalence was only 29·85% in patients without such an indication. From these data, Younossi and colleagues1 estimated that the overall prevalence of NASH is between 1·50% and 6·45%. In a meta-analysis of 11 cohort studies, Singh and coworkers59 found that among patients with biopsy-proven NAFLD, 33·6% had fibrosis progression, 43·1% had stable fibrosis, and 22·3% had improvement of fibrosis. Although most patients with NAFLD showed slow progression in their fibrosis stage (usually only by one or two stages), 17·2% of patients with non-alcoholic fatty liver and baseline stage F0 fibrosis, and 18·2% of patients with NASH and baseline stage F0 fibrosis, developed progressive fibrosis.59 These findings suggest that, even in patients with non-alcoholic fatty liver, or as often referred to, isolated steatosis, a subgroup of patients can rapidly progress to an advanced stage of fibrosis. In a study by McPherson and colleagues,60 a similar pro­ portion of patients with non-alcoholic fatty liver (37%) and NASH (43%) also had progression of fibrosis, and 44% of the patients with non-alcoholic fatty liver progressed to NASH during a mean follow-up of 6·6 years. When the authors of both studies59,60 investigated parameters predicting the progression of fibrosis, they found that diabetes60 and hypertension59 were the strongest predictive clinical parameters. The relation between hypertension and increased progression of fibrosis might predominantly be the result of insulin resistance and subclinical inflammation, which are often seen in individuals with hypertension. Diabetes is quite prevalent in patients with NAFLD (23%), and more so in those with NASH (47%); diabetes also represents a risk factor for progression to NASH, cirrhosis, and mortality.1 Because the prevalence of NASH is much higher in patients with type 2 diabetes than in the general population and NASH is believed to confer a greatly increased risk of liver fibrosis, hepatocellular carcinoma, and mortality,17,61 NASH is rightly being considered a new complication of type 2 diabetes, and will probably be screened for in the future, in the same way as diabetic retinopathy and nephropathy.

Prediction of hepatic and extrahepatic diseases and mortality in NAFLD Findings from research into the pathophysiology of hepatic and cardiometabolic diseases using different animal models62,63 highlighted the prominent role of NAFLD in the development of these diseases. Findings from several epidemiological studies have suggested that NAFLD is an independent risk factor for incident hepatic diseases, type 2 diabetes, cardiovascular disease, and chronic kidney disease.64 Compared with healthy controls, individuals with NAFLD had a 1·5-times to six times higher risk of cardiovascular disease, cardiovascular disease-related mortality, and diabetes.64 4

In most studies, these associations were independent of parameters commonly used to predict cardiometabolic risk, such as age, sex, BMI, hypertension, and dyslipidaemia, although not independent of estimates of insulin resistance.65 Because insulin resistance is strongly involved in the pathogenesis of cardiovascular disease and type 2 diabetes,66 and is strongly associated with NAFLD,14−22 the possibility that insulin resistance is a major driver of increased risk of cardiometabolic disease in people with NAFLD cannot be excluded. However, the findings that hepatokines that are being secreted from the fatty liver promote cardiometabolic diseases67 and that dysregulated hepatic lipid signalling results in hyperglycaemia and dyslipidaemia62 supports a role of hepatic lipid overload in the pathogenesis of cardio­ metabolic diseases. What are the relations between the different stages of NAFLD, such as simple steatosis, NASH, and NASH with fibrosis, and the incidence of cardiometabolic and severe liver diseases and mortality? Although patients with isolated steatosis can develop NASH and progressive fibrosis, which puts them at an increased risk of morbidity and mortality, only fibrosis, but no other histological liver characteristics, was shown to indepen­ dently predict increased all-cause and disease-specific mortality in patients with NAFLD.12,13,68 In the largest study ever done on liver biopsies, which included 646 patients with biopsy-proven NAFLD with up to 40 years of follow-up, fibrosis (stages F2−F4), but not NASH (according to the NAFLD activity score and the fatty liver inhibition of progression algorithm), strongly and independently predicted increased liver-specific morbidity, and to a lesser extent, increased overall mortality.69 Although liver-related mortality differed between patients with NAFLD (7·9%) and controls (1·4%), cardio­ vascular disease mortality was not different between the two groups (36·9% vs 39·3%). However, in the above-mentioned studies, most patients with borderline or definite NASH had fibrosis stages F1−F4 (eg, 84·9% of individuals in the study by Angulo and colleagues13 and 82·0% in the study by Hagström and colleagues69). Therefore, the group of study participants with NASH and stage F0 fibrosis was often too small to allow definite conclusions about their risk of mortality or liver-related outcomes to be drawn. Because in these studies ballooning grade, portal inflammation grade, and NASH categories also predicted increased mortality and liver-related events in univariate analyses,13 and because stating when in the natural history of NASH fibrosis develops is difficult, NASH should still be considered a condition that puts patients at an increased risk of morbidity and mortality. Furthermore, the findings from Hagström and colleagues’ study69 might suggest that, although cardiovascular disease-related mortality is high in NAFLD, advanced fibrosis in NAFLD can strongly increase liver-related, and perhaps to a lesser extent, cardiovascular disease-related, mortality.

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Precise phenotyping of NAFLD and associated comorbidities and statistical testing of the independent relation between NAFLD and incidence of cardio­ metabolic diseases are necessary to address the link between NAFLD and cardiometabolic risk. So far, data from precise cross-sectional phenotyping studies18 suggest that there is a clear threshold of liver fat content (roughly 6%), above which metabolic changes such as muscle insulin resistance, hypertriglyceridaemia, and low HDL-cholesterol concentrations are already fully established. Further accumulation of liver fat content after this threshold does not seem to be associated with more severe metabolic or histological consequences.18 However, the fact that a positive linear correlation exists between adipose tissue insulin resistance and liver fat content supports the hypothesis that NAFLD is mainly driven by dysfunctional adipose tissue and the related lipotoxic environment.18 Nevertheless, even if in such cardiometabolic outcome studies NAFLD might not be an independent marker of disease incidence, the possibility that NAFLD has a causative role cannot be excluded, because it could be involved in the pathogenesis of these diseases via the promotion of dyslipidaemia, hyper­ glycaemia, insulin resistance, and a dysregulated hepato­ kine pattern.14−22,67,70 From a clinical perspective, in most epidemiological studies NAFLD is associated with incident diabetes and cardiovascular disease, independently of established cardiometabolic risk factors, particularly high BMI and waist circumference.64 However, because increased BMI and waist circumference are only weak estimates of increased total body and visceral fat mass, both of which are strongly associated with NAFLD,14−22 adjustment for better measures of total body and visceral fat mass in those studies may have provided null results for NAFLD. Findings from phenotyping studies with precise measurements of liver fat content, total body fat mass and visceral fat mass, prediabetes, and insulin resistance could help to resolve this issue. In the Tübingen Diabetes Family Study,71 increased liver fat content, as measured by ¹H-MRS, was a stronger determinant of insulin resistance and increased intima-media thickness of the common carotid artery than total body fat mass or visceral fat mass, both measured by MRI.71 In the same study population, liver fat content was more strongly associated with different stages of prediabetes than was visceral fat mass (figure 2).72 Furthermore, Fabbrini and colleagues73 showed that increased ¹H-MRS-derived liver fat content was a stronger determinant of insulin resistance than was total body or visceral fat mass, which was measured by

p=0·03

4 Visceral fat (adjusted; kg)

Cardiometabolic risk in NAFLD

A 3 2 1 0

B 9

p<0·0001

8 Liver fat (adjusted; %)

Thus, the key questions concern whether NAFLD is an independent risk factor for cardiometabolic disease, and whether the diagnosis of NAFLD can improve the risk prediction of cardiometabolic disease in a high-risk population.

7 6 5 4 3 2 1 0

NGT

IFG

IGT

IFG + IGT

Figure 2: Relations of visceral fat mass and liver fat content with different stages of glucose tolerance Relations of adjusted (for age and sex) values of (A) visceral fat mass and (B) liver fat content in individuals with NGT, IFG, IGT, and IFG plus IGT in 330 individuals in the Tübingen Diabetes Family Study. Reproduced from Kantartzis and colleagues72 by permission of Springer Nature. Data are mean values. Error bars show SE of the mean. NGT=normal glucose tolerance. IFG=impaired fasting glucose. IGT=impaired glucose tolerance.

dual-energy x-ray absorptiometry and MRI. The investigators also showed that, for a similar increase in bodyweight and visceral fat mass, decrease in insulin sensitivity was greater in individuals with NAFLD than in those without NAFLD.74 Thus, increased fat mass and visceral obesity might not account for all of the association between NAFLD and increased cardio­ metabolic risk, which has been observed in many studies, and more research is warranted to identify the parameters that could account for this risk.

Genetics of hepatic and extra-hepatic risk in NAFLD Research has allowed substantial progress in under­ standing the role of genetics in the progression of nonalcoholic fatty liver to NASH, fibrosis, and hepato­cellular carcinoma. Using a Mendelian randomisation approach, Lauridsen and colleagues75 showed that fatty liver associated with the 148Met allele in PNPLA3 was not causally linked to ischaemic heart disease.75 Furthermore, in a large exome-wide association study of plasma lipids in more than 300 000 individuals, genetic variants located at the 148Met allele in PNPLA3 and 167Lys allele in TM6SF2 were strongly associated with increased liver fat content and progression to NASH, cirrhosis, and hepatocellular carcinoma, but also with diabetes, lower blood tri­glycerides, lower LDL-cholesterol concentrations, and protection from coronary artery disease.76 By what

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mechanisms might these two genetic variants induce more severe liver disease and type 2 diabetes, but lower blood triglycerides and LDL cholesterol? In a 2017 study, BasuRay and coworkers77 showed that the 148Met variant in PNPLA3 might disrupt ubiquitination and proteasomal degradation of PNPLA3, resulting in the accumulation of PNPLA3−148Met and impaired mobilisation of trigly­ cerides from hepatic lipid droplets. These changes might in turn result in lower blood triglyceride concentrations in individuals with the 148Met allele in PNPLA3 than in those who do not carry the allele, a hypothesis that is supported by data from a study by Liu and colleagues.76 The association between the 148Met allele in PNPLA3 and lower LDL-cholesterol con­ centrations could be explained by a possible role of PNPLA3 in the metabolism of apoB-containing lipo­proteins in the liver;78 however, no evidence directly supports this hypothesis. By contrast, TM6SF2 is thought to be involved in the regulation of cholesterol biosynthesis and triglyceride and apoB secretion.79 Additionally, in a small case-control study by Musso and coworkers80 involving 60 patients with biopsyproven NAFLD who were not obese, did not have diabetes, and were normolipidaemic and 60 matched controls genotyped for the rs58542926 C→T (Glu167Lys) polymorphism in TM6SF2, carriers of the 167Lys allele in TM6SF2 had lower postprandial lipaemia, a less atherogenic lipoprotein, and postprandial cholesterol redistribution from smaller atherogenic lipoprotein subfractions to larger intestinal and hepatic VLDL1 subfractions. Furthermore, postprandial plasma VLDL1 cholesterol response independently predicted the severity of liver histology. Because triglyceride-rich lipoprotein uptake promotes high-fat-induced liver injury,81 and because triglyceride-rich lipoproteins link cholesterol concentration in VLDL subclasses to hepatic cholesterol content, inflammation, and fibrosis,82 Musso and colleagues80 concluded that the 167Lys allele in TM6SF2 might divert toxic cholesterol away from the vessel walls into the liver and adipose tissue, thereby promoting liver injury and adipose dysfunction and protecting from cardiovascular disease. Similar mechanisms could also account for the relation between the 148Met allele in PNPLA3 and a reduced risk of cardiovascular disease. The increased risk of diabetes in carriers of the 167Lys allele in TM6SF2 and the 148Met allele in PNPLA376 might be explained by a lower concentration of LDL cholesterol in the blood. However, because genetic variants with a similarly strong ability to lower LDL-cholesterol concentrations display different effects on incident diabetes,83 other mechanisms of these alleles, other than those related to lowering LDL cholesterol, could induce diabetes. With respect to genetic determinants of NAFLD, Stender and colleagues84 showed that adiposity augments the genetic risk of NAFLD from steatosis to hepatic inflammation to cirrhosis, which supports the hypothesis that genetics might predominantly be a modifier of 6

disease severity.84 However, in the same study,84 the investigators found no evidence of strong interaction between adiposity and the same sequence variants to influence other adiposity-associated traits. These findings support the hypothesis that with a high genetic risk of NAFLD there might be a dissociation between hepatic and extrahepatic complications. The exact role that genetic testing ought to have to diagnose NAFLD remains uncertain, and it is not currently recommended by European52 or US53 clinical practice guidelines. Nevertheless, genetic testing will probably become a more important tool for risk prediction in the future because of the apparent relation between genetics and disease severity.

Cardiometabolic versus antifibrotic treatment of NAFLD Overview

The prediction of cardiometabolic risk and hepatic disease progression might differ, at least for some genetic determinants of NAFLD, but differences are also apparent in the effects of treatment on these outcomes. Guidelines recommend a holistic approach, tackling both resolution of NASH to halt fibrosis progression and mitigation of cardiovascular risk.52,53 Meaningful weight loss would be a good example of such an integrated approach, achieving both goals; but weight loss alone is rarely successful, and pharmacological therapy has often had mixed results. Targeting liver fibrosis through bile acid (farnesoid-X-receptor) pathways by use of obeticholic acid results in an unfavourable cardiometabolic profile,85 whereas treating fibrosis by modulating glucose and lipid metabolism86 or inflammation87 has had modest success. These results have led to a debate as to which approach to follow and the most appropriate outcomes to target for NASH trials,88 and whether the best strategy would be one centred on so-called antifibrotic or cardiometabolic approaches.

Antifibrotic therapies for NAFLD Generally, if approved in clinical practice, the use of treatments intended to prevent adverse liver outcomes should be focused on patients with NASH, and within this group on patients with at least moderate fibrosis (at least stage F2), because such patients have higher cardiovascular and liver-related morbidity and mortality than patients with milder disease.12,13,68,69 Although an ideal inter­vention stage (ie, where the benefit of inter­ vention to reverse NASH-fibrosis will be the greatest) has not been established, these recom­mendations help to define a patient group in which liver disease is likely to progress to meaningful outcomes (ie, cirrhosis or even hepato­ cellular carcinoma) in a substantial number of individuals unless active intervention takes place. In other words, treating the many patients with mild fibrosis (stage F1) could lead to unnecessary treatment and expenses, and in many of these people liver disease

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will not improve with treatment. These assumptions might change in the future as we learn more about disease progression in individuals at the highest risk for liver disease, such as patients with obesity and type 2 diabetes. At the other end of the disease spectrum, a strong case can be made for treating patients with cirrhosis (fibrosis stage F4), especially in the early stages; although, equally, if cirrhosis is reached, it might be too late for therapies to have a beneficial effect. No therapies that primarily target fibrosis in NASH have been approved, but many are under investigation.89−92 From available phase 2b and 3 trials, antifibrotics have had minimal effects on the unfavourable cardiometabolic milieu of NASH. As such, combination therapy might have greater success if underlying metabolic insults, believed to be driving necroinflammation and eventually fibrosis (ie, insulin resistance, lipotoxicity, glucotoxicity, and subclinical inflammation), can be mitigated—a hypothesis that has yet to be fully tested but that underlies the use of cardiometabolic therapies for treatment of NASH.

Cardiometabolic therapies for NAFLD In NAFLD, the liver becomes an indicator of cardio­ metabolic health, a barometer that allows the identification of individuals (both with and without obesity),14,16 who are insulin resistant and in a metabolic state of severe adipose-tissue dysfunction and lipo­ toxicity.19,26,27,93 Patients with prediabetes or type 2 diabetes and NAFLD have the highest cardiometabolic disease risk,13,15 with a liver-imaging ¹H-MRS threshold of at least 5·5% clearly establishing a point at which dyslipidaemia, insulin resistance, and lipotoxicity are fully established to drive steatohepatitis.18 However, from a liver-centric perspective, to avoid overdiagnosis and overtreatment,94 general practitioners should focus on the diagnosis and treatment of patients with NASH who have moderate-tosevere fibrosis. A cardiometabolic intervention for NASH relies on the central hypothesis that weight loss, either alone or combined with the use of drugs developed for the treatment of diabetes, by reversing insulin resistance and hyperglycaemia, will result in decreased cardiometabolic risk while slowing or halting steatohepatitis disease activity, and eventually, fibrosis.15,17,93 Treatment options include lifestyle modification, bariatric surgery, and pharma­ cological treatments (table 2), as reviewed elsewhere.89−92,94 Lifestyle modification should always be the first-line treatment for NASH. Histological improvement in NASH is usually proportional to the amount of weight loss.95 Although a bodyweight loss of about 5% is associated with a reduction in liver fat content of about 30% and an improvement of metabolic abnormalities, a weight loss of about 7–10% might be needed to substantially reduce hepatocyte necrosis and inflammation.29,53,96 But how effective is a large amount of weight loss on the regression of fibrosis? In a well

designed, 48-week randomised controlled trial, lifestyle intervention in patients with biopsy-proven NASH that resulted in a mean weight loss of 9·3% (vs 0·2% in the control group) significantly improved the NAFLD activity score compared with the control group, but fibrosis was not affected (p=0·62).97 Thus, in NASH-fibrosis, pharma­ cological treatment might be necessary in addition to ongoing lifestyle intervention. In another large study assessing a lifestyle intervention, fibrosis improved when weight loss of at least 10% was achieved, but the results were variable, and less than 10% of study participants lost this amount of weight.98 Most bariatric surgery studies, which also included patients with NASH, and in which weight loss is often higher than 30%, have shown improvements in NAFLD activity score, but with less conclusive or mixed results for liver fibrosis.52,53 Notably, after bariatric surgery, patients with NASH still have an increased risk of death compared with bariatric patients without NASH, although patients with NASH still benefit from bariatric surgery in so far as they have a decreased risk of mortality.99 Metformin is an insulin sensitiser, but is not believed to offer unique benefits for steatohepatitis. The glucose-lowering effect of pioglitazone is similar to that of metformin (HbA1c reduction of about 1·0−1·2%), but by contrast with metformin, the thiazolidinediones have been shown to improve insulin sensitivity and liver histology (ie, steatosis, hepatocyte necrosis, and inflammation) in patients both with100,101 and without102,103 diabetes. A fairly short-term (6 month) study by Belfort and colleagues100 established that NASH could be reversed within a relatively short period with pioglitazone. The effect of thiazolidinediones on fibrosis is more modest than their effect on NASH. However, pioglitazone was shown to reduce liver fibrosis and increase adiposetissue insulin sensitivity (highly linked to the pathophysiology of the disease) more substantially in patients with type 2 diabetes than in patients with prediabetes;104 additionally, findings from a meta-analysis of all available randomised trials100−103 showed a benefit of pioglitazone in patients with advanced fibrosis (stages F3–F4).105 These findings lend credence to the hypothesis that amelioration of the metabolic insult might help to mitigate fibrosis progression. Pioglitazone is effective for the prevention of diabetes in people with prediabetes,106 and reduces cardiovascular events in patients with metabolic syndrome or pre­ diabetes with a history of a stroke.107 The drug also improves atherogenic dyslipidaemia, an important cardio­­vascular target.108 Notably, steatosis and insulin resistance, but not steatohepatitis per se, promote atherogenic dyslipidaemia in NAFLD.109 Pioglitazone ameliorates atherogenesis and cardiovascular events in patients with type 2 diabetes,110−114 and was associated with lower mortality compared with other diabetes drugs in a large European multicohort study.115 However, no reduction in cardiovascular mortality was shown

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Cardiometabolic effects

Hepatic effects

Insulin resistance

Major cardiometabolic effects

Cardiovascular disease benefit

Steatosis

NAS

Fibrosis

Lifestyle modification

Moderate decrease

Weight loss, and mild decrease in dyslipidaemia and blood pressure

Yes

Moderate decrease

Moderate decrease

Small decrease or no effect

Bariatric surgery

Substantial decrease

Weight loss, and mild decrease in dyslipidaemia and blood pressure

Yes

Substantial decrease

Substantial decrease

Small decrease

Substantial decrease

Mild decrease in dyslipidaemia and blood pressure

Yes

Substantial decrease

Substantial decrease

Small decrease or no effect

Thiazolidinediones Pioglitazone

Glucagon-like peptide-1 receptor agonists Liraglutide

Small decrease

Weight loss

Yes

Moderate decrease

Moderate decrease

No effect

Exenatide

Small decrease

Weight loss

No

Moderate decrease

NA

NA

Dipeptidyl peptidase-4 inhibitors Sitagliptin

No effect

No effect

No

No effect

NA

NA

Vildagliptin

No effect

No effect

No

Small decrease

NA

NA

Sodium-glucose co-transporter-2 inhibitors Canagliflozin

Small decrease

Weight loss and small decrease in blood pressure

Yes

Small decrease

NA

NA

Empagliflozin

Small decrease

Weight loss and small decrease in blood pressure

Yes

NA

NA

NA

Dapagliflozin

Small decrease

Weight loss and small decrease in blood pressure

Unknown

No effect

NA

NA

Luseogliflozin*

NA

Weight loss and small decrease in blood pressure

Unknown

Small decrease

NA

NA

Ipragliflozin*

NA

Weight loss and small decrease in blood pressure

Unknown

Small decrease

NA

NA

No effect

Small decrease in oxidative stress and potential small decrease in inflammation

No (potentially harmful)

Moderate decrease

Moderate decrease†

No effect

No effect

Small decrease in oxidative stress and potential small decrease in inflammation

Unknown

Small decrease

Small decrease

No effect

Antioxidants Vitamin E Phosphodiesterase inhibitors Pentoxifylline

Data are from randomised controlled trials only. NAS=non-alcoholic steatohepatitis activity score. NA=no data available. *Randomised open-label trials. †No significant effect on NAS in patients with type 2 diabetes.

Table 2: Lifestyle, weight loss, and pharmacological interventions for the treatment of non-alcoholic fatty liver disease89-92,94

with pioglitazone compared with sulfonylureas in patients with type 2 diabetes in a large clinical trial done in Italy.116 Weight gain (less than 2 kg on average), heart failure, bladder cancer, and fractures were not significantly different between treatment groups. For a population at high cardiovascular risk, the metabolic benefits of pioglitazone are frequently overlooked because of fear of long-term weight gain (3−5% of patients),117 oedema in the lower extremities (about 5% of patients), and potential bone loss.118 Notably, weight gain with pioglitazone makes patients more metabolically healthy.100,101 This observation could help to account for the cardiovascular disease reduction associated with pioglitazone use,103,110−114 when compared with the epidemiological evidence for increased cardiovascular disease with obesity from overeating. Heart failure has been reported in patients with undiagnosed preexisting heart disease when associated with thiazolidinedione-induced fluid retention,108 but pioglita­ zone has been shown to improve cardiac function.119 Finally, controversy surrounding pioglitazone and bladder cancer diminished after a 10-year prospective study did not show an association.120 In a meta-analysis of 11 observational cohort studies and seven case-control 8

studies, no association was identified between pioglitazone and bladder cancer and when patients who had ever used the drug were compared with those who had not.121 However, there was a significant association between exposure to pioglitazone for 1–2 years (hazard ratio 1·28, 95% CI 1·08–1·55]) and more than 2 years (1·42, 1·14–1·77) with bladder cancer. On an absolute scale, development of bladder cancer in patients both exposed and not exposed to pioglitazone was lower than 0·3%. The numbers needed to treat for one additional case of bladder cancer ranged from 899 to 6380 (median 2540), whereas the numbers needed to treat for preventing one cardiovascular disease event ranged from four to 256 and for one resolution of NASH ranged from two to 12. In summary, having been incorporated as a treatment option for patients with and without diabetes in several guidelines,52,53 pioglitazone could be a crucial treatment option for patients with NASH, similar to primacy given to metformin in initial treatment of patients with type 2 diabetes—ie, pioglitazone could act as an initial low-cost therapy that provides hepatic and cardiometabolic benefits, with the potential to be combined with future antifibrotic therapies under development.

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Review

Several other glucose-lowering drugs are also under intense investigation for NASH. Dipeptidyl peptidase-4 inhibitors are neutral with respect to cardiovascular outcomes in type 2 diabetes, and are not believed to have a substantial beneficial effect on NAFLD.96 By contrast, the results of the landmark proof-of-concept LEAN trial of the glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide122 suggested the potential for use of this drug class in patients with NASH. Findings from several studies support the ability of GLP-1 receptor agonists to reduce plasma aminotransferases and hepatic steatosis, as reviewed elsewhere,17,89−91 and newer GLP-1 receptor agonists such as semaglutide are being assessed in large clinical trials in obesity and in NASH.123 Liraglutide and semaglutide have been shown to affect cardiometabolic risk and reduce cardiovascular events in patients with type 2 diabetes,124 at least in part due to weight loss, amelioration of hyperglycaemia, and enhanced insulin action. Finally, sodium-glucose co-transporter-2 (SGLT2) inhibitors are being used increasingly frequently in the treatment of type 2 diabetes.125 In animal studies of NASH, SGLT2 inhibitors have shown metabolic and liver histological benefits— these findings are also supported by results of small, uncontrolled clinical trials in which plasma amino­ transferases and steatosis were reduced.96 SGLT2 inhibitors promote weight loss (probably the main mechanism of action in NAFLD) and have been reported to reduce cardiovascular events in patients with type 2 diabetes,124 both of which are attractive properties for patients with NAFLD.

could help to predict the development of hepatic and extrahepatic disease, and perhaps even guide the choice of the pharmacological intervention with emphasis on the metabolic or antifibrotic efficacy of drug treatments, Risk factors Obesity, glucose, fructose, saturated fat, genetics, and diabetes Imaging biomarkers

Lifestyle and pharmacological intervention: >10% weight loss†, healthy diet‡, metabolic and antifibrotic drugs

Imaging biomarkers

Imaging biomarkers

Lifestyle and pharmacological intervention: >10% weight loss†, healthy diet‡, and metabolic drugs

NASH and fibrosis CVD and diabetes Genetics?

Lifestyle Intervention: 5–8% weight loss* and healthy diet

NASH

Genetics?

Fat and fibrosis brro rosis o CVD VD

Fat and fibrosis

Isolated steatosis

CVD Isolated steatosis Genetics?

Conclusions The ongoing worldwide epidemic of NAFLD and the fact that a subgroup of individuals with the disease is at an increased risk of developing advanced stages of liver disease and cardiometabolic disease means that NAFLD is an important priority for health care and research. However, NAFLD is a complex and heterogeneous disease, in which the stage of liver damage and the cardiometabolic risk parameters strongly affect risk prediction, disease progression, and liver-related mor­ bidity and mortality. Thus, a close collaboration between hepatologists and endocrinologists is necessary to provide the best medical support for patients. A multifactorial approach is required, involving a combination of diagnostic tools, stratification of risk prediction of hepatic and cardiometabolic diseases, and tailored treatment of hepatic and cardiometabolic complications. Although in the early stages of NAFLD, during which the prevalence of NASH and advanced fibrosis is low, a weight loss of 5−8% and a healthy diet might be sufficient treatment (figure 3), in more advanced stages of liver disease—associated with genetic risk and the presence of diabetes—intensified lifestyle cological treatment intervention supported by pharma­ might be necessary. In the near future, genetic testing

Cirrhosis and HCC

Fat at and at and fibrosis bbrro ros osis CVD VD VD

Lifetime

Figure 3: Natural history of NAFLD, risk of liver and cardiometabolic diseases, and treatment approaches CVD=cardiovascular disease. HCC=hepatocellular carcinoma. NASH=non-alcoholic steatohepatitis. *Weight loss of 5−8% is recommended for the prevention of cardiometabolic diseases in individuals who are overweight and obese with an increased cardiometabolic risk.23 †Weight loss of more than 10% was associated with a reduced risk of cardiovascular morbidity and mortality among patients who were overweight or obese with type 2 diabetes in the Look AHEAD study,23 and is considered necessary to improve cardiometabolic risk in individuals who are metabolically unhealthy.23 ‡A healthy diet was effective at reducing increased liver fat content and protecting from cardiovascular events and cardiovascular mortality, independent of weight loss.23

Search strategy and selection criteria We searched PubMed for full-text original studies and review articles in English published from Jan 1, 1990, to March 31, 2018, to identify reports about the causes and consequences of non-alcoholic fatty liver disease. The search terms used were “nonalcoholic fatty liver disease”, “nonalcoholic steatohepatitis” and “liver fibrosis”, together with “hepatocellular carcinoma”, “mortality”, “cardiovascular mortality”, “liver-related mortality”, “type 2 diabetes”, “insulin resistance”, “cardiovascular disease”, “prediction”, “prevention”, “lifestyle intervention”, and “treatment”. The reference lists of the identified papers were also used to identify further papers of interest. The final reference list was selected on the basis of relevance to the topic of this Review.

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including combination therapy. Much progress has been made in the scientific understanding of the natural history of NAFLD in the past decade. In the future, personalised risk prediction and individualised treatment could become a reality in the management of this disease. Contributors NS and KC reviewed the scientific literature and wrote the Review. H-UH critically reviewed the initial draft and contributed to the development of the submitted version. Declaration of interests We declare no competing interests. Acknowledgments This work was supported in part by funding from the German Research Foundation (KFO 114 and STE 1096/1−3) and the German Federal Ministry of Education and Research to the German Centre of Diabetes Research. References 1 Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease— meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology 2016; 64: 73−84. 2 Anderson EL, Howe LD, Jones HE, Higgins JPT, Lawlor DA, Fraser A. The prevalence of non-alcoholic fatty liver disease in children and adolescents: a systematic review and meta-analysis. PLoS One 2015; 10: e0140908. 3 Alisi A, Feldstein AE, Villani A, Raponi M, Nobili V. Pediatric nonalcoholic fatty liver disease: a multidisciplinary approach. Nat Rev Gastroenterol Hepatol 2012; 9: 152−61. 4 GBD 2016 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990−2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 2017; 390: 1211−59. 5 GBD 2016 Risk Factors Collaborators. Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks, 1990−2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet 2017; 390: 1345−422. 6 Pearson-Stuttard J, Zhou B, Kontis V, Bentham J, Gunter MJ, Ezzati M. Worldwide burden of cancer attributable to diabetes and high body-mass index: a comparative risk assessment. Lancet Diabetes Endocrinol 2018; 6: e6–15. 7 Younossi ZM, Otgonsuren M, Henry L, et al. Association of nonalcoholic fatty liver disease (NAFLD) with hepatocellular carcinoma (HCC) in the United States from 2004 to 2009. Hepatology 2015; 62: 1723−30. 8 Micha R, Peñalvo JL, Cudhea F, Imamura F, Rehm CD, Mozaffarian D. Association between dietary factors and mortality from heart disease, stroke, and type 2 diabetes in the United States. JAMA 2017; 317: 912−24. 9 Khera AV, Emdin CA, Drake I, et al. Genetic risk, adherence to a healthy lifestyle, and coronary disease. N Engl J Med 2016; 375: 2349−58. 10 Stefan N, Häring HU, Hu FB, Schulze MB. Divergent associations of height with cardiometabolic disease and cancer: epidemiology, pathophysiology, and global implications. Lancet Diabetes Endocrinol 2016, 4: 457−67. 11 Eslamparast T, Tandon P, Raman M. Dietary composition independent of weight loss in the management of non-alcoholic fatty liver disease. Nutrients 2017; 9: 800. 12 Ekstedt M, Hagström H, Nasr P, et al. Fibrosis stage is the strongest predictor for disease-specific mortality in NAFLD after up to 33 years of follow-up. Hepatology 2015; 61: 1547−54. 13 Angulo P, Kleiner DE, Dam-Larsen S, et al. Liver fibrosis, but no other histologic features, is associated with long-term outcomes of patients with nonalcoholic fatty liver disease. Gastroenterology 2015; 149: 389−97. 14 Yki-Järvinen H. Non-alcoholic fatty liver disease as a cause and a consequence of metabolic syndrome. Lancet Diabetes Endocrinol 2014; 2: 901−10.

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